TY - JOUR
AU - Strasser, Ruth, H
AB - Abstract Background: Ischemic preconditioning is a powerful mechanism in reducing infarct size of the heart. Protection can be performed either by an ischemic stimulus of the heart itself or by ischemia of an organ distant to the heart. To address the question whether this remote preconditioning is transduced by neuronal or humoral factors an in situ model of infrarenal occlusion of the aorta (IOA) in the rat was developed. Furthermore, the signal transduction pathways of classical and remote preconditioning regarding protein kinase C, which is one of the key enzymes in classical preconditioning, were compared. Methods and Results: Controls (30 min regional ischemia followed by 2 h of reperfusion) had an infarct size of 62±5% whereas classical preconditioning reduced it to 10±3% of the risk zone (P≤0.001). Fifteen minutes IOA without reperfusion of the aorta had no influence on infarct size (52±4%). When, however, IOA was performed for 15, 10, or 5 min, respectively, followed by a 10-min reperfusion period the size of myocardial infarction decreased significantly. This decrease was dependent on the duration of IOA (18±3%, 37±8%, 42±2%, respectively; P≤0.001 for the time-dependent linear trend in decrease of infarct size). Fifteen minutes IOA showed the strongest protection which was comparable to classical preconditioning (18±3%, P≤0.001 vs. control). Blockade of the nervous pathway by 20 mg/kg hexamethonium could not inhibit the protection afforded by IOA (14±4%). Using chelerythrine, a selective protein kinase C-inhibitor, at a dose of 5 mg/kg body weight, protection from remote (68±4%, P≤0.001 vs. 15 min IOA followed by 10 min of reperfusion without chelerythrine) as well as from classical preconditioning (56±5%, P≤0.001) was completely blocked. Conclusion: Protection of the heart by remote preconditioning using IOA is as powerful as classical preconditioning. Both protection methods share protein kinase C as a common element in their signal transduction pathways. Since hexamethonium could not block the protection from IOA and a reperfusion period has to be necessarily interspaced between the IOA and the infarct inducing ischemia of the heart, a neuronal signal transmission from the remote area to the heart can be excluded with certainty. A humoral factor must be responsible for the remote protection. Interestingly the production of the protecting factor is dependent on the duration of the ischemia of the lower limb. The protecting substance, which must be upstream of protein kinase C, remains to be identified. Receptors, Signal transduction, Preconditioning, Protein kinases, Infarction, Ischemia Time for primary review 28 days. 1 Introduction Murry et al. [1] first described the phenomenon of ischemic preconditioning (brief periods of ischemia interspaced with reperfusion) in 1986. The mechanism of reducing infarct size of the heart is well established and this protection has also been observed in a number of other organs including brain [2], liver [3], and skeletal muscles [4]. In 1993, Przyklenk et al. [5] described a mechanism of protecting the anterior wall of the heart by a brief period of ischemia in a territory of the heart which is distant to the final area of infarction. This mechanism was termed “remote preconditioning” or “preconditioning on a distance”. Recently intra-cardiac preconditioning was questioned however by Nakano [6] from Downey's group who could not find a protection in the remote myocardium after regional ischemia in situ followed by global ischemia of the rabbit heart in the Langendorff model. The phenomenon of remote preconditioning is not unique to the heart. Liauw et al. [7] have shown that ischemia of one skeletal muscle can protect the contralateral muscle from ischemia–reperfusion injury. The mechanism of remote preconditioning is not limited to one organ system either; for example McClanahan et al. [8] could show that brief ischemia of the kidney can protect the heart from infarction. Meanwhile it was also observed that a preconditioning stimulus of an area perfused by the mesenterial artery [9–11] or the femoral artery [12] confers protection to the heart. It was proposed that remote preconditioning acts through sensory nerve stimulation in the ischemic organ, since the ganglion blockade by hexamethonium may abolish this protection [9,11]. Contrary to this observation, it was shown in vitro that the effluent during preconditioning can protect virgin isolated rabbit hearts from infarction, which supports a non-neuronal action of protection [13]. Remote preconditioning was shown in several species including rat, rabbit and canine, which is comparable to classical preconditioning. Recently an indication for the presence of remote preconditioning in humans was made by Günaydin et al. [14] who showed that during coronary artery bypass surgery a tourniquet wrapped around an upper extremity to induce brief periods of ischemia resulted in an attenuated increase of lactate dehydrogenase suggesting a reduction in myocyte injury [14]. This observation also gives a potential link to the clinical and therapeutical relevance of remote preconditioning. The signal transduction pathway of both kinds of preconditioning share also similarities. Classical preconditioning can be mimicked by numerous G-protein coupled receptors (i.e. α1-adrenergic, adenosine, bradykinin, opioid). The signals from classical ischemic and pharmacological preconditioning converge on the level of protein kinase C, which seems to be a key enzyme in preconditioning [15]. After the activation of protein kinase C the signal is transmitted via other protein kinases (i.e. tyrosine kinases [16], p38 mitogen activated protein kinase [17,18]) to the yet unidentified final end-effector. In the phenomenon of remote preconditioning adenosine receptors [25], α1-adrenergic receptors [19], bradykinin receptors [9], and opioid receptors [20] have been proposed to be involved in the infarct size limiting effect since their receptor antagonists could prevent the protection obtained from preconditioning on a distance. To our knowledge nothing is known about the signal transduction cascade downstream of the receptor level, especially the role of protein kinase C in remote preconditioning has not been investigated so far. Classical preconditioning can be induced by a single brief period of ischemia followed by reperfusion prior to the infarct inducing ischemia. In order to induce protection the preconditioning ischemia must be of at least 3 min duration [21]. Longer ischemic periods below the threshold of infarction or repetitions of ischemia–reperfusion to precondition the heart do not reduce the infarct size any further. Pharmacological inhibition of ischemic preconditioning, however, is dependent on the strength of the ischemic preconditioning stimulus since for example the bradykinin receptor antagonist HOE 140 can block protection from a single preconditioning stimulus but not from three ischemia–reperfusion stimuli [22]. This was also observed for opioid receptors using naloxone as an antagonist [23]. Whether remote preconditioning is as powerful as classical preconditioning in protecting the heart and if there exist similarities in the time course of protection is not clarified yet. For protecting the heart on a distance, a model of infrarenal occlusion of the aorta was developed. It is hypothesized that remote preconditioning acts through an as yet unidentified humoral trigger signal to the heart. If so, it is proposed that the degree of protection of the heart is dependent on the amount of protecting substance which is produced by the trigger organ. If a humoral factor is responsible for the protection, a reperfusion period of the ischemic organ would have to be interspaced between the ischemia of the trigger organ and the ischemia of the heart since the protecting mediator would have to be transmitted to the heart by the circulation. To test if a neuronal pathway may be involved in the protection of remote preconditioning we used the ganglion blocker hexamethonium. Finally we compared the signal transduction pathways of classical and remote preconditioning regarding protein kinase C which is a key enzyme in classical preconditioning. 2 Methods All procedures were in conformance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996) and was approved by the local Animal Care and Use Committee. 2.1 General surgical preparation Male Wistar rats, 250–350 g, were used for the study. The rats were anesthetized via intraperitoneal administration of sodium pentobarbital (30 mg/kg). A tracheotomy was performed and the trachea was intubated with a cannula connected to a rodent ventilator (Foehr Medical Instruments, Seeheim, Germany). The rats were ventilated with room air supplemented with oxygen at 65 breaths/min. Atelectase was prevented by maintaining a positive end-expiratory pressure of 2.5 cmH2O. Arterial pH, pCO2, and pO2 were monitored at baseline, 5 and 25 min of coronary occlusion, 30 and 60 min of reperfusion by a blood gas analyzer (Ciba Corning) and maintained within a normal physiological range (pH 7.35–7.45, pCO2 30–40 mmHg, pO2 80–120 mmHg) by adjusting the respiratory rate and/or the tidal volume. Body temperature was maintained at 37 °C by using a heating pad. In some cases the administration of sodium-bicarbonate was necessary to maintain the pH in a physiological range. The left axillary artery was cannulated to measure the blood pressure and heart rate via a pressure transducer (DPT-6000, pvb Medizintechnik, Kirchseeon, Germany) connected to a MacLab/2e and a MacIntosh computer (using ADInstruments Chart V 3.6.8 data recording software). The left axillary vein was cannulated to infuse drugs or saline. The abdomen was opened in the median line and the abdominal aorta was exposed after moving the small intestine to the right side of the abdomen. For occlusion of the infrarenal aorta a Biemer vessel clip (Aesculap) was placed around the aorta for the designated times. A left thoracotomy was performed at the fourth intercostal space followed by a pericardiotomy. A 5-0 suture on a curved taper needle (5/0 Ethibond Excel, Ethicon, Germany) was passed below the left descending vein and coronary artery from the area immediately below the left atrial appendage to the right portion of the left ventricle, and the ends were pulled through a small vinyl tube to form a snare. The coronary branch was occluded by tightening the snare, which was then fixed by clamping the tube with a small hemostat. Coronary artery occlusion was verified by epicardial cyanosis and subsequent decrease in blood pressure. Reperfusion was achieved by releasing the snare and was confirmed by epicardial hyperemia. Heart rate and blood pressure were allowed to stabilize for 10 min before the following protocols were initiated. 2.2 Study groups and experimental protocols In the first protocol seven groups with 6 to 8 hearts in each group were studied. Group 1 rats served as controls and received a prolonged equilibration time of 40 min prior to the 30-min period of regional ischemia followed by 2 h of reperfusion (Fig. 1a). Group 2 was ischemically preconditioned (classical PC) with three cycles of 5 min of regional ischemia by ligation of the left descending artery followed by 5 min of reperfusion before the onset of 30 min of regional ischemia. In the third group the infrarenal aorta was occluded permanently for 165 min (IOA all the time). Regional ischemia of the heart was initiated after 15 min of IOA. In group 4 the infrarenal aorta was occluded for 15 min. At the time of the reopening of the occlusion on the aorta, regional ischemia on the heart was initiated for 30 min (15 IOA no Rep). In groups 5 to 7 the infrarenal aorta was occluded for 5, 10, or 15 min, respectively, followed by a 10-min reperfusion period of the lower limb prior to the 30 min of regional ischemia of the heart (5, 10, or 15 IOA 10 Rep). Fig. 1 Open in new tabDownload slide (a, b) Experimental protocol. See text for details. Fig. 1 Open in new tabDownload slide (a, b) Experimental protocol. See text for details. In the second protocol chelerythrine was used in three additional groups to investigate the role of PKC in remote preconditioning (Fig. 1b). Groups 8 to 10 were the same as the groups 1, 2, and 7 with the difference that chelerythrine at a concentration of 5 mg/kg body weight was infused intravenously over a period of 2 min. Five minutes later the specified treatment started (Chel+control, Chel+classical PC, Chel+15 IOA 10 Rep). In group 11 the involvement of the neurogenic pathway in remote preconditioning was tested. Hexamethonium (20 mg/kg body weight) was infused intravenously over a 3-min period 15 min prior to the IOA protocol (Hexa+15 IOA 10 Rep) according to the protocol used by Gho et al. [11]. In groups 12 and 13, 5% glucose, which serves as vehicle, was infused instead of chelerythrine (Table 2) in classically and remotely preconditioned hearts. Table 2 Infarct size data No.: group . n . Body weight (g) . Heart weight (g) . Infarction (% of risk zone) . 1: Control 6 277±8 1.54±0.08 62±5 2: Classical PC 6 285±13 1.40±0.06 10±3*** 3: IOA all the time 6 306±15 1.65±0.10 43±5 4: 15 IOA no Rep 6 309±18 1.52±0.08 52±4 5: 5 IOA 10 Rep 6 293±13 1.62±0.07 42±2 6: 10 IOA 10 Rep 6 273±10 1.64±0.04 37±8* 7: 15 IOA 10 Rep 8 284±11 1.49±0.09 18±3***§§ 8: Chel+control 6 308±14 1.41±0.05 66±6 9: Chel+classical PC 6 289±10 1.40±0.04 56±5+++ 10: Chel+15 IOA 10 Rep 6 273±4 1.31±0.03 68±4+++ 11: Hexa+15 IOA 10 Rep 4 299±5 1.28±0.04 14±4*** 12: Classical PC+vehicle 4 311±4 1.44±0.03 19±3*** 13: 15 IOA 10 Rep+vehicle 4 309±24 1.37±0.02 20±5*** No.: group . n . Body weight (g) . Heart weight (g) . Infarction (% of risk zone) . 1: Control 6 277±8 1.54±0.08 62±5 2: Classical PC 6 285±13 1.40±0.06 10±3*** 3: IOA all the time 6 306±15 1.65±0.10 43±5 4: 15 IOA no Rep 6 309±18 1.52±0.08 52±4 5: 5 IOA 10 Rep 6 293±13 1.62±0.07 42±2 6: 10 IOA 10 Rep 6 273±10 1.64±0.04 37±8* 7: 15 IOA 10 Rep 8 284±11 1.49±0.09 18±3***§§ 8: Chel+control 6 308±14 1.41±0.05 66±6 9: Chel+classical PC 6 289±10 1.40±0.04 56±5+++ 10: Chel+15 IOA 10 Rep 6 273±4 1.31±0.03 68±4+++ 11: Hexa+15 IOA 10 Rep 4 299±5 1.28±0.04 14±4*** 12: Classical PC+vehicle 4 311±4 1.44±0.03 19±3*** 13: 15 IOA 10 Rep+vehicle 4 309±24 1.37±0.02 20±5*** For abbreviations see Table 1. Mean±S.E.M. No statistical differences were found between body and heart weights. *P<0.05, ***P<0.001 vs. control, §§P<0.01 vs. 15 IOA no reperfusion, +++P<0.01 vs. the same group without chelerythrine. Open in new tab Table 2 Infarct size data No.: group . n . Body weight (g) . Heart weight (g) . Infarction (% of risk zone) . 1: Control 6 277±8 1.54±0.08 62±5 2: Classical PC 6 285±13 1.40±0.06 10±3*** 3: IOA all the time 6 306±15 1.65±0.10 43±5 4: 15 IOA no Rep 6 309±18 1.52±0.08 52±4 5: 5 IOA 10 Rep 6 293±13 1.62±0.07 42±2 6: 10 IOA 10 Rep 6 273±10 1.64±0.04 37±8* 7: 15 IOA 10 Rep 8 284±11 1.49±0.09 18±3***§§ 8: Chel+control 6 308±14 1.41±0.05 66±6 9: Chel+classical PC 6 289±10 1.40±0.04 56±5+++ 10: Chel+15 IOA 10 Rep 6 273±4 1.31±0.03 68±4+++ 11: Hexa+15 IOA 10 Rep 4 299±5 1.28±0.04 14±4*** 12: Classical PC+vehicle 4 311±4 1.44±0.03 19±3*** 13: 15 IOA 10 Rep+vehicle 4 309±24 1.37±0.02 20±5*** No.: group . n . Body weight (g) . Heart weight (g) . Infarction (% of risk zone) . 1: Control 6 277±8 1.54±0.08 62±5 2: Classical PC 6 285±13 1.40±0.06 10±3*** 3: IOA all the time 6 306±15 1.65±0.10 43±5 4: 15 IOA no Rep 6 309±18 1.52±0.08 52±4 5: 5 IOA 10 Rep 6 293±13 1.62±0.07 42±2 6: 10 IOA 10 Rep 6 273±10 1.64±0.04 37±8* 7: 15 IOA 10 Rep 8 284±11 1.49±0.09 18±3***§§ 8: Chel+control 6 308±14 1.41±0.05 66±6 9: Chel+classical PC 6 289±10 1.40±0.04 56±5+++ 10: Chel+15 IOA 10 Rep 6 273±4 1.31±0.03 68±4+++ 11: Hexa+15 IOA 10 Rep 4 299±5 1.28±0.04 14±4*** 12: Classical PC+vehicle 4 311±4 1.44±0.03 19±3*** 13: 15 IOA 10 Rep+vehicle 4 309±24 1.37±0.02 20±5*** For abbreviations see Table 1. Mean±S.E.M. No statistical differences were found between body and heart weights. *P<0.05, ***P<0.001 vs. control, §§P<0.01 vs. 15 IOA no reperfusion, +++P<0.01 vs. the same group without chelerythrine. Open in new tab 2.3 Exclusion criteria A total of 74 out of 92 rats successfully completed the above protocols. Animals were excluded from data analysis if they exhibited severe hypotension or if it was not possible to maintain adequate blood gas values within a normal physiological range due to metabolic acidosis. The exclusion of 20% of the animals from the present study was evenly distributed among the protocol groups. Five additional rats were used for the determination of the lactate levels during IOA and reperfusion. 2.4 Determination of infarct size At the end of the experiments after 2 h of reperfusion the heart was excised and quickly mounted on a Langendorff apparatus by the aortic root. The heart was perfused at constant pressure (100 cmH2O) with 0.9% saline to wash out the blood for 1 min. The coronary artery was reoccluded, and 1–10 μm zinc cadmium sulfide fluorescent particles (Duke Scientific, Palo Alto, CA) were infused into the perfusate to demarcate the risk zone as the tissue without fluorescence. The heart was then removed from the apparatus, weighed, frozen, and cut into 2-mm-thick slices. The slices were incubated in 1% triphenyltetrazolium chloride (TTC) in pH 7.4 phosphate buffer for 10 min at 37 °C followed by fixation in 10% formalin for at least 15 min. The areas of infarct (TTC negative tissue) and risk zone (nonfluorescent under ultraviolet light) were determined by planimetry. Infarct size was expressed as a percentage of the risk zone. 2.5 Lactate measurement In five additional animals ischemia of the lower limb induced by infrarenal occlusion of the aorta was verified by measuring the lactate concentration in the venous blood after releasing the clip from the aorta. After 10 min of equilibration 500 μl of venous blood were aspirated out of the inferior vena cava in a syringe filled with 50 μl heparin (baseline). The blood loss was substituted with 500 μl of 0.9% sodium chloride solution. Then the infrarenal aorta was occluded for 15 min. Ten seconds, 2 min, 5 min, and 8 min after the release of the clip from the aorta, again samples of venous blood were drawn as described above (Fig. 3). Fig. 3 Open in new tabDownload slide Augmentation of lactate in the lower limb during infrarenal occlusion of the aorta. Lactate concentration in the venous blood of the inferior vena cava. A sample was drawn after the equilibration period (baseline) and at the time points 10 s, 2 min, 5 min, and 8 min after starting the reperfusion of the lower limb after 15 min of IOA. Fig. 3 Open in new tabDownload slide Augmentation of lactate in the lower limb during infrarenal occlusion of the aorta. Lactate concentration in the venous blood of the inferior vena cava. A sample was drawn after the equilibration period (baseline) and at the time points 10 s, 2 min, 5 min, and 8 min after starting the reperfusion of the lower limb after 15 min of IOA. The lactate concentration was determined in the blood plasma according to the manufacturer's description using a colorimetric assay (Sigma Diagnostics, Deisenhofen, Germany). Determinations were done in triplicates. 2.6 Chemicals Chelerythrine chloride was purchased from Alexis Biochemicals (San Diego, CA) and was dissolved in dH2O to a concentration of 10 mg/ml. For infusion, chelerythrine was 10-fold diluted with 5% glucose solution. Hexamethonium which was dissolved in dH2O and 2,3,5-triphenyltetrazolium chloride (TTC) was purchased from Sigma Chemical (Deisenhofen, Germany). 2.7 Statistics Values are presented as means±S.E.M. One-way analysis of variance with repeated measures and Tukey–Kramer post hoc tests were used to test for differences within groups for hemodynamics, and without repeated measures to test for differences in hemodynamic values as well as in infarct size (Instat, Graphpad Software, San Diego, CA). A P value <0.05 was considered significant. 3 Results 3.1 Hemodynamics Baseline hemodynamic parameters were comparable in the 11 groups of animals (Table 1). No significant differences concerning the heart rate were found between the groups with the exception of the 15 IOA 10 Rep group which had significantly lower baseline heart rates. The mean arterial pressure (MAP) as well as the rate pressure product (RPP) were not different at baseline for all groups. At 60 and 120 min of reperfusion the MAP was significantly enhanced in the classical PC and the 15 IOA 10 Rep group compared to the control. Also the RPP as a measurement of the recovery of the heart function was significantly enhanced in both groups compared to control at 60 and 120 min of reperfusion. Additionally the RPP was significantly augmented in the 15 IOA 10 Rep group (the protected group) compared to the 15 IOA no Rep group (the non-protected group) at 60 min of reperfusion. Thus the enhanced MAP and RPP suggest an improved recovery of contractility after regional ischemia in the classically and the remotely preconditioned hearts, which also correlates with the protection status. Table 1 Hemodynamic data No.: group . Baseline . IOA . Regional ischemia . Reperfusion . . . 5 min IOA . 9 min Rep . 10 min . 29 min . 10 min . 60 min . 120 min . Heart rate, beats/min 1: Control 418±13 424±9 419±9 414±16 382±13* 376±11* 2: Classical PC 398±17 410±14 401±15 403±14 401±12 390±15 3: IOA all the time 373±8 343±13 398±13 385±12 373±11 375±6 394±12 4: 15 IOA no Rep 393±15 354±27** 384±17 391±17 388±21 369±17 375±14 5: 5 IOA 10 Rep 392±11 348±11** 360±11 384±12 370±6 366±5 364±9 371±7 6: 10 IOA 10 Rep 387±10 352±12** 354±12** 398±17 389±11 388±14 369±12 379±12 7: 15 IOA 10 Rep 367±10# 333±10** 354±11 395±11 388±10# 389±12 391±11 395±9* 8: Chel+control 396±11 415±16 412±18 386±17 387±9 378±3 9: Chel+classical PC 392±14 410±15 408±16 396±13 384±7 403±11 10: Chel+15 IOA 10 Rep 396±9 380±12§ 394±13§ 424±16 409±13 402±12 378±7 386±16 11: Hexa+15 IOA 10 Rep 374±13 312±4* 335±11 352±11 336±6 348±7 379±19 383±9 MAP, mmHg 1: Control 103±11 96±9 91±11 82±6 64±6*** 60±5*** 2: Classical PC 103±4 99±5 99±7 98±6 88±5*# 86±3**## 3: IOA all the time 108±5 103±5 106±4 95±7 85±6* 74±7*** 64±8*** 4: 15 IOA no Rep 110±4 107±6 97±6 97±7 86±4*** 75±4*** 72±4*** 5: 5 IOA 10 Rep 103±6 102±4 95±5 94±5 91±3* 82±5*** 76±3*** 72±3*** 6: 10 IOA 10 Rep 104±7 101±6 99±5 98±6 94±7 81±7*** 73±4*** 66±3*** 7: 15 IOA 10 Rep 105±3 103±3 96±3 100±4 95±3* 88±5*** 84±3***# 77±4***# 8: Chel+control 112±7 101±8 93±7 77±8*** 71±8*** 64±8*** 9: Chel+classical PC 116±7 91±11** 82±9*** 76±6***§ 62±7***§ 64±7***§§ 10: Chel+15 IOA 10 Rep 105±3 105±4 98±6 92±5 84±3**§ 78±4*** 66±3***§§ 57±6***§ 11: Hexa+15 IOA 10 Rep 92±8 70±2* 60±5*** 60±2*** 57±6*** 65±4** 71±6* 66±3** RPP, beats/min×mmHg 1: Control 50841±5523 46956±3637 45369±4550 40907±2534 31681±2669** 29658±2125*** 2: Classical PC 46242±3450 45231±3375 44479±3722 44486±3237 40866±2124# 39819±1861## 3: IOA all the time 46320±2593 41598±2401 48160±1057 42717±2505 37626±2433 34090±2614** 36405±2837 4: 15 IOA no Rep 49130±2372 44103±4370 42549±3132 43575±3560 39129±2735** 33727±1294*** 32759±820*** 5: 5 IOA 10 Rep 46719±2981 41808±2579 40044±2504* 41597±3125 39758±1751* 36332±2174*** 34158±1772*** 32925±1543*** 6: 10 IOA 10 Rep 46980±2759 42574±2487 39680±2378* 45256±3492 42827±3148 38159±3519** 33693±2496*** 32268±1874*** 7: 15 IOA 10 Rep 44849±1160 40774±1003 40204±1185 46040±1556 43724±1268 41391±2044 39423±1313#+ 37033±1592***# 8: Chel+control 49203±3480 46441±3820 43902±2999 34374±3846* 32733±3252** 29004±3062*** 9: Chel+classical PC 53868±4690 44175±5627* 41309±5712** 37442±3597*** 31050±4261*** 32900±3848*** 10: Chel+15 IOA 10 Rep 46765±2340 44537±2561 43926±3304 43620±3018 38632±2018§ 35489±2584** 29561±1330***§§§ 27172±2779***§§ 11: Hexa+15 IOA 10 Rep 39470±4074 25245±806** 24836±2278** 25283±566** 23576±1419** 27266±2015* 32630±3246 31483±1918 No.: group . Baseline . IOA . Regional ischemia . Reperfusion . . . 5 min IOA . 9 min Rep . 10 min . 29 min . 10 min . 60 min . 120 min . Heart rate, beats/min 1: Control 418±13 424±9 419±9 414±16 382±13* 376±11* 2: Classical PC 398±17 410±14 401±15 403±14 401±12 390±15 3: IOA all the time 373±8 343±13 398±13 385±12 373±11 375±6 394±12 4: 15 IOA no Rep 393±15 354±27** 384±17 391±17 388±21 369±17 375±14 5: 5 IOA 10 Rep 392±11 348±11** 360±11 384±12 370±6 366±5 364±9 371±7 6: 10 IOA 10 Rep 387±10 352±12** 354±12** 398±17 389±11 388±14 369±12 379±12 7: 15 IOA 10 Rep 367±10# 333±10** 354±11 395±11 388±10# 389±12 391±11 395±9* 8: Chel+control 396±11 415±16 412±18 386±17 387±9 378±3 9: Chel+classical PC 392±14 410±15 408±16 396±13 384±7 403±11 10: Chel+15 IOA 10 Rep 396±9 380±12§ 394±13§ 424±16 409±13 402±12 378±7 386±16 11: Hexa+15 IOA 10 Rep 374±13 312±4* 335±11 352±11 336±6 348±7 379±19 383±9 MAP, mmHg 1: Control 103±11 96±9 91±11 82±6 64±6*** 60±5*** 2: Classical PC 103±4 99±5 99±7 98±6 88±5*# 86±3**## 3: IOA all the time 108±5 103±5 106±4 95±7 85±6* 74±7*** 64±8*** 4: 15 IOA no Rep 110±4 107±6 97±6 97±7 86±4*** 75±4*** 72±4*** 5: 5 IOA 10 Rep 103±6 102±4 95±5 94±5 91±3* 82±5*** 76±3*** 72±3*** 6: 10 IOA 10 Rep 104±7 101±6 99±5 98±6 94±7 81±7*** 73±4*** 66±3*** 7: 15 IOA 10 Rep 105±3 103±3 96±3 100±4 95±3* 88±5*** 84±3***# 77±4***# 8: Chel+control 112±7 101±8 93±7 77±8*** 71±8*** 64±8*** 9: Chel+classical PC 116±7 91±11** 82±9*** 76±6***§ 62±7***§ 64±7***§§ 10: Chel+15 IOA 10 Rep 105±3 105±4 98±6 92±5 84±3**§ 78±4*** 66±3***§§ 57±6***§ 11: Hexa+15 IOA 10 Rep 92±8 70±2* 60±5*** 60±2*** 57±6*** 65±4** 71±6* 66±3** RPP, beats/min×mmHg 1: Control 50841±5523 46956±3637 45369±4550 40907±2534 31681±2669** 29658±2125*** 2: Classical PC 46242±3450 45231±3375 44479±3722 44486±3237 40866±2124# 39819±1861## 3: IOA all the time 46320±2593 41598±2401 48160±1057 42717±2505 37626±2433 34090±2614** 36405±2837 4: 15 IOA no Rep 49130±2372 44103±4370 42549±3132 43575±3560 39129±2735** 33727±1294*** 32759±820*** 5: 5 IOA 10 Rep 46719±2981 41808±2579 40044±2504* 41597±3125 39758±1751* 36332±2174*** 34158±1772*** 32925±1543*** 6: 10 IOA 10 Rep 46980±2759 42574±2487 39680±2378* 45256±3492 42827±3148 38159±3519** 33693±2496*** 32268±1874*** 7: 15 IOA 10 Rep 44849±1160 40774±1003 40204±1185 46040±1556 43724±1268 41391±2044 39423±1313#+ 37033±1592***# 8: Chel+control 49203±3480 46441±3820 43902±2999 34374±3846* 32733±3252** 29004±3062*** 9: Chel+classical PC 53868±4690 44175±5627* 41309±5712** 37442±3597*** 31050±4261*** 32900±3848*** 10: Chel+15 IOA 10 Rep 46765±2340 44537±2561 43926±3304 43620±3018 38632±2018§ 35489±2584** 29561±1330***§§§ 27172±2779***§§ 11: Hexa+15 IOA 10 Rep 39470±4074 25245±806** 24836±2278** 25283±566** 23576±1419** 27266±2015* 32630±3246 31483±1918 Mean±S.E.M. MAP, mean arterial pressure (mmHg); RPP, rate pressure product (mmHg×beats/min); n, number of animals in each group; PC, ischemic preconditioning; IOA, infrarenal occlusion of the aorta; Rep, reperfusion; Chel, chelerythrine; IOA all the time, IOA 15 min prior to the regional ischemia of the heart and during ischemia and reperfusion; 15 IOA no Rep, IOA 15 min prior to the regional ischemia which ended at the same time with the beginning of the coronary occlusion; 5, 10, 15 IOA 10 Rep, 5, 10, or 15 min IOA, respectively, followed by 10 min of reperfusion prior to the beginning of the coronary occlusion; Chel+control, 5 mg/kg chelerythrine were infused intravenously over a 2-min period starting 5 min prior to the coronary occlusion; Chel+classical PC, 5 mg/kg chelerythrine were infused over a 2-min period followed by 3 min without any intervention prior to three cycles of PC; Chel+15 IOA 10 Rep, 5 mg/kg chelerythrine were infused over a 2-min period followed by 3 min without any intervention prior to the 15 IOA 10 Rep protocol. Hexa+15 IOA 10 Rep, 20 mg/kg hexamethonium were infused over a 3-min period followed by 12 min without any intervention prior to the 15 IOA 10 Rep protocol. *P<0.05, **P<0.01, ***P<0.001 vs. baseline within the same group; statistical differences between groups were: #P<0.05, ##P<0.01 vs. control; §P<0.05, §§P<0.01, §§§P<0.001 vs. the same group without chelerythrine; +P<0.05 vs. 15 IOA no Rep. Open in new tab Table 1 Hemodynamic data No.: group . Baseline . IOA . Regional ischemia . Reperfusion . . . 5 min IOA . 9 min Rep . 10 min . 29 min . 10 min . 60 min . 120 min . Heart rate, beats/min 1: Control 418±13 424±9 419±9 414±16 382±13* 376±11* 2: Classical PC 398±17 410±14 401±15 403±14 401±12 390±15 3: IOA all the time 373±8 343±13 398±13 385±12 373±11 375±6 394±12 4: 15 IOA no Rep 393±15 354±27** 384±17 391±17 388±21 369±17 375±14 5: 5 IOA 10 Rep 392±11 348±11** 360±11 384±12 370±6 366±5 364±9 371±7 6: 10 IOA 10 Rep 387±10 352±12** 354±12** 398±17 389±11 388±14 369±12 379±12 7: 15 IOA 10 Rep 367±10# 333±10** 354±11 395±11 388±10# 389±12 391±11 395±9* 8: Chel+control 396±11 415±16 412±18 386±17 387±9 378±3 9: Chel+classical PC 392±14 410±15 408±16 396±13 384±7 403±11 10: Chel+15 IOA 10 Rep 396±9 380±12§ 394±13§ 424±16 409±13 402±12 378±7 386±16 11: Hexa+15 IOA 10 Rep 374±13 312±4* 335±11 352±11 336±6 348±7 379±19 383±9 MAP, mmHg 1: Control 103±11 96±9 91±11 82±6 64±6*** 60±5*** 2: Classical PC 103±4 99±5 99±7 98±6 88±5*# 86±3**## 3: IOA all the time 108±5 103±5 106±4 95±7 85±6* 74±7*** 64±8*** 4: 15 IOA no Rep 110±4 107±6 97±6 97±7 86±4*** 75±4*** 72±4*** 5: 5 IOA 10 Rep 103±6 102±4 95±5 94±5 91±3* 82±5*** 76±3*** 72±3*** 6: 10 IOA 10 Rep 104±7 101±6 99±5 98±6 94±7 81±7*** 73±4*** 66±3*** 7: 15 IOA 10 Rep 105±3 103±3 96±3 100±4 95±3* 88±5*** 84±3***# 77±4***# 8: Chel+control 112±7 101±8 93±7 77±8*** 71±8*** 64±8*** 9: Chel+classical PC 116±7 91±11** 82±9*** 76±6***§ 62±7***§ 64±7***§§ 10: Chel+15 IOA 10 Rep 105±3 105±4 98±6 92±5 84±3**§ 78±4*** 66±3***§§ 57±6***§ 11: Hexa+15 IOA 10 Rep 92±8 70±2* 60±5*** 60±2*** 57±6*** 65±4** 71±6* 66±3** RPP, beats/min×mmHg 1: Control 50841±5523 46956±3637 45369±4550 40907±2534 31681±2669** 29658±2125*** 2: Classical PC 46242±3450 45231±3375 44479±3722 44486±3237 40866±2124# 39819±1861## 3: IOA all the time 46320±2593 41598±2401 48160±1057 42717±2505 37626±2433 34090±2614** 36405±2837 4: 15 IOA no Rep 49130±2372 44103±4370 42549±3132 43575±3560 39129±2735** 33727±1294*** 32759±820*** 5: 5 IOA 10 Rep 46719±2981 41808±2579 40044±2504* 41597±3125 39758±1751* 36332±2174*** 34158±1772*** 32925±1543*** 6: 10 IOA 10 Rep 46980±2759 42574±2487 39680±2378* 45256±3492 42827±3148 38159±3519** 33693±2496*** 32268±1874*** 7: 15 IOA 10 Rep 44849±1160 40774±1003 40204±1185 46040±1556 43724±1268 41391±2044 39423±1313#+ 37033±1592***# 8: Chel+control 49203±3480 46441±3820 43902±2999 34374±3846* 32733±3252** 29004±3062*** 9: Chel+classical PC 53868±4690 44175±5627* 41309±5712** 37442±3597*** 31050±4261*** 32900±3848*** 10: Chel+15 IOA 10 Rep 46765±2340 44537±2561 43926±3304 43620±3018 38632±2018§ 35489±2584** 29561±1330***§§§ 27172±2779***§§ 11: Hexa+15 IOA 10 Rep 39470±4074 25245±806** 24836±2278** 25283±566** 23576±1419** 27266±2015* 32630±3246 31483±1918 No.: group . Baseline . IOA . Regional ischemia . Reperfusion . . . 5 min IOA . 9 min Rep . 10 min . 29 min . 10 min . 60 min . 120 min . Heart rate, beats/min 1: Control 418±13 424±9 419±9 414±16 382±13* 376±11* 2: Classical PC 398±17 410±14 401±15 403±14 401±12 390±15 3: IOA all the time 373±8 343±13 398±13 385±12 373±11 375±6 394±12 4: 15 IOA no Rep 393±15 354±27** 384±17 391±17 388±21 369±17 375±14 5: 5 IOA 10 Rep 392±11 348±11** 360±11 384±12 370±6 366±5 364±9 371±7 6: 10 IOA 10 Rep 387±10 352±12** 354±12** 398±17 389±11 388±14 369±12 379±12 7: 15 IOA 10 Rep 367±10# 333±10** 354±11 395±11 388±10# 389±12 391±11 395±9* 8: Chel+control 396±11 415±16 412±18 386±17 387±9 378±3 9: Chel+classical PC 392±14 410±15 408±16 396±13 384±7 403±11 10: Chel+15 IOA 10 Rep 396±9 380±12§ 394±13§ 424±16 409±13 402±12 378±7 386±16 11: Hexa+15 IOA 10 Rep 374±13 312±4* 335±11 352±11 336±6 348±7 379±19 383±9 MAP, mmHg 1: Control 103±11 96±9 91±11 82±6 64±6*** 60±5*** 2: Classical PC 103±4 99±5 99±7 98±6 88±5*# 86±3**## 3: IOA all the time 108±5 103±5 106±4 95±7 85±6* 74±7*** 64±8*** 4: 15 IOA no Rep 110±4 107±6 97±6 97±7 86±4*** 75±4*** 72±4*** 5: 5 IOA 10 Rep 103±6 102±4 95±5 94±5 91±3* 82±5*** 76±3*** 72±3*** 6: 10 IOA 10 Rep 104±7 101±6 99±5 98±6 94±7 81±7*** 73±4*** 66±3*** 7: 15 IOA 10 Rep 105±3 103±3 96±3 100±4 95±3* 88±5*** 84±3***# 77±4***# 8: Chel+control 112±7 101±8 93±7 77±8*** 71±8*** 64±8*** 9: Chel+classical PC 116±7 91±11** 82±9*** 76±6***§ 62±7***§ 64±7***§§ 10: Chel+15 IOA 10 Rep 105±3 105±4 98±6 92±5 84±3**§ 78±4*** 66±3***§§ 57±6***§ 11: Hexa+15 IOA 10 Rep 92±8 70±2* 60±5*** 60±2*** 57±6*** 65±4** 71±6* 66±3** RPP, beats/min×mmHg 1: Control 50841±5523 46956±3637 45369±4550 40907±2534 31681±2669** 29658±2125*** 2: Classical PC 46242±3450 45231±3375 44479±3722 44486±3237 40866±2124# 39819±1861## 3: IOA all the time 46320±2593 41598±2401 48160±1057 42717±2505 37626±2433 34090±2614** 36405±2837 4: 15 IOA no Rep 49130±2372 44103±4370 42549±3132 43575±3560 39129±2735** 33727±1294*** 32759±820*** 5: 5 IOA 10 Rep 46719±2981 41808±2579 40044±2504* 41597±3125 39758±1751* 36332±2174*** 34158±1772*** 32925±1543*** 6: 10 IOA 10 Rep 46980±2759 42574±2487 39680±2378* 45256±3492 42827±3148 38159±3519** 33693±2496*** 32268±1874*** 7: 15 IOA 10 Rep 44849±1160 40774±1003 40204±1185 46040±1556 43724±1268 41391±2044 39423±1313#+ 37033±1592***# 8: Chel+control 49203±3480 46441±3820 43902±2999 34374±3846* 32733±3252** 29004±3062*** 9: Chel+classical PC 53868±4690 44175±5627* 41309±5712** 37442±3597*** 31050±4261*** 32900±3848*** 10: Chel+15 IOA 10 Rep 46765±2340 44537±2561 43926±3304 43620±3018 38632±2018§ 35489±2584** 29561±1330***§§§ 27172±2779***§§ 11: Hexa+15 IOA 10 Rep 39470±4074 25245±806** 24836±2278** 25283±566** 23576±1419** 27266±2015* 32630±3246 31483±1918 Mean±S.E.M. MAP, mean arterial pressure (mmHg); RPP, rate pressure product (mmHg×beats/min); n, number of animals in each group; PC, ischemic preconditioning; IOA, infrarenal occlusion of the aorta; Rep, reperfusion; Chel, chelerythrine; IOA all the time, IOA 15 min prior to the regional ischemia of the heart and during ischemia and reperfusion; 15 IOA no Rep, IOA 15 min prior to the regional ischemia which ended at the same time with the beginning of the coronary occlusion; 5, 10, 15 IOA 10 Rep, 5, 10, or 15 min IOA, respectively, followed by 10 min of reperfusion prior to the beginning of the coronary occlusion; Chel+control, 5 mg/kg chelerythrine were infused intravenously over a 2-min period starting 5 min prior to the coronary occlusion; Chel+classical PC, 5 mg/kg chelerythrine were infused over a 2-min period followed by 3 min without any intervention prior to three cycles of PC; Chel+15 IOA 10 Rep, 5 mg/kg chelerythrine were infused over a 2-min period followed by 3 min without any intervention prior to the 15 IOA 10 Rep protocol. Hexa+15 IOA 10 Rep, 20 mg/kg hexamethonium were infused over a 3-min period followed by 12 min without any intervention prior to the 15 IOA 10 Rep protocol. *P<0.05, **P<0.01, ***P<0.001 vs. baseline within the same group; statistical differences between groups were: #P<0.05, ##P<0.01 vs. control; §P<0.05, §§P<0.01, §§§P<0.001 vs. the same group without chelerythrine; +P<0.05 vs. 15 IOA no Rep. Open in new tab Interestingly, in all groups with IOA with or without reperfusion the mean arterial blood pressure (MAP) rose about 13% immediately after the occlusion of the infrarenal aorta (Fig. 2a and b). This rise was significant only in the 15 IOA 10 Rep group (P<0.01). Within the first 2 min, however, the MAP returned to baseline values. When reopening the aorta for reperfusion of the lower limb the MAP dropped for about 34% in all IOA groups and returned to baseline values within 4 min (P<0.01 for all groups). Prior to the coronary artery occlusion all MAP values between the groups were comparable. Within the IOA groups the heart rate was slightly but significantly depressed during IOA of the aorta. Surprisingly, this heart rate depression could be prevented by inhibition of PKC by chelerythrine. Hexamethonium resulted in a significant depression of the heart rate, MAP, and RPP due to the ganglion blockade which sustained until 60 min of reperfusion. Fig. 2 Open in new tabDownload slide Open in new tabDownload slide (a) Mean arterial pressure increase with IOA and decrease with reperfusion of the aorta. Shown is a compressed exemplary original registration of the arterial pressure during IOA (upper panel) and reperfusion (lower panel). (b) Increased mean arterial pressure after the IOA initiation, pressure depression after reperfusion of the aorta. Mean arterial pressure (mmHg) was measured in the 15 IOA no Rep, 15 IOA 10 Rep, and Chel+15 IOA 10 Rep groups before IOA, immediately after the initiation of IOA, after 2, 5, and 14 min of IOA, furthermore immediately, 4, and 9 min after reopening the aorta. For comparison the MAP of the control and the PC group are shown at baseline and immediately prior to the coronary occlusion. The MAP-increase after IOA was significant in the 15 IOA 10 Rep group compared to baseline. The MAP-decrease after reperfusion of the aorta was significant compared to the MAP after 14 min of IOA in all three IOA groups. Fig. 2 Open in new tabDownload slide Open in new tabDownload slide (a) Mean arterial pressure increase with IOA and decrease with reperfusion of the aorta. Shown is a compressed exemplary original registration of the arterial pressure during IOA (upper panel) and reperfusion (lower panel). (b) Increased mean arterial pressure after the IOA initiation, pressure depression after reperfusion of the aorta. Mean arterial pressure (mmHg) was measured in the 15 IOA no Rep, 15 IOA 10 Rep, and Chel+15 IOA 10 Rep groups before IOA, immediately after the initiation of IOA, after 2, 5, and 14 min of IOA, furthermore immediately, 4, and 9 min after reopening the aorta. For comparison the MAP of the control and the PC group are shown at baseline and immediately prior to the coronary occlusion. The MAP-increase after IOA was significant in the 15 IOA 10 Rep group compared to baseline. The MAP-decrease after reperfusion of the aorta was significant compared to the MAP after 14 min of IOA in all three IOA groups. 3.2 Lactate Ischemia of the lower limb was verified by measuring the lactate, which is released during 15 min of infrarenal occlusion of the aorta. Lactate as an indicator of anaerobic glycolysis was determined in the venous blood obtained from the inferior vena cava upon reperfusion of the infrarenal aorta. The concentration of lactate increased to 248% over baseline upon reperfusion (Fig. 3) and returned to baseline values within 5 to 8 min of reperfusion. 3.3 Infarct size studies No differences in body weight or heart weight among the groups was observed (Table 2). Infarct sizes normalized as a percentage of the ischemic (risk) zone are shown in Fig. 4. Rats with 15 min IOA without reperfusion (IOA all the time, Table 2) or with reperfusion of the aorta at the time of initiation of the infarct inducing ischemia on the heart (15 min IOA no Rep, Table 2 and Fig. 4) showed an infarct size of 43±5% and 52±4%, respectively, which is comparable to that seen in control hearts (62±5%). Interspacing of a 10-min reperfusion period between 15 min of IOA and the infarct inducing ischemia of the heart (15 IOA 10 Rep), however, reduced the infarct size significantly to 18±3% (P<0.001 vs. control, P<0.01 vs. 15 IOA no Rep) which is comparable to the reduction seen in classically preconditioned hearts (classical PC, 10±3%, P<0.001 vs. control). Diminution of the duration of IOA to 10 min (10 IOA 10 Rep) still significantly reduced the infarct size. Part of the powerful protection of 15 min IOA, however, was lost (37±8%, P<0.05 vs. control). Further shortening the IOA period to 5 min (5 IOA 10 Rep) showed a trend for a reduced infarct size (42±2% P = n.s. vs. control). The reduction in infarct size with 5, 10, and 15 min of IOA showed a linear trend; this linear trend being significant (P<0.001). Fig. 4 Open in new tabDownload slide Infrarenal occlusion of the aorta remotely preconditions the heart. Infarct size normalized as a percentage of the ischemic (risk) zone in in situ rat hearts. Control hearts experienced only 30 min of regional ischemia. Hearts were ischemically preconditioned (PC) with three cycles of 5 min of regional ischemia followed by 5 min of reperfusion before the 30 min of infarct inducing regional ischemia (classical PC). 15 IOA no Rep, infrarenal occlusion of the aorta (IOA) for 15 min prior to the regional ischemia. IOA was finished at the same time when the coronary occlusion started; 5, 10, 15 IOA 10 Rep, 5, 10, or 15 min IOA, respectively, followed by 10 min of reperfusion (Rep) prior to the beginning of the coronary occlusion. Classical PC resulted in a highly significant reduction of infarct size compared to control hearts (***P<0.001). 15 IOA no Rep had no influence on infarct size, however if the IOA was followed by a 10-min reperfusion period of the lower limb (15 IOA 10 Rep) infarct size was significantly reduced (***P<0.001). This reduction was comparable to that seen in PC hearts. Shortening the IOA period to 10 min, however, followed by a 10-min reperfusion period (10 IOA 10 Rep) still significantly reduced the infarct size (*P<0.05) whereas in the 5 IOA 10 Rep group the reduction showed only a trend for a reduced infarct size. The linear trend in the loss of infarct size dependent on the duration of the IOA period length was highly significant (§P<0.001). Fig. 4 Open in new tabDownload slide Infrarenal occlusion of the aorta remotely preconditions the heart. Infarct size normalized as a percentage of the ischemic (risk) zone in in situ rat hearts. Control hearts experienced only 30 min of regional ischemia. Hearts were ischemically preconditioned (PC) with three cycles of 5 min of regional ischemia followed by 5 min of reperfusion before the 30 min of infarct inducing regional ischemia (classical PC). 15 IOA no Rep, infrarenal occlusion of the aorta (IOA) for 15 min prior to the regional ischemia. IOA was finished at the same time when the coronary occlusion started; 5, 10, 15 IOA 10 Rep, 5, 10, or 15 min IOA, respectively, followed by 10 min of reperfusion (Rep) prior to the beginning of the coronary occlusion. Classical PC resulted in a highly significant reduction of infarct size compared to control hearts (***P<0.001). 15 IOA no Rep had no influence on infarct size, however if the IOA was followed by a 10-min reperfusion period of the lower limb (15 IOA 10 Rep) infarct size was significantly reduced (***P<0.001). This reduction was comparable to that seen in PC hearts. Shortening the IOA period to 10 min, however, followed by a 10-min reperfusion period (10 IOA 10 Rep) still significantly reduced the infarct size (*P<0.05) whereas in the 5 IOA 10 Rep group the reduction showed only a trend for a reduced infarct size. The linear trend in the loss of infarct size dependent on the duration of the IOA period length was highly significant (§P<0.001). Fig. 5 presents the infarct size data for PKC-inhibition by 5 mg/kg chelerythrine. The vehicle, 5% glucose, for chelerythrine had no influence on infarct size neither in classically nor remotely preconditioned hearts (Table 2). Chelerythrine blocked protection obtained from classical PC (56±5%, P<0.001 vs. classical PC without chelerythrine). In control hearts chelerythrine had no influence on the infarct size (66±6%). Blocking PKC in hearts receiving 15 min IOA with 10 min reperfusion completely prevented the protection obtained from remote preconditioning (68±4% vs. 18±3% without chelerythrine, P<0.001). Blocking the neurogenic pathway by hexamethonium failed to block the protection from 15 IOA 10 Rep (14±4%). Fig. 5 Open in new tabDownload slide Remote preconditioning of the heart can be blocked by PKC inhibition. Infarct size normalized as a % of the ischemic (risk) zone in in situ rat hearts. For comparison the control, classical PC, and 15 IOA 10 Rep groups are shown again on this figure. Chelerythrine (5 mg/kg; Chel) was infused intravenously over a 2-min period starting immediately after the equilibration period. Three min after the infusion stop the infarct-inducing ischemia (Chel+control), the classical PC protocol (Chel+classical PC), or the IOA protocol (Chel+15 IOA 10 Rep) started. Chelerythrine had no influence on infarct size in control hearts whereas it could block protection obtained from classical PC (***P<0.001 vs. classical PC without chelerythrine). Chelerythrine in 15 IOA 10 Rep hearts completely blocks protection which is similar to the observation of the classical PC hearts (***P<0.001 vs. 15 IOA 10 Rep without chelerythrine). This suggests a PKC-dependent signal transduction pathway of remote preconditioning. Hexamethonium (20 mg/kg) was administered for 3 min, 15 min prior to the IOA protocol. Ganglion blockade by hexamethonium could not block the protection from 15 IOA 10 Rep, which excludes a neurogenic pathway for remote preconditioning. Fig. 5 Open in new tabDownload slide Remote preconditioning of the heart can be blocked by PKC inhibition. Infarct size normalized as a % of the ischemic (risk) zone in in situ rat hearts. For comparison the control, classical PC, and 15 IOA 10 Rep groups are shown again on this figure. Chelerythrine (5 mg/kg; Chel) was infused intravenously over a 2-min period starting immediately after the equilibration period. Three min after the infusion stop the infarct-inducing ischemia (Chel+control), the classical PC protocol (Chel+classical PC), or the IOA protocol (Chel+15 IOA 10 Rep) started. Chelerythrine had no influence on infarct size in control hearts whereas it could block protection obtained from classical PC (***P<0.001 vs. classical PC without chelerythrine). Chelerythrine in 15 IOA 10 Rep hearts completely blocks protection which is similar to the observation of the classical PC hearts (***P<0.001 vs. 15 IOA 10 Rep without chelerythrine). This suggests a PKC-dependent signal transduction pathway of remote preconditioning. Hexamethonium (20 mg/kg) was administered for 3 min, 15 min prior to the IOA protocol. Ganglion blockade by hexamethonium could not block the protection from 15 IOA 10 Rep, which excludes a neurogenic pathway for remote preconditioning. 4 Discussion The present study demonstrates that remote preconditioning by this newly established rat model of infrarenal occlusion of the aorta was highly protective against infarction. The protection obtained was equivalent to that seen with classical ischemic preconditioning. This protection shares PKC as a common signal in the signal transduction pathways of classical and remote preconditioning. The data presented here show a time-dependent reduction in infarct size dependent on the duration of the infrarenal occlusion of the aorta. Fifteen minutes IOA reduced infarct size to an amount which is comparable to that seen in classical preconditioned hearts. Shorter periods of IOA still decreased infarct size, but part of the protection was lost. From classical preconditioning it is known that a single ischemic stimulus has to be of at least 3 min duration to induce preconditioning [21]. Longer ischemic periods below the threshold of infarction or repetitions of ischemia–reperfusion to precondition the heart do not reduce the infarct size any further. Thus classical and remote preconditioning are somewhat different regarding the power of protection. We hypothesize that during ischemia of the lower limb a protecting factor is produced in the ischemic trigger organ. The power of protection seems to be due to the amount of protecting factor produced or/and released. This assumption is supported by the observation that with shorter periods of IOA part of the protection is lost. This is supported by the finding of Birnbaum et al. [12] who showed in a remote preconditioning model with a reduced blood supply of the lower limb there is only protection of the heart if the gastrocnemius muscle was rapidly stimulated during the remote ischemia. This protecting substance then needs a circulation time to move from the lower limb to the heart, since we found that a reperfusion period has to be necessarily interspaced between IOA and the induction of the infarction of the heart. We did not test different durations of the reperfusion period. Reperfusion of the aorta at the same time as occlusion of the coronary artery, however, does not protect the heart. This important finding clearly demonstrates that the ‘protecting substance’ has to reach the myocardial tissue prior to the heart being subjected to the infarct inducing ischemia. Dickson et al. [13,24] support the hypothesis of a humoral factor which protects the heart at a distance from infarction. They collected the effluate of isolated preconditioned rabbit hearts, which then itself could protect virgin hearts from infarction. They could show that the protecting factor binds to a hydrophobic matrix, the protecting substances, however, were neither adenosine nor norepinephrine, which is contrary to the observation made by Pell et al. [25] who found a blockade of remote preconditioning by 8-(p-sulphophenyl)theophylline, an adenosine receptor antagonist. In an in vivo rabbit model Dickson et al. found that whole blood transfusion between preconditioned and virgin animals can protect the latter from infarction [26]. It has to be evaluated if this supposed protecting substance is similar in rat and rabbit. In the rat model of remote preconditioning presented here neuronal factors can be ruled out since ganglion blockade by hexamethonium did not influence the reduction in infarct size afforded by remote preconditioning. Gho [11] and Schoemaker [9] found in another model of remote preconditioning by brief mesenterical ischemia or intramesenteric bradykinin infusion, however, controversial data, since they could prevent the protection of the heart by pretreatment with the ganglion blocker hexamethonium [9,11]. The drop in the heart rate and the mean arterial pressure with initiation of the infusion of hexamethonium can be ruled out for conflicting results since Gho [11] already excluded that the hemodynamic effects of hexamethonium do influence the infarct size. The depression of the hemodynamic parameters by hexamethonium seen here were comparable to the ones observed by Gho [11] and Schoemaker [9]. Moreover, the necessity of a reperfusion period between IOA and the coronary occlusion presented here supports the hypothesis that mainly humoral factors are responsible for the protection at a distance. Lactate was measured in the venous blood of the IOA animals to verify metabolically the ischemia of the lower limb. Lactate increased several-fold compared to baseline values. This rise in lactate, however, was compensated within 3 to 5 min of reperfusion by the buffering capacity of the blood. This short lactate peak might be one of the signals for the heart to promote preconditioning. As shown, however, a 10-min reperfusion period between IOA and the infarction of the heart has to be interspaced. Since the rise and the decline of lactate is very fast it is unlikely that lactate is responsible for the observed protection. Moreover, in fasted animals, Doenst et al. [27] showed that adding lactate to the perfusate of isolated hearts neither promotes nor blocks the protection obtained from fasting alone. Lactate not as a trigger but as a mediator of remote preconditioning is also unlikely since several studies showed that lactate did not increase but decrease in classical preconditioning [28]. Another possibility to trigger the protection of remote preconditioning could be hypothesized to be a stretch stimulus since in the model used here the infrarenal occlusion of the aorta (IOA) caused a short rise in the mean arterial pressure (MAP). It is known that brief periods—as long as 10 min—of stretch can precondition the heart [29]. We observed a 13% increase in the mean arterial pressure immediately after the initiation of the IOA (Fig. 2) which decreased to baseline values within the next 2 min. Thus the duration of stretch is much shorter and more moderate than the one used experimentally by an external stimulus to induce preconditioning [29]. After releasing the clip from the aorta the MAP decreased at about one third and returned to baseline values within 4 min of reperfusion. To sum up: hearts which received IOA without reperfusion, IOA with only reperfusion at the time of initiation of the ischemia on the heart, or 15 min IOA with reperfusion but PKC-blockade showed no reduction in infarct size despite an increase in the MAP. Thus these data rule out a pressure-derived effect of IOA. In a model of isolated perfused rabbit hearts which excludes a pressure-induced protective effect, Dickson et al. [13] found the effluent of prior preconditioned hearts to be protective in virgin isolated hearts. We observed a significant drop in the heart rate during the whole duration of infrarenal occlusion and the beginning of the reperfusion of the aorta. Interestingly this heart rate drop could be blocked by PKC-inhibition. The initial rise of the blood pressure beginning with the initiation of IOA could activate the baroreceptors thus resulting in a decreased heart rate by the baroreceptor reflex. With the initiation of the infarction of the heart we could not detect a difference in heart rate any more between the groups. Since the infarction of the heart is a strong circulation depressive stimulus the baroreceptors either could be reset independently if the animals were prior preconditioned or not or could be activated also in the non-preconditioned group. The baroreflex however had no influence on the infarct size since all IOA-hearts had a comparable drop in the heart rate but only the IOA hearts with reperfusion were protected. It is noteworthy, that in our hands remote preconditioning in the rat model was not successful when using mesenterical [9–11] or renal [8,11,25] ischemia as a protecting trigger. The passager ligation of the femoral artery in combination with rapid stimulation of the gastrocnemius muscle [12] or limb ischemia induced by placing a thin elastic tourniquet around the hind extremity [19] was not successful either. One reason for our failure using already published methods of remote preconditioning could be species differences since in most experimental settings rabbits were used. In rats, too, renal ischemia for remote preconditioning was used by different groups. However one group could show that inducing remote preconditioning by renal ischemia was only successful if the body core temperature was decreased to 30–31 °C whereas at 36–37 °C they saw no protection [11]. We therefore developed a new model of remote preconditioning by infrarenal occlusion of the aorta which is reliable to use, reproducible by different investigators and apt to investigate protecting trigger transmitters of remote preconditioning, since the venous blood of the lower limb can be easily obtained by puncture of the inferior vena cava. Classical ischemic preconditioning can be pharmacologically mimicked by stimulating α1-adrenergic-, adenosine-, bradykinin-, opioid-, and other Gq-protein coupled receptors. So far similarities on the receptor level have been investigated for α1-adrenergic- [19], adenosine- [25], bradykinin- [9], and opioid-receptors [20] in remote preconditioning. ATP-sensitive potassium channels are other important signaling elements of classical preconditioning. However, it is not clear yet if they serve as triggers, mediators, or end-effectors [30]. Classical and remote preconditioning also have the ATP-sensitive potassium channels as signal transduction element in common, since the channel blockers glibenclamide [31] and sodium 5-hydroxydecanoate [25] can abolish the protection obtained from remote preconditioning. To our knowledge so far nothing is known about the role of protein kinase C (PKC) in remote preconditioning. It is very likely that PKC is a key enzyme in the signaling mechanism of classical preconditioning, since activators of PKC mimic protection and inhibitors of PKC can block the protection obtained from ischemic or pharmacological preconditioning on a level upstream of PKC [32]. As already shown by other groups, we found that the highly selective PKC-inhibitor chelerythrine can block protection obtained from classical ischemic preconditioning without any influence on infarct size of control hearts. For the first time we could show that the powerful protection of remote preconditioning by 15 min IOA followed by 10 min of reperfusion could be completely blocked by chelerythrine. Also the functional recovery of the heart after 60 and 120 min of reperfusion was reversed by PKC-inhibition in the classically and in the remotely preconditioned hearts. Thus PKC determines this part of the signal transduction pathway both in classical and remote preconditioning. Does remote preconditioning occur in humans? This is the most important question from a clinical point of view as it could have therapeutical implications. It is known that exercise in humans can precondition the heart [33]. It could be possible that physical activity prior to a coronary occlusion has two effects: first it would accelerate the progression of myocardial ischemia because of higher metabolic demand due to physical activity, but secondary it might attenuate the degree of myocardial cell damage through remote preconditioning. An indication for the presence of remote preconditioning in humans are the recent findings of Günaydin et al. [14] who showed that during coronary artery bypass surgery a tourniquet wrapped around an upper extremity to induce brief periods of ischemia resulted in an attenuated increase of lactate dehydrogenase suggesting a reduction in myocyte injury. In summary, a new model of remote preconditioning by infrarenal occlusion of the aorta was developed which is very apt to investigate factors which trigger preconditioning on the heart. The protection obtained by this model is as effective and powerful as classical preconditioning of the heart. This protection can be completely abolished by the PKC-inhibitor chelerythrine. Whether other signal transduction elements downstream of PKC are also involved in the process of remote preconditioning has not been investigated so far. Since a recently published paper showed that remote preconditioning also occurs in humans this phenomenon could have clinical implications of perhaps greater relevance than classical ischemic preconditioning. Procedures that increase oxygen consumption or lactate production, including exercise, could become important determining factors in the protection of the heart. Acknowledgements Part of this study was presented at the Annual Meeting of the American College of Cardiology 2001 and at the German Society of Cardiology 2002. This study was supported by the Deutsche Forschungsgemeinschaft, Bonn (We 1955/2-1 and SFB 320/B4). Part of this work was sponsored by a grant of the University of Technology, Dresden (MedDrive). References [1] Murry C.E. Jennings R.B. Reimer K.A. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium Circulation 1986 74 1124 1136 Google Scholar Crossref Search ADS PubMed WorldCat [2] Kitagawa K. Matsumoto M. Tagaya M. et al. ‘Ischemic tolerance’ phenomenon found in the brain Brain Res 1990 528 21 24 Google Scholar Crossref Search ADS PubMed WorldCat [3] Peralta C. Closa D. Xaus C. et al. Hepatic preconditioning in rats is defined by a balance of adenosine and xanthine Hepatology 1998 28 768 773 Google Scholar Crossref Search ADS PubMed WorldCat [4] Lee H.T. Schroeder C.A. Shah P.M. et al. Preconditioning with ischemia or adenosine protects skeletal muscle from ischemic tissue reperfusion injury J Surg Res 1996 63 29 34 Google Scholar Crossref Search ADS PubMed WorldCat [5] Przyklenk K. Bauer B. Ovize M. Kloner R.A. Whittaker P. Regional ischemic ‘preconditioning’ protects remote virgin myocardium from subsequent sustained coronary occlusion Circulation 1993 87 893 899 Google Scholar Crossref Search ADS PubMed WorldCat [6] Nakano A. Heusch G. Cohen M.V. Downey J.M. Preconditioning one myocardial region does not necessarily precondition the whole rabbit heart Basic Res Cardiol 2002 97 35 39 Google Scholar Crossref Search ADS PubMed WorldCat [7] Liauw S.K. Rubin B.B. Lindsay T.F. Romschin A.D. Walker P.M. Sequential ischemia/reperfusion results in contralateral skeletal muscle salvage Am J Physiol 1996 270 H1407 H1413 Google Scholar PubMed OpenURL Placeholder Text WorldCat [8] McClanahan T.B. Nao B.S. Wolke L.J. et al. Brief renal occlusion and reperfusion reduces myocardial infarct size in rabbits FASEB J 1993 7 A118 Abstract OpenURL Placeholder Text WorldCat [9] Schoemaker R.G. van Heijningen C.L. Bradykinin mediates cardiac preconditioning at a distance Am J Physiol 2000 278 H1571 H1576 OpenURL Placeholder Text WorldCat [10] Petrishchev N.N. Vlasov T.D. Sipovsky V.G. Kurapeev D.I. Galagudza M.M. Does nitric oxide contribute to the mechanism of remote ischemic preconditioning Pathophysiology 2001 7 271 274 Google Scholar Crossref Search ADS PubMed WorldCat [11] Gho B.C.G. Schoemaker R.G. van den Doel M.A. Duncker D.J. Verdouw P.D. Myocardial protection by brief ischemia in noncardiac tissue Circulation 1996 94 2193 2200 Google Scholar Crossref Search ADS PubMed WorldCat [12] Birnbaum Y. Hale S.L. Kloner R.A. Ischemic preconditioning at a distance. Reduction of myocardial infarct size by partial reduction of blood supply combined with rapid stimulation of the gastrocnemius muscle in the rabbit Circulation 1997 96 1641 1646 Google Scholar Crossref Search ADS PubMed WorldCat [13] Dickson E.W. Lorbar M. Porcaro W.A. et al. Rabbit heart can be ‘preconditioned’ via transfer of coronary effluent Am J Physiol 1999 277 H2451 H2457 Google Scholar PubMed OpenURL Placeholder Text WorldCat [14] Günaydin B. Cakici I. Soncul H. et al. Does remote organ ischaemia trigger cardiac preconditioning during coronary artery surgery? Pharmacol Res 2000 41 493 496 Google Scholar Crossref Search ADS PubMed WorldCat [15] Weinbrenner C. Downey J.M. Mentzer R.M. Kitakaze M. Downey J.M. Hori M. Protein kinase C—the key-enzyme in ischemic preconditioning? Adenosine, cardioprotection and its clinical application 1997 Norwell, MA Kluwer Academic 73 91 [16] Baines C.P. Wang L. Cohen M.V. Downey J.M. Protein tyrosin kinase is downstream of protein kinase C for ischemic preconditioning's anti-infarct effect in the rabbit heart J Mol Cell Cardiol 1998 30 383 392 Google Scholar Crossref Search ADS PubMed WorldCat [17] Weinbrenner C. Liu G.S. Cohen M.V. Downey J.M. Phosphorylation of tyrosine 182 of p38 mitogen-activated protein kinase correlates with the protection of preconditioning in the rabbit heart J Mol Cell Cardiol 1997 29 2383 2391 Google Scholar Crossref Search ADS PubMed WorldCat [18] Ping P. Murphy E. Role of p38 mitogen-activated protein kinases in preconditioning. A detrimental factor or a protective kinase? Circ Res 2000 86 921 922 Google Scholar Crossref Search ADS PubMed WorldCat [19] Oxman T. Arad M. Klein R. Avazov N. Rabinowitz B. Limb ischemia preconditions the heart against reperfusion tachyarrhythmia Am J Physiol 1997 273 H1707 H1712 Google Scholar PubMed OpenURL Placeholder Text WorldCat [20] Dickson E.W. Blehar D.J. Carraway R.E. et al. Naloxone blocks transferred preconditioning in isolated rabbit hearts J Mol Cell Cardiol 2001 33 1751 1756 Google Scholar Crossref Search ADS PubMed WorldCat [21] Awan M.M. Taunyane C. Aitchison K.A. Yellon D.M. Opie L.H. Normothermic transfer times up to 3 min will not precondition the isolated rat heart J Mol Cell Cardiol 1999 31 503 511 Google Scholar Crossref Search ADS PubMed WorldCat [22] Goto M. Liu Y. Yang X.-M. et al. Role of bradykinin in protection of ischemic preconditioning in rabbit hearts Circ Res 1995 77 611 621 Google Scholar Crossref Search ADS PubMed WorldCat [23] Miki T. Cohen M.V. Downey J.M. Opioid receptor contributes to ischemic preconditioning through protein kinase C activation in rabbits Mol Cell Biochem 1998 186 3 12 Google Scholar Crossref Search ADS PubMed WorldCat [24] Dickson E.W. Blehar D.J. Lorbar M. et al. Cardioprotective factor released from rabbit heart during brief preconditioning ischemia is captured by a hydrophobic matrix Circulation 2000 102 II27 Abstract Google Scholar Crossref Search ADS WorldCat [25] Pell T.J. Baxter G.F. Yellon D.M. Drew G.M. Renal ischemia preconditions myocardium: role of adenosine receptors and ATP-sensitive potassium channels Am J Physiol 1998 275 H1542 H1547 Google Scholar PubMed OpenURL Placeholder Text WorldCat [26] Dickson E.W. Reinhardt C.P. Renzi F.P. et al. Ischemic preconditioning may be transferable via whole blood transfusion: preliminary evidence J Thromb Thrombolysis 1999 8 123 129 Google Scholar Crossref Search ADS PubMed WorldCat [27] Doenst T. Guthrie P.H. Chemnitius J.M. Zech R. Taegtmeyer H. Fasting, lactate, and insulin improve ischemic tolerance in rat heart: a comparison with ischemic preconditioning Am J Physiol 1996 270 H1607 H1615 Google Scholar PubMed OpenURL Placeholder Text WorldCat [28] Weiss R.G. Albuquerque de C.P. Vandegaer K. Chacko V.P. Gerstenblith G. Attenuated glycogenolysis reduces glycolytic catabolite accumulation during ischemia in preconditioned rat hearts Circ Res 1996 79 435 446 Google Scholar Crossref Search ADS PubMed WorldCat [29] Ovize M. Kloner R.A. Przyklenk K. Stretch preconditions canine myocardium Am J Physiol 1994 266 H137 H146 Google Scholar PubMed OpenURL Placeholder Text WorldCat [30] Downey J.M. Cohen M.V. Do mitochondrial KATP channels serve as triggers rather than end-effectors of ischemic preconditioning's protection? Basic Res Cardiol 2000 95 272 274 Google Scholar Crossref Search ADS PubMed WorldCat [31] Verdouw P.D. Gho B.C.G. Koning M.M.G. Schoemaker R.G. Duncker D.J. Cardioprotection by ischemic and nonischemic myocardial stress and ischemia in remote organs. Implications for the concept of ischemic preconditioning Ann NY Acad Sci 1996 793 27 42 Google Scholar Crossref Search ADS PubMed WorldCat [32] Ytrehus K. Liu Y. Downey J.M. Preconditioning protects ischemic rabbit heart by protein kinase C activation Am J Physiol 1994 266 H1145 H1152 Google Scholar PubMed OpenURL Placeholder Text WorldCat [33] Tomai F. Crea F. Chiariello L. Gioffré P.A. Ischemic preconditioning in humans. Models, mediators, and clinical relevance Circulation 1999 100 559 563 Google Scholar Crossref Search ADS PubMed WorldCat Copyright © 2002, European Society of Cardiology
TI - Remote preconditioning by infrarenal occlusion of the aorta protects the heart from infarction: a newly identified non-neuronal but PKC-dependent pathway
JF - Cardiovascular Research
DO - 10.1016/S0008-6363(02)00446-7
DA - 2002-08-15
UR - https://www.deepdyve.com/lp/oxford-university-press/remote-preconditioning-by-infrarenal-occlusion-of-the-aorta-protects-4dQo4EyXvd
SP - 590
EP - 601
VL - 55
IS - 3
DP - DeepDyve
ER -