TY - JOUR
AU1 - Pinto, Antonio
AU2 - Immohr, Moritz Benjamin
AU3 - Jahn, Annika
AU4 - Jenke, Alexander
AU5 - Boeken, Udo
AU6 - Lichtenberg, Artur
AU7 - Akhyari, Payam
AB - OBJECTIVES Cardiac surgery with cardiopulmonary bypass (CPB) provokes ischaemia and reperfusion injury (IRI). Superoxide is a main mediator of IRI and is detoxified by superoxide dismutases (SODs). Extracellular SOD (SOD3) is the prevailing isoform in the cardiovascular system. Its mutation is associated with elevated risk for ischaemic heart disease as epidemiological and experimental studies suggest. We investigated the influence of SOD3 on IRI in the context of CPB and hypothesized a protective role for this enzyme. METHODS Mutant rats with loss of SOD3 function induced by amino acid shift, SOD3-E124D, (SOD3 mutant; n = 9) were examined in a model of CPB with deep hypothermic circulatory arrest provoking global IRI and compared with SOD3 competent controls (n = 8) as well as sham animals (n = 7). SOD3 plasma activity was photometrically measured with a diazo dye-forming reagent. Activation of cardioprotective rescue pathways (p44–42 MAPK and STAT3), cleavage of PARP-1, expression of SOD isoforms (SOD1, 2 and 3) and nitric oxide metabolism were analysed on the protein level by western blot. To evaluate whether SOD3 inactivity directly affects the myocardium, we isolated adult cardiac myocytes, which underwent hypoxia prior to protein analyses. RESULTS Relative SOD3 plasma activity in SOD3 mutant rats was significantly decreased by at least 50% compared with that in SOD3 competent controls (prior to euthanasia P = 0.008). Effectively, physiological parameters [heart rate and mean arterial pressure (MAP)] indicated a trend toward impaired handling of ischaemia and reperfusion in SOD3 mutants: after reperfusion, mean heart rate was 46 bpm lower (P = 0.083) and MAP 8 mmHg lower (P = 0.288) than that in SOD competent controls. Decreased SOD3 activity led to reduced activation of cardioprotective rescue pathways in vivo and in vitro: relative activation of p44–42 MAPK (P = 0.074) and STAT3 (P = 0.027) was more than 30% decreased in heart and aortic tissue of SOD3 mutants (activity normalized to sham control as 1). After CPB, cleavage of PARP-1 was doubled in the control group (P = 0.017), but increased 3-fold in SOD3 mutants (P = 0.002). Furthermore, 3-nitrotyrosine as a measure of decreased nitric oxide bioavailability and other SOD isoforms (SOD1 and 2) were increased. CONCLUSIONS Collectively, SOD3 has a significant cardioprotective role in cases of IRI and directly affects the myocardium as hypothesized. Exploration of intervention strategies targeting SOD3 may provide therapeutic options against IRI and associated systemic inflammation. Cardiopulmonary bypass, Ischaemia, Reperfusion, Deep hypothermic circulatory arrest, Systemic inflammatory syndrome, Extracellular superoxide dismutase, SOD3 INTRODUCTION Cardiovascular diseases, such as coronary artery disease and acute myocardial infarction, are a main cause of mortality and morbidity worldwide. Cardiac surgery is a powerful therapeutic option in the treatment of most forms of cardiovascular disease. However, the associated need for extracorporeal circulation by cardiopulmonary bypass (CPB) translates into intraoperative contact of blood with artificial surfaces and may activate systemic inflammatory signalling cascades. Extracorporeal circulation has a 2–10% risk of systemic inflammatory response syndrome (SIRS), which in turn may induce lethal multiple organ dysfunction syndrome. Moreover, sudden reperfusion after temporary myocardial ischaemia provokes further damage in the heart [1]. Different elements of oxidative damage further extend the initial ischaemic injury [2]. Leucocytosis as well as increased IL-6 and TNF-α concentrations indicates an inflammatory component of ischaemia and reperfusion injury (IRI) on the systemic level [3]. Reactive oxygen species (ROS) such as superoxide (O2−) are a main mediator of IRI [4]. Superoxide dismutases (SODs) detoxify superoxide to hydrogen peroxide that is later metabolized to water [5, 6]. However, upon accumulation superoxide reacts with nitric oxide to form peroxynitrite [5]. Whereas nitric oxide is vasoprotective in that it has a vasodilative effect on vascular smooth muscle cells (VSMCs), peroxynitrite is a reactive nitrogen species, further extending oxidative tissue damage [5]. Because superoxide reacts six times faster with nitric oxide than with any isoform of the SOD, an adequate tissue concentration of SOD is necessary to preserve adequate nitric oxide bioavailability and to prevent pathological peroxynitrite production [7]. In the human organism, three SOD isoforms exist, of which SOD3 is the only extracellular isoform and occurs in bodily fluids such as plasma, lymph, synovial and cerebrospinal fluid [8]. In the cardiovascular system, SOD3 is the predominant isoform responsible for up to 70% of all SOD activity [9]. SOD3 is a homotetrameric secretory glycoprotein with a high affinity for sulphated glycosaminoglycans, especially heparin [8]. Therefore, SOD3 is capable of binding on cell surfaces. Hence, SOD3 is mostly located in the extracellular matrix and bound on cell surfaces [5]. VSMCs express high amounts of SOD3 and are the main source of SOD3 in the vascular system, where its expression is mediated by several tissue factors, such as angiotensin II [10]. It is suggested that overexpression of SOD3 leads to protection against arteriosclerosis by thwarting low-density lipoprotein oxidation in endothelial cells. In addition, overexpression of SOD3 in mice has been effective in decreasing infarct size and increasing cardiac regeneration after myocardial ischaemia by coronary occlusion [11]. Furthermore, it has been suggested that activation of pro-proliferative and anti-apoptotic p44-42 MAPK and AKT pathways may be linked with SOD3 activity [12]. In epidemiological studies, up to 5% of the population has been suggested to have an amino acid shift in the SOD3 enzyme due to a genetic mutation resulting in an increased risk for ischaemic heart disease and arterial hypertension [13]. A novel rat strain, based on the Dahl/SS strain (PhysGen Program, Medical College of Wisconsin), with a point mutation in SOD3 coding gene through chemical mutagenesis has become available. The mutation concerns one amino acid in the active site (E124D) of SOD3, leading to decreased enzyme activity. Enzyme expression and possible allosteric regulations are not affected, which is an advantage over regular knockout or overexpression models previously described. In one previous report, rats with SOD3-E124D mutation demonstrated an increased susceptibility to the development of pulmonary hypertension [14]. The goal of this work is to further investigate the influence of SOD3 in the context of global IRI and extracorporeal circulation by CPB in an SOD3-E124D model. As superoxide is a main mediator of IRI and SOD3 is capable of superoxide detoxification, we hypothesize a protective role for SOD3. To the best of our knowledge, this is the first report examining the effect of systemically reduced SOD3 activity in the context of oxidative challenge induced by CPB and IRI. MATERIALS AND METHODS Animals and study design This study was approved by the local authority LANUV (Landesamt für Natur, Umwelt und Verbraucherschutz NRW) and carried out in accordance with the German and European guidelines of laboratory animal care (approval number 84-02.04.2013.A365). Male SOD3-E124D rats (SOD3 mutant) and SOD3 competent controls (Dahl/SS) bred in house under licence agreements from Transposagen Biopharmaceuticals Inc. (Lexington, KY, USA) weighing between 400 and 500 g were randomly divided into six groups and subjected to CPB (CPB SOD3 competent control, n = 8; CPB SOD3 mutant, n = 9) or sham treatment (sham: sham SOD3 competent control, n = 7; sham SOD3 mutant, n = 7), whereas a further two groups served as organ donors for cardiac myocyte isolation (ISO; n = 3 for each strain, SOD3 competent control and SOD3 mutant, respectively). Prior to the experiments, all animals were kept under standard laboratory conditions with a low salt diet and water offered ad libitum. Exclusion criteria and sample size According to the permission given by the local authority for each of the groups, a maximum number of 15 animals were allowed to be involved in this study. Three animals were used for the establishment of the model and to optimize the setting as we had no data suggesting the expected effect of the SOD3 mutation in the context of the specific IRI applied by our model. The remaining animals were subjected to the procedure. To achieve comparability in the extent of IRI, a number of animals had to be excluded from further analysis. This was due to low haemoglobin values at the end of the procedure (Hb <5 mg/dl; n = 4) or prolonged periods of low systemic arterial pressure of <40 mmHg for more than 10 min (n = 1), as well as failure to convert to sinus rhythm during rewarming (n = 4). One additional animal was euthanized because of technical error of the blood gas analysis machine during the experiment. All animals displaying none of the above-mentioned exclusion criteria at the end of the reperfusion period were subjected to organ harvesting and further analyses as described in the Materials and Methods section (SOD3 mutants, n = 9; SOD competent controls n = 8). Cardiopulmonary bypass and deep hypothermic circulatory arrest Rats underwent extracorporeal circulation as described before [3]. To induce whole-body IRI, we used a model with global circulatory arrest. Deep hypothermic circulatory arrest (DHCA) was applied to prevent an exaggerated IRI effect with extended multiorgan damage during the 45-min circulatory arrest. In brief, animals were intubated, anaesthetized with isoflurane (AbbVie) and analgised by fentanyl i.m. (Rotexmedica). The sacral artery and jugular vein were cannulated for arterial and venous connection to the heart–lung machine. In addition, the right femoral artery was cannulated to monitor mean arterial pressure (MAP). Electrocardiogram, peripheral oxygen saturation and rectal temperature were also continuously monitored during the CPB procedure. After pre-filling with 13 ml of hydroxyethyl starch (Fresenius Kabi), rats were subjected to CPB using a custom-made miniaturized heart–lung machine and cooled down in 30 min. DHCA was performed at 16°C body core temperature for 45 min. Thereafter, the heart–lung machine was restarted and the rat was perfused again for 40 min of rewarming followed by an additional 60 min of reperfusion at 37°C. During reperfusion, pH value was modified by supplementation of bicarbonate (NaBiC 8.4%, B.Braun), trometamol (Tris 36.34%, B.Braun), CO2 or increased ventilation, whatever was necessary, respectively. Afterwards, rats were euthanized and organs were rinsed with ice-cold 0.9% NaCl solution (B.Braun). Heart and aorta were harvested and snap frozen in liquid nitrogen. Arterial blood gas analyses and blood count were performed before and throughout the CBP procedure (Radiometer Copenhagen ABL 700). Five blood samples were collected at predefined time-points (pre-CPB, at 25°C during cooling, at 20°C during rewarming, at 35°C during rewarming and prior to euthanasia). Sham animals underwent cannulation without the initiation of CPB. Superoxide dismutase plasma activity The activity of SOD in plasma was measured to evaluate the effects of SOD3-E124D mutation with or without oxidative challenge exerted by circulatory arrest. SOD activity was measured photometrically (Tecan Infinite M1000Pro) using a modified nitrite method with xanthine oxidase, hypoxanthine, EDTA and a diazo dye-forming reagent [15]. Enzymatic background activity of the plasma samples was thermally deactivated at 60°C for 10 min incubation time, whereas plasma catalases are inactivated at 60°C and SOD3 is stable up to 75°C [16]. All samples were treated, incubated and measured simultaneously. Western blot analyses The influence of reduced SOD3 activity on molecular biological pathways was investigated on the protein level by western blotting. Frozen heart and aortic samples were powdered, suspended in M-PER buffer (Thermo Scientific) containing protease and phosphatase inhibitor, and homogenized. After centrifugation at 15000 g for 15 min, the protein content of supernatants was determined by the Bradford assay (Sigma Life Science). Thirty micrograms of protein was applied to 10% SDS–PAGE and transferred by semi-dry blotting to PVDF membranes and incubated overnight in diluted primary antibody solution [p-p44-42 MAPK, p44-42 MAPK, pSTAT3, tSTAT3, eNOS, β-actin, PARP-1 (all Cell Signaling), 3-nitrotyrosine (abcam), SOD1, SOD3 (both Enzo Life Sciences) and SOD2 (Millipore)]. All antibodies were applied according to the manufacturers' instructions. Secondary antibodies [goat anti-rabbit HRP (Jackson Laboratory) and anti-mouse IgG (Sigma-Aldrich)] were applied for 60–180 min diluted at 1:5000–10 000 in 5% non-fat milk in TBST. Chemiluminescence (Quantum chemiluminescent HRP substrate, Advansta) was applied and recorded by the UVP CCD-Bioimaging System (Upland, CA, USA). Densitometry was performed using ImageJ v1.46 (Wayne Rasband, National Institutes of Health, USA). All samples were analysed with a specific antibody at the same time on four different PVDF membranes. Chemiluminescence of all membranes was recorded simultaneously and compared with that of a standardization sample. Adult cardiac myocyte culture For direct investigation of the SOD3 effect on myocardial tissue, in vitro studies using isolated adult rat cardiac myocytes were conducted. Isolation was performed using a Langendorff apparatus following previously published standard procedures [17]. Isolated cardiac myocytes were suspended in a culture medium (Sigma Life Sciences) and incubated at 37°C, 5% carbon dioxide and 1 or 21% oxygen for 4 h. After centrifugation at 250 g for 5 min, cells were suspended in M-PER buffer (Thermo Scientific). Phase contrast microscopy was used to confirm procedural success. Cardiac myocytes were handled for western blot as described above. Statistics Normalization of experimental parameters with relative values was always performed with a sham or a control experiment. Statistics were performed using Prism 6 (GraphPad Software, La Jolla, CA, USA). Vital signs are shown as real values. To improve the comparability of the other data, each value of all groups was divided by the mean of the sham control group. Therefore, shown results are relative values whereby the expression level of the Sham control was normalized to 1.0. Results of animals with reduced SOD3 activity were compared with those of controls and sham groups by non-parametric Mann–Whitney t-tests. Results are presented as box-and-whiskers plots, plotted from minimum to maximum. Intergroup differences were considered statistically significant at P < 0.05 and are marked by an asterisk in the text. RESULTS Operative monitoring and blood gas analysis Pre-CPB heart rates did not differ between the two groups. In both groups, heart frequency dropped to zero during cardiac arrest and regained physiological values in the reperfusion period. The heart rates of the SOD3 mutants decreased faster before DHCA and increased at a slower rate during rewarming. At euthanasia, the mean heart rate in SOD3 mutants was 46 bpm (26%) slower than in SOD3 competent controls (P = 0.083). During the course of the procedure, MAP decreased in both groups. In animals with lower SOD3 activity, MAP tended to be slightly higher at baseline but slightly lower at the end of the reperfusion, although these differences were statistically not significant (P = 0.288). Recording of rectal temperature and peripheral oxygen saturation did not reveal any differences between both groups. Blood gas analysis demonstrated a distinct increase in serum potassium and lactate levels for both groups during the reperfusion period. However, in SOD3 mutants, the increase in serum potassium (P = 0.870) and lactate (P = 0.622) levels seemed slightly, but not significantly, elevated (Fig. 1). Leucocyte counts were decreased during DHCA but recovered at later time-points. All monitored parameters are listed in Supplementary Table 1. Figure 1: Open in new tabDownload slide Intraoperative monitoring and blood gas analyses. Heart rate (A) and mean arterial pressure (B) were monitored throughout the entire procedure (Ta: pre cannulation; Tb: cannulation; Tc: CPB start; Td: 15 min cooling; Te: 25 min cooling; Tf: DHCA; Tg: 20.0°C rewarming; Th: 27.5°C rewarming; Ti: 35.0°C rewarming; Tj: 15 min reperfusion; Tk: 45 min reperfusion; Tl: euthanasia). Blood gas analyses were performed at predefined time-points (Ta: pre-CPB; Tb: 25.0°C cooling; Tc: 20.0°C rewarming; Td: 35.0°C rewarming; Te: euthanasia) for both groups (SOD3 competent control: control, n = 8; SOD3-E124D: SOD3 mutant, n = 9) and concentrations of potassium (C) and lactate (D) were measured. HR: heart rate; MAP: mean arterial pressure; SOD: superoxide dismutase; CPB: cardiopulmonary bypass. Figure 1: Open in new tabDownload slide Intraoperative monitoring and blood gas analyses. Heart rate (A) and mean arterial pressure (B) were monitored throughout the entire procedure (Ta: pre cannulation; Tb: cannulation; Tc: CPB start; Td: 15 min cooling; Te: 25 min cooling; Tf: DHCA; Tg: 20.0°C rewarming; Th: 27.5°C rewarming; Ti: 35.0°C rewarming; Tj: 15 min reperfusion; Tk: 45 min reperfusion; Tl: euthanasia). Blood gas analyses were performed at predefined time-points (Ta: pre-CPB; Tb: 25.0°C cooling; Tc: 20.0°C rewarming; Td: 35.0°C rewarming; Te: euthanasia) for both groups (SOD3 competent control: control, n = 8; SOD3-E124D: SOD3 mutant, n = 9) and concentrations of potassium (C) and lactate (D) were measured. HR: heart rate; MAP: mean arterial pressure; SOD: superoxide dismutase; CPB: cardiopulmonary bypass. Superoxide dismutase plasma activity SOD plasma activity was measured three times during the experiments (pre-CPB, at 25°C during cooling and prior to euthanasia) to quantify the differences of the two phenotypes. As expected, SOD3 plasma activity was reduced in SOD3 mutants prior to CPB (mean loss of activity = 83%; P = 0.100) and remained at a significantly lower level when compared with the SOD3 competent controls throughout the experiment (25°C during cooling, P = 0.032; prior to euthanasia, P = 0.008*; Fig. 2). Figure 2: Open in new tabDownload slide SOD plasma activity. SOD plasma activity of SOD3 competent controls (control) and SOD3-E124D (SOD3 mutant) was quantified at three predefined time-points (Ta: pre-CPB, n = 3; Tb: 25°C during cooling, n = 5; Tc: prior to euthanasia, n = 5). For quantitative analysis, all data were normalized by setting the the pre-CPB SOD activity level in the SOD3 competent control group to 1.0. SOD: superoxide dismutase; CPB: cardiopulmonary bypass. Figure 2: Open in new tabDownload slide SOD plasma activity. SOD plasma activity of SOD3 competent controls (control) and SOD3-E124D (SOD3 mutant) was quantified at three predefined time-points (Ta: pre-CPB, n = 3; Tb: 25°C during cooling, n = 5; Tc: prior to euthanasia, n = 5). For quantitative analysis, all data were normalized by setting the the pre-CPB SOD activity level in the SOD3 competent control group to 1.0. SOD: superoxide dismutase; CPB: cardiopulmonary bypass. Western blot analyses Sham groups showed a homogenous basal activation of p44-42 MAPK and STAT3 signalling and SOD3 protein expression independent of the present SOD3 activity. In case of IRI, reduced SOD3 activity caused reduced activation of the pro-proliferative and anti-apoptotic p44-42 MAPK pathway in the examined cardiovascular tissue of SOD3 mutants. In contrast, SOD3 competent controls had a 36% (P = 0.074) higher activation of p44-42 MAPK in heart tissue (Fig. 3) and a 46% (P = 0.660) higher activation in aortic tissue (Supplementary Fig. 1) when compared with SOD3 mutants. Activation of the STAT3 pathway significantly increased in both myocardial and aortic tissues of both groups, but reduced SOD3 activity impaired this effect, resulting in a 36% (P = 0.027) lower STAT3 activity in aortic samples of SOD3 mutants when directly compared with respective values of SOD3 competent controls (Fig. 3). Figure 3: Open in new tabDownload slide Activation of p44-42 MAPK and STAT3 signalling. Western blot analyses of p44-42 MAPK and STAT3 activation. Animals with impaired SOD3 activity (SOD3E-124D: SOD3 mutant) and SOD3 competent controls (control) were subjected to cardiopulmonary bypass and global IRI (CPB; CPB control, n = 8; CPB SOD3 mutant, n = 9) or to mere cannulation (sham; sham control, n = 7; sham SOD3 mutant, n = 7). p44-42 MAPK activation was determined in heart tissue (A) and STAT3 activation was quantified in aortic tissue (B). For quantitative analyses, expression level of sham control was normalized to 1.0 and compared with that of the other groups. IRI: ischaemia and reperfusion injury; SOD: superoxide dismutase; CPB: cardiopulmonary bypass. Figure 3: Open in new tabDownload slide Activation of p44-42 MAPK and STAT3 signalling. Western blot analyses of p44-42 MAPK and STAT3 activation. Animals with impaired SOD3 activity (SOD3E-124D: SOD3 mutant) and SOD3 competent controls (control) were subjected to cardiopulmonary bypass and global IRI (CPB; CPB control, n = 8; CPB SOD3 mutant, n = 9) or to mere cannulation (sham; sham control, n = 7; sham SOD3 mutant, n = 7). p44-42 MAPK activation was determined in heart tissue (A) and STAT3 activation was quantified in aortic tissue (B). For quantitative analyses, expression level of sham control was normalized to 1.0 and compared with that of the other groups. IRI: ischaemia and reperfusion injury; SOD: superoxide dismutase; CPB: cardiopulmonary bypass. IRI significantly increased SOD3 expression in aortic tissue (P = 0.029 and 0.042 in SOD3 competent controls and SOD3 mutants, respectively; Supplementary Fig. 2A). In mutant animals with reduced SOD3 plasma activity, a trend towards compensatory upregulation of the SOD3 expression by 36% was observed (P = 0.113). Analysis of myocardial samples revealed an even stronger compensatory effect in response to IRI resulting in an additional 79% increase of SOD3 expression (P = 0.074) (vs SOD3 competent controls; P = 0.047*; Supplementary Fig. 2B). To elucidate possible deleterious effects of impaired IRI handling as an effect of SOD3 deficiency, we chose to examine myocardial apoptosis after IRI. Caspase cleavage of PARP-1 as a predictor of apoptosis was altered in both groups after CPB when each group was compared with the respective sham controls. Relative PARP-1 cleavage was not significantly different between the sham groups (P = 0.788). IRI led to significantly increased apoptosis in both groups (SOD3 competent control: P = 0.017*; SOD3 mutant: P = 0.002). Although PARP-1 cleavage was doubled in the SOD3 competent controls, an impaired SOD3 function led to a 3-fold increase of apoptosis in SOD3 mutants. However, although the relative mean PARP-1 cleavage was 28% higher in SOD3 mutants, this difference did not reach statistical significance in direct comparison with the controls under the given sample size conditions (P = 0.320; Fig. 4). Figure 4: Open in new tabDownload slide Caspase cleavage of PARP-1. Western blot analyses of PARP-1 cleavage. Animals with impaired SOD3 activity (SOD3E-124D: SOD3 mutant) and SOD3 competent controls (control) were subjected to cardiopulmonary bypass and global ischaemia and reperfusion (CPB; CPB control, n = 6; CPB SOD3 mutant, n = 6) or to mere cannulation (Sham; Sham control, n = 6; Sham SOD3 mutant, n = 6). PARP-1 cleavage was determined on heart. For quantitative analyses, expression level of Sham control was normalized to 1.0 and compared with that of the other groups. SOD: superoxide dismutase; CPB: cardiopulmonary bypass. Figure 4: Open in new tabDownload slide Caspase cleavage of PARP-1. Western blot analyses of PARP-1 cleavage. Animals with impaired SOD3 activity (SOD3E-124D: SOD3 mutant) and SOD3 competent controls (control) were subjected to cardiopulmonary bypass and global ischaemia and reperfusion (CPB; CPB control, n = 6; CPB SOD3 mutant, n = 6) or to mere cannulation (Sham; Sham control, n = 6; Sham SOD3 mutant, n = 6). PARP-1 cleavage was determined on heart. For quantitative analyses, expression level of Sham control was normalized to 1.0 and compared with that of the other groups. SOD: superoxide dismutase; CPB: cardiopulmonary bypass. Effect on nitric oxide balance Sham controls had the highest eNOS protein levels. After CPB, a marked reduction in myocardial and aortic eNOS was detected in both genotypes, SOD3 mutants and SOD3 competent controls. Despite the clear trend, this reduction of eNOS after CPB and IRI was statistically significant in SOD3 competent animals when myocardial tissue was analysed (P = 0.021*; Fig. 5). Figure 5: Open in new tabDownload slide Regulation of eNOS during CPB. Western blot analyses of eNOS. Animals with impaired SOD3 activity (SOD3E-124D: SOD3 mutant) and SOD3 competent controls (control) were subjected to cardiopulmonary bypass and global ischaemia and reperfusion (CPB; CPB control, n = 8; CPB SOD3 mutant n = 9) or to mere cannulation (sham, sham control, n = 7; sham SOD3 mutant, n = 7). eNOS was determined by immunoblotting and quantified in heart (A) and aortic tissue (B). For quantitative analyses, expression level of sham control was normalized to 1.0 and compared with that of the other groups. SOD: superoxide dismutase; CPB: cardiopulmonary bypass. Figure 5: Open in new tabDownload slide Regulation of eNOS during CPB. Western blot analyses of eNOS. Animals with impaired SOD3 activity (SOD3E-124D: SOD3 mutant) and SOD3 competent controls (control) were subjected to cardiopulmonary bypass and global ischaemia and reperfusion (CPB; CPB control, n = 8; CPB SOD3 mutant n = 9) or to mere cannulation (sham, sham control, n = 7; sham SOD3 mutant, n = 7). eNOS was determined by immunoblotting and quantified in heart (A) and aortic tissue (B). For quantitative analyses, expression level of sham control was normalized to 1.0 and compared with that of the other groups. SOD: superoxide dismutase; CPB: cardiopulmonary bypass. Impact of reduced superoxide dismutase activity on adult cardiomyocytes To further investigate the direct myocardial effects of SOD3, additional experiments on primary adult cardiac myocytes were performed for semi-quantitative western blot analyses. Cardiac myocytes were incubated with either 1% or 21% oxygen for 4 h. Under normoxic conditions, SOD3 expression was distinctly increased in cardiac myocytes of SOD3 mutants when compared with SOD3 competent controls (P = 0.100). Upon hypoxic challenge, there was no further increase of SOD3 expression in cardiac myocytes of SOD3 mutants (P = 0.400), whereas SOD3 competent controls demonstrated a marked increase in the expression of SOD3 (P = 0.100; Fig. 6A). We then examined 3-nitrotyrosine as a marker of nitrosative stress. Here, we observed a less consistent response pattern in cardiac myocytes of SOD3 mutants with broader variability and a higher trend in 3-nitrotyrosine, although statistical significance was not achieved in the analysed groups (P ≥ 0.400; Fig. 6B). Figure 6: Open in new tabDownload slide Regulation of SOD3 and accumulation of 3-nitrotyrosine during hypoxia. Expression of SOD3 (A) and accumulation of 3-nitrotyrosine (B). Adult cardiac myocytes with impaired SOD3 activity (SOD3E-124D: SOD3 mutant) and SOD3 competent controls (control) were incubated either at 21% O2 (normoxia; ISO normoxia control, n = 3; ISO normoxia SOD3 mutant, n = 3) or at 1% O2 (hypoxia; ISO hypoxia control, n = 3; ISO hypoxia SOD3 mutant, n = 3). For quantitative analyses, expression level of ISO normoxia control was normalized to 1.0 and compared with that of the other groups. ISO: isolation; SOD: superoxide dismutase. Figure 6: Open in new tabDownload slide Regulation of SOD3 and accumulation of 3-nitrotyrosine during hypoxia. Expression of SOD3 (A) and accumulation of 3-nitrotyrosine (B). Adult cardiac myocytes with impaired SOD3 activity (SOD3E-124D: SOD3 mutant) and SOD3 competent controls (control) were incubated either at 21% O2 (normoxia; ISO normoxia control, n = 3; ISO normoxia SOD3 mutant, n = 3) or at 1% O2 (hypoxia; ISO hypoxia control, n = 3; ISO hypoxia SOD3 mutant, n = 3). For quantitative analyses, expression level of ISO normoxia control was normalized to 1.0 and compared with that of the other groups. ISO: isolation; SOD: superoxide dismutase. To investigate reactive gene regulation with possible compensatory effects, SOD1 and SOD2 were evaluated on the protein expression level. Hypoxia led to increased SOD1 expression in wild-type controls, but not in SOD3 mutants (SOD3 competent control P = 0.200; SOD3 mutant P = 0.700). SOD2 expression seemed to be modulated in a similar fashion, where, under hypoxic conditions, cardiac myocytes of SOD3 mutants had a distinctly lower expression when compared with SOD3 competent controls (P = 0.100; Fig. 7). Figure 7: Open in new tabDownload slide Regulation of SOD1 and SOD2 during hypoxia. Regulation of SOD1 (A) and SOD2 (B). Adult cardiac myocytes with impaired SOD3 activity (SOD3E-124D: SOD3 mutant) and SOD3 competent controls (control) were incubated either at 21% O2 (normoxia; ISO normoxia control, n = 3; ISO normoxia SOD3 mutant, n = 3) or at 1% O2 (hypoxia; ISO hypoxia control, n = 3; ISO hypoxia SOD3 mutant, n = 3). For quantitative analyses, expression level of ISO normoxia control was normalized to 1.0 and compared with that of the other groups. ISO: isolation; SOD: superoxide dismutase. Figure 7: Open in new tabDownload slide Regulation of SOD1 and SOD2 during hypoxia. Regulation of SOD1 (A) and SOD2 (B). Adult cardiac myocytes with impaired SOD3 activity (SOD3E-124D: SOD3 mutant) and SOD3 competent controls (control) were incubated either at 21% O2 (normoxia; ISO normoxia control, n = 3; ISO normoxia SOD3 mutant, n = 3) or at 1% O2 (hypoxia; ISO hypoxia control, n = 3; ISO hypoxia SOD3 mutant, n = 3). For quantitative analyses, expression level of ISO normoxia control was normalized to 1.0 and compared with that of the other groups. ISO: isolation; SOD: superoxide dismutase. DISCUSSION This work investigated the influence of SOD3 on IRI in the context of extracorporeal circulation. Using a rat strain with preserved expression but reduced systemic activity of SOD3 in combination with a delicate small animal model of CPB, we demonstrate that impaired SOD3 activity leads to impaired handling of IRI. Evaluation of physiological parameters such as heart rate and MAP already demonstrated a negative trend in animals with reduced SOD3 during reperfusion. Plasma potassium and lactate concentration as markers for severe tissue damage were distinctly increased in both groups after ischaemia and reperfusion. These high levels are an inherent aspect of the IRI in this small animal model and are regarded as a response to the procedure with whole-body IRI. The values rise distinctly at the beginning of the reperfusion phase. To maintain physiological blood pH values, we use pharmaceutical buffers and perform additional blood gas analyses throughout the reperfusion phase. Animals with a massive increase in potassium and lactate concentrations showed temporary electrocardiographic evidence of arrhythmia and ventricular fibrillation. We excluded those animals without a regular heart action from our analyses (n = 4). However, despite a clear increase in potassium levels, all animals that were processed for analyses in this study regained regular cardiac rhythm during rewarming and retained cardiac activity until the end of the reperfusion period. This finding is in agreement with previously published animal models of IRI [3, 18]. Because of these physiological parameters, impaired handling of systemic ischaemia and reperfusion in the context of extracorporeal circulation and CPB with DHCA may be suggested in both groups, with a pronounced deleterious effect observed in organisms with decreased SOD3 activity. Investigation of SOD3 levels and activity clearly illustrated that SOD3 responds to the CPB stimulus by increased expression, whereas SOD1 and SOD2 do not, suggesting a specific protective role of SOD3 for the cardiovascular system. Lob et al. [19] have previously shown that SOD3 is an important modulator of arterial blood pressure and capable of reducing MAP as an antagonist of angiotensin II. Our results are in line with these previous findings, as before CPB increased MAP was observed in SOD3 mutants. Furthermore, Obal et al. [11] described decreased infarct size and higher heart rate in transgenic mice with cardiac myocyte-restricted overexpression of SOD3. Decreased heart rate after reperfusion as observed in our experiments correlates with these findings. In our experiments, western blot analyses demonstrated that decreased SOD3 activity was accompanied by decreased activation of p44-42 MAPK and STAT3 pathways. The activation of each of the latter pathways is known to be a cardioprotective salvage response to IRI [20, 21]. These detrimental alterations in signalling pathways were observed in cardiac and aortic tissue samples as well as in vitro in cardiac myocytes. In line with these results, Obal et al. [11] have shown that overexpression of SOD3 leads to increased activation of p44-42 MAPK as well. Moreover, Laatikainen et al. [12] suggested that SOD3 is involved in different signalling pathways capable of prolonging cell proliferation and tissue recovery. As expected, cleavage of PARP-1 as a marker of apoptosis was significantly increased in both groups after the procedure. These results resemble a further favourable characteristic of the applied animal model, which parallels clinically relevant events in the context of IRI. Although apoptosis was tripled in the group with impaired SOD3 handling, the observed mean extent of apoptosis was only doubled in the control. This may suggest another important protective role of the enzyme in the context of IRI. McCully et al. [22] showed a relationship between the duration of ischaemia, the extent of cardiac tissue damage and the cleavage of PARP in a rabbit study. In human right atrium PARP-1, cleavage and other apoptotic markers significantly increased after CPB [23]. Furthermore, it has been shown that PARP-1 cleavage is decreased in human cardiac biopsies after ischaemia and reperfusion with cold crystalloid cardioplegia compared with that in samples without cardioplegia [24]. These findings indicate that PARP cleavage is dependent on the extent of cardiac tissue damage and apoptosis in the context of IRI and coincides with our results. Another important biological action of SOD3 is the support of bioavailability of nitric oxide, the main product of eNOS, which is co-localized with SOD3 in cardiac tissue [25]. eNOS protein levels were diminished after IRI induced by the CPB procedure, possibly caused by endothelial wash-out. Nevertheless, eNOS levels were lower in animals with impaired SOD3 activity. Moreover, in the same animals, 3-nitrotyrosine formation was increased. This might portend a generally decreased production of NO in SOD3 mutant animals, which is further supported by the previously demonstrated association of SOD3 deficiency with the phenotype characteristic of systemic arterial hypertension [26]. Collectively, it may be presumed that reduced SOD3 function causes increased oxidative and nitrosative stress, detectable by altered 3-nitrotyrosine. Loss of SOD3 function leads to higher ROS level in the cardiovascular system. As a consequence, superoxide reacts with NO to form peroxynitrite and leads to an increase in nitrosative stress as shown before [7, 11]. In the animal model used in this report (SOD3-E124D: SOD3 mutant), the basal gene expression level of SOD3 does not differ from that observed in the SOD3 competent control strain in vivo. After systemic challenge by IRI, SOD3 mutant rats as well as cardiac myocytes from SOD3 mutant rats showed a potentiated increase in SOD3 expression when compared with SOD3 competent controls. We suggest that the accumulation of superoxide caused by decreased SOD3 function leads to an upregulation of SOD3 expression to achieve a compensatory mechanism. This feedback mechanism has been demonstrated for ischaemic cardiomyopathy, where accumulated ROS induce the gene expression of antioxidant enzymes [27]. We also found that cardiac myocytes with decreased SOD3 activity were not capable of increasing the expression levels of SOD1 and SOD2 in response to ischaemic challenge. In contrast, cardiac myocytes with preserved SOD3 function demonstrated a compensatory increase at the gene expression level. These results strongly suggest an impaired handling of oxidative stress and a misregulation of SOD in organisms and isolated cardiac myocytes with reduced SOD3 activity. The latter observation may be explained by the fact that high expression levels of SOD3 may lower the expression of SOD1 and SOD2 isoforms. However, this hypothesis needs further proof, and published results with regard to this point are contradictory. Brahmajothi and Campbell [25] reported that SOD isoforms are heterogeneously expressed in the heart and may not modulate each other, whereas Negoro et al. [28] indicated that activation of STAT3 leads to an upregulation of SOD2 to protect cardiac myocytes from IRI. Given the fact that STAT3 expression is decreased because of decreased SOD3 activation, we presume that SOD2 activation may be expected to be affected as we observed here. Limitations of the study This study is limited in a few aspects: there are a few differences between human SOD3 and rat SOD3. Rat SOD3 has the same heparin-binding domain as human SOD3 but is missing one chromatographic fraction with high affinity to heparin that makes rat SOD3 appear as a homodimer and not a homotetramer like the human SOD3 [8]. This may affect the study in a yet unknown way. Moreover, recombinant human SOD did not improve recovery of ventricular function after acute myocardial infarction with PTCA treatment in an earlier study [29]. In contrast, we worked on a different area with a focus on extracorporeal circulation and global IRI. Besides, pharmacological development as well as adjusted doses and optimal application time(s) may lead to better mimetics focusing on the extracellular isoform of SOD. Correspondingly, new specific recombinant isoforms of SOD3 (rhEC-SOD3) have been successfully applied in a preclinical model to prevent hypoxic lung damage in vivo [30]. We used a well-established model of extracorporeal circulation to induce global IRI. However, the use of DHCA in current cardiovascular surgery is limited to extended aortic surgery and complex paediatric cardiac surgery. During the procedure, we were able to perform whole-body ischaemia for an extended period of 45 min and were able to induce IRI and global inflammation. Using DHCA enabled us to extend the ischaemia time and facilitated the resumption of regular heart action after arrest. Unfortunately, regardless of the opportunity of applying whole-body ischaemia, it is also technically very difficult and problematic to apply extracorporeal circulation and circulatory arrest without the use of hypothermia. Although DHCA is a useful model to induce systemic IRI, further elaboration and enhancement of this model to employ extracorporeal circulation and CPB without the need for DHCA may offer further insights into the signalling pathways of SOD3. In addition, deep hypothermia may affect the activation and inactivation of different enzymes and pathways in yet unknown ways. Nonetheless, in an earlier report, we demonstrated the aspects of clinically relevant IRI present in the model that were applied in this report [3]. Moreover, true and sustained cardioplegic arrest remains a challenge, mainly due to incomplete unloading of the heart by jugular venous cannulation and also due to the technical difficulties of applying selective cardioplegia via a retrograde aortic cannula in a closed-chest small animal model. Furthermore, our study is limited by the sample size, and larger experimental groups may be needed to corroborate the significance of SOD3 in the context of extracorporeal circulation-induced IRI. The later issue appears particularly noteworthy when physiological parameters such as heart rate, MAP and plasma potassium and lactate levels are analysed. On the protein level, we saw several promising effects that would likely reach statistical significance in larger group samples. Furthermore, isolation of primary cells was limited to cardiac myocytes, as they were our prime focus, but for in vivo NO bioavailability, endothelial cells and vascular smooth cells should not be neglected. Conclusions SOD3 impacts protective signal pathways on the protein level with a direct effect on myocardium as well as on aortic tissue. Decreased SOD3 activity increases tissue injury and apoptosis after extracorporeal circulation and the risk of SIRS. SOD3 has a significant cardioprotective role in the case of global IRI. Exploration of SOD3-based interventional strategies may provide novel therapeutic options for patients undergoing major cardiac surgical procedures. SUPPLEMENTARY MATERIAL Supplementary material is available at EJCTS online. ACKNOWLEDGEMENTS The authors gratefully acknowledge the support of Martin Sager, Eva Engelhardt and Peter Benten (Core Facility for Animal Research and Animal Welfare, University Hospital Duesseldorf) for the establishment and coordination of in-house breeding of Dahl/SS and SOD3-E124D strains as well as during the in vivo experiments. Moreover, the authors gratefully thank A. 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Dr Ankersmit: Because if you show these pathways, it's always good to show some western blot protein levels. So how do you envision, that would work in clinics? Dr Immohr: I think there must be much more work done, but it's known that there are new mimetics on the market now that focus on SOD3 or on chimeric SOD2 and SOD3. Also they infuse it the patient, maybe before extracorporeal circulation or slightly after the reperfusion phase, which may help patients to detoxify the superoxide dismutase in the vessel walls. It's also shown that new mimetics adapt and bind to the vessel walls, which may be a good treatment option. Dr Ankersmit: Then, of course, you've got a very nice model, because reperfusion injury is always related to inflammation. Have you looked at the inflammatory response by histologically staining with looking for NK, MC3, and CD4? Dr Immohr: In this project we did not look at inflammation histologically, but we have the sample data. But we used this model for a long time in the laboratory. In former studies we did this, and we have the publication that shows that there is inflammation. Dr Ankersmit: Decreased inflammation or increased? Dr Immohr: Increased. Increased by the extracorporeal circulation. But these are not studies based on the SOD enzyme; this is the first one with SOD3. Dr Ankersmit: It would be interesting to look at that too. Dr Immohr: Yes, that's right. Dr D Chambers(London, UK): Can I just clarify your model? It's an in vitro model? Dr Immohr: I can show it again. I think it's easier. Dr Chambers: It's an in vivo model? Dr Immohr: It's an in vivo model, yes. We have the rats and then we cannulate them. We cannulate first the median sacral artery with a standard needle. Then we cannulate the femoral artery for the pressure catheter and jugular vein. We connect it to the heart-lung machine and then we cool the rat down via the machine; then we have circulatory arrest. We stop the machine, we see that the heart rate decreases to zero. We have arrest for 45 minutes arrest, and afterwards we start the machine again, rewarm the animal, and reperfuse the animal so that we have the sinus node rhythm back. It would be very interesting to wean the animals afterwards and to do some long-term studies, but we haven't done this yet. But I think it's something for the future. Dr Chambers: So why did you do circulatory arrest rather than just global ischemia of the heart? Dr Immohr: To induce global ischemia at a higher rate. With circulatory arrest, we have global ischemia in all organs. Dr Chambers: Doesn't that initiate a massive inflammatory effect? Dr Immohr: That's the focus of the model. Dr Ankersmit: It agrees with my data. Basically if you achieve circulatory arrest, the entire body becomes a secretory machine. Dr Immohr: You see the lactate, for example, it's increasing. It's about 20 mmol/L, but we resuscitated the rat afterwards. Dr R. Ascione(Bristol, UK): Just a comment. It's a nice model but not very relevant to myocardial protection or ischemic perfusion injury. You induce total body arrest. I suppose technically it is difficult in a rat to apply an aortic cross-clamp and induce selective cardiac arrest, which is why you have not gone for that solution. Is that right? Dr Immohr: Yes, that's right. It's a very clinical model and we have no cardiac arrest, for example, but we are working on this also in that we don't need the hypothermia anymore but still have work to do with the model. It's very difficult with a rat. Dr Ascione: I see. This indeed questions the clinical relevance of the findings. Also, cooling the whole animal to 16 degrees mimics what we were doing during hypothermic circulatory arrest for cerebral protection, which is a different scenario compared to when you have a patient at 30 degrees C and you only stop and cool the heart. But anyway, it is a nice study, nice model. Author notes † Presented at the 29th Annual Meeting of the European Association for Cardio-Thoracic Surgery, Amsterdam, Netherlands, 3–7 October 2015. © The Author 2016. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com © The Author 2016. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
TI - The extracellular isoform of superoxide dismutase has a significant impact on cardiovascular ischaemia and reperfusion injury during cardiopulmonary bypass
JF - European Journal of Cardio-Thoracic Surgery
DO - 10.1093/ejcts/ezw216
DA - 2016-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-extracellular-isoform-of-superoxide-dismutase-has-a-significant-pedN9GPWvE
SP - 1035
EP - 1044
VL - 50
IS - 6
DP - DeepDyve
ER -