TY - JOUR
AB - HDL decreases MMP-9 activity in vascular smooth muscle cells via S1P3/TGFbeta cross activation M Schuchardt M Schuchardt 1 Charite - Campus Benjamin Franklin , Berlin , Germany M Toelle M Toelle 1 Charite - Campus Benjamin Franklin , Berlin , Germany T Huang T Huang 1 Charite - Campus Benjamin Franklin , Berlin , Germany A Wiedon A Wiedon 1 Charite - Campus Benjamin Franklin , Berlin , Germany M Van Der Giet M Van Der Giet 1 Charite - Campus Benjamin Franklin , Berlin , Germany Abstract Purpose: MMP-9 is the prominent isozyme of matrix metalloproteinases (MMP) known to be highly expressed in human atherosclerotic plaques. One potent inhibitor of MMP-9 production is the transforming growth factor beta (TGF-β). Similar to this, we also observed inhibitory effects with sphingosine 1-phosphate (S1P), accumulation in high density lipoprotein (HDL), which presumably suppresses MMP-9 production. The aim of this study was to reveal the inhibitory influence of HDL on MMP-9 production and the molecular mechanisms behind these processes. Methods: Rat vascular smooth muscle cells (VSMC) were stimulated with interleukin-1β (IL-1β). The following MMP-9 expression was quantified by real-time PCR and activity by zymography. For receptor knockdown studies, cells were transfected with siRNA against S1P3 receptor. Results: TGF-β significantly down-regulated IL-1β stimulated MMP-9 expression (78.6% ± 5.4 decrease; p < 0.05, n = 10). This inhibition could be significantly blocked with the TGF-β receptor inhibitor R1 (36.7% ± 2.9 vs. 78.6% ± 5.4 decrease; p < 0.05; n = 9). Like S1P, HDL also significantly suppressed the IL-1β stimulated MMP-9 expression (S1P: 42.2% ± 8.3 decrease n = 5; HDL: 44.3% ± 9.8 decrease; n = 10; p < 0.05). To reveal if there is any cross-communication between the TGF-β and S1P receptors, we also tested the TGF-β receptor inhibitor in the IL-1β/HDL and IL-1β/S1P costimulation, which significantly reversed the effect of HDL (12.2 % ± 4.6 vs. 44.3 % ± 2.4 decrease; p < 0.05; n = 7) and S1P (42.2% ± 3.5 vs. 72.3% ± 8.3 decrease; p < 0.05; n = 7). Investigation of the MMP-9 activity via zymography showed corresponding results after TGF-β and S1P treatment in the absence and presence of the inhibitor. The specific downregulation of the S1P3 receptor by siRNA abolished the HDL/S1P induced TGF-β signaling. Conclusions: HDL, S1P and TGF-β all down-regulate MMP-9 expression. The HDL and S1P mediated inhibition of MMP-9 expression could be diminished by a TGF-β receptor inhibitor and by S1P3 receptor knockdown. Therefore, HDL and its component S1P could cross activate the TGF-β signaltransduction pathway via the S1P3 receptor. Regulation of wnt5a signalling in VSMC by oxidative stress: consequences for VSMC survival C Mill C Mill 1 University Hospitals Bristol NHS FoundationTrust, Bristol Heart Institute Hospital , Bristol , United Kingdom SJ George SJ George 1 University Hospitals Bristol NHS FoundationTrust, Bristol Heart Institute Hospital , Bristol , United Kingdom JY Jeremy JY Jeremy 1 University Hospitals Bristol NHS FoundationTrust, Bristol Heart Institute Hospital , Bristol , United Kingdom Abstract Apoptosis of vascular smooth muscle cells (VSMCs) within the fibrous cap of atherosclerotic plaque causes cap thinning, which increases the incidence of plaque rupture and thereby myocardial infarction. Identification of novel mechanisms that regulate VSMC survival may be useful for the design of new treatments for increasing plaque stability. Wnt/β-catenin signalling modulates cell survival in a number of cell types however its role in VSMCs and atherosclerosis is relatively unstudied. Although we and others have observed Wnt5a protein in atherosclerotic plaques, the precise role of Wnt5a in atherosclerosis is unknown. We hypothesized that Wnt/β-catenin signalling may inhibit VSMC apoptosis induced by oxidative stress (a known inducer of apoptosis in atherosclerotic plaques). In this study treatment of mouse aortic VSMCs with 10nM Wnt5a significantly increased Wnt/β-catenin/TCF signalling by 3.4 ± 1.2 fold (n = 3, p < 0.05). This was confirmed by Western blotting for active-β-catenin and by an approximate 6-fold increase in nuclear translocation of β-catenin (n = 8, p < 0.05). Treatment of VSMCs with 100uM H2O2 to induce oxidative stress did not significantly decrease Wnt5a-induced nuclear translocation of β-catenin, even though Wnt/β-catenin/TCF signalling was reduced by 91.8 ± 19%. Treatment with Wnt5a but not Wnt5a in the presence of H2O2 significantly increased the mRNA expression of three anti-apoptotic Wnt/β-catenin regulated genes (IGF-1, WISP-2 and Survivin, 2.4 ± 0.6, 2.0 ± 0.4, 2.0 ± 0.3 fold respectively (n = 10, p < 0.05)) and this was confirmed by Western blotting. Addition of H2O2 significantly increased VSMC apoptosis by 45 ± 7% (n = 3, p < 0.05). Wnt5a significantly abolished the pro-apoptotic effect of H2O2 restoring VSMC viability to control levels. This effect was reversed using siRNA knockdown of the Wnt canonical co-receptors LRP5 and LRP6, confirming a critical role for canonical Wnt5a/β-catenin signalling in VSMC survival. In summary, we propose that Wnt5a can negate the induction of apoptosis by oxidative stress in VSMCs through canonical Wnt/β-catenin signalling (but this is independent of IGF-1, WISP-2 and Survivin expression). These findings may be important for the regulation of VSMC apoptosis within plaques, and thereby plaque stability. Deletion of the CaMK4 gene in mice determines a hypertensive phenotype. G Santulli G Santulli 1 University Hospital Federico II , Naples , Italy M Illario M Illario 2 University of Naples Federico II, Department of Biology, Cellular & Molecular Biology , Naples , Italy E Cipolletta E Cipolletta 1 University Hospital Federico II , Naples , Italy D Sorriento D Sorriento 1 University Hospital Federico II , Naples , Italy C Del Giudice C Del Giudice 1 University Hospital Federico II , Naples , Italy A Anastasio A Anastasio 1 University Hospital Federico II , Naples , Italy B Trimarco B Trimarco 1 University Hospital Federico II , Naples , Italy G Iaccarino G Iaccarino 1 University Hospital Federico II , Naples , Italy Abstract Calcium/calmodulin-dependent kinase 4 (CaMK4) belongs to the CaMK family and is expressed in selected cellular types, where it regulates calcium-dependent signaling. In particular, we recently showed its presence in the endothelium, suggesting a yet undisclosed role for this kinase in the regulation of vascular function. Furthermore, Genome-Wide Analysis (GWA) showed an association between elevated blood pressure (BP) and the rs10491334 T/C Single Nucleotide Polymorphism of human CaMK4 gene. To evaluate the role of CaMK4 in BP homeostasis, we performed an ultrasound (US, VeVo, VISUALSONICS) cardiac evaluation and an invasive (MILLAR) arterial BP measurement in mice with deletion of the CaMK4 gene (CaMK4−/−, n = 19) and wild type littermates (WT, n = 16). Vasorelaxant responses to Acetylcholine (Ach, 10 nM to 10 µM), Isoproterenol (Iso, 1 nM to 1 µM) and Nitroprusside (Np, 1 nM to 10 µM) were assessed on ex vivo aortic rings preparations, pre-constricted with phenylephrine (PE, 1 µM). CaMK4−/− presented BP levels (Systolic BP 123 ± 0.7 mmHg, Diastolic BP 90 ± 0.3 mmHg) significantly higher (ANOVA, p < 0.05) than WT (Systolic BP 110 ± 0.6, Diastolic BP 81 ± 0.3 mmHg). Interestingly, the increase in BP levels paralleled the development of cardiac size (CaMK4−/− vs WT: Heart weight/body ratio 5.5 ± 0.3 vs 4.24 ± 0.2; Heart weight/tibia lenght: 7.4 ± 0.4 vs 5.7 ± 0.3;p < 0.05), with worsening of US assessed cardiac function (CaMK4−/− vs WT: Shortening Fraction 0.36 ± 0.06 vs 0.42 ± 0.04; EndDiastolic Diameter 4.2 ± 0.3 vs 3.3 ± 0.4;p < 0.05). CaMK4−/− vessels showed an attenuated endothelium-dependent vasorelaxation to ACh (% max vasodilation; CaMK4−/− vs WT: 23.7 ± 0.05 vs 33.8 ± 0.03; p < 0.05) and to Iso (24.2 ± 0.07 vs 55.8 ± 0.05; p < 0.05), while no differences were observed in endothelium independent vasorelaxant responses to Np (not shown). We performed in vitro studies, too. In cultured endothelial cells isolated from the aorta of WT mice, CaMK4 co-immunoprecipitates with eNOS and phosphorylates it in Ser 1177 in response to ionomycin or VEGF. These responses are lost in CaMk4−/− endothelial cells. Furthermore, we found an increased production of Reactive Oxygen Species (ROS) in CaMK4−/− endothelial cells, that is typical of endothelial dysfunction. So, we conclude that CaMK4 participates in BP homeostasis through the control of eNOS activity. This could be the underlying mechanism for the association in humans of CaMK4 gene polymorphism and essential hypertension. Distinct and well distinguishable phenotypes after cell-specific deletion of Cx40 in endothelial and renin-producing cells A Jobs A Jobs 1 University of Lübeck , Lübeck , Germany C Wagner C Wagner 2 University of Regensburg , Regensburg , Germany A Kurtz A Kurtz 2 University of Regensburg , Regensburg , Germany C De Wit C De Wit 1 University of Lübeck , Lübeck , Germany Abstract Purpose: The gap junction protein connexin40 (Cx40) is important in cardiovascular and renal physiology as concluded from mice globally deficient for Cx40. These mice exhibit pronounced hypertension, altered negative feedback of renin secretion causing an increased plasma renin activity, and impaired conduction of vasodilator signals along the endothelium. However, it is unknown whether the loss of Cx40 in endothelial or renin-producing cells causes this phenotype. Methods: We interbred mice carrying a floxed Cx40 gene with those expressing the Cre recombinase driven by either the Tie2 or renin promoter, generating an endothelial cell (EC) or renin-producing cell (RPC) specific Cx40 knockout, respectively. In these mice we studied conducted responses of arterioles in the cremaster muscle by means of intravital microscopy, blood pressure using a telemetric system and the expression of Cx37 and Cx40 in vessels by immunohistochemistry. Results: Locally confined stimulation using acetylcholine, bradykinin or adenosine elicited almost instantly a vasodilation at the local site in all genotypes (control, EC-Cx40ko, RPC-Cx40ko). For acetylcholine and bradykinin dilations were also observed at upstream sites in a distance of 600 μm and 1200 μm without significant decline in amplitude and without delay in control animals and RPC-Cx40ko. However, in EC-Cx40ko the amplitude of the dilation was significantly attenuated at 1200 μm upstream compared to the local site in the case of acetylcholine (by 28 ± 3 %, P < 0.001) and bradykinin (by 31 ± 5 %, P < 0.001). In contrast, adenosine induced dilations declined similarly in all groups with increasing distance. Interestingly, mice lacking Cx40 in the endothelium did not exhibit an elevation of blood pressure (111 ± 4 mmHg versus control: 109 ± 3 mmHg, P=n.s.). However, mice deficient for Cx40 in renin-producing cells were hypertensive (139 ± 5 mmHg, P < 0.001), which was associated with an elevated heart weight. Cx37 was not retrieved in arteriolar endothelium in EC-Cx40ko, whereas it was abundantly expressed in controls and RPC-Cx40ko. Conclusion: These data demonstrate that endothelial Cx40 is important for the conduction of endothelium-dependent dilations but its loss does not cause an increase in blood pressure. Conversely, elevated pressure in RPC-Cx40ko does not affect the conduction of locally induced dilations. Surprisingly, Cx40 is required for the correct positioning of Cx37 in the plasma membrane of arterioles. Generally, Cx40 serves in different tissues specific functions and its loss causes distinguishable phenotypes between cell-specific knockout mice. ADMA by activating arteriolar renin angiotensin system elicits release of ROS and constriction A Koller A Koller 1 Dept of Physiology, Dept of Pathophysiology , Hungary Abstract There is growing evidence that ADMA, is markedly elevated in many cardiovascular diseases and recognized as a potential risk factor for cardiovascular events, such as hypertension, hyperhomocysteinemia, coronary artery disease, peripheral arterial occlusive disease, chronic heart failure, pulmonary hypertension, end-stage renal disease and preeclampsia. Yet, the underlying mechanisms have not yet been clarified. Although it seems logical that ADMA inhibits nitric oxide synthase (NOS) resulting in reduced production of NO recent studies however, proposed that ADMA may activate other mechanisms, as well. Previously we showed that elevated level of ADMA increased the release of reactive oxygen species (ROS), which reduced NO-mediation of flow dependent dilation of arterioles. In the present study, we hypothesized that ADMA activates the vascular renin angiotensin system - NAD(P)H oxidase pathway, which - by increasing ROS production - impairs NO mediated vasomotor responses. Presence of ADMA reduced flow-induced dilations of isolated rat gracilis muscle arterioles (∼160 μm at 80 mmHg, ADMA: 10-4 mol/L, in the presence of indomethacin), which was not restored by L-arginine (10-4 mol/L), but was prevented by superoxide-dismutase and catalase (SOD/CAT). Both ADMA and pyrogallol (known to generate superoxide shown by lucigenin-enhanced chemiluminescence) reduced arteriolar dilations to the NO donor, sodium nitroprusside (SNP), which was partially restored by SOD/CAT. Also, ADMA-induced alterations were prevented by SOD/CAT and the NAD(P)H oxidase inhibitor apocynin. Collectively, the novel finding of the present study is that ADMA interferes with the bioavailability of NO released from NO donors, in addition to what is released to increases in flow, suggesting that the primary action of ADMA is not related to eNOS rather by simulating RAS-dependent, AT1 receptor - NAD(P)H oxidase pathway, which elicits production of ROS. This pathomechanism may contribute to cardiovascular dysfunctions associated with elevated levels of ADMA. Grants: (OTKA) K71591, T67984 and F. Aff. 0855910D. In vivo significance of uncoupled eNOS for the development of endothelial dysfunction appears to be overestimated T Suvorava T Suvorava 1 Institute of Pharmacology and Clinical Pharmacology, University Hospital , Duesseldorf , Germany M Weber M Weber 2 Emory University School of Medicine, Division of Cardiology , Atlanta , United States of America VT Dao VT Dao 1 Institute of Pharmacology and Clinical Pharmacology, University Hospital , Duesseldorf , Germany G Kojda G Kojda 1 Institute of Pharmacology and Clinical Pharmacology, University Hospital , Duesseldorf , Germany Abstract Under certain conditions endothelial NO-synthase (eNOS) can exhibit NADPH oxidase activity to produce superoxide instead of NO, a process known as "uncoupling". Whether uncoupling of eNOS promotes cardiovascular pathology or is the consequences of the disease state is an important but unanswered question. We have generated a mutant of bovine eNOS in which cysteine 101 was replaced by alanine (C101A) resulting in uncoupling of eNOS. The effects of C101A mutation have been characterized in human embryonic kidney cells (HEK) transfected with either mutant eNOS (eNOSmu) or wild type eNOS. Transgenic mice carrying eNOSmu have been generated on a C57Bl/6 background using the endothelium-specific Tie-2 promotor. By breeding these mice with eNOS knockouts (eNOS−/−), mice that only express eNOSmu (eNOS−/−/eNOSmu+) were obtained. In vitro studies revealed typical features of uncoupled eNOS in cells transfected with eNOSmu, such as decreased eNOS activity, increased superoxide generation inhibited by L-nitroarginine (L-NA), less NO production, elevated protein oxidation and decreased eNOS dimer formation. eNOS−/−/eNOSmu+ have hypertension (138 ± 2.3 mmHg, n = 14) as compared to C57Bl/6 (p < 0.05, 118.4 ± 3.1 mmHg, n = 8), but not to eNOS−/− (135.5 ± 2.8, n = 14). Endothelial specific overexpression of uncoupled eNOS induced a large increase in superoxide and peroxynitrite formation in eNOS−/−/eNOSmu+, which was abolished by NOS-inhibitor L-NA suggesting that uncoupled eNOS is a source of elevated vascular superoxide and peroxynitrite generation. Western blot analysis confirmed the expression of eNOS in eNOS−/−/eNOSmu+ (37.1 ± 8.4 %, n = 9) in aorta and revealed an increased phosphorylation of eNOS at Ser1179 (470 ± 137 %) as compared to C57Bl/6 (p < 0.05, n = 8). Surprisingly, introduction of eNOSmu into eNOS−/− almost completely restored endothelium-dependent relaxation. There was only slight but significant rightward shift in acetylcholine dose-response curve in eNOS−/−/eNOSmu+ (p = 0.0062 vs C57Bl/6, n = 12). Experiments with L-NA and ODQ (soluble guanylyl cyclase (sGC) inhibitor) showed that endothelium-dependent relaxation in eNOS−/−/eNOSmu+ is eNOS- and cGMP-dependent. Scavenging of H2O2 by catalase revealed that endothelium-dependent relaxation in eNOS−/−/eNOSmu+ is not mediated by eNOS-catalyzed production of oxygen-derived free radicals. In striking contrast, application of NO-scavenger Fe(DETC)2 completely abolished endothelium-dependent relaxation strongly suggesting that relaxation is mediated by NO. Conclusion: In vivo significance of uncoupled eNOS for the development of endothelial dysfunction appears to be overestimated. Role of Wnt pathway in regulation of VSMC proliferation and intimal thickening A Tsaousi A Tsaousi 1 University of Bristol , Bristol , United Kingdom C Lyon C Lyon 1 University of Bristol , Bristol , United Kingdom H Williams H Williams 1 University of Bristol , Bristol , United Kingdom SJ George SJ George 1 University of Bristol , Bristol , United Kingdom Abstract Vascular smooth muscle cell (VSMC) proliferation causes intimal thickening in atherosclerosis and restenosis. We previously demonstrated that VSMC proliferation in vitro is stimulated via Wnt/beta-catenin signalling and cyclin D1 up-regulation. We aimed to determine which of the 19 known Wnts modulate VSMC proliferation. We investigated Wnt mRNA expression in quiescent and proliferating (20ng/mL PDGF for 6 hours) mouse aortic VSMCs. Microarray analysis (n = 3) on pooled VSMC mRNA detected Wnt2, 4, 6 and 10b. Quantitative-PCR demonstrated a significant increase in Wnt2 and Wnt4 mRNA in proliferating compared to quiescent VSMCs (10.8 ± 2.7 and 3.1 ± 0.5 fold, respectively, p < 0.05). Western blotting revealed this increase was only translated into protein for Wnt4 (0.46 ± 0.03 versus 0.21 ± 0.03 O.D.xmm2, n = 3, p < 0.05). This was confirmed by immunohistochemistry. Interestingly, specific siRNA knockdown (>50%) of Wnt4, but not Wnt2, significantly reduced VSMC proliferation by 54 ± 9%, n = 4. To directly compare the ability of Wnt4 versus Wnt2 protein to stimulate proliferation, quiescent VSMCs were treated with a range of concentrations (20-500ng/mL) of recombinant Wnt4 or Wnt2 protein and VSMC proliferation was assessed by bromodeoxyuridine incorporation. Wnt4, but not Wnt2, significantly increased VSMC proliferation in a dose-dependent manner with 500ng/mL of Wnt4 increasing proliferation from 1 ± 1% to 46 ± 6%, n = 4, p < 0.05). Combined treatment with 500ng/mL Wnt2 and 500ng/mL Wnt4 did not induce a synergistic effect. Immunohistochemistry revealed increased Wnt4 protein expression correlated with VSMC proliferation and cyclin D1 expression (p < 0.05 and p < 0.001, respectively, n = 18) during intimal thickening in ligated mouse and injured rat carotid arteries. Together our results show that Wnt4 stimulates VSMC proliferation and thereby contributes to intimal thickening. Fluid shear stress stimulates A kinase anchoring protein (AKAP) 12 expression and determines its intracellular localisation in human endothelial cells N Barth N Barth 1 J.W. Goethe-University, Institute for Vascular Signalling , Frankfurt/M. , Germany AE Loot AE Loot 1 J.W. Goethe-University, Institute for Vascular Signalling , Frankfurt/M. , Germany I Fleming I Fleming 1 J.W. Goethe-University, Institute for Vascular Signalling , Frankfurt/M. , Germany Abstract Continuous fluid shear stress promotes an anti-atherosclerotic endothelial phenotype, whereas areas exposed to low or oscillatory shear stress are associated with a more activated endothelial phenotype. AKAP12 is a large scaffolding protein that can integrate various signalling cascades by tethering the protein kinases A and C, as well as several phosphatases. AKAP12 is reported to promote quiescence in various cell types, but little is known about its role in the endothelium. For this reason we aimed to determine whether or not fluid shear stress influences the expression and function of AKAP12 in endothelial cells. Exposure of primary human umbilical vein endothelial cells to fluid shear stress (12 dyne/cm2) for 24 hours markedly (2.6 fold, P < 0.01) increased AKAP12 mRNA expression as assessed by RT-qPCR. Western blot analysis of cells subjected to prolonged (48-72 hours) exposure to fluid shear stress also showed a clear continuous increase in AKAP12 protein levels. Furthermore, we studied the effect of fluid shear stress on the intracellular AKAP12 localisation by immunofluorescence. In endothelial cells cultured under static conditions, AKAP12 was mainly concentrated in the perinuclear region. After exposure to fluid shear stress for 24 hours, the cells adopted a spindle shaped conformation with AKAP12 expression at the cell-cell junctions. The shear stress-dependent relocalisation of AKAP12 was prevented by the PKA inhibitor H89 (10 μM). To study the in vivo effect of shear stress on subcellular AKAP12 distribution, we performed en face staining for AKAP12 and PECAM-1 in murine aortae. In regions associated with high laminar flow such as the outer curvature of the aortic arch, AKAP12 was localised to the cell margins of spindle shaped endothelial cells. In the inner curvature of the aortic arch, an area predominantly exposed to oscillatory flow, the endothelial cells where irregularly shaped (cobble stone morphology). In these cells, AKAP12 was only detected in the perinuclear area. Taken together, our findings indicate that fluid shear stress enhances the endothelial expression of AKAP12 and its PKA-dependent localisation at cell-cell junctions, both in vitro and in vivo. These findings suggest a role for AKAP12 in the long-term responses to shear stress. Sphingosine-1 phosphate receptor 3 promotes macrophage recruitment to atherosclerotic lesions P Keul P Keul 1 University of Essen Medical School, Institute of Pathophysiology , Essen , Germany SL Lucke SL Lucke 1 University of Essen Medical School, Institute of Pathophysiology , Essen , Germany M Graeler M Graeler 2 Hannover Medical School , Hannover , Germany G Heusch G Heusch 1 University of Essen Medical School, Institute of Pathophysiology , Essen , Germany B Levkau B Levkau 1 University of Essen Medical School, Institute of Pathophysiology , Essen , Germany Abstract The causal role of the bioactive sphingolipid sphingosine 1-phosphate (S1P) in atherosclerosis is unknown. We addressed this by examining atherosclerotic lesion development in mice deficient for the S1P receptor 3 (S1P3−/−/) crossbred on an ApoE −/− background. Atherosclerotic lesion volume in the brachiocephalic artery of S1P3−/−/ApoE −/− double knockout mice fed a normal chow diet for 45 weeks was similar to that in ApoE−/− mice. However, the lesions had a dramatically reduced macrophage content (24% of ApoE−/−) and increased smooth muscle cell content (174% of ApoE−/−). To search for defects in macrophage recruitment in S1P3-deficient mice, we monitored macrophage-driven inflammation in the thioglycolate-induced peritonitis model. In this model, we observed a 38% reduction in the numbers of elicited F4/80-positive macrophages in S1P3-deficient mice compared to controls. S1P3−/−macrophages exhibited an altered cytokine signature with substantially reduced TNFβ and MCP-1 mRNA expression levels. S1P concentrations in the peritoneal cavity rapidly increased with a maximum at 48 hours, and elimination of the S1P gradient by application of the S1P analogue FTY720 resulted in an inhibition of macrophage recruitment into the peritoneal cavity. In Boyden chamber experiments, S1P was chemotactic for macrophages from control but not S1P3-deficient mice. We also observed that the S1P-synthesizing enzyme sphingosine kinase 1 was abundantly expressed in macrophages in atherosclerotic lesions. Finally, atherosclerosis studies in bone marrow chimeras showed that S1P3−/− in hematopoetic cells is important for macrophage recruitment to atherosclerotic lesions. These data suggest that S1P acts as a chemoattractant for macrophages via S1P3, and that absence of S1P3 alters the cytokine profile of monocyte/macrophages and their recruitment to lesions of atherosclerosis. Macrophage Inhibitory Cytokine-1 deficiency protects against atherosclerosis by attenuating MCP-1 dependent macrophage chemotaxis EAL Biessen EAL Biessen 1 Cardiovascular Research Institute Maastricht (CARIM) , Maastricht , Netherlands SCA De Jager SCA De Jager 2 Leiden University , Leiden , Netherlands B Bermudez-Pulgarin B Bermudez-Pulgarin 1 Cardiovascular Research Institute Maastricht (CARIM) , Maastricht , Netherlands I Bot I Bot 2 Leiden University , Leiden , Netherlands R Abia R Abia 3 CSIC , Sevilla , Spain THJC Van Berkel THJC Van Berkel 2 Leiden University , Leiden , Netherlands Abstract Macrophage inhibitory cytokine-1 (MIC-1) is a member of the transforming growth factor β (TGFβ) superfamily, which operates in acute phase responses through a currently unknown receptor. Elevated MIC-1 serum levels were recently identified as a risk factor for acute coronary syndromes. This led us to investigate the role of this cytokine on macrophage function in the context of atherogenesis. MIC-1−/−/LDLr−/− bone marrow chimeras showed markedly impaired initial lesion formation but unaltered plaque progression in the aortic sinus. Advanced plaques of MIC-1−/− chimeras nevertheless had significantly reduced macrophage infiltrates, increased collagen deposition and decreased necrotic core formation, all features of improved plaque stability. In vitro studies pointed to a prominent, regulatory role of MIC-1 in macrophage cell cycle homeostasis and necrosis but not in apoptosis or apoptotic cell handling. MIC-1 dependent cell cycle regulation was mediated through TGFbRII and SMAD3. Moreover, our studies reveal a direct link between MIC-1 and CCR2 dependent chemotaxis. MIC-1−/− macrophages displayed reduced CCR2 expression and a mitigated migratory reponse. Conversely MIC-1 induced macrophage chemotaxis in a strictly CCR2 dependent manner by attenuating TGFβRII induced GRK2 desensitization. This effect only manifests under conditions of injury and inflammation such as in atherosclerosis as CCR2 dependent stromal retention of monocytes was not altered. Collectively, our data indicate that MIC-1 exerts its pro-atherogenic effects by amplifying the MCP-1/CCR2 axis and modulating TGFβRII signaling. Our study thus unveils MIC-1 as an acute phase modifier of CCR2/TGFβ-RII mediated inflammatory responses to vascular injury. The cardiac specific beta-catenin interactor FHL2 promotes cardiac differentiaton in vitro. A Renger A Renger 1 Max Delbruck Center for Molecular Medicine , Berlin , Germany C Noack C Noack 2 Charite - Campus Berlin Buch/Experimental & Clinical Research Center, Department of Cardiology , Berlin , Germany MP Zafiriou MP Zafiriou 2 Charite - Campus Berlin Buch/Experimental & Clinical Research Center, Department of Cardiology , Berlin , Germany R Dietz R Dietz 2 Charite - Campus Berlin Buch/Experimental & Clinical Research Center, Department of Cardiology , Berlin , Germany MW Bergmann MW Bergmann 3 Asklepios Clinic St. Georg , Hamburg , Germany LC Zelarayan LC Zelarayan 1 Max Delbruck Center for Molecular Medicine , Berlin , Germany Abstract The dissection of signaling pathways involved in cardiac remodeling may lead to novel targets for heart failure therapy. Different factors involved in embryonic cardiac development are proposed to play a role in left-ventricular remodeling in the adult heart. Beta-catenin has an important role during heart development, where its regulation is essential for early embryonic heart development. Previously findings of the group showed that downregulation of beta-catenin is required for adaptive hypertrophy preserving left ventricular function in the adult heart and enhanced cardiac endogenous progenitor cell differentiation post-infarct. Since beta-catenin is an ubiquitously expressed protein in the adult organisms, we aimed to identify cardiac-specific interaction partners of beta-catenin able to modulate its transcriptional activity. Employing a human cardiac cDNA library we performed a Yeast Two-Hybrid screen using beta-catenin as bait and found the already identified four-and-a-half LIM-protein 2 (FHL2) to interact with beta-catenin in adult cardiac tissue. The specificity of the interaction was confirmed by co-immunoprecipitation and immunofluorescence assays employing H9C2 cells and neonatal cardiomyocytes. The reportergene assay, TOPflash/FOPflash-system, revealed a cardiac-specific repression of beta-catenin-mediated gene transcription in different cardiac cell lineages. Next, we observed that over-expression of FHL2 in P19 cells resulted in a significant downregulation of beta-catenin (p < 0.05) and its target gene expression (TCF4 p < 0.05; LEF1 p < 0.01; Axin2 p < 0.001) finally leading to accelerated formation of beating embryoid bodies (EB). EB originated from FHL2-transfected P19 cells, showed significant downregulation of undifferentiated markers such as the pluripotent cell lineage marker Oct3/4 (p < 0.05) and the cardiac cell lineage MesP1 (p < 0.01). Expression of Nkx2.5 and Troponin T were significantly up-regulated as demonstrated by real time PCR (p < 0.05) and immunofluorescence analysis (p < 0.05). The formation of beating areas were observed earlier in FHL2-transfected P19 cells and significantly increased in number (p < 0.001). In summary, we found FHL2, as a cardiac interaction partner of beta-catenin, to decrease beta-catenin-LEF/TCF-induced gene expression and to promote cardiac differentiation in vitro. As FHL2 is specifically abundantly expressed in the mouse and human cardiovascular system, it makes FHL2 a promising target for cardiac-specific gene regulation to improve cardiac remodeling after injury. Human beta-3 adrenoreceptor over-expression inhibits cardiac hypertrophy in vitro and in vivo JL Hammond JL Hammond 1 Université Catholique de Louvain , Brussels , Belgium J Hamelet J Hamelet 1 Université Catholique de Louvain , Brussels , Belgium T Van Assche T Van Assche 1 Université Catholique de Louvain , Brussels , Belgium C Belge C Belge 1 Université Catholique de Louvain , Brussels , Belgium A Vanderper A Vanderper 2 Katholieke Universiteit Leuven , Leuven , Belgium D Langin D Langin 3 Inserm U858, Institut of Molecular Medicine Rangueil (I2MR) , Toulouse , France P Herijgers P Herijgers 2 Katholieke Universiteit Leuven , Leuven , Belgium JL Balligand JL Balligand 1 Université Catholique de Louvain , Brussels , Belgium Abstract Background: Beta3-adrenoreceptors (AR) have been identified in human cardiac myocytes. Their acute activation has been associated with nitric oxide (NO) synthesis; however, their role in cardiac function/remodelling is unclear. Methods: Phenotypical and functional analyses were carried out in wild type (WT) and heterozygote transgenic male mice with cardiac myocyte-specific over-expression of human beta3-AR (hB3TG) treated for 10 days with isoproterenol (Iso) or saline by osmotic minipump infusion (30 mg/kg/day) or repetitive i-p injection (50 mg/kg/day). Some mice were concurrently treated with L-NAME in drinking water (2 mg/mL). In vitro hypertrophic responses to phenylephrine (PE) and Iso were analyzed in neonatal rat ventricular myocytes (NRVM) infected with a recombinant adenovirus expressing the human beta3 AR (AdV:hB3AR). Results: LV function at baseline and after acute infusion of graded doses of Iso was similar between WT and hB3TG (DP/dt max (mmHG/sec): baseline WT 5894 +/− 614 vs hB3TG 8036 +/−1332; p = ns; after Iso: 9803+/− 119 vs 10099+/− 1416; p = ns); LV function was also comparable after chronic Iso treatment. Contrary to WT (81.0 +/− 3.4 mg/TL (Iso) vs. 67.1 +/− 2.8 mg/TL (saline); p < 0.05), Iso-treated hB3TG mice did not develop LV hypertrophy by morphometric (64.7+/−1.8 mg/TL (Iso) vs 65.8 +/− 2.6 mg/TL (saline; p = ns) or echocardiographic (Delta LV mass/TL: 0.4+/− 0.14 (Iso) vs 0.10+/− 0.07 mg/mm (saline); p = 0.078) analyses. L-NAME treatment abrogated the protection from Iso-induced hypertrophy in hB3TG mice, whilst not causing hypertrophy itself. Likewise, NRVMs infected with AdV:hB3AR did not hypertrophy in response to PE or Iso, (1195 μm2 (control) vs 1252 μm2 (PE) vs 1175 μm2 (Iso): p = ns), in contrast to NRVMs infected with control AdV:GFP (1247 μm2 (control) vs 1521 μm2 (PE) vs 1381 μm2 (Iso): p < 0.001). The protection from PE-induced hypertrophy in AdV:hB3AR NRVMs was prevented by concurrent treatment with either L-NAME or KT5823 (KT - a protein kinase G inhibitor) (1187 μm2 (L-NAME alone) vs 1503 μm2 (L-NAME + PE): p < 0.001; 1273 μm2 (KT alone) vs 1439 μm2 (KT + PE): p < 0.05). In acute signalling experiments, increases in PLB and ERK phosphorylations after Iso and PE stimulation respectively were similar in AdV:GFP and AdV:hB3AR NRVMs. Conclusion: Cardiac-specific over-expression of Beta3-AR inhibits the hypertrophic response to chronic AR stimulation in vivo and in vitro, without altering LV function, in part through a NO-dependent mechanism. Cardiac Beta3 ARs may be a therapeutic target in the treatment of hypertrophic cardiomyopathies associated with elevated sympathetic tone. A novel lamin A/C mutation in hypertrophic cardiomyopathy illustrates the diversity of cardiomyopathy phenotypes in the laminopathies A Perrot A Perrot 1 Charite - Campus Berlin Buch/Experimental & Clinical Research Center , Berlin , Germany M Neubert M Neubert 1 Charite - Campus Berlin Buch/Experimental & Clinical Research Center , Berlin , Germany R Dietz R Dietz 1 Charite - Campus Berlin Buch/Experimental & Clinical Research Center , Berlin , Germany MG Posch MG Posch 2 German Heart Center Berlin , Berlin , Germany C Oezcelik C Oezcelik 1 Charite - Campus Berlin Buch/Experimental & Clinical Research Center , Berlin , Germany Abstract Introduction: The laminopathies are a diverse group of diseases caused by mutations in the LMNA gene encoding the nuclear envelope proteins lamin A and lamin C. At least 13 distinct phenotypes including myopathies, lipodystrophy, and neuropathy were described. Further, LMNA is the most frequent disease gene in familial dilated cardiomyopathy (fDCM). Methods: We screened the 12 coding exons of LMNA in a cohort of 50 patients with hypertrophic cardiomyopathy (HCM) using direct sequencing (ABI chemistry). These patients were negative for the main HCM disease genes like MYH7 and MYBPC3. Results: Besides a number of genetic polymorphisms, we identified one novel non-synonymous C>T variant in exon 7 in one HCM patient. The heterozygous mutation leads to an alanine to valine substitution at codon 441 of both lamin proteins (Ala441Val). The affected amino acid is highly conserved among different species. It is located in the Ig-like domain which is involved in LAP2-alpha and emerin-binding. The mutation was excluded in 300 control subjects without cardiomyopathy. The symptomatic male patient has a severe LV hypertrophy (IVS = 23 mm; PWT = 15 mm) and preserved systolic function. Further, he showed a complete right bundle branch block and ventricular tachycardia. This is of special interest because arrhythmias and conduction abnormalities are typical for LMNA mutation carriers with fDCM. Unfortunately, no data about the family were available until now. Conclusion: We describe here the first LMNA mutation in hypertrophic cardiomyopathy. This underlines the notion that the cardiomyopathies are true allelic diseases. Further, it is a novel aspect of the extensive phenotypic diversity of the laminopathies. Mutations in sarcomeric genes in patients with familial atrial septal defects MG Posch MG Posch 1 Charite - Campus Berlin Buch/Experimental & Clinical Research Center , Berlin , Germany S Waldmuller S Waldmuller 2 Institute for Heart and Circulation Research , Dortmund , Germany A Perrot A Perrot 1 Charite - Campus Berlin Buch/Experimental & Clinical Research Center , Berlin , Germany F Berger F Berger 3 German Heart Center Berlin , Berlin , Germany T Scheffold T Scheffold 2 Institute for Heart and Circulation Research , Dortmund , Germany P Bouvagnet P Bouvagnet 4 University Hospital of Lyon , Lyon , France C Ozcelik C Ozcelik 1 Charite - Campus Berlin Buch/Experimental & Clinical Research Center , Berlin , Germany Abstract Introduction: Congenital heart defects (CHD) are the most common inborn malformation. Ostium secundum atrial septal defects (ASDII) account for 10% of all CHD and show familial recurrence in approximately 8%. Mutations in cardiac transcription factors constitute a genetic source for inherited ASDII. Yet, the role of mutations in sarcomeric genes in pathogenesis of familial ASDII is less well defined. Methods: Sixteen patients with a proven familial aetiology of ASDII were included in the study. The number of affected individuals per family ranged from 2 to 12 (mean 3.7). Mutations in NKX2.5, GATA4, TBX20 and ZIC3 were excluded in all probands. Array based resequencing was used to analyze all exonic regions of thirteen genes. The HCM2 array (50KB) covers the coding region of ten sarcomeric genes (TCAP, TNNI3, MYH6, TPM1, MYL2, CSRP3, ACTC1, MYL3, TNNC1, TTN kinase region) and three non-sarcomeric genes (GLA, CRYAB, PRKAG2). The accuracy of the HCM2 array has previously shown to exceed 99%. Results: Five missense mutations in MYH6 (2), ACTC1 (1), TTN (1) and GLA (1) as well as one frameshift mutation in PRKAG2 were identified in five probands with familial ASDII. All mutations lie within regions highly conserved among species were not reported before. Moreover, four mutations that alter regulatory sequences in the MYH6 region were identified in four patients. Two ASDII probands were harbouring two mutations (compound heterozygous). Seven index patients were free of any mutation in the genes covered by the HCM2 array. Conclusion: In this study sarcomeric gene mutations were frequently found among patients with familial ASDII. Particularly, mutations in MYH6 may represent an important genetic origin for familial recurrence of ASDII. Functional studies remain to be done to investigate mechanistic consequences. Higher plasma levels of TIMP-1 among Marfan Syndrome patients with previous aortic root replacement: a marker of improved proteolytic balance? A Lebreiro A Lebreiro 1 Sao Joao Hospital , Porto , Portugal E Martins E Martins 1 Sao Joao Hospital , Porto , Portugal P Lourenco P Lourenco 1 Sao Joao Hospital , Porto , Portugal C Cruz C Cruz 1 Sao Joao Hospital , Porto , Portugal MJ Martins MJ Martins 2 University of Porto, Faculty of Medicine , Porto , Portugal P Bettencourt P Bettencourt 1 Sao Joao Hospital , Porto , Portugal MJ Maciel MJ Maciel 1 Sao Joao Hospital , Porto , Portugal C Abreu-Lima C Abreu-Lima 2 University of Porto, Faculty of Medicine , Porto , Portugal Abstract Introduction: Marfan Syndrome (MFS) is a connective tissue disorder that affects multiple organ systems. Cardiovascular system is typically involved, mainly through aortic dilatation or dissection, the principal cause of death in this population. Imbalances of the proteolytic cascade involving metalloproteinases (MMPs) and its inhibitors (TIMPs) have been implicated in aortic aneurysm formation and progression in Marfan Syndrome (MFS) patients. We aimed to investigate the balance between MMPs and TIMPs according to the severity of aortic disease and after aortic root replacement in a population of MFS patients. Methods: Venous blood samples of 30 MFS patients, all of them fulfilling Ghent criteria, were analyzed for MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2 and α2-macroglobulin. MMP and TIMP levels were measured by enzyme-linked immunosorbent assay and α2-macroglobulin levels were measured by immunonephelometry. All patients underwent a comprehensive echocardiographic evaluation. Results: In comparison with non operated patients (n = 18), those with past history of aortic root replacement (n = 12) had higher plasma levels of TIMP-1 [mean (SD) – 134.9 (28.5) vs. 108.2 (21.0) ng/mL; P=.006], and a decreased ratio of MMP-9/TIMP-1 [median (IQR) – 0.04 (0.03–0.09) vs. 0.07 (0.05–0.1); P=.03]. No significant differences were found between the levels of MMPs, TIMPs, α2-macroglobulin or the ratio MMP/TIMP when comparing patients with and without aortic root dilatation. Conclusions: Our results suggest that in aortic root replaced patients, a proteolytic shift towards a more homeostatic pattern occurs. This better TIMP-1 plasma profile may induce a less catabolic molecular milieu in the aortic wall contributing to disease stabilization. Our results give insight on mechanisms, beyond hemodynamic benefits, for the known prognostic improvement after aortic root replacement in Marfan patients, and may open new lines of investigation on the role of MMPs and TIMPs as biomarkers after aortic surgery in MFS patients. Structural changes of the specialized conducting tissue underlie SCN5A-linked hereditary Lenegre disease K Pilichou K Pilichou 1 University of Padua, Department of Biology , Padua , Italy B Bauce B Bauce 2 University of Padua, Department of Cardiac Thoracic and Vascular Sciences , Padua , Italy A Rampazzo A Rampazzo 1 University of Padua, Department of Biology , Padua , Italy E Carturan E Carturan 3 University of Padua, Department of Medico-Diagnostic Sciences and Special Therapies , Padua , Italy D Corrado D Corrado 2 University of Padua, Department of Cardiac Thoracic and Vascular Sciences , Padua , Italy G Thiene G Thiene 3 University of Padua, Department of Medico-Diagnostic Sciences and Special Therapies , Padua , Italy C Basso C Basso 3 University of Padua, Department of Medico-Diagnostic Sciences and Special Therapies , Padua , Italy Abstract Background: Progressive cardiac conduction (Lenègre) disease is one of the most common cardiac conduction disturbances due to degenerative changes of the specialized conducting tissue. It constitutes the substrate for bundle branch block and QRS complex widening leading to complete atrioventricular (AV) block at risk of syncope and sudden death. Recent studies indicate that SCN5A gene mutations account for a familial form of Lenègre disease. Material and Methods: Detailed conduction system investigation by serial section technique was performed in two unrelated index cases (M, 35; M, 40) with previous history of syncope and an in vivo diagnosis of AV conduction disturbances, who died suddenly. Full clinical evaluation through non- invasive and invasive diagnostic tools was carried out on both probands and their family members respectively. Finally, SCN5A genetic screening was undertaken in 23 family members (13M and 10F). Results: Severe fibrosis of the bifurcating His and branch bundles with sclerotic interruption of the right bundle branch was determined in probands cardiac tissue at post mortem examination. Genetic screening of family members revealed two different mutations, a nonsense mutation in exon 11 E473X (12 carriers, family A) and a missense one on exon 21 E1225K (3 carriers, family B). ECG of 15 SCN5A mutation carriers (2 probands were obligated carriers, 9 M and 6 F, age 38,4 ± 17 yrs, range18-72) was analyzed. None of the non carriers had PQ interval prolongation. Twelve mutation carriers (80%) presented a right ventricular conduction delay and one (6,6%) a first degree left bundle branch block. Mean PQ interval was 211 ± 36 msec. A PQ interval> 200 msec was present in 10 (67%) (mean 232 ± 20 msec, mean age 46,5 ± 15 yrs vs 22,2 ± 4,9 yrs with normal PQ interval, p = 0.0004). A QRS complex>110 msec was present in 11 subjects (mean 132 ± 16 msec, mean age 43 ± 17 yrs vs 26 ± 6 yrs with normal QRS duration, p = 0.01). A coved ST-segment elevation>2 mm in V1-V2 was present in six cases (40%). Two SCN5A mutation carriers presented normal ECG and 11 out of 13 (84%) showed late potentials in the time domain analysis of signal-averaged ECG. Conclusions: An organic substrate underlies SCN5A-associated Lenègre disease, consisting of sclerotic interruption of the specialized AV conducting tissue as to account for sudden death in young people. Family members investigation highlighted the presence of a heritable "age-related" alteration in the conduction system disease. Prolongation of the late cardiac sodium current causes right ventricular enlargement and interstitial fibrosis in deltaKPQ-Scn5a mice I Piccini I Piccini 1 University Hospital Münster , Münster , Germany L Fortmueller L Fortmueller 1 University Hospital Münster , Münster , Germany M Kuhlmann M Kuhlmann 2 European Institute of Molecular Imaging (EIMI) , Münster , Germany M Schaefers M Schaefers 2 European Institute of Molecular Imaging (EIMI) , Münster , Germany P Carmeliet P Carmeliet 3 Vesalius Research Center , Leuven , Belgium P Kirchhof P Kirchhof 1 University Hospital Münster , Münster , Germany L Fabritz L Fabritz 1 University Hospital Münster , Münster , Germany Open in new tabDownload slide Abstract 300 Figure Open in new tabDownload slide Abstract 300 Figure Abstract Background: The long QT syndrome 3 (LQT3), a "channelopathy" that prolongs ventricular action potentials and causes torsades de pointes (TdP), is caused by gain-of-function mutations in the cardiac sodium channel gene SCN5A. Whether an increased late sodium current as in LQT3 can also cause structural changes in the heart is not known. We therefore studied structural and functional alterations in a murine model of LQT3 (1505-1507 ΔKPQ-Scn5a knock-in, ΔKPQ). Methods: We investigated 3-25 months old littermate pairs of ΔKPQ and wildtype (WT) mice by means of histology (picrosirius red) and in vivo ultrasound microscopy. Results: In senescent mice (21-25 months, Figure) interstitial fibrosis was increased in left ventricular, septal, and right ventricular myocardium from ΔKPQ mice compared to WT on serial longitudinal sections (LV: ΔKPQ 3.5 + 0.8% n = 5, WT 1.2 + 0.6% n = 5, p < 0.05; Septum: ΔKPQ 1.5 + 0.5% n = 5, WT 0.4 + 0.1% n = 5, p < 0.05; RV: ΔKPQ 1.0 + 0.3% n = 5; WT 0.3 + 0.1 % n = 5, p < 0.05). Fibrosis was neither increased in ventricles from younger ΔKPQ mice (aged 3 to 10 months) nor in ΔKPQ atria. Already at 8-12 months of age, left atrial size (LA: ΔKPQ 2.5 ± 0.04mm, WT 2.3 ± 0.03mm, p < 0.05), right atrial size (RA: ΔKPQ 1.5 ± 0.07mm, WT 1.3 ± 0.06mm, p < 0.05), and right ventricular size (ΔKPQ 2.0 ± 0.05mm, WT 1.8 ± 0.04mm, p < 0.05), but not left ventricular size were enlarged in ΔKPQ mice (n = 22 ΔKPQ, n = 20 WT). We will report qPCR data on candidate genes for fibrosis, hypertrophy, and dilatation. Values are reported as mean ± SEM. Conclusions: Increase of the late cardiac sodium current causes an age-dependent increase in interstitial ventricular fibrosis and heart chamber size. This model allows to investigate signalling pathways for structural and functional alterations in LQT3 1 . Replacement of Cx43 by Cx32 induced increased arrhythmogenesis in isolated mice hearts during ischemia and reperfusion JA Sanchez JA Sanchez 1 Institut de Recerca Hospitals Vall d'Hebron, Lab. Cardiologia Experimental , Barcelona , Spain A Rodriguez-Sinovas A Rodriguez-Sinovas 1 Institut de Recerca Hospitals Vall d'Hebron, Lab. Cardiologia Experimental , Barcelona , Spain E Agullo E Agullo 1 Institut de Recerca Hospitals Vall d'Hebron, Lab. Cardiologia Experimental , Barcelona , Spain D Garcia-Dorado D Garcia-Dorado 1 Institut de Recerca Hospitals Vall d'Hebron, Lab. Cardiologia Experimental , Barcelona , Spain Abstract Purpose: A reduction in intercellular communication may disrupt cardiac electrical propagation and promote arrhythmogenesis. However, we have previously demonstrated that replacement of Cx43 by Cx32, a connexin isoform with reduced conductivity, in a knock-in mice model, does not induce major changes in myocardial electrical properties under normoxic conditions. The aim of this study was to analyze in this model the incidence of both spontaneous and inducible arrhythmias during ischemia-reperfusion. Methods: Langendorff-perfused hearts from Cx43KI32 and wild-type mice were submitted to either 60 min of LAD occlusion or 20 min of regional ischemia followed by 30 minutes of reperfusion. Hearts were paced from the left ventricular apex using rectangular pulses. Baseline conduction velocity and electrical impedance were determined in hearts from both genotypes. Inducibility of ventricular tachyarrhythmias was tested by programmed electrical stimulation, following measurement of effective refractory period, by adding 1-5 premature beats after a train of 16 beats at cycle lengths of 100 ms, and by a burst pacing protocol at cycle lengths ranging from 80 to 30 ms. Results: Replacement of Cx43 by Cx32 did not modifiy baseline conduction velocity or myocardial electrical impedance. However, under normoxic conditions, the number of premature ventricular complexes (PVCs) during continuous pacing at 133 ms (58.73 ± 13.48 vs.16.40 ± 3.94, p < 0.05, n = 10-11), and the number of inducible ventricular tachycardia (VT) was higher in hearts from Cx43KI32 mice than in wild-type animals (1.07 ± 0.45 vs. 0.15 ± 0.10, p < 0.05, n = 13-14). Similarly, Cx43KI32 hearts showed an increased incidence during ischemia of spontaneous PVCs (436.43 ± 51.14 vs. 93.43 ± 27.43, p < 0.001, n = 7), spontaneous VTs (15.29 ± 3.99 vs. 3.57 ± 1.80, p < 0.05, n = 7), and inducible VTs (8.50 ± 2.76 vs. 2.38 ± 0.60, p < 0.05, n = 8), as compared with wild-type animals. Moreover, induced VTs were longer in Cx43KI32 mice hearts (214.79 ± 40.69 vs. 67.45 ± 59.39 s, p < 0.01). Similar findings were observed during reperfusion (spontaneous PVCs: 706.40 ± 206.08 vs. 92.80 ± 33.30, p < 0.05; spontaneous VTs: 33.40 ± 8.48 vs. 2.60 ± 0.93, p < 0.01; inducible VTs: 5.00 ± 2.86 vs. 1.20 ± 0.80, p-NS, n = 5). Conclusions: Replacement of Cx43 by Cx32 induced a marked increase in arrhythmogenesis both under normoxic conditions and during ischemia-reperfusion, despite no major changes in baseline electrical properties. These results suggest that modifications in gap junctional communication not apparent under normal conditions may be pathophysiologically important during ischemia-reperfusion. Genetic deletion of beta-arrestin-1 improves function of the infarcted heart by reducing cardiotoxic neurohormonal overstimulation A Lymperopoulos A Lymperopoulos 1 Nova Southeastern University , Ft. Lauderdale , United States of America G Rengo G Rengo 2 University of Naples Federico II , Naples , Italy E Gao E Gao 3 Thomas Jefferson University , Philadelphia , United States of America C Zincarelli C Zincarelli 2 University of Naples Federico II , Naples , Italy WJ Koch WJ Koch 3 Thomas Jefferson University , Philadelphia , United States of America Abstract Chronic heart failure (HF) is characterized by enhanced circulating catecholamines (CAs) and other cardiotoxic hormones, such as aldosterone, which contribute to the increased morbidity and mortality of the disease. Excessive catecholaminergic stimulation of the failing heart leads to maladaptive changes that further compromise cardiac function and increase cardiac workload, while aldosterone significantly accelerates cardiac adverse remodeling post-myocardial infarction (MI). Cardiac β-adrenergic receptor (AR) desensitization and down-regulation is a hallmark abnormality in HF at the molecular level and is due to the concerted action of cardiac G-protein coupled receptor kinase-2 (GRK2), which is up-regulated in HF, together with its co-factors in receptor desensitization, the β-arrestins (βarrs). Consistent with this, βarr1 knockout (KO) mice exhibit enhanced cardiac contractile responsiveness to catecholaminergic stimulation. Additionally, we recently reported that βarr1 promotes angiotensin II-dependent aldosterone production in the adrenal cortex, and this leads to elevated circulating aldosterone levels in vivo. In the present study, we sought to investigate the effects of genetically deleting βarr1 on post-MI cardiac function and neurohormonal status in mice progressing to HF. We took advantage of the available βarr1KO mouse model and we studied these mice at 4 weeks after surgically-induced MI, in parallel with C57/B6 wild type (WT) controls. Cardiac function was assessed by echocardiography and in vivo catheterization. Plasma CAs and aldosterone were measured by ELISA and cardiac βAR signaling status was examined, as well. Cardiac function is markedly improved in βarr1KOs at 4 weeks post-MI, as evidenced by increased ejection fraction compared to WT mice (41.5 + 2.8 % vs. 21.8 + 2.4 %, respectively, n = 9, p < 0.0001) and increased isoproterenol-induced contractility. Additionally, cardiac dimensions are significantly reduced compared to WTs, indicating attenuation of adverse cardiac remodeling. Moreover, plasma circulating norepinephrine, epinephrine and aldosterone levels are also significantly reduced and cardiac cAMP (the main second messenger produced by βARs) and βAR plasma membrane density are robustly elevated in post-MI βarr1KOs compared to WTs. Thus, genetic deletion of βarr1 prevents post-MI HF-associated cardiac βAR desensitization/down-regulation and substantially improves the adverse neurohormonal burden of the post-MI heart. As a result, the function of the post-MI heart is significantly improved. Widespread reduction of sodium hydrogen exchanger isoform 1 across the heart after a single and local injection of siRNA in the mouse myocardium. PE Morgan PE Morgan 1 Centro de Investigaciones Cardiovasculares , La Plata , Argentina AA Diez AA Diez 1 Centro de Investigaciones Cardiovasculares , La Plata , Argentina NG Perez NG Perez 1 Centro de Investigaciones Cardiovasculares , La Plata , Argentina HE Cingolani HE Cingolani 1 Centro de Investigaciones Cardiovasculares , La Plata , Argentina Abstract Sodium hydrogen exchangers belong to a family of ten members. NHE isoform 1 (SLC9A1) is the main isoform expressed in the heart. Cardiac pathologies like ischemia/reperfusion injury and hypertrophy have been associated to NHE1 hyperactivity, whose pharmacological inhibition proved to be beneficial. RNA interference technology allows, specifically and locally, to reduce protein expression. Small interference RNA was designed against a 25 pb long mRNA target sequence specific for NHE1 (siRNANHE1). The specificity was confirmed by NCBI BLAST tool. siRNA was produced in vitro by hybridization of complementary single strand RNAs generated by a T7 RNA polymerase. As a negative control was generated dsRNA with the scrambled target sequence (siRNASCR). siRNA effectiveness was studied in HEK293 cells transfected with NHE1 cDNA alone or co-transfected with either siRNANHE1 or siRNASCR. After 48 hs of transfection, HEK293 cells were lysated, electrophoresed and inmunoblotted for NHE1 and GAPDH. HEK293 cells transfected with increasing amounts of siRNANHE1 (μg): 0, 1, 4, 10, 40 reduced NHE1 expression in a dose dependent manner and compared with siRNASCR transfected cells; respectively: 100; 56.6 ± 3.5; 41.6 ± 6.8; 17.15 ± 0.2; 8.72 ± 7.4; 78.06 ± 3.1 (n = 3 each, ANOVA One-way p < 0.05). Thereafter, siRNA was studied in vivo in the mouse heart. Anesthetised mice had a small opening in the chest and the siRNA (40 μg) was injected site at the apex in the myocardial wall of the left ventricle. Subsequently, the chest was closed and 48/72 hs later the mice was sacrificed, hearts were excised and analysed for protein expression on inmunoblots. NHE1 expression studied in ventricle lysates showed a reduction for the siRNANHE1 treated mice compared with the ones injected with siRNASCR (37.16 ± 5, n = 5 and 93.7 ± 13.2, n = 4; t-test p < 0.05). Due to the extensive reduction of NHE1 expression it was studied in three different parts of the left ventricle, namely apex, medium body and base. NHE1 expression (reported as NHE1/GAPDH ratio) showed a widespread reduction across the ventricle: Apex 0.126 ± 0.0331, n = 3; Medium 0.225 ± 0.0788, n = 3; Base 0.343 ± 0.0984 n = 3; siRNASCR 1.520 ± 0.1700, n = 3 (ANOVA One-way, P < 0.05). In this report we conclude that only one injection of naked siRNANHE1 in the heart was enough to successfully reduce the NHE1 expression. A second and remarkable finding is the ability of the siRNA molecule to achieve a reduction of the NHE1 expression far away from its injection site. This finding corresponds with a novel notion that siRNA molecules could diffuse through the heart syncytium. Calcium spikes in cardiac myocytes involve only a few open RyR channels. A Zahradnikova, Jr A Zahradnikova, Jr 1 Slovak Academy of Sciences, Institute of Molecular Physiology and Genetics , Bratislava , Slovak Republic E Polakova E Polakova 1 Slovak Academy of Sciences, Institute of Molecular Physiology and Genetics , Bratislava , Slovak Republic I Zahradnik I Zahradnik 1 Slovak Academy of Sciences, Institute of Molecular Physiology and Genetics , Bratislava , Slovak Republic A Zahradnikova A Zahradnikova 1 Slovak Academy of Sciences, Institute of Molecular Physiology and Genetics , Bratislava , Slovak Republic Abstract During the excitation-contraction coupling in cardiac myocytes, calcium needed for contraction is released from the sarcoplasmic reticulum via a large number of calcium release units (CRU) comprising calcium releasing ryanodine receptor (RyR) channels. Although the estimated number of RyRs per CRU is relatively large, about 180 – 270, only a small fraction of them opens during a single calcium release event to produce the observed amplitudes of calcium release current in calcium sparks. Recently, using a RyR gating model that includes allosteric Ca(2+) and Mg(2+) dependent regulation of RyR, we provided explanation for the low number of RyR channels activated in calcium sparks. In this work, we used direct measurement of calcium release fluxes from individual CRUs - calcium spikes - to estimate the number of RyR channels activated by calcium currents in rat ventricular myocytes under conditions close to physiological. Isolated myocytes were stimulated by voltage pulses to 0 mV from a holding potential of −50 mV using whole-cell patch clamp technique. Calcium spikes were measured using laser scanning confocal microscopy, with 0.1 mM fluo −3 as the Ca(2+) indicator, and 1 mM EGTA to limit Ca(2+) diffusion. Calcium spikes were analyzed by fitting individual fluorescence intensity profiles with a phenomenological function. We have observed two distinct populations of calcium spikes. The early spikes occurred during the peak of the calcium current and were characterized by high fluorescence amplitudes. The late calcium spikes were activated during the whole time of the voltage stimulus and their amplitudes were low. The amplitude histogram of early calcium spikes could be approximated by the sum of 4 Gaussian functions. Although the amplitudes of individual components were not constrained during the fitting procedure, they turned out to be close to multiples of the smallest one. Only one amplitude modus was found in the group of late calcium spikes. These results suggest that in early calcium spikes, only a small number of RyR channels opens under our experimental conditions. While the single amplitude modus detected in late calcium spikes would imply that only one RyR channel opens during late releases, the possibility that lower amplitude levels are blurred in the noise should be considered. Beneficial effects of CaMKII inhibition in the human failing heart N Fluschnik N Fluschnik 1 Department of Cardiology and Pneumology, Georg-August-University , Goettingen , Germany ST Sossalla ST Sossalla 1 Department of Cardiology and Pneumology, Georg-August-University , Goettingen , Germany K Ort K Ort 1 Department of Cardiology and Pneumology, Georg-August-University , Goettingen , Germany S Neef S Neef 1 Department of Cardiology and Pneumology, Georg-August-University , Goettingen , Germany G Hasenfuss G Hasenfuss 1 Department of Cardiology and Pneumology, Georg-August-University , Goettingen , Germany LS Maier LS Maier 1 Department of Cardiology and Pneumology, Georg-August-University , Goettingen , Germany Abstract Increased activity and amount of the CaMKII (Ca2+/ calmodulin-dependent protein kinase II) are known to be involved in the pathogenesis of heart failure. But detailed expression analysis and functional effects of the CaMKII have never been investigated in human myocardium. Western Blots were performed using 25 human end-stage failing and 12 nonfailing (NF) hearts. CaMKIIδ expression levels were significantly increased by 128.9 ± 9.5% in right ventricular (RV) myocardium of patients suffering from ischemic cardiomyopathy and by 135.8 ± 6.1% suffering from dilated cardiomyopathy (DCM) compared to NF myocardium. These results were associated with significant elevations of both splice variants of the CaMKII- δb and δc in DCM and ICM RV as well as LV failing myocardium. Experiments with isometrically twitching trabeculae (37°C, pH 7.4, Ca2+ 2 mM, n = 14 each) revealed significantly improved force-frequency-relationships during CaMKII inhibition using KN-93 compared to trabeculae treated with KN-92 as control (2 μM each). Twitch force amplitude increased by 92 ± 20% for KN-93 and only by 10 ± 13% for KN-92 at 2 Hz and by 101 ± 33 vs. -4 ± 15% at 3 Hz (both frequencies P < 0.05 compared to KN-92, n = 14 each). Furthermore, inhibition of CaMKII improved PR behavior (steady state/post-rest) after a rest interval of 120s by 2.5 ± 0.4 (KN-93) vs. 1.5 ± 0.3 (KN-92) (n = 12 each, p < 0.05) indicating an improved SR Ca2+ behavior. This positive inotropic effect was further investigated using Fluo 3 loaded isolated human ventricular myocytes. During caffeine applications the F/F0 was 2.1 ± 0.3% for KN-93 treated myocytes compared to those in the presence of KN-92 1.3 ± 0.1% (n = 5 vs. n = 9 cells, p < 0.05). Most importantly, confocal microscopy obtained a reduced Ca2+-spark frequency of 137 ± 16 pL-1*s-1 in the presence of KN-93 compared to 212 ± 27 pL-1*s-1 (KN-92) (1 μM, n = 28 vs. n = 31, p < 0.05) resulting in a significantly reduced calculated Ca2+ leak per cell by 30%. These findings indicate that improved contractility due to CaMKII inhibition is caused by a reduced SR Ca2+ leak. This improvement was also associated with a significant reduction of the RyR2 receptor phosphorylation status in trabeculae that were treated with KN-93 during the functional experiments. CaMKII inhibition leads to a phosphorylation decrease at the specific binding site for CaMKII at Ser-2815 by 74.0 ± 11.3% (n = 3 each, p < 0.05). In conclusion, this study shows improved cardiac contractility in human failing myocardium due to CaMKII inhibition. This may be attributable to a reduced RyR2 phosphorylation which leads to a reduced SR Ca2+ leak causing an increased SR Ca2+ content. Conversion of smooth muscle cells into a foam cell like phenotype by transport of LDL from macrophages into smooth muscle cells during cell-cell-contacts S Weinert S Weinert 1 Hospital Magdeburg, Department of Cardiology , Magdeburg , Germany DM Poitz DM Poitz 2 Dresden University of Technology, Heart Center, Department of Cardiology and Intensive Care , Dresden , Germany J Herold J Herold 1 Hospital Magdeburg, Department of Cardiology , Magdeburg , Germany A Schmeisser A Schmeisser 1 Hospital Magdeburg, Department of Cardiology , Magdeburg , Germany JH Strasser JH Strasser 2 Dresden University of Technology, Heart Center, Department of Cardiology and Intensive Care , Dresden , Germany RC Braun-Dullaeus RC Braun-Dullaeus 1 Hospital Magdeburg, Department of Cardiology , Magdeburg , Germany Abstract The local interaction of mono-nuclear cells [Ma] with vascular smooth muscle cells [VSMC] is known to play a key role in atherosclerotic plaque development and destabilization. An in-vitro co-culture model was established to study how Ma interact and modulate the behavior of VSMC. The MA derived macrophages [MP] were exposed to fluorescence-labeled acetylated LDL [FL-LDL] prior co-culture. Semi-confluent VSMC were treated with Mitomycin C to stop proliferation and co-cultured with the FL-LDL tagged MP in a ratio of 1:3, respectively. Immune cytochemistry revealed that, within 4 weeks of co-culture,> 20% of the sm-actin positive cells (VSMC) were positive for Fl-LDL. Cell separation in a trans-well co-culture system resulted in complete absence of double-labeled cells. Instead, time-lapse microscopy demonstrated that VSMC acquired the FL-ac-LDL from MP in co-culture by cell-cell-contact. Three dimensional high magnification microscopy visualized that the entire cytosol of single VSMC was filled with FL-ac-LDL particles within 24 h. The usage of the acidotropic fluorescence dye LysoTracker demonstrated that some, but not all Fl-ac-LDL was bioactive and reached the VSMC lysosomes. Infection of the VSMC prior to co-culture with a virus coding for Lamp1-RFP fusion protein demonstrated that the remaining FL-ac-LDL was located in non acidic lysosomes. Fluorescent and classical oil-red O staining showed a significant increase of neutral lipid droplets in co-cultured Fl-ac-LDL pos. VSMC compared to their Fl-ac-LDL neg. neighbours (internal control). In a next step VSMC were infected prior co-culture with a virus coding for a Rab5a-GFP fusion protein to mark all early endosomes. In contrast to endothelial cells, which were used as pos. control, no co-localisation between Rab5a and the transported Fl-ac-LDL in co-cultured VSMC were found. Thus, phagocytotis and autophagy were excluded which implies a direct cell-cell exchange of LDL particles. Rescue of the VSMC from co-culture and subsequent analysis showed an increase in phagocytotic activity compared to VSMC which had been co-cultured with MP in a trans-well system (as control). Xenogenic cell composure (rat VSMC, human MP) and subsequent quantitative RT-PCR with rat specific primers demonstrated induction of genes typical for MP (CD68 and Mac2) and expectable alteration of cholersterol sensitv genes (HMG-CoA reductase down) in VSMC. These results demonstrate an active and phagocytosis independent exchange of LDL/Cholesterol from MP into VSMC and imply that MP are capable to transform VSMC into plaque-destabilizing foam cells. Profiling of microRNAs following vascular injury in ApoE−/− mice M Nazari-Jahantigh M Nazari-Jahantigh 1 Institute of Molecular Cardiovascular Research, University Hospital Aachen, RWTH , Aachen , Germany C Weber C Weber 1 Institute of Molecular Cardiovascular Research, University Hospital Aachen, RWTH , Aachen , Germany A Schober A Schober 1 Institute of Molecular Cardiovascular Research, University Hospital Aachen, RWTH , Aachen , Germany Abstract We aimed to identify microRNAs involved in neointima formation in mice with an atherogenic background. Wire-induced carotid injury was performed in ApoE−/− mice on western-type diet. Uninjured carotid arteries served as control. RNA was isolated after 1, 7, 14, and 28 days (n = 3-4 each group) and hybridized to an Agilent microRNA microarray (Sanger v12). Significantly regulated microRNAs (>2-fold) over time (P < 0.05; ANOVA and Benjamini-Hochberg correction) were clustered by K-means algorithm. QRT-PCR was performed for validation. 95 out of 611 microRNAs were differentially expressed and seven groups were separated by cluster analysis. Although all microRNAs in group 1-3 were increased from day 7 on, expression at day 1 was unchanged or decreased in group 1 (e.g. miR-155) and 2 (e.g. miR-199a-3p), respectively, or slightly up-regulated in group 3 (e.g. miR-21). Group 4 (e.g. miR-188-5p) was characterized by highest expression from day 1 on. Following down-regulation at 1 day, microRNAs in group 5, such as let-7i, were elevated again to control levels thereafter. In contrast, persistent down-regulation as early as day 1 was specific for group 6 (e.g. miR-378). The expression of microRNAs in group 7 (e.g. miR-129-3p), however, was not significantly different from control at day 1, but declined predominantly after 7 and 14 days. The expression profile of 6 miRs from different groups was verified by qRT-PCR. Distinct groups of similarly regulated microRNAs were detected in the course of neointima formation in hyperlipidemic mice. These groups might be functionally related to specific cellular and molecular processes of vascular wound healing. Endothelial dysfunction in LOX-1 overexpressing mice A Leuner A Leuner 1 Medical Clinic III, Department of Vascular Endothelium and Microcirculation , Dresden , Germany B Eichhorn B Eichhorn 2 Dresden University of Technology, Department of Pharmacology and Toxicology , Dresden , Germany U Ravens U Ravens 2 Dresden University of Technology, Department of Pharmacology and Toxicology , Dresden , Germany H Morawietz H Morawietz 1 Medical Clinic III, Department of Vascular Endothelium and Microcirculation , Dresden , Germany Abstract Oxidized LDL-cholesterol (oxLDL) is a risk factor for atherosclerosis. The major receptor for uptake of oxLDL is the lectin-like oxidized low-density lipoprotein receptor-1 LOX-1. This receptor is preferentially expressed on endothelial and vascular smooth muscle cells. LOX-1 is induced in atherosclerotic vessels of experimental and clinical studies. In this study, we analyzed the impact of endothelial LOX-1 receptor overexpression on vascular function in the thoracic aorta and on metabolic parameters. LOX-1 transgenic mice (LOX-1tg) were fed with standard diet (Con) or high-fat diet (HFD) for 20 weeks. C57BL/6 (wild-type; WT) mice served as control. No differences in body weight were found between WT and LOX-1tg mice after 20 weeks on standard or high-fat diet. Vascular function was determined in the thoracic aorta using a wire-myograph. Endothelium-dependent relaxation was impaired in LOX-1tg + Con mice, but not further decreased after high-fat diet. Endothelium-dependent relaxation was completely blocked by the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME). In WT and LOX-1tg vessels, endothelium-independent relaxation was not altered on standard or high-fat diet. Organ weight of heart, kidney and liver was similar in LOX-1tg + HFD and WT + HFD. The absolute amount of white epididymal adipose tissue was significantly increased in LOX-1tg compared to WT. This was further increased after high-fat diet. In addition, we analyzed the mRNA expression in white epididymal adipose tissue by RT-PCR. Leptin and Resistin mRNA expression was increased in LOX-1tg mice + HFD compared to WT. Adiponectin, TNF-alpha, IL-6 and CD36 mRNA expression was unchanged. Interestingly, PPAR-gamma was downregulated in LOX-1tg mice. In conclusion, LOX-1 overexpression causes endothelial dysfunction in the thoracic aorta. In addition, the expression of adipokines like leptin or resistin is increased in the adipose tissue and might affect endothelial function. Simvastatin therapy and endothelial function in young adults with subclinical atherosclerosis EE Babes EE Babes 1 Faculty of Medicine , Oradea , Romania VV Babes VV Babes 1 Faculty of Medicine , Oradea , Romania MI Popescu MI Popescu 1 Faculty of Medicine , Oradea , Romania A Ardelean A Ardelean 1 Faculty of Medicine , Oradea , Romania M Rus M Rus 1 Faculty of Medicine , Oradea , Romania C Bustea C Bustea 1 Faculty of Medicine , Oradea , Romania Abstract Background: Endothelial dysfunction is an early reversible stage in the development of atherosclerosis and has predictive value for future cardiovascular events. Statins have been shown to reverse endothelial dysfunction in dyslipidemic patients. The aim of the study was to evaluate the effect of simvastatin therapy on endothelial function in young asymptomatic adults with subclinical atherosclerosis. Methods: At baseline, endothelial function was assessed as flow-mediated-dilation(FMD) of the brachial artery, in 270 asymptomatic adults aged between 20-40 y. Subjects with FMD values in the lowest tertile (n = 78), were divided in two groups: 45 subjects treated with simvastatin 40 mg/day and 33 subjects treated with placebo. Subjects were reevaluated after 1 year of therapy. Results: There were no significant differences between two groups regarding baseline lipids and FMD. After 1 year FMD was significantly higher in the Simvastatin group vs placebo group- difference 10,11(95%CI 9,079 to 11,144) (p < 0,0001). After 1 year, in the Simvastatin group FMD significantly increased from baseline-4,48 ± 1,87% to 13,77 ± 2,48%; difference-9,28 (CI 8,21 to 10,36) (p < 0,0001). In the placebo group, after 1 year FMD significantly decreased from baseline-4,54 ± 1,87% to 3,66 ± 1,61% (p = 0,045). After 1 year, LDL was significantly lower -80,06 ± 7,29mg/dl in the Simvastatin group vs 140,33 ± 18,38mg/dl in the placebo group(p < 0,0001). Post treatment LDL in the Simvastatin group significantly decreased from baseline value-141,81 ± 18,4mg/dl(p < 0.0001). Conclusions: Our data show significant improvement of endothelial dysfunction towards normal levels after 1 year of simvastatin therapy in young asymptomatic adults. These results emphasize the relevance of statin therapy at an early stage, when the atherosclerotic process is still reversible. Endothelial dysfunction and circulating platelet activation in apoE/LDLR /- mice along the development of atherosclerosis P Gwozdz P Gwozdz 1 Jagiellonian University Medical College, Department of Experimental Pharmacology , Krakow , Poland G Csanyi G Csanyi 1 Jagiellonian University Medical College, Department of Experimental Pharmacology , Krakow , Poland B Luzak B Luzak 2 Department of Haemostatic Disorders, Medical University of Lodz , Lodz , Poland M Gajda M Gajda 3 Chair of Histology, Jagiellonian University Medical College , Krakow , Poland L Mateuszuk L Mateuszuk 1 Jagiellonian University Medical College, Department of Experimental Pharmacology , Krakow , Poland A Chmura-Skirlinska A Chmura-Skirlinska 4 Nencki Institute of Experimental Biology , Warsaw , Poland C Watala C Watala 2 Department of Haemostatic Disorders, Medical University of Lodz , Lodz , Poland S Chlopicki S Chlopicki 1 Jagiellonian University Medical College, Department of Experimental Pharmacology , Krakow , Poland Abstract Endothelial dysfunction, impaired endothelial antiplatelet defence and circulating platelet activation accompanies hypercholesterolemia and precedes the development of atherosclerosis in humans.However, the primary role of endothelial dysfunction in hypercholesterolemic mice with atherosclerosis was questioned. The aim of the present study was to analyze NO- and PGI2-dependent endothelial function in conduit and resistance vessels as well as activation of circulating platelets in relation to the onset and progression of atherosclerosis in apoE/LDLR−/− mice. Endothelial function of conduit and resistance vessels was determined respectively in isolated aorta and isolated heart in 2, 4 and 8-month old apoE/LDLR−/− mice.Atherosclerotic lesion size using oil red-O staining and monocyte accumulation using anti-CD68 immunostaining was determined in aortic roots.HbNO level in erythrocytes was analyzed using electron spin resonance and circulating platelet expression of CD40L and P-selectin was analyzed using flow cytometry. In the aorta of 2-month-old apoE/LDLR−/− mice there was no evident lipid deposition and subendothelial macrophage accumulation.However, the acetylcholine-induced NO-dependent relaxation in the aorta was already decreased, with concomitant up-regulation of PGI2 production.In aorta from 4 and 8-month-old apoE/LDLR−/− mice, lipid deposition and macrophage accumulation were gradually increased, with augmented impairment of NO-dependent function and up-regulation of PGI2 production.In contrast, in the isolated heart of 2, 4 and 8-month-old apoE/LDLR−/− mice, NO-dependent vasodilatation evoked by bradykinin was preserved, while vasodilatatory response to NO-donors SNAP and SNP was augmented.PGI2 production was also augmented, in COX-2-dependent way.Endothelial dysfunction was accompanied by decreased level of HbNO in erythrocytes in all age groups.Finally, serum sCD40L concentration and circulating platelet CD40L, but not P-selectin, expression, was increased in apoE/LDLR−/− mice of all age groups. In conclusion, in apoE/LDLR−/− mice impairment of endothelial NO bioavailability and circulating platelet activation precedes the development of atherosclerotic plaques and is reflected by decreased systemic level of HbNO.Impairment of endothelial NO-dependent function is better compensated in coronaries then in aorta: up-regulation of PGI2 production is present in aorta and coronaries, but increased vessel wall sensitivity to NO only in coronaries.The robust compensatory mechanisms in coronary circulation of apoE/LDLR−/− seem to be responsible for the preservation of exercise capacity in these mice. Endothelial function and arterial stiffness in adult patients with primary immunodeficiencies- pilot study I Kierzkowska I Kierzkowska 1 Jagiellonian University, Medical College, Department of Internal Medicine and Gerontology , Cracow , Poland J Sulicka J Sulicka 1 Jagiellonian University, Medical College, Department of Internal Medicine and Gerontology , Cracow , Poland A Kwater A Kwater 1 Jagiellonian University, Medical College, Department of Internal Medicine and Gerontology , Cracow , Poland M Strach M Strach 1 Jagiellonian University, Medical College, Department of Internal Medicine and Gerontology , Cracow , Poland A Surdacki A Surdacki 2 Jagiellonian University, Medical College, 2nd Department of Cardiology , Cracow , Poland M Siedlar M Siedlar 3 Jagiellonian University Medical College, Department of Clinical Immunology , Cracow , Poland T Grodzicki T Grodzicki 1 Jagiellonian University, Medical College, Department of Internal Medicine and Gerontology , Cracow , Poland Abstract 321 Table . IDS (n = 7) . Control (n = 11) . BMI 24,1 + 3 23,4 + 2,8 WHR O,83 + 0,08 O,82 + 0,07 FG 4,6 + 60,3 4,7 + 0,4 FIBRINOGEN 4,2 + 1,0* 3 + 0,7 HSCRP 2,3 + 2,8 2,8 + 0,8 FMD 6,9 + 7,9 7,25 + 10,4 NID 31 + 12,6 22,5 + 9,8 PVW 12,3 + 2,66 11,5 + 2,07 . IDS (n = 7) . Control (n = 11) . BMI 24,1 + 3 23,4 + 2,8 WHR O,83 + 0,08 O,82 + 0,07 FG 4,6 + 60,3 4,7 + 0,4 FIBRINOGEN 4,2 + 1,0* 3 + 0,7 HSCRP 2,3 + 2,8 2,8 + 0,8 FMD 6,9 + 7,9 7,25 + 10,4 NID 31 + 12,6 22,5 + 9,8 PVW 12,3 + 2,66 11,5 + 2,07 Open in new tab Abstract 321 Table . IDS (n = 7) . Control (n = 11) . BMI 24,1 + 3 23,4 + 2,8 WHR O,83 + 0,08 O,82 + 0,07 FG 4,6 + 60,3 4,7 + 0,4 FIBRINOGEN 4,2 + 1,0* 3 + 0,7 HSCRP 2,3 + 2,8 2,8 + 0,8 FMD 6,9 + 7,9 7,25 + 10,4 NID 31 + 12,6 22,5 + 9,8 PVW 12,3 + 2,66 11,5 + 2,07 . IDS (n = 7) . Control (n = 11) . BMI 24,1 + 3 23,4 + 2,8 WHR O,83 + 0,08 O,82 + 0,07 FG 4,6 + 60,3 4,7 + 0,4 FIBRINOGEN 4,2 + 1,0* 3 + 0,7 HSCRP 2,3 + 2,8 2,8 + 0,8 FMD 6,9 + 7,9 7,25 + 10,4 NID 31 + 12,6 22,5 + 9,8 PVW 12,3 + 2,66 11,5 + 2,07 Open in new tab Abstract Objective: Connection between inflammation and atheromatosis and higher cardiovascular risk is known for last several years. Recurrent severe infections are axial diagnostic criteria of immunodeficiency syndromes (IDS). The object of this study is comparative assessment of cardiovascular (CV) risk profile and endothelial function in adult patients with primary immunodeficiencies chronically treated with substitutional immunoglobulins doses. Methods: Adults with primary immunodeficiency syndrome during sufficient human immunoglobulins substitution were assessed for CV risk factors, arterial function and compared to control group. Medical history, physical examination with anthropological measurements, levels of fasting glucose (FG), cholesterol (TC), fibrinogen, hsCRP were performed. Endothelial function was assessed using endothelium dependent flow-mediated dilatation (FMD%) and nitroglycerin induced dilatation (NID %) of brachial artery and arterial stiffness by carotid -femoral pulse wave velocity (PWV). Results: We compared preliminary results of 7 IDS subjects (4 men, age: 36,5 + 13,5) years) with the control group of 11 volunteers (7 men, age: 34+ 5,6). One of 7 assessed IDS patients and 2 of 11 from control group were smokers. There was significant difference in fibrinogen level (p < 0.05) but we did not find any significant differences between compared groups in BMI, WHR, FG, hsCRP, TC, endothelial function measured by FMD and arterial stiffness in PVW values. Conclusions: Preliminary results indicate that adult patients with chronically treated with IVIG do not present significant differences in endothelial function and arterial stiffness in comparison with age-matched healthy subjects 1 . Unraveling downstream TGF-beta1 signaling required for monocyte motility and the effect of diabetes mellitus hereon. S Olieslagers S Olieslagers 1 Maastricht University Medical Center, Department of Cardiology , Maastricht , Netherlands L Pardali L Pardali 2 Leiden University Medical Center, Department of Molecular and Cellular Biology , Leiden , Netherlands V Tchaikovski V Tchaikovski 1 Maastricht University Medical Center, Department of Cardiology , Maastricht , Netherlands P Ten Dijke P Ten Dijke 2 Leiden University Medical Center, Department of Molecular and Cellular Biology , Leiden , Netherlands J Waltenberger J Waltenberger 1 Maastricht University Medical Center, Department of Cardiology , Maastricht , Netherlands Abstract Arteriogenesis is stimulated in the ischemic heart and can enhance regional blood flow to improve impaired myocardial function. Monocytes contribute to arteriogenesis by infiltration to sites of collateral growth and subsequent production and release of growth factors. TGF-β1 mediates monocyte motility and can stimulate arteriogenesis. TGF-β1 exerts its action via SMAD-dependent and independent signaling mechanisms including the PI3K/AKT, p38 and ERK pathways. The TGF-β-induced mechanisms mediating monocyte movement are unknown so far. Moreover, the chemotactic response of monocytes to TGF-β under the pathological conditions of diabetes mellitus (DM) has never been assessed previously. DM is a predominant cardiovascular risk factor known to deteriorate arteriogenesis by abrogation of cellular movement. Monocytes were isolated through gradient centrifugation and subsequent immunological magnet-isolation. Selective functional inhibition of TGF-β1-related downstream kinases was achieved by pharmacological intervention using the inhibitors LY364947 (ALK5), Wortmannin (PI3K), AKT-VIII (AKT), SB239063 (p38), PD98059 (MEK) and RNAi directed against SMAD proteins. Monocyte motility was quantified in the modified Boyden chamber. Kinase activation was assessed by immunoblotting. Stimulation of monocytes with TGF-β1 resulted in the activation of the PI3K/AKT, p38 and ERK1/2 pathways, next to the activation of canonical SMAD2. The type 1 TGF-β receptor ALK5 was required for activation of the non-SMAD signaling elements AKT, p38 and ERK1/2. Moreover, blockage of the ALK5 receptor activity prevented monocyte chemotaxis towards TGF-β1. Individual inhibition of PI3K and AKT both diminished TGF-β1-mediated monocyte motility without affecting the phosphorylation level of ERK1/2, p38 or SMAD2. With regard to MAPKs, TGF-β1-induced monocyte chemotaxis did not rely on ERK1/2, but rather on p38, as detected by specific MAPK inhibition. Furthermore, individual genetic knock-downs for different SMAD molecules did not negatively affect chemotaxis toward TGF-β1. Remarkably, TGF-β1 was able to strongly stimulate chemotaxis in monocytes freshly isolated from DM patients. These novel data provide a molecular basis of TGF-β1-induced monocyte migration, which is mediated via non-canonical pathways. TGF-β1-induced monocyte chemotaxis is fully functional in diabetic conditions. TGF-β1-induced chemotactic signaling is distinct from the ones used by VEGF-A and MCP-1, which are impaired in DM. Therefore, TGF-β1 is a promising candidate for stimulating collateral growth in patients with DM. Reinfusion of spleen cells is correcting altered thrombus organisation after splenectomy MK Renner MK Renner 1 Medical University of Vienna , Vienna , Austria B Redwan B Redwan 1 Medical University of Vienna , Vienna , Austria MP Winter MP Winter 1 Medical University of Vienna , Vienna , Austria A Panzenboeck A Panzenboeck 1 Medical University of Vienna , Vienna , Austria J Jakowitsch J Jakowitsch 1 Medical University of Vienna , Vienna , Austria R Sadushi-Kolici R Sadushi-Kolici 1 Medical University of Vienna , Vienna , Austria D Bonderman D Bonderman 1 Medical University of Vienna , Vienna , Austria IM Lang IM Lang 1 Medical University of Vienna , Vienna , Austria Abstract Purpose: Splenectomy is associated with complicated venous thromboembolism, such as recurrent deep venous thrombosis, portal vein thrombosis, and chronic thromboembolic pulmonary hypertension (CTEPH). It is believed that the loss of mechanical filtering function of the spleen permits the accumulation of senescent blood cells entailing a hypercoagulable state and altered thrombus resolution. The aim of our study was to decipher the population of spleen cells responsible for misguided thrombus resolution. Methods: We utilized a mouse model of stagnant flow venous thrombosis to characterize venous thrombus resolution. Vena cava ligation was performed one month after splenectomy. In defined groups, whole spleens or spleens depleted of leukocyte subpopulations were reinfused intraperitoneally into autologous mice. On days 3,7,14 and 28 after vena cava ligation thrombi were harvested for histology. Results: Thrombus areas of splenectomized mice were significantly larger than those of controls at all time points (ANOVA, n = 8, p < 0.03). Reinfusion of autologous whole spleen homogenates reconstituted a normal pattern of thrombus organisation. The depletion of neutrophils and macrophages led to a significant increase in thrombus size. Conclusion: Reinfusion of spleen cells can restore the normal process of venous thrombus organisation in a mouse model. Cells of the innate immune system appear to be key mediators of thrombus resolution. Lymphocyte populations in aspirated thrombus and peripheral blood sample in patients with ST elevation myocardial infarction A Toso A Toso 1 Misericordia e Dolce Hospital, Department of Cardiology , Prato , Italy L Tanini L Tanini 2 Flow Cytometry Laboratory , Prato , Italy T Pizzetti T Pizzetti 2 Flow Cytometry Laboratory , Prato , Italy M Leoncini M Leoncini 1 Misericordia e Dolce Hospital, Department of Cardiology , Prato , Italy M Maioli M Maioli 1 Misericordia e Dolce Hospital, Department of Cardiology , Prato , Italy D Tedeschi D Tedeschi 1 Misericordia e Dolce Hospital, Department of Cardiology , Prato , Italy C Oliviero C Oliviero 2 Flow Cytometry Laboratory , Prato , Italy F Bellandi F Bellandi 1 Misericordia e Dolce Hospital, Department of Cardiology , Prato , Italy Abstract 324 Table . B cells (CD19+) . T cells(CD3+) . T cellsCD4+ . T cellsCD8+ . CD4+/CD8+Ratio . NK cells(CD3-CD16+CD56+) . Peripheral Blood 10 ± 6 % 64 ± 15 % 40 ± 15 % 23 ± 14 % 2,5 ± 1,5 % 25 ± 15 % Aspirated Thrombus 7 ± 6 % 68 ± 17 % 41 ± 21 % 21 ± 14 % 3,1 ± 3 % 25 ± 17 % . B cells (CD19+) . T cells(CD3+) . T cellsCD4+ . T cellsCD8+ . CD4+/CD8+Ratio . NK cells(CD3-CD16+CD56+) . Peripheral Blood 10 ± 6 % 64 ± 15 % 40 ± 15 % 23 ± 14 % 2,5 ± 1,5 % 25 ± 15 % Aspirated Thrombus 7 ± 6 % 68 ± 17 % 41 ± 21 % 21 ± 14 % 3,1 ± 3 % 25 ± 17 % Open in new tab Abstract 324 Table . B cells (CD19+) . T cells(CD3+) . T cellsCD4+ . T cellsCD8+ . CD4+/CD8+Ratio . NK cells(CD3-CD16+CD56+) . Peripheral Blood 10 ± 6 % 64 ± 15 % 40 ± 15 % 23 ± 14 % 2,5 ± 1,5 % 25 ± 15 % Aspirated Thrombus 7 ± 6 % 68 ± 17 % 41 ± 21 % 21 ± 14 % 3,1 ± 3 % 25 ± 17 % . B cells (CD19+) . T cells(CD3+) . T cellsCD4+ . T cellsCD8+ . CD4+/CD8+Ratio . NK cells(CD3-CD16+CD56+) . Peripheral Blood 10 ± 6 % 64 ± 15 % 40 ± 15 % 23 ± 14 % 2,5 ± 1,5 % 25 ± 15 % Aspirated Thrombus 7 ± 6 % 68 ± 17 % 41 ± 21 % 21 ± 14 % 3,1 ± 3 % 25 ± 17 % Open in new tab Abstract A deviant lymphocyte response was observed in the peripheral blood of patients suffering from an acute coronary event. We sought to analyze the distribution of lymphocyte populations both in the aspirated intracoronary thrombus and in the peripheral blood in a consecutive series of ST elevation myocardial infarction (STEMI) patients submitted to primary percutaneous coronary intervention (PCI). Methods: Seventeen consecutive STEMI patients who underwent primary PCI with an effective aspiration of atherothrombotic material by a manual-aspiration catheter (Export System, Medtronic), were included. Venous blood sampling was drawn at the beginning of PCI. The filtered, aspirated material has been dipped in culture medium (RPMI 1640) enriched with 10% foetal calf serum and then minced with scalpels in Petri dish and re-suspended in culture medium. Peripheral blood and tromboaspirated lymphocytes were compared for the distribution of lymphocyte subsets by flow cytometric analysis. Results: No significant differences were found between peripheral blood and aspired thrombus for all the lymphocyte subset frequencies (Table). Conclusions: At our knowledge this is the first report that analyze the lymphocyte subset frequency in the atherothrombotic material aspirated during primary PCI. Our results show that, in the acute phases of STEMI, peripheral blood and coronary thrombus express an identical lymphocyte population frequency. In this context, for clinical research Purposes, the simple analysis of peripheral blood can be used 1 . Lymphocyte subsets according to clinical presentation of acute coronary syndromes A Toso A Toso 1 Misericordia e Dolce Hospital, Department of Cardiology , Prato , Italy L Tanini L Tanini 2 Flow Cytometry Laboratory , Prato , Italy T Pizzetti T Pizzetti 2 Flow Cytometry Laboratory , Prato , Italy M Leoncini M Leoncini 1 Misericordia e Dolce Hospital, Department of Cardiology , Prato , Italy M Maioli M Maioli 1 Misericordia e Dolce Hospital, Department of Cardiology , Prato , Italy D Tedeschi D Tedeschi 1 Misericordia e Dolce Hospital, Department of Cardiology , Prato , Italy P Casprini P Casprini 2 Flow Cytometry Laboratory , Prato , Italy F Bellandi F Bellandi 2 Flow Cytometry Laboratory , Prato , Italy Abstract 325 Table Lymphocyte distribution by groups . B cells (CD19+) . T cells(CD3+) . T cellsCD4+ . T cellsCD8+ . CD4+/CD8+Ratio . NK cells(CD3-CD16+CD56+) . STEMI 10 ± 6 % 64 ± 15 % 40 ± 15 % 23 ± 14 % 2,5 ± 1,5 25 ± 15 % * NSTEMI 10 ± 6 % 77 ± 7 % 46 ± 13 % 31 ± 17 % 2 ± 1 13 ± 4 % Controls 10 ± 4 % 72 ± 6 % 48 ± 10 % 27 ± 9 % 1.9 ± 1.1 12 ± 6 % . B cells (CD19+) . T cells(CD3+) . T cellsCD4+ . T cellsCD8+ . CD4+/CD8+Ratio . NK cells(CD3-CD16+CD56+) . STEMI 10 ± 6 % 64 ± 15 % 40 ± 15 % 23 ± 14 % 2,5 ± 1,5 25 ± 15 % * NSTEMI 10 ± 6 % 77 ± 7 % 46 ± 13 % 31 ± 17 % 2 ± 1 13 ± 4 % Controls 10 ± 4 % 72 ± 6 % 48 ± 10 % 27 ± 9 % 1.9 ± 1.1 12 ± 6 % *p < 0,05 STEMI group vs NSTEMI group. Open in new tab Abstract 325 Table Lymphocyte distribution by groups . B cells (CD19+) . T cells(CD3+) . T cellsCD4+ . T cellsCD8+ . CD4+/CD8+Ratio . NK cells(CD3-CD16+CD56+) . STEMI 10 ± 6 % 64 ± 15 % 40 ± 15 % 23 ± 14 % 2,5 ± 1,5 25 ± 15 % * NSTEMI 10 ± 6 % 77 ± 7 % 46 ± 13 % 31 ± 17 % 2 ± 1 13 ± 4 % Controls 10 ± 4 % 72 ± 6 % 48 ± 10 % 27 ± 9 % 1.9 ± 1.1 12 ± 6 % . B cells (CD19+) . T cells(CD3+) . T cellsCD4+ . T cellsCD8+ . CD4+/CD8+Ratio . NK cells(CD3-CD16+CD56+) . STEMI 10 ± 6 % 64 ± 15 % 40 ± 15 % 23 ± 14 % 2,5 ± 1,5 25 ± 15 % * NSTEMI 10 ± 6 % 77 ± 7 % 46 ± 13 % 31 ± 17 % 2 ± 1 13 ± 4 % Controls 10 ± 4 % 72 ± 6 % 48 ± 10 % 27 ± 9 % 1.9 ± 1.1 12 ± 6 % *p < 0,05 STEMI group vs NSTEMI group. Open in new tab Abstract Considerable evidence supports the crucial role of lymphocytes in the initiation and progression of atherosclerotic process while, in more advanced stages, they contribute to the destabilization of the atherosclerotic lesions. Recent analysis postulated that a deviant lymphocyte response mediate plaque instabilization and recurrence of acute coronary events. In the present study, we compared the peripheral lymphocyte populations of patients with acute coronary syndrome (ACS) according to the clinical presentation of the process. Methods: Peripheral blood samples from 30 consecutive patients suffering from ACS were taken at the time of the index event and analyzed for the distribution of lymphocyte subsets by flow cytometry. Patients were subgrouped according to the clinical presentation of the disease: 17 patients (72 ± 13yrs) with ST elevation myocardial infarction (STEMI group); 13 patients (71 ± 12yrs) with non ST elevation myocardial infarction (NSTEMI group). As a control population, we studied 50 consecutive healthy donors (36 male, mean age 58 ± 8 yrs). Results: In the overall population Natural Killer (NK) cell frequency was significantly higher with respect to control subjects (20 ± 14 vs 12 ± 6%, p < 0,05). In the table are reported the frequencies of total B cells, CD4+, CD8+ and CD16+CD56+ T cells and of NK cells for the two study groups and for control subjects. Among the two study groups, NK-cell frequency was significantly higher in STEMI than in NSTEMI patients. No significant differences were found between other lymphocyte subset frequencies. Conclusions: In patients with ACS we observed a higher frequency of peripheral NK cells than in the control group. Stratifying the patient population according to the ACS clinical presentation, NK cell frequency was significantly higher in STEMI than in NSTEMI patients, without difference between other lymphocyte subsets 1 . Relationship between natural killer cells and renal function in acute coronary syndromes A Toso A Toso 1 Misericordia e Dolce Hospital, Department of Cardiology , Prato , Italy L Tanini L Tanini 2 Flow Cytometry Laboratory , Prato , Italy T Pizzetti T Pizzetti 2 Flow Cytometry Laboratory , Prato , Italy M Leoncini M Leoncini 1 Misericordia e Dolce Hospital, Department of Cardiology , Prato , Italy M Maioli M Maioli 1 Misericordia e Dolce Hospital, Department of Cardiology , Prato , Italy D Tedeschi D Tedeschi 1 Misericordia e Dolce Hospital, Department of Cardiology , Prato , Italy M Amato M Amato 3 Misericordia e Dolce Hospital, Department of Nephrology , Prato , Italy F Bellandi F Bellandi 1 Misericordia e Dolce Hospital, Department of Cardiology , Prato , Italy Open in new tabDownload slide Abstract 326 Figure NK cells and Creatinine Clearance in ACS Open in new tabDownload slide Abstract 326 Figure NK cells and Creatinine Clearance in ACS Abstract Recent experimental studies suggest that NK cells are involved in the pathogenesis of atherosclerosis and one clinical report documented higher circulating levels of NK cells in patients with severe atherosclerotic disease. Moreover, impaired renal function is an important risk factor for patients with symptomatic coronary artery disease. Aim of this study was to evaluate a possible relationship between renal function and the frequency of peripheral NK cells in patients with acute coronary syndrome (ACS). Methods: The study group included 30 pts (17 with STEMI and 13 with NSTEMI ACS). All study patients were submitted to coronary revascularization at our institution. On admission peripheral blood sample was taken in all patients to measure the distribution of lymphocyte subsets by flow cytometry. Creatinine clearance (CrCL) was calculated on admission by applying the Cockroft-Gault formula to the serum creatinine. Results: In the overall group (mean age 71 ± 12.3 yrs, 20 men) mean estimated CrCL was 59 ± 21ml/min; 53% (16/30) of patients had an estimated CrCL < 60 ml/min. NK cell frequencies were higher in patients with CrCL < 60 ml/min than in patients with CrCL > = 60 ml/min (24 ± 16% vs 16 ± 10%, p = 0.05). A significant correlation was found between CrCL values and frequency of NK cells (R = −0,58; p = 0,001). Conclisions: Among patients with ACS peripheral NK cell frequencies are increased in patients with a impaired renal function and a significant correlation exists between NK cell frequencies and extimated creatinine clearance 1 . Opposed effects of c-reactive protein isoforms on platelet activation and VASP phosphorylation B Molins B Molins 1 Hospital de la Santa Creu i Sant Pau, Cardiovascular Research Center, CSIC-ICCC , Barcelona , Spain E Pena E Pena 1 Hospital de la Santa Creu i Sant Pau, Cardiovascular Research Center, CSIC-ICCC , Barcelona , Spain L Badimon L Badimon 1 Hospital de la Santa Creu i Sant Pau, Cardiovascular Research Center, CSIC-ICCC , Barcelona , Spain Abstract Purpose: The lowering of C-reactive protein (CRP) has been related to reduction of cardiovascular events (JUPITER trial) suggesting that CRP may be a direct causative factor of atherothrombosis. We have previously reported that CRP bioactivity on thrombogenesis and platelet deposition seems based on loss of its pentameric symmetry (natCRP), resulting in formation of monomeric CRP (mCRP). Here our aim was to provide mechanistical information on direct and opposed effects of CRP isoforms on platelet activation. Methods: Fresh human blood was incubated with CRP isoforms (0-50 ug/mL) and platelet activation was evaluated by flow cytometry measuring surface P-selectin and CD63 expression. The responses to CRP were also evaluated in the presence of mitogen-activated protein (MAP) kinases inhibitors, CD36 and CD16 blocking-function antibodies, abciximab or clopidogrel pretreatment. The extent of platelet aggregation was also measured in platelet rich plasma containing CRP isoforms. Washed platelets were incubated with CRP isoforms (25 ug/mL) and VASP phosphorylation was analyzed by western blot of platelet lysates and immunofluorescence. Results: Platelet aggregation and flow cytometry analysis revealed that high levels of mCRP significantly induced platelet aggregation and surface P-selectin and CD63 exposure in a dose-dependent manner, whereas natCRP was unable to produce any effect at any tested dose. p38 MAPK and JNK inhibitors as well as CD36 blocking antibody partially inhibited the effect of mCRP in P-selectin expression, whereas MEK1/2 inhibitor and CD16 blocking antibody did not exert any effect. Moreover, treatment with abciximab (GPIIb-IIIa antibody) almost completely inhibited mCRP-induced platelet activation, while mCRP showed no effect on platelets from volunteers who had previously taken clopidogrel (150 mg). mCRP significantly induced vasodilator-stimulated phosphoprotein (VASP) dephosphorylation by reducing PKG activity contributing to platelet shape change. natCRP, in contrast, significantly induced VASP phosphorylation by increasing PKG activity. Conclusions: These data indicate that whereas serum natCRP may no affect platelet activation, the tissue-associated form of CRP, mCRP, displays a prothrombotic phenotype contributing to platelet activation and atherothrombotic complications. This work has been possible thanks to PNS-MICINN SAF2006-10091, CIBERobn CB06/03 Instituto de Salud Carlos III, and Lilly Foundation. Impact of cangrelor on P2Y12 mediated platelet reactivity in patients with diabetes mellitus and coronary artery disease JL Ferreiro Gutierrez JL Ferreiro Gutierrez 1 University of Florida-Shands Jacksonville , Jacksonville , United States of America M Ueno M Ueno 1 University of Florida-Shands Jacksonville , Jacksonville , United States of America R Alissa R Alissa 1 University of Florida-Shands Jacksonville , Jacksonville , United States of America K Dharmashankar K Dharmashankar 1 University of Florida-Shands Jacksonville , Jacksonville , United States of America D Capodanno D Capodanno 1 University of Florida-Shands Jacksonville , Jacksonville , United States of America B Desai B Desai 1 University of Florida-Shands Jacksonville , Jacksonville , United States of America TA Bass TA Bass 1 University of Florida-Shands Jacksonville , Jacksonville , United States of America DJ Angiolillo DJ Angiolillo 1 University of Florida-Shands Jacksonville , Jacksonville , United States of America Open in new tabDownload slide Abstract 328 Figure Open in new tabDownload slide Abstract 328 Figure Abstract Purpose: Patients with diabetes mellitus (DM) have high platelet reactivity and reduced response to oral P2Y12 inhibitors compared with non-DM patients. Cangrelor is a novel intravenous P2Y12 inhibitor with potent and reversible effects. Our hypothesis is that cangrelor overcomes high platelet reactivity in DM patients and induces a similar degree of P2Y12 inhibitory effects compared with non-DM patients. Methods: Platelet reactivity was evaluated in 80 clopidogrel naïve patients with coronary artery disease on aspirin therapy. Functional assays included flow cytometric analysis of the status of phosphorilation of the vasodilator-stimulated phosphoprotein (VASP) and multiple electrode aggregometry (MEA) following ADP stimuli. Samples were evaluated at baseline and after in vitro incubation with cangrelor (500 nM). VASP results were expressed in percentage of P2Y12 reactivity index (PRI) and MEA results as area under the curve of arbitrary units (AU*min). Results: No differences in baseline levels of PRI and ADP-induced platelet aggregation were observed in patients with (n = 40) and without (n = 40) DM (PRI: 84.8% [79.2-88.0] vs. 85.9% [82.5-88.8]; AU*min: 650 [457-903] vs. 627 [440-793]; p = NS for both). A marked decrease in PRI and AU*min was observed in both groups after incubation with cangrelor (DM: 13.6% [9.6-22.4] and 108 AU*min [70-153]; non-DM: 11.9% [7.9-23.8] and 101 AU*min [64-123]; p < 0.0001 for every comparison with baseline values). A similar degree of platelet inhibitory effects, defined as the absolute difference before and after incubation with cangrelor, was achieved in DM compared to non-DM patients using both assays (Figure). Conclusions: Potent blockade of the P2Y12 signaling pathway with cangrelor enables achieving similar inhibitory effects in DM and non-DM patients 1 . The mechanism of aldosterone prothrombotic action in rats E Chabielska E Chabielska 1 Medical University of Bialystok, Department of Biopharmacy , Bialystok , Poland A Gromotowicz A Gromotowicz 1 Medical University of Bialystok, Department of Biopharmacy , Bialystok , Poland J Szemraj J Szemraj 2 Medical University of Lodz, Department of Medical Biochemistry , Lodz , Poland A Stankiewicz A Stankiewicz 1 Medical University of Bialystok, Department of Biopharmacy , Bialystok , Poland A Zakrzeska A Zakrzeska 1 Medical University of Bialystok, Department of Biopharmacy , Bialystok , Poland Abstract Our previous study showed that acute aldosterone (ALDO) infusion enhances venous thrombosis in normotensive rats in the mechanism related to reduced nitric oxide synthesis and increased oxidative stress. The aim of the present study was to investigate the possible changes in haemostasis and hemodynamic parameters during one-hour ALDO infusion. Moreover, the role of mineralocorticoid receptor (MR), as well as angiotensin receptor AT1, since some of ALDO effects could be related to angiotensin II action, was determined. Male Wistar normotensive rats (300-350g) were used in this study (n = 12-15). ALDO, (30 µg/kg/h) or control (VEH, 0.4% ethanol; 2 ml/kg/h) was infused into the femoral vein 5 minutes before venous thrombosis induction and was continued for 1 hour. Eplerenone (EPL, 100 mg/kg), a selective MR antagonist, was administered p.o., 30 minutes before ALDO infusion. Valsartan (VAL, 10 mg/kg), a selective AT1 antagonist, was infused 5 minutes before ALDO infusion. The hemodynamic parameters: carotid blood flow, systolic and diastolic blood pressure and heart rate were measured continuously throughout the study. Template bleeding time (BT) was also measured. Platelet adhesion to collagen ex vivo and in vitro (10-8M to 10-5M) was carried out. Tissue factor (TF), plasminogen activator (t-PA), plasminogen activator inhibitor type-I (PAI-I) and thrombin activatable fibrinolysis inhibitor (TAFI) plasma levels were measured by enzyme immunoassay. Marked increases in thrombus mass and thrombosis incidences were observed after ALDO infusion. ALDO infusion significantly shortened BT and increased platelet adhesion ex vivo. TF, PAI-1 and TAFI plasma levels were increased while t-PA level was reduced in ALDO-treated group. Co-administration of EPL significantly, although partially reduced thrombosis and diminished the effects of ALDO on BT, platelet adhesion ex vivo, TF, t-PA, PAI-1 and TAFI. VAL administered together with ALDO also reduced thrombus formation. Moreover, pretreatment with VAL reversed partially the ALDO-induced fibrinolysis impairment. The hemodynamic parameters were unaffected throughout the ALDO infusion. ALDO did not change platelet adhesion in vitro. In conclusion, short-term ALDO infusion enhances experimental venous thrombosis in the mechanism involving primary haemostasis, coagulation and fibrinolysis-dependent pathways. Our results suggest the involvement of indirect mechanism(s) in ALDO-increased platelet adhesion. The prothrombotic effect of ALDO is partially mediated by MR, as well as AT1 receptor. Macrophage depletion using clodronate liposomes after transmural myocaridal infarction leads to intraventricular thrombosis in mice SAA Mohammed SAA Mohammed 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy F Molla F Molla 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy A Soldo A Soldo 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy I Russo I Russo 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy G Germano G Germano 2 Clinical Institute Humanitas IRCCS , Milan , Italy G Balconi G Balconi 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy L Staszewsky L Staszewsky 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy R Latini R Latini 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy Open in new tabDownload slide Abstract 330 Figure Echocardiography,H & E & VonKossa staining. Open in new tabDownload slide Abstract 330 Figure Echocardiography,H & E & VonKossa staining. Abstract Background: Role of macrophages, which play a central role in wound healing, can be studied in MI with tools such as clodronate liposomes (CL) which are reported to eliminate macrophage/monocytes with low toxicity and high specificity. Aim: To study the role of macrophages post MI after depleting them using CL. Methods: Circulating monocytes were depleted using CL in intact mice & confirmed 24h later by FACS analysis. MI was induced by coronary artery ligation (CAL) in 37 C57 mice, injected with 0.2ml (5mg/ml) CL (i.v & i.p) or liposomes or PBS immediately after CAL & on day 3. Echocardiography was performed 4 weeks later, and sacrificed hearts were harvested for histology. Results and Conclusion: Percentage & absolute number (per microlitre of blood) of circulating monocytes in intact mice were 5.1 ± 0.3% & 212 ± 26 before, and 1 ± 0.3% & 47.3 ± 15.6 24h after CL respectively. No significant differences were observed among CAL groups after echocardiography & infarct size analysis. LVEF in intact mice was 57 ± 3.42%, while it was similar for all CAL groups (38 ± 3.35% in Liposome, 28 ± 3.79% in CL & 24 ± 2.99% in PBS). CD68-stained macrophage density in peri-infarct zone (296 ± 21.4/mm 2 in CL, 426 ± 56/mm 2 in Liposome, 405 ± 18.1/mm 2 in PBS) was significantly lower in CL group vs Liposome (p = 0.02) or PBS (p = 0.001) groups. All CL treated CAL mice with large transmural infarcts showed large intraventricular thrombi attached to the endocardium with extensive calcium deposition. Thrombi were never found neither in liposome or untreated CAL mice, nor in CL treated intact mice. The unexpected finding while deserves further investigation, prevents the use of clodronate liposome after MI induced by CAL in the mouse 1 . The effects of hypertension on human coronary micro vascular structure F Lynch F Lynch 1 Univeristy of Manchester, Cardiovascular Research Group, Biomedical Sciences , Manchester , United Kingdom C Austin C Austin 1 Univeristy of Manchester, Cardiovascular Research Group, Biomedical Sciences , Manchester , United Kingdom B Prendergast B Prendergast 2 Manchester Royal Infirmary, Manchester Heart Centre , Manchester , United Kingdom D Keenan D Keenan 2 Manchester Royal Infirmary, Manchester Heart Centre , Manchester , United Kingdom R Malik R Malik 1 Univeristy of Manchester, Cardiovascular Research Group, Biomedical Sciences , Manchester , United Kingdom A Izzard A Izzard 1 Univeristy of Manchester, Cardiovascular Research Group, Biomedical Sciences , Manchester , United Kingdom AM Heagerty AM Heagerty 1 Univeristy of Manchester, Cardiovascular Research Group, Biomedical Sciences , Manchester , United Kingdom Open in new tabDownload slide Abstract 331 Figure Open in new tabDownload slide Abstract 331 Figure Abstract Changes in micro vascular morphology have been reported in subcutaneous arteries from hypertensive (HT), diabetic, hypertensive and diabetic and normotensive (NT) patients. To date there have been no studies examining the structure of isolated human coronary resistance arteries. We isolated human coronary resistance arteries taken from the left atrial appendage taken at the time of coronary artery bypass graft surgery in NT (n = 12) and HT (n = 32) patients. Arteries were studied in vitro using a pressure arteriograph system to quantify their structural characteristics. They were pressurized to 60 mmHg. Following equilibration all vessels studied was exposed to calcium free PSS and a pressure curve (3mmHg–100mmHg) was performed. Lumen diameter and wall thickness were measured and wall:lumen ratio and cross sectional area were calculated. Values are expressed as mean (+/− SEM). Data were analysed using 2 way ANOVA and unpaired t tests. Blood pressure of treated hypertensive patients (138/78mmHg) was similar to that of normotensive (138/76mmHg) patients. There was no significant differences (P = 0.6) in diameter, HN 128(7)μm vs NT 120(12)μm and wall:lumen ratio HN 28(3) vs NT 33(5) at 60mmHg. The stress strain relationship (Fig 1 ) was significantly shifted (P < 0.05) to the left in arteries from hypertensive patients compared to those from normotensive patients. These results demonstrate that hypertension increases arterial stiffness but successful control of blood pressure may prevent hypertension associated structural changes. Pharmacological characterization of arteriolar smooth muscle located vanilloid receptor-1 (TRPV1) A Czikora A Czikora 1 Institute of Cardiology, University of Debrecen , Debrecen , Hungary E Lizanecz E Lizanecz 1 Institute of Cardiology, University of Debrecen , Debrecen , Hungary I Rutkai I Rutkai 1 Institute of Cardiology, University of Debrecen , Debrecen , Hungary J Boczan J Boczan 2 University of Debrecen, Department of Neurology , Debrecen , Hungary R Porszasz R Porszasz 3 University of Debrecen, MHSC-Faculty of Medicine, Department of Pharmacology and Pharmacotherapy , Debrecen , Hungary Z Papp Z Papp 1 Institute of Cardiology, University of Debrecen , Debrecen , Hungary I Edes I Edes 1 Institute of Cardiology, University of Debrecen , Debrecen , Hungary A Toth A Toth 1 Institute of Cardiology, University of Debrecen , Debrecen , Hungary Abstract The vanilloid receptor 1 (TRPV1) plays a role in the activation of sensory neurons by various painful stimuli; therefore TRPV1 antagonists become a promising therapeutic candidate to target the pain pathway. However, functional TRPV1 expression was observed in the arteriolar smooth muscle cells affecting tissue blood distribution. This latter finding suggests not only a possible side effect of TRPV1 antagonist therapy for analgesia, but also presents a possible new drug target to regulate microvascular diameter. Here we characterized the vascular smooth muscle located TRPV1 mediated vasoconstriction to establish its pharmacological properties. Vascular diameter of isolated, pressurized skeletal (m. gracilis) muscle arterioles (diameter: 207 ± 7 μm, n = 56) possessing spontaneous myogenic tone was measured upon treatment by various TRPV1 agonists/partial agonists. Potency, efficacy, kinetics of action, receptor desensitization and in some cases changes in intracellular Ca2+ contrentracion was determined. Some of the tested TRPV1 agonists evoked substantial constrictions (capsaicin, MSK-195, JYL-79), while others were without effect (resiniferatoxin, JYL-273). A complete desensitization of arteriolar TRPV1 was observed after agonist treatments (with the exception of capsaicin). A partial agonist (JYL-1511) was used to estimate the sufficient level of TRPV1 stimulation which evokes desensitization of arteriolar TRPV1. Finally, a TRPV1 antagonist, AMG9810, suggested that the antagonism is competitive. Our data suggests that the arteriolar smooth muscle located TRPV1 has different structure-activity relationship than the sensory neuron located receptor. These differences may help to design antagonists devoid of vascular side effects as well as new drug candidates regulating microvascular diameter. Geometric and functional characteristics of pial arterioles in obese Zucker rats. A Colantuoni A Colantuoni 1 University Hospital Federico II , Naples , Italy S Vagnani S Vagnani 2 Department of Oncology, Transplants and Advanced Technologies in Medicine, University , Pisa , Italy D Lapi D Lapi 1 University Hospital Federico II , Naples , Italy Abstract The mean objective of this study was to assess the in vivo geometric features of pial microvascular networks in obese Zucker rats (OZR) because the vascular architecture plays a key role in the regulation of the cerebral microvascular blood flow. Previous studies have suggested that cerebral network arrangement contributes to the outcome of stroke. Furthermore, the present study was aimed to evaluate the rhythmic diameter changes of pial arterioles, because type II diabetes mellitus is known to determine an abnormal reactivity of the resistance vasculature leading to inadequate control of capillary blood flow. Rat pial microcirculation was observed using fluorescence microscopy through a cranial window. The arteriolar network was mapped by stop-frame images. Diameters and lengths were measured with a computer-assisted method. Pial arterioles were classified according to Strahler method modified according to diameter. Arteriolar segments and elements were used to express the series-parallel features of the pial arteriolar networks and a connectivity matrix was elaborated to describe the connection of blood vessels from one order to another. The rhythmic variations in diameter of pial arterioles were evaluated with computer- assisted power spectrum techniques. Pial microcirculation in OZR presented four orders of branchings, from order 1 (diameter: 14.2 ± 2.4 μm) to order 4 (43 ± 3 μm). Order 1 arterioles gave origin to capillaries (order 0). The diameter, length and branching of the arteriolar orders grew as a geometric sequence with the order number in accordance with Horton's law. The segments/elements ratio was higher in order 4 and 3 arterioles, indicating the greatest asymmetry of ramifications as observed in Wistar rats. The connectivity matrix showed that order 4 arterioles gave origin to most order 2 and 1 arterioles while few order 3 vessels derived from these parent arterioles: branching patterns significantly different compared with those calculated in Wistar rats. OZR order 3 and 2 arterioles showed a lower fundamental frequency compared with the frequency detected in Wistar rats (0.015 ± 0.005 Hz and 0.08 ± 0.02 Hz versus 0.03 ± 0.01 Hz and 0.12 ± 0.01 Hz, respectively, p < 0.01). Therefore, OZR pial microvascular networks appeared arranged in four arteriolar orders and these branching patterns presented several smaller vessels with vasomotion activity slower when compared with Wistar rats oscillations. These cerebral microvascular features observed in OZR may contribute to further clarify the multiple hemodynamic and neuronal complications observed in the diabetic brain pathophysiology. Impairment of finger arteriolar functions in patients with ischemic heart failure and high post open-heart surgery complication score N Maroz-Vadalazhskaya N Maroz-Vadalazhskaya 1 Scientific and Practical Center of Cardiology , Minsk , Belarus I Koslov I Koslov 1 Scientific and Practical Center of Cardiology , Minsk , Belarus V Shumavetz V Shumavetz 1 Scientific and Practical Center of Cardiology , Minsk , Belarus T Glibovskaya T Glibovskaya 1 Scientific and Practical Center of Cardiology , Minsk , Belarus Y Ostrovskiy Y Ostrovskiy 1 Scientific and Practical Center of Cardiology , Minsk , Belarus Abstract Poor heart systolic function leads to impairment of micricirculation.The aim was the estimation of link between the finger microcirculation and early postoperative complications in patients with ischemic heart failure, whom coronary bypass grafting (CABG) was performed. Material and Methods: Cohort of 52 patients (age 55.3 ± 8.48 yrs, 45 male) with coronary artery disease (mean 2.7 ± 0.4 vessels) and heart failure (NYHA class 3.14 ± 0.11), LVEF 29.5 ± 7.3%, BNP 375 ± 341.7 pg/ml (12-1560 pg/ml), depressed heart rate variability with SDNNi 40.2 ± 19.9, RMSD 27 ± 20.8 (at 24h ECG) had undergone echo (SONOS 5500) and duplex ultrasound exam of finger arteriola (FA) (probe 6-15 MHz), 24 hours ECG-monitoring (Holter) with heart rate variability (HRV) analysis, BNP level (Triage Bioset) before surgery, at discharge and 1 year after surgery). All patients had undergone CABG and LV plasty. Mean cross-clamping and total ischemia time were 145.0 ± 49.5 min and 101.0 ± 38.5 min resp. A 30-day mortality was 10% (5 pts) and 1-year – 15.4% (8 pts). Event complication score (ECS) was applied as 1-perioperative inotropic support, 2-perioperative inotropic support and intraaortic balloon pumping (IABP), 3- death, 0- neither support nor death. Patients were divided in two groups: 1 – 10 patients with ECS = 0, 2 group – 20 patients with ECS>0. Groups were comparable in age, volume of operation and LVEF. Results: At base patients of both groups had depressed LVEF (p = NS), increased BNP level (163.8 pg/ml and 264.8 pg/ml, between groups p < 0.005), depressed HRV (SDNNi 53.2 ± 15.1 and 32.1 ± 12.7, p < 0.01, Mean FA diameter was larger in group 2 (7.61 mm vs 5.38 mm, p < 0.0001). Endothelium dependent (ED) and endothelium non dependent (END) dilatation was more significant in patients of group 1 without postoperative complications (ED % of FA diameter increasing 55.4% (p < 0.004 vs basal) vs 30.9% (p < 0.02 vs basal) in group 1 and 2 resp., between groups p = NS; END % of FA diameter increasing 71.2% (p < 0.001 vs basal) vs 20.0% (p = NS vs basal) in group 1 and 2 resp., between groups p < 0.01). Strong negative correlation was found in all patients between BNP level and FA basal diameter (r = −0.92, p < 0.01). ED and END diameter and flow volume correlated different in patients of groups: patients without postoperative complications had positive correlation of BNP and FA circulation (diameter and volume flow increasing) (r = 0.89, p < 0.01), but patients of gr.2 had negative one (r = −0.20 p = 0.05). Conclusion: patients with complicated postoperative period is characterized impaired FA circulation, which correlated with BNP level before surgery. How much is the pulsation of the blood flow in the human precapillary arterioles of the eye? AG Koutsiaris AG Koutsiaris 1 TEI of Larissa, Department of Medical Laboratories , Larissa , Greece SV Tachmitzi SV Tachmitzi 2 University Hospital of Larissa, Department of Ophthalmology , Larissa , Greece MG Kotoula MG Kotoula 2 University Hospital of Larissa, Department of Ophthalmology , Larissa , Greece A Giannoukas A Giannoukas 3 University Hospital of Larissa, Department of Vascular Surgery , Larissa , Greece E Tsironi E Tsironi 2 University Hospital of Larissa, Department of Ophthalmology , Larissa , Greece Abstract Purpose: One popular way of quantifying the blood flow pulsation is the resistive index (RI) which is used for the flow resistance assessment downstream the site of measurement. However, the normal range of RI values is not known throughout the vascular tree and especially at the lowest diameter arterioles leading to the capillary bed. The purpose of this work was a preliminary quantification of the RI at the precapillary arterioles of human conjunctiva in vivo. Methods: The velocity pulse was estimated from digital images of blood flow in 12 arterioles of 5 healthy persons. The images were acquired with a high speed digital camera attached to a common slit lamp microscope with a special objective lens. After image registration, the velocity was estimated off-line. Results: The arteriolar diameters ranged between 6.3 and 11.6 µm. The average value of the velocity throughout the cardiac cycle (AVV) ranged between 1.3 and 3.3 mm/s and the overall mean value for all 12 microvessels (< AVV >) was 2.71 ± 0.2 (SE) mm/s. The RI ranged between 31.5% and 81.8% and the overall mean value (< RI >) was 58% ± 2.5% (SE). Conclusions: Characteristic parameters of the blood velocity pulse were quantified for the first time just before the human capillary bed. The RI seems to be surprisingly high just before human capillaries. Possible changes of these characteristics in pathologic conditions (e.g. carotid stenosis) remain to be investigated. Contribution of Ca2+ sensitization to contractions in basilar artery of the rat I Rutkai I Rutkai 1 University of Debrecen, MHSC-Faculty of Med., Institute of Cardiology, Division Clinical Physiology , Debrecen , Hungary A Czikora A Czikora 1 University of Debrecen, MHSC-Faculty of Med., Institute of Cardiology, Division Clinical Physiology , Debrecen , Hungary A Darago A Darago 1 University of Debrecen, MHSC-Faculty of Med., Institute of Cardiology, Division Clinical Physiology , Debrecen , Hungary P Orosz P Orosz 1 University of Debrecen, MHSC-Faculty of Med., Institute of Cardiology, Division Clinical Physiology , Debrecen , Hungary Z Megyesi Z Megyesi 1 University of Debrecen, MHSC-Faculty of Med., Institute of Cardiology, Division Clinical Physiology , Debrecen , Hungary I Edes I Edes 1 University of Debrecen, MHSC-Faculty of Med., Institute of Cardiology, Division Clinical Physiology , Debrecen , Hungary Z Papp Z Papp 1 University of Debrecen, MHSC-Faculty of Med., Institute of Cardiology, Division Clinical Physiology , Debrecen , Hungary A Toth A Toth 1 University of Debrecen, MHSC-Faculty of Med., Institute of Cardiology, Division Clinical Physiology , Debrecen , Hungary Abstract Hypertension afflicts about 40% of the adult population in developed countries. Although successful therapy can usually be established, the number of idiopathic cases is high. Nevertheless, hypertension may occurs via two major vascular mechanisms: (i) higher intracellular Ca 2+ concentration in smooth muscle resulting in increased myogenic tone and (ii) increased Ca 2+ sensitivity of the contractile system leading to increased vasoconstrictive response to agonists. The aim of this study was to characterize the Ca 2+ sensitivity of force production in isolated arteries. Methods: Arteria basilaris was isolated from the rat. Arterial rings (3-5 mm in length) were fixed on an isometric contractile measurement system. Contractile force (in mN) and intracellular Ca 2+ concentrations ([Ca 2+ ]i) of vascular rings expressed as Fura-2 (340/380 ratio) were measured simultaneously in the presence of agonists (KCl, serotonin, U-46619) and inhibitors of PKC (GF19209, 1 μM) and Rho kinase (fasudil, 2.5 μM). Results: KCl evoked a maximal contraction of 3.2 ± 0.2 mN (n = 97) with a 0.17 ± 0.01 (340/380 ratio) increase in [Ca2+]i (n = 97). The Ca 2+ sensitivity of force production was 18.5 mN/340/380 ratio, which was independent of the dose. Serotonin evoked a higher contraction 6.7 ± 0.5 mN (n = 19) with similar changes in [Ca 2+ ]i 0.15 ± 0.02 (n = 19). The Ca 2+ sensitivity of force production increased parallely with the dose (EC50 = 32 nM) reaching its maximum at 38.7 mN/340/380 ratio. U-46619, a thromboxane A2 (TxA2) receptor agonist evoked a contraction of 4.2 ± 0.5 mN with significantly lower increase in [Ca2+]i (0.04 ± 0.01 340/380 ratio, n = 23) than that of for KCl. As a result the increase in Ca2+ sensitivity of force production was more prominent (maximum: 97.9 mM/340/380 ratio, EC50 = 7,9 nM). The Ca 2+ sensitivity of force production was not affected by PKC inhibition upon serotonin and U-46619 treatments (39.7 and 111.0 mM/340/380 ratio, respectively) but decreased upon Rho-kinase inhibition (to 24.2 and 42.4 mM/340/380 ratio for serotonin and U-46619, respectively). Summary: We developed and validated a method for the determination of Ca 2+ sensitivity of force production in isolated vascular preparations in vivo. It was revealed that Rho kinase, but not PKC mediated increase in Ca2+ sensitivity of force production contributes to vascular constriction to serotonin and TxA2 receptor stimulation. Adipose tissue modulates vascular response B Eichhorn B Eichhorn 1 Dresden University of Technology, Department of Pharmacology and Toxicology , Dresden , Germany S Schudeja S Schudeja 2 Dresden University of Technology, Department of Physiology , Dresden , Germany K Matschke K Matschke 3 Dresden University of Technology, Department of Cardiosurgery , Dresden , Germany A Deussen A Deussen 2 Dresden University of Technology, Department of Physiology , Dresden , Germany U Ravens U Ravens 1 Dresden University of Technology, Department of Pharmacology and Toxicology , Dresden , Germany Open in new tabDownload slide Abstract 338 Figure Open in new tabDownload slide Abstract 338 Figure Abstract Adipose tissue is not only an energy depot, but also an endocrine organ secreting vasoactive molecules. Purpose: Here we examined the effect of perivascular adipose tissue on contraction and relaxation of arteries from mouse (A. saphena) and human (A. mammaria). Methods: Vessels were intersected into two pieces with perivascular adipose tissue (PVAT) and without (control, CTL). Contractile force of these artery rings was measured in a myograph and normalized to length. Contractions were induced by increasing concentrations of phenylephrine (PE) and relaxation in PE-precontracted arteries was examined using acetylcholine (ACh) and sodium-nitroprusside (SNP). Results: In murine Aa. saphenae the PE-induced force of contraction was reduced in presence of PVAT (3.4 ± 0.1 mN/mm) compared to CTL (3.9 ± 0.1 mN/mm, n = 4, p = 0.012). The potency of PE was lower in arteries surrounded by adipocytes (-logEC50[M] 5.3 ± 0.1 vs. CTL 5.8 ± 0.1, n = 4, p = 0.012). The endothelium-dependent relaxation with ACh was more efficient in presence of PVAT compared to the CTL (21 ± 3 % vs. CTL 35 ± 4 %, n = 5, p = 0.023), whereas EC50 values were similar. The endothelium-independent relaxation was not affected by the adipocytes. In human Aa. mammariae the contraction amplitude induced by PE was also lower in vessels surrounded by PVAT than in CTL (4.9 ± 0.8 mN/mm vs. 7.7 ± 0.7 mN/mm, n = 4, p = 0.039). The efficacy was unaltered (-logEC50[M] 5.8 ± 0.1 vs. 5.5 ± 0.4, n = 4). Most notably, PVAT blunted the spontaneous contraction of human arteries (Figure 1 : Original recordings of contraction force from human A. mammaria with (top) and without (bottom) PVAT in response to increased PE concentrations). Conclusions: Perivascular adipose tissue releases an anticontractile factor during vascular contraction and relaxation. Matrix metalloproteinase-2 may cleave calponin in vascular smooth muscle during endotoxemia MM Castro MM Castro 1 University of Alberta, Heritage Medical Research Centre (HMRC) , Edmonton , Canada JJ Cena JJ Cena 1 University of Alberta, Heritage Medical Research Centre (HMRC) , Edmonton , Canada MP Walsh MP Walsh 2 University of Calgary, Department of Biochemistry and Molecular Biology , Calgary , Canada R Schulz R Schulz 1 University of Alberta, Heritage Medical Research Centre (HMRC) , Edmonton , Canada Abstract Sepsis is one the most important causes of death in the world. Its cardiovascular manifestations include myocardial dysfunction and severe arterial hypotension, caused in part by vascular hyporeactivity to vasoconstrictors. Matrix metalloproteinases (MMPs) are known to play an important role in vascular dysfunction and tissue remodeling in many cardiovascular diseases. We have shown that MMP-2 activation in aortas exposed to bacterial lipopolysaccharide (LPS) contributes to sepsis-induced vascular hypocontractility. The mechanisms by which MMPs mediate hypocontractility need to be studied. Calponin, a 34 kDa protein located in the contractile apparatus of smooth muscle cells, contributes to vascular smooth muscle contraction by vasoconstrictors. It is structurally similar to troponin I, a regulatory protein involved in cardiac myocyte contraction, which is cleaved by MMP-2 during pro-inflammatory stress injury. Purpose: Since troponin I is degraded by MMP-2, calponin may represent an intracellular target of MMP-2 in vascular smooth muscle and contribute to MMP-2-mediated vascular dysfunction in sepsis. Methods and Results: Rats were given either an i.p. injection of a non-lethal dose of LPS (4 mg/kg) or pyrogen-free water vehicle (control). After 30 min some animals were also given an i.p. injection of MMP inhibitor doxycycline (4 mg/kg). At 6 h, plasma nitrate + nitrite levels rose from 8 ± 3 μM in control rats (n = 12) to 141 ± 75 μM in LPS rats (n = 11), indicating activation of inducible nitric oxide synthase. Protein levels of calponin and MMP-2 were analyzed in aortas by western blot. We observed a trend towards an increase in MMP-2 (1.65 fold) and a decrease in calponin levels (2.4 fold) in LPS (n = 7) vs. control rats (n = 8), the latter which was attenuated by doxycycline. Aorta extracts of control and LPS rats were immunoprecipitated with either IgG1 or anti-calponin antibodies. Immunoblot analysis with anti-MMP-2 of anti-calponin, but not IgG1, showed a 72 kDa band corresponding to MMP-2 especially in LPS rats. Analysis of the immunoprecipitate with anti-calponin using gelatin zymography also revealed a 72 kDa MMP-2 which was not detected in immunoprecipitate with IgG1. In vitro incubation of purified calponin with MMP-2 (100:1 molar ratio) for 2 h at 37°C led to calponin degradation and the appearance of an ∼15 kDa cleavage product, which was prevented by GM6001 (100 nM), an inhibitor of MMP activity. Conclusion: Calponin may be a target of MMP-2 in vascular smooth muscle of LPS-injected rats, thus possibly contributing to sepsis-induced vascular hypocontractility. Is acetylcholine induced coronary spasm associated with fibrinogen level? KL Poddar KL Poddar 1 Korea University Guro Hospital , Seoul , Republic of Korea SW Rha SW Rha 1 Korea University Guro Hospital , Seoul , Republic of Korea S Ramasamy S Ramasamy 1 Korea University Guro Hospital , Seoul , Republic of Korea JY Park JY Park 1 Korea University Guro Hospital , Seoul , Republic of Korea CU Choi CU Choi 1 Korea University Guro Hospital , Seoul , Republic of Korea HS Seo HS Seo 1 Korea University Guro Hospital , Seoul , Republic of Korea CG Park CG Park 1 Korea University Guro Hospital , Seoul , Republic of Korea DJ Oh DJ Oh 1 Korea University Guro Hospital , Seoul , Republic of Korea Abstract 340 Table Variables, n (%) . Spasm (+), n = 525 pts . Spasm (−), n = 381 pts . P-value . OR (95% CI) . Fibrinogen 285.62 ± 108.42 279.25 ± 93.57 0.248 1.00 (0.99-1.00) Age 57.55 ± 11.88 53.36 ± 13.41 <0.001 1.03 (1.01-1.04) Male gender 301 (57.3) 179 (47.0) 0.008 0.59 (0.40-0.87) PVD 64 (12.2) 22 (5.8) 0.078 0.53 (0.26-1.08) CCB 111 (21.1) 52 (13.6) 0.018 0.60 (0.39-0.91) Variables, n (%) . Spasm (+), n = 525 pts . Spasm (−), n = 381 pts . P-value . OR (95% CI) . Fibrinogen 285.62 ± 108.42 279.25 ± 93.57 0.248 1.00 (0.99-1.00) Age 57.55 ± 11.88 53.36 ± 13.41 <0.001 1.03 (1.01-1.04) Male gender 301 (57.3) 179 (47.0) 0.008 0.59 (0.40-0.87) PVD 64 (12.2) 22 (5.8) 0.078 0.53 (0.26-1.08) CCB 111 (21.1) 52 (13.6) 0.018 0.60 (0.39-0.91) Open in new tab Abstract 340 Table Variables, n (%) . Spasm (+), n = 525 pts . Spasm (−), n = 381 pts . P-value . OR (95% CI) . Fibrinogen 285.62 ± 108.42 279.25 ± 93.57 0.248 1.00 (0.99-1.00) Age 57.55 ± 11.88 53.36 ± 13.41 <0.001 1.03 (1.01-1.04) Male gender 301 (57.3) 179 (47.0) 0.008 0.59 (0.40-0.87) PVD 64 (12.2) 22 (5.8) 0.078 0.53 (0.26-1.08) CCB 111 (21.1) 52 (13.6) 0.018 0.60 (0.39-0.91) Variables, n (%) . Spasm (+), n = 525 pts . Spasm (−), n = 381 pts . P-value . OR (95% CI) . Fibrinogen 285.62 ± 108.42 279.25 ± 93.57 0.248 1.00 (0.99-1.00) Age 57.55 ± 11.88 53.36 ± 13.41 <0.001 1.03 (1.01-1.04) Male gender 301 (57.3) 179 (47.0) 0.008 0.59 (0.40-0.87) PVD 64 (12.2) 22 (5.8) 0.078 0.53 (0.26-1.08) CCB 111 (21.1) 52 (13.6) 0.018 0.60 (0.39-0.91) Open in new tab Abstract Purpose: It had been studied that coronary spasm event is followed by an increase in fibrinopeptide A level. hs-CRP is considered as significant risk factor for coronary spasm. But it is still not clear whether acetylcholine(Ach) induced coronary spasm is associated with higher fibrinogen level. Methods: Between Nov 2004 and June 2009, a total 906 patients (pts) presented with suspected variant angina underwent coronary angiography and Ach provocation test (20, 50 and 100 μg to left coronary artery) and also had assessment of fibrinogen level in blood. Pts with diffuse or focal significant spasm of more then 70% were grouped under spasm(+) group (n = 525 pts) while patient with no or insignificant spasm were grouped under spasm(−) group (n = 381 pts). Multivariate analysis using SPSS software was done to see whether pts with spasm postive patients have difference in fibrinogen level as compared with spasm negative pts. Results: Higher Age, male sex, presence of peripheral vascular disease and use of calcium channel blocker were found to be associated with coronary spasm positive event after Ach administration while peripheral vascular disease was seen as having trend towards significance towards predicting possibility of coronary spasm event with Ach. Fibrinogen level was not found to be an predictor for an event of Ach induced coronary spasm.(Table). Conslusion: Fibrinogen level in blood during event of variant angina, does not seems to be related to the Ach induced coronary spasm. Further studies needs to be done in this regard to confirm the existence of association of fibrinogen level with coronary spasm 1 . Utility of molecular diagnosis in a family with Marfan Syndrome and an atypical vascular phenotype. A Lebreiro A Lebreiro 1 Sao Joao Hospital , Porto , Portugal E Martins E Martins 1 Sao Joao Hospital , Porto , Portugal J Almeida J Almeida 1 Sao Joao Hospital , Porto , Portugal S Pimenta S Pimenta 1 Sao Joao Hospital , Porto , Portugal JM Bernardes JM Bernardes 1 Sao Joao Hospital , Porto , Portugal JC Machado JC Machado 2 Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) , Porto , Portugal C Abreu-Lima C Abreu-Lima 3 University of Porto, Faculty of Medicine , Porto , Portugal Open in new tabDownload slide Abstract 341 Figure –3D reconstruction of the magnetic resonance angiography of 3 brothers with clinical features of MFS, showing, in all of them, a peculiar mild narrowing at the level of the isthmus and a post-isthmic aortic ectasia. (Abstract 426 Figure). Open in new tabDownload slide Abstract 341 Figure –3D reconstruction of the magnetic resonance angiography of 3 brothers with clinical features of MFS, showing, in all of them, a peculiar mild narrowing at the level of the isthmus and a post-isthmic aortic ectasia. (Abstract 426 Figure). Abstract Diagnosis of Marfan Syndrome (MFS) usually relies on clinical criteria, but phenotypic presentation varies widely among affected subjects. Aortic of dissection or rupture, are the cause of death in over 90% of untreated patients. Although less frequently than in the ascending aorta, the first aortic event (dilatation or dissection) can also occur in the descending thoracic aorta. Early recognition of at risk individuals is important in view of the available medical and surgical treatments that significantly improve life expectancy. Molecular studies may provide an etiologic diagnosis in patients with milder or atypical clinical presentations and contribute to preventive management and genetic counseling of carriers, as well as in reassurance of unaffected individuals. In this paper, we describe a family that came to our attention because of two cases of type B aortic dissections, occurring in young first degree relatives, with clinical features suggestive of MFS, but with aortic diameters smaller than expected in these kind of complications. Before dissection, one of these patients and other two sisters with similar phenotypes, preformed an angioresonance that showed a peculiar anatomy of the descending aorta (Fig. 1 ), not previously described as being associated with MFS. Mutation screening and the identification of a FBN1 mutation was determinant to establish a definitive diagnosis of MFS. By describing this family with MFS and an atypical clinical presentation, we highlight the utility of FBN1 mutation's screening in selected cases. Vascular toxicity of microbeam irradiation depends on the stage of capillary maturation S Sabatasso S Sabatasso 1 University of Fribourg, Department of Medicine, Vascular Biology , Fribourg , Switzerland JA Laissue JA Laissue 2 University of Bern, Institute of Anatomy , Bern , Switzerland R Hlushchuk R Hlushchuk 1 University of Fribourg, Department of Medicine, Vascular Biology , Fribourg , Switzerland E Brauer-Krisch E Brauer-Krisch 3 European Synchrotron Radiation Facility (ESRF) , Grenoble , France A Bravin A Bravin 3 European Synchrotron Radiation Facility (ESRF) , Grenoble , France H Blattmann H Blattmann 4 Niederwiesstrasse 13 C, CH-5417 , Untersiggenthal , Switzerland K Michaud K Michaud 5 University Center of Legal Medicine , lausanne , Switzerland V Djonov V Djonov 1 University of Fribourg, Department of Medicine, Vascular Biology , Fribourg , Switzerland Abstract Purpose: The aim of this study is to explore the effects of Microbeam Radiation Therapy (MRT) on vascular biology using the chick chorio-allantoic membrane (CAM). The model represents an almost pure vascular system with immature vessels (with no periendothelial coverage) at day 8 and mature (with coverage) vessels at day 12 of CAM embryonic development. Methods: CAMs were irradiated with multiplanar beams (width: 25 μm; interbeam spacing: 200 μm) at doses between 100 and 300 Gy and were evaluated morphologically and in vivo at different time points. As comparison, seamless radiation (classical beam) of 1, 2, 5 and 10 Gy was administered to growing CAMs. Results: In vivo monitoring of day 8 CAM immature vasculature 6 hr after MRT revealed a near total destruction of the capillary plexus. Surprisingly the supplying vessels, i.e. arteries and draining vessels (veins) were not affected. Conversely, at day 12, only well defined lesions in the microvasculature (mature ones) were observed. The lesions distributed typically in parallel along the beams. Morphological investigation revealed demarcated apoptotic capillary segments within the beams' width. Time course of the changes at day 12 of development revealed that 15 minutes after a 300 Gy MRT, the CAM thickness increased dramatically. TEM revealed only an enlargement of the interendothelial cell junctions which could explain the oedema. Thirty minutes after irradiation, CAM thickness was still increased, but less pronounced. The microvessels along the beam path showed first signs of disruption. Sixty minutes after MRT, the capillary segments along the beams underwent apoptosis. The remaining vasculature recovered rapidly and CAM regained its normal thickness. Between 1 hr and 6 hr no additional morphological changes were observed. A seamless 10 Gy classical irradiation suppressed embryonic angiogenesis in the CAM. Vascular growth was inhibited, with the vasculature corresponding to earlier developmental stages. Conclusions: The effects of microbeam irradiation are most likely mediated by capillary damage. Tissue injury is due to insufficient blood supply. The vascular toxicity of microbeam irradiation depends on the stage of capillary maturation: the uncovered capillaries are much more vulnerable in comparison to the mature ones, which overcome the MRT in a range of 100 to 300 Gy. The physiological effects of MRT appear within a short time, with the most important structural alterations being present during the first 15-60 minutes after irradiation. The effects of seamless irradiation are most likely mediated by inhibition of endothelial cell division. Cinaciguat prevents neointima formation after arterial injury by decreasing proliferation and extracellular matrix metabolism K Hirschberg K Hirschberg 1 University Hospital of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany V Tarcea V Tarcea 1 University Hospital of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany SZ Pali SZ Pali 2 Heart Center, Semmelweis University , Budapest , Hungary S Korkmaz S Korkmaz 1 University Hospital of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany S Loganathan S Loganathan 1 University Hospital of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany B Merkely B Merkely 2 Heart Center, Semmelweis University , Budapest , Hungary M Karck M Karck 1 University Hospital of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany G Szabo G Szabo 1 University Hospital of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany Abstract Objective: Vascular smooth muscle cell (VSMC) proliferation and remodeling of the extracellular matrix contribute to lumen loss after arterial injury leading to restenosis. Matrix metalloproteinases (MMP) play an important role in this process by degrading structural proteins and enabling VSMCs to migrate. We previously showed that upregulation of cGMP-signaling prevents neointimal hyperplasia. In the present study, we investigated the effects of cinaciguat, a soluble guanylate cyclase (sGC)-activator on gene regulation during neointima formation. Methods and Results: Eighty male Sprague-Dawley rats underwent endothelial denudation by wire-injury of the right common carotid artery. Cinaciguat (10mg/kg/day orally) were administered to 40 rats (1-, 2-, 3-day and 1-, 2-, 3-week treatment time), while 40 rats received placebo. Left carotids served as sham controls. Cinaciguat-treatment significantly reduced neointima/media area ratio (0.45 ± 0.32 in the treatment group vs. 1.09 ± 0.69 in the control wire-injury group) and percent stenosis (17.53 ± 10.84% in the treatment group vs. 43.25 ± 30.83% in the control wire-injury group) 3 weeks after endothelial denudation, as assesed by haematoxylin-eosin stained cross-sections. By using quantitative real-time PCR, MMP-9 was significantly upregulated in the control-injured carotids at each of the six time intervals, whereas proliferating cell nuclear antigene (PCNA) gene expression increased mainly within the first week. Cinaciguat significantly reduced PCNA expression from the third day post-injury, whereas MMP-9 expression was downregulated during the whole follow-up. Plasma cGMP-concentration –as assessed by competitive enzyme immunoassay- did not change in the first three post-injury days in treated animals, and were elevated at 1- and 2-week follow-up, however the difference was not significant. Conclusion: Our results show that pharmacologic activation of sGC prevents injury-induced neointimal hyperplasia by regulating proliferation and MMP-9 expression. Therapeutic angiogenesis with recombinant bFGF: a potential strategy for the treatment of critical lower limb ischemia L Pagliani L Pagliani 1 University of Padua , Padua , Italy E Faggin E Faggin 1 University of Padua , Padua , Italy M Rattazzi M Rattazzi 1 University of Padua , Padua , Italy M Puato M Puato 1 University of Padua , Padua , Italy M Presta M Presta 2 University of Brescia , Brescia , Italy F Grego F Grego 1 University of Padua , Padua , Italy GP Deriu GP Deriu 1 University of Padua , Padua , Italy P Pauletto P Pauletto 1 University of Padua , Padua , Italy Abstract Background: Therapeutic angiogenesis represents a potential strategy for the treatment of critical lower limb ischemia (CLLI). Angiogenesis can be exerted by using recombinant proteins of pro-angiogenic factors such as basic Fibroblast Growth Factor (bFGF). Aims: 1) to produce bFGF for human use, using a properly engeneered non-pathogenic strain of E. Coli; 2) to test the in vitro and in vivo angiogenic efficacy of the recombinant bFGF; 3) to estabilish tolerability and safety of treating patients suffering from CLLI, and no chance of revascularization, with in situ bFGF administration. Methods: bFGF was isolated and purified from the bacterial cells. Growth tests in vitro on endothelial cells were made to verify the angiogenic effect of the recombinant bFGF. The bFGF efficacy was also evaluated in vivo using NZ rabbits with surgically induced acute LLI. After 15 days of tratment with either bFGF or saline the skeletal muscles of sacrificed animals were analyzed for small vessel density by histology and immunocytochemistry. The preliminary clinical testing was carried out as compassionate therapy after permission of the Ethic Committee in 2 patients with CLLI who were candidates for amputation. Follow up studies included periodically clinical-instrumental studies. Results: In vitro experiments on endothelial cells showed significant cell growth. Compared to controls, the morphometric analyses of the skeletal muscle from rabbits treated with bFGF showed a better preserved muscle structure and an increase in small vessel density (n° of capillaries/unit area 0.59 ± 0.3 vs 0.31 ± 0.5; p = 0.0031). Patients who were given bFGF in the ischemic limb had no evidence of acute adverse effects. In the first patient, the treatment with bFGF was not accompanied by clinical improvement and limb amputation was performed two weeks later. The second patient underwent discrete, progressive clinical improvement and did not undergo amputation. After a 3-month period, his walking capacity rose up to 250 meters and the RMN-angiography showed growth of collaterals within the ischemic muscle area. After a 5-year follow-up a steady clinical state is present. Conclusion: The recombinant bFGF was effective in inducing an angiogenic effect in both in vitro and in vivo. In the clinical setting, i.m. administration of our bFGF was safe and well tolerated in two patients suffering from CLLI. In one of them clinical conditions improved so that amputation was avoided. Further large-scale studies are warranted to estabilish the efficacy of bFGF as an alternative to amputation in patients with CLLI. Thrombospondin-1 in subtypes of pulmonary hypertension R Kaiser R Kaiser 1 Saarland University Hospital , Homburg , Germany Abstract Objective: One of the main treatment principles of pulmonary hypertension (PH) is vasorelaxation of pulmonary resistancevessels by accumulation of cGMP in the vascular smooth muscle cells. This pathway can be counteracted by the binding of thrombospondin-1(TSP-1) to its receptors CD47 and CD36 in high concentrations. TSP-1 can be released by any cell type under cellular stress. In endothelial cells increased shearstress and hypoxia are known to induce TSP-1. Furthermore binding of TSP-1 to CD47 on endothelial cells leads to increased apoptosis. The NO pathway of both shearstres and VEGFR is inhibited by CD47 mediated reduction of guanylate cyclase and protein kinase G. This pathway and conditions are crucial in pathogenesis and treatment of PH. We examined the clinical relevance of TSP in patients with PH. Methods: After informed consent, blood was drawn in 62 patients with pulmonary hypertension of various types. TSP-1, PDGF-ββ and big-Endothelin was determined by ELISA according to vendor instructions. Results: Patients with pulmonary arterialhypertension (n = 50) or chronic thromboembolic pulmonary hypertension (n = 5) showed a significant elevation of TSP-1 (491 ± 133 bzw.416 ± 119ng/ml), PDGF-ββ (809 ± 166 vs. 652 ± 326pg/ml) and big-Endothelin (4.45 ± 0.69 vs. 2.56 ± 1.80pg/ml) as compared to the control group (n = 21, TSP-1: 113 ± 22ng/ml, PDGF-ββ: 279 ± 107pg/ml, big-Endothelin: 1.35 ± 0.37pg/ml). Patient with a PH related to COPD (n = 5) showed a normal TSP-1 level (212 ± 54ng/ml). Although the control group was slightly younger, there was no correlation of age and TSP-level. Furthermore, a trend of decreased circulating TSP was observed after ilomedin testing. No relationship between TSP-1 levels and established hemodynamic parameters could be found. Discussion: This data suggest that pathological processes in PH as increased shearstress and chronic hypoxia may lead to an induction of TSP-1 in pulmonary hypertension. Furthermore TSP-1 could interfere with therapies using the cGMP or PKG pathways. The predictive value of TSP-1 remains to be elucidated. Secretoneurin gene therapy improves cardiac function in a model of myocardial infarction K Albrecht K Albrecht 1 Innsbruck Medical University, Department of Internal Medicine I , Innsbruck , Austria W Schgoer W Schgoer 1 Innsbruck Medical University, Department of Internal Medicine I , Innsbruck , Austria M Theurl M Theurl 1 Innsbruck Medical University, Department of Internal Medicine I , Innsbruck , Austria AGE Beer AGE Beer 1 Innsbruck Medical University, Department of Internal Medicine I , Innsbruck , Austria D Wiedemann D Wiedemann 2 Innsbruck Medical University, Department of Cardiac Surgery , Innsbruck , Austria CM Steger CM Steger 2 Innsbruck Medical University, Department of Cardiac Surgery , Innsbruck , Austria N Bonaros N Bonaros 2 Innsbruck Medical University, Department of Cardiac Surgery , Innsbruck , Austria R Kirchmair R Kirchmair 1 Innsbruck Medical University, Department of Internal Medicine I , Innsbruck , Austria Abstract Purpose: The angiogenic neuropeptide Secretoneurin (SN) is upregulated by hypoxia in several tissues like neurones and skeletal muscle cells. Recently it was shown that SN gene therapy restores tissue integrity, function and perfusion in a mouse hind-limb ischemia model by induction of angiogenesis, arteriogenesis and vasculogenesis. Therefore we hypothesized that this cytokine might also affect function of cardiac cells and exert beneficial effects in a rat model of myocardial infarction (MI). Methods & Results: To investigate the effect of SN in vitro, a matrigel assay using human coronary artery endothelial cells (HCAEC) in the absence or presence of different concentrations of SN was performed. SN dose dependently induced angiogenesis with a maximum effect at 10 ng/ml. A specific SN- antibody and interestingly a VEGF- antibody too, abolished the observed effect. The same was true for the migratory behaviour of coronary endothelial cells, as the chemotactic effect was significantly increased with 1 to 100 ng/ml SN compared to control. SN- and VEGF- antibodies abrogated these observed effects. Western Blot analysis of HCAEC extracts revealed stimulation of ERK1/2 by SN 100 ng/ml after 2 to 20 minutes, indicating activation of this signal transduction pathway. Furthermore, SN acted antiapoptotic on HCAECs as well as on human cardiac myocytes (HCMs), which was investigated using TUNEL and DAPI staining. Western blot analysis of HCM extracts provided activation of the ERK1/2 and PI3-Kinase/AKT signal transduction pathway after 5 and 10 minutes. In an model of acute MI in rats, SN gene therapy was performed by injecting 200ug plasmid intramyocardially 10 min after LAD ligation. An empty vector was used as control. Left ventricular function was evaluated post-operatively and on day 28 after LAD ligation by echocardiography. SN treated rats showed a better outcome with significantly improved ejection fraction (EF) and left ventricular end-diastolic diameter (LVEDD). Conclusion: In vitro data suggest angiogenic and antiapoptotic effects of SN on cardiac cells via activation of the MAPK and PI3-Kinase/AKT signal transduction pathway. Interestingly, the effects of SN on cardiac cells seem to be VEGF- dependent, which will be further investigated in the future. In vivo, SN gene therapy significantly improved left ventricular function in a rat MI model. These findings suggest that SN gene therapy might be a promising new therapeutic strategy to improve ischemic cardiac dysfunction. Photothermal nanodestruction with silica-gold nanoshells versus stroma ablation with lentiviral vector expressing diphtheria toxin for the management of atherosclerotic plaque AN Kharlamov AN Kharlamov 1 Ural State Medical Academy , Yekaterinburg , Russian Federation Abstract Background: Some modern angioplasty techniques generally just manipulate the form of the plaque and have a relatively high complication rate and restenosis risk. Objective: We designed it to compare two nanobiotechniques of atherodestruction in quest of more optimal approach for the angioplasty with more high level of efficacy and safety. Methods: A total of 27 white transgenic (Apo E−/−) swines (on Western diet) were assigned to the three groups (60/20 nm silica-gold nanoshells, lentivirus, and saline control). The stroma-specified lentivirus vector was constructed on the basis of long-term bone marrow/ fibroblasts cell culture. An ultrasound-mediated albumin-coated gas-filled microbubbles system has used for the noninvasive delivery of nanoshells and viral vectors into carotid arteries (in silastic collar model). The primary combined outcome was the plaque volume (PV) and the atherothrombosis rate. Results: A change of the PV (mm 3 ; on data of IVUS) immediately after the laser irradiation/ in 3 months in the group with using of nanoshells (1st group) was 18.1%/ 34.4% (p < 0.01 for all comparisons [FAC]), and in the group with stroma ablation reached 11.6%/ 44.5% (p < 0.05 FAC), and in the saline control assessed as 2.9%/ 4.3% (p < 0.01 as compared with another groups). There were some cases of atherothrombosis (3 cases) but in the nanoshell group only. The histological analysis has confirmed the ‘burning’ effect of nanophotodestruction with multiple ruptures, necrosis, homogenization of tissue, and consequent possible functional insufficiency of vessel. The average square of the wounded surface of intima was after the procedure in the 1st group about 74.9 mm 2 / 1 cm 2 (p < 0.001 FAC), in the 2nd one – about 25.1 mm 2 / 1 cm 2 (p < 0.005 FAC), and in the control is 11.3 mm 2 / 1 cm 2 (p < 0.005 FAC). In the toxin ablation group we have seen only a degradation of adventitia with signs of the plaque rebuilding, revascularization and degradation of the plaque nucleus. An influence over the non-organic part of the plaque was predominated in the groups with nanoshells’ using as compared to lentiviral ablation (61.2% vs. 44.3%, p < 0.001). Conclusions: The toxin ablation technique with viral vectors is more effective as a tool for the angioplasty with more high level of safety opening new opportunities for the following studies and reflex a clinical relevance of this idea. The nanophotodestruction approach can reason acute rupture and atherothrombosis limiting any clinical perspectives. The murine femoral artery cuff model: phenotypic assesment of the developing neointima M Cabaravdic M Cabaravdic 1 Center of Biomolecular Medicine and Pharmacology , Vienna , Austria J Breuss J Breuss 1 Center of Biomolecular Medicine and Pharmacology , Vienna , Austria P Uhrin P Uhrin 1 Center of Biomolecular Medicine and Pharmacology , Vienna , Austria BR Binder BR Binder 1 Center of Biomolecular Medicine and Pharmacology , Vienna , Austria Abstract Restenosis has remained a major limitation for use of the arterial reconstruction procedures, including balloon angioplasty, stenting and coronary artery bypass procedures, especially in the case of balloon angioplasty. Restenosis develops mainly through two processes: neointima formation and vessel remodeling. Several mouse models of neointima formation have been developed. One of the most used models is the femoral artery cuff model. In spite of its frequent use, the time course of neointima formation this models has not yet been described in detail. We have performed a series of experiments assessing especially the early time points of this model and have also tested a substance blocking neointima formation. We found that the cell death, as a consequence of the surgical injury, is much more extensive in this model than previously thought. The TUNEL assay showed death of almost all cells in the tunica media and intima 3 days after vascular injury, whereas the test substance (Indirubin-3′-monoxime [I3′MO], a known CDK inhibitor) could lower/postpone the cell death rate at 3 day time point and also reduced neointima formation (P < 0,05) 28 days after the cuff placement. I3′MO could significantly reduce the number of proliferating cells in cross sectional area after 3,7, and 14 days (P < 0,05), probably via inhibition of STAT3 phosphorilation, since we found a significant reduction of the number of pSTAT3 positive cells at all these time points. Repair mechanisms after the cuff induced perivascular injury include CD11b, Gr-1 and F4/80 positive cell mobilisation, which is influenced by I3′MO as well. These drastic effects on cell survival seen early after cuffing might help to elucidate the chain of events leading to neointima formation in this model. Effects of dipeptidyl peptidase-4 (DPP-4) inhibition on angiogenesis and hypoxic injury in type 2 diabetes F Fiordaliso F Fiordaliso 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy G Balconi G Balconi 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy SAA Mohammed SAA Mohammed 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy MS Maggioni MS Maggioni 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy A Biondi A Biondi 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy S Masson S Masson 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy L Cervo L Cervo 2 The Mario Negri Institute for Pharmacological Research, Department of Neuroscience , Milan , Italy R Latini R Latini 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy Abstract Decreased microvascular density, one of major vascular complications in diabetes, is accompanied by increased susceptibility to myocardial ischemia and by progressive left ventricular dysfunction in diabetic patients. Circulating Endothelial Progenitor Cells (EPCs) are incorporated into new vessels and promote neo-vascularization. Insufficient numbers of EPCs are reported in both type 1 and type 2 diabetic patients. The present study aims at evaluating whether the administration to diabetic ob/ob mice of a dipeptidyl peptidase-4 (DPP-4) inhibitor, an antihyperglycemic drug, with the ability to preserve the active form of the mediator of progenitor cell mobilization SDF-1 (Stromal cell-Derived Factor-1), increases EPC circulating levels, which, in turn, may contribute to the regeneration of blood vessels and reducing myocardial ischemic stress induced by strenuous exercise. Eight six-week-old male diabetic/obese ob/ob mice were orally treated with a DPP-4 inhibitor (PKF, 39 mg/kg/day) for 2 weeks while 8 age-matched, lean control and ob/ob mice received the vehicle. Before sacrifice, all mice underwent oral glucose tolerance test and a protocol of 10 min forced swimming. At sacrifice, capillary density, circulating EPCs and hypoxic areas positive to HypoxyprobeTm-1 were measured. Ob/ob mice were hyperinsulinemic, exhibited mild hyperglycemia and were glucose intolerant compared with lean age-matched control mice. Treatment with the DPP-4 inhibitor improved significantly glucose tolerance of ob/ob mice (31.3% decrease in glucose AUC compared to untreated ob/ob, p < 0.05). The number of circulating EPCs was reduced by 3.4 fold in diabetic ob/ob mice (178 ± 14 EPCs/mm 2 in controls vs 52 ± 11 in ob/ob, p < 0.05) while treatment with the DPP-4 inhibitor doubled the number of circulating EPCs in ob/ob mice (115 ± 17 EPCs/mm 2 , p < 0.05 vs untreated ob/ob). Similarly, capillary rarefaction by 24% in ob/ob mice (p < 0.05 vs controls) was compensated by the pro-angiogenic effect provided by DPP-4 inhibition (2089 ± 83 capillaries/mm 2 vs 1818 ± 52 in ob/ob untreated ob/ob, p < 0.05). Finally, hypoxic stress condition determined by forced swimming in ob/ob mice (38 ± 3% of myocardial hypoxic areas vs 3.0% in control mice, p < 0.05) was significantly reduced when ob/ob mice were treated with the DPP-4 inhibitor (24.4 ± 2.4%, p < 0.05 vs untreated ob/ob). In conclusion, DPP-4 inhibition may reestablish an adequate capillary network in the myocardium of diabetic ob/ob mice by the mobilization of endogenous EPCs and, as a result, reducing the susceptibility to myocardial ischemic injury provoked by strenuous exercise in diabetic ob/ob mice. The potential role of CD4 t-cells in therapeutic monocyte transplantation for augmentation of arteriogenesis A Francke A Francke 1 Dresden University of Technology, Heart Center, Department of Cardiology and Intensive Care , Dresden , Germany J Herold J Herold 2 Otto-von-Guericke University of Magdeburg, Departement of Cardiology , Magdeburg , Germany W Soenke W Soenke 2 Otto-von-Guericke University of Magdeburg, Departement of Cardiology , Magdeburg , Germany RH Strasser RH Strasser 1 Dresden University of Technology, Heart Center, Department of Cardiology and Intensive Care , Dresden , Germany RC Braun-Dullaeus RC Braun-Dullaeus 2 Otto-von-Guericke University of Magdeburg, Departement of Cardiology , Magdeburg , Germany Abstract Purpose: Therapeutic augmentation of collateral vessel growth (arteriogenesis) is of tremendous clinical importance for alleviation of ischemic vascular diseases. In contrast to autologous/syngeneic (same/inbred animal) monocyte transplantation (synMo), allogenic Mo (alMo) transplantation strongly promotes collateral growth. However, the mechanism has not yet been elucidated. We tested the hypothesis that alMo transplantation augments arteriogenesis through a CD4 depended T-cell response. Methods: For transplantation Mo were generated from bone marrow of C57BL6 (alMo) and Balb/c mice (synMo). Transplantation was done 24h after ligation of the femoral artery of Balb/c mice and a perfusion index (PI= ratio of un-ligated to ligated leg) was calculated by laser Doppler perfusion imaging immediately, 1, 2 and 3 wks after ligation. Furthermore, histological evaluation of collateral vessel number, diameter and capillary density within the muscle was done. For CD4-cell depletion experiments, a specific anti-CD4 antibody (100 μg) was injected intraperitoneally before and 24 h after ligation of the femoral artery. Verification of CD4-deplentation was done by longitudinal FACS analysis. In additional experiments, inter T-cell cross talk was suppressed by cyclosporine treatment, which was injected daily (20 μg/kg bw) over a period of 3 wks. Results: Transplantation of 2.5 Mio synMo resulted in a non-significant increase of PI (0.57 ± 0.09) in comparison to un-treated animals (control: PI = 0.48 ± 0.09). However, transplantation of 2.5 Mio alMo was able to augment arteriogenesis remarkably by 60% to a PI of 0.83 ± 0.09 (P < 0.001). Histological workup demonstrated a 2.5 fold increase of capillary density within the calf muscle, a measure of ischemia-driven angiogenesis, from 22 ± 2 to 50 ± 6 vessels/mm 2 (p < 0.001) in animals which had been treated with synMo but not in recipients of alMo (29.5 ± 5, n.s.) implying that arteriogenesis had resulted in sufficient blood supply to the distal limb. CD-4 depletion resulted in a 97,4% reduction of native CD4-cells after 2 wks; the arteriogenic response to alMo transplantation was completely abolished (PI:0.46 ± 0.08). As well, application of cyclosporine A inhibited the positive effect of alMo transplantation on arteriogenesis. The PI was found significantly reduced to 0.43 ± 0.12 in alMo plus cyclosporine-treated animals. Cyclosporin A treatment alone did not affect endogenous arteriogenesis (PI 0.45 ± 0.11). Conclusions: Our data implies that augmentation of arteriogenesis by alMo is dependent on a CD4-dependent immunological response of the host to the graft. Collateral vessel growth after compromised cerebral blood supply in C57/BL6 mice N Hecht N Hecht 1 Charite - University Medicine Berlin , Berlin , Germany P Vajkoczy P Vajkoczy 1 Charite - University Medicine Berlin , Berlin , Germany J Woitzik J Woitzik 1 Charite - University Medicine Berlin , Berlin , Germany Abstract Purpose: Experimental research has provided first evidence for spontaneous and therapeutic collateral vessel growth in the hypoperfused rat brain. To further explore mechanisms of collateral vessel growth and novel therapeutic approaches regarding hemodynamic perfusion deficits in the brain, an equivalent model of chronic cerebral hypoperfusion for the investigation of transgenic mouse strains is needed. Methods: Chronic impairment of cerebral blood supply was induced by uni- or bilateral carotid artery occlusion. Thereafter, we performed measurements of the cerebrovascular reserve capacity by Laser Speckle Contrast Analysis after acetazolamide stimulation on day 0, 3, 7, 14 and 21. On day 21 the mice underwent a latex/carbon black perfusion to assess collateral vessel growth. Results: Permanent bilateral carotid artery occlusion yielded a mortality rate of 90% within the first 24 hours. After unilateral carotid artery occlusion all animals survived and at all time points the cerebrovascular reserve capacity was significantly reduced by 60% on the ipsilateral and 20% on the contralateral side. Twenty-one days after vessel occlusion, the diameters of the ACA and ICA were significantly increased by 25% compared to sham and control groups. Additionally, we noted a 50% increase in the number of leptomeningeal vessels with a 40% higher number of large-caliber vessels (30-50 μm),compared to sham and control. Conclusion: Unilateral occlusion of one internal carotid artery is a successful model to study natural or induced collateral vessel growth in a model of chronic cerebral ischemia in C57/BL6 mice. Protein tyrosine phosphatases as negative regulators and novel drug targets in collateral growth D Hackbusch D Hackbusch 1 Charite - University Medicine Berlin, Center for Cardiovascular Research , Berlin , Germany N Gatzke N Gatzke 2 Charite - Campus Virchow-Klinikum, Department of Cardiology , Berlin , Germany A Duelsner A Duelsner 2 Charite - Campus Virchow-Klinikum, Department of Cardiology , Berlin , Germany O Tsuprykov O Tsuprykov 1 Charite - University Medicine Berlin, Center for Cardiovascular Research , Berlin , Germany S Slavic S Slavic 1 Charite - University Medicine Berlin, Center for Cardiovascular Research , Berlin , Germany I Buschmann I Buschmann 2 Charite - Campus Virchow-Klinikum, Department of Cardiology , Berlin , Germany K Kappert K Kappert 1 Charite - University Medicine Berlin, Center for Cardiovascular Research , Berlin , Germany Abstract Platelet-derived growth factor (PDGF)-signaling is known to be essential in vascular remodeling processes, such as restenosis and atherosclerosis. Its relevance in collateral growth, arteriogenesis, however, has not been explored. Purpose: We investigated the role of the PDGF receptors and its associated ligands, and of endogenous PDGF receptor-antagonizing protein tyrosine phosphatases (PTPs) in cerebral collateral remodeling. Methods: Arteriogenesis was induced in a 3-vessel-occlusion (3-VO) model in male Sprague Dawley rats, involving occlusion of one carotid plus both vertebral arteries. Rats underwent 3-VO surgery and were sacrificed after 3, 7, 21, and 35 days, respectively, with or without additional pharmacological PTP-blockade. Cerebral vessel diameter and cerebrovascular reserve (CVR) were assesed, in combination with immunohistochemical stainings and quantitative RT-PCR based gene expression profiling. Results: A significant and time-dependent enlargement of cerebral arteries was evident in 3-VO animals. All components of PDGF signaling and arterial growth displayed dynamic expression alterations. The most significant changes in both gene expression and vessel diameter increase were present in the ipsilateral posterior cerebral artery (PCAipsi). In particular, 3 days after 3-VO a higher proliferative activity was present compared to control animals as measured by PCNA-transript levels, which was in line with immunohistochemical analyses. This proliferation-profile during early collateral growth was accompanied by a 3-fold increase in PDGF-B and PDGF-D gene expression. While the PDGF β-receptor was also dynamically regulated after 3-VO, the PDGF α-receptor was unaffected. PDGF-receptor antagonizing PTPs PTP-1B, SHP-2, and DEP1 were found to be upregulated 2, 2.5 and 5.5 fold, respectively, in the late phase (21 days after 3-VO) of vessel remodeling, which was also characterized by lower proliferation. Finally, animals treated with the pan-PTP inhibitor bis-maltolato-oxovanadium revealed a significant increase (+19%) in the PCAipsi diameter. The CVR was also elevated by 8% compared to control animals, clearly demonstrating a beneficial impact of PTP inhibition on collateral growth. Conclusions: Our data indicate PDGF β-receptor and its cognate ligands PDGF-B and PDGF-D to be involved in arteriogenic processes. In addition, this is the first description of endogenous receptor-antagonizing PTPs being dynamically regulated during cerebral arteriogenic remodeling. PTP inhibition leading to enhanced collateral growth suggests PTPs as novel targets as anti-ischemic treatment. Targeting of cyclooxygenase-2-dependent metalloproteinase-9 production by human vascular endothelium as an explanation for the anti-angiogenic properties by statins M Massaro M Massaro 1 Institute of Clinical Physiology-CNR , Lecce , Italy E Scoditti E Scoditti 1 Institute of Clinical Physiology-CNR , Lecce , Italy MA Carluccio MA Carluccio 1 Institute of Clinical Physiology-CNR , Lecce , Italy C Storelli C Storelli 2 University of Salento, Department of Biological and Environmental Science and Technology (DiSTeBA) , Lecce , Italy A Distante A Distante 1 Institute of Clinical Physiology-CNR , Lecce , Italy R De Caterina R De Caterina 3 G. D'Annunzio University , Chieti , Italy Abstract 353 Table Treatment . MMP-9 . COX-2 . Control 10 ± 3 30 ± 7 PMA 70 ± 8* 300 ± 35* PMA + S 1 μmol/L 19 ± 5* 75 ± 12* Treatment . MMP-9 . COX-2 . Control 10 ± 3 30 ± 7 PMA 70 ± 8* 300 ± 35* PMA + S 1 μmol/L 19 ± 5* 75 ± 12* Open in new tab Abstract 353 Table Treatment . MMP-9 . COX-2 . Control 10 ± 3 30 ± 7 PMA 70 ± 8* 300 ± 35* PMA + S 1 μmol/L 19 ± 5* 75 ± 12* Treatment . MMP-9 . COX-2 . Control 10 ± 3 30 ± 7 PMA 70 ± 8* 300 ± 35* PMA + S 1 μmol/L 19 ± 5* 75 ± 12* Open in new tab Abstract Statins exert pleiotropic vasculoprotective actions that include anti-inflammatory and anti-angiogenic activities. Many of these effects are linked to their property to inhibit isoprenoid synthesis such as Ras and Rho. Cyclooxygenase(COX)-2 expression has been found increased in inflammation and angiogenesis and recently in atherosclerotic lesions where it colocalizes with metalloproteinase(MMP)-9. Recent insights into the regulation of COX-2 and MMP-9 expression suggest the involvement of a common Rho-dependent pathway, as well as the existence of a link between COX-2 and MMP-9. This prompted us to investigate the effect of statins on MMP-9- and COX-2-stimulated expression in human umbilical vein endothelial cell (HUVEC). Methods: HUVEC were incubated with activated simvastatin (S) for 12 h and then stimulated with phorbol myristate acetate (PMA) for 12 h. MMP production, COX-1/2 activity and expression were assessed by zymography, RIA for 6-keto-PGF1α and Western analysis, respectively. In parallel, we assayed the effect of statins and COX-2 inhibitors on angiogenesis performing Matrigel assay. Results: At 0.1-10 μmol/L, S reduced MMP-9 production, COX-2 activity and expression, without affecting COX-1. Densitometric analysis of MM-9 and COX-2 are shown in the Table (as unit of optical density, *p < 0.01 vs PMA). The effect of statins on COX-2 protein expression and MMP-9 production was mimicked by the Rho inhibitor C3 transferase and totally reversed by the addition of mevalonate and geranylgeranylpyrophosphate. The existence of a link between COX-2 activity and MMP-9 production was also observed since COX-2 activity inhibition by NS-398 reduced the stimulated expression of MMP-9. As a functional consequence of statin inhibition of COX-2 expression and MMP-9 production, S also reduced endothelial tubular-like differentiation on Matrigel. Conclusions: S reduces the induced expression of COX-2, as well as the stimulated release of MMP-9 in HUVEC, and through this mechanism exert an anti-angiogenic effect. This may contribute to cholesterol lowering-unrelated protective effects of statins 1 . Electrophysical effects of ropinirole in canine ventricular myocardium LV Barandi LV Barandi 1 University of Debrecen, MHSC, Department of Physiology , Debrecen , Hungary G Harmati G Harmati 1 University of Debrecen, MHSC, Department of Physiology , Debrecen , Hungary J Simko J Simko 2 MISEK nonprofit kft. , Miskolc , Hungary B Horvath B Horvath 1 University of Debrecen, MHSC, Department of Physiology , Debrecen , Hungary N Szentandrassy N Szentandrassy 1 University of Debrecen, MHSC, Department of Physiology , Debrecen , Hungary T Banyasz T Banyasz 1 University of Debrecen, MHSC, Department of Physiology , Debrecen , Hungary J Magyar J Magyar 1 University of Debrecen, MHSC, Department of Physiology , Debrecen , Hungary PP Nanasi PP Nanasi 1 University of Debrecen, MHSC, Department of Physiology , Debrecen , Hungary Abstract Dopamine agonist drugs applied in the treatment of the Parkinson's disease may significantly augment the risk of QT prolongation, as a result of which torsade de pointes ventricular tachycardia may eventuate. In an in vivo canine model ropinirole reduced the PR, while increased QTc time. hERG potassium channels expressed on CHO-K1 cells were inhibited and the action potential(AP) of canine Purkinje fibres was elongated by ropinirole. The aim of the present study was to analyse the effects of ropinirole on the AP of isolated ventricular cardiomyocytes. Experiments were done on canine isolated ventricular cardiomyocytes using conventional mikroelectrode technique. Rising concentrations of the drug were applied in a cummulative manner. Ropinirole had no effect on the resting membrane potential and on the AP amplitude. The maximum rate of depolarization (Vmax), and AP's Phase1 magnitude were reduced on concentration dependent manner. 100 μM ropinirole reduced Vmax to 83,9 ± 2,5%, while Phase1 decreased to 70,3 ± 4,6% of control. The ropinirole had biphasic effect on the action potencial duration(APD). The APD measured at 25% of repolarisation (APD25) was increased by 5,8 ± 3,6 ms on the effect of 10 μM ropinirole, but this parameter was reduced by 43,7 ± 10,9 ms in the presence of 300 μM drug. Similar biphasic action was observed in case of APD90. The APD90 changed from 221,9 ± 8,3 ms to 239,8 ± 10,8 ms in 10 μM, and to 214,5 ± 1 ms in the presence of 300 μM ropinirole. The effects of drug were reversible except on the changes of the Phase 1 magnitude. We can declare that ropinirole increased AP duration in concentrations between 3 and 100 μM, but in 300 μM the APD was decreased. These concentrations significantly exceed the therapeutic plasma level of the drug (max. 80 nM), therefore in normal conditions the foresaid side effects do not occur, but on a sensitized heart (e.g. in disturbance of repolarization) are more likely to happen. Novel oxidative markers are associated with acute myocardial injury due to radiofrequency ablation A Kaya A Kaya 1 Istanbul University,Cardiology Institute , Istanbul , Turkey I Uzunhasan I Uzunhasan 1 Istanbul University,Cardiology Institute , Istanbul , Turkey A Yildiz A Yildiz 1 Istanbul University,Cardiology Institute , Istanbul , Turkey Z Yigit Z Yigit 1 Istanbul University,Cardiology Institute , Istanbul , Turkey C Turkoglu C Turkoglu 1 Istanbul University,Cardiology Institute , Istanbul , Turkey Open in new tabDownload slide Abstract 355 Figure Distrubition of Female and male Open in new tabDownload slide Abstract 355 Figure Distrubition of Female and male Abstract Purpose: The aim of this study is to evaluate the utility of novel oxidative markers of TAS and TOS in detecting minor myocardial injury following radiofrequency ablation. Methods: We analyzed the data of consecutive 19 patients who underwent radiofrequency ablation.TAS and TOS levels were measured by new Erel's automated method. Blood samples were obtained before and 24 h after the procedure. Results: All patients had succesful radiofrequency ablation.There were significant differences between TAS and TOS levels before and after radiofrequency ablation (TAS1 = 0689 vs TAS2 = 0198 p = 0,243; TOS1 = 1,023 TOS2 = 0,907p = 0,001). Conclusion: Radiofrequency ablation may be associated with acute oxidative stress due to myocardial injury.TAS and TOS levels successfully determines the amount of oxidative stress in this setting.These novel markers may be important in other situations associated with oxidative stress due to myocardial injury 1 . Adrenergic regulation of the resting membrane potential in the left atrium and thoracic veins of the rat N Doisne N Doisne 1 University Francois-Rabelais, Faculty of Sciences PCCV (EA 4433) , Tours , France N Zannad N Zannad 2 Regional Hospital and University of Tours, Hospital Trousseau, Department of Cardiology , Tours , France B Hivert B Hivert 1 University Francois-Rabelais, Faculty of Sciences PCCV (EA 4433) , Tours , France P Cosnay P Cosnay 1 University Francois-Rabelais, Faculty of Sciences PCCV (EA 4433) , Tours , France V Maupoil V Maupoil 1 University Francois-Rabelais, Faculty of Sciences PCCV (EA 4433) , Tours , France I Findlay I Findlay 1 University Francois-Rabelais, Faculty of Sciences PCCV (EA 4433) , Tours , France Abstract The physiology of cardiac muscle in thoracic veins is of interest as a source of ectopic activity which can lead to paroxysmal atrial fibrillation. We have shown that noradrenalin can induce automatic activity in the pulmonary vein of the rat. Here we describe the effects of adrenergic receptor agonists upon the resting membrane potential recorded with intracellular microelectrodes from cardiac muscle in the left atrium (LA), pulmonary veins (PV) and superior vena cava (SVC) of the rat. Isolated tissues were continuously perfused with Krebs-Heinseleit solution gassed with 95% O2, 5% CO2 and maintained at 37°C. The quiescent resting membrane potentials were: LA -84 ± 1 mV (n = 14), SVC -74 ± 1 mV (n = 14), PV -69 ± 1 mV (n = 17). In each tissue noradrenalin (10 μM) induced a biphasic response with an initial hyperpolarisation (LA -3 ± 1 mV, SVC -9 ± 1 mV, PV -8 ± 1 mV) followed by a depolarisation (LA +1 ± 2 mV, SVC +15 ± 2 mV, PV +24 ± 1 mV). Only in PV did noradrenalin induce automatic electrical activity when the membrane potential reached ∼-50 mV. The α-adrenergic receptor agonist cirazoline (1 μM) induced a monophasic depolarisation of the resting membrane potential in each tissue: to -80 ± 2 mV in LA, to -49 ± 1 mV in SVC and to -34 ± 1 mV in PV. In no case did cirazoline induce automatic electrical activity. The β adrenergic receptor agonist isoprenaline (0.1 μM) induced a monophasic hyperpolarisation of the resting membrane potential in each tissue: to -87 ± 1 mV in LA, to -79 ± 2 mV in SVC and to -80 ± 2 mV in PV. Conclusions: 1. There are significant differences between the resting membrane potentials of the left atrium and both thoracic veins. 2. The responses of the resting membrane potential to adrenergic receptor stimulation are qualitatively similar but quantitatively different with LA. 3. Depolarisation is not sufficient to induce automatic electrical activity. Effects of diclofenac on the repolarization in dog ventricular muscle L Virag L Virag 1 University of Szeged, Faculty of Medicine, Department of Pharmacology & Pharmacotherapy , Szeged , Hungary A Kristof A Kristof 2 University of Szeged, Hungarian Academy of Sciences, Division for Cardiovascular Pharmacology , Szeged , Hungary I Koncz I Koncz 1 University of Szeged, Faculty of Medicine, Department of Pharmacology & Pharmacotherapy , Szeged , Hungary T Szel T Szel 1 University of Szeged, Faculty of Medicine, Department of Pharmacology & Pharmacotherapy , Szeged , Hungary N Jost N Jost 2 University of Szeged, Hungarian Academy of Sciences, Division for Cardiovascular Pharmacology , Szeged , Hungary P Biliczki P Biliczki 1 University of Szeged, Faculty of Medicine, Department of Pharmacology & Pharmacotherapy , Szeged , Hungary JGY Papp JGY Papp 1 University of Szeged, Faculty of Medicine, Department of Pharmacology & Pharmacotherapy , Szeged , Hungary A Varro A Varro 1 University of Szeged, Faculty of Medicine, Department of Pharmacology & Pharmacotherapy , Szeged , Hungary Abstract The nonsteroidal anti-inflammatory drug diclofenac are widely used for chronic pain. There is, however, little information about the effects of this drug on the ventricular repolarization and potassium currents in general. Therefore, the aim of our study was to characterize the cellular electrophysiological effect of diclofenac in dog right ventricular papillary muscle, Purkinje fiber and also in isolated dog ventricular myocytes by applying the standard microelectrode and whole-cell patch clamp techniques at 37 C. At a stimulation cycle length of 1000 ms diclofenac at 10 μM and 20 μM concentrations did not lengthen significantly ventricular repolarization (APD90, control: 208 ± 10 ms, 10 μM diclofenac: 205 ± 16 ms, 20 μM diclofenac: 225 ± 12 ms, n = 4-5) and at 20 μM slightly decreased the maximal rate of depolarization (Vmax, control: 271 ± 24 V/s, drug: 195 ± 18 V/s, n = 4) in dog papillary muscle indicating sodium channel blocking property of the drug. However, after impairment of the repolarization reserve by adding 30 μM BaCl2, diclofenac at the concentration of 20 μM moderately and reverse rate-dependently prolonged the action potential duration (APD90, control: 222 ± 12 ms, 30 μM BaCl2: 268 ± 14 ms, 20 μM diclofenac: 297 ± 14 ms, n = 11) in dog papillary muscle. In dog Purkinje fibers 20 μM diclofenac slightly shortened the APD90 (control: 246 ± 8 ms, drug: 226 ± 9 ms, n = 6) at basic cycle length of 500 ms, which imply the late sodium channel blocking property of the drug. The effects of diclofenac on the rapid component of the delayed rectifier (IKr), the transient outward (Ito) and on the inward rectifier (IK1) potassium currents were also investigated. The drug (50 μM) did not influence the Ito (control: 3427 ± 854 pA, drug: 3604 ± 999 pA, test potential: 50 mV, n = 6) and IK1 (control: 112 ± 16 pA, drug: 108 ± 22 pA, test potential: -60 mV, n = 3) currents. However, the IKr tail current amplitude was significantly decreased by 10 μM diclofenac (control: 68.9 ± 8.3 pA, drug: 52.3 ± 5.6 pA, test potential: 20 mV, n = 7). The estimated EC50 value of IKr blockade by the drug was approx. 50 μM. It was concluded that diclofenac at therapeutic concentration did not influence the ventricular repolarization in the heart. However, at high dose and at impaired repolarization reserve, the possible proarrhythmic side effects of the drug are can not be excluded. In vitro effects of Aliskiren on structural atrial remodeling during rapid pacing A Bukowska A Bukowska 1 Otto-von-Guericke University of Magdeburg, Institute of Clinical Pharmacology , Magdeburg , Germany K Skopp K Skopp 1 Otto-von-Guericke University of Magdeburg, Institute of Clinical Pharmacology , Magdeburg , Germany M Hammwoehner M Hammwoehner 2 St. Vincenz-Krankenhaus , Paderborn , Germany C Huth C Huth 3 Otto-von-Guericke University of Magdeburg, Department of Cardiovascular Surgery , Magdeburg , Germany S Bode-Boeger S Bode-Boeger 1 Otto-von-Guericke University of Magdeburg, Institute of Clinical Pharmacology , Magdeburg , Germany A Goette A Goette 2 St. Vincenz-Krankenhaus , Paderborn , Germany Abstract Background: Recent studies have clearly shown the activation of angiotensin II dependent molecular pathways in fibrillating human atrial tissue leading to myocardial hypertrophy and fibrosis. Thus, inhibition of profibrotic processes has been identified as a novel nonionchannel drug target to treat or prevent atrial fibrillation (AF). The aims of the present study are to study the effects of Aliskiren (a specific renin inhibitor) on pacing induced molecular changes using an organotypic human atrial tissue model. Methods: Human atrial tissue slices (n = 23) were cultivated for 24h. Electrical field stimulation was applied at 3 Hz to resemble AF with or without administration of Aliskiren. Furthermore, experiments were also performed in the presence of elevated levels of renin (0.1nM). Molecular changes were assessed on the transcriptional level using RT-PCR. Results: In nonstimulated human atrial tissue cultures, Aliskiren (1.2 to 12 nM) does not affect the expression of adhesion molecules and collagens. Aliskiren therapy alone downregulates the transcript level of CTGF (connective-tissue growth factor) in tissue cultures from patients with AF by 42% (p < 0.05). Electrical stimulation up-regulates adhesion molecules (ICAM-1, VCAM-1), PAI-1 (plasminogen activator inhibitor) and profibrotic molecules: CTGF collagens (Ia, III and IV). Aliskiren per se does not regulate the expression of these molecules. However, the addition of renin (0.1 nM) during rapid pacing enables Aliskiren to downregulate the expression of profibrotic molecules CTGF, collagen Ia (p = 0.057), and collagen IV (p < 0.01). Conclusion: Aliskiren has no effects on the expression of profibrotic genes in atrial tissue from patients in sinus rhythm. However, Aliskiren seems to have protective antifibrotic and antithrombotic effects during acute episodes of atrial tachyarrhythmia in the presence of elevated renin levels. Mathematical modelling of human atrial ion current changes associated with chronic beta-blocker therapy AJ Workman AJ Workman 1 University of Glasgow, BHF Glasgow Cardiovascular Research Centre , Glasgow , United Kingdom J Dempster J Dempster 2 University of Strathclyde, Strathclyde Institute of Pharmacy and Biomedical Sciences , Glasgow , United Kingdom GE Marshall GE Marshall 1 University of Glasgow, BHF Glasgow Cardiovascular Research Centre , Glasgow , United Kingdom AC Rankin AC Rankin 1 University of Glasgow, BHF Glasgow Cardiovascular Research Centre , Glasgow , United Kingdom Abstract Purpose: The long term treatment of patients with beta-adrenoceptor antagonists (β-blockers) has been associated with a potentially antiarrhythmic prolongation of atrial cellular action potentials (AP), and altered K+ currents; so-called pharmacological remodelling. The aim was to investigate the potential contribution of these K+ current changes to the AP-prolongation, using mathematical modelling. Methods: Action potentials were simulated using commercially available software interfaced with an established mathematical model of human atrial electrophysiology. Inward rectifier (IK1) and/or transient outward (ITO) K+ current densities were scaled according to changes previously found, in human atrial cells from our laboratory, to be associated with chronic β-blocker therapy. The contribution of these changes to the AP configuration and duration at 90% repolarisation (APD90) was assessed from the last AP of a 1 Hz 8-AP train. Results: Simulated APs had a marked "spike and dome" (Type 1) configuration, whereas the APs from our laboratory are typically of Type 3; having a less prominent plateau and shorter APD90. Therefore, the model was first modified to mimic the AP configuration and APD90 as recorded in cells from non-β-blocked patients, by appropriate scaling of the two main AP plateau currents; L-type Ca2+ current (scaled to 50%) and ultra-rapid delayed rectifier K+ current (scaled to 150%). Repeating this AP simulation with IK1 reduced by the same magnitude as reported in atrial cells from β-blocked patients (34% at -120 mV) resulted in a 26% increase in APD90, from 186 ms to 234 ms. A simulated voltage clamp of IK1, using a linear voltage ramp increasing from -120 mV to +60 mV at 26 mV/s, indicated that this AP change was due to a reduction in outward IK1, e.g., of 34% at the voltage of peak outward current, -67 mV. By reducing ITO by 41%, as associated with β-blocker therapy, but without changing IK1, the APD90 was again prolonged, but by a lesser degree (9%) than with the IK1 reduction. Simultaneously imposing these reductions in IK1 and ITO increased APD90 by 28%, compared with the 21% increase associated with β-blocker therapy. Conclusion: The use of a mathematical model modified to mimic APs recorded in human atrial cells from our laboratory has aided our understanding of potential ionic mechanisms of pharmacological remodelling. The atrial AP-lengthening associated experimentally with long term treatment of patients with β-blockers may result from the observed combined reduction in IK1 and ITO, with a potentially greater contribution from IK1. 1H NMR studies of aging rat heart with experimental atherosclerosis CR Revnic CR Revnic 1 C.C.Iliescu Cardiology Dpt , Bucharest , Romania C Ginghina C Ginghina 1 C.C.Iliescu Cardiology Dpt , Bucharest , Romania F Revnic F Revnic 2 National Institute of geriatrics and Gerontology , Bucharest , Romania Abstract Abstract: the aim of our study was related with investigation of biophysical aspects of heart membrane permeability in old rats feed on cholesterol reach diet and on rats which received also Aslavital (100 mg procaine chlorhydrate, 6 mg benzoic acid, 30 mg piridoxine chlorhydrate) and Procaine treatment. Material and method: 24 rats aged 32 month old have been divided in 4 groups of 6 rats each: group A control, group B feed for 6 weeks with cholesterol reach diet only, group C feed on cholesterol reach diet and treated for 6 weeks with Aslavital (4mg/kg body weight Procaine) and group D feed on cholesterol reach diet treated with Procaine(4mg/kg body weight) for 6 weeks. 1H RMN permeability studies of plasma membrane from control and treated rats hearts have been done with an Aremi`78 Spectrometer at 25MHz frequency by measuring T21 and T22 proton transverse relaxation times. Results: there is a decrease in proton transverse relaxation time in plasma membrane of cardiomyocyte of rats fed on cholesterol reach diet which suggest an accelerated proton exchange. Membrane permeability to water can be taken into consideration as an index of cardiovascular system recovery, important in maintaining a dynamic equilibrium with vascular destruction phenomenon due to high blood pressure. Conclusion: our results using 1H RMN proton transverse relaxation time method, pointed out that Aslavital and Procaine treatment in rats feed on reach cholesterol diet brought about the stabilization of water exchange. Aslavital is considered as a buffered Procaine with a moderate effect upon water permeability. Molecular mechanisms in control of sodium-potassium atpase funciton in hypoxic heart S Yakushev S Yakushev 1 Vetsuisse Faculty at the University of Zurich , Zurich , Switzerland IY Petrushanko IY Petrushanko 2 Engelgardt Institute of Molecular Biology RAS , Moscow , Russian Federation A Makhro A Makhro 1 Vetsuisse Faculty at the University of Zurich , Zurich , Switzerland M Segato Komniski M Segato Komniski 1 Vetsuisse Faculty at the University of Zurich , Zurich , Switzerland VA Mitkevich VA Mitkevich 2 Engelgardt Institute of Molecular Biology RAS , Moscow , Russian Federation AA Makarov AA Makarov 2 Engelgardt Institute of Molecular Biology RAS , Moscow , Russian Federation M Gassmann M Gassmann 1 Vetsuisse Faculty at the University of Zurich , Zurich , Switzerland A Bogdanova A Bogdanova 1 Vetsuisse Faculty at the University of Zurich , Zurich , Switzerland Abstract Oxygen deprivation in the heart is followed by the ATP depletion, oxidative stress and slow accumulation of Na. Successful adaptation to hypoxia includes suspension of the energy-consuming processes. NAK inhibition by a redox-sensitive liable cytosolic factor in ischemic heart was reported earlier. The nature of this liable inhibitor is currently unknown. This study focuses on the mechanisms of oxygen-induced regulation of the NAK in rat myocardium. Hypoxia was induced in isolated blood-perfused rat hearts (SO2 decrease from 98 to 35%). NAK hydrolytic activity was assessed as a function of oxygenation in heart homogenate, membrane fraction and in purified enzyme. Enzyme modifications were characterized by WB, IP, mass spectrometry (MS) and isothermal titration calorimetry (ITC). Deoxygenation resulted in a dose-dependent inhibition of the NAK in ventricular tissue homogenate. In the membrane fraction hypoxia caused dose-dependent up-regulation of NAK activity followed by the changes in phosphorylation of regulatory subunit phospholemman. NAK function in hypoxic tissue homogenate could be rescued when 300 µM L-NAME was present in the circulating blood. L-NAME had no effect on the NAK in normoxic heart. The oxygen-dependent changes in NAK activity were mediated by multiple thiol modification steps in the catalytic subunit. Deoxygenation shifted reduced or nitrosylated thiols to glutathionylated form thus shielding them from irreversible oxidation. Massive conformational changes following glutathionylation resulted in the complete NAK inhibition due to the inability to bind ATP. Thus, ATP consumption was reduced in hypoxic myocardium. Being more sensitive to oxidation α2 isoform was glutathionylated at lower GSSG levels than α1. NOS inhibition aggravated oxidative stress and resulted in restoration of the NAK activity due to de-glutathionylation and thiol oxidation. The role of glutathionylation in control of NAK in hypoxic myocardium was verified using purified α1β1 enzyme treated with GSSG and glutathionylated Cys residues were identified using MS. Extensive conformational changes in the α1 subunit triggered by glutathionylation characterized using ITC. We conclude that NAK activity is a function of NO and GSSG level in the myocardium. Hypoxia triggers decrease in NO production and mild oxidation resulting in GSSG accumulation and partial reversible deactivation of the NAK in "hibernating" myocardium. Aggravation of oxidative stress results in de-glutathionylation and irreversible oxidation of Cys residues and restoration of the NAK function. This in turn may facilitate ATP depletion and myocardial damage. Osmotic pressure regulates expression levels of cardiac aquaporins 1 and 4 A Rutkovskiy A Rutkovskiy 1 Oslo University Hospital, Ulleval , Oslo , Norway LH Mariero LH Mariero 1 Oslo University Hospital, Ulleval , Oslo , Norway KO Stenslokken KO Stenslokken 2 Institute for Molecular Biosciences, University of Oslo , Oslo , Norway G Valen G Valen 3 Institute of Basic Medical Sciences, University of Oslo , Oslo , Norway J Vaage J Vaage 1 Oslo University Hospital, Ulleval , Oslo , Norway Abstract Purpose: Cardiac aquaporins (AQPs) 1 and 4 are expressed in cardiomyocytes and endothelial cells. We hypothesized that altered blood osmolarity will cause changes in expression of AQP1 and 4 to reduce edema formation, which affects cell volume and contractile function. Methods: In series 1, we exposed HL-1 cultured cardiomyocytes to non-isoosmotic and control medium (200, 250, 300, 350, 400 mOsm/L) for 1 hour. Cells were harvested 3 and 24 hours later. In series 2, we injected 1.5 ml normo- and 0.5 ml hyperosmotic saline (1M and 2M NaCl) intraperitoneally in mice. Blood plasma and hearts were harvested 1, 4, 8 and 24 hours after the injections, n = 5 for each timepoint. Results: Western blot analysis showed an increase in glycosylated fraction of AQP1 three hours after hyperosmotic treatment with 400 mOsmol/L in HL-1 cells. In mice, blood osmolarity and aquaporin expression were only affected by injections of 2M NaCl. rtPCR analysis revealed increase in AQP1 mRNA expression 4 hours and decrease in AQP4 mRNA expression 1 hour after the injection. Western blot showed a decrease in AQP4 protein expression at 4 hours, while total AQP1 protein expression was unchanged. However, glycosylated fraction of AQP1 was upregulated 1 hour. Conclusion: High blood osmolarity influences AQP1 and 4 expression in the heart. Galphai2-specific regulation of voltage-dependent L-type calcium channels (L-VDCC) in heart S Dizayee S Dizayee 1 Department of Pharmacology , Cologne , Germany S Kaestner S Kaestner 1 Department of Pharmacology , Cologne , Germany F Kuck F Kuck 2 Department of Biochemistry and Molecular Biology II , Dusseldorf , Germany R Piekorz R Piekorz 2 Department of Biochemistry and Molecular Biology II , Dusseldorf , Germany P Hein P Hein 1 Department of Pharmacology , Cologne , Germany J Matthes J Matthes 1 Department of Pharmacology , Cologne , Germany B Nurnberg B Nurnberg 3 Department Pharmakology and Toxicology , Tubingen , Germany S Herzig S Herzig 1 Department of Pharmacology , Cologne , Germany Abstract Purpose: Voltage-dependent L-type calcium channels (L-VDCC) play an essential role in heart physiology and heart disease. They are regulated by G protein-coupled receptor (GPCR) signalling. Two inhibitory and pertussis toxin (PTX)-sensitive G proteins (Gαi2 and Gαi3) are present in the heart with Gαi2 being the predominant one. Here we elucidate their role and effects on cardiac L-VDCC. Methods: Ventricular tissue and isolated myocytes were obtained from mice with targeted deletion of either Gαi2 or Gαi3. mRNA expression levels of G proteins and L-VDCC subunits were quantified by real-time PCR. G protein expression levels were measured by immunoblotting and ADP ribosylation. Calcium channel function was assessed by whole-cell calcium current measurements. Results: In Gαi2−/− mice, the expression of Gαi3 mRNA and protein is markedly up-regulated to 187 ± 21 % and 567 ± 59 %, respectively. Likewise, Gαi2 mRNA (127 ± 5 %) and protein (131 ± 10 %) levels are enhanced in Gαi3−/− mice. Cav ± 1 subunit mRNA levels are not changed in Gαi2−/− and Gαi3−/− mice. However, Cavβ subunit expression profile in Gαi2−/− mice is different compared to wild-type mice. L-VDCC current density in cardiomyocytes from Gαi2−/− mice is significantly lowered (-7.9 ± 0.6 pA/pF, n = 11, p < 0.05) compared to wild-type. In contrast, the current density is transiently increased (-14.3 ± 0.8 pA/pF, n = 14, p < 0.05) in myocytes from Gαi3−/− mice. Steady-state inactivation is shifted to negative potentials, and recovery kinetics are slowed by gene ablation of Gαi2 (but not of Gαi3) and by acute treatment with PTX. Conclusions: These data provide new insights into isoform-specific modulation of L-VDCC by inhibitory G proteins. Gαi2 plays a pivotal role in channel regulation. This may importantly affect L-VDCC function, especially under pathophysiological conditions. A universal tool for investigating ion channel regulation by PIP2 in cardiac myocytes F Hertel F Hertel 1 Ruhr University Bochum, Department of Cellular Physiology , Bochum , Germany A Switalski A Switalski 1 Ruhr University Bochum, Department of Cellular Physiology , Bochum , Germany K Bender K Bender 1 Ruhr University Bochum, Department of Cellular Physiology , Bochum , Germany M-C Kienitz M-C Kienitz 1 Ruhr University Bochum, Department of Cellular Physiology , Bochum , Germany L Pott L Pott 1 Ruhr University Bochum, Department of Cellular Physiology , Bochum , Germany Abstract The majority of ion channels is regulated by phosphatidylinositol-(4,5)-bisphosphate (PIP2) in the cell membrane. The abundance of PIP2 in turn, is regulated by different kinases, phosphatases, and PLC in a cell-type specific manner. Ci-VSP, a depolarization-activated phosphatase, which dephosphorylates PIP2 to PI(4)P combined with fluorescent (FRET) probes for optical monitoring of changes in plasma membrane PIP2 is an emerging tool to study regulation of ion channels and transporters by PIP2 without interfering with cellular signaling pathways. For expression of Ci-VSP and a pair of FRET-generating PIP2 binding probes (PH-domains of PLCδ1 fused to CFP or YFP) a multicistronic adenoviral vector was constructed. This vector contained the cDNAs for Ci-VSP and the fluorescent PH-domains separated by viral 2A-peptide sequences in a single ORF. The 2A-sequences result in cotranslational dissociation of the polyprotein into the singular proteins. In isolated adult rat atrial myocytes infected with this construct, endogenous GIRK channel current and the simultaneously recorded CFP/YFP FRET ratio were reduced by depolarizing voltage steps in a graded voltage and time-dependent fashion. Both signals recover with half times of <10 s, corresponding to replenishment of PIP2. Our data demonstrate versatility of 2A-peptide based expression vectors for manipulating and monitoring changes of membrane phosphoinositides and effects on ion channels in cardiac myocytes. A new perspective to the heart's structure: direct tissue analysis using secondary ion mass spectrometry L Fornai L Fornai 1 University of Padua Medical School-cardiovascular Pathology , Padova , Italy A Angelini A Angelini 1 University of Padua Medical School-cardiovascular Pathology , Padova , Italy ER Erika Amstalden Van Hove ER Erika Amstalden Van Hove 2 FOM- Institute for Atomic and Molecular Physics , Amsterdam , Netherlands M Fedrigo M Fedrigo 1 University of Padua Medical School-cardiovascular Pathology , Padova , Italy G Thiene G Thiene 1 University of Padua Medical School-cardiovascular Pathology , Padova , Italy RMA Heeren RMA Heeren 2 FOM- Institute for Atomic and Molecular Physics , Amsterdam , Netherlands Abstract Secondary ion mass spectrometry is commonly used to study many different types of complex surfaces. SIMS has been used to produce biologically sensible images of organic secondary ion emission both from individual cells and from tissue samples. While it is quite possible to observe the anatomical structure of heart sample with a conventional microscope, it is impossible to identify the chemical composition of particular features. A series of secondary ion images obtained from successive sections of animal heart could, in principle, be used to produce a molecular atlas. Such atlases (based optical images) are widely used for anatomical and physiological reference. This work describes the normal anatomical structures of the rat heart using secondary ion mass spectrometry (SIMS). Rats hearts were flash-frozen, and stored at −80°C until sectioning. 10 μ thick rat heart tissue sections were cut using a cryomicrotome. All SIMS experiments were performed on a Physical Electronics (Eden Praire, MN) TRIFT-II time-of-flight SIMS (TOF-SIMS). All experiments were performed using Au+ primary ions with a primary ion beam current of 3 nA, a primary pulse length of 30 ns, and a primary ion energy of 22 14;KeV. To correlate heart's molecular profile with morphological features, the tissue sections were stained with Haematoxylin and Eosin after SIMS analysis. Surface rastering of heart tissue sections generated multiple secondary ions in a mass range up to 900 m/z. In all the experiments the several heart structures can be clearly seen, due to different lipid composition and signal intensity. In the mass range between m/z 250 and 500, a very abundant pseudomolecular ion signal of cholesterol (m/z 385) [M-H] together with the dominant fragment ion (m/z 369)[M-OH] can be observed. Several localization pattern can be distinguished. Cholesterol related ions show high intensity in both atrias, the aorta, the pulmonary artery and the outline both ventricles. The ion at m/z 523 (diacylglycerol) localized very precisely within the pulmonary artery, tricuspid and mitral valve. This ion is not localized in aortic valve. The m/z 105 (choline) signal localizes in both atrias, aorta, pulmonary artery, in the atrioventricular valves and semilunar valves but is not present in ventricles surface. The aim of our study is to identify novel and useful fragments of biomolecules in four specific major areas of the heart: the pericardium, the myocardium, the mural and valvular endocardium, and the great vessels. Novel Aspects: First time identification of characteristic molecules that map the spatial organization in heart's structure. Altered surface density of TRPM4 channel associated with cardiac conduction disorders M Kruse M Kruse Center for Molecular Neurobiology Hamburg, Institute for Neural Signal Transduction , Hamburg , Germany O Pongs O Pongs Center for Molecular Neurobiology Hamburg, Institute for Neural Signal Transduction , Hamburg , Germany Abstract TRPM4, a member of the transient receptor potential melastatin (TRPM) family, encodes a calcium-activated non-selective cation (CAN) channel. The TRPM4 channel matches the native cardiac CAN channel NSCCa, for example ion selectivity, Ca2+-dependence, single channel conductance and open probability. The CAN-channel has been suggested to contribute to the transient inward current (Iti) initiated by Ca-waves and to limit the driving force for Ca2+ entry in cardiac and other excitable cells. Genetic analysis of members of several independent families affected by cardiac conduction disorders like progressive familial heart block type I or isolated cardiac conduction disease revealed four mutations in the TRPM4 gene. Genes for other cardiac ion channels were not altered. Quantitative analysis of TRPM4-mRNA content in human cardiac tissue showed highest expression level in Purkinje fibers. Whole-cell patch-clamp recordings from HEK293-cells transiently transfected with mutant TRPM4-channels showed a significant gain-of-function associated with an increase in TRPM4 current density. Coexpression with TRPM4-wt channel displayed a dominant phenotype of mutant channels. Ca2+-dependence, single channel conductance and open probability remained unaffected in all mutant channels in comparison to wild-type. The data indicated that the observed mutational effect on TRPM4-current amplitude was most likely due to a change in channel surface density. In order to evaluate TRPM4 channel surface density, we introduced a Myc-tag into an extracellular site of TRPM4 for analysis of TRPM4 surface density using fluorescence-activated cell-sorting (FACS) and immunofluorescence analysis by confocal laser microscopy. In agreement with our electrophysiological results, both FACS and confocal laser microscopy analysis showed for Myc-tagged mutant TRPM4 channels an increase in surface density compared to wild-type. Additionally, we found an increased steady-state concentration of mutant protein in Western-blots. Cellular expression studies showed that the mutation interferes with dynein-dependent endocytosis of TRPM4 channel. We propose that increased cardiac TRPM4-channel activity slows or even blunts cardiac conduction because of its effects on membrane depolarization and Ca2+ influx. The influence of expression of mutant TRPM4-channels in primary cardiac cells is under investigation. Arginine/asymmetric dimethylarginine ratio as a novel prognostic marker in ICD patients HI Lehmann HI Lehmann 1 Otto-von-Guericke University, Departement of Cardiology , Magdeburg , Germany J Martens-Lobenhoffer J Martens-Lobenhoffer 2 Otto-von-Guericke University of Magdeburg, Institute of Clinical Pharmacology , Magdeburg , Germany M Hammwoehner M Hammwoehner 1 Otto-von-Guericke University, Departement of Cardiology , Magdeburg , Germany FW Roehl FW Roehl 3 Otto-von-Guericke University, Institute of Biometry and Medical Informatics , Magdeburg , Germany A Bukowska A Bukowska 2 Otto-von-Guericke University of Magdeburg, Institute of Clinical Pharmacology , Magdeburg , Germany SM Bode-Boeger SM Bode-Boeger 2 Otto-von-Guericke University of Magdeburg, Institute of Clinical Pharmacology , Magdeburg , Germany A Goette A Goette 4 St. Vincent's Hospital , Paderborn , Germany Abstract Purpose: Patients with severe heart failure (left ventricular ejection fraction<35%) are at high risk for the occurrence of malignant ventricular arrhythmias (VT/VF). Non-invasive and invasive risk stratification has failed to predict SCD in this cohort of patients. The aim of the present study was to evaluate, if biomarkers (arginine/asymmetric dimethylarginine) can predict the occurrence of VT/VF. Methods: Plasma levels of asymmetric dimethylarginine (ADMA) and arginine were examined in 106 patients with ischemic or dilated cardiomyopathy, who received an ICD device for primary prevention. Patients were assessed for appropriate ICD-therapy of VT/VF with a mean follow-up time of 344 ± 86 days. Results: Patients with appropriate ICD-therapy had higher mean ADMA values (0.56 ± 0.08 µmol/l vs. 0.51 ± 0.09 µmol/l; p < 0.05) and lower values of the Arginine/ADMA-ratio than patients without appropriate ICD therapy (144.71 ± 32.50 µmol/l vs. 175.30 ± 41.30 µmol/l; p < 0.01). ADMA levels weakly negatively correlated with LVEF (mean LVEF: 24 ± 6%; r = −0.23; p < 0.05). Univariate analysis revealed that ADMA, arginine, and their quotient could increase prediction of appropriate ICD therapy. An Arginine/ADMA-Ratio below 142.21 µmol/l was associated with a sevenfold increased risk of VT/VF during follow-up. Logistic regression ratified that coherency. Conclusions: The arginine/ADMA-ratio is a novel biomarker that helps to predict the occurrence of VT/VF in high risk patients. Intra- and extracellular domain of DPP10 beta-subunit are important for molecular interaction with Kv4.3 channel complex S Radicke S Radicke 1 University of Leipzig , Leipzig , Germany C Cotella C Cotella 2 University of Eastern Piedmont , Novara , Italy D Sblattero D Sblattero 2 University of Eastern Piedmont , Novara , Italy M Schaefer M Schaefer 1 University of Leipzig , Leipzig , Germany U Ravens U Ravens 3 Dresden University of Technology , Dresden , Germany E Wettwer E Wettwer 3 Dresden University of Technology , Dresden , Germany C Santoro C Santoro 2 University of Eastern Piedmont , Novara , Italy Abstract Purpose: Accessory β-subunits modulate channel function of transient outward current Ito encoded by Kv4.3. Thus, the cytosolic β-subunit KChIP2 is required for Kv4.3 integration into the plasma membrane and binds to the intracellular N-terminus of Kv4.3. DPP10 is a transmembrane glycosylated protein containing a short intracellular and a huge extracellular domain. In heterologous expression systems DPP10 hastens Kv4.3 inactivation and recovery from inactivation and shifts the half-maximum voltages of steady-state activation and inactivation to more negative potentials. The molecular interaction of DPP10 with Kv4.3 channels is poorly understood. Here, we investigated the relevance of the intra- and extracellular moieties of DPP10 for modulation of Kv4.3 channel function. Methods: DPP10 truncation mutants were co-expressed in CHO cells with stable expression of the Kv4.3/KChIP2 channel complex. The effects of the mutants on Ito current were studied with standard voltage-clamp techniques and were compared to the wild type controls. Results: Deletion of DPP10 intracellular domain completely abolished DPP10 hastening effects and resulted in Ito currents with slowed kinetics of activation, inactivation and recovery from inactivation. Steady-state activation and inactivation curves were shifted to more positive potentials. In addition, Ito channel complex reacted more sensitive to 4-aminopyrindine when the intracellular terminus of DPP10 was truncated. The deletion of the extracellular domain had only slight effects on current properties with slowed inactivation recovery and shift of steady-state activation to more positive potentials. However, eight predicted DPP10 glycosylation sites are located in the extracellular domain. Complete inhibition of N-glycosylation by tunicamycin prevented a proper trafficking of Kv4.3 subunit to the cell membrane. Mutations of four single glycosylation sites resulted in a reduction of DPP10 surface expression with functional consequences for Ito channel interaction as slowing of current kinetics. Conclusion: We conclude that both parts of DPP10 molecule are important for the interaction with Kv4.3 channels. The intracellular N-terminus of DPP10 is responsible for the effects on Ito kinetics and voltage-dependence and for sensitivity to channel blockers. The extracellular glycosylated C-terminus of DPP10 is required for translocation of the protein and for functional interaction. Beta 3-adrenoceptor antagonist SR59230 is a novel blocker of cloned cardiac Kir2.x channels C Seyler C Seyler 1 The Medical Faculty of Heidelberg , Heidelberg , Germany M Kulzer M Kulzer 1 The Medical Faculty of Heidelberg , Heidelberg , Germany E Zitron E Zitron 2 University Hospital of Heidelberg , Heidelberg , Germany EP Scholz EP Scholz 2 University Hospital of Heidelberg , Heidelberg , Germany F Welke F Welke 1 The Medical Faculty of Heidelberg , Heidelberg , Germany D Thomas D Thomas 2 University Hospital of Heidelberg , Heidelberg , Germany CA Karle CA Karle 2 University Hospital of Heidelberg , Heidelberg , Germany Abstract Purpose: Pharmacological inhibition of the cardiac inwardly rectifying potassium current IK1 can prolong action potential duration and has been shown to terminate reentry mechanisms and fibrillation under experimental conditions. However, specific IK1 inhibitors have been lacking to date. Here we show that the beta 3-adrenoreceptor antagonist SR59230 is an antagonist at cloned Kir2.x channel homo- and heteromers which form the molecular basis of cardiac IK1 current and hence might be a promising compound for the pharmacological termination of ventricular fibrillation. Methods: Human Kir2.x channels were heterologously expressed in Xenopus oocytes and current recordings were performed using the double-electrode voltage-clamp technique. Results: SR59230 inhibited cloned Kir2.x channels within the low micromolar range. Inhibitory effects varied between Kir2.1, Kir2.2, and Kir2.3 channel homomers: i.e. SR59230 blocked Kir2.3 channels with an IC50 value of 14.6 µM being 2.3-fold selective over Kir2.1 homomers and 3.2-fold selective over Kir2.2 homomers (IC50 of 33.2 µM and 46.4 µM, respectively). Onset of block was slow and only partially reversible upon washout. Analyzing inhibitory effects on Kir2.x channel heteromers, similar IC50 values could be observed for Kir2.1/2.2 and Kir2.1/2.3 heteromers (IC50 of 30.0 µM and 32.2 µM). In contrast, affinity to Kir2.2/2.3 heteromers was low, yielding an IC50 value of 49.5 µM. SR59230 lacked major effects on other potassium channels involved in repolarization (i.e. hERG, KvLQT1) even in a high concentration of 200 µM. Conclusions: Here we show that beta 3-adrenoreceptor antagonist SR59230 is a novel and specific antagonist at Kir2.x homo- and heteromers with the highest affinity to Kir2.3 homomers. Considering therapeutic effects of IK1 blockade on reentrant circuits, SR59230 may be a promising compound for the treatment of atrial and ventricular fibrillation. The new beta-subunit DPP10 may contribute to Ito regulation in heart disease K Schmidt K Schmidt 1 Department of Pharmacology and Toxicology , Dresden , Germany S Radicke S Radicke 2 Department of Pharmakology and Toxicology , Leipzig , Germany D Dobrev D Dobrev 1 Department of Pharmacology and Toxicology , Dresden , Germany U Ravens U Ravens 1 Department of Pharmacology and Toxicology , Dresden , Germany E Wettwer E Wettwer 1 Department of Pharmacology and Toxicology , Dresden , Germany Abstract Purpose: Down-regulation of transient outward current Ito is considered as an important contributor to the development of ventricular and atrial arrhythmias in heart failure and atrial fibrillation (AF), respectively. Recent studies demonstrated that dipeptidyl-aminopeptidase-like proteins (DPPLs) like DPP6 that accelerate Ito inactivation are up-regulated in human failing hearts, potentially accounting for the reduction of Ito. In this study we provide evidence for the presence of an additional member of the DPPL-family, DPP10, in human heart. Methods: The presence of DPP10 in human atrial and ventricular tissue was studied with standard RT-PCR, immunofluorescence staining and Western blot analysis. Results: DPP10 mRNA could be detected in human heart, brain, lung, liver, kidney, pancreas and spleen. At the protein level DPP10 was detectable in freshly isolated human atrial myocytes on immunofluorescence staining and showed preferential plasma membrane location. Quantification of DPP10 expression in atrial tissue revealed no significant differences between sinus rhythm and AF patients. Since there is a unique transmural gradient of Ito current in the human ventricle, we determined the transmural distribution of DPP10. As shown for DPP6, the mRNA levels of DPP10 were comparable in endo- and epicardial layers of ventricular tissue samples. Expression of DPP10 was higher in failing versus non-failing ventricles at both mRNA and protein level, clearly suggesting that increased DPP10 expression may potentially contribute to reduced Ito current in heart failure. Conclusions: Our results suggest that DPP10 is expressed in the human atrium and ventricle. DPP10 could play a role in the physiological regulation of cardiac Ito and may contribute to the reduced Ito in heart failure, with uncertain contribution to decreased Ito in AF. This novel information may have important implications for the development of new antiarrhythmic approaches. Is SAP97 the main modulator of regulating Kv ion channels in dilated cardiomyopathy? N Houshmand N Houshmand 1 University of Szeged, Faculty of Medicine, Department of Pharmacology & Pharmacotherapy , Szeged , Hungary D Menesi D Menesi 2 Hungarian Academy of Sciences, Center for Biological Research , Szeged , Hungary U Ravens U Ravens 3 Dresden University of Technology, Department of Pharmacology and Toxicology , Dresden , Germany E Wettwer E Wettwer 3 Dresden University of Technology, Department of Pharmacology and Toxicology , Dresden , Germany D Cotella D Cotella 4 University of Eastern Piedmont, Department of Clinical & Experimental Medicine , Novara , Italy JG Papp JG Papp 1 University of Szeged, Faculty of Medicine, Department of Pharmacology & Pharmacotherapy , Szeged , Hungary A Varro A Varro 1 University of Szeged, Faculty of Medicine, Department of Pharmacology & Pharmacotherapy , Szeged , Hungary V Szuts V Szuts 1 University of Szeged, Faculty of Medicine, Department of Pharmacology & Pharmacotherapy , Szeged , Hungary Abstract Background: Dilated cardiomyopathy is a multifactorial disease therefore it can be expected that several gene expression is altered in the heart. The exact nature of those changes has not been resolved yet. Voltage-dependent potassium channels regulate membrane excitability and cell communication between cardiomyocytes. Cardiac ion channels are assembled as a homomeric and heteromeric tetramers composed of α- and β-subunits providing the molecular basis for the transmembrane ionic currents. The synapse-associated protein 97 (SAP97) can associate with different potassium ion channels modulating and anchoring these proteins at the plasma membrane. Aim and Methods: The aim of this study was to compare the expression of Kv ion channels and the SAP97 in the cardiomyocytes of failing heart. We investigated the expression of the Kv ion channels by molecular biological techniques (real-time qPCR and immunoblotting) in the heart of failing and non-failing human ventricular preparations. Results: The α-subunits of mRNA of channels did not change significantly or we measured mild up- or down-regulation. HERG and KvLQT1 were slightly up regulated but the KChIP2 gene significantly decreased 50% as the MIRP4 in the left ventricle tissues of DCM patients comparing to those of undiseased controls. Immunoblotting studies revealed that the protein expression of these α-subunits is significantly decreased in the samples of patients. Conclusion: DCM remodel the expression of delayed rectifier potassium channel genes and proteins. Down-regulation was significant in the anchoring-subunits and little changed in the pore forming α-subunits. The decreased SAP97 expression suggests that alterations in regulation of potassium ion channel expression may play a main role in the development of pathological cardiac repolarization in cardiomyopathy. SAP97 modulates the Kir2.x ion channels in cardiomyocytes of failing human heart V Szuts V Szuts 1 University of Szeged, Faculty of Medicine, Department of Pharmacology & Pharmacotherapy , Szeged , Hungary N Houshmand N Houshmand 1 University of Szeged, Faculty of Medicine, Department of Pharmacology & Pharmacotherapy , Szeged , Hungary LG Puskas LG Puskas 2 Hungarian Academy of Sciences, Center for Biological Research , Szeged , Hungary N Jost N Jost 1 University of Szeged, Faculty of Medicine, Department of Pharmacology & Pharmacotherapy , Szeged , Hungary L Virag L Virag 1 University of Szeged, Faculty of Medicine, Department of Pharmacology & Pharmacotherapy , Szeged , Hungary I Kiss I Kiss 2 Hungarian Academy of Sciences, Center for Biological Research , Szeged , Hungary F Deak F Deak 2 Hungarian Academy of Sciences, Center for Biological Research , Szeged , Hungary A Varro A Varro 1 University of Szeged, Faculty of Medicine, Department of Pharmacology & Pharmacotherapy , Szeged , Hungary Abstract Background: The synapse-associated protein 97 (SAP97) is a member of the membrane-associated guanylate kinases and associate with ion channels anchoring these proteins at the plasma membrane (PM). SAP97 is able to co-expressing with Kir2x isoforms in the PM of cardiomyocytes. Inward rectifier current (IK1) is underlying Kir2x and important in the modulation of cell excitability, repolarization of action potential and determination of cellular resting membrane potential. Aim and Methods: Hypothetically the SAP97 binding to Kir2x isoforms and distribution are changed in the cardiomyocytes of failing heart. Therefore we investigated the parameters of IK1 current applying the whole cell patch-clamp and the Kir2x ion channels by molecular biological techniques in the heart of failing and non-failing human ventricular preparations. We examined the pore forming α-subunit-coding mRNA expression of Kir2.1, Kir2.2 and Kir2.3 isoforms such us Kir2.4 (distributed in Golgi) by real-time qPCR and determined the Kir2x protein by immunoblotting. Results: Expression of coding genes for Kir2.1(KCNJ2), Kir2.3(KCNJ4) and Kir2.4(KCNJ4) increased significantly (226 % , 194 % and 160%) while Kir2.2(KCNJ12) (66%) down-regulated in ventricle tissues of cardiomyopathic patients. mRNA expression of SAP97 also significantly less (30%) in the ventricular tissues of patients with dilated cardiomyopathy. Immunoblotting studies revealed that the protein expression of Kir2.1 and Kir2.3 changed but the Kir2.2 does not. Conclusion: These results suggest that the IK1 current properties are altered because the expression SAP97 bind Kir2x isoforms are changed and may be the conformation are different of these heteromeric Kir2x channel in failing human compare to the non-failing heart. Erythrocyte membrane omega-3 fatty acid status and ventricular premature complexes in asymptomatic adolescents with structurally normal hearts: a case-control mass spectrometry study SERGEY Tereshchenko SERGEY Tereshchenko 1 Medical Research Institute for Northern Problems , Krasnoyarsk , Russian Federation M Gladyshev M Gladyshev 2 Institute of Biophysics of Siberian Branch of Russian Academy of Sciences , Krasnoyarsk , Russian Federation G Kalachova G Kalachova 2 Institute of Biophysics of Siberian Branch of Russian Academy of Sciences , Krasnoyarsk , Russian Federation N Syshchik N Syshchik 2 Institute of Biophysics of Siberian Branch of Russian Academy of Sciences , Krasnoyarsk , Russian Federation N Gogolashvili N Gogolashvili 1 Medical Research Institute for Northern Problems , Krasnoyarsk , Russian Federation E Dedok E Dedok 1 Medical Research Institute for Northern Problems , Krasnoyarsk , Russian Federation L Evert L Evert 1 Medical Research Institute for Northern Problems , Krasnoyarsk , Russian Federation Abstract Experimental studies have indicated that n-3 fatty acids increase the arrhythmia threshold and effectively prevent ventricular fibrillation in animal and in vitro models. VPCs are a common form of arrhythmia that are themselves innocent but that may trigger more serious arrhythmic events, such as ventricular tachycardia or ventricular fibrillation. It supposed, that ω-3 fatty acids might intervene in the occurrence of PVCs by slowing down the spontaneous beating rate or by prolonging the refractory period. However, to our knowledge, no studies have examined the relation between fatty acid status and ventricular ectopy in healthy adolescents.It supposed that ω-3 fatty acids might intervene in the occurrence of ventricular premature complexes (PVCs) by slowing down the spontaneous beating rate or by prolonging the refractory period. However, to our knowledge, no studies have examined the relation between fatty acid status and ventricular ectopy in healthy adolescents. Aim: To examine the association between erythrocyte membrane ω-3 fatty acids content and number of PVCs over 24-h period in asymptomatic adolescents with structurally normal hearts. Methods: The total number of PVCs over 24-h period was determined by 24-h Holter monitoring in 36 healthy adolescents (11-17 years, boy/girl ratio was 19/17). We studied absolute (mg/l) content of erythrocyte membrane fatty acids using a gas chromatograph–mass spectrometer. Data are shown as median (25-75% quartiles), the Mann–Whitney test was used. Results: According to Holter monitoring two groups were generated: with PVCs>100/24-h (case group, PVCs median – 400.5/24-h, n = 8) and PVCs<100/24-h (control group, PVCs median – 1.5/24-h, n = 28). 35 fatty acids were detected (from 9:0 to 24:1ω11) in erythrocyte membrane. From ω-3 fatty acids group only for docosahexaenoic acid (DHA, 22:6ω3) we revealed decreasing in case group (28.5 (14.6-55.5) mg/l) as compared with control group (58.7 (36.4-73.2) mg/l), p = 0.017. Conclusion: Thus, a greater content of DHA in erythrocyte membrane was associated with lower ventricular ectopy in adolescents with structurally normal hearts. We suppose that DHA may have the direct antiarhythmic activity by modulation of calcium ion fluxes and/or stabilization of cardiac myocytes membranes. An electrophysiological long-term model of human adult myocardium J Wenzel J Wenzel 1 University of Lubeck, Institute for clin. and exp. Pharmacology and Toxicology , Lubeck , Germany M Brandenburger M Brandenburger 1 University of Lubeck, Institute for clin. and exp. Pharmacology and Toxicology , Lubeck , Germany R Bogdan R Bogdan 2 R&D TD Cardiovascular, Sanofi-Aventis , Frankfurt am Main , Germany D Richardt D Richardt 3 University of Lubeck, Department of Cardiac and Thoracic Vascular Surgery , Lubeck , Germany M Reppel M Reppel 4 University of Lubeck, Medical Clinic II / Cardiology , Lubeck , Germany J Hescheler J Hescheler 5 University of Cologne, Institute for Neurophysiology , Cologne , Germany A Dendorfer A Dendorfer 1 University of Lubeck, Institute for clin. and exp. Pharmacology and Toxicology , Lubeck , Germany H Terlau H Terlau 1 University of Lubeck, Institute for clin. and exp. Pharmacology and Toxicology , Lubeck , Germany Abstract Purpose: Electrophysiological myocardial research is hampered by the lack of relevant experimental models. Animal models differ from human physiology in terms of ion channel specificity and pathophysiological processes. Single human cardiomyocytes do not reflect an intact tissue environment, display variable integrity, and lose differentiation during culture. To overcome these limitations, we established slice preparations from human ventricular myocardium as an electrophysiological model and tested their pharmacological properties during long-term culture. Methods: Myocardial tissue, which was removed during heart-valve replacement surgery, was cut into 300 µm slices under cardioplegic conditions. Slices prepared within 3 hours after surgery were either used in acute experiments or cultured for up to 28 days. Action potentials were recorded in an organ bath under physiological conditions and mRNA expression levels were determined by real-time RT PCR. Results: Intracellular recordings of cardiomyocytes in fresh slices revealed typical characteristics of human endocardial action potential in terms of resting membrane potential (RMP: -77 ± 2 mV), action potential amplitude (APA: 105 ± 4 mV), max. upstroke velocity (dV/dt: 149 ± 26 V/s), action potential duration at 20% repolarisation (APD20: 164 ± 10 ms) and at 90% repolarisation (APD90: 384 ± 20 ms). Excitation conduction after focal electrical stimulation was 0.35 ± 0.04 m/s, reflecting the velocity in vivo. Treatment with dofetilide (hERG inhibition) or rilmakalim (KATP opener) changed APD90 significantly (to 151 ± 3 % and 30 ± 5 % respectively). Throughout the culture period mRNA expression of cardiac ion channels and transporters (Nav1.5, Cav1.2, hERG1, NCX) was constant. Electrophysiological properties of tissue-embedded cardiomyocytes were well preserved after 28 days (RMP: -77 ± 3 mV, APA: 95 ± 3 mV, dV/dt: 110 ± 14 V/s, APD20: 81 ± 18 ms, APD90: 354 ± 7 ms, conduction velocity 0.22 ± 0.06 m/s), indicating a fully functional state of ion currents and intercellular coupling. Since responses to pharmacological manipulation of hERG and KATP were similarly reproduced, cultured slices were suitable for pharmacologic assessment during the whole culture period. Conclusions: Overall, we introduce a new multicellular model of human myocardium, which opens up new opportunities for functional research and drug development. The method of tissue cultivation presented here makes this model available for routine electrophysiology and examinations of patient specific pathomechanisms. Changes in qrs duration and r wave amplitude in leads with st segment elevation differentiate epicardial from transmural myocardial injury R Wiegerinck R Wiegerinck 1 Hospital de Sant Pau , Barcelona , Spain C Galvez-Monton C Galvez-Monton 1 Hospital de Sant Pau , Barcelona , Spain E Jorge E Jorge 1 Hospital de Sant Pau , Barcelona , Spain R Martinez R Martinez 1 Hospital de Sant Pau , Barcelona , Spain E Ricart E Ricart 1 Hospital de Sant Pau , Barcelona , Spain J Cinca J Cinca 1 Hospital de Sant Pau , Barcelona , Spain Abstract Purpose: qrs duration and r wave amplitude are increased in acute transmural ischemic injury due to a depressed intramyocardial activation. Theoretically, when myocardial injury is confined to the epicardium, the intramyocardial activation will not be affected and thus no changes in the qrs complex will occur. We assessed whether analysis of the qrs complex can differentiate epicardial from transmural myocardial injury. Methods: The effects of local or diffuse epicardial myocardial injury induced respectively by topical or intrapericardial exposure to potassium solutions were compared with transmural myocardial injury induced by acute coronary artery occlusion in 17 pigs. The effects were measured in local electrograms from transmural needles inserted in the left ventricular free wall or in the peripheral 12-lead ECG. Results: Local epicardial application of 50 mM potassium (7 pigs) induced st segment elevation in local electrograms in the epicardial (from 0.2 ± 0.06 mV to 0.5 ± 0.09 mV; p < 0.05) but not underneath in the midmyocardial layer (from 0.1 ± 0.07 mV to -0.1 ± 0.09 mV). Intrapericardial injection of potassium (10 pigs) induced st segment elevation on average in 9/12 ECG-leads. Acute left anterior descending coronary artery occlusion induced st segment elevation associated with increased qrs duration (69 ± 1.2 ms to 89 ± 3.6 ms; p < 0.001) and increased r wave amplitude (0.1 ± 0.01 mV to 0.7 ± 0.09 mV; p < 0.001), but this association did not occur after exposure to potassium. In addition, local midmyocardial activation times were not affected by epicardial application of potassium (from 182 ± 5.9 ms to 183 ± 5.8 ms), but increased significantly during acute left anterior descending coronary artery occlusion (246 ± 20.9 ms; p < 0.01). Conclusion: Transmural but not epicardial myocardial injury is associated with qrs prolongation and increased r wave amplitude in leads with st segment elevation. This differential qrs pattern is due to delayed local activation in transmural but not in epicardial injury. Cardiac sodium channel variants and fyn tyrosine kinase modulation GS Bagavananthem Andavan GS Bagavananthem Andavan 1 University of Vienna, Dept. of Pharmacology and Toxicology , Vienna , Austria ROSA Lemmens Gruber ROSA Lemmens Gruber 1 University of Vienna, Dept. of Pharmacology and Toxicology , Vienna , Austria Abstract Cardiac sodium channel (SCN5A) splice variant Q1077 deleted (hH1c1) and Q1077 present (hH1c3) mutants are present in 45% and 25 % of human population. Arginine at position 1027 in hH1c1 and hH1c3 is replaced by glutamine in SCN5A and glutamine at 1077 is only present in SCN5A and hH1c3. In previous studies, src tyrosine kinase (Fyn) showed opposite effects on cardiac (SCN5A) and neuronal sodium (SCN2A) channel inactivation. Steady state half maximum inactivation of cardiac sodium channels was shifted to more positive potentials, whereas in neuronal channels it was shifted towards more negative potentials, despite having conserved Tyrosine at position 1495, which is the site of phosphorylation in both channels. Steady state activation was not affected in both sodium channel isoforms. In our study we found that Fyn has a different action on the cardiac sodium channel variants hH1c1 and hH1c3. Experiments were performed by means of the patch clamp technique in the whole cell mode. Catalytic active (FynCA or ΔTyr527) or catalytic dead Fyn (FynKD or K299M) was transiently expressed with CD8 in stably expressed HEK293 cells embodying hH1c1 and hH1c3 clones. In hH1c1, FynCA shifted the activation (Vmid −51.6 ± 1.5 to −63.9 ± 1.0) and inactivation curves (Vmid −64.4 ± 0.7 to −72.5 ± 0.4) to more negative potentials, which was reversed by the tyrosine kinase inhibitor PP2 (activation: Vmid −63.9 ± 1.0 to −52.0 ± 1.9, and inactivation: Vmid −72.5 ± 0.4 to −63.7 ± 0.7). In contrast, in hH1c3 Fyn shifted both activation (Vmid −86.2 ± 2.3 to −65.8 ± 0.5) and inactivation (Vmid −85.1 ± 0.7 to -63.4 ± 0.3) curves to more positive potentials. PP2 reversed the shift of both, activation (Vmid −63.4 ± 0.3 to −88.2 ± 1.0) and inactivation (Vmid −63.4 ± 0.3 to −87.2 ± 1.7). When hH1c1 and hH1c3 were transfected with FynKD mutant, there was no shift observed (hH1c1: activation Vmid −62.98 ± 0.82 and inactivation Vmid −56.07 ± 1.08, hH1c3: activation Vmid −84.86 ± 0.18 and inactivation Vmid −85.23 ± 0.25). Above result enunciates that hH1c1 and hH1c3 variants encoding for Nav1.5 are differently regulated by Fyn. These data will be pertinent in understanding the role of Q1077, which is present in the transport associated region. This region is in the cytoplasmic loop connecting domain II-III (LDII-III), which implies that LDII-III might play a pivotal role in regulating Fyn function. Modulation of ventricular fibrillation initiation and action potential duration restitution by acetylcholine, effect of varying extracellular calcium concentration K Brack K Brack 1 University of Leicester , Leicester , United Kingdom J Coote J Coote 2 University of Birmingham , Birmingham , United Kingdom GA Ng GA Ng 1 University of Leicester , Leicester , United Kingdom Open in new tabDownload slide Abstract 378 Figure Open in new tabDownload slide Abstract 378 Figure Abstract Purpose: We have shown that ventricular fibrillation (VF) is modulated by autonomic nerve activation. The underlying mechanisms are unknown but may involved action potential restitution (APDR). The L-type calcium current activity is affected by sympathetic stimulation. There is no data on the interaction between cholinergic activation and calcium levels on the effects of APDR and VF initiation. We investigated the effect of acetylcholine (Ach) at different levels of extracellular [Ca2+] on APDR and VF initiation. Methods: Rabbit Langendorff perfused hearts were used (n = 6, 2.9 ± 0.2 kg). Monophasic Action Potentials were recorded from the apex and base of the left ventricle and duration (MAPD) measured at 90% repolarisation. APDR was studied with single ventricular extrastimuli (S2) down to the effective refractory period (ERP). S2-MAPD was plotted against preceding diastolic intervals and maximum Slope measured. VF threshold (VFT) was determined as the minimum current required to induce VF with rapid pacing. APDR, ERP and VFT were studied during at baseline, during acetylcholine perfusion (Ach, 0.1 µM), and 0.9 mM (Low), 1.8 mM (normal) and 3.6 mM (High) extracellular [Ca2+]. Data are mean ± SEM, statistics using 2-factor repeated ANOVA. Results: Low [Ca2+] increased ERP and VFT whilst high [Ca2+] had opposite effects. The relationship between ERP and VFT was shifted up and to the right by Ach (Fig A). APDR slope was not affected by different levels of [Ca2+] but was reduced by Ach at normal and high [Ca2+] (Fig B). Concluions: Ventricular ERP and VFT are affected by different [Ca2+] whilst APDR slope is not. The antifibrillatory effect of Ach on the ventricle appears to be mediated via effects on APDR but the interaction between Ach and [Ca2+] on VF initiation needs further exploration 1 . Molecular bases of Brugada syndrome and possible roles of microRNAs in SCN5A modulation H Daimi H Daimi 1 University of Jaen, Department of Experimental Biology , Jaen , Spain A Haj Khelil A Haj Khelil 2 Molecular Biology Laboratory, Faculty of Pharmacy, University of Monastir , Monastir , Tunisia A Neji A Neji 3 Fattouma Bourguiba University Hospital, Dept of Cardiology , Monastir , Tunisia K Ben Hamda K Ben Hamda 3 Fattouma Bourguiba University Hospital, Dept of Cardiology , Monastir , Tunisia S Maaoui S Maaoui 3 Fattouma Bourguiba University Hospital, Dept of Cardiology , Monastir , Tunisia A Aranega A Aranega 1 University of Jaen, Department of Experimental Biology , Jaen , Spain J Chibani J Chibani 2 Molecular Biology Laboratory, Faculty of Pharmacy, University of Monastir , Monastir , Tunisia D Franco Jaime D Franco Jaime 1 University of Jaen, Department of Experimental Biology , Jaen , Spain Abstract The Brugada syndrome was described in 1992 as a new clinical entity characterized by a typical electrocardiogram pattern (right bundle branch block and persistent ST-segment elevation in right precordial leads) and sudden cardiac death. This disorder is often inherited with an autosomal dominant transmission mode. Loss-of-function mutations in SCN5A, which encodes the α-subunit of the Nav1.5 sodium ion channel conducting the depolarizing INa current, causes 18–30% of BrS cases. Based on findings to date, a few mutations have been described in other candidate genes such as GPD1L, cardiac potassium channels, and auxillary cardiac sodium channels subunits. Gene sequencing of the whole SCN5A, SCN1B, PITX2, SCN4B, KCNE3, MOG1 and GPD1L coding region and 3′UTR in patients belonging to two unrelated Tunisian families with typical clinical manifestations of BrS in more than one member, including also a control cohort (n = 200), unraveled the presence of many molecular abnormalities with an heterogeneous distribution among families members. The analysis of the SCN5A 3′UTR region demonstrate that some of the already cited molecular abnormalities create a new binding site of microRNA which could be of a great effect on the SCN5A gene regulation. DNA sequencing of the SCN5A conserved miRNAs revealed the presence of a nucleotide substitution CàT 36 base pair upstream the precursor sequence of one of the studied microRNAs. Our functional studies demonstrate that microRNA increase the SCN5A gene expression in HL1 transfected cells as well as partially inhibits Nav1.5 migration from ER to the plasma membrane. Our findings give more evidence of the incomplete penetrance of BrS genetic bases since we have witnessed an heterogeneous distribution of the genetic abnormalities among the family members with partial correlation with the clinical data. In this genetic background we have described a new mutation which is the first declared in BrS patients from Tunisian origin. Besides, we have noticed that more than 70% of the identified SCN5A variants are localized in the 3′UTR and that this could not be hazardous. More evidences of the putative role of the 3′UTR mutations in generating or at least rising the risk of BrS in our study population were given by our functional essays. The overexpression of some microRNAs in HL1 cells model affects the SCN5A expression level and gives a strong evidence of the non neglected role of microRNAs in modulating SCN5A gene expression and proposing a reasonable hypothesis implicating microRNAs mutations in generating BrS in our study population. Heterogeneous effects of left-sided and right-sided sympathetic outflows on the whole heart: studies in the isolated innervated rabbit heart A-S Tanko A-S Tanko 1 University of Leicester, Glenfield Hospital , Leicester , United Kingdom KE Brack KE Brack 1 University of Leicester, Glenfield Hospital , Leicester , United Kingdom JH Coote JH Coote 2 University of Birmingham , Birmingham , United Kingdom GA Ng GA Ng 1 University of Leicester, Glenfield Hospital , Leicester , United Kingdom Abstract 380 Table LSS vs. RSS . . Baseline . Low . Med . High . HR (bpm) LS 158.7 ± 9.9 175.0 ± 9.6 184.12 ± 6.5* 193.0.±6.8* RS 155.3 ± 9.5 185.14 ± 11.5*† 217.6.±10.6*† 223.7 ± 11.8*† LVP (mmHg) LS 34.7 ± 3.6 39.6 ± 3.7 45.3 ± 4.7***† 48.9 ± 4.8***† RS 35.1 ± 3.5 36.5 ± 3.9 38.2 ± 3.8 38.7 ± 3.6* VAC (ms) LS 143.0 ± 10 134.5 ± 10.2* 129 ± 10.3***† 125.3 ± 9.9***† RS 143.7 ± 9.4 140.4 ± 9.7 139.6 ± 9.6** 139.9 ± 9.4** . . Baseline . Low . Med . High . HR (bpm) LS 158.7 ± 9.9 175.0 ± 9.6 184.12 ± 6.5* 193.0.±6.8* RS 155.3 ± 9.5 185.14 ± 11.5*† 217.6.±10.6*† 223.7 ± 11.8*† LVP (mmHg) LS 34.7 ± 3.6 39.6 ± 3.7 45.3 ± 4.7***† 48.9 ± 4.8***† RS 35.1 ± 3.5 36.5 ± 3.9 38.2 ± 3.8 38.7 ± 3.6* VAC (ms) LS 143.0 ± 10 134.5 ± 10.2* 129 ± 10.3***† 125.3 ± 9.9***† RS 143.7 ± 9.4 140.4 ± 9.7 139.6 ± 9.6** 139.9 ± 9.4** Table *P < 0.05 vs. BL: †P < 0.05 LS vs. RS. Open in new tab Abstract 380 Table LSS vs. RSS . . Baseline . Low . Med . High . HR (bpm) LS 158.7 ± 9.9 175.0 ± 9.6 184.12 ± 6.5* 193.0.±6.8* RS 155.3 ± 9.5 185.14 ± 11.5*† 217.6.±10.6*† 223.7 ± 11.8*† LVP (mmHg) LS 34.7 ± 3.6 39.6 ± 3.7 45.3 ± 4.7***† 48.9 ± 4.8***† RS 35.1 ± 3.5 36.5 ± 3.9 38.2 ± 3.8 38.7 ± 3.6* VAC (ms) LS 143.0 ± 10 134.5 ± 10.2* 129 ± 10.3***† 125.3 ± 9.9***† RS 143.7 ± 9.4 140.4 ± 9.7 139.6 ± 9.6** 139.9 ± 9.4** . . Baseline . Low . Med . High . HR (bpm) LS 158.7 ± 9.9 175.0 ± 9.6 184.12 ± 6.5* 193.0.±6.8* RS 155.3 ± 9.5 185.14 ± 11.5*† 217.6.±10.6*† 223.7 ± 11.8*† LVP (mmHg) LS 34.7 ± 3.6 39.6 ± 3.7 45.3 ± 4.7***† 48.9 ± 4.8***† RS 35.1 ± 3.5 36.5 ± 3.9 38.2 ± 3.8 38.7 ± 3.6* VAC (ms) LS 143.0 ± 10 134.5 ± 10.2* 129 ± 10.3***† 125.3 ± 9.9***† RS 143.7 ± 9.4 140.4 ± 9.7 139.6 ± 9.6** 139.9 ± 9.4** Table *P < 0.05 vs. BL: †P < 0.05 LS vs. RS. Open in new tab Abstract Purpose: Previous studies carried out investigating differential effects between left and right sympathetic outflows to the heart have employed in vivo canine models with possible confounding factors and effects from circulating catecholamines. The aim of this study was to examine these differences in the rabbit using a novel preparation. Methods: Adult male NZW rabbits (2.1-3.1kg; n = 9) were used and heart isolated with intact autonomic nerves. Left ventricular pressure (LVP) was measured using fluid-filled balloon. The left (LS) and right (RS) sympathetic chains were isolated (T4 to T1/2) and stimulated (x2 threshold voltage) at 2Hz (Low), 5Hz (Med) and 10Hz (High) during sinus rhythm and during right ventricular pacing at 250bpm to examine chronotropic, ionotropic and retrograde ventriculo-atrial conduction (VAC) effects. Data are mean± SEM; statistical analysis was performed using 2-factor ANOVA. Results: RS increased peak heart rate (HR) more significantly than LS whilst LS increased LVP greatly than RS (Table). Sympathetic stimulation produced a frequency dependent decrease in VAC which was more significant with LS than RS. Conclusion: In summary, stimulation the right sympathetic outflow to the heart has a more prominent effect on the sino-atrial node but a weaker on the ventricle, whereas the reverse is true when stimulating the left sympathetic outflow. This may reflect regional distribution differences on the sympathetic innervations of the heart 1 . Influence of aging on catecholaminergic automatic activity and responses to alpha-adrenergic stimulation in rat pulmonary vein N Doisne N Doisne 1 University Francois-Rabelais, Faculty of Sciences PCCV (EA 4433) , Tours , France B Hivert B Hivert 1 University Francois-Rabelais, Faculty of Sciences PCCV (EA 4433) , Tours , France P Cosnay P Cosnay 1 University Francois-Rabelais, Faculty of Sciences PCCV (EA 4433) , Tours , France I Findlay I Findlay 1 University Francois-Rabelais, Faculty of Sciences PCCV (EA 4433) , Tours , France V Maupoil V Maupoil 1 University Francois-Rabelais, Faculty of Sciences PCCV (EA 4433) , Tours , France Abstract Paroxysmal atrial fibrillation can be induced by ectopic activity occuring in cardiac muscle of pulmonary veins (PV) in human. Prevalence of this arrhythmia has been shown to increase with aging. Here we describe the influence of aging on the occurrence of catecholaminergic automatic activity induced by noradrenalin in cardiac muscle of rat PV. We isolated strips of PV from young (10 ± 2 weeks) and old (24 ± 2 months) Wistar rats and we recorded responses to α-adrenergic stimulation upon the resting membrane potential (RMP), with intracellular microelectrodes, and upon contraction, with a force transducer, from cardiac muscle of these strips. Isolated tissues were continuously superfused with Krebs-Henseleit solution gassed with 95% O2, 5% CO2 and maintained at 37°C. RMP of cardiac muscle of PV was less negative in the old rats (-64 ± 3 mV vs -71 ± 1 mV in the young, p < 0.05). Noradrenalin at 1O μM induced automatic activity occuring in bursts in 100% of young PV (n = 30) versus 85% of old PV (n = 20, p < 0.05). Cirazoline at 1 μM induced a strong depolarization of the membrane potential in both groups. This depolarization was more pronounced in the young rats (37 ± 1 mV vs 30 ± 4 mV in the old, p < 0.005). However, the end-point of this depolarization was not significantly different between the young and the old rats (-35 ± 1 mV vs -38 ± 3 mV, respectively). Immunoblotting confirmed the presence of alpha-adrenergic receptor in cardiac muscle of PV from both groups. Application of 1 μM cirazoline resulted in a strong decrease in contraction amplitude of cardiac muscle of PV, which was larger in the young rats (-79 ± 5 % vs -51 ± 14 % in the old, p < 0.05). Surprisingly, cirazoline facilitated the occurrence of a pacing associated spontaneous activity in PV from old but not in PV from young rats. In conclusion, despite blunted responses to adrenergic agonists in PV from old rats, the stimulation of α1-adrenoceptors facilitated the occurrence of a pacing associated spontaneous activity in old but not in young rat PV. Bone marrow-derived progenitor cells do not differentiate into definite vascular cells in a time course analysis of neointima formation. J-M Daniel J-M Daniel 1 Justus Liebig University Giessen, Med. Clinic I, Cardiology , Giessen , Germany W Bielenberg W Bielenberg 1 Justus Liebig University Giessen, Med. Clinic I, Cardiology , Giessen , Germany P Stieger P Stieger 1 Justus Liebig University Giessen, Med. Clinic I, Cardiology , Giessen , Germany H Tillmanns H Tillmanns 1 Justus Liebig University Giessen, Med. Clinic I, Cardiology , Giessen , Germany DG Sedding DG Sedding 1 Justus Liebig University Giessen, Med. Clinic I, Cardiology , Giessen , Germany Abstract Purpose: Bone marrow derived progenitor cells (BMPCs) contribute to neointima (NI) formation, but the time-points of differentiation into vascular cells and, most importantly, the long term contribution of BMPCs to the vascular lesion remain undefined. Methods: Wire-induced injury of the femoral artery was performed on chimeric C57BL/6 mice transplanted with bone marrow from transgenic mice expressing enhanced green fluorescence protein (EGFP). Vessels were harvested at 3 days, 1, 2, 3, 4, 6 and 16 weeks after dilatation (n = 8 animals per time point) and analyzed using high resolution microscopy. Results: We unexpectedly found that only ∼1% of the neointimal EGFP+-cells co-expressed α-smooth muscle actin (α-SMA) as a marker for vascular smooth muscle cells (VSMCs) at 4 weeks after injury, whereas smooth muscle myosin heavy chain or calponin were not expressed in BM-derived cells at all. The absolute number of EGFP+-cells in the NI declined from 110.81 ± 18.98 at 2 weeks to only 5.31 ± 2.21 at 16 weeks after dilatation. During this time period, staining for CD68 identified most of the EGFP+-cells as monocytes/ macrophages. Interestingly, the majority of the BM-derived cells expressing α-SMA also stained positive for myeloid lineage markers (CD68 or CD11b) and very likely represent "smooth muscle-like macrophages". In addition, BM-derived cells expressing CD31 as a marker for endothelial cells (ECs) were only rarely detected and accounted for<0.3% of the ECs in the NI. However, perivascular progenitor cells might be involved in NI formation, since a substantial fraction of stem cell antigen (Sca)-1 was detected in proliferating EGFP negative cells in the adventitial layer. Conclusions: We provide evidence that BMPCs in the NI rarely co-express markers for vascular cells. Since BM-derived cells did not contribute to the neointimal cellular mass beyond the inflammatory response, it is very unlikely that BMPC differentiate into definite vascular lineages during NI formation. Different classes and sub-types of stem cell mobilization in patients with heart failure C Fortini C Fortini 1 Chair of Cardiology, University of Ferrara and Salvatore Maugeri Foundation, IRCCS , Ferrara , Italy B Toffoletto B Toffoletto 2 Center for Regenerative Medicine, University of Udine , Udine , Italy A Fucili A Fucili 1 Chair of Cardiology, University of Ferrara and Salvatore Maugeri Foundation, IRCCS , Ferrara , Italy AP Beltrami AP Beltrami 2 Center for Regenerative Medicine, University of Udine , Udine , Italy V Fiorelli V Fiorelli 1 Chair of Cardiology, University of Ferrara and Salvatore Maugeri Foundation, IRCCS , Ferrara , Italy G Francolini G Francolini 1 Chair of Cardiology, University of Ferrara and Salvatore Maugeri Foundation, IRCCS , Ferrara , Italy R Ferrari R Ferrari 1 Chair of Cardiology, University of Ferrara and Salvatore Maugeri Foundation, IRCCS , Ferrara , Italy CA Beltrami CA Beltrami 2 Center for Regenerative Medicine, University of Udine , Udine , Italy Abstract 383 Table Cytokines . Related cells . r . p . PDGF-BB CD34 − CD45 − CD90 + 0.36 0.0001 CD34 − CD45 − CD105 + 0.3 0.0012 CD34 − CD45 − CD90 + CXCR4 + 0.5 <0.0001 CD34 + CD45 + CD90 + CXCR4 + 0.23 0.0158 CD34 + CD45 − CXCR4 + 0.52 <0.0001 bFGF CD34 − CD45 − CD90 + CXCR4 + 0.29 0.0026 CD34 + CD45 − CXCR4 + 0.35 0.0002 TNF-a CD34 − CD45 − CD105 + 0.26 0.0056 CD34 − CD45 − CD90 + CXCR4 + 0.23 0.0165 Cytokines . Related cells . r . p . PDGF-BB CD34 − CD45 − CD90 + 0.36 0.0001 CD34 − CD45 − CD105 + 0.3 0.0012 CD34 − CD45 − CD90 + CXCR4 + 0.5 <0.0001 CD34 + CD45 + CD90 + CXCR4 + 0.23 0.0158 CD34 + CD45 − CXCR4 + 0.52 <0.0001 bFGF CD34 − CD45 − CD90 + CXCR4 + 0.29 0.0026 CD34 + CD45 − CXCR4 + 0.35 0.0002 TNF-a CD34 − CD45 − CD105 + 0.26 0.0056 CD34 − CD45 − CD90 + CXCR4 + 0.23 0.0165 Open in new tab Abstract 383 Table Cytokines . Related cells . r . p . PDGF-BB CD34 − CD45 − CD90 + 0.36 0.0001 CD34 − CD45 − CD105 + 0.3 0.0012 CD34 − CD45 − CD90 + CXCR4 + 0.5 <0.0001 CD34 + CD45 + CD90 + CXCR4 + 0.23 0.0158 CD34 + CD45 − CXCR4 + 0.52 <0.0001 bFGF CD34 − CD45 − CD90 + CXCR4 + 0.29 0.0026 CD34 + CD45 − CXCR4 + 0.35 0.0002 TNF-a CD34 − CD45 − CD105 + 0.26 0.0056 CD34 − CD45 − CD90 + CXCR4 + 0.23 0.0165 Cytokines . Related cells . r . p . PDGF-BB CD34 − CD45 − CD90 + 0.36 0.0001 CD34 − CD45 − CD105 + 0.3 0.0012 CD34 − CD45 − CD90 + CXCR4 + 0.5 <0.0001 CD34 + CD45 + CD90 + CXCR4 + 0.23 0.0158 CD34 + CD45 − CXCR4 + 0.52 <0.0001 bFGF CD34 − CD45 − CD90 + CXCR4 + 0.29 0.0026 CD34 + CD45 − CXCR4 + 0.35 0.0002 TNF-a CD34 − CD45 − CD105 + 0.26 0.0056 CD34 − CD45 − CD90 + CXCR4 + 0.23 0.0165 Open in new tab Abstract Background: stem cells (SC) can contribute to heart repair. Aim: to evaluate the correlation between different classes [mesenchymal SC (MSC), hematopoietic SC (HSC), endothelial progenitor cells (EPCs) and the tissue-committed SC (TCSC)] of SC recruitment and cytokine levels in patients (P) with different stages of heart failure (HF). Methods: 85 HF patients (22 in NYHA class I, 40 in NYHA class II, 15 in NYHA class III and 8 in NYHA class IV) and 23 controls were enrolled. Their circulating SC and cytokines (PDGF-BB, bFGF, HGF, VEGF, SDF-1α, TNF-α) were analysed by flow-cytometry multicolor staining and specific radio immunoassays, respectively. Results: Compared with healthy subjects, MSC were significantly mobilized in NYHA class 4 P: CD34-CD45-CD105+, CD34-CD45-CD90+ and CD34-CD45-CXCR4+ cells were increased, 10.7, 25.9 and 2.1 fold respectively. Mobilization of CD34-CD45-CD90+CXCR4+cells progressively increased from class I to class IV (fold increases: 8.5, 12.3 and 18.2 respectively). A significant involvement of CXCR4+ subpopulation of HSC (CD34+CD45+CD90+CXCR4+, 1.4 vs. 14.2 cells/microliter in controls and NYHA class III patients respectively) and TCSC (CD34+CD45-CXCR4+, 1.5 cells/microliter in controls vs. 12, 11.1 and 20.8 cells/microliter in NYHA class II, III and IV respectively) were also observed. All tested cytokines were enhanced in HF P. Table 1 shows the correlations with related cells. Conclusions: In P with HF, MSC are primarily mobilized and strongly correlated with the tested cytokines, with significant involvement of the CXCR4 specific subpopulation of HSC and EPC/EC. Stem cells homing, ventricular remodelling and cytokines profile in an animal model of heart failure C Castellani C Castellani 1 University of Padua, Department of Medico-Diagnostic Sciences and Special Therapies , Padua , Italy B Ravara B Ravara 2 CNR, Institute of Neuroscience, University of Padua , Padua , Italy R Tavano R Tavano 3 University of Padua, Department of Experimental Biomedical Science , Padua , Italy G Thiene G Thiene 1 University of Padua, Department of Medico-Diagnostic Sciences and Special Therapies , Padua , Italy R Vettor R Vettor 4 University of Padua , Padua , Italy P De Coppi P De Coppi 5 Dept. of Oncohaematology , Padua , Italy E Papini E Papini 3 University of Padua, Department of Experimental Biomedical Science , Padua , Italy A Angelini A Angelini 1 University of Padua, Department of Medico-Diagnostic Sciences and Special Therapies , Padua , Italy Abstract Purpose: Aim of our study was to investigate the homing and differentiation of human amniotic stem cell (hAFC, like embryonic stem cells) and rat vascular stromal adipocite GFP positive cells (rGFP, vascular progenitor cells) in a rat model of heart failure. Methods and Materials: 28 male Sprague-Dowley rats were injected with monocrotaline to develop right heart failure. Four millions hAFC or rGFP cells were injected via tail vein three weeks after MCT injection. Stem cells differentiation was studied by double immunofluorescence technique. The occurrence of heart failure syndrome was confirmed on transverse histologic sections of he heart looking at RV hypetrophy /dilatation and measuring BNP plasma levels. Cytokine profile was assessed by Multiplex array on sera. Results: RVM/Vol was lower in MCT-stem cells treated rats (0.34 ± 0.02) than MCT rats (1.58 ± 0.51) and similar to controls (0.49 ± 0.29, p < 0.001). MCT-stem cells treated rats showed plasma brain natriuretic peptide (BNP) of 2.0 ± 1.3* ng/ml lower than MCT rats (5.2 ± 1.2*# ng/ml) and similar to the controls 1.56 ± 0.5 ng/ml# controls, p < 0.001*#. hAFC cells were detected in the lung, heart and skeletal muscle with a homing percentage of 2.26 ± 0.23, 0.5 ± 0.42 and 0.36 ± 0.41 respectively. rGFP cells were detected in the lung, heart and skeletal muscle with a homing percentage of 2.01 ± 0.31, 0.6 ± 0.3 and 0.8 ± 0.4 respectively. Stem cells showed a differentiation toward smooth muscle and endothelial cells. Protein suspension array of rats sera showed that cytokines profile in rats with heart failure had depressed levels of IL10 and significantly higher levels of pro-inflammatory cytokines, while those injected with stem cells had a pattern similar to controls and in particular we found high levels of anti-inflammatory IL10 and low level of pro-inflammatory TNF alfa and IFN-γ. Conclusions: hAFC and rGFP cells are engrafted in the lung, heart and skeletal muscle where they differentiate mainly in vascular and endothelial cells. This brings about a positive cardiac remodelling accompanied by decreased levels of BNP suggesting a lower degree of heart stress. The IL10 pathway activation which leads to reduction of the pro-inflammatory cytokines cascade, together with the formation of new vascular cells may be the cause of these positive haemodynamic changes.Alternatively the engrafted cells may have an endocrine/paracrine function with production of beneficial anti-inflammatory cytokines. Ex-vivo expanded bone marrow human CD34+ hematopoietic stem cells for repairing myocardial ischemic injury in immunodeficient mice F Molla F Molla 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy A Soldo A Soldo 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy A Biondi A Biondi 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy L Staszewsky L Staszewsky 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy I Russo I Russo 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy M Gunetti M Gunetti 2 Stem Cell Transplantation and Cellular Therapy Unit; Pediatric Onco-Hematology Division, Regina Marg , Turin , Italy F Fagioli F Fagioli 2 Stem Cell Transplantation and Cellular Therapy Unit; Pediatric Onco-Hematology Division, Regina Marg , Turin , Italy R Latini R Latini 1 The Mario Negri Institute for Pharmacological Research, Department of Cardiovascular Research , Milan , Italy Abstract 385 Table Echocardiocardiographic anlysis . SHAM . CAL+PBS . CAL+hCD34 + . CAL+hCD34 + . CAL+hCD34 + . CAL+PBS vs CAL+hCD34 + . . n = 9 . n = 16 . fresh n = 8 . fresh+exp. n = 24 . fresh vs fresh+exp. (p=) . fresh (p=) . Mortality 2/11, 18% 10/26, 38% 0/8, 0% 26/50, 52% − − BW (g) 22.80 ± 1.34 24.19 ± 0.85 22.23 ± 1.48 25.03 ± 0.72 0.07 0.23 HR (bpm) 653 ± 13 604 ± 15 635 ± 16 634 ± 10 0.94 0.21 LVIDd (mm) 2.63 ± 0.04 4.84 ± 0.20 4.96 ± 0.17 4.20 ± 0.17 0.02 0.70 LVIDs (mm) 0.72 ± 0.08 3.98 ± 0.25 4.16 ± 0.15 3.19 ± 0.24 0.03 0.63 SF (%) 71.88 ± 2.81 18.87 ± 2.28 14.75 ± 1.22 27.31 ± 2.89 0.02 0.23 LVEF (%) 83.98 ± 1.09 19.96 ± 2.27 21.68 ± 2.94 32.88 ± 3.79 0.11 0.66 AWThd (mm) 0.91 ± 0.03 0.76 ± 0.08 0.40 ± 0.03 0.88 ± 0.08 0.06 0.13 PWThd (mm) 0.97 ± 0.03 0.70 ± 0.06 0.83 ± 0.08 0.94 ± 0.05 0.21 0.24 . SHAM . CAL+PBS . CAL+hCD34 + . CAL+hCD34 + . CAL+hCD34 + . CAL+PBS vs CAL+hCD34 + . . n = 9 . n = 16 . fresh n = 8 . fresh+exp. n = 24 . fresh vs fresh+exp. (p=) . fresh (p=) . Mortality 2/11, 18% 10/26, 38% 0/8, 0% 26/50, 52% − − BW (g) 22.80 ± 1.34 24.19 ± 0.85 22.23 ± 1.48 25.03 ± 0.72 0.07 0.23 HR (bpm) 653 ± 13 604 ± 15 635 ± 16 634 ± 10 0.94 0.21 LVIDd (mm) 2.63 ± 0.04 4.84 ± 0.20 4.96 ± 0.17 4.20 ± 0.17 0.02 0.70 LVIDs (mm) 0.72 ± 0.08 3.98 ± 0.25 4.16 ± 0.15 3.19 ± 0.24 0.03 0.63 SF (%) 71.88 ± 2.81 18.87 ± 2.28 14.75 ± 1.22 27.31 ± 2.89 0.02 0.23 LVEF (%) 83.98 ± 1.09 19.96 ± 2.27 21.68 ± 2.94 32.88 ± 3.79 0.11 0.66 AWThd (mm) 0.91 ± 0.03 0.76 ± 0.08 0.40 ± 0.03 0.88 ± 0.08 0.06 0.13 PWThd (mm) 0.97 ± 0.03 0.70 ± 0.06 0.83 ± 0.08 0.94 ± 0.05 0.21 0.24 Values expressed as mean ± SE. BW: body weight, HR: heart rate, LVIDd: left ventricular end diastolic diameter, LVIDs: left ventricular end systolic diameter, SF: shortening fraction, LVEF: left ventricular ejection fraction, AWThd: anterior wall diastolic thickness, PWThd: posterior wall diastolic thickness. Open in new tab Abstract 385 Table Echocardiocardiographic anlysis . SHAM . CAL+PBS . CAL+hCD34 + . CAL+hCD34 + . CAL+hCD34 + . CAL+PBS vs CAL+hCD34 + . . n = 9 . n = 16 . fresh n = 8 . fresh+exp. n = 24 . fresh vs fresh+exp. (p=) . fresh (p=) . Mortality 2/11, 18% 10/26, 38% 0/8, 0% 26/50, 52% − − BW (g) 22.80 ± 1.34 24.19 ± 0.85 22.23 ± 1.48 25.03 ± 0.72 0.07 0.23 HR (bpm) 653 ± 13 604 ± 15 635 ± 16 634 ± 10 0.94 0.21 LVIDd (mm) 2.63 ± 0.04 4.84 ± 0.20 4.96 ± 0.17 4.20 ± 0.17 0.02 0.70 LVIDs (mm) 0.72 ± 0.08 3.98 ± 0.25 4.16 ± 0.15 3.19 ± 0.24 0.03 0.63 SF (%) 71.88 ± 2.81 18.87 ± 2.28 14.75 ± 1.22 27.31 ± 2.89 0.02 0.23 LVEF (%) 83.98 ± 1.09 19.96 ± 2.27 21.68 ± 2.94 32.88 ± 3.79 0.11 0.66 AWThd (mm) 0.91 ± 0.03 0.76 ± 0.08 0.40 ± 0.03 0.88 ± 0.08 0.06 0.13 PWThd (mm) 0.97 ± 0.03 0.70 ± 0.06 0.83 ± 0.08 0.94 ± 0.05 0.21 0.24 . SHAM . CAL+PBS . CAL+hCD34 + . CAL+hCD34 + . CAL+hCD34 + . CAL+PBS vs CAL+hCD34 + . . n = 9 . n = 16 . fresh n = 8 . fresh+exp. n = 24 . fresh vs fresh+exp. (p=) . fresh (p=) . Mortality 2/11, 18% 10/26, 38% 0/8, 0% 26/50, 52% − − BW (g) 22.80 ± 1.34 24.19 ± 0.85 22.23 ± 1.48 25.03 ± 0.72 0.07 0.23 HR (bpm) 653 ± 13 604 ± 15 635 ± 16 634 ± 10 0.94 0.21 LVIDd (mm) 2.63 ± 0.04 4.84 ± 0.20 4.96 ± 0.17 4.20 ± 0.17 0.02 0.70 LVIDs (mm) 0.72 ± 0.08 3.98 ± 0.25 4.16 ± 0.15 3.19 ± 0.24 0.03 0.63 SF (%) 71.88 ± 2.81 18.87 ± 2.28 14.75 ± 1.22 27.31 ± 2.89 0.02 0.23 LVEF (%) 83.98 ± 1.09 19.96 ± 2.27 21.68 ± 2.94 32.88 ± 3.79 0.11 0.66 AWThd (mm) 0.91 ± 0.03 0.76 ± 0.08 0.40 ± 0.03 0.88 ± 0.08 0.06 0.13 PWThd (mm) 0.97 ± 0.03 0.70 ± 0.06 0.83 ± 0.08 0.94 ± 0.05 0.21 0.24 Values expressed as mean ± SE. BW: body weight, HR: heart rate, LVIDd: left ventricular end diastolic diameter, LVIDs: left ventricular end systolic diameter, SF: shortening fraction, LVEF: left ventricular ejection fraction, AWThd: anterior wall diastolic thickness, PWThd: posterior wall diastolic thickness. Open in new tab Abstract Background: Bone marrow derived progenitor cells are the most widely used for cardiac repair after MI. Benefit in humans appear to depend on the number of cells administered. Bone marrow progenitor cells are difficult to expand while preserving their stemness. Aims: To compare in experimental infarcts the repair potential of fresh hCD34+ cells with that of hCD34+ expanded in vitro. Methods: The expansion procedure of hCD34+ cells, using cocktail of growth factors, committed to endothelial lineage. MI was induced by coronary ligation (CAL) in 87 SCIDbg mice. After 4 hours, purified hCD34+ (300000/100ul) or PBS were injected into the left ventricular chamber. 7 days later expanded hCD34+ or PBS were re-injected. Transthoracic echocardiography was performed 6 weeks later; after sacrifice hearts were harvested for histology and RT-PCR. Results and Conclusion: LV systolic function improved (48%) and LV internal diastolic diameter was smaller in animals double injected with hCD34+ compared to PBS group. RT-PCR and lamin AC+ staining revealed evidence of hCD34+ fresh + expanded group. Double injection after MI but not single injection of fresh hCD34+ favorably affects cardiac function and LV remodeling. The effectiveness of expanded human hCD34+ creates new opportunities for administering large numbers of cells with limited sampling of bone marrow 1 . Embryonic stem cells as a model to study the effect of antineoplastic drugs S Suffredini S Suffredini 1 University of Florence, Interuniversity Center of Molecular Medicine and Applied Biophysics (CIMMBA) , Florence , Italy L Sartiani L Sartiani 1 University of Florence, Interuniversity Center of Molecular Medicine and Applied Biophysics (CIMMBA) , Florence , Italy F Stillitano F Stillitano 1 University of Florence, Interuniversity Center of Molecular Medicine and Applied Biophysics (CIMMBA) , Florence , Italy A Mugelli A Mugelli 1 University of Florence, Interuniversity Center of Molecular Medicine and Applied Biophysics (CIMMBA) , Florence , Italy E Cerbai E Cerbai 1 University of Florence, Interuniversity Center of Molecular Medicine and Applied Biophysics (CIMMBA) , Florence , Italy Abstract Purpose: Embryonic stem cells represent a suitable model to study cardiotoxic effect of endogenous or exogenous substances. Indeed the cardiotoxic effect of antineoplastic therapy is a common hitch in treated patients and limits the efficacy of therapy. Recently it has been proposed that these effects, being cardiomyocytes terminally differentiated, may be due to an "extra-sensitive" population represented by resident progenitors of cardiomyocytes. This may also explain the relatively high and delayed incidence of cardiac complications in patients treated for childhood cancers. However, the sensitivity of stem cells to antineoplastic drugs has not been investigated. We aimed to study the effect of doxorubicin and imatinib on commitment, differentiation and maturation of stem cells toward cardiomyocytes. Methods: Mouse ES (mES, CGR8) cells were propagated in medium and differentiated into cardiomyocytes via formation of embryoid body (EB). We evaluated the effect of imatinib (1 and 0.1 µM) and doxorubicin (Doxo, 0.1, 0.5 and 1 µM) at different stage of the differentiation process. The effect of drugs on viability was studied by MTT test. The effect of drugs on function were evaluated by measuring the percentage of beating EBs. To get information on the molecular mechanisms underlying or associated with the functional results, we used quantitative RT-PCR to investigate the expression of transcription factor involved in cardiac differentiation (GATA-4, Nkx2.5, MEF2C). Results: Adding Doxo at the first day of differentiation process hindered EB formation. Thus, we added Doxo onto already formed EBs, by exposing EBs for 48 hours, starting from day 2 of EB formation. Also in this condition, Doxo showed a marked toxicity (MTT test) and prevented cell growth and the expression of cardiac specific markers. Surprisingly, when added to beating EBs (day 7-8), a 48-hour exposure to Doxo did not influence EB contractility. On the contrary, imatinib did not show any toxic effect on EB formation, cell growth and contractility. Conclusion: Antineoplastic drugs exert different effects on cardiac commitment and differentiation of mES. Moreover, the window for Doxo toxicity seems to be restricted to the early stages of cell commitment, namely EB formation. Thus, it seems important to verify the effects of Doxo on cell aggregation and proliferation as well as expression of early mesodermic transcription factors. Loss of purified embryonic stem cell derived cardiomyocytes after intramyocardial injection into syngeneic mouse hearts occurs early and can be delayed but not prevented by coinjection of Matrigel(TM) B Krausgrill B Krausgrill 1 University Hospital of Cologne , Cologne , Germany M Halbach M Halbach 1 University Hospital of Cologne , Cologne , Germany SP Soemantri SP Soemantri 1 University Hospital of Cologne , Cologne , Germany K Schenk K Schenk 1 University Hospital of Cologne , Cologne , Germany N Lange N Lange 1 University Hospital of Cologne , Cologne , Germany J Hescheler J Hescheler 1 University Hospital of Cologne , Cologne , Germany T Saric T Saric 1 University Hospital of Cologne , Cologne , Germany J Muller-Ehmsen J Muller-Ehmsen 1 University Hospital of Cologne , Cologne , Germany Abstract Purpose: Objective: In cardiac cell replacement therapy, embryonic stem cell derived cardiomyocytes (ES-CM) integrate into host myocardium and improve cardiac function. Here, we quantified engraftment, persistence and survival of transplanted ES-CM which are crucial factors for this therapy's effectiveness and tested coinjection of Matrigel to prevent early cell loss. Methods: Male murine ES-CM (alpha-PIG44) were generated and highly purified (>97%) using an antibiotic selection strategy and a genetic resistance under cardiac specific promoters. 300,000 ES-CM were transplanted with 2 direct intramyocardial injections (10 μl each) into healthy regions at the border of previous cryo-injury (CRYO) or into sham-operated (SHAM) hearts of syngeneic female adult mice. Hearts were explanted immediately (0h) or after 6h, 24h, 48h, 5d, 21d (randomized assignment). Additionally, similar cell suspensions were mixed with Matrigel in either low (M-low) or high concentration (M-high) prior to transplantation into sham-operated hearts which were explanted immediately (0h) or after 6h. Genomic DNA was isolated from explanted hearts and the number of transplanted cells was determined by quantitative real-time PCR for Y-chromosome. Results: Engraftment efficiency was low in both groups with 13.1 ± 5.2% (SHAM) and 12.1 ± 4.2% (CRYO) of the transplanted ES-CM at 0h. At 6h, numbers remained unchanged in SHAM (10.7 ± 2.5%) but tended to decrease in CRYO (1.9 ± 0.6%, P < 0.1 vs 0h, P = 0.015 vs SHAM). At 24h, numbers declined significantly in SHAM (0.9 ± 0.2%, P < 0.01 vs 6h and P < 0.05 vs 0h) to similar levels as observed in CRYO (1.5 ± 0.6%). At later time points, numbers remained unchanged in both groups with 1.7 ± 0.7% (SHAM) and 1.5 ± 0.6% (CRYO) at 48h, 0.9 ± 0.2% (SHAM) and 2.4 ± 2.1% (CRYO) at 5d and 0.4 ± 0.3% (SHAM) and 1.4 ± 1.1% (CRYO) at 21d. Coinjection of Matrigel increased engraftment efficiency at 0h to 19.2 ± 6.3% (M-low) and 36.1 ± 11.6% (M-high, P < 0.05 vs 0h without Matrigel), but numbers were similar at 6h with 16.6 ± 6.6% (M-low) and 14.5 ± 3.1% (M-high, both P=n.s. vs 6h without Matrigel). Conclusions: Loss of ES-CM after intramyocardial injection occurs early and is similarly high in both cryo-injured and sham-operated hearts. Coinjection of Matrigel increased engraftment efficiency in a dose-dependent manner but failed to increase persistence of transplanted cells at later time points. Strategies to improve engraftment, persistence and survival of transplanted cells must be identified to optimize the effectiveness of cardiac cell replacement therapy. Hematopoeitic stem cells are recruited to the injured microcirculation by site specific adhesive mechanisms DPJ Kavanagh DPJ Kavanagh 1 University of Birmingham , Birmingham , United Kingdom Y Zhao Y Zhao 1 University of Birmingham , Birmingham , United Kingdom AI Yemm AI Yemm 1 University of Birmingham , Birmingham , United Kingdom N Kalia N Kalia 1 University of Birmingham , Birmingham , United Kingdom Abstract Purpose: HSCs can be recruited to injury sites and subsequently aid tissue repair. Mechanisms governing recruitment following injury are poorly understood. Further, it is not known whether these are site specific. We therefore utilized intravital microscopy to elucidate the mechanisms involved in HSC recruitment to gut and cremaster muscle microcirculation following ischemia-reperfusion (IR) injury. Methods: For these studies we utilized the HSC line, HPC-7. HPC-7 have been well documented as excellent surrogates for HSCs. The ileum or cremaster of anaesthetised (ketamine/xylazine) mice was subjected to ischemia. IgG, anti-CD18 or anti-CD49d pre-treated HPC-7s were introduced during reperfusion and their adhesion monitored. Some HPC-7s were incubated (20 min) with homogenised IR injured gut conditioned media (IR-CM) prior to determining their adhesion to frozen IR injured gut sections in vitro. Results: IR injury significantly enhanced HPC-7 adhesion in ileum (p < 0.05) and cremaster (p < 0.05) compared to controls. Adhesion was significantly reduced by anti-CD18 (p < 0.05) pre-treatment in ileum whereas anti-CD18 (p < 0.05) and anti-CD49d (p < 0.01) reduced adhesion in cremaster. Incubation of cells with IR-CM significantly (p < 0.05) increased their adhesion to gut sections compared to naïve cells. Conclusion: We have previously demonstrated that CD49d is critical for hepatic HSC recruitment. Collectively, these studies suggest site specific homing mechanisms govern HSC recruitment to injured tissue. Furthermore, as yet unidentified factors released during injury can enhance HSC adhesion – this has important therapeutic implications. Effects of GLP-1 eluting stem cell therapy (GLP-1 CellBeads) on collagen remodelling, infarct size and apoptosis in a porcine embolisation model EJ Wright EJ Wright 1 University of Manchester, School of Medicine , Manchester , United Kingdom KA Farrell KA Farrell 1 University of Manchester, School of Medicine , Manchester , United Kingdom C Wallrapp C Wallrapp 2 CellMed AG , Alzenau , Germany P Geigle P Geigle 2 CellMed AG , Alzenau , Germany AL Lewis AL Lewis 3 Biocompatibles UK Ltd , Farnham, Surrey , United Kingdom PW Stratford PW Stratford 3 Biocompatibles UK Ltd , Farnham, Surrey , United Kingdom N Malik N Malik 1 University of Manchester, School of Medicine , Manchester , United Kingdom CM Holt CM Holt 1 University of Manchester, School of Medicine , Manchester , United Kingdom Abstract Introduction: Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone, which also has cardioprotective effects and has recently emerged as a promising treatment for limiting post- myocardial infarction (MI) injury. Human mesenchymal stem cells engineered to secrete a GLP-1 fusion protein and encapsulated in an alginate matrix (GLP-1 CellBeads) have been developed as a novel therapeutic agent post-MI. The aim of this study was to investigate the effects of GLP-1 CellBeads on post-MI healing in a porcine model. Methods: GLP-1 CellBeads were delivered to the left anterior descending coronary artery to create several micro-infarcts. Cell-free beads were delivered as controls. Hearts were explanted at 1 and 4 weeks post-MI and infarct zone, border zone and non-infarct (remote) regions were identified. Gross infarct size was measured as a percentage of total left ventricular area. Tissue was analysed for: inflammation (number of MAC 387 positive cells per mm2), apoptosis (% of TUNEL positive cells) and collagen (% picrosirius red staining). Results: Compared to the cell-free beads group (n = 4), the GLP-1 treated group (n = 6) exhibited less infarct at both one week (6.21 ± 0.64 vs. 9.78 ± 1.80% LV, p = NS) and four weeks post-MI (4.7 ± 2.1 vs. 21.8 ± 4.8% LV, p = 0.02). Within the infarct regions there was increased inflammation in the GLP-1 treated groups at both time points (1 week: 97.22 ± 19.62 vs. 36.67 ± 7.78, p = 0.01; 4 weeks: 24.2 ± 4.57 vs. 12.3 ± 3.16, p = 0.03). At one week, apoptosis rates in the infarct area was similar in the two groups (1.46 ± 0.57 vs. 1.47 ± 0.17, p = NS) and at 4 weeks was lower in the GLP-1 treated group (0.51 ± 0.18 vs.1.84 ± 0.62, p = NS). Collagen content at one week was lower (5.14 ± 1.19 vs. 9.95 ± 1.42, p = 0.05) and at 4 weeks higher in the GLP-1 treated group (20.89 ± 8.25 vs. 6.87 ± 2.92, p = NS). Conclusion: GLP-1 CellBeads have an effect on post-MI infarct size, inflammation and ventricular remodelling. These findings require further validation prior to clinical translation Optimizing persistence and survival of rat neonatal cardiomyocytes after transplantation into infarcted syngeneic rat hearts B Krausgrill B Krausgrill University Hospital of Cologne , Cologne , Germany M Raths M Raths M Halbach M Halbach K Schenk K Schenk J Hescheler J Hescheler J Muller-Ehmsen J Muller-Ehmsen Abstract 390 Table intramyocardially injected total cell number, total volume, number of injection sites . persistence and survival (absolute number) . persistence and survival (% of injected cells) . 5mill., 100 μl, 1 inj. site 0.7 ± 0.1mill.** 14.1 ± 2.9% 15mill., 100 μl, 1 inj. site 0.9 ± 0.2mill.* 6.0 ± 1.3% 15mill., 300 μl, 1 inj. site 1.0 ± 0.2mill.* 6.7 ± 1.3% 5mill., 100 μl, 3 inj. sites 0.3 ± 0.1mill.*** 6.5 ± 1.5% 15mill., 100 μl, 3 inj. sites 1.0 ± 0.1mill.* 6.7 ± 0.9% 15mill., 300 μl, 3 inj. sites 2.0 ± 0.5mill. 13.4 ± 3.2% intramyocardially injected total cell number, total volume, number of injection sites . persistence and survival (absolute number) . persistence and survival (% of injected cells) . 5mill., 100 μl, 1 inj. site 0.7 ± 0.1mill.** 14.1 ± 2.9% 15mill., 100 μl, 1 inj. site 0.9 ± 0.2mill.* 6.0 ± 1.3% 15mill., 300 μl, 1 inj. site 1.0 ± 0.2mill.* 6.7 ± 1.3% 5mill., 100 μl, 3 inj. sites 0.3 ± 0.1mill.*** 6.5 ± 1.5% 15mill., 100 μl, 3 inj. sites 1.0 ± 0.1mill.* 6.7 ± 0.9% 15mill., 300 μl, 3 inj. sites 2.0 ± 0.5mill. 13.4 ± 3.2% *P < 0.05 **P < 0.01 ***P < 0.001 vs 15mill., 300 μl, 3 inj. sites. Open in new tab Abstract 390 Table intramyocardially injected total cell number, total volume, number of injection sites . persistence and survival (absolute number) . persistence and survival (% of injected cells) . 5mill., 100 μl, 1 inj. site 0.7 ± 0.1mill.** 14.1 ± 2.9% 15mill., 100 μl, 1 inj. site 0.9 ± 0.2mill.* 6.0 ± 1.3% 15mill., 300 μl, 1 inj. site 1.0 ± 0.2mill.* 6.7 ± 1.3% 5mill., 100 μl, 3 inj. sites 0.3 ± 0.1mill.*** 6.5 ± 1.5% 15mill., 100 μl, 3 inj. sites 1.0 ± 0.1mill.* 6.7 ± 0.9% 15mill., 300 μl, 3 inj. sites 2.0 ± 0.5mill. 13.4 ± 3.2% intramyocardially injected total cell number, total volume, number of injection sites . persistence and survival (absolute number) . persistence and survival (% of injected cells) . 5mill., 100 μl, 1 inj. site 0.7 ± 0.1mill.** 14.1 ± 2.9% 15mill., 100 μl, 1 inj. site 0.9 ± 0.2mill.* 6.0 ± 1.3% 15mill., 300 μl, 1 inj. site 1.0 ± 0.2mill.* 6.7 ± 1.3% 5mill., 100 μl, 3 inj. sites 0.3 ± 0.1mill.*** 6.5 ± 1.5% 15mill., 100 μl, 3 inj. sites 1.0 ± 0.1mill.* 6.7 ± 0.9% 15mill., 300 μl, 3 inj. sites 2.0 ± 0.5mill. 13.4 ± 3.2% *P < 0.05 **P < 0.01 ***P < 0.001 vs 15mill., 300 μl, 3 inj. sites. Open in new tab Abstract Purpose: In cardiac cell replacement therapy, the number of new contractile cells within the injured heart is crucial for this therapy's effectiveness. Therefore, we quantified and optimized persistence and survival of rat neonatal cardiomyocytes (NCM) after intramyocardial (IM) injection into infarcted rat hearts. Methods: 1 week after myocardial infarction (MI, LAD ligation) in female F344 rats, syngeneic male NCM were IM injected into the infarct area. Different injection conditions including cell number (5 or 15mill.), volume (100 or 300 μl) and number of injection sites (1 or 3 inj. sites) were investigated. 4 weeks after MI, numbers of transplanted cells were determined using quantitative real-time PCR (genomic DNA of whole hearts, Y chromosome). Results: After IM injection into 1 inj. site, the absolute number of detected male cells was 0.7-1.0mill. for 5 or 15mill. NCM in 100 or 300 μl. Distribution to 3 inj. sites significantly increased this value only for 15mill. NCM in 300 μl. Persistence and survival in % of the injected cell number was dependent on the injected cell number per inj. site (approx. 12% for 5mill. vs. 6-7% for 1.8 or 15mill., both P < 0.05) and on the injected volume per inj. site (approx. 12% for 100 μl vs. 6-7% for 33 or 300 μl, both P < 0.05). IM injection of exactly 5mill. NCM in exactly 100 μl per inj. site was optimal with approx. 13% (vs. 6-7% for 1.8mill. in 33 μl or 15mill. in 300 μl, both P < 0.05). Conclusions: Persistence and survival of transplanted NCM is limited and dependent on injected cell number and volume per inj. site. Using the optimal conditions, the absolute number of persisting and surviving transplanted cells can be increased by repeated IM injections. Such optimal conditions must be determined and considered for all kinds of cardiac cell replacement therapy 1 . Controlled cardiac differentiation of human embryonic stem cell-derived embryoid bodies in scalable bioreactors M Zagallo M Zagallo 1 Venetian Institute of Molecular Medicine , Padua , Italy C Luni C Luni 2 University of Padua , Padua , Italy E Serena E Serena 1 Venetian Institute of Molecular Medicine , Padua , Italy E Cimetta E Cimetta 2 University of Padua , Padua , Italy S Zatti S Zatti 1 Venetian Institute of Molecular Medicine , Padua , Italy G Giobbe G Giobbe 1 Venetian Institute of Molecular Medicine , Padua , Italy N Elvassore N Elvassore 2 University of Padua , Padua , Italy Abstract Cell transplantation is a promising approach to replace scarred or nonfunctional myocardium in a diseased heart. At the moment the major obstacle is the generation of the amount of functional cardiomyocytes required for clinical applications, which is hindered by the limited availability and proliferative capacity of these cells. Human embryonic stem (hES) cells may be a promising source but their use has been limited by the lack of robust expansion and differentiation protocols. In this scenario, we aim at culturing human embryonic stem cell-derived embryoid bodies (hEBs) onto microstructured photopolymerizable polyacrilamide hydrogels coupled to milli-liter stirred bioreactors to enhance the efficiency of cell proliferation and direct the cardiac differentiation. The hydrogels support long term culture of size-controlled hEBs within an array of 60 microwells, each representing data points in parallel. The milli-liter bioreactors, with volumes of 1-10ml, ensure dynamic and controlled cell culture conditions. To verify the feasibility of using bioreactor technology to the culture of hEBs, we compared the culture of hEBs in stirred bioreactors to those maintained in static conditions. We derived hEBs from the hES cell lines H9 and HES2, which show high potential towards cardiac differentiation. Multiparametric analyses were carried out modulating the physic-chemical properties of the hydrogel, the growth factors concentration in the medium and varying stirring rates and oxygen tension of the culture system. Cell proliferation was investigated using BrdU incorporation by cytofluorimetric and immunofluorescence analysis. To study the differentiation profile of hEBs, the expression of cardiac-specific genes was investigated by Real Time PCR, while spatial distribution of the markers in the hEBs was analyzed by immunofluorescence on 10 μm hEBs cryosections. Under proper culture conditions, hEBs differentiate into cell types of the three germ layers, including cardiomyocytes, as verified by immunostaining for cardiac markers including Troponin T, connexin 43 and NKX 2.5. The yield in cardiomyocytes achieved in bioreactors was significantly higher than that in static conditions, ranging from 5 to 10%. In conclusion, the technology we developed ensures a scalable culture environment and allow to obtain highly specialized cardiac cells. The small system volumes permit to simultaneously screen a wide range of cell culture conditions thus representing an important step in translating from small to clinically-relevant scale cultivation of cells for their applications in patients and drug screening in cardiology. An in vitro model for cardiac cell therapy: coupling a microfluidic platform with arrayed human embryonic stem cells-derived cardiomyocytes for screening pathological conditions E Serena E Serena 1 Venetian Institute of Molecular Medicine , Padua , Italy E Cimetta E Cimetta 2 University of Padua, Chemical Engineering Department , Padua , Italy T Zaglia T Zaglia 3 University of Padua, Department of Biomedical Sciences , Padua , Italy S Zatti S Zatti 1 Venetian Institute of Molecular Medicine , Padua , Italy A Zambon A Zambon 2 University of Padua, Chemical Engineering Department , Padua , Italy K Gordon K Gordon 4 McEwen Centre for Regenerative Medicine , Toronto , Canada N Elvassore N Elvassore 2 University of Padua, Chemical Engineering Department , Padua , Italy Abstract Cell therapy is a promising approach for the treatment of infarcted hearts, but is hampered by the limited survival and engraftment of transplanted cells. Besides, the development of new therapies is hindered by the lack of in vitro systems reproducing the infarcted heart environment. We aimed at developing an in vitro model for testing cardiac cell therapy under physiopathological conditions. We designed a microfluidic platform (µFP) to be coupled with human cardiomyocytes (hCMs) arrayed in uniformly sized circular islands for studying their viability and functional contractility upon exposure to pathological stimuli: reactive oxygen species, hypoxia and increasing substrate stiffness. hCMs, derived from HES2, were cultured onto a poly-acrylamide hydrogel with tissue-like mechanical properties, and arrayed through micropatterning (300 µm diameter, 500 µm spacing islands). The multilayered µFP, interfaceable with the arrayed hCMs, was fabricated using lithographic techniques and molded in PDMS. It comprised: a membrane-based vacuum system for the reversible sealing, a gas exchange unit for the maintenance of defined O2 partial pressures, the microfluidic channels delivering fluids to the 12x12mm culture chamber, electrodes for electrical stimulation. It is compatible with standard microscopy, allowing on line observations. We obtained highthroughput data on multi-parametric experiments performed selectively treating defined sections of the arrayed hCMs via delivery of compartmentalized contiguous flow domains at different composition. Our system was first validated in static culture. Arrayed hCMs maintained the expression of cardiac Troponin I and T, Connexin 43, NKX2.5, α-actinin. The spontaneous contraction frequency was 0.83 ± 0.23Hz, while a pacing induced via exogenous electrical stimulation led to an increase up to 4Hz. Cell viability and contractility were not affected by substrate stiffness up to E≈35kPa. Exposure to H2O2 concentrations>0.2mM induced cell death. hCMs viability was not compromised with exposure to 0.1mM, while their contractility was reduced. We then succeeded in culturing the arrayed hCMs within the µFP reproducing the temporal pattern of ischemia/reperfusion, with exposure to hypoxia and oxidative stress. Real time imaging allowed a quantitative analysis of spontaneous and electrically-stimulated contractile activity and viability. Such system could be a useful tool for screening in a highthroughput fashion the effects of multiple conditions on an in vitro cell model representative of human heart physiology, thus potentially helping the processes of therapy and drug development. Cardiomyocytes derived from human embryonic stem cells as an alternative model for cardiotoxicity and contractility studies. M Mioulane M Mioulane 1 Imperial College London , London , United Kingdom G Foldes G Foldes 1 Imperial College London , London , United Kingdom NN Ali NN Ali 1 Imperial College London , London , United Kingdom SE Harding SE Harding 1 Imperial College London , London , United Kingdom Abstract Human embryonic stem cell-derived cardiomyocytes (hESC-CM) are being examined as an alternative source of human cardiac myocytes to current animal cardiomyocyte models for drug development and toxicity assays, as well as cell-based therapy. In vitro cardio-differentiation of hESCs recapitulates early cardiogenesis and generates spontaneously beating cells that express cardio-specific genes and contractile proteins. Here we asked whether the response of hESC-CM to neuro-hormones and ischemia differ from current animal cardiomyocyte models, or from human adult ventricular myocytes (AVM), in terms of contractility and cell death. Spontaneous contraction of clusters of hESC-CM generated from H7 hESC line by embryoid body method was measure by a video edge detection system (IonOptix). The negative chronotropic effect of adenosine (ADO, 10 µM) in hESC-CM was mediated through the activation of the Gi coupled A1-adenosine receptor subtype (-38.7 ± 11.7% (ADO) vs. -3.7 ± 3.4% (ADO+DPCPX (A1R antagonist)), p < 0.05, n = 6). A bimodal distribution of chronotropic response to ADO developed in hESC-CM over 125 days, with strong negative responses possibly linked to maturation of IK, Ach. After A1R blockade, adenosine had a positive inotropic effect on isolated hESC-CM but not on dog AVM (34.9 ± 9.3%, P < 0.05, and 0.5 ± 6.6% respectively, n = 5 and n = 6). This resembles human AVM from failing (but not non-failing) hearts, where the A2a-AR agonist CGS 21680 had a positive inotropic effect (42.9 ± 16.7%, n = 8, P < 0.001). Similar to human AVM, adenosine had an anti-adrenergic effect in hESC-CM (p < 0.001, n = 3 in hESC-CM and p < 0.01, n = 4 in adult). Apoptosis assays were performed on hESC-CM differentiated from hESC exposed to Activin-A and BMP-4 in a monolayer. Single cardiomyocytes were stained with anti-MHC, anti-active caspase-3 antibodies and analyzed by automated fluorescent microscope (Cellomics). β1AR stimulation induced an increase in caspase-3 intensity in rat neonatal cardiomyocytes (40 ± 7.5%, p < 0.01, n = 6) but not in hESC-CM (3 ± 3%, p = 0.45, n = 3). Caspase-3 activation with the PKC inhibitor Chelerythrine was lower than in hESC-CM than neonatal preparations (% apoptotic cells: 14.5 ± 4%, n = 6 vs. 61.5 ± 4%, n = 3, P < 0.001). Our data show that hESC-CMs are more resistant to apoptosis than rat neonatal cardiomyocytes. Taken together, our comparative study suggest that hESC-CM respond differently in term of contractility and cell death to animal models and can be more closely related to the human adult failing cardiomyocyte phenotype. Nitric oxide protects mouse embrionic stem cell-derived cardiomyocytes against simulated ischemia/reoxygenation injury A Gorbe A Gorbe 1 University of Szeged, Faculty of Medicine, Cardiovascular Research Group, Department of Biochemistry , Szeged , Hungary A Szunyog A Szunyog 1 University of Szeged, Faculty of Medicine, Cardiovascular Research Group, Department of Biochemistry , Szeged , Hungary ZV Varga ZV Varga 1 University of Szeged, Faculty of Medicine, Cardiovascular Research Group, Department of Biochemistry , Szeged , Hungary MK Pirity MK Pirity 2 Biotalentum Ltd. , Gödöllo , Hungary S Rungaruniert S Rungaruniert 3 Molecular Animal Biotechnology Lab, Szent István University , Gödöllo , Hungary A Dinnyes A Dinnyes 3 Molecular Animal Biotechnology Lab, Szent István University , Gödöllo , Hungary T Csont T Csont 1 University of Szeged, Faculty of Medicine, Cardiovascular Research Group, Department of Biochemistry , Szeged , Hungary P Ferdinandy P Ferdinandy 1 University of Szeged, Faculty of Medicine, Cardiovascular Research Group, Department of Biochemistry , Szeged , Hungary Abstract Embryonic stem (ES) cell-derived cardiomyocytes are a promising cell source for cardiac repair after myocardial infarction. Therefore, it is of great importance to protect stem cells against ischemia/reperfusion (I/R) injury. The aim of this study was to test, whether a nitric oxide (NO) - donor is protective against I/R injury of mouse ES cell-derived cardiomyocytes. Therefore, we used a combination of hypoxic chamber and chemical simulation of ischemia and reoxygenation. Mouse ES cell line (HM-1) derived embryonic bodies (EB) were seeded onto 24-well plates (1 EB/well) and were kept in growing medium (containing ascorbic acid) under normoxic condition (at 37°C, 5% CO2) for 5 days. Then medium of the cultures were replaced with a "hypoxic" solution and plates were kept in a hypoxic chamber (gased with 95% N2 and 5% CO2 at 37°C) for 150 minutes in the presence or absence of the NO-donor S-nitrozo-penicillamine (SNAP, 10-6 M). Next step was the reoxygenation, when cells were covered with growing medium (without ascorbic acid) and kept in a normoxic incubator for 120 minutes. Finally, viability tests were done with Trypane blue staining and cells were counted in a hemocytometer. After simulated ischemia the cell death was 22.0 ± 2.1%, which was attenuated by using the NO donor SNAP (13.3 ± 1.9%, p < 0.05). As a control experiment, other series of EBs were covered with a "normoxic" solution and plates were kept in the normoxic incubator for 150 minutes. All cells were subjected to reoxygenation and viability tests were performed similarly to simulated ischemia groups. In normoxic condition 12.5 ± 0.1% of the cells died. In conclusion, our present results show that NO-donors may protect ESC-derived cardiomyocytes against ischemia/reoxygenation injury. Thus, NO-donors can be useful candidates for the enhancement of ES cell-derived cardiomyocyte survival after implantation into the ischemic myocardium. Growth and proliferative activity of human embryonic stem cell-derived cardiomyocytes is modulated in a calcineurin-NFAT-dependent manner G Foldes G Foldes 1 Imperial College London, National Heart & Lung Institute , London , United Kingdom M Mioulane M Mioulane 1 Imperial College London, National Heart & Lung Institute , London , United Kingdom A Iqbal A Iqbal 1 Imperial College London, National Heart & Lung Institute , London , United Kingdom M.D. Schneider M.D. Schneider 1 Imperial College London, National Heart & Lung Institute , London , United Kingdom NN Ali NN Ali 1 Imperial College London, National Heart & Lung Institute , London , United Kingdom SE Harding SE Harding 1 Imperial College London, National Heart & Lung Institute , London , United Kingdom Abstract Cardiac cell replacement therapy using human embryonic stem cell-derived cardiomyocytes (hESC-CM) remains a potential approach to regenerate myocardium. The major hurdles to their clinical application are post-transplantation cell loss and immunogenicity. Here we studied the effects of calcineurin-targeting immunosuppressants cyclosporine (CsA; 0.2 µM) and FK-506 (10 ng/mL) on the growth and proliferative activity of hESC-CM in culture. H7 hESCs were treated with Activin A and bone morphogenetic protein-4 to generate hESC-CM, which were dissociated into single cells after 30 days. As shown by automated microscopy, treatments with CsA and FK-506 decreased proliferation (ratio of mitotic marker Ki67+ / myosin heavy chain-α/β+ cells, from 35 ± 1% to 15 ± 0.7% and 21 ± 1%, respectively, p < 0.001) and number of single hESC-CM (by 40 ± 5% and 50 ± 4%, p < 0.01). Both immunosuppressants reduced α1-adrenoceptor phenylephrine (10 µM, 48 h)-mediated increase in cell size (p < 0.001) and percentage of hESC-CM with organised sarcomere (p < 0.01). Transfection of hESC-CM with a dominant-negative vector of downstream NFAT transcription factor inhibited hESC-CM hypertrophy (p < 0.05 vs. empty expression vector pBJ5). Transfection with a GFP-VIVIT construct or administration of cell permeable 11R-VIVIT protein, selective inhibitors of calcineurin-NFAT interaction, without affecting calcineurin phosphatase activity, blocked phenylephrine-induced hypertrophy but not the proliferative activity of hESC-CM. This suggests that NFAT mediates hypertrophic signalling while non-NFAT-dependent calmodulin-calcineurin pathways may play a role proliferative activity of hESC-CM. Chelerythrine-induced increase in caspase-3 activation of the apoptotic machinery was inhibited by CsA (p < 0.01), suggesting that CsA offsets cell loss to a smaller extent by reduction in apoptosis. Our results show that immunosuppressants reduce number, size and maturation of hESC-CM via calcineurin-NFAT pathways. The resulting reduction in graft expansion capabilities would potentially necessitate implantation of increased hESC-CM numbers when immunosuppressants are given. VE-cadherin-positive endothelial derived microparticles and NT-proBNP can detect silent myocardial ischemia in diabetic patients EE Babes EE Babes 1 Faculty of Medicine , Oradea , Romania VV Babes VV Babes 1 Faculty of Medicine , Oradea , Romania Abstract Background: Previous studies have demonstrated that CD144 endothelium-derived microparticles(EMP) can be a specific marker for quantifying endothelial dysfunction and/or injury. Increased levels of CD144EMP were found in patients with diabetes mellitus, especially in those individuals with coronary artery disease. NT-proBNP is a neurohormone that in recent studies proved useful in detecting ischemic heart disease. The aim of the study was to evaluate whether CD144EMP and NT –proBNP may be useful for identifying silent myocardial ischemia in type 2 diabetes mellitus patients. Methods: Study was performed on 32 diabetes mellitus patients in whom silent ischemia was identified through exercise stress test and Holter ECG compared with 27 diabetic patients and no silent ischemia respectively-negative exercise stress test and Holter ECG. Complete clinical and paraclinical evaluation (lipid profile, glycated Hb, microalbuminuria, NT-proBNP, CD144EMP and echocardiography) were also performed in all patients. Results: There were no significant differences between two groups regarding: age, body mass index, lipid profile, systolic and diastolic blood pressure, left ventricular mass and left ventricular ejection fraction. Duration of diabetes was greater in the group with silent ischemia(p = 0,002), glycated Hb was higher(p = 0,001); microalbuminuria was more frequent (p = 0,0008). NT-proBNP was higher in patients with silent ischemia-135,26 ± 25,28pg/ml vs 79,25 ± 24,5pg/ml in patients without silent ischemia(p < 0,0001). CD144EMP were higher in patients with silent ischemia-0,747 ± 0,152x106/ml vs 0,482 ± 0,726x106/ml in patients without silent ischemia(p < 0,0001). In multiple regression analysis for all patients, CD144EMP-(p = 0,02,r = 0,75) and NT-proBNP-(p = 0,02,r = 0,75) remained independent predictors for silent ischemia(R2 = 0,59). Conclusions: CD144EMP and NT-proBNP are valuable predictors for silent ischemia in diabetic patients and probably can be used in screening for silent ischemia in patients with diabetes. Heart failure development predictors at patients with hypertrophic cardiomyopathy EM Khodjaeva EM Khodjaeva 1 Republic Specialised Center of Surgery , Tashkent , Uzbekistan RA Ibadov RA Ibadov 1 Republic Specialised Center of Surgery , Tashkent , Uzbekistan KH Khalikulov KH Khalikulov 1 Republic Specialised Center of Surgery , Tashkent , Uzbekistan AA Mansurov AA Mansurov 1 Republic Specialised Center of Surgery , Tashkent , Uzbekistan Abstract Purpose: estimation of heart failure development risk factors at patients with hypertrophic cardiomyopathy. Methods: we have observed 50 patients with the confirmed diagnosis of hypertrophic cardiomyopathy. Average age of patients were 28,3 ± 1,2 years old. 47 patients were men and the other 3 were women. All patients were examined by standard complex diagnostic procedures – ECG, chest x-ray, 2D transthoracic EchoCG. The final verification of diagnosis was made by heart catheterization. At 2 cases we have revealed signs which has appropriate to the incompact "spongeous" trabecular myocardium with the lacunization. The classic signs of hypertrophic cardiomyopathy with the average systolic wall thickness of 3,1 ± 0,8 sm were confirmed at 80% cases. The clinical symptoms were revealed at 6 patients, which were appeared as the breathlessness, low tolerance to the physical exertion, T wave inversion at IV-Vth ECG chest leads. One year follow-up at 60% patients showed that heart failure varied at all patients and had a tendency to the hardening. Results: the use of 2D transthoracic EchoCG, stress-tests, stress EchoCG and transesophageal EchoCG on the early stages of this disease gave us possibility to reveal of heart failure development predictor factors and turnover the tactics of management of these patients. In association of the evolution of myocardial wall thickness on the conventional transthoracic ultrasound study, the reduction of tolerance in physical exertion in this category of patients, and positive results of stress echocardiography and the lack of symptoms at rest at the same time, we have performed radio ablation or surgical excision of hypertrophic myocardial mass. Conclusion: determination of heart failure development predictors in asymptomatic patients with hypertrophic cardiomyopathy will prevent the progression circulatory failure and reduce amount of patients with the sub-and decompensated stage of heart failure. Prognostic value of exercise capacity in postinfarction period in patients treated with thrombolytics A Astvatsatryan A Astvatsatryan 1 European Regional Educational Academy, Faculty of Medecine , Yerevan , Armenia M Senan M Senan 1 European Regional Educational Academy, Faculty of Medecine , Yerevan , Armenia Abstract We wanted to clarify prognostic value of exercise capacity in postinfarction period in pts treated with thrombolysis (Streptokinase). Methods: 76 pts (33 female, aged 42 ± 21) with first acute myocardial infarction with ST segment elevation (STEMI) were randomized to early thrombolysis (less than 3 hours of the onset of symptoms of infarction; n = 37) and relatively late thrombolysis (more than 3 but less than 6 hours; n = 39). All of them performed standard Bruce Treadmill Test on 30th day of STEMI. The primary endpoint was a composite of death and reinfarction and follow-up was up to 2 years. Results: As it was expected, pts with relatively late thrombolysis had a lower exercise capacity (5.9 [2.1-11.7] METs versus 7.7 [2.8-14.5], p < 0.001) and more frequently significant ischemic ST segment depression (25% versus 12%), p < 0.002) compared to pts with early thrombolysis. Exercise capacity was of prognostic importance for death and/or reinfarction, (hazard ratio per one MET increase was 0.71 (0.61-0.97), p < 0.05) and for death alone (0.72 [0.55-0.93], p < 0.05) in pts with relatively late thrombolysis. Unexpectedly, exercise capacity had no prognostic importance for death and/or reinfarction, (hazard ratio per one MET increase was 0.38 [0.77-0.99], p = NS) and for death alone (0.53 [0.65-0.89], p = NS) in pts with early thrombolysis. Conclusion: Exercise capacity has long-term prognostic value in pts with STEMI and relatively late thrombolysis, but not in pts with early thrombolysis. Hyperglycemia on admission in patients with unstable angina is a predictor of cardiac mortality A Astvatsatryan A Astvatsatryan 1 European Regional Educational Academy, Faculty of Medecine , Yerevan , Armenia M Senan M Senan 1 European Regional Educational Academy, Faculty of Medecine , Yerevan , Armenia Abstract Aim: We studied the phenomena of early elevation of plasma glucose level in patients (pts) with unstable angina pectoris (UA). Methods: 54 pts (age 50.2 ± 13.2, males 32) with UA and unexpected hyperglycemia on admission (without history of diabetes mellitus) were involved in our study. Troponin-T (Tn-T, quantitative method) levels were measured 24 hours after admission in these pts. Pts with unexpected hyperglycemia were divided in two groups: G1 (n = 30) consists of pts with subsequent Tn-T elevation, G2 (n = 24) consists of pts with negative Tn-T. The endpoints were acute myocardial infarction (AMI), non-fatal AMI and mortality during one year follow up. Results: In G1 tertile distribution showed that plasma glucose level was related to AMI mortality (6.4%, 5.7% and 9.1%, p < 0.005) but not related to non-fatal AMI (12.8%, 12.4% and 14.9%, p = NS). In G2 tertile distribution showed that plasma glucose level was related to non-fatal AMI (12.5%, 14.6% and 21.1%, p < 0.005) but not related to cardiac mortality (3.2%, 2.8% and 3.9%, p = NS). In G1 to the third tertile by Cochran-Cox regression analysis level of abnormal high plasma glucose level was an independent predictor of cardiac mortality (HR = 2.3, 95%, CI 1.3-3.8; p < 0.01), in G2 level of plasma glucose level was independent predictor of non-fatal AMI (HR = 1.3, 95%, CI 1.1- 2.2; p < 0.03) but not cardiac mortality. Conclusion: Unexpected hyperglycemia with subsequent Tn-T elevation in pts with UA is a predictor of cardiac mortality. In the absence of Tn-T elevation unexpected hyperglycemia predicts non-fatal AMI. Asymmetric dimethylarginine in patients with myocardial infarction after stenting A Nemeth A Nemeth 1 Heart Institute, University of Pecs , Pecs , Hungary Z Lenkey Z Lenkey 1 Heart Institute, University of Pecs , Pecs , Hungary Z Ajtay Z Ajtay 1 Heart Institute, University of Pecs , Pecs , Hungary A Cziraki A Cziraki 1 Heart Institute, University of Pecs , Pecs , Hungary E Sulyok E Sulyok 2 Institute of Public Health and Health promotion, Faculty of Health Sciences, University of Pecs , Pecs , Hungary I Horvath I Horvath 1 Heart Institute, University of Pecs , Pecs , Hungary JM Lobenhoffer JM Lobenhoffer 3 Otto-von-Guericke University of Magdeburg, Institute of Clinical Pharmacology , Magdeburg , Germany SM Bode-Boger SM Bode-Boger 3 Otto-von-Guericke University of Magdeburg, Institute of Clinical Pharmacology , Magdeburg , Germany Abstract Purpose: NO metabolism can be characterized by asymmetric dimethylarginine (ADMA) which is an endogenous competitive inhibitor of nitric oxide synthase. Increased ADMA plasma levels are associated with cardiovascular disease. Methods: Two groups of patients undergoing percutaneous coronary intervention (PCI) with stenting were enrolled into the study. Group I included 16 patients with, whereas group II consisted of 24 patients without STEMI (controls). Before PCI and at 1 hour, 5 days and 30 days after reperfusion blood samples were taken for measurement of l-arginine, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and l-ornithine. L-arginine and methylarginines were measured by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Arginine methylation index (Arg-MI) was calculated according to the formula: Arg-MI = (ADMA+SDMA)/MMA. Results: In patients with elective stent implantation stenting induced a prompt and sustained depression of ADMA (F = 9.594, p < 0.001), and l-ornithine (F = 9.107, p < 0.001) with simultaneous increase of l-arginine (F = 6.715, p < 0.001) and l-arginine/ADMA ratio (F = 8.250, p < 0.001). By contrast, STEMI patients responded to stent placement with a variable increase in l-arginine (F = 4.556, p < 0.01) and l-ornithine (F = 14.975, p < 0.001), whereas there was an early fall of Arg-MI after stenting followed by a steady increase to approach the initial values. ADMA remained constant for STEMI patients after stent placement (F = 2.982, p < 0.069). The differences in the time-course for ADMA (F = 9.431, p < 0.001), Arg-MI (F = 4.415, p < 0.012) and l-ornithine (F = 5.105, p < 0.003) proved to be significant between the STEMI and control group. Conclusions: Stent placement improves endothelial dysfunction in patients with coronary heart disease (CHD). Long-term follow-up of ADMA may be suitable marker to monitor the improvement of coronary endothelial function after revascularisation. Can we make a diognosis of acute myocardial infarction just depending on once cardiac biomarker examination? J Li J Li 1 Soochow University , Suzhou , People's Republic of China Y He Y He 1 Soochow University , Suzhou , People's Republic of China X Yang X Yang 1 Soochow University , Suzhou , People's Republic of China F Wang F Wang 1 Soochow University , Suzhou , People's Republic of China H Xu H Xu 1 Soochow University , Suzhou , People's Republic of China X Li X Li 1 Soochow University , Suzhou , People's Republic of China X Zhao X Zhao 1 Soochow University , Suzhou , People's Republic of China Y Lin Y Lin 1 Soochow University , Suzhou , People's Republic of China Abstract Introduction: Development of cardiac biomarkers, such as myoglobin and cardiac troponin I, is a prerequisite for diagnosis of acute myocardial infarction. In the real world, however, a variety of diseases can lead to the rise of these biomarkers; what is more, cardiac doctors are usually requested to make a diagnosis of acute myocardial infarction just depending on once cardiac biomarker examination. There is obviously a disparity between the "theoretical"criteria (development changes) and the "real world" requirement in the diagnosis of acute myocardial infarction. We hypothesized that resetting the cut-off values of these biomarkers may contribute the diagnosis of acute myocardial infarction in the "real world"and narrow this disparity between the "theoretical criteria" and the "real world". Methods: One thousand and seventy-five consecutive patients underwent myoglobin and cardiac troponin I test from Jan. 1st, 2006 to Dec 31st, 2008. Out of which 463 cases were with acute myocardial infarction and 612 with non-acute myocardial infarction. Logistic regression analysis was used to study the diagnostic values of the cardiac troponin I, the ratio of troponin I/myoglobin and the myoglobin in acute myocardial infarction. Receiver operating characteristic curve analysis studied the diagnostic power of the above-mentioned 3 indices. Results: ROC demonstrated that the area under the curve (AUC) of the cardiac troponin I was 0.918, 95% confidence interval (CI) 0.898 to 0.936,P was 0.0001, the cut-off value was 0.21,sensitivity was 96.32%, 95%CI is 93.3–98.2%, specificity was 77.45, and 95%CI was 73.8–80.8% the AUC of myoglobin was 0.635,95%CI was 0.602 to 0.668%, P was 0.0001, the cut-off value was 112, sensitivity was 51.47%, 95%CI was 45.4–57.5%, specificity was 71.26%, 95% CI was 67.4–74.9%. the AUC of the ratio of cardiac troponin I/myoglobin was 0.815,95% CI 0.787–0.840%,P was 0.0001,the cut-off value was 0.0112, sensitivity was 72.79%, 95%CI was 67.1–78.0%, specificity was 82.79%, 95% CI 79.5–85.8%. Conclusions: Resetting the cut-off values of the cardiac troponin I, the ratio of troponin I/myoglobin and the myoglobin may contribute the diagnosis of acute myocardial infarction in the "real world" and narrow the disparity between the "theoretical criteria" and the "real world". Lymphangiogenesis in the fetal and early postnatal mouse heart M Juszynski M Juszynski 1 Medical University of Warsaw, Center of Biostructure Research, Department of Pathological Anatomy , Warsaw , Poland B Ciszek B Ciszek 2 Medical University of Warsaw, Center of Biostructure Research, Department of Anatomy , Warsaw , Poland A Jablonska A Jablonska 1 Medical University of Warsaw, Center of Biostructure Research, Department of Pathological Anatomy , Warsaw , Poland E Stachurska E Stachurska 1 Medical University of Warsaw, Center of Biostructure Research, Department of Pathological Anatomy , Warsaw , Poland A Ratajska A Ratajska 1 Medical University of Warsaw, Center of Biostructure Research, Department of Pathological Anatomy , Warsaw , Poland Abstract Purpose: Until now not much has been known about the structure of lymphatic vessels in the heart. Moreover, the knowledge on lymphangiogenesis in fetal heart was negligible. The research on lymphangiogenesis became possible after the discovery of markers specific for lymphatic vessels. We performed the examination of the development of lymphatic vessels in the mouse heart during embryonic and early postnatal life and the analysis of its course and location. Methods: The research was performed on normal mice hearts, fetal and mature, from 11 ED to 8 PD. The hearts were collected from fetuses and newborn mice, frozen, cut serially and stained with anti-LYVE antibody. Some of the hearts were doubly stained with anti-LYVE and anti-SMA antibodies. Other hearts were immersed in formaldehyde solution and stained with anti-LYVE antibody using the whole-mount method. Ink with gelatine or latex were injected subepicardially into mature mouse hearts. Results: On ED 11 we found some diffused aggregations of cells with very week LYVE assay located among myocardial trabecules of both ventricles and in the atria, but their number was negligible. In the following developing stages LYVE(+) cells are located subepicardially forming primitive tubes. Subepicardial assay is dispersed, but present also in the atria. It is more sensitive in the ventriculo-atrial dorsal groove, where LYVE(+) cells form significant aggregations. LYVE(+) cells begin to form the web of vessels, which is not yet continuous at this stage. In some of the areas, the tubes form appendices, sprouting into myocardial wall. On ED 15 it is possible to distinguish the main lymphatic vessels: on the surface of intraventricular grooves, anterior and posterior, on the surface of the atrioventricular groove, on the posterior surface of the left atrium and left ventricle. The diameter of the vessels is significant, but the vessels lack lamina media. There are also many single and aggregated LYVE (+) cells apart from the vessels. Moreover LYVE(+) cells form vessel web locally among the myocardial trabecules. In the postnatal hearts lymphatic vessels grow reaching myocardium and locating in the vicinity of the coronary arteries on the dorsal surface of the cone. A great number of the vessels is also present subepicardially. The course and location of lymphatic vessels in the mature mice hearts are similar. Conclusions: The lymphatic vessels in the heart develop from the diffused buds, which mature by developing into a thin web and enlarging their diameter. The aggregations of the greatest lymphatic vessels appear on ED 15 and correspond with the vessels of the mature mouse heart. Anatomical and molecular mapping of rabbit his-purkinje system A Atkinson A Atkinson 1 University of Manchester , Manchester , United Kingdom S Inada S Inada 1 University of Manchester , Manchester , United Kingdom J Li J Li 1 University of Manchester , Manchester , United Kingdom R Sleiman R Sleiman 1 University of Manchester , Manchester , United Kingdom H Zhang H Zhang 1 University of Manchester , Manchester , United Kingdom M Boyett M Boyett 1 University of Manchester , Manchester , United Kingdom H Dobrzynski H Dobrzynski 1 University of Manchester , Manchester , United Kingdom Abstract The Purkinje fibres (PFs) form the terminal portion of the cardiac conduction system (CCS) and provide a rapid conduction pathway for signal transduction. They have distinct action potential characteristics from ventricular muscle and they have been linked to a number of ventricular arrhythmias including torsade de pointes arrhythmias associated with long-QT syndrome. Whole-mount immunohistochemical method employing middle neurofilament (NF-M) as a marker of CCS in the rabbit heart was used to map anatomical distribution of the left and right bundle branches (LBB, RBB) and free-running PFs (LPFs, RPFs) in three rabbits. The LBB appeared as a ribbon like structure composed of many fine fibres running in parallel on the septal surface. The fibres formed denser strands traversing the ventricular chamber before branching into a network of fine fascicles covering the left ventricular free wall. The RBB appeared to be narrower than LBB running over the septal surface. It has a number of fascicles branching from it along its length that extend across the ventricle and form a dense network on the right ventricular free wall. qPCR was used to analyse expression of ∼30 transcripts in eight rabbits. The results show that PFs have a distinct expression profile from left ventricle (LV). Our key findings in LPF and/or RPF versus LV are a higher expression of Cx40 (GJ), HCN1 and HCN4 (If), Nav1.1 (INa), Cav1.3 (ICaL), Kir3.1 (IKAch), Kv4.3 and DPP6 (Ito), but lower expression of Cx45 (GJ), Cav1.2 (ICaL), Kv1.4 and KChIP2 (Ito), Kv1.5, ERG and KvLQT1 (IK), Kir2.1 (IK1), Kir6.2 (IKATP), RYR2, NCX1 and SERCA2a (Ca2+ handling). Computer modelling was employed to create a 3D reconstruction of the PF network and to model the action potential characteristics of PFs based on the qPCR data. We conclude that PFs form a complex asymmetrical network in the left and right ventricular chambers of the rabbit heart and they have a unique pattern of expression of ion and gap junction channels and Ca 2+ handling proteins. 3D anatomical model of the cardiac conduction system and atrioventricular ring tissue in the rat heart O Fedorenko O Fedorenko 1 University of Manchester , Manchester , United Kingdom G Hao G Hao 1 University of Manchester , Manchester , United Kingdom A Atkinson A Atkinson 1 University of Manchester , Manchester , United Kingdom J Yanni J Yanni 1 University of Manchester , Manchester , United Kingdom D Buckley D Buckley 2 University of Leeds , Leeds , United Kingdom RH Anderson RH Anderson 1 University of Manchester , Manchester , United Kingdom MR Boyett MR Boyett 1 University of Manchester , Manchester , United Kingdom H Dobrzynski H Dobrzynski 1 University of Manchester , Manchester , United Kingdom Abstract Introduction and Purpose: The cardiac conduction system (CCS) is responsible for the initiation and propagation of the action potential through the heart and it consists of the sinus node (SN), inferior nodal extension (INE), atrioventricular node (AVN)/penetrating bundle (PB), His bundle, right and left bundle branches (RBB, LBB), and the Purkinje fibres (PFs). The atrioventricular ring tissue (AVRT) consists of the left, right and aortic rings (LR, RR, AR) and retroaortic node (RAN). Morphologically, the AVRT is made of nodal-like myocytes around the tricuspid, mitral and aortic valves. Although the AVRT is anatomically continuous with the CCS, it is not involved in the normal electrical activity of the heart, but it can be responsible of atrial tachycardias. The aim of this study was to make a 3D anatomical model of the CCS and AVRT. Methods: Hearts from 6 adult rats were harvested, frozen and cryosectioned in the coronal plane through the long axis from the dorsal to ventral surface. Every 100 μm, tissue sections (25 μm in thickness) were stained with Masson's trichrome and immunolabelled for HCN4, Cx40 (markers of the CCS), Cx43 (marker of the working myocardium) and caveolin3 (marker of cardiac myocytes). One heart was subject to MRI prior to freezing and the 3D anatomical model was constructed using Matlab. Results: Our data show that the anatomical location of all components of the CCS and AVRT was consistent in all hearts. The dorso-ventral extent of different structures was: SAN ∼2.3 mm, INE ∼0.4 mm, AVN/PB ∼0.7 mm, His bundle ∼2.3 mm; RAN ∼0.5 mm; RR ∼1.8 mm; LR ∼1.3 mm. Our first 3D anatomical model shows the full extent of the CCS and AVRT and confirms the relationship of the AVRT with the CCS observed in previous studies from our laboratory. For example, it shows that the AVRT is discrete from the CCS yet continuous with it. It also shows that the SN is an extensive structure as observed before in the rabbit heart. Conclusions: Our model provides an anatomical reconstruction of the whole CCS and AVRT and, therefore, is a useful tool for teaching and research Purposes. Investigation of the prevalence and distributing feature of cardiovascular risk factors in Kazak populations in Xinjiang YT Ma YT Ma 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of XIANG Ma XIANG Ma 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of YH Hu YH Hu 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of YN Yang YN Yang 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of DING Huang DING Huang 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of FEN Liu FEN Liu 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of YING Huang YING Huang 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of CHENG Liu CHENG Liu 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of Abstract Objective: The cardiovascular disease has become a major public health issue worldwide. Previous studies have shown that the prevalence of hypertension was significantly different between the Han and the Kazak populations, the two major populations in Xinjiang. The current study aims to investigate the status of primary risk factors of cardiovascular disease (CVD) in Kazak populations in Xinjiang. Methods: In total, the participants aged ≥35 years and lived for more than 5 years were selected with stratified random sampling and investigated with epidemiological Methods. All the participants were interviewed by trained and certified observers under a structured questionnaire. Fasting plasma glucose (FPG), blood lipids, body mass index, blood pressure (BP) of all sample population were measured. Height, weight, and waistline and hip circumference were obtained from each participant with standard protocols. The risk of CVD in aged from 35 to 59 was evaluated by the Methods and tools of 10-Year's risk estimation of ischemic cardiovascular disease (ICVD) in Chinese. The data were analyzed with SPSS11.0 statistical software. P < 0.05 was considered statistically significant. Results: 1,265 Kazak subjects (537 males and 727 females) from Xinjiang were enrolled. The prevalence rate of hypertension, diabetes mellitus and metabolic syndrome in males was significantly higher than females in the Kazak population (51.70% vs. 37.5%, P < 0.001; 3.87% vs. 1.11%, P < 0.002 and 15.66% vs. 11.96%, P < 0.002, respectively). However, there are not significantly difference of total cholesterol (TC) level (4.91 ± 1.03 mmol/L vs. 4.67 ± 0.99 mmol/L, P < 0.838) between the males and the females. The rate of smoking was 10.43% in males and 7.98% in females, P < 0.001. There are 86.22 % of male and 89.0 % of female that 10-year absolute risk of ICVD less than 10%(P < 0.38). And 8.19% of male and 3.85 % of female had a 10-year absolute risk of ICVD higher than 20%(P < 0.09). Conclusions: The risk factors of CVD was high in males of Kazak populations in Xinjiang. To control body weight and blood pressure actively was the most important index. Anti-tobacco intervention addressed for pregnant women in 400 city project TJ Jedrzejczyk TJ Jedrzejczyk 1 Medical University of Gdansk , Gdansk , Poland L Balwicki L Balwicki 1 Medical University of Gdansk , Gdansk , Poland L Wierucki L Wierucki 1 Medical University of Gdansk , Gdansk , Poland T Zdrojewski T Zdrojewski 1 Medical University of Gdansk , Gdansk , Poland Abstract Purpose: The purpose of our research was to measure phenomenon of active and passive smoking among pregnant women in the 400 smallest cities with surrounding villages. The aim was also to trace social features associated with smoking and develop the strategy for future antitabacco interventions. Method: The research was conducted along with intervention based on American College of Obstetricians and Gynecologists 5 A's model adjusted to Polish organizational limitations. The model include 1) asking about tobacco smoking during every contact with pregnant women, 2) advising not to smoke or avoid passive exposure, 3) assessing the determination to make a quit attempt, 4) assist with the trial, 5) arrange next meeting with the focus on the problem. The research itself was based on questionnaire used by midwives and measuring level of carbon monoxide in exhaled air with Micro Co devices. The intervention was targeted at actively or passively smoking women, her partner if he smoke, and women whose quit smoking after they become pregnant. The intervention was followed after the delivery. Results : During the research 919 pregnant women were questioned and examined with micro Co device. Among that number were 22% of active smokers and 31% exposed on passive smoking in their home or work. The prevalence of smoking were higher among lower educated -46% of women with only primary school compared with 7% of women with university diploma. Smoking was more frequent among women with lower per capita personal income. 80% of smoking pregnant women were motivated to make a quit attempt. Conclusions : The prevalence of smoking among women in the smallest Polish cities and their surroundings is very important problem of public health. The awareness of the problem in the target group allow for successful intervention. The 5a's intervention model can be successfully adopted in Polish health care system. NMDA receptors function in rat heart: does age and O2 availability matter? A Makhro A Makhro 1 Institute of Veterinary Physiology, UZH , Zurich , Switzerland I Agarkova I Agarkova 2 University Hospital Zurich , Zurich , Swaziland J Vogel J Vogel 1 Institute of Veterinary Physiology, UZH , Zurich , Switzerland M Gassmann M Gassmann 1 Institute of Veterinary Physiology, UZH , Zurich , Switzerland A Bogdanova A Bogdanova 1 Institute of Veterinary Physiology, UZH , Zurich , Switzerland Abstract Purpose: NMDAR is a ligand-gated nonselective cation channel with a ten-fold preference for Ca2+ over Na+ and K+. It is highly abundant in neurons, but is also expressed in non-neuronal tissues including mammalian heart. Among the agonists of the NMDAR is homocysteic acid (HCA). High concentration of plasma HCA is associated with high risk of CVD in human patients. The present study is designed to assess localization and function of the NMDAR with a special focus on age-dependence and hypoxic stress. Methods: Experiments were performed in isolated neonantal and adult rat cardiomyocytes and blood-perfused isolated hearts of Wistar rats ranging from young (2-3 mo) to senescent (6-18 mo). The effects of agonists and antagonists on the heart function (HR, LVP), ATP, and GSH levels, intracellular Ca2+ were assessed along with evaluation of the NMDA expression pattern. Results: The NMDAR in the adult rat ventricular tissue was represented by the following subunits: NR1, NR2A, B, C and D. The expression levels increased with age from nondetectible in neonatal to maximal in senescent heart. Presence of 300 µM NMDA/HCA in blood caused up to 20% increase in HR and an increase in LVP in young animals. Administration of antagonists (100 µM MK-801 or memantine) reduced HR by 35% or 28% respectively but had no effect on LVP. In young hearts NMDAR agonists increased LVP by 15% whereas antagonists had no effect. In senescent hearts NMDAR agonists induced moderate tachycardia and arrhythmia along with a 20% decrease in LVP whereas antagonists (100 µM MK-801/memantine) restored rhythmicity and had a subtle bradycardic effect. The exposure of young rat hearts to the hypoxia (35% instead of 98% SO2) was well-tolerated. Treatment of hypoxic young hearts with 300 µM NMDA/HCA resulted in ATP depletion, GSH oxidation and heart stunning. Senescent hearts responded to O2 deprivation with ATP depletion, GSH oxidation, acute severe bradycardia followed by arrhythmia and cessation of contractility. Administration of the NMDAR antagonists prior to hypoxic exposure rescued contractile function, and prevented oxidation. Conclusion: NMDAR is involved in regulation of Ca2+ levels and Ca2+-dependent function of the young and senescent, but not in neonatal heart. High NMDAR abundance in senescent rat heart makes it hypoxia-intolerant. Increase in plasma HCA levels may cause arrhythmia and destabilize function of the senescent heart especially under conditions of limited oxygen availability. Our findings make myocardial NMDAR into a potential target of pharmacological intervention. Effect of atorvastatin on the inflammatory cytokine response in young and senescent endothelial cells K Korybalska K Korybalska 1 University of Medical Science Department of Pathophysiology , Poznan , Poland M Pyda M Pyda 2 University of Medical Science Department of Cardiology , Poznan , Poland J Witowski J Witowski 1 University of Medical Science Department of Pathophysiology , Poznan , Poland Abstract 409 Table . Control . TNFα . TNFα + AT 1 µM . TNFα + AT 10 µM . Early-passage cells IL-6 0.45 ± 0.21 1.9 ± 0,5 (a) 1.3 ± 0,2 (a) 1.4 ± 0,3 (a) CXCL8/IL-8 19.0 ± 2.0 198 ± 59 (a) 158 ± 22 (a) 132 ± 18 (a) CCL2/MCP-1 7.3 ± 3.4 125 ± 32 (a) 72 ± 14 (a) 84 ± 10 (a) sICAM-1 1.2 ± 0.3 17.9 ± 6.2 (a) 10.71 ± 2.2 (a) 12.5 ± 2.6 (a) Senescent cells IL-6 1.58 ± 0.74 (c) 1.3 ± 0.9 1.3 ± 0.6 0.8 ± 0.2 (c) CXCL8/IL-8 50.5 ± 5.4 (c) 75 ± 41 (c) 80 ± 29 (c) 57 ± 12 (c) CCL2/MCP-1 13.0 ± 4.5 (c) 28 ± 5 (b,c) 24 ± 14 (c) 14 ± 3 (c) sICAM-1 38.5 ± 14.0 (c) 21.5 ± 3.6 (b) 17.5 ± 5.4 (b) 20.2 ± 8.2 (b) . Control . TNFα . TNFα + AT 1 µM . TNFα + AT 10 µM . Early-passage cells IL-6 0.45 ± 0.21 1.9 ± 0,5 (a) 1.3 ± 0,2 (a) 1.4 ± 0,3 (a) CXCL8/IL-8 19.0 ± 2.0 198 ± 59 (a) 158 ± 22 (a) 132 ± 18 (a) CCL2/MCP-1 7.3 ± 3.4 125 ± 32 (a) 72 ± 14 (a) 84 ± 10 (a) sICAM-1 1.2 ± 0.3 17.9 ± 6.2 (a) 10.71 ± 2.2 (a) 12.5 ± 2.6 (a) Senescent cells IL-6 1.58 ± 0.74 (c) 1.3 ± 0.9 1.3 ± 0.6 0.8 ± 0.2 (c) CXCL8/IL-8 50.5 ± 5.4 (c) 75 ± 41 (c) 80 ± 29 (c) 57 ± 12 (c) CCL2/MCP-1 13.0 ± 4.5 (c) 28 ± 5 (b,c) 24 ± 14 (c) 14 ± 3 (c) sICAM-1 38.5 ± 14.0 (c) 21.5 ± 3.6 (b) 17.5 ± 5.4 (b) 20.2 ± 8.2 (b) a – statistical difference vs. control group in young cells b – statistical difference vs. control group in senescent cellsc – statistical difference between young and senescent cells in corresponding groups Open in new tab Abstract 409 Table . Control . TNFα . TNFα + AT 1 µM . TNFα + AT 10 µM . Early-passage cells IL-6 0.45 ± 0.21 1.9 ± 0,5 (a) 1.3 ± 0,2 (a) 1.4 ± 0,3 (a) CXCL8/IL-8 19.0 ± 2.0 198 ± 59 (a) 158 ± 22 (a) 132 ± 18 (a) CCL2/MCP-1 7.3 ± 3.4 125 ± 32 (a) 72 ± 14 (a) 84 ± 10 (a) sICAM-1 1.2 ± 0.3 17.9 ± 6.2 (a) 10.71 ± 2.2 (a) 12.5 ± 2.6 (a) Senescent cells IL-6 1.58 ± 0.74 (c) 1.3 ± 0.9 1.3 ± 0.6 0.8 ± 0.2 (c) CXCL8/IL-8 50.5 ± 5.4 (c) 75 ± 41 (c) 80 ± 29 (c) 57 ± 12 (c) CCL2/MCP-1 13.0 ± 4.5 (c) 28 ± 5 (b,c) 24 ± 14 (c) 14 ± 3 (c) sICAM-1 38.5 ± 14.0 (c) 21.5 ± 3.6 (b) 17.5 ± 5.4 (b) 20.2 ± 8.2 (b) . Control . TNFα . TNFα + AT 1 µM . TNFα + AT 10 µM . Early-passage cells IL-6 0.45 ± 0.21 1.9 ± 0,5 (a) 1.3 ± 0,2 (a) 1.4 ± 0,3 (a) CXCL8/IL-8 19.0 ± 2.0 198 ± 59 (a) 158 ± 22 (a) 132 ± 18 (a) CCL2/MCP-1 7.3 ± 3.4 125 ± 32 (a) 72 ± 14 (a) 84 ± 10 (a) sICAM-1 1.2 ± 0.3 17.9 ± 6.2 (a) 10.71 ± 2.2 (a) 12.5 ± 2.6 (a) Senescent cells IL-6 1.58 ± 0.74 (c) 1.3 ± 0.9 1.3 ± 0.6 0.8 ± 0.2 (c) CXCL8/IL-8 50.5 ± 5.4 (c) 75 ± 41 (c) 80 ± 29 (c) 57 ± 12 (c) CCL2/MCP-1 13.0 ± 4.5 (c) 28 ± 5 (b,c) 24 ± 14 (c) 14 ± 3 (c) sICAM-1 38.5 ± 14.0 (c) 21.5 ± 3.6 (b) 17.5 ± 5.4 (b) 20.2 ± 8.2 (b) a – statistical difference vs. control group in young cells b – statistical difference vs. control group in senescent cellsc – statistical difference between young and senescent cells in corresponding groups Open in new tab Abstract Endothelial cell senescence is increasingly implicated in cardiovascular pathology. It is characterized by an increase in spontaneous secretion of inflammatory cytokines and altered response to pro-inflammatory stimuli. Since senescent cells may accumulate in tissues with age, they may contribute to the loss of local homeostasis and the development of chronic imflammatory state. Statins have been advocated as agents that may restore endothelial cell function and thus decrease the incidence of cardiovascular complications. We have compared the impact of atorvastatin (AT) on the release of several cytokines by young and senescent endothelial cells. Experiments were performed using human umbilical vein endothelial cells (HUVECs) that were cultured to senescence by serial passaging. Cytokine release was assessed in young and senescenct HUVECs exposed for 24 hours to AT (1-10 µM) in the presence or absence of TNFα (10 ng/ml). Selected cytokines were quantified using specific immunoassays. The results are express as means (pg/µg cell protein) ± SD; n = 6. These results indicate that - compared to young endothelial cells - the release of cytokines by senescent cells is increased under basal conditions but decreased in response to stimulation. AT reduces TNFα-stimulated cytokine release by HUVECs; in senescent cells this decrease reaches the levels seen in unstimulated cells. Such a pattern may point to a different sensitivitiy of aged tissues to the effect of statins 1 . The emotional status of men and women with chronic heart failure A Ibatov A Ibatov 1 Sechenov Moscow medical academy , Moscow , Russian Federation Abstract Purpose: to study gender-specific emotional status of patients with ischemic heart disease (IHD) and chronic heart failure (CHF). Methods: 56 patients (men and women) with ischemic heart disease and chronic heart failure at the age of 42 to 65 years were examined. Patients are divided into two groups. The first group included 18 women, average age – 58.3 ± 1.2 years. The second group included 48 men, average age – 56.8 ± 1.5 years. The level of anxiety and depression was investigated by the Hospital Anxiety and Depression Scale (HADS), personality characteristics - by the MMPI questionnaire. Results: the groups did not differ in age, duration of IHD and CHF, functional class of heart failure, therapy. The level of anxiety and depression were in 1st group accordingly -9.3 ± 0.7 and 6.9 ± 0.7 scores, in 2nd group accordingly -5.6 ± 0.7 (p < 0.05) and 5.7 ± 0.7 (p > 0.05) scores. MMPI test parameters in the first and second group were, accordingly: on scale of Hypochondriasis – 60.2 ± 1.2 and 55.8 ± 1.4 (p < 0.05) scores; on scale of Depression – 55.7 ± 3 , 6 and 50.6 ± 2.0 (p > 0.05) scores; on scale of Hysteria – 56.4 ± 1.5 and 50.1 ± 1.3 (p < 0.01) scores; on scale of Psychopathic Deviate – 53.4 ± 2.7 and 45.5 ± 1.7 (p < 0.05) scores; on scale of Paranoia – 58.5 ± 3.0 and 52.1 ± 1.8 (p > 0.05) scores; on scale of Psychasthenia – 53.5 ± 2.3 and 49.7 ± 2.1 (p > 0.05) scores; on scale of Schizophrenia – 59.7 ± 3.5 and 47.1 ± 1.7 (p < 0.01) scores; on scale of Hypomania – 58.1 ± 1.6 and 44.3 ± 2.4 points (p < 0.001) scores. Conclusions: women with chronic heart failure had a more high level of anxiety and more expressed personality characteristics, as compared to men with chronic heart failure. Beneficial effects of beta-blocker treatment on alterated basal cardiac function and responses to beta-adrenoceptor stimulation in female rats during maturation NN Sozmen NN Sozmen 1 Ankara University, Faculty of Medicine , Ankara , Turkey A Seymen A Seymen 1 Ankara University, Faculty of Medicine , Ankara , Turkey E Tuncay E Tuncay 1 Ankara University, Faculty of Medicine , Ankara , Turkey B Turan B Turan 1 Ankara University, Faculty of Medicine , Ankara , Turkey Abstract Beta-adrenergic receptor (βAR) blockers are one of the most frequently prescribed drugs for the treatment of cardiovascular dysfunction showing different effects either short or long term usages. Results of the studies also reveal sex differences in cardiac performance and responses to pathological conditions. There are numerous physiologic changes with aging that affect function of cardiovascular system. The number of patients with diagnosed heart failure continues to grow worldwide and the search for new and effective therapies for this condition remains a priority yet. The use of beta-blockers is also well established in the clinical context of aging heart. Therefore, in this study, first hemodynamic and electrophysiological parameters of heart from female rats are examined and compared at aged 3 and 9 months. We first observed a significant decrease in left ventricular developed pressure (LVDP), marked prolongation in late-repolarization phases of APD (APD75 and APD90), significant decrease in the L-type Ca2+-channel current density (ICaL) and marked alterations in the parameters of intracellular free Ca2+ ([Ca2+]i) in heart preparations from aged rats compared to those of young rats. Second, long-term effect of a non-selective beta-blocker, timolol treatment (TIM; 5 mg/kg/day, intragastrically for 8 months) on both mechanical and electrical activities of heart from aged and young rats are investigated. Timolol treatment completely restored 1) prolonged APD75 and APD90, 2) decreased LVDP, and 4) inhibited ICaL. Furthermore, we examined age-dependent changes in responses to βAR stimulation (isoproterenol, ISO), the adenylate cyclase (AC) activity and βAR affinity to ISO. TIM treatment prevented age-dependent increase in cell size and decrease in βAR density, significantly besides normalization of age-dependent decrease in AC activity. Our results showed that beta-blocker treatment of aging samples could protect the hearts against age-induced alterations in both basal activity and βAR responses of the heart. Taken together, these data demonstrate that treatment of aging female rats with βAR blockers for 8-month appears to confirm the role of beta-adrenergic pathway in aging-induced cardiac dysfunction. In addition, although prevention of these types of alterations in cardiac function by using beta-blockers might present them to be a useful pharmacological strategy for the treatment of aging heart, it can be clearly suggested to take into consideration of particular clinical benefits due to the possible differences in the effect of individual βAR blocking agent. (Supported by TUBITAK-SBAG-107S427) Effects from age to Wnt and NF kappaB signal pathway in heart of mice YING Huang YING Huang 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of YT Ma YT Ma 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of YINING Yang YINING Yang 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of FEN Liu FEN Liu 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of BD Chen BD Chen 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of XM Li XM Li 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of Abstract Background: Wnt and NF-κB signal pathway are all related to cardiac remodeling post -infarction and there were age-related differences of outcome post infarction.We study changes of these two signal pathways in heart in old mice to prepare some materials for advanced study. Methods: We used 20 mice to study changes of these two signal pathways:10 in young (3-mo) and old group(18-mo) respectively.They were detected expression of dvl-1, β-catenin, p/t GSK-3β and connexin 43 in the left ventricle (LV) by western-blot. And they were detected expression of p65 and p50 by immunohistochemistry and western-blot. At the same time they were detected expression of ICAM-1 and VCAM-1. Results: (1) Expression of dvl-1 increased by 2.41 fold in old group compared with young mice in the LV (P = 0.000).There were no statistically differences of expression of β-catenin between the old and young group (P = 0.647) in LV. Ratio of p/t GSK-3β is much lower in old compared with young(P = 0.000).When talking about connexin 43,there were statistically significant differences in old group compared with young (P = 0.001) in the LV. (2) There was no difference of numbers of p65 and p50 positive cells between old and young group by immunohistochemistry (P < 0.05).But expression of p65 and p50 in nuclear protein in old group is higher than it in young group (P = 0.000).There was no difference of ICAM-1 between old and young group(P = 0.401).Expression of VCAM-1 in old group was higher than it in young group (P = 0.000). Conclusion: There were age-related differences of protein associated with Wnt and NF-κB signal pathway in heart.Progressing study about changes in these two signal pathway post myocardial-infarction might find new mechanisms about age-related differences of cardiac remodeling. Long term outcomes of a comprehensive cardiac rehab program in women with coronary artery disease L Houston-Feenstra L Houston-Feenstra 1 Loma Linda University Medical Center , Loma Linda , United States of America J.R. Chiong J.R. Chiong 1 Loma Linda University Medical Center , Loma Linda , United States of America K Jutzy K Jutzy 1 Loma Linda University Medical Center , Loma Linda , United States of America Abstract 413 Table . Control . . . . Cardiac Rehab . . . . . . Baseline . 12 mos. . 24 mos. . P value * . Baseline . 12 mos. . 24 mos. . P Value * . . . . . . TCL 195.9 194.6 207.4 N/S 191.9 164.7 164.6 0.021 TRIG 214 200 212 N/S 265 173.7 175.7 0.006 HDL 45.6 44 43.11 N/S 42.9 46.7 43.4 N/S LDL 114 115 122.7 N/S 104 84.93 92.2 0.02 Hosp Days 1.4 0.667 0.818 .03 1.4 0.333 0.4 0.001 ED Visits 0.469 0.7 0.758 0.01 0.133 0.08 0.000 0.002 . Control . . . . Cardiac Rehab . . . . . . Baseline . 12 mos. . 24 mos. . P value * . Baseline . 12 mos. . 24 mos. . P Value * . . . . . . TCL 195.9 194.6 207.4 N/S 191.9 164.7 164.6 0.021 TRIG 214 200 212 N/S 265 173.7 175.7 0.006 HDL 45.6 44 43.11 N/S 42.9 46.7 43.4 N/S LDL 114 115 122.7 N/S 104 84.93 92.2 0.02 Hosp Days 1.4 0.667 0.818 .03 1.4 0.333 0.4 0.001 ED Visits 0.469 0.7 0.758 0.01 0.133 0.08 0.000 0.002 Note: * P value baseline to 24 months. Hospital days = number days hospitalized per year per patient, ED Visits = number of ED visits per patient per year. Open in new tab Abstract 413 Table . Control . . . . Cardiac Rehab . . . . . . Baseline . 12 mos. . 24 mos. . P value * . Baseline . 12 mos. . 24 mos. . P Value * . . . . . . TCL 195.9 194.6 207.4 N/S 191.9 164.7 164.6 0.021 TRIG 214 200 212 N/S 265 173.7 175.7 0.006 HDL 45.6 44 43.11 N/S 42.9 46.7 43.4 N/S LDL 114 115 122.7 N/S 104 84.93 92.2 0.02 Hosp Days 1.4 0.667 0.818 .03 1.4 0.333 0.4 0.001 ED Visits 0.469 0.7 0.758 0.01 0.133 0.08 0.000 0.002 . Control . . . . Cardiac Rehab . . . . . . Baseline . 12 mos. . 24 mos. . P value * . Baseline . 12 mos. . 24 mos. . P Value * . . . . . . TCL 195.9 194.6 207.4 N/S 191.9 164.7 164.6 0.021 TRIG 214 200 212 N/S 265 173.7 175.7 0.006 HDL 45.6 44 43.11 N/S 42.9 46.7 43.4 N/S LDL 114 115 122.7 N/S 104 84.93 92.2 0.02 Hosp Days 1.4 0.667 0.818 .03 1.4 0.333 0.4 0.001 ED Visits 0.469 0.7 0.758 0.01 0.133 0.08 0.000 0.002 Note: * P value baseline to 24 months. Hospital days = number days hospitalized per year per patient, ED Visits = number of ED visits per patient per year. Open in new tab Abstract Objective: A comparison of long term outcomes of a comprehensive cardiac rehab (CR) program vs. routine follow up care in women hospitalized with Cardiovascular disease(CVD). Background: CVD is the leading cause of death in women in the US. CR programs document short-term reductions in CVD adverse outcomes in male dominated research populations, benefit in females remains controversial. Methods: This study compares 2 groups of female patients hospitalized for CVD who were followed for 24 months post hospital discharge. Both groups received education and counseling in cardiac risk reduction and were discharged on aspirin, beta-blocker, angiotensin converting enzyme-inhibitor and statin therapy. One group identified as CR (15 patients: average age 65.7) participated in a 12-week comprehensive CR program and one group identified as Control (34 patients: average age 65.2) received routine follow up care associated with CVD. Patients participating in CR received an individualized program including optimization of medications and specific cardiac risk interventions. CR program lasted 12 wk with 3 visits per week. Conclusions: 1. Women participating in a comprehensive CR program demonstrated long-term improvement in Cardiac risk profile including LDL less than 100, compared to control patients with LDL of 122.7 2. Women in CR demonstrated significantly fewer Hospitalizations related to CVD at 24 months. 3. Women in CR had a decrease in ED visits while the control group showed a significant increase in ED visits at 24 mos 1 . Pro-integrin processing by furin-convertase regulates VSMC signaling and function V Furundzija V Furundzija 1 German Heart Center Berlin , Berlin , Germany J Kaufmann J Kaufmann 1 German Heart Center Berlin , Berlin , Germany K Kappert K Kappert 2 Charite - University Medicine Berlin, Campus Mitte , Berlin , Germany H Meyborg H Meyborg 1 German Heart Center Berlin , Berlin , Germany E Fleck E Fleck 1 German Heart Center Berlin , Berlin , Germany P Stawowy P Stawowy 1 German Heart Center Berlin , Berlin , Germany Abstract Serving as molecular bridges, integrins transmit signaling between the ECM and the cytoskleleton in both directions. The pro-alpha v integrin is synthesized as a larger proprotein, which undergoes endoproteolytic cleavage by the proprotein convertase furin in the trans-Golgi network. Pro-integrin cleavage is neither required for heterodimerization nor membrane expression, but regulates ligand-induced outside-in phosphorylation of non-receptor tyrosine kinases and adhesion/migration. The precise role of pro-integrin alpha v maturation on integrin-dependent signaling cascades and cell functions, however, is largely unknown in inside-out signaling in vascular smooth muscle cells (VSMCs). Results: The phorbol ester phorbol 12-myristate 13-acetate (PMA; 100nM) induced remodeling of focal adhesion sides as well as migration and adhesion on vitronectin (10 μg/mL) in VSMCs. Immunoblotting demonstrated that this was accompanied by increased phosphorylation of focal adhesion kinase (FAK) and paxillin. PMA-mediated activation of integrin adapter proteins and cell adhesion/migration was inhibited by pharmacological inhibitors of PKC (Ro32-0432) and ERK1/2 (PD98059), whereas PI3-kinase (wortmannin) was not required. Furthermore, blocking integrins with cyclic arginin-glycine-aspartate containing hemxamers (100nM) inhibited cell functions, and phosphorylation of FAK/paxillin, demonstrating that integrin-activation is part of PMA-induced inside-out signaling. To explore the function of pro-alpha v maturation, VSMCs were treated with decanoyl-RVKR-chloromethylketone (50 μM), which inhibited furin and thus integrin cleavage in PMA-treated cells. Even though furin-inhibtion and thus the inhibition of integrin maturation inhibited VSMC migration, it did not affect the inside-out mediated PKC, ERK1/2 or FAK/paxillin phosphorylation. However, double labelling immunofluorescence revealed that the status of pro-integrin activation by furin inhibited alpha v beta 3, as well as phospho-paxillin targeting to focal adhesion sites in integrin inside-out signaling. Furthermore, furin-dependent integrin activation was required for actin polymerization and stress fiber formation in VSMCs. Conclusion: Furin-dependent pro-integrin processing modulates rapid adaptive integrin/cytoskeleton changes, essential to VSMC motility in inside-out signaling, which represents a crucial component in atherogenesis and restenotic tissue remodeling. Low testosterone level correlates with detrimental cardiovascular risk profile in premature atherosclerosis in men E Ksiezycka-Majczynska E Ksiezycka-Majczynska 1 National Institute of Cardiology, Department of Coronary Heart Disease , Warsaw , Poland B Lubiszewska B Lubiszewska 1 National Institute of Cardiology, Department of Coronary Heart Disease , Warsaw , Poland M Kruk M Kruk 1 National Institute of Cardiology, Department of Coronary Heart Disease , Warsaw , Poland P Kurjata P Kurjata 1 National Institute of Cardiology, Department of Coronary Heart Disease , Warsaw , Poland W Ruzyllo W Ruzyllo 1 National Institute of Cardiology, Department of Coronary Heart Disease , Warsaw , Poland Abstract Objectives: Risk factors for premature CAD in men are not well recognized. Recent data suggest that low testosterone level may be linked to metabolic risk factors for CAD and were shown to be related to cardio vascular death in a broad male population. Therefore we assessed the relationship of endogenous testosterone levels and CV risk factors with particular attention to their metabolic component in male patients with premature CAD. Methods: 241 male with early onset of CAD (acute MI, angina confirmed by angiography or revascularization ≤45 years of age) enrolled between January 2006 to November 2008 were studied. They were divided into two subgroups, below and above median value (15.5 nmol/l) for total testosterone (TT) measured at admission. All other parameters age, BMI, waist circumference, glucose, insulin, calculated homeostasis model assessment index- insulin resistance (HOMA-IR), total cholesterol, HDL, LDL and TG were analyzed according to that division. Results: Pts with lower TT had worse metabolic risk factors profile: higher BMI (29.4 vs 27.1 p < 0.0001), larger waist circumference (103.0cm vs 99.0 p < 0.001), higher level of insulin (12.4 μU/ml vs 9.8 p < 0.001), and calculated HOMA-IR (3.07 vs 2.36 p < 0.0007), more often hypertension (53.3% vs 40.7 p = 0.05). Comparing pts with lowest quartile of TT ≤ 12,4 nmol/l with highest> 19,3 significant difference in TG level was detected, being higher in group with low TT (1.77 mmol/l vs 1.32 p < 0.002). There was no difference in age 42.8 y vs 42.9, cigarette smoking, diabetes and history of dyslipidemia, fasting glucose and cholesterol level. It should be mentioned that 25% of pts were already treated with statins. Our study found an inverse relation between male hypotestosteronaemia and metabolic syndrome components: BMI, waist circumference, presence of hypertension, level of insulin, insulin resistance and unfavorable direction for TG level. Conclusions: Among male patients with premature CAD lower testosterone level is correlated with cardio vascular metabolic risk factors. Low TT may be a predictive marker for those at higher risk of cardiovascular disease and may indicate those who may potentially benefit from additional hormonal therapeutic intervention. Prospective studies are carried to found relationship between those findings and clinical features and outcome. Gender characteristics of heart rate variability in patients, suffering chronic heart failure A Ibatov A Ibatov 1 Sechenov Moscow medical academy , Moscow , Russian Federation Abstract The Purpose: to study heart rate variability (HRV) in women and men with chronic heart failure (CHF). Materials and methods: 56 patients with ischaemic heart disease (IHD) and CHF were examined. Patients are divided into two groups. The first group included 18 patients - women, average age 58.3 ± 1.2 years. The second group included 48 patients, men, average age 56.8 ± 1.5 years. Heart rate variability was examined on 5-min recordings of electrocardiogram at rest. Results: the groups did not differ in age and therapy. The average functional class of heart failure (by NYHA) was in first group −1.78 ± 0.22, in the second group -2.05 ± 0.11 (p > 0.05). Parameters of heart rate variability at rest in 1st and 2nd group were, accordingly: heart rate – 70.0 ± 2.7 and 62.6 ± 1.7 beats / min (p < 0.05), SDNN – 18.5 ± 2.2 and 28.9 ± 2.5 msec (p < 0,05); pNN50% -1.3 ± 0.5 and 3.0 ± 1.2 (p > 0,05); TP – 383.1 ± 73.4 and 777.8 ± 199.0 msec2 (p > 0.05); HF – 96.0 ± 25.1 and 112.5 ± 19.3 msec2 (p > 0.05); LF – 74.6 ± 16.4 and 190.4 ± 29.9 msec2 (p < 0.01); VLF – 212.6 ± 41.7 and 474.8 ± 165.9 msec2 (p > 0.05); HF% -25.1% and 14.5% (p > 0.05), LF% -19.5% and 24.5% (p > 0.05), VLF% -55.5% and 61.1% (p > 0.05). The patients of 1st group had vegetative balance at rest: 66.7 % predominance of parasympathetic nervous system, 11.1 % balance between parasympathetic and sympathetic nervous system and 22.2 % prevalence sympathetic nervous system. The patients of 2nd group had vegetative balance at rest accordingly: 42.1 % predominance of parasympathetic nervous system (p > 0.05), 34.2 % balance between parasympathetic and sympathetic nervous system (p < 0.05) and 23.7 % prevalence sympathetic nervous system (p > 0.05). Conclusions: Thus, women suffering chronic heart failure, compared with men suffering chronic heart failure, had a lower heart rate variability at rest, which is a bad prognostic sign. Angiotensin converting enzyme expression is switched on and off during reversible differentiation of cardiac fibroblasts to myofibroblasts RB Driesen RB Driesen 1 Catholic University of Leuven, Department of Cardiovascular Diseases , Leuven , Belgium T Coenen T Coenen 1 Catholic University of Leuven, Department of Cardiovascular Diseases , Leuven , Belgium RH Fagard RH Fagard 1 Catholic University of Leuven, Department of Cardiovascular Diseases , Leuven , Belgium KR Sipido KR Sipido 1 Catholic University of Leuven, Department of Cardiovascular Diseases , Leuven , Belgium VV Petrov VV Petrov 1 Catholic University of Leuven, Department of Cardiovascular Diseases , Leuven , Belgium Abstract Purpose: In diseased myocardium, myofibroblasts (MyoFb) produce high levels of angiotensin converting enzyme (ACE); whereas in normal myocardium it is nearly absent. In contrast, fibroblasts (Fb) in 2D-culture show high levels of ACE. Therefore, we investigated whether ACE expression in 2-D and 3-D cultured Fb is related to specific (de)-differentiation. Methods: Adult rat Fb (Wistar) were cultured for 4 days in DMEM with 10% serum in the absence (C-control) and presence (C-SD-208) of SD-208 (3 µmol/L), an inhibitor of TGF-β1 receptor 1 kinase. Also cells were incubated with TGF-β1 (400 pmol/L) for 6 days (C-TGF-β1). We replated these cultures in three dimensional collagen matrices (3-DCM), either in floating (3 days) or in restrained ring form with or without static stretch (14 days). ACE and α-smooth muscle actin (α-SMA, marker for MyoFb) were determined in 2-D cultures and 3-DCM by immunostaining, WB and PCR. Cell proliferation was measured in 2-D cultures by cell counting and in 3-DCM by proliferating cell nuclear antigen (PCNA) immunostaining. Contraction of floating 3-DCM was determined by measuring gel volume with H3+-H2O. Results: In 2-D cultures, spontaneous Fb differentiation (C-control) is associated with increased cell size, retardation of cell proliferation, formation of stress fibers and induction of α-SMA in 75,6% of the cells. These MyoFb were found to express ACE mRNA (1972 ± 68). C-SD-208 greatly reduced number of α-SMA positive MyoFb up to 6% and stimulated cell proliferation indicating inhibition of Fb differentiation. This coincided with a reduction in ACE expression (1155 ± 209). C-TGF-β1 stimulated differentiation of non-proliferating MyoFb with a markedly enlarged cell size, highly developed stress fibers and high α-SMA expression in 96% of the cells. This stimulation coincided with high ACE expression levels (4414 ± 746). In 3-D cultures, floating 3-DCM populated with C-control or C-TGF-β1 cells revealed a 2-fold and 6-fold stronger contraction than 3-DCM populated with C-SD-208. C-SD-208 seeded in restrained 3-DCM showed no expression for ACE and α-SMA but preservation of cell proliferation. Similar observations were made in 3-DCM populated with C-control indicating dedifferentiation of MyoFb into Fb. C-TGF-β1 seeded cells maintained their phenotype as indicated by positive α-SMA and ACE staining and loss of proliferation. Conclusion: We conclude that Fb are not involved in ACE production confirming the important role of MyoFb in the renin-angiotensin system activation. We also propose that dedifferentiation may contribute to reduction of MyoFb after post-myocardial infarction. High energy phosphotransfer in the failing mouse heart-role of adenylate kinase and glycolytic enzymes D Aksentijevic D Aksentijevic 1 University of Oxford , Oxford , United Kingdom C Lygate C Lygate 1 University of Oxford , Oxford , United Kingdom K Makinen K Makinen 1 University of Oxford , Oxford , United Kingdom L Sebag-Montefiore L Sebag-Montefiore 1 University of Oxford , Oxford , United Kingdom D Medway D Medway 1 University of Oxford , Oxford , United Kingdom J Schneider J Schneider 1 University of Oxford , Oxford , United Kingdom S Neubauer S Neubauer 1 University of Oxford , Oxford , United Kingdom Abstract Interaction between creatine kinase (CK), adenylate kinase (AK) and glycolytic enzymes mediates intracellular high-energy phosphotransfer. While a reduction in CK activity is characteristic of the failing heart, little is known about the potential for compensatory changes in AK and glycolytic enzymes, with no published data for the failing murine heart. The aim of this study was to measure activity of these key phosphotransfer enzymes in two common mouse models of chronic heart failure (CHF). C57BL/6 mice were subjected to transverse aortic constriction (TAC; n = 12), myocardial infarction induced by coronary artery ligation (CAL; n = 15), or sham operation (n = 8-12). Cardiac function was characterised 5-8 weeks post-surgery by echocardiography, left ventricular (LV) haemodynamics assessment and cine-MRI. Mice were selected for the presence of congestive heart failure. Activities of phosphotransfer enzymes CK, AK and glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK) and pyruvate kinase (PK) were assessed in LV tissue extracts spectrophotometrically. Western blotting was used for the detection of the total protein LV expression of the predominant AK isoform AK1. All mice had severe LV hypertrophy (LV weight TAC 172% of sham, CAL 132% of sham; p < 0.01), impaired systolic function (fractional area change TAC 33% vs. 59% sham; p < 0.01, ejection fraction CAL 19% vs. 66% sham; p < 0.01) and pulmonary congestion (TAC 120%, CAL 61% increase in lung weight/body weight; p < 0.01) compared to sham controls. A significant decrease in myocardial CK (TAC 5.6 ± 1.2 vs. sham 6.8 ± 0.9 U/mg; CAL 4.5 ± 0.9 vs. sham 5.6 ± 0.8 U/mg p < 0.05) and maximal CK reaction velocity (TAC 345.4 ± 69 vs. sham 490.8 ± 102 p < 0.001; CAL 260.3 ± 67 vs. sham 374.8 ± 45, p < 0.05) was observed in both experimental models. However, despite a significant reduction in AK1 protein expression in both CHF groups (TAC 51%, CAL 39% vs. sham; p < 0.05), the activity of AK (TAC 2.8 ± 0.5 vs. sham 2.6 ± 0.5 U/mg; CAL 2.5 ± 0.5 vs. sham 2.4 ± 0.8 U/mg) and its isoforms (AK1 TAC 2.1 ± 0.5 vs. sham 2.1 ± 0.5 U/mg; CAL 1.6 ± 0.4 vs. sham 1.6 ± 0.4 U/mg) remained unchanged. In contrast, the activities of glycolytic phosphotransfer mediators GAPDH and PGK were 19% and 12% higher in TAC (p < 0.05) and 31% and 23% higher in CAL models (p < 0.05). In conclusion, murine CHF is characterised by impaired CK function, unaltered AK activity, and increased activity of glycolytic phosphotransfer enzymes, independent of CHF aetiology. These results support the concept of increased contribution to phosphotransfer by alternative non-CK pathways during cardiac failure. Shift from adult to fetal metabolic phenotype during experimental ischemia reiterates the epigenetic plasticity of the molecular networks associated with myocardial metabolism in the aging heart R Gasser R Gasser 1 Medical University of Graz, Department of Cardiology , Graz , Austria E Holzwart E Holzwart 1 Medical University of Graz, Department of Cardiology , Graz , Austria PP Rainer PP Rainer 1 Medical University of Graz, Department of Cardiology , Graz , Austria D Von Lewinski D Von Lewinski 1 Medical University of Graz, Department of Cardiology , Graz , Austria H Maechler H Maechler 2 Medical University of Graz, Department of Cardiac Surgery , Graz , Austria S Gasser S Gasser 1 Medical University of Graz, Department of Cardiology , Graz , Austria U Roessl U Roessl 1 Medical University of Graz, Department of Cardiology , Graz , Austria BM Pieske BM Pieske 1 Medical University of Graz, Department of Cardiology , Graz , Austria Abstract The fetal heart predominantly uses glucose for its metabolism, whereas the adult mainly metabolises fatty acids. During special conditions, like hypoxia and exercise, the adult metabolic phenotype converts to the fetal one mainly using glucose as a substrate. It has been shown, that a glucose oriented cardiac metabolism is beneficial in myocardial ischemia. Our microarray experiments confirm those data. We find that gene-expression of biological processes which are associated with glucose metabolism are up-regulated during hypoxia, whereas those associated with fatty acid and amino-acid metabolism are downregulated. Testing the effects of β-blockers (atenolol and nebivolol) we find a similar shift in well oxygenised preparations, suggesting that the cardioprotective action of β-blockers is effected by a shift from the adult to the fetal phenotype. Myocardial ischemia increases glucose uptake through translocation of GLUT1 and GLUT4 to the sarcolemma. This appears to be beneficial during ischemia and recovery. We find no significant regulation by β-blockers during myocardial ischemia - there is, however, a significant difference between the expression of GLUT1 in well oxygenised preparations with (0.087 ± 0.02) and without nebivolol (0.62 ± 0.02 SEM; P < 0.05). Similarly, atenolol led to an increase of GLUT1-expression in well oxygenated preparations compared to controls: 1.18 ± 0.08 and 0.62 ± 0.02 SEM; P < 0.05, resp. While there is no significant regulation by beta-blockers during myocardial ischemia, there is a significant difference between the expression of GLUT4 in well oxygenised preparations with (0.52 ± 0.01) and without nebivolol (0.29 ± 0.02 SEM; P < 0.05). Similarly, atenolol led to an increase of GLUT4-expression in well oxygenated preparations compared to controls: 0.92 ± 0.10 and 0.29 ± 0.02 SEM; P < 0.05), resp. These results mirror the increased demand of glucose in the presence of β-blockers. Shifting myocardial metabolism to the fetal phenotype has become a new target for anti-anginal treatment in the aging heart. Either by augmentation of glucose metabolism or by inhibiting fatty acid metabolism. The latter has been successfully targeted by drugs like trimetazidine and ranolazine. In summary, it has been shown for the first time that some of the anti-anginal effects of beta blockers may possibly be conveyed by their action on GLUT 1/4 expression in myocardial cells as well as by facilitating glucose metabolism and in turn causing a shift to the fetal metabolic phenotype in the adult human heart. Role of protein tyrosine phosphatases in pathway-selective insulin resistance J Krueger J Krueger 1 Charite - University Medicine Berlin, Center for Cardiovascular Research , Berlin , Germany U Kintscher U Kintscher 1 Charite - University Medicine Berlin, Center for Cardiovascular Research , Berlin , Germany K Kappert K Kappert 1 Charite - University Medicine Berlin, Center for Cardiovascular Research , Berlin , Germany Abstract Insulin resistance plays a major role in the development of type 2 diabetes, and exerts great impact on the progression of vascular inflammation. Purpose: A post-receptor defect in insulin signalling has been suggested at the molecular level to contribute to insulin resistance. The signalling of the insulin receptor is initiated by receptor ligation, but is critically controlled and antagonized by the activity of protein tyrosine phosphatases (PTPs). The precise role of PTPs in insulin resistance, however, has not yet been explored. Methods/Results: Here we investigated the tissue specific gene expression of PTPs in a mouse model of insulin resistance. Male C57BL/6J mice were fed a high-fat diet (HFD, 60% kcal from fat), or a low-fat diet (LFD, 10% kcal from fat) for 10 weeks. HFD mice exhibited a significant increase in body weight, and were characterized by impaired insulin tolerance, as assessed by i.p. insulin tolerance test. The PTPs SHP-1 and PTP1B have recently been implicated in insulin signalling. Organ-based gene expression analyses in insulin-resistant mice demonstrated upregulation of SHP-1 and PTP1B in epididymal fat tissue and the liver, respectively. Furthermore, DEP-1, a PTP essentially reducing tyrosine-phosphorylation of various receptor tyrosine kinases, was significantly upregulated in epididymal fat. These candidate PTPs negatively regulating insulin receptor signalling were further explored in insulin sensitive cell lines. SiRNA-mediated approaches against SHP-1 in the mouse liver cell line AML12 reduced SHP-1 gene expression and protein levels by 80% and 70%, respectively. This resulted in significantly impaired dephosphorylation of a tyrosine-phosphopeptide (DADE(pY)LIPQQG). Insulin stimulation induced site-selective tyrosine phosphorylation patterns in the insulin receptor at the sites Y972, Y1158, and Y1361 after siRNA mediated downregulation of SHP-1 or by administration of the SHP-1 inhibitor sodium stibogluconate. Furthermore, SHP-1 impairment time-dependently enhanced insulin-induced Akt- and Erk-phosphorylation. Conclusions: These results indicate an important role of PTPs in general, and of SHP-1 in particular, as endogenous antagonists of the insulin receptor and its intracellular binding sites. In addition, this is the first description of endogenous insulin receptor-antagonizing PTPs being organ-specifically regulated in a model of high-fat diet induced insulin resistance. Activity-modification of these insulin receptor targeting PTPs may alter metabolic diseases as well as cardiovascular morbidity and mortality. Human atrial dilatation is associated with increased diffusion restriction for adenine nucleotides T Podramagi T Podramagi 1 Department of Pathophysiology, University of Tartu , Tartu , Estonia K Paju K Paju 1 Department of Pathophysiology, University of Tartu , Tartu , Estonia A Piirsoo A Piirsoo 2 Department of Human Biology and Genetics, University of Tartu , Tartu , Estonia M Roosimaa M Roosimaa 1 Department of Pathophysiology, University of Tartu , Tartu , Estonia L Kadaja L Kadaja 1 Department of Pathophysiology, University of Tartu , Tartu , Estonia E Orlova E Orlova 1 Department of Pathophysiology, University of Tartu , Tartu , Estonia A Ruusalepp A Ruusalepp 3 Department of Cardiac Surgery, University of Tartu , Tartu , Estonia E Seppet E Seppet 1 Department of Pathophysiology, University of Tartu , Tartu , Estonia Abstract Purpose: Chronic volume overload causing stretching of atrial wall leads to remodeling of the myocardium at the molecular and cellular level which eventually manifests as atrial dilatation, a frequent consequence of cardiac failure. Despite some indications of mitochondrial involvement in pathogenesis of cardiac failure, the relationships between their function and remodeling are insufficiently studied. Therefore, we compared oxidative phosphorylation (OXPHOS) and its regulation in extensively dilated and normal-sized human atria. Methods and Results: Control (C) group consisted of patients undergoing coronary artery bybass surgery but having normal atrial size determined on the basis of echocardiographically measured right atrial area and body surface area ratio (RAA/BSA), whereas the second group consisted of patients undergoing valve replacement and having dilated atria (DA), with RAA/BSA ratio more than 2-fold higher than in C group. Oxygraphic measurements revealed that saponin-permeabilized myocardial fibers from right atria of DA group exhibited decreased maximal respiratory rate in the presence of ADP. Both groups were characterized by effective coupling of mitochondrial creatine kinase (CK) to OXPHOS, as suggested by a 4–5-fold creatine (Cr)-dependent decrease in apparent Km for ADP (KmADP). However, in DA group the values of KmADP were increased by 55% and 69% in the absence and presence of Cr, respectively. This change gave rise to severely reduced apparent catalytic efficiency of the system of OXPHOS, as the ratio of Vmax to KmADP in DA group was decreased by 38% and 46%, in the absence and presence of Cr, respectively. The altered regulation of OXPHOS by ADP was not associated with damage of mitochondria, as their ultrastructure explored by electron microscopy (EM) was normal and the respiratory control index measured was similar for both groups, irrespectively of the presence of Cr. Total CK activity measured spectrophotometrically as well as distribution of isoforms of CK and expression levels of the genes encoding CK isoforms were similar in both groups. At the same time, EM revealed that some type of fibrillar protein was extensively accumulated in the cytosol of DA cells whereas it was not seen in C atria. Conclusions: The results uncover a novel mechanism underlying the atrial remodeling in failing heart – increased intracellular diffusion restrictions for ADP, possibly due to accumulation of fibrillar protein. Whether this type of alteration may affect interaction of mitochondria with ATPases and how it is associated with decreased capacity of OXPHOS is currently under study. The activation of the AMP-activated protein kinase increases cardiac insulin sensitivity via multiple molecular mechanisms J Auquier J Auquier 1 Université catholique de Louvain , Brussels , Belgium A Ginion A Ginion 1 Université catholique de Louvain , Brussels , Belgium L Hue L Hue 1 Université catholique de Louvain , Brussels , Belgium S Horman S Horman 1 Université catholique de Louvain , Brussels , Belgium C Beauloye C Beauloye 1 Université catholique de Louvain , Brussels , Belgium JL Vanoverschelde JL Vanoverschelde 1 Université catholique de Louvain , Brussels , Belgium L Bertrand L Bertrand 1 Université catholique de Louvain , Brussels , Belgium Abstract We previously shown that activation of the AMP-activated protein kinase (AMPK) by biguanides like metformin and phenformin increases insulin sensitivity and cardiac glucose uptake in the heart. It has been proposed that the p70 S6 protein kinase (p70S6K) pathway plays a role in these effects. p70S6K pathway is known to reduce insulin signalling via a negative feedback loop involving the serine phosphorylation of the insulin receptor substrate-1 (IRS-1). Knowing that AMPK inhibits p70S6K, we postulated that AMPK increases insulin sensitivity by inhibiting this p70S6K-dependent negative feedback loop. The present study attempted to verify this hypothesis in primary cultured cardiomyocytes. The stimulation of cardiac glucose uptake by phenformin (0.6 ± 0.1 vs control: 0.1 ± 0.05 μmoles/mg.h, p ≤ 0.05) correlated with AMPK phosphorylation, whereas that by insulin (0.9 ± 0.1 μmoles/mg.h, p ≤ 0.05) correlated with Akt phosphorylation. Both phenformin and insulin induced the phosphorylation of the Akt-substrate 160 (AS160) known to regulate glucose uptake. Together, insulin and phenformin induced the overstimulation of both Akt and AS160. This resulted to the overstimulation of glucose uptake (2.0 ± 0.2 μmoles/mg.h). This phenformin-dependent increase in insulin effect correlated with the inhibition of p70S6K phosphorylation (control: 0.0 ± 0.0; insulin:1.0 ± 0.0; insulin+phenformin: 0.68 ± 0.11 AU, p ≤ 0.05) and with reduced phosphorylation of IRS-1 on serine (control: 0.06 ± 0.04; insulin: 1.0 ± 0.0; insulin+phenformin: 0.51 ± 0.12 AU, p ≤ 0.05). To verify the role of the p70S6K inhibition in phenformin effects, we used rapamycin, a known inhibitor of the p70S6K pathway. Rapamycin mimicked phenformin on p70S6K (insulin+rapamycin: 0.0 ± 0.0 AU, p ≤ 0.05), IRS-1 (insulin+rapamycin: 0.14 ± 0.04 AU, p ≤ 0.05), Akt and AS160 phosphorylation state. By contrast, rapamycin was enabled to amplify the effect of insulin on glucose uptake (insulin+rapamycin: 1.1 ± 0.2 μmoles/mg.h). Similar results have been obtained in insulin-resistant cardiomyocytes. In conclusion, the insulin-sensitizing effect of AMPK on insulin signalling (including Akt and AS160) acts, as hypothesized, through the inhibition of the negative feedback loop involving p70S6K and IRS-1. In contrary, the insulin-sensitizing effect of AMPK on glucose uptake is independent of this p70S6K inhibition and should be explained by another, still unidentified, molecular mechanism. Whatever the precise events, our results suggest that AMPK activation could be a potential therapeutic approach to treat insulin resistance in the heart. Effect of type 2 diabetes on the gene expression pattern of rat hearts: a DNA microarray study V Fekete V Fekete 1 Department of Biochemistry, University of Szeged , Szeged , Hungary A Zvara A Zvara 2 Department of Functional Genomics, Biological Research Center, Hungarian Academy of Sciences , Szeged , Hungary J Pipis J Pipis 1 Department of Biochemistry, University of Szeged , Szeged , Hungary C Konya C Konya 3 Béres Pharmaceuticals , Budapest , Hungary C Csonka C Csonka 1 Department of Biochemistry, University of Szeged , Szeged , Hungary L Puskas L Puskas 2 Department of Functional Genomics, Biological Research Center, Hungarian Academy of Sciences , Szeged , Hungary T Csont T Csont 1 Department of Biochemistry, University of Szeged , Szeged , Hungary P Ferdinandy P Ferdinandy 4 Pharmahungary Group , Szeged , Hungary Abstract Purpose: Diabetes, obesity, and dyslipidemia are major cardiovascular risk factors. Therefore, here we examined the possible alterations in cardiac gene expression pattern due to metabolic syndrome. Methods: Zucker diabetic fatty (ZDF) and their control lean rats were used for the study at 25 weeks of age. Fasting blood glucose, serum cholesterol and triglycerid levels were measured by colorimetric assay. Hearts were then isolated and subjected to 10 min ex vivo perfusion to wash out blood. Then total RNA was isolated from the myocardium and assayed by rat oligonucleotide microarray for 15000 genes. Expression of selected genes were confirmed by quantitative real-time PCR. Results: Fasting blood glucose, serum cholesterol and triglycerid levels were elevated in ZDF rats compared to leans (10.0 ± 2.7 mmol/L vs. 5.4 ± 0.1 mmol/L, 3.5 ± 0.2 mmol/L vs. 2.1 ± 0.1 mmol/L and 9.5 ± 1.1 mmol/L vs. 1.1 ± 0.1 mmol/L, respectively). Thirty-six genes showed upregulation and fourty-nine genes showed downregulation in ZDF hearts compared to leans. Some of these genes have already been shown to play a role in the metabolic changes of diabetic heart (e.g.: upregulation of cytosolic acyl-CoA thioesterase 1 and downregulation of argininosuccinate synthetase, 3-hydroxybutirate dehydrogenase type 1, Hsp70 and Hsp60). However, we have found several genes with altered expression in ZDF rats that have not been related to the diabetic heart (e.g.: downregulation of activating transcription factor 3, neuronatin, nicotinic cholinergic receptor gamma polypeptid, connective tissue growth factor and upregulation of transglutaminase 1, ubiquitin carboxy-terminal hydrolase L1 and aggrecan1). Conclusions: Metabolic syndrome alters the gene expression profile of the myocardium which may be involved in development of cardiac pathologies in the presence of metabolic syndrome. PDK (Pyruvate dehydrogenase kinase) is differentially regulated by beta-blockers in experimental myocardial hypoxia S Gasser S Gasser 1 Medical University of Graz, Department of Cardiology , Graz , Austria PP Rainer PP Rainer 1 Medical University of Graz, Department of Cardiology , Graz , Austria E Holzwart E Holzwart 1 Medical University of Graz, Department of Cardiology , Graz , Austria U Roessl U Roessl 1 Medical University of Graz, Department of Cardiology , Graz , Austria E Kraigher-Krainer E Kraigher-Krainer 1 Medical University of Graz, Department of Cardiology , Graz , Austria D Von Lewinksi D Von Lewinksi 1 Medical University of Graz, Department of Cardiology , Graz , Austria BM Pieske BM Pieske 1 Medical University of Graz, Department of Cardiology , Graz , Austria R Gasser R Gasser 1 Medical University of Graz, Department of Cardiology , Graz , Austria Abstract Pyruvate dehydrogense kinase isoforms inhibit pyruvate dehydrogenase, which constitutes an important step in glucose metabolism. It is involved in various phenomena of aging and its expression changes with age. Glucose metabolism is activated in early myocardial ischemia and in response to an increased need of high-energy-phosphate in the healthy heart during extreme physical activity. In myocardial ischemia, inhibition of PDK expression would be beneficial in order to shift myocardial metabolism from adult towards the fetal phenotype, thus metabolising more glucose than fat in order to preserve myocardial integrity. Myocardial tissue probes derive from right auricles of patients undergoing cardiac surgery. A small part of the right auricle is removed when the heart is put on extra-corporal circulation. This sample is then placed in cooled Tyrode's solution and hypoxia is simulated by switching 100% oxygen to 100% nitrogen (hypoxia) in one of the two chambers. By doing so, we are able to compare ischemic and non-ischemic tissue of the same patient. Snap frozen samples are stored at -70°C until RNA isolation. Quality of isolated RNA is analysed by Bioanalyzer 2100 system. Arrays are scanned with the AB1700 chemiluminescence array reader and data are processed by PANTHER software. In our microarray experiments we find that, in particular, PDK isoform 4 is significantly less expressed under nebivolol both during O2 perfusion and simulated ischemia, an effect practically negligible under atenolol. Here, nebivolol also exhibits a unique cardio-protective property, different from standard β-blockers. We find that, without the influence of beta-blockers, there is no significant regulation of PDK-expression during myocardial ischemia. There is a trend towards a decrease in PDK-Gene expression. There is, however, a significant difference between the expression of PDK during myocardial ischemia in the presence of atenolol (3.62 ± 0.18) and nebivolol (1.97 ± 0.06 SEM; P < 0.05): PDK-expression is decreased during normoxia (trend) and ischemia (significant) in the presence of nebivolol. Here, confirmed by real time PCR, the finding that PDK gene expression is down-regulated by nebivolol compared to atenolol in normoxia (trend, not statistically significant) and simulated ischemia/hypoxia (statistically significant) may argue for a higher protective, anti-ischemic but also anti-anginal metabolic potential of nebivolol compared to standard β-blockers like atenolol. Especially patients with angina may profit from this particular property of nebivolol over atenolol. 13C NMR metabolic studies of connexin 43 deficient mice A Gonzalez-Loyola A Gonzalez-Loyola 1 University Hospital Vall d'Hebron , Barcelona , Spain I Barba I Barba 1 University Hospital Vall d'Hebron , Barcelona , Spain A Rodriguez-Sinovas A Rodriguez-Sinovas 1 University Hospital Vall d'Hebron , Barcelona , Spain C Fernandez-Sanz C Fernandez-Sanz 1 University Hospital Vall d'Hebron , Barcelona , Spain E Agullo E Agullo 1 University Hospital Vall d'Hebron , Barcelona , Spain M Ruiz-Meana M Ruiz-Meana 1 University Hospital Vall d'Hebron , Barcelona , Spain D Garcia-Dorado D Garcia-Dorado 1 University Hospital Vall d'Hebron , Barcelona , Spain Open in new tabDownload slide Abstract 425 Figure 13C NMR spectra Open in new tabDownload slide Abstract 425 Figure 13C NMR spectra Abstract Connexin 43 (Cx43), the main gap junction forming protein, plays an important role in myocardial ischemia-reperfusion injury. Cx43 has been found in the inner membrane of mitochondria which could explain that replacement of Cx43 by Cx32 in Cx43KI32 mice increases resistance to ischemia-reperfusion injury and reduces ATP levels as compared to wild type (WT) animals. The aim of the present work was to investigate metabolic differences between WT and Cx43 deficient mice by using Langendorf hearts perfused with 1-13C-Glucose. After 20 minutes of labeling, hearts were frozen and metabolites were extracted using the methanol: chloroform method. The resulting extracts were analyzed by means of NMR spectroscopy. We observed that the 13C from 1-13C-Glucose was incorporated, among others, into glucose-6-P, glucose-1-P (Fig A, C), glutamate C3 and C4 (Fig B, D) lactate and alanine. No differences were found in the singlet to doublet ratio of the glutamate C4 peak suggesting that the malate-aspartate shuttle activity is similar in WT and Cx43 deficient mice. On the other hand, the ratio between glucose-6P and glucose was higher in wild type than in Cx43 deficient mice suggesting that the hexokinase activity in wild type is elevated as compared to Cx43 deficient mice (Figures A, C). Moreover, the analysis of the glutamate peaks allows us to deduce that the TCA cycle turnover seems to be more active in mice lacking Cx43. Out of the present results we can conclude that there are metabolic differences between both genotypes. The high TCA turnover in Cx43 deficient mice could be explained as a mechanism to compensate for inefficient energy production or elevated expendure. These results further support the importance of gap-junction independent mechanisms of Cx43 1 . Metabolomics allows for a fast and accurate diagnosis of myocardial ischemia. A translational approach MJ Forteza MJ Forteza 1 Hospital Clinico Universitario, INCLIVA, Universidad de Valencia , Valencia , Spain V Bodi Peris V Bodi Peris 1 Hospital Clinico Universitario, INCLIVA, Universidad de Valencia , Valencia , Spain D Monleon D Monleon 1 Hospital Clinico Universitario, INCLIVA, Universidad de Valencia , Valencia , Spain L Mainar L Mainar 1 Hospital Clinico Universitario, INCLIVA, Universidad de Valencia , Valencia , Spain JM Morales JM Morales 1 Hospital Clinico Universitario, INCLIVA, Universidad de Valencia , Valencia , Spain D Moratal D Moratal 2 Polytechnic University of Valencia , Valencia , Spain I Trapero I Trapero 1 Hospital Clinico Universitario, INCLIVA, Universidad de Valencia , Valencia , Spain FJ Chorro FJ Chorro 1 Hospital Clinico Universitario, INCLIVA, Universidad de Valencia , Valencia , Spain Open in new tabDownload slide Abstract 426 Figure Open in new tabDownload slide Abstract 426 Figure Abstract Purpose: Reliable biochemical diagnosis of myocardial ischemia is a major challenge in Cardiology. We evaluated the usefulness of the metabolic profiling of blood plasma by nuclear magnetic resonance (NMR) to detect coronary ischemia in swine and patients. Methods: Ischemia was experimentally induced in 9 swine by means of balloon inflation in the proximal left anterior descending artery. Twenty patients with stable angina scheduled for coronary angioplasty and 10 control patients who underwent diagnostic angiography were also studied. Blood plasma NMR spectra (500 μL) was drawn immediately before and 10 min after intervention (after angiography in control patients). Partial Least Squares (PLS) multivariate analysis library was used. Principal components chosen explained at least 70% of the variance. Metabolite quantification was achieved by in-house peak-fitting routine over most relevant signals. Results: In swine and patients, multivariate analysis showed striking differences before and 10-min post-ischemia in signals belonging mainly to ketonic bodies and fatty acids. Highly significant differences were detected between controls (after diagnostic angiography) and patients (after angioplasty-related ischemia) in these signals. Based on these metabolites we built a simple PLS discriminant model for distinguishing patients from controls. This model was validated with 10 random data replicates and with new data and accurately detected myocardial ischemia (Figure 1 ). Conclusions: The different metabolic profile detected here between plasma of ischemic and non-ischemic subjects may provide the basis for a novel biochemical diagnosis of myocardial ischemia. Myocardial iron load and homeostasis in failing human heart P Leszek P Leszek 1 National Institute of Cardiology , Warsaw , Poland B Sochanowicz B Sochanowicz 2 Institute of Nuclear Chemistry & Technology, Dept. Radiobiology & Health Protection , Warsaw , Poland M Szperl M Szperl 1 National Institute of Cardiology , Warsaw , Poland P Kolsut P Kolsut 1 National Institute of Cardiology , Warsaw , Poland W Piotrowski W Piotrowski 1 National Institute of Cardiology , Warsaw , Poland T Rywik T Rywik 1 National Institute of Cardiology , Warsaw , Poland B Danko B Danko 2 Institute of Nuclear Chemistry & Technology, Dept. Radiobiology & Health Protection , Warsaw , Poland M Kruszewski M Kruszewski 2 Institute of Nuclear Chemistry & Technology, Dept. Radiobiology & Health Protection , Warsaw , Poland Abstract Objectives: To assess myocardial iron (Iron-M), ferritin (FR-M), transferrin receptor (sTfR-M) in heart failure (HF) in relation to serum Iron markers. Background: Correction of anemia and/or iron deficiency with erythropoietin and/or iron in HF seems promising, yet little is known about myocardial iron load and homeostasis. Improperly shielded Iron catalyses reactive free radicals, causing deleterious effects. Methods: Study group 33 patients, left/right ventricle (LV/RV) (LVEDV 245 ± 84 ml; LVESV 189 ± 85 ml; LVEF 22 ± 11%; RVD 32 ± 10 mm), NTproBNP (5464 ± 4825 pg/ml). Iron homeostasis assessment serum: iron, FR, transferrin/saturation (TSAT), sTfR; myocardial: Iron-M (Instrumental Neutron Activation Analysis, μg/g), FR-M, sTfR-M (ELISA – ng/mg protein) in the explanted failing hearts (FH), compared to non-failing hearts (NFH n = 11). Results: In FH as compared to NFH, Iron-M was reduced in RV (174 ± 45 vs 233 ± 97, respectively, p = 0.07), LV (189 ± 58 vs 265 ± 119, p = 0.04), without significant changes in FR-M/sTfR-M. Out of all serum Iron markers only sTfR was negatively correlated with Iron-M in either ventricle (RV r = −0.44, p = 0.03, LV r = −0.38, p = 0.07). With regard to serum iron status, based on TSAT, patients were divided into two subgroups: TSAT<15% (n = 11) and TSAT≥15% (n = 22). Both subgroups had similar grade of LV/RV dysfunction, NT-proBNP levels. FR-M was lower in TSAT<15% than in TSAT≥15% (LV −31 ± 26 vs 46 ± 29; p = 0.07) and (RV – 24 ± 24 vs 43 ± 29; p = 0.02), without differences in Iron-M and sTfR-M. Conclusions: In HF, Iron-M levels were reduced. Serum iron markers did not reflect Iron-M levels, except for serum sTfR. In low serum iron group, decrease in myocardial storage protein FR-M was observed. Dietary supplementation with docosahexaenoic acid, but not eicosapentanoic acid, profoundly remodels cardiac mitochondrial phospholipid fatty acid composition and prevents permeability transition W Stanley W Stanley 1 University of Maryland , Baltimore , United States of America R Khairallah R Khairallah 1 University of Maryland , Baltimore , United States of America N Khanna N Khanna 1 University of Maryland , Baltimore , United States of America K O'shea K O'shea 1 University of Maryland , Baltimore , United States of America T Kristian T Kristian 1 University of Maryland , Baltimore , United States of America P Hecker P Hecker 1 University of Maryland , Baltimore , United States of America R Des Rosiers R Des Rosiers 2 Montreal Heart Institute affiliated with the University of Montreal , Montreal , Canada G Fiskum G Fiskum 1 University of Maryland , Baltimore , United States of America Abstract Treatment with the ω-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) from fish oil exerts cardioprotective effects. We recently showed supplementation with DHA+EPA suppresses Ca2+-induced opening of the mitochondrial permeability transition pore (MPTP) in the rat heart (J Mol Cell Cardiol, 2009). These effects are associated with increased DHA and EPA and lower arachidonic acid (ARA) in cardiac phospholipids (PL). Little is known about the independent effects of DHA and EPA on cardiac mitochondria. While clinical studies suggest the triglyceride lowering effects of DHA and EPA are equivalent, there is growing evidence that DHA may be superior at remodeling mitochondrial phospholipids and preventing MPTP. To compare the effects of dietary supplementation with the ω-3 PUFA DHA and EPA on cardiac mitochondrial PL fatty acid composition and Ca2+-induced MPTP opening. Rats were fed a standard lab chow with either normal low levels of ω-3 PUFA, or DHA or EPA at 2.5% of energy intake for 8 weeks, and cardiac mitochondria were isolated and analyzed for respiration, Ca2+-induced MPTP opening and PL fattyacyl composition. DHA and EPA lowered plasma triglycerides to a similar extent (∼45%), and both increased EPA in mitocondrial phospholipid from undectable levels to ∼5% of total PL fatty acid(p < 0.05). On the other hand, DHA supplementation increased DHA in mitochondrial PL (8.7 % to 14.8%) (p < 0.05)while EPA supplementation had no effect on DHA levels. ARA has been shown to promote MPTP and apoptosis. DHA supplementation decreased ARA in mitochondrial phospholipid from 15 6% to 5.6%, while EPA supplemenation has significantly less effect (9.8%)(p < 0.05). Importantly, supplemetnation with DHA increased the amount of Ca2+ required to trigger MPTP opening (from 82 to 136 nmols/mg mito prot)(p < 0.05), while supplementation with EPA supplementation had no effect (80 nmols/mg mito prot). Protein levels of VDAC 1 & 2 and cyclophilin D were not affected. DHA treatment decreased state 4 respiration by 30% and the increased the respiratory control ratio by 70% with pyruvate+malate as the substrate in both the absence and presence of oligomycin (p < 0.05); treatment with EPA had no effect. In conclusion, high dietary DHA but not EPA, profoundly altered mitochondrial phospholipid fatty acid composition, and delayed Ca2+-induced MPTP opening. These findings suggest that clinical treatment with DHA may exert greater cardioprotective benefit than treatment with ω-3 PUFA formulations containing high amounts of EPA. Cardiac carnitine palmitoyltransferase to malonyl-CoA is regulated by leptin MS Fernandez-Alfonso MS Fernandez-Alfonso 1 Complutense University of Madrid , Madrid , Spain R Guzman-Ruiz R Guzman-Ruiz 2 Universidad CEU-San Pablo , Madrid , Spain B Somoza B Somoza 2 Universidad CEU-San Pablo , Madrid , Spain M Gil-Ortega M Gil-Ortega 2 Universidad CEU-San Pablo , Madrid , Spain C Attane C Attane 3 Université Paul Sabatier , Toulouse , France I Castan-Laurell I Castan-Laurell 3 Université Paul Sabatier , Toulouse , France P Valet P Valet 3 Université Paul Sabatier , Toulouse , France M Ruiz-Gayo M Ruiz-Gayo 2 Universidad CEU-San Pablo , Madrid , Spain Abstract Fatty acid (FA) metabolism in non-adipose tissues is activated by acute leptin increase as well as by endogenous hyperleptinemia evoked by high-fat diets (HF). This supports the notion that hyperleptinemia is pivotal to prevent and/or delay steatosis during periods of high energy intake. Since carnitine palmitoyltransferase I (CPT-I) allows mitochondrial uptake/oxidation of FA, the hypothesis of this study is that leptin drives cardiac CPT-I activity. Hyperleptinemia was induced in C57BL/6J mice either by exogenous leptin administration or by means of HF (45% calories from fat) for 32 weeks. The ability of malonyl-CoA (the main endogenous inhibitor of CPT-I) to inhibit cardiac CPT was analyzed. Half-maximun inhibitory concentrations of malonyl-CoA were 8.1 ± 1.5 μmol/l in controls vs 69.3 ± 5.2 μmol/l (p < 0.01) in leptin-treated mice. This effect was also observed in cardiac explants incubated with leptin. Acute leptin evoked an increase of cardiac pAkt levels which correlated with CPT sensitivity to malonyl-CoA. In accordance, triciribine, an inhibitor of proteinkinase B (Akt) phosphorylation (pAkt), blocked the effect of leptin. Moreover, the inhibitory effect of malonyl-CoA was hindered in hearts from HF hyperleptinemic mice, which exhibited a normal FA content. In these hearts pAkt levels also correlated with CPT sensitivity to malonyl-CoA. Our data show that leptin reduces the sensitivity of cardiac CPT-I to malonyl-CoA and suggest the involvement of an Akt-related signaling pathway in this effect. This mechanism appears to be sensitive to both acute and chronic hyperleptinemia suggesting a pivotal role of leptin to drive cardiac metabolism under situations associated to increased FA intake. Preoperative biochemical predictors of ventricular fibrillation in patients with ichemic heart failure N Maroz-Vadalazhskaya N Maroz-Vadalazhskaya 1 Scientific and Practical Center of Cardiology , Minsk , Belarus T Denissevich T Denissevich 1 Scientific and Practical Center of Cardiology , Minsk , Belarus V Shumavetz V Shumavetz 1 Scientific and Practical Center of Cardiology , Minsk , Belarus Y Ostrovskiy Y Ostrovskiy 1 Scientific and Practical Center of Cardiology , Minsk , Belarus Abstract The study was designed to determine a preoperative predictors of early postoperative ventricular fibrillation (VF) in patients whom the coronary artery bypass grafting (CABG) due to ischemic heart failure was performed. Cohort of 36 patients (age 49,1 ± 6,7 yrs; 2F; LVEF 35,1 ± 3,1%; LVEDDI 112,7 ± 16,4 ml/m 2 ; NYHA 2,6 ± 0,7; stable angina 2,4 ± 0,7) with stable angina, LVEF<40% and class NYHA>2 was undergone low dose dobutamine echo (LDE) and CABG with mitral valve and left ventricular plasty. Blood volume samples were taken during LDE from vena (at base (V1) and at 15 mkg/kg/min (V2)) and during operation from arteria and coronary sinus (0-deep anesthesia before heparin infusion, 1-before clamping of aorta, 2- after declamping of aorta, 3- after shunt reperfusion). In each sample the plasma free fatty acid (FFA), lactate (L) and glucose (Gl) were prospectively analyzed as absolute volume (mmol/L) and coefficient of differences between arteria and coronary sinus (Coef, %). Results: Total 576 segments were estimated at base and peak LDE: 48,14% of segments were akinetic, 28,47%-hypokinetic, 4,17%-dyskinetic. Dobutamine infusion leaded to significant improving of LVEF (p < 0,001) and decreasing of akinetic segments (p < 0,02). There was no episode of VF or A-V block preoperatively (Holter ECG) or during stress-echo (12 leads ECG). Immediately after the shunt reperfusion point 13 patients had VF with unstable hemodynamics and hypotonia with concerned multiple electrical defibrillation. Due to intraoperative VF patients were selected in two groups: gr. 1- 23 pts with no VF and gr. 2-13 pts with intraoperative VF. Groups were similar in age, class NYHA, LVEDDI, basal Gl level, intraoperative cross-clamping and ischemia time, but pts of gr.2 had lower LVEF (p < 0,05) and higher venous basal plasma level of FFA (p < 0,04). At peak LDE the FFA increasing was found in gr.1 (p 0,02), but not in gr.2 (p 0,4). During followed intraoperative points biochemical dynamics was similar in both groups, but absolute volumes were lower in gr.2 (p < 0,05). Stepwise forward analysis showed, that the high venous basal FFA plasma level (p = 0,0032) and low preoperative LVEF (p = 0,012) predicted the development VF during operation. Conclusion: Preoperative LDE with simultaneous assessment of intracardiac hemodynamics and venous plasma level of FFA lets predict the early postoperative VF in patients with low pump function and ischemic heart failure. The additional data may be helpful for more adequate preoperative and intraoperative management. Differential effects of pressure overload on mitochondrial function during the development of heart failure A Schrepper A Schrepper 1 University of Leipzig, Heart Center , Leipzig , Germany M Schwarzer M Schwarzer 1 University of Leipzig, Heart Center , Leipzig , Germany PA Amorim PA Amorim 1 University of Leipzig, Heart Center , Leipzig , Germany M Schoepe M Schoepe 1 University of Leipzig, Heart Center , Leipzig , Germany FW Mohr FW Mohr 1 University of Leipzig, Heart Center , Leipzig , Germany T Doenst T Doenst 1 University of Leipzig, Heart Center , Leipzig , Germany Abstract Background: We previously demonstrated a defect in mitochondrial respiratory capacity in pressure overload-induced heart failure. Since hypertrophy is initially a compensating mechanism, we expected differential changes in respiratory capacity at different time points during the development of heart failure and assessed respiratory capacity in mitochondria from heart and skeletal muscle. Methods: Pressure overload was induced by transverse aortic constriction (TAC). 2, 6, 10 and 20 weeks after TAC, heart and skeletal muscle (Gastrocnemius (Gas) and Soleus (Sol)) mitochondria were isolated and maximal respiratory capacity (state 3 respiration) and ATP/O ratio were assessed. Results: Systolic function was normal after 2, 6 and 10 weeks of TAC. At 20 weeks of pressure overload, contractile dysfunction (FS: 42,4 ± 1,6 vs. 25,1 ± 0,4%, p ≤ 0,05) and LV dilation (LVEDD: 7,8 ± 0,2 vs. 9,0 ± 0,2mm, p ≤ 0,05) were present. Respiratory capacity was significantly increased after 2 and 6 weeks of pressure overload. This increase was present in both heart (natomsO/min/mg: 224 ± 21 vs. 562 ± 35; p ≤ 0,05) and skeletal muscle (natomsO/min/mg: Gas 108 ± 22 vs. 227 ± 22; Sol 19 ± 2 vs. 198 ± 29; p ≤ 0,05). At 10 weeks after TAC, state 3 respiration was pseudo-normalized and at 20 weeks a significant decrease in maximal respiratory capacity of both cardiac (natomsO/min/mg: 623 ± 109 vs. 186 ± 12; p ≤ 0,05) and skeletal muscle (natomsO/min/mg: Gas 222 ± 36 vs. 54 ± 5; Sol 111 ± 10 vs. 69 ± 14; p ≤ 0,05) was observed. Coupling between ATP production and oxygen consumption (ADP/O) was preserved at all time points. Conclusion: Pressure overload differentially effects mitochondrial respiratory capacity. An initial increase in mitochondrial function is followed by a decline, associated with contractile dysfunction. When comparing results from different studies temporal changes should be considered. In addition, the similarity of changes between heart and skeletal muscle suggest a workload independent mechanism. 3-Iodothyronamine cardiac metabolism: new insights resulting from liquid chromatography tandem mass spectrometry analysis G Chiellini G Chiellini 1 University of Pisa , Pisa , Italy S Ghelardoni S Ghelardoni 1 University of Pisa , Pisa , Italy A Saba A Saba 1 University of Pisa , Pisa , Italy M Marchini M Marchini 1 University of Pisa , Pisa , Italy S Frascarelli S Frascarelli 1 University of Pisa , Pisa , Italy A Raffaelli A Raffaelli 1 University of Pisa , Pisa , Italy TS Scanlan TS Scanlan 2 Oregon Health & Science University , Portland , United States of America R Zucchi R Zucchi 1 University of Pisa , Pisa , Italy Abstract 3-Iodothyronamine (T1AM) is a naturally occurring derivative of thyroid hormone that can potentially activate the orphan G protein-coupled receptor (GPCR), known as trace amine-associated receptor 1 (TAAR1). Significant functional effects have been observed after administration of exogenous T1AM: in the isolated heart, a negative inotropic and chronotropic action was produced, and the resistance to ischemic injury was increased, possibly as a consequence of an action on intracellular calcium homeostasis. In the present study we investigated the uptake and catabolism of exogenous T1AM in cardiac preparations using liquid chromatography tandem mass spectrometry (HPLC-ESI-MS-MS) analysis. Isolated working rat hearts were perfused with T1AM (50 nM) and analysis was performed in the recirculating buffer and in cardiac homogenate. Similar experiments were performed in isolated cardiomyoblats (H9c2 cells). In the latter model both incubation medium and cell lysate were collected at different times and submitted to analysis. The analytical method included reverse phase HPLC coupled to tandem mass spectrometry (ESI-MS-MS), and enabled contemporary detection of T1AM, 3-iodothyroacetic acid (TA1), thyronamine (T0AM) and thyroacetic acid (TA0). Experiments were repeated in the presence of 0.1 mM iproniazid, an inhibitor of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO). In both models T1AM concentration in the perfusion buffer decreased exponentially over time (the half life was on the order of 20 min in isolated heart perfused with 200 ml of recirculating buffer). T1AM could be detected in cardiac homogenate and in cell lysate and total cellular or tissue concentration was approximately 20 fold higher than extracellular concentration, showing that significant uptake and/or protein binding occurred. We also detected TA1, a product of T1AM oxidative deamination, which significantly accumulated in cell lysate and cardiac homogenate. Pretreatment of H9c2 cells or isolated hearts with iproniazid abolished T1AM conversion to TA1. Deiodinated derivates (i.e. T0AM and TA0) were not detected in any model. We concluded that T1AM is taken up by cardiomyocytes and can be catabolized to TA1 through iproniazid-sensitive amine oxidases. HPLC-ESI-MS-MS proved to be an effective and quantitative technique to elucidate T1AM metabolism. Porcine bone marrow-derived mesenchymal stem cells as source for medial but not intimal cells in small diameter vascular tissue engineered grafts NMS Van Den Akker NMS Van Den Akker 1 Cardiovascular Research Institute Maastricht (CARIM) , Maastricht , Netherlands DGM Molin DGM Molin 1 Cardiovascular Research Institute Maastricht (CARIM) , Maastricht , Netherlands FF Kolk FF Kolk 1 Cardiovascular Research Institute Maastricht (CARIM) , Maastricht , Netherlands F Jeukens F Jeukens 1 Cardiovascular Research Institute Maastricht (CARIM) , Maastricht , Netherlands RHG Olde Engberink RHG Olde Engberink 1 Cardiovascular Research Institute Maastricht (CARIM) , Maastricht , Netherlands J Waltenberger J Waltenberger 1 Cardiovascular Research Institute Maastricht (CARIM) , Maastricht , Netherlands MJ Post MJ Post 1 Cardiovascular Research Institute Maastricht (CARIM) , Maastricht , Netherlands Abstract Purpose: To facilitate the use of vascular grafts for small diameter vessels in patients, it is a prerequisite to minimize the risk for thrombosis, neointima formation and compliance mismatch. Seeding and preconditioning of a synthetic graft with medial and intimal cells is considered the best option to obtain functional vascular grafts. Preferably, these cells are derived from a patient-autologous source to minimize inflammation and rejection. In the current research, we investigated in a porcine model whether bone marrow-derived mesenchymal cells (MSCs) can be isolated, cultured and instructed to gain sufficient endothelial cell (EC) or vascular smooth muscle cell (SMC) phenotype in a reproducible manner. Methods: MSCs were isolated from bone marrow of female domestic Yorkshire pigs of 40-50 kg (n = 6). Mononuclear cells were separated using gradient centrifugation and cultured on fibronectin-coated flasks in MSC, EC or SMC-defined culture media. Between passage 3 and 7, cells were analysed for EC and SMC phenotype and function. For the analysis of EC characteristics, mRNA (RT-qPCR) and protein (FACS, IHC) expression patterns of CD31, CD34 and VEGFR2 were determined, while NO and PGI2-production and thrombogenicity were assessed as functional parameters. To determine SMC differentiation, mRNA and protein levels were analyzed for alpha-SMA, calponin, desmin and collagen. Results: Porcine MSCs can be reproducibly isolated and cultured. Up to passage 15, no large alterations in growth dynamics (doubling time, morphology) were observed. Porcine MSCs cultured in EC-defined medium showed clear cobblestone morphology and grew in a monolayer comparable to endothelial cells. In contrast, these cells did not show any characteristics of EC function or phenotype. Porcine MSCs cultured in MSC or SMC-defined culture media presented with elongated morphology with multiple protrusions and grew in multiple layers. When looking at the expression of SMC markers, cells grown in MSC-defined medium showed 2 to 70 times higher expression levels compared to cells cultured in SMC defined medium. Conclusions: We conclude that bone marrow-derived MSCs can be reproducibly isolated and cultured from porcine bone marrow. These cells can readily be expanded and tested for application in the medial part of a small diameter vascular graft. Care should, however, be taken when using MSCs as a source for intimal cells, especially due to lack of vasodilator-production and pro-thrombogenic properties. (NA and DM contributed equally to this research) Isolation and in vitro performance of blood-derived endothelial outgrowth cells for vascular tissue engineering NMS Van Den Akker NMS Van Den Akker 1 Cardiovascular Research Institute Maastricht (CARIM) , Maastricht , Netherlands DGM Molin DGM Molin 1 Cardiovascular Research Institute Maastricht (CARIM) , Maastricht , Netherlands S Verbruggen S Verbruggen 1 Cardiovascular Research Institute Maastricht (CARIM) , Maastricht , Netherlands HM Schulten HM Schulten 1 Cardiovascular Research Institute Maastricht (CARIM) , Maastricht , Netherlands MJ Post MJ Post 1 Cardiovascular Research Institute Maastricht (CARIM) , Maastricht , Netherlands J Waltenberger J Waltenberger 1 Cardiovascular Research Institute Maastricht (CARIM) , Maastricht , Netherlands Abstract Purpose: Bare synthetic vascular grafts for replacement of small diameter vessels in patients are complicated by high thrombogenic and immunogenic risks. To minimize these risks endothelial coverage is considered an essential design parameter for tissue engineered vascular grafts. Experimentally, different subpopulations of cells derived from the mononuclear fraction of peripheral blood have been proposed as a cellular source for this Purpose. In the past decade both early and late outgrowth endothelial progenitor cells from peripheral blood have been identified, the latter also referred to as endothelial outgrowth cells (EOCs). Methods: Mononuclear cells were isolated from blood of female domestic Yorkshire pigs of 40-50 kg using gradient centrifugation, seeded on fibronectin-coated flasks and cultured in endothelial cell-defined culture medium. Analysis of the endothelial phenotype was performed on early passage porcine cells (below P4) through mRNA (RT-qPCR) and protein (FACS, IHC) expression analysis for CD31, CD34 and VEGFR2. Endothelial function was determined by means of Ac-LDL uptake-assay and determination of NO and PGI2-production. Results: Very few cells had attached after 4 days and only after 5 additional days small numbers of colonies were seen. After subsequent passaging, however, they grew to full confluence in a monolayer. A strong endothelial phenotype was confirmed on mRNA and protein level, suggesting these cells to be EOCs. Additionally, all 3 functional assays confirmed endothelial properties of these EOCs. Conclusions: In conclusion, EOCs isolated from porcine blood show profound endothelial characteristics already one week after isolation with characteristics of differentiated rather than of progenitor cells. This suggests these cells to be outgrowing circulating endothelial cells. Although these cells hold highly qualified endothelial potentials, further research will be required to identify the origin of these cells, to optimize their expansion capacity in culture and to successfully translate the techniques to the human setting in order to test their possibilities in clinical vascular tissue engineering. Regulation of cardiac progenitor cells during heart development F Rochais F Rochais University of the Mediterranean , Marseille , France RG Kelly RG Kelly University of the Mediterranean , Marseille , France Abstract Cardiac progenitor cells of the second heart field (SHF) contribute to the poles of the elongating embryonic heart. Situated in pharyngeal mesoderm, cells of the SHF express genes encoding the fibroblast growth factor Fgf10 and transcription factor Isl1. Failure or perturbation of SHF development leads to congenital heart defects. Recent studies have demonstrated the existence, in the fetal and early postnatal heart, of resident cardiac progenitor cells that specifically express Isl1. Such cells have the potential to differentiate into cardiomyocytes, smooth muscle and endothelial cells and are thought to represent residual progenitor cells derived from the SHF. Through analysis of a transgene integration site position effect we have identified the transcriptional repressor Hes1 as a novel regulator of SHF development. Hes1, a target gene of the Notch signaling pathway, regulates diverse progenitor cell populations in the developing embryo and is expressed in the SHF during heart tube elongation. Analysis of Hes1−/− hearts reveals outflow tract alignment defects including overriding aorta and ventricular septal defects. Hes1−/− embryos display SHF proliferation defects associated with elevated levels of the cyclin-dependant kinase inhibitor p27kip1, a reduction in cardiac neural crest cells and fail to completely extend the outflow tract. These data reveal a role for Hes1, and potentially Notch signaling, in SHF development. Given the importance of Isl1 as a marker of resident progenitor cells in the later heart we investigated whether other regulators of the SHF (Fgf10 and Hes1) contribute to myocardial progenitor cell fate in the fetal heart. While heart tube extension occurs normally in Fgf10−/− embryos, the ventricles of mutant hearts are highly dysmorphic suggesting a role for this gene in growth of the fetal heart. Our initial observations have found that proliferation of fetal cardiomyocytes is impaired in Fgf10−/− hearts while Isl1-positive cell numbers are increased. In contrast, Hes1 deletion impacts negatively on Isl1-positive cell numbers. Together, our results identify Hes1 as a novel regulator of SHF progenitor cell deployment and reveal a potential role of Fgf10 and Hes1 in regulating cardiac progenitor cell fate and cardiac growth during the fetal period. This study will increase our understanding of the molecular mechanisms governing the maintenance and differentiation of cardiac progenitor cells. The anti-apoptotic signalling by TF/FVIIa governs apoptosis by regulating the tumor suppressor death associated protein kinase 1 (DAPK1) M Aberg M Aberg 1 Uppsala University Hospital, Department of Medical Sciences, Clinical Chemistry , Uppsala , Sweden M Johnell M Johnell 1 Uppsala University Hospital, Department of Medical Sciences, Clinical Chemistry , Uppsala , Sweden M Wickstrom M Wickstrom 1 Uppsala University Hospital, Department of Medical Sciences, Clinical Chemistry , Uppsala , Sweden A Siegbahn A Siegbahn 1 Uppsala University Hospital, Department of Medical Sciences, Clinical Chemistry , Uppsala , Sweden Abstract Purpose: Tissue factor (TF) initiates coagulation but is also a signalling receptor involved in the migration and survival of cells. TF is expressed within the atherosclerotic plaque on inflammatory cells (monocytes/macrophages) and on plaque stabilizing cells (smooth muscle cells, fibroblasts), but also on malignant cells which often are used as model systems when examining the biological effects of TF signalling. Death receptor-induced apoptosis within the plaque may contribute to its destabilization. This study determines the impact of anti-apoptotic TF signalling on the extrinsic pathway of apoptosis. Methods: Apoptosis was induced in MDA-MB-231 breast and PC3 prostate cancer cells by serum starvation or treatment TNF-related apoptosis inducing ligand (TRAIL) for 24 or 6 h respectively. The cells were also treated with 10 or 100 nM FVIIa, 5 nM FVIIa/130 nM FX, 100 nM active site-inhibited FVIIa, the PI3-kinase inhibitor LY294002, or TF-blocking antibodies. mRNA was analyzed by real-time PCR and protein expressions by flow cytometry or western blot. Caspase-8 and -3 levels, cell size, and altered nuclear morphology were recorded using an automated fluorescence imaging microscope. Finally, 84 apoptosis-related genes were screened by using a real-time PCR array. Results: In serum starved MDA-MB-231 cells apoptosis was recorded at 24 h. Addition of FVIIa reduced the caspase-8 activation, the cell shrinkage and the chromatin condensation/nuclear fragmentation. Similarly, TRAIL-induced apoptosis was regulated by TF/FVIIa (10 and 100 nM) and TF/FVIIa/FXa but not by active site-inhibited FVIIa. The real-time PCR array and western blot showed 10-100 nM FVIIa-treatment to significantly decrease the transcription of DAPK1 and its association with caspase-8 activating proteins. The regulation of both the apoptosis and DAPK1 expression by FVIIa was dependent on the PI3-kinase/AKT signalling pathway and inhibited by TF antibodies. Conclusions: We hereby report for the first time that TF/FVIIa-induced signalling governs the extrinsic pathway of apoptosis by reducing the levels of DAPK1 in a PI3-kinase dependent manner. The proteolytic activity of FVIIa was also mandatory. Death receptor induced apoptosis may be of importance for the atherosclerotic plaque rupture. TF/FVIIa induced down-regulation of DAPK1, or other specific inhibitors, might play a role in future treatment of coronary artery disease in the context of stabilizing the plaque. More research in other biological settings is though needed. Doxorubicin cancer therapy induces autophagy in adult cardiomyocytes P Dimitrakis P Dimitrakis 1 Bern University Hospital , Bern , Switzerland V Groppalli V Groppalli 2 Department of Evolutionary and Functional Biology , Parma , Italy D Ott D Ott 1 Bern University Hospital , Bern , Switzerland F Seifriz F Seifriz 1 Bern University Hospital , Bern , Switzerland T Suter T Suter 1 Bern University Hospital , Bern , Switzerland C Zuppinger C Zuppinger 1 Bern University Hospital , Bern , Switzerland Abstract Objective: Doxorubicin (Doxo) is a robust chemotherapy drug used in the treatment of various cancers. Its usefulness in the clinic has been hindered by its side effects, most notably that of dilated cardiomyopathy and congestive heartfailure due to myocardial cell death. The role of autophagy in doxorubicin-inducedcardiotoxicity is not fully understood. Our hypothesis is that Doxo induces autophagy, as a maladaptive response, leading to cell death. Methods: Chronic effects of Doxo were studied in isolated adult rat cardiomyocytes cultured for a total of 12 days in medium containing 20% FCS andexposed to the cancer therapy for 48 hours. Measurement of mitochondrialactivity and membrane potential (MTT), release of lactate-dehydrogenase (LDH)and DNA degradation were used for testing apoptosis and necrosis. Autophagicactivity was monitored by Western blotting for the autophagosome marker LC-3I/II and accumulation of poly-ubiquitinylated proteins and Cathepsin-D by immunofluorescence microscopy. Results: LC-3 I/II protein was found to be increased in a dose-dependent mannerby Doxo. Accumulation of polyubiquitin-positive aggregates, Cathepsin-D-positive vesicles andmyofibrillar disarray were observed at 1 µM Doxo. Typical markers for apoptosis (TUNEL) and necrosis (LDH-release, MTT) showed a significant increase of cell death only atsupraclinical Doxo concentrations of >20 µM for 48 hours. Conclusions: We conclude that low doses of Doxo cause autophagy and the accumulation of oxidatively damaged macromolecules in the absence of DNA-degradation inadult ventricular cardiomyocytes. Higher doses of Doxo further increaseautophagy stimulation leading to cell death. Manipulating autophagic activity inthe myocardium challenged by cytotoxic therapies may emerge as meaningful intervention in order to enhance cardiomyocyte survival. Effect of nitrates on clotting at the rats with L-NAME introduction Y Kashcheyeu Y Kashcheyeu 1 Grodno State Medical University , Grodno , Belarus Abstract 440 Table Parameters of the rats blood coagulation Group . R, min . k, min . T, min . Ma (sm) . Control (n = 10) 3,0 ± 0,85 3,5 ± 0,65 18,0 ± 4,53 4,98 ± 0,62 L-NAME (n = 8) 2,2 ± 0,27** 2,5 ± 0,3** 14,0 ± 0,42* 5,60 ± 0,26* L-NAME+ NaNO3 0,05 mg/kg (n = 8) 2,5 ± 0,25## 2,9 ± 0,11## 16,1 ± 1,4# 5,30 ± 0,09# L-NAME+ NaNO3 0, 5 mg/kg (n = 8) 3,1 ± 0,51## 3,1 ± 0,3## 19,8 ± 2,1## 5,08 ± 0,31# Group . R, min . k, min . T, min . Ma (sm) . Control (n = 10) 3,0 ± 0,85 3,5 ± 0,65 18,0 ± 4,53 4,98 ± 0,62 L-NAME (n = 8) 2,2 ± 0,27** 2,5 ± 0,3** 14,0 ± 0,42* 5,60 ± 0,26* L-NAME+ NaNO3 0,05 mg/kg (n = 8) 2,5 ± 0,25## 2,9 ± 0,11## 16,1 ± 1,4# 5,30 ± 0,09# L-NAME+ NaNO3 0, 5 mg/kg (n = 8) 3,1 ± 0,51## 3,1 ± 0,3## 19,8 ± 2,1## 5,08 ± 0,31# Open in new tab Abstract 440 Table Parameters of the rats blood coagulation Group . R, min . k, min . T, min . Ma (sm) . Control (n = 10) 3,0 ± 0,85 3,5 ± 0,65 18,0 ± 4,53 4,98 ± 0,62 L-NAME (n = 8) 2,2 ± 0,27** 2,5 ± 0,3** 14,0 ± 0,42* 5,60 ± 0,26* L-NAME+ NaNO3 0,05 mg/kg (n = 8) 2,5 ± 0,25## 2,9 ± 0,11## 16,1 ± 1,4# 5,30 ± 0,09# L-NAME+ NaNO3 0, 5 mg/kg (n = 8) 3,1 ± 0,51## 3,1 ± 0,3## 19,8 ± 2,1## 5,08 ± 0,31# Group . R, min . k, min . T, min . Ma (sm) . Control (n = 10) 3,0 ± 0,85 3,5 ± 0,65 18,0 ± 4,53 4,98 ± 0,62 L-NAME (n = 8) 2,2 ± 0,27** 2,5 ± 0,3** 14,0 ± 0,42* 5,60 ± 0,26* L-NAME+ NaNO3 0,05 mg/kg (n = 8) 2,5 ± 0,25## 2,9 ± 0,11## 16,1 ± 1,4# 5,30 ± 0,09# L-NAME+ NaNO3 0, 5 mg/kg (n = 8) 3,1 ± 0,51## 3,1 ± 0,3## 19,8 ± 2,1## 5,08 ± 0,31# Open in new tab Abstract Introduction: Inhibition of NO-sinthase has reducing the anticoagulant properties of vascular wall, increasing the coagulation potential and the risk of thrombosis. The aim was to study the parameters of coagulation in terms of inhibition of NO-synthase and the introduction of nitrates. Materials and methods: The study was conducted on 32 female rats (200-220 gr) -4 groups: 1 control and 3 experimental. The rats of experimental groups have receiving L-NAME (5 mg/kg) - nonselective inhibitor of NO-synthase. The rat of the 2-nd experimental group (n = 7) additionly were receive sodium nitrate (NaNO3) at a dose of 0,05 mg/kg, rats of the 3-d experimental group (n = 7) - at dose of 0,5 mg/kg. At the day studies at the rats under general anesthesia in the blood samples receiving by catheterization of the common carotid artery the parameters (R, k, T, Ma) of coagulation was studied at coagulograph (euthanasia – thiopental, 100 mg/kg). Data were processed statistically. Results: In rats, 1-st experimental group observed a shortening of the R, k, T and increasing Ma, compared with rates in the control (p < 0,001), indicating that activation of blood coagulation by the introduction of L-NAME. In the 2-nd experimental group a tendency to decrease in activity indices of coagulation was observed, in the 3-rd experimental group - correction the coagulation parameters, compared with control (p > 0,05). Conclusion: Introduction of NO-synthase nonselective inhibitor L-NAME activates the coagulation hemostasis at rats, and the introduction of sodium nitrate was normalize the indicators of secondary hemostasis 1 . LC/MS-based metabolomic analysis of isoprenaline- and carbachol-mediated effects in stem cell-derived cardiomyocytes R Mueller R Mueller 1 Department of Pharmacology , Cologne , Germany MHJ Wiesen MHJ Wiesen 1 Department of Pharmacology , Cologne , Germany T Saric T Saric 2 Department of Neurophysiology , Cologne , Germany D Gruendemann D Gruendemann 1 Department of Pharmacology , Cologne , Germany J Hescheler J Hescheler 2 Department of Neurophysiology , Cologne , Germany S Herzig S Herzig 1 Department of Pharmacology , Cologne , Germany Abstract Purpose: In this study we introduce a qualitative and semi-quantitative assay aimed to identify substances involved in regulating cardiac signaling pathways in stem cell-derived murine cardiomyocytes. As a starting point for validation of our approach, we used isoprenaline (ISO) and carbachol (CCh) application, followed by subsequent LC/MS analysis of cell lysates. Methods: Cardiomyocytes were derived from murine embryonic stem cells. To analyze ISO (1 μM) and CCh (30 μM) mediated effects, cardiomyocytes were stimulated at day 16 of differentiation. Subsequently, LC-MS/MS analysis of cell lysates was performed. Full scan mass spectra of stimulated and unstimulated (control) cell lysates were analyzed using LC-MS difference shading which is based on color-coded comparative image analysis. After initial studies of MS/MS fragmentation, multiple reaction monitoring (MRM) was applied for quantification of cAMP. Results: Sample normalization by means of quantification of total protein confirmed a linear relationship between basal cAMP levels and total protein content (r2 = 0.992) and basal cAMP levels and number of cardiomyocytes (r2 = 0.997). As expected, cAMP concentrations in ISO-stimulated cell lysates were detected at higher levels when compared to control cell lysates (ratio: 5.44 ± 1.51, n = 8). In the presence of ISO, CCh reduced elevation of cAMP levels compared to control (ratio: 3.72, n = 2). cAMP levels in CCh-stimulated cell lysates were found slightly decreased (ratio: 0.84 ± 0.16, n = 5). Several m/z signal differences were found in full scan spectra of stimulated and control cell lysates by LC-MS difference shading analysis. These findings are currently validated by means of database searches and direct measurements of matched metabolites playing a role in cardiac signaling. Conclusion: Signal transduction via small molecules can be monitored in murine stem-cell derived cardiomyocytes by LC-MS/MS. Using adrenergic and cholinergic agonists we validated our experimental approach based on cAMP levels in cell lysates. Using LC-MS difference shading, several additional m/z signal differences were detected matching small molecules. Hence, our assay appears to be suited to identify yet unknown substances involved in cardiac signalling pathways ("metabolomics"). Modulation of myocardial stiffness by beta-adrenergic stimulation on rabbit papillary muscles-its role in normal and failing heart I Falcao-Pires I Falcao-Pires 1 University of Porto, Faculty of Medicine , Porto , Portugal AP Fontes-Sousa AP Fontes-Sousa 1 University of Porto, Faculty of Medicine , Porto , Portugal L Lopes-Conceicao L Lopes-Conceicao 1 University of Porto, Faculty of Medicine , Porto , Portugal C Bras-Silva C Bras-Silva 1 University of Porto, Faculty of Medicine , Porto , Portugal AF Leite-Moreira AF Leite-Moreira 1 University of Porto, Faculty of Medicine , Porto , Portugal Abstract Purpose: We investigated the acute effects of β-adrenergic stimulation on the myocardial stiffness in normal and heart failing hearts. Methods: New-Zealand white rabbits were treated with doxorubicin (1 mg/kg, intravenously twice weekly for 8 weeks) to induce HF (DOX-HF) or saline (control). Effects of isoprenaline (10-10-10-5 M), a non-selective β-adrenergic agonist, were tested in papillary muscles from control and DOX-HF group. In the former, the effects of isoprenaline were also evaluated in the presence of a damaged endocardial endothelium, atenolol (β1-adrenoceptor antagonist), ICI-118551 (β2-adrenoceptor antagonist), KT-5720 (PKA inhibitor), L-NNA (NO-synthase inhibitor), or indomethacin (cyclooxigenase inhibitor); also, passive length-tension relations were constructed before and after adding isoprenaline (10-5 M). Results: In the control group, isoprenaline increased resting muscle length up to 1.017 ± 0.006L/Lmax. Correcting it to its initial value resulted in a 28.5 ± 3.1% decrease of resting tension (RT), indicating decreased muscle stiffness, as confirmed by the isoprenaline-induced right-downward shift of the passive length-tension relation. These effects were modulated by β1- and β2-adrenoceptor, PKA and prostaglandins. In DOX-HF group, the effect on myocardial stiffness was attenuated (1.004 ± 0.002L/Lmax versus 16.2 ± 4.6% decrease of RT). Conclusion: This study highlights the importance of β-adrenergic stimulation as a relevant mechanism of acute neurohumoral modulation of diastolic function and reveals its underlying mechanisms. Intracellular pH-dependent modulation of cell-cell coupling and voltage-gating in connexin45 homotypic and connexin45/connexin43 heterotypic gap junctions F Bukauskas F Bukauskas 1 Albert Einstein College of Medicine , New York , United States of America N Palacios-Prado N Palacios-Prado 1 Albert Einstein College of Medicine , New York , United States of America Abstract Intracellular acidification has a broad effect on the heart function including inhibition of intercellular communication through gap junction (GJ) channels formed of connexins (Cxs) m30.2, 40, 43 and 45. These changes may be critical under ischemic conditions, since the uncoupling can reduce acid-dissipation through the surrounding myocardial network. In this study, we used HeLa cells expressing wild type Cx45 as well as Cx45 and Cx43 tagged with EGFP. We examined junctional conductance (gj) and voltage gating by measuring gj dependence on transjunctional voltage (Vj). To analyze gj-Vj plots, we used a stochastic four-state model that describes the GJ channel composed of two hemichannels each containing a gating mechanism. This allowed us to find Vo,H, the voltage across the hemichannel at which half of the hemichannels are open, and AH, a constant reflecting the steepness of Vj-gating. In homotypic Cx45 GJs, alkalization from pHi = 7.2 to 8.0 increased gj ∼1.8-fold and reduced sensitivity to Vj mainly due to an increase in Vo,H. There were no changes in the number of channels that are operational/functional (NF,O). Acidification reduced gj and increased sensitivity to Vj mainly due to a reduction of Vo,H and NF,O. Summarized gj-pHi dependencies for Cx45 and Cx45-EGFP were sigmoidal with pKas of ∼7. Correlation between a number of channels assembled in Cx45-EGFP junctional plaques and maximal number GJs that were open at the alkalization showed that only a small fraction (K≈0.04) of channels are operative. Therefore, in Cx45, Cx43 (K = 0.1) and Cx57 (K = 0.01), for which K values were reported, gj can be largely regulated by factors (phosphorylation, pHi, etc) that have potential to change this ratio without de-novo formation or degradation of GJs. Heterotypic Cx45/Cx43-EGFP GJs exhibited asymmetric Vj-gating, which was reduced during alkalization and increased during acidification. pKa was ∼6.7, which can be predicted from pKas of Cx45 and Cx43-EGFP (pKa≈6.5) homotypic GJs assuming that series hemichannels respond to pHi independently. In summary, dynamic pH-dependent modulation of cell-cell coupling of Cx45 homotypic and Cx45/Cx43 heterotypic GJ channels at moderate changes in pHi can be largely explained by changes in the voltage-gating of the Cx45 hemichannel. However, stronger acidification (<6.5) closes the slow gate that leads to the reduction of NF,O. These changes have potential to disturb cell-to-cell metabolic communication and electrical signaling in the conduction system of the heart, blood vessels and neurons where Cx45 is co-expressed with Cx43 and other Cx isoforms. The human skeletal muscle secretome - identification of the most abundantly secreted proteins in vitro and their regulation in response to training in vivo F Norheim F Norheim 1 Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo , Oslo , Norway T Raastad T Raastad 2 The Norwegian School of Sport Sciences , Oslo , Norway B Thiede B Thiede 1 Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo , Oslo , Norway CA Drevon CA Drevon 1 Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo , Oslo , Norway F Haugen F Haugen 1 Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo , Oslo , Norway Abstract Background: It is overwhelming evidence that regular physical activity protects against several types of diseases including certain types of cancer, type 2 diabetes and dementia. These beneficial effects of physical activity may be due to improved energy balance, but they might also be explained by peptide signals released from the skeletal muscles. Aim: In this project we focused on identification of proteins secreted at a high level from cultured human skeletal muscle myotubes and investigate their regulation at the mRNA level in response to training. Methods/results: Proteins in serum-free culture medium released from human skeletal myotubes from 3 different donors, were separated using SDS-PAGE, digested with trypsin, and analyzed using Nano-LC-LTQ Orbitrap-MS/MS. A total of 282 proteins were detected, and by using different filtering Methods, we classified 120 of these as potential muscle secretory proteins. A published computational reconstruction of the human skeletal muscle secretome derived from the transcriptome, was used to exclude proteins with transmembrane regions or known intracellular localization. This refinement led to the identification of 22 muscle secretory proteins. To confirm that the secretory proteins originated from the muscle cells, we quantified mRNA in cell lysates by RT-PCR. Notably, 21 muscle secretory proteins were expressed at the mRNA level in the cell cultures, and of these SPARC, cathepsin D and PAI-1 had the highest expression level. To test if enhanced gene expression of muscle secretory proteins were associated with fully differentiated myotubes we measured mRNA of 12 secretory proteins at different stages of differentiation. The mRNA level of 9 muscle secretory proteins were increased during differentiation. Human muscle biopsies (m. vastus lateralis) were analyzed by RT-PCR and showed that 19 muscle secretory proteins were expressed at the mRNA level. To gain insight into how training influences their expression, 10 healthy human subjects were strength-trained for 11 weeks. Biopsies were taken before start of the intervention, after two weeks of training, and at the end of the intervention, and analyzed by RT-PCR. Compared to baseline, 15 muscle secretory proteins, including SPARC, showed significantly increased expression after 2 and/or 11 weeks with strength-training. Conclusions: We have identified several novel secretory proteins highly secreted from human myotubes. Some of these proteins are secreted from skeletal muscle in response to training. Regulation of the extracellular matrix proteins in human cardiac fibroblasts derived from endomyocardial biopsies of patients with dilated cardiomyopathy D Lindner D Lindner 1 Charite - University Medicine, Campus Benjamin Franklin, Department of Cardiology and Pulmonology , Berlin , Germany D Westermann D Westermann 1 Charite - University Medicine, Campus Benjamin Franklin, Department of Cardiology and Pulmonology , Berlin , Germany C Zietsch C Zietsch 1 Charite - University Medicine, Campus Benjamin Franklin, Department of Cardiology and Pulmonology , Berlin , Germany H-P Schultheiss H-P Schultheiss 1 Charite - University Medicine, Campus Benjamin Franklin, Department of Cardiology and Pulmonology , Berlin , Germany C Tschoepe C Tschoepe 1 Charite - University Medicine, Campus Benjamin Franklin, Department of Cardiology and Pulmonology , Berlin , Germany Abstract Background: The extracellular matrix is critical for maintaining the structural integrity of the heart. An increase in interstitial collagen concentration is associated with a stiffer myocardium whereas disruption of the myocardial ECM is associated with ventricular dilatation. The primary role of cardiac fibroblasts is the synthesis of collagen, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Degradation of extracellular matrix (ECM) is mediated by MMPs and antagonized by TIMPs. A novel role is the activation or degradation of chemokines after the proteolytic cleavage by MMPs. We aim to access the expression level of cardiac fibroblasts in the presence and in the absence of TGF-β1, which is expressed by inflammatory cells and mediate the differentiation to myofibroblasts, which leads to an increased collagen accumulation and finally to fibrosis. Methods and Results: Primary cardiac fibroblasts were obtained by outgrowth from biopsies of the heart from patients with dilated cardiomyopathy (DCM). The fibroblasts were incubated in the presence and the absence of 5 ng/ml TGF-β1 for 6 and 24 hours. Total RNA was extracted from the cells and transcribed to cDNA, before analyzing the gene expression by quantitative PCR. We investigated the expression of collagen and a-smooth-muscle actin, which should be increased in activated fibroblasts as well as the expression of different MMPs and TIMPs. MMP2 is the highest expressed MMP in cardiac fibroblasts and the expression was further increased after TGF-β1 treatment, which was also detected for the MMP14. The expression of MMP3, 7, 8 and 9 is hardly detectable in untreated fibroblasts. However, TGF-β1 treatment resulted in a 3-fold increased MMP3, whereas the expression of MMP7, 8, and 9 was not affected. Furthermore, an increased expression of the endogenous inhibitors of MMPs TIMP1-4 could be demonstrated after TGF-β1 treatment. Conclusion: We can clearly demonstrate that TGF-β1 activates cardiac fibroblast, which leads to a differentiation to myofibroblasts resulting in a higher expression of collagen and a-smooth-muscle actin in patients with DCM. Furthermore, all MMP inhibitors TIMP1-4 show higher expression levels after fibroblast activation leading to an inhibition of collagen degradation and further to fibrosis. However, especially the expression of MMP2, which is known to activate latent TGF-β1, and MMP3 is increased after TGF- β1 treatment. These results indicate the key role of cardiac fibroblasts in the rearrangement (remodelling) of the myocardium which occurs in heart disease. Extracellular matrix remodelling in an ovine model of ageing and heart failure MA Horn MA Horn 1 University of Manchester , Manchester , United Kingdom HK Graham HK Graham 1 University of Manchester , Manchester , United Kingdom MA Hall MA Hall 2 Liverpool Heart and Chest Hospital , Liverpool , United Kingdom MA Richards MA Richards 1 University of Manchester , Manchester , United Kingdom JD Clarke JD Clarke 1 University of Manchester , Manchester , United Kingdom KM Dibb KM Dibb 1 University of Manchester , Manchester , United Kingdom AW Trafford AW Trafford 1 University of Manchester , Manchester , United Kingdom Abstract We sought to establish a model of heart failure (HF) encompassing young and aged animals to determine if the amount and regulation of the cardiac extracellular matrix (ECM) occur in ageing, and whether these changes are similar to those in HF. HF was induced in sheep aged 18 months (young) and those over 8 years (old) by 4 weeks rapid right ventricular pacing (RVP) at 3.5Hz. Echocardiography was performed to determine left ventricular (LV) diameter (EDID) and contractile function. Paraffin-embedded LV samples were stained with picro-sirius red and collagen visualised by polarised light microscopy. MMP activity and TIMP expression were assessed from LV extracts using gelatin zymography and western blotting, respectively. Statistical significance was calculated using the mean ± SEM and a t-test or by 2-way ANOVA where appropriate. Ageing resulted in increased EDID (24%, P < 0.01) and decreased fractional shortening (14%, P < 0.05) compared to young controls. LV collagen content increased in ageing compared to young controls (2.3 ± 0.4% vs. 1.0 ± 0.14%, n = 5, P < 0.01) coinciding with increased MMP-2 activity (1.5 ± 0.08 vs. 1.0 ± 0.22, n = 7-8, P < 0.05). RVP resulted in increased EDID in young (3.8 ± 0.07cm vs. 2.5 ± 0.12cm, n = 15, P < 0.001) and aged animals (4.2 ± 0.13cm vs. 3.3 ± 0.19cm, n = 10, P < 0.001). Decreased fractional shortening occurred in young (0.3 ± 0.01 vs. 0.7 ± 0.02, n = 15, P < 0.001) and old (0.2 ± 0.02 vs. 0.6 ± 0.02, n = 10, P < 0.001) paced animals compared to pre-pacing. LV collagen content increased with RVP in young (2.6 ± 0.28% vs. 1.0 ± 0.14%, n = 5-8, P < 0.001) and decreased in aged paced animals vs. pre-pacing (1.2 ± 0.25% vs. 2.3 ± 0.4%, n = 5-6, P < 0.05). MMP-2 activity increased with RVP (young, 1.7 ± 0.14 and old, 1.7 ± 0.17, n = 6-7) compared to pre-pacing (young, 1.0 ± 0.22 and old, 1.5 ± 0.08, n = 7-8), P < 0.05. TIMP-3 and TIMP-4 expression was reduced by 27% (P < 0.05) and 39% (P < 0.05), respectively after RVP in aged animals only compared to pre-pacing. The commonality of these changes seen in ageing and HF indicate that cardiac ECM remodelling is important in the predisposition of ageing to the development of HF. All procedures accord to The UK Animals (Scientific Procedures) Act, 1986. ATF3 induce Beclin1-mediated autophagy against trans-aortic banding (TAB) induced cardiomyopathy C-F Cheng C-F Cheng 1 Tzu Chi General Hospital , Hualien , Taiwan H Lin H Lin 2 Institute of Toxicology and Pharmacology, Tzu Chi University , Hualien , Taiwan Abstract Activating transcription factor 3 (ATF3) is a member of the CREB/ATF family of transcription factors. Prior transgenic mice study demonstrated that the expression of ATF3 gene had cardiac conduction abnormalities and contractile dysfunction. However, little information has been reported from knockout experiments. The aim of this study was to explore the molecular mechanisms of ATF3 against trans-aortic banding (TAB) induced cardiac dilatation using ATF3−/− mice study. ATF3−/− (KO) and wild-type (WT) mice were subjected to TAB animal model for four weeks. TAB treatment resulted in cardiac dilatation assessed by echocardiography. In addition, several markers including apoptosis level (TUNEL staining), activated caspase-3 and calpain expression, were higher in KO mice in compared with WT littermates. We then restored ATF3 expression to the KO heart using AAV8-ATF3 techniques, in which recovery of prior abnormal cardiac function were found in KO mice post TAB treatment. Molecular and biochemical analysis revealed that ATF3 can increase autophagy expression via Beclin dependent pathway. Interaction of ATF3 with ATF/CRE on the beclin 1 promoter was demonstrated in nuclear extracts from Adv-ATF3-treated neonatal cardiomyocytes and further confirmed by chromatin immune-precipitation. We then pretreated KO mice with AAV8-ATF3 and subjected them to intra-peritoneal injection of beclin inhibitor, 3MA following TAB treatment. ATF3−/− mice received AAV8-ATF3 injection and 3MA developed heart failure with elevated levels of activated caspase-3, apoptotic bodies, and decreased survival in compared to KO mice post TAB but without 3MA treatment, implying ATF3 cannot exert its protective effect without beclin existence. Together, our studies reveal a novel epigenetic regulation mediated by the stress-inducible gene ATF3 under TAB-induced cardiac dilatation and suggest that ATF3 can exert beclin mediated autophagy protection against TAB induced cardiomyopathy. Electroporation-mediated delivery of functional genes: a promising approach for nonviral-based gene therapy of the failing heart S Eigeldiger-Berthou S Eigeldiger-Berthou 1 University Hospital Bern, Clinic for Cardiovascular Surgery , Bern , Switzerland P Buntschu P Buntschu 1 University Hospital Bern, Clinic for Cardiovascular Surgery , Bern , Switzerland A Frobert A Frobert 1 University Hospital Bern, Clinic for Cardiovascular Surgery , Bern , Switzerland M Flueck M Flueck 2 Institute for Biomedical Research into Human Movement and Health, Manchester Metropolitan University , Manchester , United Kingdom H Tevaearai H Tevaearai 1 University Hospital Bern, Clinic for Cardiovascular Surgery , Bern , Switzerland A Kadner A Kadner 1 University Hospital Bern, Clinic for Cardiovascular Surgery , Bern , Switzerland Abstract Objective: Alterations of β-adrenergic receptor signaling is a hallmark of heart failure (HF). Gene therapy may represent a promising therapeutic strategy. Here, we aim to establish and to utilize an electroporation-based viral-free system to transfect the β-adrenergic receptor kinase inhibitor (βARKct) in myocardial muscles in vivo to restore β-adrenergic receptor normal function. Methods: Electroporation (EP) was performed with the reporter plasmid (pCMV GFP), as control, and the gene of interest (pUB βARKct). The genes were electroporated in vitro in neonatal rat cardiomyocyts, and in vivo, on the beating heart of anesthetized Lewis rats. In vitro, EP efficiency was quantified by FACS and quantitative RT-PCR. Expression of the proteins was also assessed by Western blot. In vivo, gene expression was assessed seven days and three weeks post-intramyocardial EP by fluorescence, immunohistology and Western blot. Results: Electroporation was technically easy to apply and comparatively safe. Serial assessments of gene expression demonstrated cellular and intramyocardial expression of the reporter gene and βARKct. Expression pattern showed in vivo strongest expression areas around electrode placement zones. Conclusion: Electroporation is a promising therapeutic approach for non-viral-based gene therapy for HF. It is easy to apply and without harmful side-effects. However further functional analyses are necessary to demonstrate βARKct expression up to three weeks and to confirm the positive effects of restoring the long-term β-adrenergic signaling for improvement of HF. Targeting myocardin signaling pathway for heart failure A Mikhailov A Mikhailov 1 University of La Corufplusmn;a , La Corufplusmn;a , Spain M Torrado M Torrado 1 University of La Corufplusmn;a , La Corufplusmn;a , Spain A Centeno A Centeno 2 University Hospital Center of La Corufplusmn;a , La Corufplusmn;a , Spain E Lopez E Lopez 2 University Hospital Center of La Corufplusmn;a , La Corufplusmn;a , Spain L Lourido L Lourido 1 University of La Corufplusmn;a , La Corufplusmn;a , Spain A Castro Beiras A Castro Beiras 2 University Hospital Center of La Corufplusmn;a , La Corufplusmn;a , Spain Abstract Background: The myocardin (myocd) signaling pathway is important for cardiac and smooth muscle cell (SMC) differentiation during fetal life, but the consequences of myocd over-expression in heart failure (HF) remain poorly defined. Aims: To develop and validate the in vivo approaches aimed at manipulations of myocd signaling, either via the over-expression or the blockage of myocd expression, in a large-animal HF model. Methods: A HF model was established in neonatal piglets by injection of cardio-toxic agent, Doxorubicin (Dox). In vivo plasmid delivery was performed in normal and Dox-injected piglets using a catheter-based procedure developed by us for injections of plasmid DNA into target points of the left ventricle (LV). The animals were randomly selected to receive intramyocardial injections of either plasmid vectors carrying the pig myocd or short-hairpin RNA plasmid DNAs designated to target myocd variants expressed in pig heart, along with animals injected with empty vectors or constructs expressing non-effective short-hairpins. Molecular, immunochemical, and functional Methods were used to examine the expression and functional consequences of myocd expression modulations in in-vivo settings. Results: We present the results of the target validation experiments, the most important among which are the following: (1) an in vivo forced expression of myocd in normal LV myocardium results in up-regulation of a set of myocd-regulated cardiac fetal and SMC genes that is associated with an impaired systolic function (decrease of LV-end systolic pressure values) suggesting that exaggerated myocd expression might be maladaptive to myocardial remodeling, (2) a selective silencing of myocd signaling in LV myocardium of normal piglets results in downregulation of myocd-dependent gene expression and in augmented systolic performance (increase of LV-end systolic pressure values), uncovering that could alleviate HF conditions, (3) in neonatal piglets, forced myocd expression in LV myocardium followed by Dox-induction of HF-phenotype results in a significant lowering of survival rates, and (4) an inhibition of myocd signaling, at advanced stages of HF, downregulates expression of the myocd-dependent SMC genes in failing LV myocardium and attenuates a premature mortality of animals. Conclusions: The results reveal that either over- or under-expression of myocd could contribute to dysregulated cardiac function, and illustrate how the same signaling pathway that promotes SMC and heart defects when perturbed in the embryo might be therapeutically redeployed for the treatment of postnatal myocardial damage. Dilated cardiomyopathy caused by hereditary hemochromatosis T Popov T Popov 1 Institute of Cardiovascular Diseases Vojvodina , Novi Sad , Serbia I Srdanovic I Srdanovic 1 Institute of Cardiovascular Diseases Vojvodina , Novi Sad , Serbia M Petrovic M Petrovic 1 Institute of Cardiovascular Diseases Vojvodina , Novi Sad , Serbia T Canji T Canji 1 Institute of Cardiovascular Diseases Vojvodina , Novi Sad , Serbia M Kovacevic M Kovacevic 1 Institute of Cardiovascular Diseases Vojvodina , Novi Sad , Serbia A Jovelic A Jovelic 1 Institute of Cardiovascular Diseases Vojvodina , Novi Sad , Serbia M Sladojevic M Sladojevic 1 Institute of Cardiovascular Diseases Vojvodina , Novi Sad , Serbia G Panic G Panic 1 Institute of Cardiovascular Diseases Vojvodina , Novi Sad , Serbia Abstract Introduction: Haemochromatosis is one of the most common autosomal recessive disorder in which inappropriate increase in intestinal iron absorption results in deposition of excessive amounts of iron in parenchymal cells with eventual tissue damage and impaired function of organs, especially the liver, pancreas, heart, and pituitary. Purpose: Purpose of this case report is to demonstrate the clinical manifestation of myocardial haemochromatosis, significance of early recognition and prevention of disease manifestation, non-invasive diagnostic procedures and causative treatment. Case report: A male patient, age 27, admitted to hospital because of signs of congestive heart failure, rapid atrial fibrillation and malignant rhythm disorders. Transthoracic echocardiography showed right ventricular dilatation and severely reduced left ventricular systolic function, and significant tricuspid reflux. The serum levels of iron, ferritin, transferrin saturation were markedly elevated. Right ventricular biopsy and cardiac magnetic resonance revealed myocardial haemochromatosis. To remove systemic iron, venesection of 500ml was undertaken 3-4 times monthly. Two years later, patient is without signs and symptoms of heart failure and with normal transthoracic echocardiographic findings. Genotypisation of hereditary haemochromatosis gene (HFE) and hemojuvelin gene (HJV) was undertaken and revealed that examinated patient is homozygote for G320V mutation in the HJV gene. Family study revealed that mother was G320V heterozygous carrier, father and brother were also G320V heterozygous carriers and heterozygous carriers for H63D mutation in gene for HFE. No one of them has signs and symptoms of hereditary haemochromathosis. Conclusion: Early recognition and treatment (phlebotomy) of hemochromathosis is essential to prevent irreversible complication. Nowdays blood tests which include iron metabolism and genotypisation are sufficient for diagnosis and begining of treatment. Role of 17beta-oestradiol and oestrogen receptor beta in the genomic response to hypertrophy G Kararigas G Kararigas 1 Charite - University Medicine Berlin, Campus Mitte , Berlin , Germany D Fliegner D Fliegner 1 Charite - University Medicine Berlin, Campus Mitte , Berlin , Germany V Regitz-Zagrosek V Regitz-Zagrosek 1 Charite - University Medicine Berlin, Campus Mitte , Berlin , Germany Abstract Purpose: Left ventricular hypertrophy is a major risk factor for the development of cardiovascular disease. We and others have clearly indicated an important role for 17beta-oestradiol (E2) and oestrogen receptor beta (ERβ) in attenuating pressure overload (PO)-induced hypertrophy. However, the underlying molecular mechanisms are poorly understood. This study was undertaken to investigate the role of E2/ERβ signalling in PO-induced hypertrophy at a genome-wide level and to identify the pathways involved in potential protection against hypertrophy in female mice. Methods: Female wild-type (WT) and ERβ knockout (ERβ−/−) cycling C57Bl6 mice were subjected to transverse aortic constriction (TAC) or sham surgery. At nine weeks post operation, left ventricles were harvested and RNA was hybridised to the Mouse Genome 430 2.0 Array profiling>39,000 transcripts. The microarray data were analyzed with R version 2.8.1 and the Bioconductor packages. Results: Assessment of single gene differential expression with linear models, an empirical Bayesian method and a false discovery rate-adjusted P < 0.05 identified 197 probe sets regulated in WT, 941 in ERβ−/−, and 366 in both WT and ERβ−/− mice. Additionally, gene set enrichment analysis using the Kyoto Encyclopaedia of Genes and Genomes (KEGG) public pathway database and based on 1000 permutations revealed 26 pathways modulated in WT, while almost the double, i.e. 49, were modulated in ERβ−/− mice. Of these, 16 were modulated in both WT and ERβ−/− mice. Among others, the cytochrome P450 pathway was repressed in WT/TAC mice, the apoptosis pathway was induced in ERβ−/−/TAC mice, while the ECM-receptor interaction pathway was induced in both WT and ERβ−/−/TAC mice. Conclusions: In conclusion, we provide evidence that the E2/ERβ axis plays an important role in the genomic response to hypertrophy modifying the expression of several genes and particularly whole pathways. Lastly, our novel findings offer further insight into the numerous effects of E2 and ERβ observed in animal studies. Cardiac conduction system anomalies in fetal and adult hearts of a transgenic model of Long QT-Syndrome associate to hypertrophic cardiomyopathy AJ De La Rosa Sanchez AJ De La Rosa Sanchez 1 University of Jaen , Jaen , Spain J Dominguez J Dominguez 1 University of Jaen , Jaen , Spain D Sedmera D Sedmera 2 Laboratory of Cardiovascular Morphogenesis , Prague , Czech Republic D Franco D Franco 1 University of Jaen , Jaen , Spain A Aranega A Aranega 1 University of Jaen , Jaen , Spain Abstract The mouse transgenic line α-MHC-KvLQT1-iso2-T7 shares phenotypic characteristics with the Long QT syndrome (LQTS), which is a human cardiovascular disorder characterised by an increase of the cardiac action potential duration and it has been related to cases of sudden death in newborns. LQTS has been linked to mutations in distinct ion channel-coding genes, such as KCNQ1 and SCN5A. Our results of a detailed analysis of this LQTS-model evidenced an increase in the heart/body weight ratio as well as increased expression of hypertrophic molecular markers (β-MHC, α-SMA) indicating the presence of a hypertrophy cardiomyopathic (HCM) in this transgenic mice. Because hypertrophic process in heart has been previously related with sodium channel remodeling, we analyzed sodium channel gene expression in this LQTS mouse model showed that Scn5a and Scn1b up-regulation in trangenic hearts occurs at early developmental stage (i.e. E13.5), before the HCM phenotype is established. Moreover, functional analysis by optical mapping evidenced a delayed electric impulse propagation thought the cardiac conduction system in adult as well as in embryonic and fetal transgenic hearts respect to wild-type. Intercrossing this transgenic mice with Cx40-GFP mice, we found an increase of spread out fibers in the apical part of the Left Bundle Branch (LBB) as well as changes in the configuration of fibers from LBB that achieve the free LV wall in transgenic Cx40-GFP+/− α-MHC-KvLQT1-iso2-T7 respect to Cx40-GFP+/− mice. Additionally, we found a decrease in Purkinje fibers number in the Right Bundle Brunch (RBB) in transgenic Cx40-GFP+/− α-MHC-KvLQT1-iso2-T7 respect to Cx40-GFP+/−. These changes occur in adult such as in fetal stages, actually we are analyzing LBB and RBB in embryonic stages, to elucidate if the cardiac conduction system anomalies are already present in embryonic hearts. This study might shed new insights into understanding the mechanisms underlying cardiac electrophysiological disorders in newborns and may open new ways to better understand clinical pathologies such as cardiac congenital anomalies, arrhythmias and perinatal sudden death. Regulation of vascular smooth muscle cell proliferation by DNA-dependent protein kinase through the orphan nuclear receptor NOR1 S Medunjanin S Medunjanin 1 Otto-von-Guericke University of Magdeburg, Departement of Cardiology , Magdeburg , Germany F Burgbacher F Burgbacher 2 Dresden University of Technology, Heart Center, Department of Cardiology and Intensive Care , Dresden , Germany A Schmeisser A Schmeisser 1 Otto-von-Guericke University of Magdeburg, Departement of Cardiology , Magdeburg , Germany RH Strasser RH Strasser 2 Dresden University of Technology, Heart Center, Department of Cardiology and Intensive Care , Dresden , Germany RC Braun-Dullaeus RC Braun-Dullaeus 1 Otto-von-Guericke University of Magdeburg, Departement of Cardiology , Magdeburg , Germany Abstract Background: NOR1 is a member of the NR4A subfamily of nuclear receptors, which plays a central role in vascular smooth muscle cell (VSMC) proliferation and in vascular proliferative processes such as neointimal formation. Given its importance for DNA repair, cell cycle progression and survival, we hypothesized that the DNA-dependent protein kinase (DNA-PK) modulates NOR1 signalling. Results: Cultured human aortic VSMC were treated with the specific DNA-PK inhibtor NU7026. NU7026 treatment resulted in a 70% inhibition of FCS-induced cell cycle progression of VSMC (P < 0.05), as measured by BrdU incorporation studies. As well, FCS-stimulated upregulation of NOR1 protein as well as the cell cycle promoting proteins proliferating cell nuclear antigen (PCNA) and cyclin D1 and hyperphosphorylation of the retinoblastoma protein (RB) were prevented by DNA-PK inhibition with NU7026. Immuno-fluorescence and co-immuno-precipitation studies from VSM cell lysates demonstrated that DNA-PK forms a complex with NOR-1. Furthermore, DNA-PK's ability to physophorylate NOR1 was investigated. Mutational analysis, kinase assays and mass spectrometric characterization demonstrated that NOR1 is a substrate of DNA-PK and is strongly phosphorylated in the N-terminal domain. Finally, immune histochemistry demonstrated that both, the DNA-PK subunits and NOR1 are expressed in human atherosclerotic plaque specimens, predominantly within the neointimal VSMC. Conclusion: DNA-PK directly phosphorylates NOR-1 and, this way, modulates VSMC proliferation. The identification of new key molecules involved in VSMC function will help to understand cellular responses to vascular injury and may provide future targets for therapeutic interventions. Changes of ERK1/2, PI3K-Akt signaling pathway in pressure overload-Induced Cardiac Hypertrophy in Mice XM Li XM Li 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of Y Ma Y Ma 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of Y Yang Y Yang 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of F Liu F Liu 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of W Han W Han 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of BD Chen BD Chen 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of JF Zhang JF Zhang 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of XM Gao XM Gao 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of Abstract Objective: To explore the changes in extracellular regulated protein kinase (ERK1/2), phosphoinositid 3-kinase/ protein kinase B (PI3K-Akt) in the pressure overload induced hypertrophy at the different time course and to determine molecular mechanism from hypertrophy to heart failure. Methods: C57/BL 12wk old mice were subjected to sham-operation(SH) or transversing aortic constriction (TAC) to establish left ventricular hypertrophy. Echocardiographic assessments, hemodynamic determination, organ weight measurement, morphological and histological examination were performed at 1, 4, 8, 12 and 16 wks after surgery. mRNA levels of atrial natriuretic peptide (ANP), α-myosin heavy chain (α-MHC), Bcl-2 and Bax were determined by RT-PCR and ERK1/2, and Akt levels were detected by Western blot. Results: (1)Compared with SH group, left ventricular (LV) end-systolic (LVESd) and LV end-diastolic diameters (LVEDd), LV posterior wall thickness at diastole (Pwdth), posterior wall thickness at systole (Pwsth), anterior wall thickness at diastole (Awdth), anterior wall thickness at systole (Awsth) progressive increased after TAC. LV fractional shortening (FS%) significantly decreased at 16 wk (P < 0.05). LV systolic pressure (LVSP), rate of rise of left ventricular pressure (dp/dtmax and dp/dtmin) in TAC group were progressive increased after 4 wk. From 8∼12 wk these parameters maintained stabilization and then sharply decreased at 16 wk (all P < 0.05). However LV end-diastolic pressure (LVEDP) was increased at 8 wk and there was statistically difference from 12 wk (P < 0.05). (2) Histologically, cell size measured by cross-section area and cardiac collagen measured by percentage of Sirius Red positive stained area showed a progressive increase from 4 to 16 wk. (3) Compared with SH group, mRNA levels of ANP was time-dependently increased while α-MHC and Bcl-2 were time-dependently decreased. The ratio of Bcl-2 /Bax was decreased. Phosphorylation of ERK1/2 was increased at 4 wk, then decreased with age of TAC (all P < 0.05). The total and phosphorylated Akt did not change after TAC. Conclusion: (1) Pressure-overload induced by TAC resulted in a development of LVH from early concentric hypertrophy to late eccentric hypertrophy. And eventually toward cardiac dysfunction or heart failure. Those changes were associated with increase of cell size and cardiac fibrosis. (2)The changes of ERK1/2 and Bcl-2 at the different time course after TAC indicated that ERK1/2 signaling pathway may involve in the regulation of coyocyte apoptosis in hypertrophic heart. Molecular mechanisms of apical hypertrophic cardiomyopathy due to ACTC E99K mutation studied in man and mouse CR Bayliss CR Bayliss 1 Imperial College London, National Heart & Lung Institute , London , United Kingdom W Song W Song 1 Imperial College London, National Heart & Lung Institute , London , United Kingdom D Stuckey D Stuckey 2 University of Oxford , Oxford , United Kingdom E Dyer E Dyer 1 Imperial College London, National Heart & Lung Institute , London , United Kingdom M-C Leung M-C Leung 1 Imperial College London, National Heart & Lung Institute , London , United Kingdom L Monserrat L Monserrat 3 University Hospital La Coruna, Hospital Juan Canalejo , Corunna , Spain S Marston S Marston 1 Imperial College London, National Heart & Lung Institute , London , United Kingdom Abstract The E99K mutation of the cardiac actin gene (ACTC) has been studied in 76 mutation carriers from 10 different families. The phenotype is apical hypertrophic cardiomyopathy (HCM) with hypertrabeculation. We have investigated the functional properties of mutant actin from two patient biopsies and have also generated a transgenic (TG) mouse expressing the ACTC E99K mutation. The expression level of the mutant gene was measured by 2-D Electrophoresis. The human biopsy expressed 39% mutant actin and the TG mice expressed 50% E99K actin. Biopsy tissue was compared to donor heart as were TG and non-transgenic (nTG) tissue. The phosphorylation profile of sarcomeric proteins was measure and in both the human and mouse samples there was no significant difference between mutant-containing and control samples. The overall actin content of the samples was compared and no differences were found. The TG mouse model replicates many features of HCM in human heart. 48% of TG females and 22% of TG males died suddenly between 28 and 45 days. At 7 months, abnormal cardiac morphology (including apical hypertrophy), impaired relaxation, significantly lower ejection fractions and stroke volume and a blunted response to isoprenaline were observed (MRI and echocardiography). In ECG, TG mice had more frequent atrial ectopic beats, atrial flutter and increased P wave duration. The in vitro motility assay was used to investigate the Ca2+-regulation of reconstituted thin filaments composed of human cardiac tropomyosin and troponin, and actin purified from both the biopsy/donor human heart tissue or TG/nTG mice. There was no difference in Ca2+-sensitivity when donor troponin (1.62 molsPi/molsTnI) was recombined into thin filament (Donor/E99K 1.04) and only a small difference when failing heart troponin (0.26 molsPi/molsTnI) was used (Donor/E99K 1.27). This is in contrast to the mouse tissue which showed a marked difference in Ca2+-sensitivity when donor troponin was used (nTG/E99K 2.54) and a much smaller difference with phosphatase treated troponin (nTG/E99K) 1.21). The Ca2+-sensitivity of the mutant mouse actin demonstrated a blunted response to TnI phosphorylation. Unregulated E99K actin showed a reduced sliding speed compared to control actin in both human (15%) and mouse (8%) experiments. In mouse the main effect of the mutation is an increase in Ca2+-sensitivity, in common with other HCM causing mutations and this may be sufficient to generate the HCM phenotype; however in our human samples the increased Ca2+-sensitivity was not as evident indicating a more complex situation in human heart. The RH domain of GRK5 regulates cardiac hypertrophy in vivo D Sorriento D Sorriento 1 University of Naples Federico II, Dpt of Clinical Medicine, Cardiovascular & Immunological Science , Naples , Italy G Santulli G Santulli 1 University of Naples Federico II, Dpt of Clinical Medicine, Cardiovascular & Immunological Science , Naples , Italy A Fusco A Fusco 1 University of Naples Federico II, Dpt of Clinical Medicine, Cardiovascular & Immunological Science , Naples , Italy B Trimarco B Trimarco 1 University of Naples Federico II, Dpt of Clinical Medicine, Cardiovascular & Immunological Science , Naples , Italy G Iaccarino G Iaccarino 1 University of Naples Federico II, Dpt of Clinical Medicine, Cardiovascular & Immunological Science , Naples , Italy Abstract We have recently demonstrated that the G protein coupled receptor kinase, GRK5, regulates the activity of the transcription factor NFkB in endothelial cells. In particular, GRK5, by means of its RH domain, binds and stabilizes the complex NFkB /IkB in the nucleus thus inhibiting NFkB activity. Several studies suggest the key role of NFkB in cardiac hypertrophy by regulating hypertrophic gene expression and we have demonstrated that in cardiomyoblasts GRK5-NT, comprising the RH domain, inhibits both phenylephrine (PE)-induced NFkB and ANF promoter activity evaluated by luciferase assay. Here we evaluated the ability of GRK5-NT to modulate hypertrophic response in vivo by inhibiting NFkB activity. To this aim we used two different rat models of cardiac hypertrophy, the spontaneously hypertensive (SHR) and the normotensive rats (WKY) in which hypertrophy is induced by chronic administration of PE. GRK5-NT overexpression in SHR and WKY was induced by direct cardiac injection of 10 an adenovirus encoding GRK5-NT. Echocardiographic analysis shows that the intra-cardiac injection of GRK5-NT is able to reduce cardiac mass in hypertensive SHR and to reduce the development of PE-induced hypertrophy in WKY. This associates with an inhibition of NFkB transcription activity, evaluated by EMSA, and the associated signaling (IkB and NFkB levels) and phenotypes (fibrosis, apoptosis). Moreover, we have demonstrated that such phenomenon is independent from blood pressure levels, since in SHR, in which hypertrophy strictly depends on BP, GRK5-NT did not cause a reduction of BP levels but even an increase probably through ameliorating cardiac contractility. In conclusion, our study underlines the role of NFkB in the development of cardiac hypertrophy and the potential role of the RH domain of GRK5 as inhibitor of hypertrophy in vivo. Experimental cardiac hypertrophy induced by isoproterenol in rat: physiological,biochemical and ultrastructural aspects CR Revnic CR Revnic 1 C.C.Iliescu Cardiology Dpt , Bucharest , Romania C Ginghina C Ginghina 1 C.C.Iliescu Cardiology Dpt , Bucharest , Romania F Revnic F Revnic 2 National Institute of geriatrics and Gerontology , Bucharest , Romania Abstract The aim of our study was to induce cardiac hypertrophy in Wistar rats following treatment with Isoproterenol(IPR), and to see the effect of 45 minutes ischemia followed by 60 minutes reperfusion upon physiological parameters:coronary flow (C.F.),heart rate (H.R.) and left ventricular developed pressure (LVPD), versus controls. Material and method: Isoproterenol-induced cardiac hypertrophy. A group of 12 rats received isoproterenol (-sympathomimetic, 5 mg/kg body wt/day sc) for 7 days. Isolated retrograde perfused heart preparation. The rats have been killed by cervical dislocation and hearts from Control and (IPR) treated group, have been quickly removed and placed on ice bath ,then mounted and perfused with Krebs Hanseleit buffer at 37C in Langendorff retrograde perfusion system at a constant pressure.45 minutes ischemia has been followed by 60 minutes reperfusion and physiological parameters(coronary flow (C.F.),heart rate (H.R.) and left ventricular developed pressure(LVS.P.) of rat heart from the two groups have been measured. TACS DNA laddering kit has been used to assess ventricular cells from (IPR) treated rat and from Controls, for apoptosis and fragments of left ventricles of both groups have been processed for Electron Mycroscopy(E.M) investigations. Results: Evaluation of (C.F.), (H.R.) and (L.V.S.P.) in Controls and (IPR) treated rats have pointed out a 50% reduction in (C.F). in (IPR) treated rats versus controls during stabilization period. In treated rats, LVSP is depressed at the beginning and then is gradually increasing, while in controls is almost constant during the whole experiment.Protection of myocardium against oxidative stress is significantly depressed following (IPR) treatment, synthesis and utilization of GSH is limited versus controls.Cardiac hypertophy experimentally induced with (IPR) is related with a decrease in(C.F.) and contractile capacity associated with an increased level of LDH and CK enzymes. Myocite apoptosis has been present , pointed out by DNA laddering on gel electrophoresis. in IS treated rats.In (IPR)treated rats,(E.M.). studies have pointed out mitochondrial derangement as well as desorganisation of myofibres. Conclusion: (IPR) treatment in rats induces cardiac hypertophy and changes in physiological parameters of heart with a negative impact upon synthesis of antioxidant enzymes as well as changes in ultrastructure of heart muscle and at the molecular level(DNA laddering is present) as a sign of apoptosis in ventricular myocites. Sphingosine kinase-2 mediated production of sphingosine-1-phosphate: a new function in mitochondrial respiration M Paillard M Paillard 1 VCU-VAMC; U886 Cardioprotection, Lyon-FRANCE , Richmond , United States of America J Liang J Liang 2 Virginia Commonwealth University , Richmond , United States of America GM Strub GM Strub 2 Virginia Commonwealth University , Richmond , United States of America L Gomez L Gomez 1 VCU-VAMC; U886 Cardioprotection, Lyon-FRANCE , Richmond , United States of America NC Hait NC Hait 2 Virginia Commonwealth University , Richmond , United States of America JC Allegood JC Allegood 2 Virginia Commonwealth University , Richmond , United States of America EJ Lesnefsky EJ Lesnefsky 3 Virginia Commonwealth University-VAMC , Richmond , United States of America S Spiegel S Spiegel 2 Virginia Commonwealth University , Richmond , United States of America Abstract 458 Table Oxidative Phosphorylation . Glutamate/Malate . Succinate . TMPD-ascorbate . . state 3 . state 4 . RCR . state 3 . state 4 . RCR . 2 mM ADP . WT 251 ± 6 29 ± 3 9 ± 1 355 ± 18 100 ± 7 3.6 ± 0.1 897 ± 32 SphK2-KO 195 ± 18* 19 ± 5 13 ± 3 274 ± 18* 84 ± 9 3.3 ± 0.2 710 ± 54* . Glutamate/Malate . Succinate . TMPD-ascorbate . . state 3 . state 4 . RCR . state 3 . state 4 . RCR . 2 mM ADP . WT 251 ± 6 29 ± 3 9 ± 1 355 ± 18 100 ± 7 3.6 ± 0.1 897 ± 32 SphK2-KO 195 ± 18* 19 ± 5 13 ± 3 274 ± 18* 84 ± 9 3.3 ± 0.2 710 ± 54* Values are mean ± SEM, n = 5-6 per group; RCR= respiratory control ratio. *p < 0.05 vs. WT Open in new tab Abstract 458 Table Oxidative Phosphorylation . Glutamate/Malate . Succinate . TMPD-ascorbate . . state 3 . state 4 . RCR . state 3 . state 4 . RCR . 2 mM ADP . WT 251 ± 6 29 ± 3 9 ± 1 355 ± 18 100 ± 7 3.6 ± 0.1 897 ± 32 SphK2-KO 195 ± 18* 19 ± 5 13 ± 3 274 ± 18* 84 ± 9 3.3 ± 0.2 710 ± 54* . Glutamate/Malate . Succinate . TMPD-ascorbate . . state 3 . state 4 . RCR . state 3 . state 4 . RCR . 2 mM ADP . WT 251 ± 6 29 ± 3 9 ± 1 355 ± 18 100 ± 7 3.6 ± 0.1 897 ± 32 SphK2-KO 195 ± 18* 19 ± 5 13 ± 3 274 ± 18* 84 ± 9 3.3 ± 0.2 710 ± 54* Values are mean ± SEM, n = 5-6 per group; RCR= respiratory control ratio. *p < 0.05 vs. WT Open in new tab Abstract Sphingosine-1-phosphate (S1P) is a potent lipid mediator that regulates diverse physiological and pathological processes acting through five specific cell surface receptors. The majority of research to date has focused on the activation of these receptors, but there is now evidence to suggest that S1P also exerts intracellular functions independent of its cell surface receptors. We sought to determine if knock-out of sphingosine kinase 2 (SphK2-KO), one of the isoenzymes that forms S1P, will impact mitochondrial function. Mouse heart mitochondria were isolated by differential centrifugation from both SphK2-KO and WT mice. Oxidative phosphorylation (states 3 and 4, nAO/min/mg) was assessed using a Clark-type electrode (RCR: state 3/state 4). We found by Western Blot that SphK2 was present in WT mitochondria (but not KO) and that SphK2-KO mitochondria displayed a lower content of S1P. SphK2-KO mitochondria displayed a significant decrease in oxidative phosphorylation with complex I (23%), II (23%) and cytochrome oxidase complex IV (21%) substrates with preserved coupling of respiration (RCR, p = ns). A new, aberrant band of complex IV was detected by Blue-Native PAGE in SphK2 KO mitochondria, suggesting a disorganization of complex IV. Thus, the absence of SphK2 leads to a dysfunction in mitochondrial oxidative phosphorylation with the primary defect at complex IV. This finding supports novel actions of S1P in mitochondria 1 . Differential effects of preconditioning and postconditioning on postischemic microvascular function and leukocyte recruitment C Zuchi C Zuchi 1 Cardiology, University of Perugia , Perugia , Italy S Coiro S Coiro 1 Cardiology, University of Perugia , Perugia , Italy M Bettini M Bettini 1 Cardiology, University of Perugia , Perugia , Italy G Ciliberti G Ciliberti 1 Cardiology, University of Perugia , Perugia , Italy I Mancini I Mancini 1 Cardiology, University of Perugia , Perugia , Italy I Tritto I Tritto 1 Cardiology, University of Perugia , Perugia , Italy LC Becker LC Becker 2 Johns Hopkins University , Baltimore , United States of America G Ambrosio G Ambrosio 1 Cardiology, University of Perugia , Perugia , Italy Open in new tabDownload slide Abstract 459 Figure Open in new tabDownload slide Abstract 459 Figure Abstract Purpose: Brief intermittent ischemia (I)/reperfusion(R), occurring either before prolonged ischemia (preconditioning; preC) or upon reflow (postconditioning; postC) reduces infarct size. PreC also protects vasculature, improving endothelial function and reducing leukocyte recruitment in postischemic tissues. We sought to ascertain whether PostC also protects postischemic microvasculature, by directly evaluating whether it reduces microvascular dysfunction and leukocyte recruitment during postischemic reflow. Methods: Rat cremaster microcirculation was viewed in vivo by videomicroscopy; rolling and adherence of acridine red-labeled leukocytes were monitored. Muscles underwent 90 min of I and 90 min of R. Endothelium-dependent and -independent vasodilating reserve was then evaluated by local muscle superfusion with 10-4 M acetylcholine and 10-4 M nitroprusside. Control muscles were subjected to I and R, without interventions (n = 15). PreC was induced before prolonged ischemia (1 cycle of 5 min ischemia followed by 10 min reflow; n = 11). PostC was induced upon reflow (5 cycles of 10 sec ischemia intermingled with 10 sec reflow; n = 15). Sham-operated muscles were observed for the same length of time, without I (n = 13). Results: I/R markedly impaired vasodilation and induced leukocyte recruitment; PreC significantly improved all indices of vascular injury, while PostC reduced leukocyte recruitment, but did not improve microvascular function (Figure). Conclusion: Thus, preC improved both microvascular function and leukocyte recruitment; in contrast, postC was effective in reducing leukocyte recruitment but it did not protect postischemic microvascular function. These findings suggest that microvascular function and leukocyte recruitment may be influenced by different protective pathways 1 . Sirtuin 1 and its role in ischemic preconditioning T Adam T Adam University of Cape Town , Cape Town , South Africa S Sharp S Sharp LH Opie LH Opie S Lecour S Lecour Abstract Purpose: Sirtuin 1 (Sirt 1), a histone deacetylase and anti-aging factor, protects the heart from apoptosis, oxidative stress, cardiac hypertrophy, fibrosis and ventricular dysfunction. Interestingly, the polyphenol resveratrol (RSV) potently activates Sirt 1 and pharmacologically mimics ischemic preconditioning (IPC). We propose that Sirt 1 may play a role in IPC-mediated cardioprotection. Methods: Cultured C2C12 mouse myotubes were preconditioned for 30 min either by exposure to hypoxia or with RSV (10 μM), in the presence or absence of the sirtuin inhibitors nicotinamide (NAM, 50 μM) or Sirt 1 inhibitor III (SIII, 100nM). After a 1 hour wash-out period, myotubes were subjected to a simulated ischemic insult for 8 hours. Cell viability was assessed using the trypan blue exclusion test. In addition, Sirt 1 activity was determined in cytosolic extracts after 30 min of simulated IPC. Similarly, isolated rat hearts were subjected to 30 min of global ischemia and 1 hour of reperfusion. IPC was elicited with two cycles of 5 min ischemia and reperfusion, with or without NAM prior to the prolonged ischemic insult. Triphenyltetrazolium chloride staining was performed to evaluate infarct size, and Western blot analysis for measuring cytosolic levels of Sirt 1. Results: Both simulated IPC (66.9 ± 2.9%) and RSV (66.8 ± 2.5%) improved cell viability compared to the simulated ischemic control (47.2 ± 3.6%) (p < 0.001, n = 6). Addition of NAM or SIII to the cells did not abolish the cytoprotective effect of simulated IPC. Furthermore, Sirt 1 activity was unchanged by simulated IPC (1.8 ± 0.38 arbitrary units (AU) for simulated IPC vs. 1.6 ± 0.16 AU for normoxic control, p = ns, n ≥ 6). In the isolated heart, IPC significantly reduced infarct size from 62.4 ± 7.9% to 29.1 ± 6.5% (p < 0.05 vs. ischemic control, n ≥ 6), but had not effect on Sirt 1 protein levels (43.5 ± 7.4 AU for IPC vs. 34.9 ± 3.4 AU for ischemic control, p = ns, n = 4). The presence of NAM did not abrogate the cardioprotective effect of IPC (32.3 ± 12% for NAM+IPC vs. IPC, p = ns). Conclusion: These data do not support a role for the cytoprotective factor Sirt 1 in IPC-induced cardioprotection. Consecutive activation of PKA and PKC mediates the signalling pathways of temperature preconditioning to provide a highly effective cardioprotective mechanism I Khaliulin I Khaliulin 1 University of Bristol , Bristol , United Kingdom JE Parker JE Parker 1 University of Bristol , Bristol , United Kingdom AP Halestrap AP Halestrap 1 University of Bristol , Bristol , United Kingdom Abstract Purpose: We have recently shown that temperature preconditioning (TP) is a powerful cardioprotective mechanism and that protein kinase C activation is critical for this cardioprotective effect. The purpose of the present study was to investigate further the signalling mechanisms underlying the cardioprotective effect of TP and to evaluate whether this mechanism could be a basis for pharmacologically induced cardioprotection. Methods: In the first set, isolated rat hearts were subjected to TP by 3 cycles of 2 min hypothermic perfusion (26°C) interspersed with 6 min normothermic perfusion (37°C). Recovery of haemodynamic function and lactate dehydrogenase (LDH) release during reperfusion were used to evaluate the cardioprotection. In some of these hearts cyclic AMP concentration and PKA activity were measured at the end of pre-ischaemic period. In the second series of experiments, control and TP hearts were perfused in the presence or absence of either the β-adrenergic blocker sotalol (10 µM) or the PKA inhibitor H-89 (10 µM). Recovery during reperfusion was assessed. PKC activity was measured in control and TP hearts treated with H-89. In the third series of experiments, hearts were treated with either β-adrenergic agonist isoproterenol (200 nM), PKC activator adenosine (30 µM) or with isoproterenol followed by perfusion with adenosine prior to ischaemia. Cardioprotective effects, including Ca2+-induced MPTP opening, were assessed during reperfusion. In other hearts from these groups MPTP opening, Ca2+ retention by mitochondria and glycogen content were measured at the end of the pre-ischaemic period. Results: TP was highly cardioprotective as assessed by hemodynamic function and LDH release during reperfusion. Cyclic AMP concentrations and PKA activity were increased in TP hearts. PKA inhibitor H-89 completely abolished and sotalol partially blocked the cardioprotective effect of TP. H-89 blocked TP-induced PKC activation indicating that PKC was activated downstream of PKA in TP hearts. Both isoproterenol and adenosine alone protected hearts against ischemia/reperfusion injury but the consecutive isoproterenol/adenosine perfusion resulted in much greater protection. Isoproterenol-treated but not adenosine–treated hearts showed a significant reduction in glycogen content before ischaemia. Conclusions: PKA activation is an important component of the signal transduction pathway of TP preceding the activation of PKC. Pharmacologically induced consecutive PKA/PKC activation mimics TP in providing potent cardioprotection. Modulation of oxidative stress induced necrosis and apoptosis by peroxynitrite or nitric oxide donors in cardiac myocytes A Kandasamy A Kandasamy 1 University of Alberta , Edmonton , Canada R Schulz R Schulz 1 University of Alberta , Edmonton , Canada Abstract Purpose: A contributing factor to some cardiac diseases is the loss of myocardial cells. Enhanced oxidative stress caused by excessive reactive oxygen species has been implicated in cell death in cardiac pathologies such as ischemia/reperfusion injury and heart failure. Reactive nitrogen species are implicated to both mediate and protect from apoptosis. Their role in cardiac myocyte necrosis is unknown. We evaluated the effects of the peroxynitrite donor SIN-1 and compared it against the nitric oxide (NO) donors SNAP or DETA-NONOate on necrosis and/or apoptosis induced by oxidative stress stimuli in neonatal rat ventricular myocytes. Methods: Neonatal rat ventricular myocytes were cultured and serum starved cells were treated with either hydrogen peroxide (200 µM) or doxorubicin (1 µM) in the presence or absence of peroxynitrite or NO donors (all at 1 mM). Lactate dehydrogenase release was measured in the conditioned media as a marker of necrotic cell death. Cell homogenates from different treatments were subjected to immunoblotting to detect the appearance of cleaved caspase-3 and poly ADP-ribose polymerase (PARP). Caspase-3 activity in the cell lysates were measured by incubation with 7-amino-4-trifluoromethyl coumarin. Nitrite levels in the conditioned media were quantified by Griess reagent. Results: Hydrogen peroxide induced the greatest and earliest (1 h) increase in lactate dehydrogenase release (3.5 fold at 24 h), followed by doxorubicin (twofold increase observed only at 24 h). Hydrogen peroxide did not cause apoptosis. SIN-1 and SNAP similarly, and DETA-NONOate to a lesser extent, reduced LDH release caused by hydrogen peroxide. SIN-1 and SNAP, but not DETA-NONOate, reduced doxorubicin-induced lactate dehydrogenase release. Only SIN-1 reduced the appearance of a 17 kDa caspase-3 cleavage fragment and 89 kDa PARP cleavage fragment caused by doxorubicin at 6 and 24 h. Similarly, only SIN-1 attenuated the doxorubicin-induced increase in caspase-3 activity at 6 and 24 h. SIN-1 and DETA-NONOate produced ∼3-4 fold higher nitrite levels in cell culture media compared to SNAP. Conclusions: Hydrogen peroxide causes rapid cell necrosis whereas doxorubicin induces apoptosis and late necrosis in neonatal cardiac myocytes. The peroxynitrite donor SIN-1 and NO donors SNAP and DETA-NONOate differentially modulate necrosis and apoptosis in cardiac myocytes. Exercise may cause contractile dysfunction characterized by mitochondrial dysfunction at the level of complex I M Schoepe M Schoepe 1 University of Leipzig, Heart Center, Department of Cardiac Surgery , Leipzig , Germany M Schwarzer M Schwarzer 1 University of Leipzig, Heart Center, Department of Cardiac Surgery , Leipzig , Germany A Schrepper A Schrepper 1 University of Leipzig, Heart Center, Department of Cardiac Surgery , Leipzig , Germany M Osterholt M Osterholt 1 University of Leipzig, Heart Center, Department of Cardiac Surgery , Leipzig , Germany P Amorim P Amorim 1 University of Leipzig, Heart Center, Department of Cardiac Surgery , Leipzig , Germany FW Mohr FW Mohr 1 University of Leipzig, Heart Center, Department of Cardiac Surgery , Leipzig , Germany T Doenst T Doenst 1 University of Leipzig, Heart Center, Department of Cardiac Surgery , Leipzig , Germany Abstract Background: Exercise is considered a method to induce physiological hypertrophy. We compared two exercise protocols in rats, differing in their intensity and their potential to improve respiratory capacity. Methods: We treadmill trained rats for 10 weeks with either 10% (moderate) or 16% incline (intensive). Training intervals were 4 times a week and increased from 20 to 120 min per interval within 6 weeks. At 6 and 10 weeks we performed echocardiography and assessed maximal respiratory capacity and complex activities in isolated mitochondria. Results: Within ten weeks animals trained with 16% incline developed hypertrophy (LVPWD: 1,6 ± 0,1 vs. 2,4 ± 0,1mm; p < 0,05) with normal function (Ejection fraction: 75,2 ± 2,5 vs. 75,6 ± 2,1%; n.s.). However, after 6 weeks contractile function was impaired (EF: 74,5 ± 1,67 vs. 65,8 ± 2,3%; p < 0,05; eFS%: 44,92 ± 1,53 vs. 38,12 ± 2,04%; p < 0,05), which was associated with decreased maximal respiratory capacity of isolated mitochondria (state 3 respiration: 326 ± 71 vs. 161 ± 22 natomsO/min/mg; p < 0,05) and a gene expression shift from the adult (α) to the fetal (β) isoform of myosin heavy chain (α MHC: from 1,49 ± 0,27 to 0,62 ± 0,07; p < 0,05; β MHC: from 0,38 ± 0,06 to 0,64 ± 0,28; p < 0,05). Interestingly, heart failure markers ANF and BNP gene expression were reduced at both time points. Isolated assessment of complex activities revealed a defect at complex I after six weeks of exercise (1030 ± 96 vs. 758 ± 71 nmol/min/mg protein; p < 0,05), while no changes were detected for the other complexes. In contrast, at 10% incline, there was no hypertrophy at ten weeks (LVPWD: 1,7 ± 0,1 vs. 1,8 ± 0,1mm; n.s.), normal function (EF: 74,3 ± 1,5 vs. 69,0 ± 1,0%; n.s.; eFS%: 45,3 ± 1,4 vs. 40,0 ± 0,8%; n.s.) and maximal respiratory capacity was normal at both time points. Furthermore, there were no changes in complex activities after six weeks of moderate exercise (1030 ± 96 vs. 1084 ± 152 nmol/min/mg protein; n.s.). Conclusion: Exercise, as intermittent treadmill running, may cause contractile dysfunction in heart muscle depending on running intensity. This dysfunction appears to be reversible and is associated with mitochondrial dysfunction located at the level of complex I of the respiratory chain. Lower intensity training does not cause contractile or mitochondrial function but, in our hands, does also not cause hypertrophy. Lack of Cx43 impairs mitochondrial respiration and reduces sarcoplasmic reticulum calcium content C Fernandez-Sanz C Fernandez-Sanz 1 University Hospital Vall d'Hebron , Barcelona , Spain M Ruiz-Meana M Ruiz-Meana 1 University Hospital Vall d'Hebron , Barcelona , Spain E Miro-Casas E Miro-Casas 1 University Hospital Vall d'Hebron , Barcelona , Spain E Agullo E Agullo 1 University Hospital Vall d'Hebron , Barcelona , Spain K Boengler K Boengler 2 University of Essen Medical School, Institute of Pathophysiology , Essen , Germany R Schulz R Schulz 2 University of Essen Medical School, Institute of Pathophysiology , Essen , Germany D Garcia-Dorado D Garcia-Dorado 1 University Hospital Vall d'Hebron , Barcelona , Spain Abstract We have previously shown that Cx43 localizes at the inner mitochondrial membrane and that Cx43 deficiency impairs mitochondrial K+ uptake. Anatomical microdomains between mitochondrial and sarcoplasmic reticulum (SR) allow a close functional interaction between both organella. In this study we investigated whether replacement of Cx43 by Cx32 modifies mitochondrial respiration and membrane potential, and the consequences of these effects on SR Ca2+ uptake and release. Methods: Mitochondria were isolated from Cx43KI32 mouse hearts and respiration was assessed on glutamate-malate (complex I) or succinate-rotenone (complex II) substrates using a Clark-type electrode. Isolated cardiac myocytes from homozygous (Cx32/32) and wild-type animals (Cx43/43) were incubated with JC-1, and mitochondrial membrane potential was determined using an Ar-Kr confocal imaging system. SR Ca2+ content was quantified by a ratiofluorescence system in Fura-2 loaded cardiac myocytes after the addition of 5mM caffeine. Results: Lack of Cx43 was associated with an increase in state 2 respiration (7.8 ± 0.1 vs 5.7 ± 0.1 nmol O/min × UCS, p = 0.03), and a decrease in state 3 respiration (39 ± 0.2 vs 45 ± 0.1 nmol O/min × UCS, p = 0.05) using substrates of complex I, which resulted in decreased respiratory control rate (5.0 ± 0.1 vs 7.8 ± 0.3, p < 0.01). CII-mediated respiratory control rate was not modified by the absence of Cx43, but state 2 and 3 were both significantly reduced (p = 0.02 and p = 0.003, respectively). Mitochondria from animals lacking Cx43 presented a slight depolarization under control conditions (1.7 ± 0.1 vs 2.2 ± 0.1 a.u. of JC-1 ratiofluorescence, p = 0.02). SR Ca2+ transient amplitude induced by caffeine was significantly reduced in cardiac myocytes from animals lacking Cx43 (0.21 ± 0.03 vs 0.52 ± 0.1 F/Fo Fura-2, p = 0.03). Time to Ca2+ peak and relaxation time were not modified by Cx43, but in 68.2% of myocytes lacking Cx43 caffeine failed to induce SR Ca2+ release (vs 0% failure in wild-type myocytes). Conclusion: These results underscore the importance of Cx43 in mitochondrial function, and suggest that reduced mitochondrial respiration associated with Cx43 deficiency impairs SR Ca2+ handling. Oxidation of myofibrillar proteins causes contractile dysfunction in human heart failure S Menazza S Menazza 1 University of Padua, Department of Biomedical Sciences , Padua , Italy M Canton M Canton 1 University of Padua, Department of Biomedical Sciences , Padua , Italy FL Sheeran FL Sheeran 2 The University of Melbourne, Murdoch Childrens Research Institute , Melbourne , Australia F Di Lisa F Di Lisa 1 University of Padua, Department of Biomedical Sciences , Padua , Italy S Pepe S Pepe 2 The University of Melbourne, Murdoch Childrens Research Institute , Melbourne , Australia Abstract Background: Previous studies in microembolized pig hearts (Eur. Heart. J. 27, 875-881, 2006) demonstrated that augmented intracellular accumulation of reactive oxygen species causes oxidative modifications at the level of the contractile machinery. This study was aimed at investigating the role of myofibrillar protein (MP) oxidation\nitrosylation and the relationship between MP oxidation and contractile impairment in human failing myocardium (NYHA class IV). Methods and Results: As compared to samples from non-failing donor hearts (NF-group, n = 15), left ventricular biopsies from explanted failing hearts (NYHA class IV, HF-group, n = 33) displayed a 2.3 ± 0.29- and 2.6 ± 0.53-fold increase in actin and tropomyosin (Tm) carbonylation respectively, and a 2.2 ± 0.47-fold higher level of high-molecular-mass complexes of Tm due to disulphide cross-bridge formation. MP were also modified by reactive nitrogen species. The extent of S-nitrosylation was 1.3 ± 0.15-fold higher in the HF-group. Interesting, actin and Tm carbonylation along with Tm oxidation significantly correlated with both loss of viability (r 2 = 0.646, P = 6.16E-12; r 2 = 0.453, P = 1.58E-07; r 2 = 0.221, P = 0.001, respectively), as indicated by plasma TnI levels and contractile impairment (r 2 = 0.599, P = 6.88E-112; r 2 = 0.457, P = 1.37E-07; r 2 = 0.646, P = 6.16E-12, respectively), as shown by reduced left ventricular ejection fraction (LVEF). Conclusion: This study demonstrates that HF-related oxidative and nitrosative stresses induce covalent changes of MP and that these MP changes play a relevant role in contractile impairment as suggested by the inverse correlation between MP oxidation and LVEF. Monoamine oxidase activity in end-stage heart failure associated with dilated and ischemic cardiomyopathies: a comparison between left and right ventricles E Borchi E Borchi 1 University of Florence , Florence , Italy ME Manni ME Manni 1 University of Florence , Florence , Italy V Bargelli V Bargelli 1 University of Florence , Florence , Italy C Giordano C Giordano 2 Sapienza University of Rome , Rome , Italy G D'amati G D'amati 2 Sapienza University of Rome , Rome , Italy E Cerbai E Cerbai 1 University of Florence , Florence , Italy C Nediani C Nediani 1 University of Florence , Florence , Italy L Raimondi L Raimondi 1 University of Florence , Florence , Italy Abstract Purpose: Dilated (DCM) and ischemic (IHD) cardiomyopathies are the most common causes of heart failure, which is characterized by a progressive remodeling and dysfunction and has a poor prognosis. Among the multiple mechanisms involved, the increase in monoamine oxidases (MAOs)-derived H2O2 and aldehyde may play an important role in the maladaptive evolution towards failure. At homeostatic conditions, the amount of aldehydes and H2O2 are regulated by aldehyde dehydrogenase (ALDH) and catalase (CAT) activities, respectively, both indicated as rescuing enzymes of the heart. Possibly, different levels of such enzymes might be present in DMC and IHD failing hearts and they may be proposed as discriminating markers of the two cardiomyopathies. Therefore, we investigated the enzymatic activities of MAO-A and MAO-B, of cytosolic and mitochondrial ALDH and of CAT and the amount of carbonylated proteins and malondialdehyde (MDA), indirect markers of oxidative stress, in right (RV) and left ventricles (LV) from end-stage failing hearts secondary to IHD and DMC. Methods: MAO activity was measured radiochemically, using specific substrates for MAO-A and MAO-B isoforms in the presence of semicarbazide, inhibitor of other amine oxidases. CAT and ALDH activities were determined spectrophotometrically. MDA and carbonylated proteins levels were assayed using specific kits. Results: Both MAO isoforms were detected in failing hearts, with a total MAO activity ten times higher in IHD than in DMC. Interestingly, the activity of MAO-A, the more representative isoform, was similar in LV and RV for IHD but it was higher in LV (0.74 ± 0.18) than in RV (0.32 ± 0.12; p < 0.05) for DCM, with no differences in MAO-B activity. On the other hand, CAT activity was significantly increased in LV (27.53 ± 1.94; p < 0.05) compared to RV (17.18 ± 3.52), only for DMC. The same hearts showed MDA levels significantly enhanced in RV (3.51 ± 0.44), compared to LV (2.70 ± 0.45). No differences were observed in total ALDH activity and in the content of carbonylated protein. Conclusion: Both MAO isoforms, with a prevalence of MAO-A, were observed in failing hearts. Nevertheless, we reported a significant increase in total MAO activity in IHD compared to DCM. In DMC specimens MAO-A was preferentially active in LV compared to RV, in concomitance with a higher level of CAT activity and a lower level of MDA amount, suggesting a role for CAT in scavenging MAO-derived H2O2. Our results indicate that the evaluation of MAO activity, in particular MAO-A, might help to discriminate between IHD and DCM associated with heart failure. Effect of chronic intermittent and continuous hypoxia on the expression of phospholipase A2 and manganese superoxide dismutase in rat hearts P Micova P Micova 1 Charles University Prague, Faculty of Science , Prague , Czech Republic P Balkova P Balkova 1 Charles University Prague, Faculty of Science , Prague , Czech Republic F Kolar F Kolar 2 Academy of Sciences of the Czech Republic, Institute of Physiology , Prague , Czech Republic J Neckar J Neckar 2 Academy of Sciences of the Czech Republic, Institute of Physiology , Prague , Czech Republic F Novak F Novak 1 Charles University Prague, Faculty of Science , Prague , Czech Republic O Novakova O Novakova 1 Charles University Prague, Faculty of Science , Prague , Czech Republic Abstract Adaptation to chronic hypoxia is associated with increased generation of reactive oxygen species which is implicated in the induction of the improved cardiac tolerance against acute ischemia/reperfusion injury. Phospholipases A2 (PLA2) hydrolyze fatty acids from the sn-2 position of membrane phospholipids and help to keep the stability and integrity of cell membranes via deacylation/reacylation cycle during increased oxidative stress. They preferentially hydrolyze peroxidized fatty acids from the membranes and produce lipid signalling molecules, e.g. lysophospholipids and free fatty acids serving also as precursors of eicosanoids. The aim of this study was to compare effects of intermittent hypobaric hypoxia (IHH, 8h/day, 5 weeks, 7000 m), continuous normobaric hypoxia (CNH, 24 h/day, 3 weeks, 10.5 % O2) and intermittent normobaric hypoxia (INH, 23 h/day, 3 weeks, 10.5 % O2) on the expression of secretoric (sPLA2), cytosolic (cPLA2) and calcium-independent (iPLA2) phospholipases and a key mitochondrial antioxidative enzyme, manganese superoxide dismutase (MnSOD), in left ventricular myocardium of adult male Wistar rats by Western blotting. IHH decreased the expression of sPLA2, cPLA2 and iPLA2 to 54 %, 87 % and 72 %, respectively, and increased MnSOD expression only in the mitochondrial fraction to 123% compared with the normoxic group. CNH increased cPLA2, iPLA2 and MnSOD expressions in homogenates to 120 %, 157 % and 124 %, respectively, compared with the normoxic group. Surprisingly, PLA2 and MnSOD expressions were not affected by INH. These data show that various models of chronic hypoxia and adaptation protocols exert diverse effects on the expression of PLA2 and MnSOD. The dinucleotide Up4A induce monocyte chemoattractant protein-1 production via NADPH oxidase activation M Schuchardt M Schuchardt 1 Charite - Campus Benjamin Franklin , Berlin , Germany M Toelle M Toelle 1 Charite - Campus Benjamin Franklin , Berlin , Germany N Pruefer N Pruefer 1 Charite - Campus Benjamin Franklin , Berlin , Germany J Pruefer J Pruefer 1 Charite - Campus Benjamin Franklin , Berlin , Germany V Jankowski V Jankowski 1 Charite - Campus Benjamin Franklin , Berlin , Germany J Jankowski J Jankowski 1 Charite - Campus Benjamin Franklin , Berlin , Germany M Van Der Giet M Van Der Giet 1 Charite - Campus Benjamin Franklin , Berlin , Germany Abstract Purpose: There is a growing body of evidence that nucleotides and purinergic signaling play a crucial role in the development of vascular diseases such as atherosclerosis. Uridine adenosine tetraphosphate (Up4A), an endothelium-derived dinucleoside polyphosphate, seems to have atherogenic properties. The production and secretion of chemokines, e.g. monocyte chemoattractant protein-1 (MCP-1) is a crucial condition in inflammatory response which is often triggered by NADPH oxidase. Here, we investigated the effect of Up4A on MCP-1 production in vascular smooth muscle cells. Furthermore, the activation of NADPH oxidase after Up4A treatment of the cells is investigated. Methods: Rat vascular smooth muscle cells (VSMCs) were used for experiments. MCP-1 and Nox1/4 expression was measured by real-time PCR. Rac1 phosphorylation was measured by ELISA. Translocation of p47phox was detected by Western Blot technique in membrane fraction and cytosolic protein fraction of the cells. H2O2 production was examined by loading VSMCs with 5,6-chloromethyl-2′,7′-dichlorodihydrofluorescein-dieacetate-acetylester (CM-H2DCFDA). Results: Up4A treatment dose-dependently induce MCP-1 expression and secretion in VSMCs (expression: max. 52 ± 5fold [100 µmol/l]; logEC50 [mol/l]: -5.3 ± 0.2; n = 5; secretion: max. MCP-1 [100 µmol/l]: 1641 ± 42 pg/mg; logEC50 [mol/l]: -5.8 ± 0.4; n = 6). Tiron, a vitamin E analog, is able to significantly diminish the Up4A-induced MCP-1 expression (48 ± 23% decrease, n = 5). Furthermore, incubation of CM-H2DCFDA-labeled VSMCs with Up4A resulted in a significant and dose-dependent increase in DCF fluorescence intensity (n = 6). NADPH oxidase is composed of different subunits. The expression of Nox1 is increased after Up4A stimulation of the cells, whereas Nox4 expression is decreased. The activation of NADPH oxidase requires phosphorylation of Rac1 and translocation of p47phox to the plasma membrane. Up4A induced Rac1 phosphorylation in a significant and time-dependent manner. P47phox amount in membrane fraction increased time-dependently after Up4A stimulation, whereas the amount in the cytosolic protein fraction decreased, respectively. Conclusions: In this study we could show that Up4A is a potent inductor of MCP-1 expression and secretion in VSMCs. NADPH oxidase, the predominant source of ROS in the vasculature, is activated by Up4A and known to regulate MCP-1 production in VSMCs. Therefore, the endothelial-derived factor Up4A is not only a potent vasoconstrictor but in addition a potent inductor of pro-inflammatory response in the vascular wall. Hyperoxia increases peroxynitrite production due to increased association of eNOS with beta-actin in pulmonary artery endothelial cells W Han W Han 1 Medical College of Georgia , Augusta , United States of America Y Su Y Su 1 Medical College of Georgia , Augusta , United States of America Abstract Oxygen toxicity is the most severe side-effect of oxygen therapy in neonates and adults. The cellular damage of oxygen toxicity is related to over-production of reactive oxygen species (ROS) and subsequent formation of reactive nitrogen species, such as peroxynitrite. In the present study, we investigated the effect of hyperoxia on the production of peroxynitrite in pulmonary artery endothelial cells (PAEC). PAEC were exposed to normoxia (21% oxygen, 74% nitrogen, and 5% CO2) and hyperoxia (95 % oxygen and 5% CO2) at 37°C for 24 h, then peroxynitrite, NO release, endothelial NO synthase (eNOS) activity, superoxide production, and association of eNOS with beta-actin were measured. Incubation of PAEC with 95% oxygen for 24 h resulted in an increase in the intracellular level of peroxynitrite. The presence of uric acid, a peroxynitrite scavenger, prevented hyperoxia-induced increase in peroxynitrite. The increase in the production of peroxynitrite is accompanied by increases in eNOS activity, NO release, superoxide production, and association of eNOS with beta-actin in PAEC. To study whether beta-actin-eNOS association contributes to increased peroxynitrite production and NO release, beta-actin-eNOS interaction were inhibited by reducing beta-actin availability or by using a synthetic peptide (P326TAT) containing a sequence corresponding to actin binding site on eNOS (aa 326-333 of human and porcine eNOS). We found that disruption of beta-actin-eNOS interaction prevented hyperoxia-induced increase in beta-actin-eNOS association, eNOS activity, NO release, and peroxynitrite production. Hyperoxia failed to induce the increases in eNOS activity, NO release, and peroxynitrite formation in COS-7 cells transfected with plasmids containing cDNA for eNOS mutant in which amino acids leucine and tryptophan were replaced with alanine in the actin binding site on eNOS. Taken together, our data indicate that increased association of eNOS with beta-actin in PAEC contributes to hyperoxia-induced increase in the production of peroxynitrite which may cause nitrosative stress in pulmonary vasculature. Creatine reduces mitochondrial permeability transition pore opening in response to oxidative stress in vitro S Zervou S Zervou 1 University of Oxford , Oxford , United Kingdom D Aksentijevic D Aksentijevic 1 University of Oxford , Oxford , United Kingdom C Lygate C Lygate 1 University of Oxford , Oxford , United Kingdom S Neubauer S Neubauer 1 University of Oxford , Oxford , United Kingdom Abstract Purpose: Creatine (Cr) plays an important role in energy metabolism inthe heart via the creatine kinase (CK) energy transfer and buffering system. Formation of the mitochondrialpermeability transition pore (MPTP) is involved in the releaseof mitochondrial components triggering apoptotic/necrotic cell death, forexample, in response to ischaemia/reperfusion in vivo, or to exposure to oxidative stress in vitro. Due to the localization of mitochondrial-CK to the inter-mitochondrial membrane space we hypothesized that creatine could have a modulatory role in MPTP pore opening. Methods: Intracellular creatine levels are dependent on activity of a specific plasma membrane creatine transporter (CrT), we therefore elevated intracellular Cr by stably transfecting 3T3 fibroblastswith the rat CrT and adding Cr (250 μM) to the culture media. Cells were loaded with 2-5 μM calcein-AM, for 15 min at 37°C and nuclei counter-stained with propidium iodide. Cobalt chloride and ionomycin were used to quench cytoplasmicand total cell fluorescence, respectively. Under these conditions fluorescenceis attributable to calcein in the mitochondria, and signal loss is indicative of MPTP opening. Cells were exposed to either hydrogen peroxide (300 μM) or calcium chloride (5 μM) to trigger MPTP activation in the presence or absence of Cr, and analysed by a flow cytometer equipped with a 488-nm argon laser. Results: Exposure of cells to calcium chloride caused a marked increase in pore activation, by 2-fold, and exposure to hydrogen peroxide a 4-fold increase (P < 0.01). In control experiments, this effect was blocked by Cyclosporin A (CsA; 0.2 μM) demonstrating specificity for MPTP activation. Pore activation by hydrogen peroxide could be partly inhibited by pre-incubation with Cr, which reduced the levels of MPTP activation by 50% (P < 0.01). Conclusions: Our preliminary results suggest that creatine can protect against oxidative stress in vitro by inhibition of MPTP opening. Further work is required to elucidate the mechanism of action, and to determine whether Cr can modulate the MPTP in vivo. Effects of cinaciguat on endothelial dysfunction induced by peroxynitrite in rat aorta B Seidel B Seidel University of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany S Korkmaz S Korkmaz University of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany T Radovits T Radovits University of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany K Hirschberg K Hirschberg University of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany S Loganathan S Loganathan University of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany E Barnucz E Barnucz University of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany M Karck M Karck University of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany G Szabo G Szabo University of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany Abstract Reactive oxygen and nitrogen species (e.g. peroxynitrite) induce nitro-oxidative stress and DNA injury leading to endothelial dysfunction. Elevated intracellular cyclic GMP (cGMP) may contribute to an effective cytoprotection against nitro-oxidative stress. The novel soluble guanylate cyclase (sGC)-activator cinaciguat, has been reported to elevate intracellular cGMP-levels and activate the NO-cGMP-PKG-pathway in vivo. We investigated the effects of cinaciguat treatment, on endothelial dysfunction induced by peroxynitrite. In organ bath experiments, we investigated the endothelium-dependent (acetylcholine, ACh) and -independent (sodium nitroprusside, SNP) vasorelaxation of isolated aortic rings of rats. Endothelial dysfunction was induced by peroxynitrite (200 μM). In the treatment-group, rats were treated orally 2 times with cinaciguat (10 mg/kg). Immunohistochemical analysis was performed for cGMP and nitrotyrosine. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) was used for detection of DNA-strand breaks. In-vitro exposure of aortic rings to peroxynitrite resulted in an impairment of endothelium-dependent vasorelaxation (maximal relaxation (Rmax) to ACh: 93 ± 2% control vs. 45 ± 6% peroxynitrite). Cinaciguat-treatment led to an improvement of endothelial function (Rmax to ACh: 45 ± 6% peroxynitrite vs. 67 ± 3% cinaciguat+peroxynitrite). The endothelium-independent vascular smooth muscle function, indicated by the vasorelaxation to SNP in peroxynitrite and cinaciguat+peroxynitrite-groups showed a significant shift of the concentration-response curves to the right and a decreased sensitivity without any alterations of the Rmax. Cinaciguat-treatment significantly increased cGMP-score, reduced nitro-oxidative stress and reduced DNA-breakage in the aortic wall. Our results support the view that impairment of intracellular cGMP-signalling plays a role in the pathogenesis of endothelial dysfunction induced by peroxynitrite which can be effectively reversed by pharmacological sGC-activation. HOX-1 and COX-2: two key mediators regulating C2 myoblast tolerance to oxidative stress IK Aggeli IK Aggeli 1 University of Athens, School of Biology, Dept. Animal and Human Physiology , Athens , Greece E Kefaloyianni E Kefaloyianni 1 University of Athens, School of Biology, Dept. Animal and Human Physiology , Athens , Greece I Beis I Beis 1 University of Athens, School of Biology, Dept. Animal and Human Physiology , Athens , Greece C Gaitanaki C Gaitanaki 1 University of Athens, School of Biology, Dept. Animal and Human Physiology , Athens , Greece Abstract Purpose: A plethora of clinical trials have proven the role of skeletal myoblasts in cell-based therapy for cardiac repair and regeneration. In particular, their engraftment into post-infarcted myocardium has been found to exert salutary effects but the mediating therapeutic mechanisms remain obscure. Given that oxidative stress is strongly related to myocardial ischemia/reperfusion as well as infarction, an effort was made in the present study to investigate C2 skeletal myoblasts' tolerance against hydrogen peroxide treatment and unravel the respective regulatory transduction mechanisms. Evidently, it is important to identify paracrine factors released by skeletal myoblasts that could potentially mediate their beneficial cardiotropic actions during reactive oxygen species (ROS) imbalances. Methods: To this end, we decided to investigate HOX-1 potential involvement, along with cyclooxygenase-2 (COX-2) response since H2O2 is known to stimulate prostaglandin (PG) production. Therefore, Western blot and RT-PCR were initially performed in order to examine HOX-1 and COX-2 protein and mRNA levels after exposure to 0.5mM of H2O2 while, with the use of inhibitors and EMSA, the mediators of these signalling cascades were also investigated. Measurement of PGE2 levels was used as an indicator of COX-2 activity and cell viability was also assessed by MTT assay, under the interventions studied. Results: H2O2 treatment (0.5 mM) resulted in a time- and dose-dependent response of HOX-1 and COX-2 mRNA and protein levels, with ERK1/2, p38-MAPK and MSK1 found to mediate these effects. Furthermore, Src and JNKs blockade attenuated COX-2 response. AP1 DNA binding activity was also found to be modulated in a time-dependent manner after H2O2 treatment, with a profile demonstrated to be JNK- and Src-mediated. The physiological roles of COX-2 and HOX-1 were next evaluated by use of their respective inhibitors (sc236 and ZnPP) and their salutary effect on myoblasts survival under these interventions was demonstrated. Conclusions: Collectively, our novel findings highlight the signalling cascades regulating HOX-1 and COX-2 expression and their fundamental contribution to skeletal myoblasts' tolerance under oxidative stress. Furthermore, increased biosynthesis and release of PGE2 was revealed to mediate COX-2 beneficial role. With skeletal myoblasts use as a therapeutic intervention, it is evident that further studies are needed in order to delineate the mechanism involved in HOX-1 and COX-2 potential role in myocardial remodelling after redox imbalances related to infarction. Bradykinin, insulin and opioids mimic ischaemic postconditioning via the SAFE pathway L Lacerda L Lacerda University of Cape Town , Cape Town , South Africa S Somers S Somers LH Opie LH Opie S Lecour S Lecour Abstract Purpose: Ischaemic postconditioning (IPostC) confers protection via the SAFE (Survivor Activating Factor Enhancement) pathway that involves the cytokine, tumour necrosis factor alpha (TNFα), the TNF Receptor 2 (TNFR2) and the transcription factor, Signal Transducer and Activator of Transcription-3 (STAT-3). We propose that activation of this pathway is also crucial for bradykinin, insulin and opioids to confer protection against reperfusion injury. Methods: Langendorff retrograde perfusion was performed on isolated hearts from TNFα deficient (TNFα−/−), TNFR1 deficient (TNFR1−/−), TNFR2 deficient (TNFR2−/−) and cardiomyocyte- specific STAT-3 deficient (STAT-3−/−) mice and their respective wildtypes (WT). Pharmacological postconditioning with each cardioprotective agent was performed by 6 alternating 10 sec cycles of reperfusion alone or reperfusion with either bradykinin (Bk-PostC), insulin (Ins-PostC), or DADLE, the delta opioid agonist, (D-PostC). Infarct size (IS) was evaluated as an endpoint by triphenyltetrazolium staining. Results: Bk-PostC, Ins-PostC and D-PostC reduced infarct size by 30%, 31% and 29%, respectively in WT, p < 0.01 versus control and by 28%, 32% and 26%, respectively in TNFR1−/−, p < 0.01 versus control. However, all three agents failed to protect TNF−/− and TNFR2−/− hearts. Similarly, bradykinin failed to protect against reperfusion injury in STAT-3−/− mice. Conclusion: Activation of TNFα via its receptor 2 and STAT-3 is crucial for cardioprotection afforded by bradykinin, insulin and opioids, therefore emphasizing the essential role of the SAFE pathway in pharmacological postconditioning. Pharmacological manipulation of the vagally-mediated release of nitric oxide: role of post-ganlionic neurons, muscarinic receptors, vasoactive intestinal peptide and the endothelium K Brack K Brack 1 University of Leicester , Leicester , United Kingdom JH Coote JH Coote 2 University of Birmingham , Birmingham , United Kingdom GA Ng GA Ng 1 University of Leicester , Leicester , United Kingdom Abstract Propose: We have previously shown that nitric oxide (NO) is released during vagus nerve stimulation (VS) in the cardiac ventricle which modulates the inducibility of ventricular fibrillation via effects on electrical restitution. There is no information on the signaling pathways involved or the origin of NO that is released. In this study we measured NO release during muscarinic receptor and vasoactive intestinal peptide (VIP) inhibition, ganglionic blockade and endothelium removal in the isolated rabbit heart preparation with intact autonomic nerves. Methods: NO was measured using 4,5-diaminofluorescein diacetate (DAF) fluorescence measured at 490 nm during constant right ventricular pacing at 200 bpm. DAF fluorescence was measured at baseline (BL) and during VS (10-15 Hz, 10-15 V), before and during perfusion with 0.1 µM Atropine, 20 nM VIP(6-28), 0.5 mM Hexamethonium and after perfusion with 0.2% bolus injection of Triton-X (0.1 ml). Data are mean ± SEM, statistical analysis using 2-factor ANOVA, n = 11, weight 2.8 ± 0.1 kg. Results: The VS mediated increase in NO-dependent fluorescence was not affected by perfusion of atropine (increase of 2.1 ± 0.4% [BL] & 1.7 ± 0.2% [VS], NS, n = 8), VIP(6-28) (increase of 1.8 ± 0.3% [BL] & 1.9 ± 0.5% [VS], NS, n = 3) or endothelium denudation (increase of 1.5 ± 0.3% [BL] & 2.1 ± 0.8% [VS], NS, n = 5) but was abolished during perfusion with hexamethonium (increase of 2.4 ± 0.4% [BL] & -0.1 ± 0.5% [VS], P < 0.05, n = 5). Conclusions: These data suggest that the release of NO during VS originates from post-ganglionic efferent nerves in the ventricle but not due to antegrade stimulation of afferent vagal fibres. The NO release is muscarinic receptor and VIP independent and does not involve the endothelium as a source of NO. The context of these findings needs further exploration in relation to the possibility of a tentative parallel vagus-NO pathway in the ventricle. Stress cardiomyopathy: a role for stimulus trafficking at the beta-2 adrenoceptor in cardiac depression? HE Paur HE Paur 1 Imperial College London, National Heart & Lung Institute , London , United Kingdom V Nikolaev V Nikolaev 2 University of Wurzburg, Institute of Pharmacology and Toxicology , Wurzburg , Germany A Lyon A Lyon 1 Imperial College London, National Heart & Lung Institute , London , United Kingdom SE Harding SE Harding 1 Imperial College London, National Heart & Lung Institute , London , United Kingdom Abstract Background: Stress cardiomyopathy (SC) is characterised by acute akinetic apical and hyperkinetic basal myocardium. At supraphysiological adrenaline levels, protein kinase A (PKA)-mediated phosphorylation of the β2-adrenoceptor (β2AR) may induce a switch in coupling from the Gs-PKA pathway to the inhibitory Gi protein pathway. Higher β2AR levels in the apical myocardium would explain the regional changes observed in SC. Methods: Left-ventricular (LV) cardiomyocytes were isolated from adult rat myocardium. β2AR-mediated contraction to isoproterenol was tested in the presence of the β1AR blocker CGP20712A, either with or without pre-incubation with the Gi inhibitor pertussis toxin. β2AR and β1AR densities were quantified from binding curves of apical and basal cardiomyocyte membranes constructed in the presence of the βAR radioligand [I125]CYP and increasing concentrations of the β2AR blocker ICI118551. In vivo left ventricular responses to intravenous catecholamines were recorded using echocardiography. Results: Rapid intravenous infusion of 10-4M adrenaline, but not an equivalent dose of noradrenaline, induced a significant negative inotropic effect (change in fractional shortening (FS)) at the base (-7.89 ± 11.7% p < 0.05 vs untreated (adrenaline); 9.58 ± 0.52% p < 0.001 vs untreated (noradrenaline)). This effect of adrenaline was more pronounced at the apex (-19.7 ± 5.13% p < 0.05 vs untreated, p = 0.051 vs base). Minimal change to apical FS was observed with noradrenaline (-1.21 ± 8.44% p>0.05 vs untreated) (n = 5 (adrenaline) n = 4 (noradrenaline)). Apical cardiomyocytes showed a larger positive inotropic β2AR response compared to basal cardiomyocytes (0.97 ± 0.21 fold increase from untreated (apex) vs 0.60 ± 0.13 fold increase from untreated (base)(p < 0.01 n = 10 (apex), n = 13 (base)). Prolonged in vitro β2AR stimulation presents as a maximum positive inotropy preceding a steady fall towards pre-treatment levels, an effect alleviated by Gi inhibition. A larger proportion of β2ARs exist at the apex (65.2 ± 4.3%) compared to the base (41.1 ± 5.4%) (p < 0.01; n = 4 binding experiments). Conclusion: Rapid intravenous infusion of high doses of adrenaline, which has a higher affinity for the β2AR and Gi signalling pathways than noradrenaline, tends to induce a greater negative inotropic effect in the apical myocardium compared to basal regions. This is consistent with a larger β2AR component within the apex of the mammalian ventricle, compared to the base. We propose that excessive β2AR activation following surges in plasma adrenaline leads to coupling of β2ARs to Gi-signalling that may mediate the apical akinesis observed in SC. The acute effects of urocortin 2 on the diastolic properties of the myocardium C Bras-Silva C Bras-Silva 1 University of Porto, Faculty of Medicine , Porto , Portugal AP Fontes-Sousa AP Fontes-Sousa 1 University of Porto, Faculty of Medicine , Porto , Portugal S Silva S Silva 1 University of Porto, Faculty of Medicine , Porto , Portugal M Gomes M Gomes 1 University of Porto, Faculty of Medicine , Porto , Portugal P Ferreira P Ferreira 1 University of Porto, Faculty of Medicine , Porto , Portugal AF Leite-Moreira AF Leite-Moreira 1 University of Porto, Faculty of Medicine , Porto , Portugal Abstract Purpose: The urocortin (Ucn) peptides are recently isolated members of the highly conserved corticotrophin-releasing factor (CRF) family. Ucn2 enhances contractility via CRF2 receptor-mediated stimulation of protein kinase (PK) A. As PKA is a well known modulator of diastolic function, we investigated the acute effects of Ucn2 on myocardial diastolic properties and the underlying mechanisms. Methods: The effects of increasing concentrations of Ucn2 (10-10 to 10-6M) were evaluated in right ventricular papillary muscles isolated from male New Zealand White rabbits (Krebs-Ringer: 1,8mM CaCl2, 35°C), in the absence (n = 12) and in the presence of: (i) PKA inhibitor (H89, 10-6M, n = 9) or (ii) PKC inhibitor (Chelerythrine, 10-5M, n = 9). Passive length-tension relations were constructed before and after a single concentration of Ucn 2 (10-6M; n = 7). Reported parameters include passive tension (PT; mN/mm2) and muscle length (L; L/Lmax). Results: Ucn2 induced a concentration-dependent increase in resting muscle length up to 1.012 ± 0.004 L/Lmax at 10-6M. Correcting muscle length to its initial value resulted in a 29.6 ± 8.9% decrease of PT, indicating a decrease in muscle stiffness, also evident in the down and rightward shift of the passive length-tension relation. This effect was attenuated in the presence of either PKA or PKC inhibitor. Ucn2 (10-6M) induced an increase in resting muscle length of 1.005 ± 0.002 L/Lmax, in the presence of H89, and of 1.006 ± 0.002 L/Lmax in the presence of Chelerythrine, corresponding to a decrease of PT of only 11.0 ± 4.0%, and 13.3 ± 6.4%, respectively. Conclusions: Ucn2 acutely decreases myocardial stiffness, an effect that is dependent on PKA and PKC activation. This is a potentially powerful physiologic mechanism, as it may allow the heart to reach the same diastolic volume with up to 30% lower filling pressures. These findings reinforce the relevance of Ucn2 in the pathophysiology of heart diseases. Steroids regulate cacna1g expression and T-type-generated calcium signaling V Capuano V Capuano 1 INSERM U999, hopital Marie Lannelongue , le plessis robinson , France L Ferron L Ferron 2 University College London , London , United Kingdom Y Ruchon Y Ruchon 1 INSERM U999, hopital Marie Lannelongue , le plessis robinson , France F Ben Mohamed F Ben Mohamed 1 INSERM U999, hopital Marie Lannelongue , le plessis robinson , France J-F Renaud J-F Renaud 1 INSERM U999, hopital Marie Lannelongue , le plessis robinson , France Abstract The Ca2+ entry through T-type channels is reported to have two main roles, one dedicated to cell excitability and the other dedicated to the stimulation of a range of Ca2+ dependent biological events. However, little is known about the mechanism involved in the regulation of T-type channel expression and the dependent Ca2+ signaling pathway remains to be explored. We have investigated the effect of the steroids on the regulation of the T-type Ca2+ channels expression in cultures of neonatal rat ventricular myocytes. We found that aldosterone and dexamethasone increased T-type Ca2+ current (ICaT) associated with an increase in Cav3.1 mRNA. We isolated the upstream region from Cav3.1 encoding gene (cacna1g) and tested the activity of the promoter in ventricular myocytes. We found a minimal steroid-responsive region that displayed putative glucocorticoid receptor (GR) and nuclear factor kappa-B (NFκB) targets. The GR selective antagonist, RU38486 (10 µM), almost turned off the transcriptional activity of Cav3.1 encoding gene and a NFκB inhibitor (pyrrolodine dithiocarbonate, 10 µM), completely abolished the steroid-induced mRNA increase. Although aldosterone and dexamethasone triggered similar response on cacna1g promoter, we found that this response was mediated through specific glucocorticoid responsive elements likely required for the binding with mineralocorticoid- or glucocorticoid-related signaling targets. We looked for the signaling pathways that regulated cana1g expression and we found that steroid-induced P38-MAPK and ERK1/2 signaling pathways exhibited opposite effects. Indeed, a P38-MAPK inhibitor (SB203580, 10 µM), abolished dexamethasone-induced increase in cacna1g promoter activity whereas the inhibitor of the ERK1/2 activation, PD98059 (10 µM), induced a substantial increase in the promoter activity. And finally, we have shown that ICaT blockers (mibefradil, 1 µM; flunarizine, 10 µM; fluoxetine, 5 µM) increased the amount of Cav3.1 mRNA and promoter activity. In conclusion, we have defined several signaling pathways and steroid-related responsive elements involved in the regulation of cacna1g expression. Also, we have shown that ICaT-generated Ca2+ signaling exerts a negative feedback on gene expression and future experiments will focus on the link between T-type Ca2+ influx and gene expression. Changes in the apelinergic system and correlation between plasma levels of apelin and myocardial hypertrophy in rats and humans I Falcao-Pires I Falcao-Pires 1 University of Porto, Faculty of Medicine , Porto , Portugal N Goncalves N Goncalves 1 University of Porto, Faculty of Medicine , Porto , Portugal C Gavina C Gavina 2 Sao Joao Hospital , Porto , Portugal S Pinho S Pinho 1 University of Porto, Faculty of Medicine , Porto , Portugal C Moura C Moura 1 University of Porto, Faculty of Medicine , Porto , Portugal MJ Amorim MJ Amorim 2 Sao Joao Hospital , Porto , Portugal P Pinho P Pinho 2 Sao Joao Hospital , Porto , Portugal AF Leite-Moreira AF Leite-Moreira 1 University of Porto, Faculty of Medicine , Porto , Portugal Open in new tabDownload slide Abstract 478 Figure Apelin levels and correlation with LVMI Open in new tabDownload slide Abstract 478 Figure Apelin levels and correlation with LVMI Abstract Purpose: Cardiac hypertrophy is a (de)compensatory response to haemodynamic overload that takes place during ventricular remodelling. The apelinergic system is an emerging neurohumoral system in cardiovascular pathophysiology which has been shown to be altered in hypertrophied and diabetic hearts. We aimed to investigate the impact of left ventricular (LV) pressure-overload and diabetes on the apelinergic system. Methods: Pressure-overload was established in rats by supra-renal aortic-banding. Six-weeks later, diabetes was induced by streptozotocin (65mg/kg,ip), resulting in 4 groups: SHAM, banded (BA), diabetic (DM) and diabetic-banded (DM-BA). Twelve weeks later, LV function and structure were evaluated by echocardiography and biopsies and plasma samples collected. Furthermore, plasma samples and LV-endomyocardial biopsies were procured from aortic stenosis and mitral stenosis patients during surgery to evaluate myocardial expression of apelin and APJ-receptor and plasma levels of apelin. Results: Direct correlations between apelin plasma levels and LV-mass index and between apelin and APJ myocardial expression were observed both in humans and rats (Fig A). Expression of apelin and APJ was not significantly altered by pressure-overload in humans, being downregulated by pressure-overload and even more by diabetes in rats (Fig B). Finally, an inverse correlation between apelin rat plasma levels and its myocardial expression was observed. Conclusions: While apelin/APJ myocardial expression decreases, apelin plasma levels increase in LV-hypertrophy. Considering apelin's positive inotropic and vasodilator properties, this elevation in apelin plasma levels may represent a compensatory mechanism to maintain inotropism and cardiac output during pressure-overload or diabetic cardiomyopathy 1 . Evidence against b3-adrenoceptor (b3AR)-mediated increases in L-type Ca2+ current ICa,L and contractile force in human atrial myocardium at physiological temperature T Christ T Christ 1 Dresden University of Technology, Department of Pharmacology and Toxicology , Dresden , Germany P Molenaar P Molenaar 2 Queensland University of Technology , Brisbane , Australia U Ravens U Ravens 1 Dresden University of Technology, Department of Pharmacology and Toxicology , Dresden , Germany AJ Kaumann AJ Kaumann 3 University of Cambridge , Cambridge , United Kingdom Abstract Purpose: Stimulation of cardiac β3ARs was suggested to increase human atrial L-type Ca2+ currents (ICa,L) and force of contraction (Fc). Since β3AR agonists are being developed for the treatment of various diseases, cardiostimulatory effects could be potentially harmful and compromise clinical application. Methods: ICa,L was measured in voltage-clamped human atrial myocytes, isometric Fc was recorded in electrically stimulated trabeculae. The putative β3AR-agonists SR58611, BRL37344 and CGP12177 were used, effects via β1/β2AR were excluded by addition of nadolol. Experiments were performed at 24°C and 37°C. Results: We confirmed that ICa,L increased in response to the 3 βAR agonists (1 µM each) at 24°C, but unlike in the earlier report, the effects were small compared to isoprenaline. Increases in ICa,L with SR58611 and BRL37344 were antagonized by the β3AR-selective blocker L-748,337, the response to CGP12177, however, was antagonized only by bupranolol. Unlike nadolol, this β1/β2/β3AR antagonist also blocks the low affinity site of the β1AR (β1LAR), suggesting that CGP12177 effects are mediated via β1LAR. The small increase in ICa,L obtained by β3AR stimulation with SR58611 was not accompanied by an increase in Fc, whereas the same increase in ICa,L obtained with β1LAR stimulation by CGP12177 produced a small, but significant positive inotropic effect suggesting that β3AR-mediated responses are restricted to Ca2+ channels. At 37°C we could not detect any increase in ICa,L and Fc by SR58611 and BRL37344 that were mediated by β3AR, even in the presence of IBMX (10 µM). The positive inotropic effects with high concentrations of BRL37344 were not antagonized by L748,337, but only by the β2-selective blocker ICI118,551, suggesting that BRL37344 had surmounted the β2AR block by nadolol. At physiological temperature IBMX was required for facilitation of the small β1LAR-mediated increases of ICa,L and FC by CGP12177. Both effects were sensitive to bupranolol but not to L748,337. Conclusions: Increase in ICa,L by β3AR stimulation is restricted to unphysiologically low temperature, is of small size and is not accompanied by a positive inotropic effect. Chest pain due to coronary artery disease alters stress neuropeptide levels:Potential implications for clinical assessment E Kletsiou E Kletsiou 1 University of Athens, Attikon Hospital, 2nd Department of Cardiology , Athens , Greece M Giannakopoulou M Giannakopoulou 2 University of Athens, Faculty of Nursing , Athens , Greece E Bozas E Bozas 2 University of Athens, Faculty of Nursing , Athens , Greece E Iliodromitis E Iliodromitis 1 University of Athens, Attikon Hospital, 2nd Department of Cardiology , Athens , Greece M Anastasiou-Nana M Anastasiou-Nana 1 University of Athens, Attikon Hospital, 2nd Department of Cardiology , Athens , Greece EDE Papathanassoglou EDE Papathanassoglou 3 Department of Nursing, Cyprus University of Technology , Nicosia , Cyprus Abstract Purpose: Upon stress, specific neuropeptides are released into the systemic circulation. Substance P (SP) is a neuropeptide involved in nociception, and stress perception along with neuropeptide Y (NPY). We sought to determine: a) alterations in plasma SP and NPY levels upon the experience and alleviation of angina pectoris, b) associations between stress neuropeptide levels and intensity of chest pain as well as the severity of the underlying disease and c) possible differences between patients and gender - age matched controls. Method: A descriptive correlation repeated measures design was employed. A group of coronary critical care adults with retrosternal pain, fulfilling the criteria of acute coronary syndrome and a control group of healthy gender-age matched volunteers were studied. Blood samples were collected during an episode of pain associated with ECG changes and 24 hours later, where no pain or ECG changes were reported. Neuropeptide levels were quantified by an immunosorbent (ELISA) assay. The level of pain was assessed by behavioural pain scales (Payen's 2001- Puntillo's 1997 pain scale) and the numeric visual analogue scale. Clinical severity was quantified by APACHE-II and the Multiple Organ Dysfunction Score (MODS). Results: NPY and SP levels were significantly increased in patients with coronary artery disease during the episode of pain versus no pain (p = 0.03, 0.04) respectively. In patients' group, a positive correlation between SP and NPY levels respectively and VAS pain ratings (r = 0.474, 0.565, p = 0.02), ratings at the Puntillo (r = 0.474, 0.563, p = 0,010) and the Payen scales (r = 0.562, 0.737, p < 0.0001) was observed. SP and NPY levels exhibited significant inverse association with MODS scores (r = −0.404 -0.450, p < 0.03). NPY levels in both pain and free of pain conditions were positively correlated with troponin (r = 0.469 0.64 p = 0.009 0.001) and other circulating biomarkers such as γGT, CK, LDH, SGOT, SGPT (r = 0.42–0.74 p < 0,001). Significant differences between AMI patients and controls were found in SP (Δ M = −199.97 pg/ml, p = 0.04) while significant differences in NPY levels were observed not only in AMI but in all patients with coronary artery disease compared to the controls (p < 0.04). Conclusions: Elevated SP and NPY levels in critically ill patients suffering from angina pectoris or AMI and their association with pain perception, indicate that they could be used as reliable markers of the level of chest pain and the degree of distress. Further studies are necessary to elucidate the effectiveness and feasibility of plasma SP and NPY levels as useful biomarkers. Effects of two types of restraint stress on the expression of oxytocin receptor in rat heart M Chottova Dvorakova M Chottova Dvorakova 1 Charles University Prague, 1st Faculty of Medicine, Institute of Medical Biochemistry , Prague , Czech Republic E Mistrova E Mistrova 2 Charles University Prague, Faculty of Medicine in Pilsen , Pilsen , Czech Republic J Slavikova J Slavikova 2 Charles University Prague, Faculty of Medicine in Pilsen , Pilsen , Czech Republic S Hynie S Hynie 1 Charles University Prague, 1st Faculty of Medicine, Institute of Medical Biochemistry , Prague , Czech Republic P Sida P Sida 1 Charles University Prague, 1st Faculty of Medicine, Institute of Medical Biochemistry , Prague , Czech Republic V Klenerova V Klenerova 1 Charles University Prague, 1st Faculty of Medicine, Institute of Medical Biochemistry , Prague , Czech Republic Abstract Oxytocin (OT), recognized traditionally as a hormone with a major role in childbirth and lactation, is produced in hypothalamic supraoptic and paraventricular nuclei. However, evidence has emerged suggesting its involvement in the regulation of the cardiovascular system, since OT-containing axons terminate in several brain stem nuclei known to be involved in cardiovascular control. Additionally, OT and its receptors (OTR) are known to be present in the heart. During stressful conditions several cardiovascular parameters are altered. Here, we have tested hypothesis, whether OTR mRNA expression in the heart could be affected by stress. Heart preparations were analyzed by real-time RT-PCR. Relative expression of OTR mRNA was expressed as a ratio of target gene CT value to CT value of housekeeping gene – beta-actin. The results were considered significantly different when p < 0.05. We used Sprague-Dawley (S-D) and Lewis (LEW) rat strains, the latter being known to have a deficient responsiveness of the hypothalamic-pituitary-adrenal axis. A restraint stressor (immobilization, IMO) and restraint stressor combined with partial immersion of rats into water (IMO+C) were applied to rats for 1 hour. One or 3 hours after the stress termination rats were killed by decapitation, heart was removed, and atria and ventricles were separated. OTR mRNA expressions were determined in four groups of animals (IMO1, IMO3, IMO+C1, IMO+C3) and compared to control animals. While the expression of OTR mRNA in control animals was more than 30 times higher in the atria than in the ventricles, atria of all experimental groups were subjected to quantitative analysis of OTR mRNA. Expression of OTR mRNA was similar in the left atria in both rat strains but in the right atria was about four times higher in LEW rats than in S-D rats. IMO3 as well as IMO+C3 caused in both strains significant decrease of the mRNA expression in the left atrium, however, there were no significant changes in the right atria. In summary, exposure to two types of restraint stressors caused significant changes in OTR mRNA production in the left atrium, which may have relevance in cardiovascular control. Granted by MSM 0021620806 and MSM 0021620819. n-3 fatty acids contribute to plaque stability differentially affecting the release of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human monocytes/macrophages in culture M Massaro M Massaro 1 Institute of Clinical Physiology-CNR , Lecce , Italy E Scoditti E Scoditti 1 Institute of Clinical Physiology-CNR , Lecce , Italy MA Carluccio MA Carluccio 1 Institute of Clinical Physiology-CNR , Lecce , Italy C Storelli C Storelli 2 University of Salento, Department of Biological and Environmental Science and Technology (DiSTeBA) , Lecce , Italy A Distante A Distante 1 Institute of Clinical Physiology-CNR , Lecce , Italy R De Caterina R De Caterina 3 G. D'Annunzio University , Chieti , Italy Abstract 482 Table . MMP-1 . MMP-2 . MMP-9 . TIMP-1 . TIMP-2 . Control 4.6 ± 0.5 3.0 ± 1.0 5.0 ± 0.8 60.0 ± 7.0 18.5 ± 2.1 PMA+TNF 28.3 ± 5.2 3.5 ± 0.8 26.5 ± 1.5 300.5 ± 15.2 16.0 ± 1.3 DHA+PMA+TNF 25.0 ± 3.2 3.3 ± 0.9 12.0*±0.9 297.4 ± 10.0 27.8*±2.1 EPA+PMA+TNF 27.3 ± 2.5 3.1 ± 0.8 9.8*±1.4 295 ± 12.4 30*±2.5 . MMP-1 . MMP-2 . MMP-9 . TIMP-1 . TIMP-2 . Control 4.6 ± 0.5 3.0 ± 1.0 5.0 ± 0.8 60.0 ± 7.0 18.5 ± 2.1 PMA+TNF 28.3 ± 5.2 3.5 ± 0.8 26.5 ± 1.5 300.5 ± 15.2 16.0 ± 1.3 DHA+PMA+TNF 25.0 ± 3.2 3.3 ± 0.9 12.0*±0.9 297.4 ± 10.0 27.8*±2.1 EPA+PMA+TNF 27.3 ± 2.5 3.1 ± 0.8 9.8*±1.4 295 ± 12.4 30*±2.5 Values are given as ng/106 cells/48h and presented as means ± standard deviation *P < 0.05 as compared with the respective control. Open in new tab Abstract 482 Table . MMP-1 . MMP-2 . MMP-9 . TIMP-1 . TIMP-2 . Control 4.6 ± 0.5 3.0 ± 1.0 5.0 ± 0.8 60.0 ± 7.0 18.5 ± 2.1 PMA+TNF 28.3 ± 5.2 3.5 ± 0.8 26.5 ± 1.5 300.5 ± 15.2 16.0 ± 1.3 DHA+PMA+TNF 25.0 ± 3.2 3.3 ± 0.9 12.0*±0.9 297.4 ± 10.0 27.8*±2.1 EPA+PMA+TNF 27.3 ± 2.5 3.1 ± 0.8 9.8*±1.4 295 ± 12.4 30*±2.5 . MMP-1 . MMP-2 . MMP-9 . TIMP-1 . TIMP-2 . Control 4.6 ± 0.5 3.0 ± 1.0 5.0 ± 0.8 60.0 ± 7.0 18.5 ± 2.1 PMA+TNF 28.3 ± 5.2 3.5 ± 0.8 26.5 ± 1.5 300.5 ± 15.2 16.0 ± 1.3 DHA+PMA+TNF 25.0 ± 3.2 3.3 ± 0.9 12.0*±0.9 297.4 ± 10.0 27.8*±2.1 EPA+PMA+TNF 27.3 ± 2.5 3.1 ± 0.8 9.8*±1.4 295 ± 12.4 30*±2.5 Values are given as ng/106 cells/48h and presented as means ± standard deviation *P < 0.05 as compared with the respective control. Open in new tab Abstract Objectives: High intakes of omega-3 fatty acids have been associated with protection from plaque rupture. The secretion of metalloproteinases (MMPs) by macrophages plays a key role in matrix degradation underlying plaque instability. Conversely, tissue inhibitors of metalloproteinases (TIMPs) appear to contribute to plaque stability. We therefore studied the effects of omega-3 fatty acids on the release and activity of MMPs and TIMPs in cultured human monocytoid cells. Methods: Human U937 monocytoid cells were differentiated into macrophages by exposure for 24 h to 30 ng/mL phorbol myristate acetate (PMA) and 10 ng/mL tumor necrosis factor(TNF)-α. Both monocytoid cells and macrophages were treated for 48 h with DHA (22:6 n-3) or EPA (22:6 n-3) (25-100 μmol/L) before stimulation for 24 h with 10 ng/ml TNF-α. Cell supernatates were used to test the release of gelatinase A (MMP-2), gelatinase-B (MMP-9), collagenase-1 (MMP-1), TIMP-1 and TIMP-2, by ELISAs, and total gelatinase and anti-gelatinase activities by zymography and retro-zymography techniques, respectively. Results: The long term exposure to 50 μmol/L EPA and DHA, but not to arachidonic acid (20:4 n-6), significantly reduced MMP-9 protein release without affecting the release of MMP-1, MMP-2 and TIMP-1. Conversely, TIMP-2 protein release was significantly increased by EPA and DHA (Table). Zymography for MMP-9 and retro-zymography for TIMP-1 and -2 reproduced the same results. Conclusions: The long term exposure to omega-3 fatty acids significantly reduces MMP-9 release without affecting the release of MMP-1 and -2. This effect, associated with the increase of TIMP-2 protein production and activity, may contribute to explaining the plaque-stabilizing effect by omega-3 fatty acid observed in humans treated with omega-3 fatty acids 1 . Zinc finger protein 580: A novel factor in an endothelial pathway distinct for lipoprotein-induced response of IL 8 A Zakrzewicz A Zakrzewicz 1 Charite - Campus Benjamin Franklin , Berlin , Germany C Hoffmann C Hoffmann 1 Charite - Campus Benjamin Franklin , Berlin , Germany M Hohberg M Hohberg 1 Charite - Campus Benjamin Franklin , Berlin , Germany S Chlench S Chlench 1 Charite - Campus Benjamin Franklin , Berlin , Germany J Maroski J Maroski 1 Charite - Campus Benjamin Franklin , Berlin , Germany M Drab M Drab 1 Charite - Campus Benjamin Franklin , Berlin , Germany G Siegel G Siegel 1 Charite - Campus Benjamin Franklin , Berlin , Germany AR Pries AR Pries 1 Charite - Campus Benjamin Franklin , Berlin , Germany Abstract The response of endothelial cells to lipoproteins has well known implications for the development of atherosclerosis. Here we analysed a role for zinc finger protein 580 (ZNF 580) in the response of endothelial cells to physiological concentrations of lipoproteins. In Human umbilical vein endothelial cells (HUVEC) ZNF 580 levels were examined by real-time RT-PCR, immunoblotting, and immunostaining, levels of Interleukin 8 (IL 8) were examined by real-time RT-PCR and ELISA. ZNF 580 was located in the nucleus and induced by native LDL but not by HDL or TNFa. Especially LDL from patients with low oxLDL/LDL ratios increased ZNF 580 but it was even suppressed by LDL and HDL from patients with high oxLDL/LDL ratios. Lipoprotein-mediated increases of IL 8 were less pronounced with high concentration of ZNF 580 et vice versa. A transient knockdown of ZNF 580 led to an increase of IL 8. Our data support the hypothesis, that ZNF 580 protects endothelial cells by suppression of IL 8 when exposed to nLDL. However, this self-protection fails with increasing oxLDL/LDL ratios. Expression of the transcription factor c-Myb in atherosclerotic aortae of apoE−/− mice K Farrell K Farrell University of Manchester , Manchester , United Kingdom CM Holt CM Holt University of Manchester , Manchester , United Kingdom Abstract Atherosclerosis involves the proliferation and death of vascular smooth muscle cells. C-Myb is a transcription factor that functions in cell cycle progression, proliferation and apoptosis of various cell types. Several studies have shown that c-Myb plays an important role following vascular injury; however, its function in atherosclerosis remains unclear. Atherosclerotic lesions from aortic roots of ApoE−/− mice fed a high-fat or standard chow diet from 8 to 24 weeks post-weaning were analysed. C-Myb was observed in smooth muscle-like cells in the aorta and scattered throughout plaques. C-Myb was detected in isolated aortic cells, some of which were c-kit+, however the majority of c-Myb+ cells were α-smooth muscle actin+. Western blot analysis was performed on aortae from ApoE−/− and ApoE+/+ mice from 8-24 weeks (n = 3), to assess changes in c-Myb expression. There was no significant difference in c-Myb levels between ApoE−/− and ApoE+/+ mice at 8 weeks (432.5 ± 45.1 OD/mm2 vs. 254.5 ± 23.4 OD/mm2, ns). However, c-Myb expression was significantly higher in ApoE+/+ mice at 16 (896 ± 129.7 OD/mm2 vs. 156 ± 13.0 OD/mm2; P < 0.01) and 24 weeks (1248 ± 188.4 OD/mm2 vs. 337.2 ± 29.9 OD/mm2; P < 0.05), compared to ApoE−/− mice. TUNEL staining was performed in the aortic root to determine levels of apoptosis, and was expressed as a percentage of total cells in both the medial layer of the vessel wall and the plaque. The apoptotic index increased with plaque progression in ApoE−/− aortae; however this was not significant (8 weeks 6.48 ± 2.2%; 16 weeks 11.97 ± 3.5%; 24 weeks 14.09 ± 3.2%; P < 0.06). Current work involves comparison of apoptosis rates between ApoE−/− and ApoE+/+ mice at these timepoints. Data suggests that apoptosis levels correlate with c-Myb expression in ApoE−/− aortae. Decreased c-Myb levels are correlated with an increase in apoptosis, suggesting a putative role of c-Myb as a survival factor. Future work will investigate the mechanistic involvement of c-Myb in signalling pathways that may influence atherosclerosis. In addition, we are generating mice in which c-Myb can be conditionally deleted in smooth muscle cells, to clarify the contribution of c-Myb to atherosclerotic plaque development. A new method to evaluate the hydrophility of serum-lipoproteins G Schrot G Schrot 1 Academie of science, Institute of cardiovascular research (1989) , Berlin , Germany Abstract Purpose: Serum lipoproteins are micellar structured particles with a hydrophobic core and a hydrated hydrophilic surface, caused by the dipolar character of water molecules. The structure of vascular endothelium surface is similar: a hydrated layer with hydrophilic parts inside and hydrophobic parts outside the blood stream. Based on the second thermodynamic law, the duration of contacts and the adherence between lipoproteins and the endothelium are prolonged at lower hydrophilic lipoproteins. To predict the micell stability in blood and the contact behaviour (duration, adherence,permeation) with biological membranes it is important to know their hydrophility. Methods: Serum lipoproteins are incubated with a micellar indicator, a nonylphenol ethoxylate, which hydrophility is known. Formation of mixed micelles were following. The optical behaviour (cloud point) of these mixed micelles is changing depending on the hydrophility of lipoproteins. The hydrophility of lipoproteins from 15 patients with occlusive arterial disease and 96 healthy persons were determined. Results: For the first time a micellar indicator was found to determine the hydrophility of lipoproteins. In relation to the molar concentration of cholesterols, triglycerids and phospolipids the hydrophility of lipoproteins from patients with occlusive arterial disease was significantly lower than the hydrophility of healthy persons. Conclusions: In theory the thermodynamic interactions between lipoproteins and the endothelium surface of the vessel wall can be prolonged by hydrophobic lipoproteins. Thus, there is a high possibility of more receptor contacts and more deposits of cholesterol and triglycerids. This may contribute to the pathogenesis of atherosclerosis. Further studies must show if lower hydrophility of serum lipoproteins is a pathogenetic factor of atherosclerosis. Experimental atherosclerosis at rabbits aorta induced by acetylcholine microinjections in the reticular formation of the midbrain A Ibatov A Ibatov 1 Sechenov Moscow medical academy , Moscow , Russian Federation Abstract The Purpose: to study influence of acetylcholine microinjections in the reticular formation of the midbrain rabbits on development of experimental atherosclerosis. Materials and methods: 48 rabbits (8 months of age and weighing 2.0-2.5 kg) were examined. All rabbits ate vegetative diet without cholesterol. 25 rabbits with injection of acetylcholine were experimental group. 23 rabbits without injection of acetylcholine were control group. Acetylcholine injected in a day during 30 days in a dose 100 microgram in the reticular nucleus of mesencephalon. Results: After 30 days of acetylcholine injections the increase of cholesterol level in plasma was estimated. The macroscopic and microscopic examination of aortas of these rabbits showed lipomatosis, liposclerosis, atheromatosis and atherocalcinosis. The level of cholesterol did not change in control group and atherosclerosis changes in aortas of control group were not estimated. Conclusion: the long activation of central cholinergic receptors of mesencephalon leads to development of atherosclerosis. It once again evidences role of central nervous system in pathogenesis of atherosclerosis. Attenuation of early atherogenesis in LDLR-knockout mice by proteasome inhibition N Wilck N Wilck 1 Charite - University Medicine Berlin, Campus Mitte , Berlin , Germany M Fechner M Fechner 1 Charite - University Medicine Berlin, Campus Mitte , Berlin , Germany A Arias A Arias 1 Charite - University Medicine Berlin, Campus Mitte , Berlin , Germany S Meiners S Meiners 1 Charite - University Medicine Berlin, Campus Mitte , Berlin , Germany G Baumann G Baumann 1 Charite - University Medicine Berlin, Campus Mitte , Berlin , Germany V Stangl V Stangl 1 Charite - University Medicine Berlin, Campus Mitte , Berlin , Germany K Stangl K Stangl 1 Charite - University Medicine Berlin, Campus Mitte , Berlin , Germany A Ludwig A Ludwig 1 Charite - University Medicine Berlin, Campus Mitte , Berlin , Germany Abstract Purpose: Low and non-toxic proteasome inhibition has anti-inflammatory, anti-proliferative and anti-oxidative effects on vascular cells in vitro and in vivo. We therefore hypothesized that low-dose inhibition of the proteasome might provide anti-atherogenic protection. To test this hypothesis, LDL-receptor (LDLR) – knockout (KO) mice were treated with bortezomib - a potent inhibitor of the proteasome approved for therapy of multiple myeloma. Methods and Results: Male LDLR-KO mice, 10 weeks of age, were fed a Western diet for 6 weeks with intraperitoneal injections of bortezomib (WD+Bor) or solvent NaCl (WD). Bortezomib was injected at a low dose of 50 μg/kg body weight. Chymotrypsin-like proteasomal activity measured in liver homogenates was decreased to 67.2% compared to solvent-treated control group. Cholesterol and triglyceride plasma levels were not affected by the treatment. En face Oil red O staining of aortae and aortic root cryosections demonstrated significant reduction of atherosclerotic lesion coverage in bortezomib-treated animals. Bortezomib treatment significantly reduced vascular cellular adhesion molecule-1 expression and macrophage infiltration as shown by histological analysis of cryosections. Significant reduction of lipid peroxidation products and DHE fluorescence revealed potent anti-oxidative effects of low-dose bortezomib treatment. Moreover, serum levels of monocyte chemoattractant protein-1 and interleukin-6 were decreased by bortezomib treatment. Conclusion: Low-dose inhibition of the proteasome exerts anti-oxidative and anti-inflammatory effects and attenuates development of atherosclerotic lesions in LDLR-KO mice. The role of dendritic cells in human atherosclerotic lesions A Christ A Christ 1 Cardiovascular Research Institute Maastricht (CARIM) , Maastricht , Netherlands W Eijgelaar W Eijgelaar 1 Cardiovascular Research Institute Maastricht (CARIM) , Maastricht , Netherlands M Daemen M Daemen 1 Cardiovascular Research Institute Maastricht (CARIM) , Maastricht , Netherlands Abstract Introduction: Dendritic Cells (DC) are specialized antigen-presenting cells with the unique ability to initiate a primary immune response by activating naïve T-lymphocytes. So far, the presence of two different DC-subtypes, conventional (cDC) and plasmacytoid (pDC) DC, was described in human atherosclerotic plaques. Until know it is not clear what functional role DC play in atherosclerosis and whether they contribute to plaque destabilization through an interaction with colocalizing cells. Purpose: The aim of the current study was to investigate (1) how DC-subtypes are distributed in different plaque tissue samples and (2) with which cell types they colocalize. Methods: Immunohistochemistry on early (intimal thickening or xanthoma; n = 13), advanced stable (thick fibrous cap atheroma; n = 12) and advanced vulnerable (thin fibrous cap atheroma or intraplaque hemorrhage; n = 13) lesions from human carotid autopsy specimens was used for the detection of human cDC (CD11c+, CD1c+), pDC (CD123+, BDCA-2+) and colocalizing cell types such as T cells (CD3+), B cells (CD19+), macrophages (CD68+) and granulocytes (BM-2+). Anti-CD83 was used as an antibody to stain mature DC. Results: cDC and pDC were predominantly localized in the plaque shoulder region of advanced lesions. cDC were also found at the plaque base and in the media-adventitia layer. There was a significant increase in the number of CD11c+ labeled cDC from 2.07 ± 0.3 cells/mm2 in early to 10.27 ± 2.9 cells/mm2 in advanced vulnerable lesions (p < 0.05). pDC numbers increased significantly from 3.87 ± 0.9 cells/mm2 to 32.29 ± 11.2 cells/mm2 in vulnerable compared to stable lesions (p < 0.05). Both DC populations were mainly colocalized with T cells and macrophages in plaque shoulder regions. cDC showed significant elevated levels of the maturation marker CD83 in advanced compared to early lesions (p < 0.05). Granulocytes and B cells were mainly found in the adventitia and did not cluster with DC. Conclusion: The findings of this study demonstrate the presence of two DC populations in atherosclerotic lesions, mainly localized in plaque shoulder regions in close contact with T cells and macrophages. cDC show an elevated expression level of the maturation marker CD83 in advanced lesions. To identify a specific gene expression profile of plaque-infiltrating DC, DC-enriched and non-enriched areas of advanced stable and ruptured lesions will be isolated by laser capture microdissection. Due to this a genome-wide microarray approach will be generated to establish a disease-associated gene signature of DC. The CXCR7 ligand CCX771 reduces neointima formation after vascular injury and atherosclerosis in ApoE−/− mice X Li X Li 1 Institute of Molecular Cardiovascular Research, University Hospital Aachen, RWTH , Aachen , Germany M Penfold M Penfold 2 ChemoCentryx Inc. , California , United States of America T Schall T Schall 2 ChemoCentryx Inc. , California , United States of America C Weber C Weber 1 Institute of Molecular Cardiovascular Research, University Hospital Aachen, RWTH , Aachen , Germany A Schober A Schober 1 Institute of Molecular Cardiovascular Research, University Hospital Aachen, RWTH , Aachen , Germany Abstract The chemokine receptor CXCR7 plays a role in cell survival, adhesion, tumor growth and development by binding to CXCL12 (SDF-1) and CXCL11 (I-TAC). CCX771 is a highly specific CXCR7 ligand, which has been shown to affect SDF-1α/I-TAC binding to CXCR7 and modulate β-arrestin coupling. We studied the role of CCX771 in atherosclerotic vascular disease, such as neointima formation after vascular injury and diet-induced atherosclerosis. Wire-induced injury of the carotid artery was performed in Apoe−/− mice on western-type diet. Mostly endothelial CXCR7 immunostaining was evident at 1 week after injury. Mice were treated with CCX771 (10 mg/kg/d, s.c., n = 8) or the solvent Captisol (10%, n = 8) for 28 days. Plasma CCX771 levels were sufficiently high to provide full receptor coverage at trough. CCX771 reduced neointima formation by 35% due to a diminished neointimal macrophage content. SMC and T-cell content, however, were not affected by CCX771. CXCL12-mediated mobilization of Sca-1+/Lin- progenitor cells 24 h after injury was similar in CCX771- and Captisol-treated mice. In the atherosclerosis model, Apoe−/− mice on high-fat diet were treated with CCX771 (10mg/kg/d, s.c., n = 10) or Captisol (10%, n = 10) for 3 months. Analysis of the en face prepared throcaoabdominal aorta after Oil-Red-O staining demonstrated a 41% decrease of aortic lipid deposition in CCX771-treated mice (P < 0.05). Furthermore, the plaque area in the aortic root was reduce by 39% through CCX771 (P < 0.05). Determination of body weight, serum AST, ALT, creatinine, and C-reactive protein revealed no differences between CCX771- and Captisol-treated mice in the wire-injury and atherosclerosis model. We demonstrate a protective role of the CXCR7 ligand CCX771 in atherosclerotic vascular disease. Interleukin-33 is up regulated by oncostatin M and leptin in human smooth muscle cells in vitro R Hintenberger R Hintenberger 1 Medical University of Vienna, Department of Internal Medicine II, Division of Cardiology , Vienna , Austria C Kaun C Kaun 1 Medical University of Vienna, Department of Internal Medicine II, Division of Cardiology , Vienna , Austria S Pfaffenberger S Pfaffenberger 1 Medical University of Vienna, Department of Internal Medicine II, Division of Cardiology , Vienna , Austria G Maurer G Maurer 1 Medical University of Vienna, Department of Internal Medicine II, Division of Cardiology , Vienna , Austria K Huber K Huber 2 Wilhelminen Hospital , Vienna , Austria J Wojta J Wojta 1 Medical University of Vienna, Department of Internal Medicine II, Division of Cardiology , Vienna , Austria S Demyanets S Demyanets 1 Medical University of Vienna, Department of Internal Medicine II, Division of Cardiology , Vienna , Austria Abstract Background: The new interleukin 1 family member interleukin 33 (IL-33) - first described in 2005 - plays an important role in different diseases. While IL-33 seems to be protective against atherosclerosis and helminthic infection, it can promote asthma, atopic dermatitis, anaphylaxis and joint inflammation. Later inflammatory effects are most likely caused by expanding T helper type 2 (Th2) cells and by activation of mast cells. Human coronary artery smooth muscle cells (HCASMC) constitutively express IL-33 mRNA. The regulation of IL-33 is poorly described and seems to differ between various cell types. The aim of our study was to investigate the regulation of IL-33 in HCASMC by the pro-inflammatory cytokine oncostatin M (OSM) and by the adipokine leptin, which both are known to be involved in the pathogenesis of atherosclerosis and metabolic syndrome. Methods: HCASMC were isolated from pieces of coronary arteries obtained from patients undergoing heart transplantation. These cells were treated with OSM at concentrations between 100 ng/ml and 0.01 ng/ml or leptin at concentrations between 500 ng/ml and 31.25 ng/ml. Specific mRNA levels for IL-33 were determined by real-time PCR. IL-33 protein in cell lysates and culture supernatants was measured by specific ELISA. Results: We found that HCASMC constitutively expressed IL-33 protein intracellular. In cell culture supernatants only trace levels of IL-33 were detected using a specific ELISA. Both OSM and leptin significantly (p ≤ 0.05) increased intracellular IL-33 protein expression. IL-33 protein was increased up to 4-fold after 48 hours (h) of incubation with 100 ng/ml of OSM and up to 2-fold after 48 h of incubation with 500 ng/ml of leptin. The effects of OSM and leptin on IL-33 protein production were dose-dependent. These cytokines also upregulated mRNA specific for IL-33 in HCASMC after an incubation time of 12 h. The results were reproducible in HCASMC isolated from different donors. Conclusions: We found that IL-33 protein is expressed by human coronary artery SMC intracellular. IL-33 is up regulated by the cytokines OSM and leptin. Further investigations to understand the pathophysiological role of these regulations are warranted. Vector proteins in targeted transport of fatty acids in vivo in various classes of lipoproteins V Titov V Titov 1 Russian Cardiology Research & Production Center , Moscow , Russian Federation Abstract Under the baseline conditions fatty acids (FA) are transported to cells as polar lipids (phospholipids and diglycerides) by high-density lipoproteins (apoAI-LP); as nonpolar lipids (triglycerides and cholesterol esters), by very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL), apoB-100 LP. There are specific circulating proteins which direct FA to certain cell classes depending on activation of certain biological functions. We believe that these proteins are represented by apoE, C-reactive protein (CRP) and apo(a). When the biological function of locomotion is activated (high muscular activity), the flow of FA, a substrate for ATP production, is directed to the myocytes by apoE in the form of apoE/B-100 receptor-mediated endocytosis of saturated and unsaturated FA in VLDL. Phylogenetically, apoE is much younger than apoA-I and apoB-100, and in contrast to them, it is synthesized by peripheral cells. ApoE production was initiated in vivo during the development of the biological function of locomotion. CRP activates the interstitial tissue cells which are involved in the biological function of inflammation. By binding to lysophosphatidylcholine on the VLDL surface CRP ????blocks, overlaps, covers, masks??????apoE/B-100 and becomes a ligand, after which CRP+VLDL is internalized only by cells that expose CRP "receptors" on the plasma membrane. An increase in the phospholipase A2 activity in VLDL is a phase of CRP activity and an inflammation test. Lipid spots in the intima result from functional macrophagal lipoidosis initiated by CRP as a proinflammatory mediating agent; cells are overloaded with triglycerides and do not pertain to atheromatosis. When cell proliferation, a local reparation reaction, is activated, an additional amount of essential polyenic FA is required. Proliferating cells produce apo(a) which binds to circulating VLDL and masks apoB-100B ligand, becomes a ligand per se and is internalized by cells that expose LP(a) "receptors" on the plasma membrane. The effects of CRP and apo(a) are unspecific, which accounts for an increase in their plasma content by tens of times; an increase in apo(a) content can reflect both cell proliferation in the intima and development of restenosis. When CRP blocks myocyte internalization of FA as triglycerides in VLDL by apo E/B-100 endocytosis, cells have to absorb nonesterified FA from associates with albumin. This lays the basement for myocytic resistance to insulin. As long as blood content of CRP remains high ant the biological reaction of inflammation is activated, normal metabolism of FA and glucose in myocytes cannot be expected. Expression of microRNAs during development of atherosclerosis in ApoE−/− mice M Nazari-Jahantigh M Nazari-Jahantigh 1 Institute of Molecular Cardiovascular Research, University Hospital Aachen, RWTH , Aachen , Germany C Weber C Weber 1 Institute of Molecular Cardiovascular Research, University Hospital Aachen, RWTH , Aachen , Germany A Schober A Schober 1 Institute of Molecular Cardiovascular Research, University Hospital Aachen, RWTH , Aachen , Germany Abstract We aimed to identify micro RNAs involved in the generation of atherosclerotic lesions. Female ApoE−/− mice (6-8 weeks) were fed regular chow or high fat diet for 3 or 10 months (n = 3-4 each group). Total RNA was isolated from the thoracoabdominal aorta (mirVana microRNA isolation kit). After verifying high RNA quality (Agilent 2100 Bioanalyzer), reverse transcription and preamplification (Megaplex RT & Preamp Rodent Pool Set, Applied Biosystems) was performed. Samples were loaded on preconfigured 384-well microfluidic cards (TaqMan Array MicroRNA Cards) for real-time analysis (7900HT RT-PCR System) of 518 mouse microRNAs (Sanger miRBase v10). Results were analyzed by using quantitative RT-PCR StatMiner software (Integromics). Time course of miR expression was determined by qBase software (Biogazelle) and statistically analyzed by One-way ANOVA (P < 0.05, GraphPad Prism). In ApoE−/− mice on regular chow (control) 363 microRNAs were found to be expressed in the aortic vessel wall. After high fat diet, 27 and 35 miRs were newly expressed at 3 and 10 months, respectively. In contrast, 11 miRs were no longer detectable after 3 months and 5 miRs were absent after 10 months. At 10 months compared with 3 months of high fat diet, 26 miRs were exclusively expressed, whereas 12 miRs were not detectable anymore. Interestingly, three main different regulation patterns of microRNA expression in the development of atherosclerosis have been observed. In the first group, 16 miRs were transiently down-regulated at 3 months of high fat diet and returned to control levels again after 10 months. The second regulation pattern, including 9 miRs, is characterized by increased expression solely after 10 months. Additionally, sustained down-regulation of 5 miRs was found at 3 and 10 months constituting the third group. We could identify miRs, which are differentially regulated in atherogenesis. Up-regulation of miRs was rather unique in advanced atherosclerosis, whereas transient down-regulation of a subset of miRs is characteristic for early atherosclerosis. These results demonstrate for the first time distinct regulation of specific miRs in different stages of plaque development. Overexpression of arginase II worsens endothelial dysfunction and atherosclerosis while arginase I overexpression may have cardioprotective effects JPF Chin-Dusting JPF Chin-Dusting 1 Baker IDI Heart and Diabetes Institute , Melbourne , Australia BL Vaisman BL Vaisman 2 NIH/NHLBI , Bethesda , United States of America SML Khong SML Khong 1 Baker IDI Heart and Diabetes Institute , Melbourne , Australia AT Remaley AT Remaley 2 NIH/NHLBI , Bethesda , United States of America KL Andrews KL Andrews 1 Baker IDI Heart and Diabetes Institute , Melbourne , Australia Abstract Cardiovascular disorders associated with endothelial dysfunction, like atherosclerosis, have decreased endothelial nitric oxide (NO) bioavailability. L-arginine, the primary substrate for endothelial nitric oxide synthase (eNOS), is important in the regulation of NO production. Arginase, found in the vasculature as arginase I and II, competes with eNOS for L-arginine and has been implicated in the endothelial dysfunction of many disease states. Since few studies have identified the arginase isoform responsible, the aim of this study was to assess the role of increased arginase I and II expression on endothelial function in the development of atherosclerosis. Transgenic mice on a C57BL/6 background with endothelial overexpression of either human arginase I (hArgI) or II (hArgII) gene (under control of endothelial-specific Tie2 promoter) were studied. Both strains of transgenic mice had elevated arginase activity: 4-10 fold in the aorta, heart and kidney of the transgenic arginase I (Tie2hArgI) mice and 4.6-62 fold in all organs of the transgenic arginase II (Tie2hArgII) mice except the liver. Resident peritoneal macrophages from transgenic mice and controls did not differ in the level of arginase activity, confirming endothelial specificity. Overexpression of hArg did not lead to significant changes in plasma amino acids lipid levels in either strain of mice when compared to their controls. ROS levels, assessed by lucigenin-enhanced chemiluminescence in the aortae of Tie2hArgI and Tie2hArgII mice remained unchanged. Using small vessel myography, endothelial dysfunction was evidenced as a diminished response to endothelium-dependent dilator, acetylcholine (ACh; P < 0.05) in aorta from Tie2hArgII, but not Tie2hArgI mice. When crossed with ApoE knockout mice there was a significant increase of atherosclerotic lesions in the Tie2hArgII mice (P < 0.05). Interestingly, in mesenteric arteries, inhibition of endothelium dependent hyperpolarizing factor (EDHF) abolished responses to ACh in the mesenteric arteries of control and Tie2hArgII but not the Tie2hArgI mice. In addition, the NOS inhibitor, L-NAME reduced the ACh response in the Tie2hArgI mice, suggesting an apparent increase in NO production in the mesentery of these mice. Furthemore, a significant decrease in atherosclerotic lesions was seen in the Tie2hArgI mice. Collectively, these results suggest that overexpression of arginase II leads to endothelial dysfunction and an increase in atherosclerotic lesion development while overexpression of arginase I in the endothelium may, conversely, have a cardio-protective role. Visceral fat deposition is attenuated by calanus oil supplementation in rats on a high fat diet AC Hoeper AC Hoeper 1 University of Tromsø , Tromsø , Norway AM Khalid AM Khalid 1 University of Tromsø , Tromsø , Norway BN Fuglested BN Fuglested 1 University of Tromsø , Tromsø , Norway E Aasum E Aasum 1 University of Tromsø , Tromsø , Norway TS Larsen TS Larsen 1 University of Tromsø , Tromsø , Norway Abstract Introduction: Epidemiological data have shown a clear association between visceral adiposity and insulin resistance/type 2 diabetes, and changes in lifestyle leading to reduced fat mass and/or altered fat distribution have been shown to delay the onset of the disease in individuals at high risk. In this study we examined the effect of calanus oil, which was extracted from the marine zooplankton calanus finmarchicus, on visceral adiposity, blood chemistry, glucose tolerance, and myocardial metabolism in normal rats fed a high fat (HF) diet. Methods: Male Wistar rats were fed either normal chow or HF diet with or without calanus oil (1.5%, w/w) for a total period of 16 weeks. At the end of the feeding period, blood samples were taken and a glucose tolerance test was performed. Finally, the animals were sacrifised and the hearts cannulated for working heart perfusion and determination of myocardial substrate oxidation (using radioactive glucose and palmitate). Perirenal adipose tissue was carefully dissected out and weighed. It is important to note that the contribution of omega-3 fatty acids (FA) from the calanus oil in the present diet was much lower (0.183%, w/w) than that reported in other studies with omega-3 FA supplementations. Results: Rats fed HF diet showed increased body weight and visceral fat mass, as reflected by a significant increase in perirenal fat. They also showed insulin resistance and a shift in myocardial metabolism in favor of FA oxidation. Supplementation of the diet with calanus oil resulted in a significant reduction of perirenal fat, which was associated with a modest attenuation of insulin resistance. The reduction in visceral fat did not, however, influence myocardial substrate oxidation or the mechanical function of the heart. Conclusion: These data show that calanus oil has the capacity to reduce visceral fat and improve glucose homeostasis in obese rats, suggesting that it also may reduce inflammation and disease states associated with obesity. The active component in the calanus oil remains to be determined. Phylogenesis, anatomic, functional heterogeneity of the arterial bed and pathogenesis of arterial hypertension V Titov V Titov 1 Russian Cardiology Research & Production Center , Moscow , Russian Federation Abstract During early phylogenesis, each forming paracrine regulated community (future structural and functional units of body organs) included a pool of specialised cells, elements of the interstitial tissue, and local peristaltic pump. In the community the pump performed the biological functions of homeostasis and endoecology (purity of the intercellular medium); the paracrine community regulated the pump via humoral factors. The nephron whose constituents are renal capillary, interstitial tissue and afferent arteriole can be regarded as a paracrine regulated cell community. The activity of the muscle type arteriole (local pump) is regulated by the renal capillary cells on the basis of glomerulo-tubular direct and reverse relationships, thus establishing a "municipal" regulation. Later on, the development of the biological function of locomotion (movement) required a closed circulation system in which the heart and elastic arteries, as a single functional unit, have united millions of much more ancient local peristaltic pumps. We suppose that muscular arterioles are these peristaltic pumps, and the number of paracrine communities equals the number of arterioles in the body. Phylogenetically, arterial bed consists of two segments: proximal (heart and elastic arteries) and distal (muscular arterioles). Proximal segment is regulated at the "federal" level by the vasomotor center via sympathetic and parasympathetic innervation. Distal segment (peripheral peristaltic pumps) is regulated at the "municipal" level of paracrine communities. When the biological function of locomotion is performed, "federal" and "municipal" regulations are coordinated via the "mechanosensitivity" of the muscular arteriolar endothelium, thus providing intense cell perfusion. In the realisation of the biological function of homeostasis "federal" regulation often comes in opposition with "municipal" regulation, which is crucial for the pathogenesis of arterial hypertension. Arterial pressure measurement provides information on proximal segment of the arterial bed, while the endothelium-dependent vasodilation test characterises the condition of the distal segment, i.e., of peripheral pumps (muscular arterioles). In vivo, pathological alterations in paracrine communities and distal segment of the arterial bed are always compensated by increased hydrodynamic pressure in the proximal segment with the development of arterial hypertension. Discoordination (conflict, opposition???) between "federal" and "municipal" regulations in the paracrine community of the nephron is the major cause of glomerulosclerosis and tubulo-interstitial inflammation. Hydroxytyrosol suppresses MMP-9 activity and expression in human monocytes. A mechanism for plaque stabilization by an olive oil component of Mediterranean diets MA Carluccio MA Carluccio 1 Institute of Clinical Physiology-CNR , Lecce , Italy E Scoditti E Scoditti 1 Institute of Clinical Physiology-CNR , Lecce , Italy M Massaro M Massaro 1 Institute of Clinical Physiology-CNR , Lecce , Italy C Storelli C Storelli 2 University of Salento, Department of Biological and Environmental Science and Technology (DiSTeBA) , Lecce , Italy A Distante A Distante 1 Institute of Clinical Physiology-CNR , Lecce , Italy R De Caterina R De Caterina 3 “G. d'Annunzio” University and Center of Excellence on Aging , Chieti , Italy Abstract Purpose: Mediterranean diets, of which olive oil is an important component, are associated with low prevalence of cardiovascular diseases. The production of inflammatory mediators, such as prostaglandin (PG) E2, the overexpression of the inducible cyclooxygenase (COX)- 2 isoform and the activation of matrix metalloproteinase(MMP)-9 by macrophages likely contributes to plaque instability leading to acute coronary events. We studied the effects of the olive oil phenolic antioxidant hydroxytyrosol (HT) on MMP-9 and COX-2 activity and expression in human monocytes and explored underlying mechanisms. Methods: Human monocytes were treated either with 1-50 μmol/L HT for 60 min or with selective inhibitors of PKC or COX isoenzymes for 30 min before stimulation with 30 nmol/L phorbol myristate acetate (PMA) for 24 h. Cell supernatants were tested for the release of MMP-9, PGE2 and TIMP-1 and -2 by ELISA and MMP-9 activity by zymography. Cell protein extracts were analyzed by Western analysis for COX-2 expression and for membrane translocation of PKCs and the NADPH oxidase p47phox subunit. We analyzed the activity of COX-2 promoter by transient transfection experiments and the.activation of the transcription factor Nuclear Factor(NF)-kappaB by EMSA. Results: PMA and, to a lesser extent, PGE2, induced the release of MMP-9 in monocytes. Cell exposure to HT before PMA stimulation reduced MMP-9 activity and expression (IC50 for HT of 10 micromol/L p < 0.01) without affecting the release of TIMP-1 and -2. Correspondingly, HT inhibited PMA-induced PGE2 production (by 54 ± 7%) and COX-2 expression (by 43 ± 5%) without affecting COX-1. Inhibition by HT was mediated by the suppression of NF-kappaB and the NADPH oxidase p47phox and PKCα/β1 activation. Conclusions: Our findings show that HT, at concentrations nutritionally achievable, inhibits the expression and the release of MMP-9 at least in part by the suppression of COX-2 dependent PGE2 pathway. Such effect occurs through the attenuation of PKCα/β1 and NADPH oxidase activation. Overall, such results contribute to explaining the vascular protective effects exerted by olive oil in Mediterranean diets. Rac-1 is a novel HIF-1 target gene. Implications for vascular remodeling I Diebold I Diebold 1 German Heart Center, Clinic at the Technical University of Munich , Munich , Germany A Petry A Petry 1 German Heart Center, Clinic at the Technical University of Munich , Munich , Germany T Djordjevic T Djordjevic 1 German Heart Center, Clinic at the Technical University of Munich , Munich , Germany RS Belaiba RS Belaiba 1 German Heart Center, Clinic at the Technical University of Munich , Munich , Germany S Fratz S Fratz 1 German Heart Center, Clinic at the Technical University of Munich , Munich , Germany J Hess J Hess 1 German Heart Center, Clinic at the Technical University of Munich , Munich , Germany T Kietzmann T Kietzmann 2 University of Kaiserslautern , Kaiserslautern , Germany A Goerlach A Goerlach 1 German Heart Center, Clinic at the Technical University of Munich , Munich , Germany Abstract The GTPase Rac-1 is an important regulator of the cytoskeleton and an essential activator of reactive oxygen species (ROS) generation by NADPH oxidases. We recently showed that activated Rac-1 can promote proliferation of vascular smooth muscle cells suggesting a role for Rac-1 in vascular remodeling. However, the regulatory pathways controlling Rac-1 levels in vascular smooth muscle cells and their consequences for vascular remodeling are not well resolved and have been addressed in this study. To this end, pulmonary artery smooth muscle cells (PASMC) were stimulated with the coagulation factor thrombin, known to activate ROS generation by NADPH oxidases and PASMC proliferation. Interestingly, thrombin not only activated Rac-1 involving the protease-activated receptor PAR-1, Gαi and calcium, but also enhanced mRNA and protein levels of this GTPase via a transcriptional mechanism. Surprisingly, bioinformatic analysis of the Rac-1 promoter revealed several putative consensus sites for the hypoxia-inducible transcription factor HIF-1, which has been implicated in the pathogenesis of hypoxia-induced pulmonary vascular remodeling. In fact, depletion of HIF-1α by shRNA prevented induction of Rac-1 by thrombin, and chromatin immunoprecipitation confirmed binding of HIF-1α to the Rac-1 promoter. Furthermore, HIF-1α was also involved in the upregulation of the Rac-1 target p21-activated kinase-1 (PAK-1). Subsequently, we found enhanced levels of Rac-1 and PAK-1 in remodeled pulmonary vessels in patients or a lamb model of congenital heart disease. Importantly, HIF-1α levels were also increased in these vessels independently of hypoxia. Interestingly, the HIF-1 target genes Rac-1 and PAK-1 themselves were able to induce and activate HIF-1α via an NFkappaB-dependent mechanism. Since HIF-1α was also required for PASMC proliferation, our data suggest that Rac-1 and PAK-1 provide an essential interface connecting HIF-1 and NFkappaB signaling and thereby contributing to vascular remodeling also independently of hypoxia. Adiponectin plays a permissive role for structural and metabolic remodeling in mice subjected to pressure overload K O'shea K O'shea 1 University of Maryland , Baltimore , United States of America D Chess D Chess 1 University of Maryland , Baltimore , United States of America R Khairallah R Khairallah 1 University of Maryland , Baltimore , United States of America K Walsh K Walsh 2 Boston University , Boston , United States of America W Stanley W Stanley 1 University of Maryland , Baltimore , United States of America Abstract Recent data suggest adiponectin, an adipocyte-derived hormone, affects development of heart failure in response to hypertension. Severe short-term pressure overload (1-3 weeks of transverse aortic constriction (TAC)) in adiponectin −/− mice causes greater LV hypertrophy than in wild type (WT) mice, but conflicting results are reported regarding LV remodeling, with either increased or decreased LV end diastolic volume compared to WT mice. Here we assessed the effects of prolonged TAC on LV hypertrophy and remodeling. WT and adiponectin −/− mice were subjected to TAC and maintained for 6 weeks. Regardless of strain, TAC induced similar LV hypertrophy (∼70%) and up-regulation of mRNA for heart failure marker genes (∼11-fold for atrial natriuretic factor and ∼40-fold for myosin heavy chain beta/alpha)(p < 0.05). On the other hand, LV chamber remodeling was dramatically different, with classic LV dilation in WT TAC mice, but concentric LV hypertrophy in adiponectin −/− mice. Compared to WT mice, adiponectin −/− TAC mice had lower LV end diastolic volume (63 ± 6 vs. 142 ± 21 µL) and end systolic volume (35 ± 5 vs. 111 ± 19 mL), and ejection fraction was higher compared to WT (25.9 ± 3.6 vs. 46.5 ± 3.6%)(all p < 0.05). LV relative wall thickness was unaffected by TAC in the WT mice (0.35 ± 0.03 in sham vs.0.37 ± 0.02in WT) but was significantly increase in the adiponectin −/− mice (from 0.30 ± 0.03 to 0.55 ± 0.05) (p < 0.05). Thus adiponectin deletion prevented LV chamber remodeling and deterioration in systolic function in response to TAC. The activities of marker enzymes of mitochondrial oxidative capacity (citrate synthase and medium chain acyl-CoA dehydrogenase) were reduced in WT TAC mice by ∼35% (p < 0.05), but were maintained at sham levels in adiponectin −/− TAC mice. In conclusion, in WT mice, long-term pressure overload caused dilated LV hypertrophy accompanied by decreased activity of mitochondrial oxidative enzymes. While adiponectin deletion did not affect LV hypertrophy, it prevented LV chamber remodeling and preserved mitochondrial oxidative capacity, suggesting that adiponectin plays a permissive role in mediating changes in cardiac structure and metabolism in response to pressure overload. Diabetes mellitus and pressure-overload: distinct mechanisms for diastolic dysfunction I Falcao-Pires I Falcao-Pires 1 University of Porto, Faculty of Medicine , Porto , Portugal N Goncalves N Goncalves 1 University of Porto, Faculty of Medicine , Porto , Portugal J Van Der Velden J Van Der Velden 2 VU University Medical Center , Amsterdam , Netherlands D Moreira-Goncalves D Moreira-Goncalves 1 University of Porto, Faculty of Medicine , Porto , Portugal WJ Paulus WJ Paulus 2 VU University Medical Center , Amsterdam , Netherlands HWM Niessen HWM Niessen 2 VU University Medical Center , Amsterdam , Netherlands S Perlini S Perlini 3 Foundation IRCCS Polyclinic San Matteo - University of Pavia , Pavia , Italy AF Leite-Moreira AF Leite-Moreira 1 University of Porto, Faculty of Medicine , Porto , Portugal Abstract Aims: We aimed to characterize the changes in myocardial structure and function induced by two frequent co-existing causes of diastolic heart failure, chronic pressure-overload and diabetes mellitus. Methods: Pressure-overload was established in rats by supra-renal aortic banding. Six weeks later, diabetes was induced by streptozotocin (65 mg/kg,ip), resulting in 4 groups: SHAM, banded (BA), diabetic (DM) and diabetic-banded (DB). Twelve weeks later, pressure-volume loops were obtained and left ventricular (LV) samples were collected to evaluate function and phosphorylation of myofilamentary proteins and extracelular matrix alterations. Results: Compared to SHAM, pressure-overload increased cardiomyocyte's diameter (BA: 22.0 ± 0.4 µm, SHAM: 18.2 ± 0.3 µm) and myofilament maximal-force (BA: 25.7 ± 3.6kN/m2, SHAM: 18.6 ± 1.4kN/m2), Ca2+-sensitivity (pCa50; BA: 5.56 ± 0.02, SHAM: 5.50 ± 0.02) and MyBP-C phosphorylation, while decreasing rate of force-redevelopment (Ktr; BA: 14.9 ± 1.1s-1, SHAM: 25.2 ± 1.5s-1). At the extracellular matrix level, fibrosis (BA: 10.8 ± 0.9%, SHAM: 5.3 ± 0.6%), pro-MMP-2 and MMP-9 activity were augmented and, in vivo, relaxation was impaired (Tau, BA: 14.0 ± 0.9ms, SHAM: 12.9 ± 0.4ms). Diabetes increased cardiomyocyte's diameter (DM: 21.4 ± 0.4 µm, DB: 20.6 ± 0.4 µm), fibrosis (DM: 13.9 ± 1.8%, DB: 13.8 ± 0.8%), advanced glycation end-products deposition (DM: 4.9 ± 0.6score/mm2, DB: 5.1 ± 0.4 score/mm2, SHAM: 2.1 ± 0.3score/mm2), pCa50 (DM: 5.57 ± 0.02, DB: 5.57 ± 0.01) and TIMP-1 concentration, while decreased Ktr (DM: 13.5 ± 1.9s-1, DB: 15.2 ± 1.4s-1) and MMP-9/TIMP-1 ratio. Diabetic hearts were stiffer (higher end-diastolic-pressure: DM: 7.0 ± 1.2mmHg, DB: 6.7 ± 0.7mmHg, SHAM: 5.3 ± 0.4mmHg; steeper end-diastolic-pressure-volume relation: DM: 0.59 ± 0.18, DB: 0.83 ± 0.17, SHAM: 0.41 ± 0.10), and hypo-contractile (decreased heart rate and cardiac output). The association of diabetes and pressure-overload combined relaxation abnormalities and increased stiffness inducing further pulmonary congestion (Lungs/body weight; DB: 5.23 ± 0.21g/Kg, SHAM: 3.80 ± 0.14 g/Kg). Conclusions: Diabetes mellitus and pressure overload led to distinct diastolic dysfunction phenotypes: while diabetes impaired relaxation and promoted ventricular stiffening, pressure overload predominantly impaired relaxation. Their association amplified these changes in a way that precipitates to a faster progression to heart failure. Cardiac hyperaldosteronism and fetal gene program in arterial hypertension F Azibani F Azibani 1 INSERM U942 , Paris , France F Tournoux F Tournoux 1 INSERM U942 , Paris , France L Fazal L Fazal 1 INSERM U942 , Paris , France E Polidano E Polidano 1 INSERM U942 , Paris , France R Merval R Merval 1 INSERM U942 , Paris , France C Chatziantoniou C Chatziantoniou 2 INSERM U702 , PARIS , France JL Samuel JL Samuel 1 INSERM U942 , Paris , France C Delcayre C Delcayre 1 INSERM U942 , Paris , France Abstract Our Purpose was to determine the role of intracardiac aldosterone in the development of cardiac hypertrophy and re-expression of fetal gene program in a context of arterial hypertension. Thus, we created AS-RenTgKC mice by crossing strains of mice overexpressing the aldosterone synthase (AS) gene in cardiomyocytes with RenTgKC mice overexpressing the renin gene in liver. AS mice have an increase (x 2) of intracardiac aldosterone and an altered coronary function; RenTgKC mice have an 8-fold increase of plasmatic renin and AngII, and sustained hypertension. Methods: Blood pressure of mice was measured by plethysmography at 6 months of age. Echocardiographies were performed at 9 months. mRNA expression was measured by RT-qPCR, that of the proteins by western blot. Cardiac fibrosis was measured on LV sections stained by Sirius red. Results: The heterozygous RenTgKC (Ren +/−) mice were hypertensive (139 ± 4 mm Hg) compared to WT controls (98 ± 7 mm Hg, P < 0.05). The association with the cardiac hyperaldosteronism did not modify this parameter. The echocardiography did not reveal major changes of cardiac function whatever the groups. The hypertension-induced cardiac hypertrophy (HW / BW), which developed in Ren +/− mice, was enhanced in AS-Ren +/− mice (+19.6 % ± 5.7 and +41.4 % ± 6.4; P < 0.05). Regarding the fetal gene program, as expected, βMyHC (x 3; P < 0.01), BNP (x 2.5; P < 0.01) or ANP (x 2.5; P < 0.01) mRNAs were up-regulated in left ventricles of Ren +/− mice compared to controls. Conversely in AS-Ren+/− mice, these transcripts were not or moderately up-regulated. Interestingly, a short treatment with eplerenone (1week) reversed the AS-Ren+/− mouse phenotype towards classical fetal phenotype with up-regulation of BNP, ANP and βMyHC mRNAs. Furthermore, MCIP-1 mRNA expression, which was up-regulated in Ren+/− mice (x1.9; p < 0.01), was not increased in AS-Ren+/− mice suggesting an inhibition by aldosterone of the hypertension-induced calcineurin pathway. Conclusion: The data indicate that in a context of arterial hypertension, a cardiac hyperaldosteronism altered the expression of the cardiac fetal gene program through a MR specific pathway. Role of cardiac aldosterone on the development of cardiac fibrosis in response to hypertension F Azibani F Azibani 1 INSERM U942 , Paris , France F Tournoux F Tournoux 1 INSERM U942 , Paris , France L Fazal L Fazal 1 INSERM U942 , Paris , France E Polidano E Polidano 1 INSERM U942 , Paris , France R Merval R Merval 1 INSERM U942 , Paris , France C Chatziantoniou C Chatziantoniou 2 INSERM U702 , PARIS , France JL Samuel JL Samuel 1 INSERM U942 , Paris , France C Delcayre C Delcayre 1 INSERM U942 , Paris , France Abstract The development of fibrosis is known to depend on the balance between profibrotic (CTGF, BMP4) and antifibrotic factors (BNP, ANP, BMP7). The Purpose of this study was to determine 1) the role of intracardiac aldosterone in the development of cardiac fibrosis in a context of arterial hypertension, and 2) the involved signalling pathways. To this aim, we created AS-RenTgKC mice by crossing strains of mice overexpressing the aldosterone synthase (AS) gene in cardiomyocytes with RenTgKC mice overexpressing the renin gene in liver. AS mice have an increase (x 2) of intracardiac aldosterone and an altered coronary function; RenTgKC mice have an 8-fold increase of plasmatic renin and AngII, and sustained hypertension. Methods: Blood pressure of AS/ RenTgKC mice was measured by plethysmography at 6 months of age. Echocardiographies were performed at 9 months. CTGF, BMP4, BMP7, TGFβ, and BNP mRNA expressions were measured by RT-qPCR, that of the proteins by western blot. Cardiac fibrosis was measured on LV sections stained by Sirius red. Results: 1) the heterozygous RenTgKC (Ren +/−) mice were hypertensive (139 ± 4.9 mm Hg) compared to WT controls (98 ± 6.5 mm Hg). The association with the aldosterone did not modify this parameter. The echocardiographic data did not reveal major changes of cardiac function. 2) At the molecular level, we showed in ventricles: a) A significant increase of the CTGF mRNA (x 1.9; p < 0.05) and of the TGF-β 1 mRNA (x 1.5; p < 0.01), in both Ren +/− and AS-Ren +/− mice compared to WT and AS mice, respectively. b) A significant increase of BMP 7 mRNA (x 2.2; p < 0.05), of BMP 4 mRNA (x 1.7; p < 0.001) of BNP (x3; p < 0.001) in Ren+/− compared to WT mice. c) A decrease of the BMP4 mRNA (x0.77; p < 0.05) and of BNP (x0.39; p < 0.01) in AS-REN +/− mice compared with Ren +/−. 4).The Sirius red staining showed a significant increase of cardiac interstitial fibrosis (x 2.5) in Ren +/− and AS-REN +/− mice compared to WT and AS, respectively. Conclusion: These results indicate that during arterial hypertension, a cardiac aldosteronism worsens the development of myocardial fibrosis via changes in the balance (CTGF, TGF-β / BMP4, BNP) Metformin mediated restoration of mitochondrial integrity is associated with improved cardiac contractility in isoproterenol-induced heart failure: a role for mitochondrial biogenesis. P Mgandela P Mgandela 1 University of the Witwatersrand , Johannesburg , South Africa R Brooksbank R Brooksbank 1 University of the Witwatersrand , Johannesburg , South Africa T Maswanganyi T Maswanganyi 1 University of the Witwatersrand , Johannesburg , South Africa AJ Woodiwiss AJ Woodiwiss 1 University of the Witwatersrand , Johannesburg , South Africa GN Norton GN Norton 1 University of the Witwatersrand , Johannesburg , South Africa S Makaula S Makaula 1 University of the Witwatersrand , Johannesburg , South Africa Abstract Purpose: We investigated whether isoproterenol-induced heart failure (HF) is associated with loss of mitochondrial integrity and whether metformin ameliorates LV dysfunction in non-diabetic failing hearts. Moreover, we investigated the role of the cellular energy-sensing enzyme AMPK, as well as the mitochondrial regulatory genes, the nuclear respiratory factor 1 (NRF-1) and the mitochondrial transcription factor (Tfam), in the regulation of mitochondrial biogenesis in the isoproterenol model. Methods: Control rats received no treatment, ISO rats were injected with 0.04 mg/kg isoproterenol (ISO) daily for 4 months, METF rats received 300mg/kg metformin in drinking water and ISO+METF received isoproterenol and metformin. Mitochondrial and myofibrilar changes were assessed using transmission electron microscopy and HF was determined using echocardiography. Changes in gene expression for the AMPK, NRF-1 and Tfam genes were determined using real-time PCR. Results: We observed mitochondrial and myofibrilar derangements in ISO-treated rats. However, metformin treatment reversed these ISO-induced deleterious effects. Moreover, echocardiography revealed significant increases in LVEDD (11.9 ± 0.4 mm, p < 0.05) and LVESD (4.90 ± 0.40 mm, p < 0.05), and a reduction in FSend (41.46 ± 1.68 %, p < 0.05) compared to controls. ISO+METF treated rats showed a significant reduction in LVESD (3.64 ± 0.37 mm, p < 0.05 vs ISO) and improved FSend (52.10 ± 3.70 %, p < 0.05 vs ISO). However, moderate changes were observed in LVEDD (10.43 ± 0.7 mm, NS vs ISO). Our PCR data showed upregulation of the AMPK (3.55 ± 1.22), NRF1 (6.35 ± 3.20) and Tfam (2.89 ± 0.55) genes in ISO-treated rats, compared to controls (1.09 ± 0.47, 2.17 ± 0.78 and 1.00 ± 0.20, respectively). The AMPK gene was upregulated in the METF (4.77 ± 1.33) and ISO+METF (5.38 ± 2.29) groups. Conclusion: Metformin mediated restoration of mitochondrial integrity is associated with improved cardiac contractility in isoproterenol-induced heart failure. There is strong evidence to suggest AMPK activation as a causative factor of the changes. Transthyretin aggregates affect viability and electrical properties of cardiomyocytes. L Sartiani L Sartiani 1 University of Florence, Interuniversity Center of Molecular Medicine and Applied Biophysics (CIMMBA) , Florence , Italy M Bucciantini M Bucciantini 2 University of Florence, Department of Biochemical Sciences , Florence , Italy V Spinelli V Spinelli 1 University of Florence, Interuniversity Center of Molecular Medicine and Applied Biophysics (CIMMBA) , Florence , Italy R Coppini R Coppini 1 University of Florence, Interuniversity Center of Molecular Medicine and Applied Biophysics (CIMMBA) , Florence , Italy E Russo E Russo 2 University of Florence, Department of Biochemical Sciences , Florence , Italy A Mugelli A Mugelli 1 University of Florence, Interuniversity Center of Molecular Medicine and Applied Biophysics (CIMMBA) , Florence , Italy E Cerbai E Cerbai 1 University of Florence, Interuniversity Center of Molecular Medicine and Applied Biophysics (CIMMBA) , Florence , Italy M Stefani M Stefani 2 University of Florence, Department of Biochemical Sciences , Florence , Italy Abstract Senile systemic amyloidosis (SSA), is a sporadic disease affecting aged people whose main symptom is a severe cardiomyopathy associated with arrhythmias. SSA is characterized by the presence of extracellular amyloid fibrillar aggregates of transthyretin (TTR), a plasma protein carrying the thyroid hormone and the retinol binding protein. The aggregates are deposited in several tissues and, together with their oligomeric precursors, are responsible for tissue functional impairment. To date, liver and heart transplantation are the only medical treatments of SSA; accordingly, a thorough investigation of the molecular basis of cell/tissue functional and viability impairment induced by TTR aggregates is expected to provide knowledge needed to identify new pharmacological targets and to develop novel therapeutic strategies. We studied the effect on electrophysiology and viability of HL-1 cardiomyocytes of prefibrillar and fibrillar aggregates of TTR supplemented to the culture media. Only the prefibrillar aggregates were able to interact with the cell membrane and were internalized. This resulted in a moderate impairment of cell viability at the lowest aggregate concentration (10 µM). In the same cells exposed to TTR prefibrillar aggregates, the cytosolic calcium content showed a slow, progressive rise over time; it did not reach a steady state level and came back to its basal levels upon TTR removal from bath solution. By the patch-clamp technique we investigated the effect of the enhanced intracellular calcium on the electrical properties of isolated mouse ventricular myocytes. Action potential recordings were performed at increasing rate of stimulation (0.5, 1 and 2 Hz) before and after application of TTR prefibrillar aggregates. The results showed a progressive prolongation of the action potential that was associated with a marked increase of the duration of the plateau phase; eventually, early and delayed afterdepolarizations occurred. These effects were seen at any frequency of stimulation. Altogether, our data indicate the presence of electrical abnormalities in the exposed cells with pro-arrhythmic potential induced by aggregated TTR. On-going investigation will assess the ionic basis of the altered intracellular calcium and clarify the mechanisms of TTR aggregate cytotoxicity particularly at the level of the cell membrane, the endoplasmic reticulum and the mitochondria. To our knowledge, this is the first mechanistic demonstration of a direct proarrhythmic effect of TTR aggregates in cardiomyocytes, a possible cause of SSA cardiomyopathy. Telmisartan, an angiotensin-II receptor blocker ameliorate cardiac remodeling in rats with dilated cardiomyopathy V Sukumaran V Sukumaran 1 Niigata University , Niigata , Japan K Watanabe K Watanabe 1 Niigata University , Niigata , Japan M Ma M Ma 1 Niigata University , Niigata , Japan R Thandavarayan R Thandavarayan 1 Niigata University , Niigata , Japan W Azrozal W Azrozal 1 Niigata University , Niigata , Japan F Sari F Sari 1 Niigata University , Niigata , Japan H Shimazaki H Shimazaki 1 Niigata University , Niigata , Japan Y Kobayashi Y Kobayashi 1 Niigata University , Niigata , Japan Abstract Multiple trials over the past several years have examined indications for angiotensin receptor blockers (ARBs) in the treatment of left ventricular (LV) dysfunction, both acutely after myocardial infarction and in chronic heart failure (CHF). However, the effects of telmisartan, an ARB in rats with CHF after experimental autoimmune myocarditis (EAM) have not yet been investigated. CHF was elicited in Lewis rats by immunization with cardiac myosin, and twenty eight days after immunization, the surviving Lewis rats were divided into two groups and treated with either telmisartan (10 mg/kg/day) or vehicle. Age matched normal rats without immunizations were also included in this study. After four weeks of treatment, we investigated the effects of telmisartan on cardiac function, proinflammatory cytokines and cardiac remodeling in EAM rats. Myocardial functional parameters measured by hemodynamic and echocardiographic studies were significantly improved by telmisartan treatment in rats with CHF compared with that of vehicle treated rats with CHF. Telmisartan significantly reduced cardiac fibrosis, and hypertrophy and its marker molecules (LV mRNA expressions of transforming growth factor beta1, collagen I and III, and atrial natriuretic peptide) compared with vehicle treated rats. CHF-induced increased myocardial mRNA expressions of proinflammatory cytokines, [interleukin (IL)-6, IL-1β], monocyte chemoattractant protein-1, and matrix metalloproteinases (MMP-2 and -9) were also suppressed by the treatment with telmisartan. Moreover, the plasma level of angiotensin-II was significantly elevated in telmisartan treated rats. Our results indicate that telmisartan treatment significantly improved LV function and ameliorated the progression of cardiac remodeling in rats with CHF after EAM. Cardioprotection of the perivascular adipose tissue against the simulated ischemia/reperfusion injury is nitric oxide dependent, but mitochondrial ATP(K) channels independent in the human heart T Roleder T Roleder 1 Slaski Uniwersytet Medyczny w Katowicach , Katowice , Poland KS Golba KS Golba 1 Slaski Uniwersytet Medyczny w Katowicach , Katowice , Poland MA Deja MA Deja 1 Slaski Uniwersytet Medyczny w Katowicach , Katowice , Poland M Malinowski M Malinowski 1 Slaski Uniwersytet Medyczny w Katowicach , Katowice , Poland S Wos S Wos 1 Slaski Uniwersytet Medyczny w Katowicach , Katowice , Poland Abstract Objective: The adipose tissue serves not only as a lipid storage, but moreover as the endocrine organ. Recently it was revealed that the perivascular adipose tissue of the internal thoracic artery (PVT) has relaxing activity on this artery in humans, probably paracrine in nature. Aims: Whether PVT or the visceral adipose tissue (VAT) can elicit cardioprotection in a human hypoxic myocardial tissue in vitro and whether nitric oxide or the activation of mitochondrial ATP-sensitive potassium channels (mitoK(ATP)) mediate its cardioprotection formed the aims of the current study. Methods: PVT, VAT and muscular trabeculae of the right atrial were harvested from patients subjected to the coronary artery bypass grafting surgery and then incubated in the organ bath. Electrically driven atrial trabeculae were subjected to a simulated ischemia/reperfusion (hypoxia/reoxygenation) protocol (60min/60min); n = 28. In the first experiment, PVT or VAT was applied in the first 15 min. of reoxygenation (PVT and VAT group respectively). In the second experiment the non-selective inhibitor of nitric oxide synthase isoforms (100uM of LNMMA - L-NG-monomethyl Arginine) or mitoK(ATP) inhibitor (100 uM of glibenclamide) were applied with PVT at the first 15 min. of reoxygenation as well (PVT+LNNMA and PVT+Glyb group respectively). 10 uM norepinephrine (NE) application ended each experiment. Two trabeculae were studied simultaneously; ones subjected only to hypoxia/reoxygenation injury severed as a control. The maximal inotropic response (Amax) was recorded before hypoxia, during the reoxygenation period and after NE application. The results are presented as a percent of Amax values before hypoxia (mean ± SEM). Results: Amax was higher in the PVT group and in the PVT+Glyb group vs. control in the last 30 min. of reoxygenation and after NE application (Amax: PVT30-60min = 33,43 ± 2,6% vs. Control30-60min = 24.91 ± 2,6%*, PVTNE = 69,05 ± 13,3% vs. ControlNE = 42,74 ± 10,6%*, PVT+Glyb30-60min = 52,98 ± 3,1% vs. Control30-60min = 43,83 ± 3,1%, PVT+GlybNE = 94,08 ± 11,0% vs. ControlNE = 72,50 ± 13,3%). In turn Amax was lower for the VAT group and for the PVT+LNNMA group vs. control group during whole reoxygenation period and after NE application (Amax: VAT = 45,90 ± 4,5% vs. Control = 59.20 ± 5,87%*; VATNE = 70,1 ± 19,2%* vs. ControlNE = 93,14 ± 19,8%; PTV+LNMMA = 26,25 ± 1,7% vs. Control = 42,68 ± 1,7%; PVT+LNNMANE = 45,69 ± 5,3% vs. ControlNE = 81,53 ± 9,1%; *p < 0,05). Conclusion: Only PVT not VAT exerts cardioprotection which is nitric oxide depended and mitoK(ATP) independent. Depleting extracellular RNA reduces vascular permeability, edema formation and myocardial infarction size P Stieger P Stieger 1 University Hospital Giessen and Marburg, Medical Clinic I, Cardiology and Angiology , Giessen , Germany M Grebe M Grebe 1 University Hospital Giessen and Marburg, Medical Clinic I, Cardiology and Angiology , Giessen , Germany H Tillmanns H Tillmanns 1 University Hospital Giessen and Marburg, Medical Clinic I, Cardiology and Angiology , Giessen , Germany KT Preissner KT Preissner 2 Biochemisches Institut , Giessen , Germany D Sedding D Sedding 1 University Hospital Giessen and Marburg, Medical Clinic I, Cardiology and Angiology , Giessen , Germany Abstract Cell injury due to myocardial infarction (MI) leads to exposure of intracellular material and is associated with increased permeability of vessels in the vicinity of the damage, a process that contributes to tissue injury throughout the ventricle. We previously demonstrated, that released, natural extracellular RNA significantly increases the permeability across microvascular endothelial cells through a vascular endothelial growth factor (VEGF)-dependent mechanism. In the present study we therefore evaluated the impact of RNase-application in vivo after induction of MI in mice. The left coronary artery (LAD) of C57/Bl 6 was ligated and RNase (100 μg) or control buffer was administered intravenously 30 min, 3 and 6 hours following LAD ligation. Wet and dry weight of heart slices were measuerd for analysis of myocardial edema, and evans blue dye and tetrazolium were used to delineate the area at risk and infarction size within the myocardium 24h after ligation. Cardiac function as measured by fractional shortening was assessed by echocardiography, MRT- and Micro CT-imaging. RNAse treatment had no effect on blood pressure, total plasma protein or albumin levels, peripheral blood cell counts or glucose levels. However, edema formation was significantly decreased in RNAse treated mice as measured by wet/dry ratio (3.38 ± 0.19 vs. 3.93 +- 0.27; P < 0.05). Since risk zone sizes were similar between groups, the percentage infarction of the risk zone was significantly smaller in RNAse treated mice (P < 0.05). Moreover, Fractional shortening analysis determined by echocardiography revealed a significantly enhanced myocardial contractility and showed a prolongation in survival after MI in RNAse treated mice (25.3 ± 2.6% vs. 13.8 ±2.6 %; P < 0.05). These results identify extracellular RNA as a novel natural permeability factor which augments ischemia-induced edema formation and myocardial infarction. Moreover, RNase treatment may serve as a novel vessel-protective modality, preventing MI-induced edema formation and tissue injury and thus improving survival after MI. No additional benefit from higher dose erythropoietin to reduce infarct size after coronary artery ligation in wistar rats E Ercan E Ercan 1 Canakkale Onsekiz Mart University Faculty of Medicine Dept of Cardiology , Canakkale , Turkey A Guven A Guven 2 Canakkale Onsekiz Mart University Faculty of Medicine Dept of Histology and Embriology , canakkale , Turkey F Asgun F Asgun 3 Canakkale Onsekiz Mart University Faculty of Medicine Dept of Cardiovascular Surgery , Canakkale , Turkey M Ickin M Ickin 2 Canakkale Onsekiz Mart University Faculty of Medicine Dept of Histology and Embriology , canakkale , Turkey F Ercan F Ercan 4 Marmara University Faculty of Medicine Dept of Histology and Embriology , Istanbul , Turkey A Kaplan A Kaplan 5 Aile Hekimligi Egitim Arastirma , istanbul , Turkey O Yavuz O Yavuz 6 Duzce University, Faculty of Medicine, Department of Medical Biochemistry , Duzce , Turkey S Bagla S Bagla 7 Gene technology , Ankara , Turkey Abstract 507 Table Infarct sizes of the groups . . Rat groups n = 19 Control group Erythropoietin (10000 U/kg i.p.) . Erythropoietin (5000 U/kg i.p.) . Infarct size (%) Mean ± SD 51,99 ± 8,14 31,38 ± 10,52 31,00 ± 12,30 Percentile 05 38,00 19,72 18,45 Percentile 95 61,40 45,00 47,02 . . Rat groups n = 19 Control group Erythropoietin (10000 U/kg i.p.) . Erythropoietin (5000 U/kg i.p.) . Infarct size (%) Mean ± SD 51,99 ± 8,14 31,38 ± 10,52 31,00 ± 12,30 Percentile 05 38,00 19,72 18,45 Percentile 95 61,40 45,00 47,02 Open in new tab Abstract 507 Table Infarct sizes of the groups . . Rat groups n = 19 Control group Erythropoietin (10000 U/kg i.p.) . Erythropoietin (5000 U/kg i.p.) . Infarct size (%) Mean ± SD 51,99 ± 8,14 31,38 ± 10,52 31,00 ± 12,30 Percentile 05 38,00 19,72 18,45 Percentile 95 61,40 45,00 47,02 . . Rat groups n = 19 Control group Erythropoietin (10000 U/kg i.p.) . Erythropoietin (5000 U/kg i.p.) . Infarct size (%) Mean ± SD 51,99 ± 8,14 31,38 ± 10,52 31,00 ± 12,30 Percentile 05 38,00 19,72 18,45 Percentile 95 61,40 45,00 47,02 Open in new tab Open in new tabDownload slide Abstract 507 Figure Open in new tabDownload slide Abstract 507 Figure Abstract Background: Erythropoietin (Epo) can reduce the infarct size and have a cardioprotective effect in animal models of myocardial infarction (MI). We hypotezed that higher dose of Epo can reduce myocardial infarct size more efficiently. Method: We compared the impact of conventional and higher dose of single systemic administration of recombinant human Epo alpha (5000 U/kg versus 10.000 U/kg i.p.) on left ventricular (LV) infarct size immediately after the induction of a myocardial infarction (MI) by permanent ligation of the left descending coronary artery. Serum epo levels, immunohistochemistry (Epo-R, Caspase 3, HIF) and electronmicroscopy were studied. Results: Injection of 5.000 U/kg Epo alpha (Figure-middle) reduces the infact size compared to the control group (Figure-top). However, 10000U/kg Epo (Figure-buttom) showed no additional benefit over 5000 U/kg Epo i.p.after coronary ligation (p:0.7). Conclusion: Epo reduces the infact size after MI in rats. Optimal dose is needed to be defined to achieve maximal myocardial protection. Higher dose of Epo have trend to lower infarct size but no additional significant benefit was found. 5000 U/kg may be the higher upper limit to reduce the infarct size 11 . Effects caused by postconditioning of mouse heart suffering from reperfusion injury and mechanisms induced by reperfusion injury salvage kinase. YINING Yang YINING Yang 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of YT Ma YT Ma 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of FEN Liu FEN Liu 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of XM Li XM Li 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of YING Huang YING Huang 1 Cardiovascular Institute of 1st affiliated hospital in Xinjiang Medical University , Urumuqi , China, People's Republic of Abstract Objective: Explore cardioprotective mechanisms induced by reperfusion injury salvage kinase(ERK1/2) signal transduction pathways changes in isolated mouse hearts. Methods: Established Langendorff perfused mouse heart model, 96 C57BL mouse hearts were divided to 4 groups: 1) I/R control: 30 min global ischemia and 2 h reperfusion; 2) IPC : IPC with 3 episodes of 10 s of ischemia and 10 s reperfusion after 30 min ischemia and before 2 h reperfusion; 3) IPC+ERK1/2 inhibitor PD98059 (10-5 mol/L for 15 min); 4) I/R+ERK1/2 inhibitor PD98059 (10-5 mol/L for 15 min). The effects of IPC on Ischemic size, myocardial cell apoptosis index, phospho-protein kinase(P-ERK1/2) and phospho-protein kinase B(P-Akt) were measured. Expressions of apoptosis related proteins, including Bax, Bcl-2 and Cyt.C, in cytosolic and membrane fraction were detected by Western blot. Results: Following 180min reperfusion, infarct size was significantly reduced in postconditioning group (32.8 ± 3. 8)% compared with I/R group hearts [(48.1 ± 4.8)%, P <0.05], and equally improved myocardial function compared with I/R group (all P < 0.05). Compared with ischemia reperfusion group, ischemic postconditioning group markedly decreased apoptotic index(AI)(P < 0.05), increased the mRNA expression of Bcl-2 gene(P < 0.05), the ratio of Bcl-2 to Bax was incidentally up-regulated (P < 0.05). Although IPC did not induce changes in the level of Bcl-2 expression in cytosolic fraction compared with I/R groups, the expression of Bcl-2 in membrane fraction was up regulatedin IPC group compared with I/R group. The increase in there lease of mitochondrial Cyt.C into cytosol induced by I/R was significantly reduced by IPC. The histopathological changes in mitochondria and myocardium destroyed by ischemia repefusion were alleviated markedly by ischemic postconditioning. IPC significantly increased myocardial ERK1/2 phosphorylation, PD98059 inhibited the phosphorylation of ERK1/2 and abolished the cardioprotective effects induced by IPC. Conclusions: Postconditioning plays a pivotal role in reducing myocardial infarct size after ischemia/ reperfusion injury. IPC right before reperfusion but not after reperfusion attenuated I/R injury of isolated mouse hearts via the upstream target, ERK1/2-MAPK signal transduction pathway which mediated the expression of Bcl-2/Bax and the release of mitochondrial Cyt.C. Effects of regulation of L-carnitine concentration in experimental heart infarction model J Kuka J Kuka 1 Riga Stradins University , Riga , Latvia R Vilskersts R Vilskersts 2 Latvian Institute of Organic Synthesis , Riga , Latvia E Vavers E Vavers 2 Latvian Institute of Organic Synthesis , Riga , Latvia E Liepins E Liepins 2 Latvian Institute of Organic Synthesis , Riga , Latvia M Dambrova M Dambrova 2 Latvian Institute of Organic Synthesis , Riga , Latvia Abstract L-carnitine is essential for the transport of long-chain fatty acids into the mitochondria, supporting normal functioning of cardiomyocytes. Mildronate is a cardioprotective drug whose mechanism of action is based on inhibition of both biosynthesis of L-carnitine and reabsorbtion of it in kidneys, while direct correlation between carnitine concentration and anti-infarction activity of mildronate has not been shown experimentally before. Therefore, the aim of this study was to investigate how changes in L-carnitine concentration affect myocardial ischemia-reperfusion injury in isolated rat heart model. Male Wistar rats were perorally treated daily for 14 days with L-carnitine (100 mg/kg), mildronate (100 mg/kg) and a combination of both substances (100+100 mg/kg). Experimental isolated rat heart ischemia-reperfusion (I-R) experiment and carnitine concentration measurement by UPLC-MS/MS were performed. Administration of mildronate induced a 3-fold decrease in plasma L-carnitine concentration. L-carnitine supplementation induced a 1.5-fold increase in L-carnitine plasma concentration, while treatment with combination did not induce significant changes in L-carnitine concentration after 14-day treatment. In heart tissues, after mildronate and combination treatment, L-carnitine concentrations were decreased by 68% and 29%, respectively; while L-carnitine treatment did not induce any changes. Mildronate and L-carnitine treatment significantly decreased the infarct size for 34% and 28%, respectively. Combination decreased infarct size by only 9% and this effect was not significant. In conclusion, our results provide direct evidence that the cardioprotective effect of mildronate is related to the metabolic actions due to decrease in L-carnitine heart tissue concentration. In addition, our study shows the beneficial role of regulated L-carnitine availability in cardioprotective therapy. May hyperoxic ventilation during early reperfusion increase infarct size? LH Mariero LH Mariero 1 Oslo University Hospital, Ulleval , Oslo , Norway A Rutkovskiy A Rutkovskiy 1 Oslo University Hospital, Ulleval , Oslo , Norway KO Stenslokken KO Stenslokken 2 Institute for Molecular Biosciences, University of Oslo , Oslo , Norway J Vaage J Vaage 1 Oslo University Hospital, Ulleval , Oslo , Norway Abstract Purpose: Oxygen is routinely administered to patients undergoing acute myocardial infarction and also during acute revascularization. Since reactive oxygen species are key mediators of ischemia-reperfusion injury, increased oxygen availability might theoretically aggravate myocardial injury during revascularization and reperfusion. Therefore, we hypothesized that hyperoxic ventilation during early reperfusion will increase infarct size. Methods: Anesthetized rats were exposed to 40 minutes of regional myocardial ischemia and 120 minutes of reperfusion. Control animals (n = 14) were ventilated with 40 % oxygen throughout and test animals were ventilated with normobaric hyperoxia (95% O2) during the last ten minutes of ischemia and the first ten minutes of reperfusion. Due to lethal reperfusion arrhythmias, one animal in the hyperoxia group and six animals in the control group were excluded. Hearts (n = 8 in the control group and n = 10 in the test group) were harvested for measurements of area at risk and infarct size. Results: The incidence of lethal arrhythmias was 1/11 in the test group and 6/14 in the control group, p = 0.06. Reperfusion with hyperoxia did not influence infarct size compared to the normoxia group (20 ± 8 % and 24 ± 8, respectively, mean ± SD, p>0.2). There was no difference in hemodynamics between the groups. Conclusion: Normobaric hyperoxia during early reperfusion did not increase infarct size and is safe to use during revascularization. CB2 receptor influences scar formation after murine myocardial infarction GD Duerr GD Duerr 1 University of Bonn, Dept. of Cardiac Surgery , Bonn , Germany G Suchan G Suchan 1 University of Bonn, Dept. of Cardiac Surgery , Bonn , Germany T Heuft T Heuft 1 University of Bonn, Dept. of Cardiac Surgery , Bonn , Germany T Klaas T Klaas 1 University of Bonn, Dept. of Cardiac Surgery , Bonn , Germany A Zimmer A Zimmer 2 University of Bonn, Institute of Molecular Psychiatry , Bonn , Germany A Welz A Welz 1 University of Bonn, Dept. of Cardiac Surgery , Bonn , Germany BK Fleischmann BK Fleischmann 3 University of Bonn, Institute of Physiology I , Bonn , Germany O Dewald O Dewald 1 University of Bonn, Dept. of Cardiac Surgery , Bonn , Germany Abstract Purpose: Myocardial infarction induces transient inflammatory response with subsequent tissue remodelling and scar formation. We showed macrophage infiltration-dependent formation of granulation tissue and rapid scar development in mice. Since endocannabinoid receptor CB2 has been associated with regulation of macrophage function, we investigated its role in murine myocardial infarction. Methods: One hour LAD-occlusion was followed by reperfusion over 6 hrs, 1, 3 and 7 days in C57/Bl6 (WT)- and CB2−/−-mice (n = 8/group). Hearts were processed for functional, morphological, and mRNA-studies. Results: We found transient CB2 mRNA-expression in reperfused infarction in WT-mice. WT-mice showed a rapid phagocytosis of dead cardiomyocytes with formation of granulation tissue after 3 days reperfusion. At the same time, a transient macrophage infiltration was observed with a peak after 3 days. Myofibroblast differentiation followed after 3 days and compacted collagen deposition was observed in completed scar formation after 7 days reperfusion. In contrast, CB2-deficiency led to postponed phagocytosis of dead cardiomyocytes resulting in delayed granulation tissue formation. Macrophage infiltration was comparable between the strains. CB2−/−-mice showed in contrast to WT-mice myofibroblast persistence in the scar after 7 days reperfusion. Collagen deposition appeared also less compacted at this time point. The catheter-based left ventricular function measurements showed significantly worse left ventricular function in CB2−/−-mice. The mRNA-analysis showed significantly lower induction of TNF-α, CCL2 and CCL4 in CB2−/−-mice. Anti-inflammatory IL-10 and remodelling related TGF-β1 and tenascin C were not induced in CB2−/−-hearts. Conclusions: Endocannabinoid receptor CB2 seems to regulate expression of cytokines and chemokines in order to modulate the macrophage phagocytosis in infracted heart. It also appears to be involved in differentiation of myofibroblasts through regulation of tenascin and TGF-β with significant impact on myocardial remodeling and scar formation. Cardiac imaging in mice using routine clinical MRI scanners JGJ Voelkl JGJ Voelkl 1 Innsbruck Medical University , Innsbruck , Austria B Haubner B Haubner 2 Innsbruck Medical University, Department of Internal Medicine III, Cardiology , Innsbruck , Austria C Kremser C Kremser 1 Innsbruck Medical University , Innsbruck , Austria A Mayr A Mayr 1 Innsbruck Medical University , Innsbruck , Austria G Klug G Klug 2 Innsbruck Medical University, Department of Internal Medicine III, Cardiology , Innsbruck , Austria M Reiner M Reiner 2 Innsbruck Medical University, Department of Internal Medicine III, Cardiology , Innsbruck , Austria O Pachinger O Pachinger 2 Innsbruck Medical University, Department of Internal Medicine III, Cardiology , Innsbruck , Austria B Metzler B Metzler 2 Innsbruck Medical University, Department of Internal Medicine III, Cardiology , Innsbruck , Austria Abstract Background: To enable cardiac imaging in mice without having to invest in expensive dedicated equipment, we adapted a clinical 1.5T magnetic resonance imaging (MRI) scanner for use in a murine ischemia/reperfusion model. We aimed to determine correlation between infarct size evaluated by late gadolinium enhancement (LGE) in comparison with histology and functional parameters. Method: Mice subjected to ischemia/reperfusion procedure were randomized into groups of 0, 30 and 60 min of ischemia. LGE facilitated the determination of in vivo infarct sizes which were compared to histological infarct areas. In addition, fractional area change (FAC) assessed with cine MRI was matched to infarct size and fractional shortening (FS) measured with echocardiography. Results: A significant difference in LGE imaging of infarct size was found between all groups (infarct area after 1 week: 30min: 18.3 ± 4.4%; 60min: 38.3 ± 10.2%). There was good correlation between infarct size measured by adapted phase-sensitive inversion recovery (PSIR) sequence and histological infarct size (after 1 week: r = 0.807 p < 0.001). Moreover, functional parameters derived from cine MRI imaging (FAC) showed good correlation with infarct size (r= −0.837) and echocardiography (FS) (r = 0.860); both p < 0.001. Conclusions: Here, we demonstrate that cardiac MRI in mice using clinical 1.5T MRI scanners enables quantification of in vivo infarct dimensions as well as assessment of cardiac functional parameters in an ischemia/reperfusion mouse model. This adapted scanner obviously cannot perform as well as specialized rodent scanners, but can provide a valuable imaging alternative for basic phenotyping with advantages over echocardiography. Cardioprotective activity of apelin-12 against ischaemia-reperfusion injury in isolated rat heart O Pisarenko O Pisarenko 1 Russian Cardiology Research-and-Production Complex , Moscow , Russian Federation V Shulzhenko V Shulzhenko 1 Russian Cardiology Research-and-Production Complex , Moscow , Russian Federation Y Pelogeykina Y Pelogeykina 1 Russian Cardiology Research-and-Production Complex , Moscow , Russian Federation D Khatri D Khatri 1 Russian Cardiology Research-and-Production Complex , Moscow , Russian Federation I Studneva I Studneva 1 Russian Cardiology Research-and-Production Complex , Moscow , Russian Federation Abstract This work was designed to explore efficacy of apelin-12 (A-12) as a cardioprotective agent using the isolated working heart model. Hearts of male Wistar rats perfused with Krebs-Henseleit buffer (KHB) with 11 mM glucose were subjected to 35-min global ischaemia and 30-min reperfusion. A short-term infusion of KHB containing A-12 (35, 70, 140, 280 and 560 µM) was applied prior to ischemia (A-12-I) or at onset of reperfusion (A-12-R). KHB infusion was used in control. Metabolic state of reperfused hearts was assessed by tissue contents of adenine nucleotides (AN), lactate, pyruvate and phosphocreatine (PCr). Lactate dehydrogenase (LDH) leakage assay in perfusate was employed for assessment of membrane integrity. A-12 infusion induced a dose-dependent gradual increase in recovery of coronary flow, contractile and pump function during reperfusion, with the largest augmentation of these indices in A-12-I group. Thus, after infusion of 140 µM A-12, recovery of coronary flow, the LVDP-HR product and minute volume were 92 ± 4%, 81 ± 5% and 77 ± 5% of the initial values, respect., in A-12-I group, 83 ± 3%, 61 ± 4% and 52 ± 3% in A-12-R group, and 76 ± 2%, 42 ± 2%, 32 ± 2 % in control by the end of reperfusion. Both A-12 groups exhibited a significant reduction of I/R contracture. Enhanced functional recovery in A-12-I group was combined with a decrease in LDH leakage in perfusate on early reperfusion. Preischaemic infusion of 140 µM A-12 markedly increased myocardial ATP content, twice decreased AMP accumulation and did not affect ADP content at the end of reperfusion compared with these indices in control. These alterations resulted in enhanced preservation of the total AN pool and a better recovery of the energy charge potential in reperfused hearts. There was a trend towards increase in myocardial PCr by the end of reperfusion in A-12-I group; however this benefit did not reach statistical significance. At the end of experiment, myocardial lactate and lactate/pyruvate ratio were on average 5-fold lower in A-12-I treated hearts compared with control ones and did not differ significantly from the initial values. This finding implies that a better restoration of energy metabolism in the hearts protected with A-12 before ischemia might be attributed to ameliorated glucose oxidation during reperfusion. Therefore, enhanced functional recovery of ischemic heart and a lesser cell membrane damage induced by A-12 are associated with maintaining high energy phosphates, particularly ATP, in reperfused myocardium. Changes in energy metabolism may play a role in the mechanism of cardioprotection afforded by A-12 during I/R stress. Q50, a novel zinc complex, reduces ischemia/reperfusion injury in a rat model of heterotopic heart transplantion E Barnucz E Barnucz 1 University of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany S Loganathan S Loganathan 1 University of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany K Hirschberg K Hirschberg 1 University of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany S Korkmaz S Korkmaz 1 University of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany B Merkely B Merkely 2 Semmelweis University Heart Center , Budapest , Hungary M Karck M Karck 1 University of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany G Szabo G Szabo 1 University of Heidelberg, Department of Cardiac Surgery , Heidelberg , Germany Abstract Myocardial ischemia/reperfusion characterized by both significant oxidative stress and characteristic changes in the antioxidant defense, occurs during heart transplantation and alters myocardial function. Recent studies have suggested that the zinc-homeostasis plays an important role in ischemia/reperfusion injury. The novel zinc-complex Q50 has a major effect on this zinc-homeostasis. In the present study we investigated the effects of the cytoprotective Q50 on myocardial function after ischemia/reperfusion in a rat model of heterotopic heart transplantation. Heterotopic heart transplantation was performed in Lewis rats. Two experimental groups were assigned for this study: one transplanted control group and one Q50-treated group. In the treatment group the donors were injected with Q50 (30 mg/kg) intravenously 1 hour before onset of ischemia. The ischemia time was standardized to 1 hour. The target parameters were assessed after 1 hour of reperfusion. Left ventricular pressures, its first derivative and left ventricular end-diastolic pressure were evaluated. Adenosine triphosphate (ATP) content of the myocardial tissue was measured, energy charge potential (ECP), as marker for the energy state of the myocardial tissue, was calculated. After 1 hour, left ventricular systolic pressure (transplanted control vs. Q50-treated group: 82 ± 4 vs. 105 ± 5 mmHg; p < 0.05) and its first derivative (transplanted control vs. Q50-treated group: 1740 ± 116 mmHg/s vs. 3219 ± 190; p < 0.05) were significantly higher in the Q50-treated group compared with the transplanted control group. Moreover, myocardial ATP content (transplanted control vs. Q50-treated group: 1.86 ± 0.41 vs. 6.66 ± 0.63 μmol/g; p < 0.05) and ECP (transplanted control vs. Q50- treated group: 0.49 ± 0.04 vs. 0.85 ± 0.08; p < 0.05) were significantly higher in the Q50-treated group. Thus, we conclude that the novel Q50 shows cardioprotective effects and improves myocardial function during the critical phase of ischemia/reperfusion after heart transplantation. Human recombinant EPO reduces myocardial ischemia/reperfusion injury P Bencsik P Bencsik 1 University of Szeged , Szeged , Hungary A Gorbe A Gorbe 1 University of Szeged , Szeged , Hungary GF Kocsis GF Kocsis 1 University of Szeged , Szeged , Hungary C Csonka C Csonka 1 University of Szeged , Szeged , Hungary T Csont T Csont 1 University of Szeged , Szeged , Hungary M Shamloo M Shamloo 2 Stanford University , Palo Alto , United States of America KW Woodburn KW Woodburn 3 Affymax, Inc , Palo Alto , United States of America P Ferdinandy P Ferdinandy 1 University of Szeged , Szeged , Hungary Abstract Purpose: Little is known about the efficacy of erythropoietin (EPO) on myocardial reperfusion injury. Therefore, we aimed to study whether EPO and darbepoetin (a modified synthetic EPO-analogue with longer circulating half life) are able to protect the heart against reperfusion injury. Methods: Primary cultures of neonatal cardiomyocytes were treated with 1, 10, 100 or 500 U/ml EPO and subjected to 2.5 hrs of hypoxia and 2 hrs of reoxygenation followed by trypan blue viability test to estimate the effective dose range of EPO. In separate experiments, in mechanically ventilated anesthetized male Wistar rats (250-350 g) myocardial infarction was induced by 30 min coronary occlusion followed by 2 hours of reperfusion. Rats (n = 8-10/each group) were given either vehicle, B-type natriuretic peptide (10 nmol/kg + 2 nmol/kg/15 min infusion; positive control) or EPO (5000 U/kg) 5 min before the onset of reperfusion. Darbepoetin (2.5, 5, 15 and 25 μg/kg) was administered 15 min before onset of reperfusion. Infarct size was determined by the standard Evans blue/TTC method. Results: EPO in 100 U/ml concentration increased cell viability significantly when compared to control group (from 59.6 ± 1.5% to 66.2 ± 2.3%). EPO at 5000 U/kg reduced infarct size (45.3 ± 4.8%, p < 0.05) when compared to vehicle (65.1 ± 2.5%). Darbepoetin showed a U-shaped dose-response curve, with maximal infarct size limiting effect at 5 μg/kg (44.6 ± 6.1%, p < 0.05). The positive control BNP reduced infarct size to 40.3 ± 7.4%. Conclusions: Both EPO and darbepoetin are able to decrease infarct size when administered before the onset of reperfusion. EPO analogues may be promising cardioprotective agents that alleviate acute ischemia/reperfusion injury when applied before revascularization in patients with acute myocardial infarction. Postconditioning fails to limit infarct size in rat hearts with global ischemia. G Szucs G Szucs 1 Cardiovascular Research Group, University of Szeged , Szeged , Hungary K Kupai K Kupai 1 Cardiovascular Research Group, University of Szeged , Szeged , Hungary C Csonka C Csonka 1 Cardiovascular Research Group, University of Szeged , Szeged , Hungary C Csont C Csont 2 Pharmahungary Group , Szeged , Hungary P Ferdinandy P Ferdinandy 2 Pharmahungary Group , Szeged , Hungary Abstract Purpose: The cardioprotective efficacy of postconditioning (PostC), i.e. intermittent interruption of coronary flow in the very early phase of reperfusion, is influenced by the PostC protocol used. Therefore, our aim was to study the infarct size limiting efficacy of PostC after global or regional ischemia and to test the involvement of matrix metalloproteinase-2 (MMP-2) in PostC. Methods: Hearts isolated from male Wistar rats were randomized into four groups. Hearts were subjected to either 30-min global ischemia or coronary occlusion-induced regional ischemia followed by 120-min reperfusion with or without postconditioning protocol induced by six intermittent periods of ischemia/reperfusion of 10-s duration each. At the end of reperfusion, infarct size was determined by standard TTC staining. In separate experiments cardiac MMP-2 activities are measured at the 10th min of reperfusion in all groups. Results: We found that postconditioning applied after coronary occlusion significantly decreased infarct size (12.6 ± 3.2% vs. 43.0 ± 3.1%, p < 0.05) and attenuated MMP-2 activity while the same postconditioning protocol after global ischemia failed to affect infarct size (41.3 ± 3.6% vs. 42.9 ± 2.5%) and did not influence MMP-2 activity. Conclusion: PostC offered protection after regional ischemia by a mechanism that likely involves a decreased activation of MMP-2. However, PostC remains ineffective when applied after global ischemia in isolated rat hearts. The influence of chronic renal failure on the cardioprotective effect of postconditioning in rats with partial nephrectomy. G Kocsisne Fodor G Kocsisne Fodor 1 University of Szeged, Faculty of Medicine, Cardiovascular Research Group , Szeged , Hungary P Bencsik P Bencsik 2 Pharmahungary Group , Szeged , Hungary V Fekete V Fekete 1 University of Szeged, Faculty of Medicine, Cardiovascular Research Group , Szeged , Hungary ZV Varga ZV Varga 1 University of Szeged, Faculty of Medicine, Cardiovascular Research Group , Szeged , Hungary P Monostori P Monostori 3 University of Szeged, Faculty of Medicine, Department of Pediatrics , Szeged , Hungary S Turi S Turi 3 University of Szeged, Faculty of Medicine, Department of Pediatrics , Szeged , Hungary P Ferdinandy P Ferdinandy 2 Pharmahungary Group , Szeged , Hungary T Csont T Csont 2 Pharmahungary Group , Szeged , Hungary Abstract Purpose: Cardioprotection by postconditioning may be influenced by some co-morbidities, such as e.g. hyperlipidemia. It is not known if chronic renal failue may interact with cardioprotective mechanisms. Therefore, here we aimed to examine the effect of chronic renal failure on the infarct size limiting effect of ischemic postconditioning. Methods: Male Wistar rats underwent 5/6 nephrectomy. Ten weeks later, blood was collected to measure serum carbamide and hearts were isolated and perfused according to Langendorff. Hearts were subjected to 10 min aerobic perfusion followed by 30 min of normothermic regional ischemia and 120 min of reperfusion with or without a postconditioning protocol. Postconditioning was induced by six intermittent periods of ischemia/reperfusion cycles of 10-s duration each. At the end of the perfusion, infarct size was determined by standard TTC staining and expressed as % infarct of the area at risk. Results: Serum carbamide significantly elevated at week 10 after 5/6 nephrectomy when compared to sham operated controls (8.1mmol/l vs.12.0 mmol/l p < 0.0001). Postconditioning significantly decreased infarct size in both the sham operated, and in the nephrectomized rat hearts when compared to non-postconditioned ischemic controls (11.3 ± 2.4% vs. 29.4 ± 5.3% and 14.4 ± 3.5% vs. 22.8 ± 4.9% p < 0.005 2-way ANOVA). Infarct size was not affected significantly by the nephrectomy itself in the non-postconditioned groups. Discussion: We conclude that the infarct size limiting effect of postconditioning is still effective in chronic renal failure induced by 5/6 nephrectomy, however, the magnitude of protection by postconditioning seems to be attenuated. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2010. For permissions please email: journals.permissions@oxfordjournals.org Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2010. For permissions please email: journals.permissions@oxfordjournals.org
TI - Sunday, 18 July 2010
JF - Cardiovascular Research
DO - 10.1093/cvr/cvq176
DA - 2010-07-15
UR - https://www.deepdyve.com/lp/oxford-university-press/sunday-18-july-2010-YCGbWixlA0
SP - S89
EP - S135
VL - 87
IS - suppl_1
DP - DeepDyve
ER -