Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α(2)-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α(2)-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α(2)-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α(2)-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α(2)-adrenergic receptor stimulation.     

Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α₂-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α₂-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α₂-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α₂-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α₂-adrenergic receptor stimulation.     

The role of C-reactive protein in ischemia/reperfusion injury and preconditioning in a rat model of myocardial infarction

For the first time the involvement of C-Reactive protein (CRP) in early (acute) and delayed ischemic (IPC) and pharmacological (chemical) preconditioning (CPC) in an in vivo model of rat myocardial infarction was presented. Acute IPC was produced by three 5 minute occlusion (ischemia) periods interspersed with 5 minute reperfusion, followed by 30 minute occlusion of the left coronary artery and 2 hour reperfusion injury. Acute CPC was produced by a k-opioid receptor agonist U50488H (5 mg/kg) applied i.v. 15 minutes before 30 minute ischemia/ 2 hour reperfusion. Delayed preconditioning was produced by 30 minute ischemia/ 2 hour reperfusion, induced 24 hour after either ischemic or pharmacological preconditioning. The myocardial ischemia/reperfusion injury was evaluated on the basis of total and cardiac creatine kinase isoenzyme activity, functional recovery of the heart (ECG), infarct size (% IS/RA) and mortality at the end of the experiments. The results obtained showed that: k-opioid receptor agonist U50488H mimics both the acute and delayed IPC in the above experimental protocol; Both acute IPC and most probably CPC act by opening of K(ATP) channels (the effects were blocked by nonspecific ATP-sensitive K channel blocker glybenclamide), and via activation of protein kinase C (a selective protein kinase C inhibitor chelerythrine blocked the efects); C-reactive protein (CRP) was significantly elevated by 54% in non-preconditioned acute ischemia/reperfusion injury. The elevation was more pronounced (82% increase) 24 hour after non-preconditioned ischemia/reperfusion injury. It reflected very well the increase in cardiac isoenzymes, infarct size and mortality of the rats, and can be used as a marker of the severity of myocardial injury in this model; The increase of CRP was prevented by both IPC and CPC in early, and especially in late preconditioning. This confirms the involvement of CRP as a marker in cardiac ischemic/reperfusion injury. It was concluded that in addition to the established involvement of adenosine, bradykinin, opioid and other receptors, a suppression of myocardial CRP/complement production might be involved in the biological mechanism of preconditioning. This could be a promising perspective in clinical interventions against ischemia/reperfusion injuries of the heart.     

Activation of signaling pathways and regulatory mechanisms of mRNA translation following myocardial ischemia-reperfusion.

Protein expression in the heart is altered following periods of myocardial ischemia. The changes in protein expression are associated with increased cell size that can be maladaptive. There is little information regarding the regulation of protein expression through the process of mRNA translation during ischemia and reperfusion in the heart. Therefore, the purpose of this study was to identify changes in signaling pathways and downstream regulatory mechanisms of mRNA translation in an in vivo model of myocardial ischemia and reperfusion. Hearts were collected from rats whose left main coronary arteries had either been occluded for 25 min or reversibly occluded for 25 min and subsequently reperfused for 15 min. Following reperfusion, both the phosphoinositide 3-kinase and mitogen-activated protein kinase pathways were activated, as evidenced by increased phosphorylation of Akt (PKB), extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase. Activation of Akt stimulated signaling through the protein kinase mammalian target of rapamycin, as evidenced by increased phosphorylation of two of its effectors, the ribosomal protein S6 kinase and the eukaryotic initiation factor eIF4E binding protein 1. Ischemia and reperfusion also resulted in increased phosphorylation of eIF2 and eIF2B. These changes in protein phosphorylation suggest that control of mRNA translation following ischemia and reperfusion is modulated through a number of signaling pathways and regulatory mechanisms.     

Soluble epoxide hydrolase inhibition and gene deletion are protective against myocardial ischemia-reperfusion injury in vivo

Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids. EETs are formed from arachidonic acid during myocardial ischemia and play a protective role against ischemic cell death. Deletion of sEH has been shown to be protective against myocardial ischemia in the isolated heart preparation. We tested the hypothesis that sEH inactivation by targeted gene deletion or pharmacological inhibition reduces infarct size (I) after regional myocardial ischemia-reperfusion injury in vivo. Male C57BL\6J wild-type or sEH knockout mice were subjected to 40 min of left coronary artery (LCA) occlusion and 2 h of reperfusion. Wild-type mice were injected intraperitoneally with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE), a sEH inhibitor, 30 min before LCA occlusion or during ischemia 10 min before reperfusion. 14,15-EET, the main substrate for sEH, was administered intravenously 15 min before LCA occlusion or during ischemia 5 min before reperfusion. The EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE) was given intravenously 15 min before reperfusion. Area at risk (AAR) and I were assessed using fluorescent microspheres and triphenyltetrazolium chloride, and I was expressed as I/AAR. I was significantly reduced in animals treated with AUDA-BE or 14,15-EET, independent of the time of administration. The cardioprotective effect of AUDA-BE was abolished by the EET antagonist 14,15-EEZE. Immunohistochemistry revealed abundant sEH protein expression in left ventricular tissue. Strategies to increase 14,15-EET, including sEH inactivation, may represent a novel therapeutic approach for cardioprotection against myocardial ischemia-reperfusion injury.     

Ischemia induced activation of heat shock protein 27 kinases and casein kinase 2 in the preconditioned rabbit heart.

Protein kinase C (PKC), p38 MAP kinase, and mitogen-activated protein kinase-activated kinases 2 and 3 (MAPKAPK2 and MAPKAPK3) have been implicated in ischemic preconditioning (PC) of the heart to reduce damage following a myocardial infarct. This study examined whether extracellular signal-regulated kinase (Erk) 1, p70 ribosomal S6 kinase (p70 S6K), casein kinase 2 (CK2), and other hsp27 kinases are also activated by PC, and if they are required for protection in rabbit hearts. CK2 and hsp27 kinase activities declined during global ischemia in control hearts, whereas PC with 5 min ischemia and 10 min reperfusion increased their activities during global ischemia. Resource Q chromatography resolved two distinct peaks of hsp27 phosphotransferase activities; the first peak (at 0.36 M NaCl) appeared to correspond to the 55-kDa MAPKAPK2. Erk1 activity was elevated in both control and PC hearts after post-ischemic reperfusion, but no change was observed in p70 S6K activity. Infarct size (measured by triphenyltetrazolium staining) in isolated rabbit hearts subjected to 30 min regional ischemia and 2 h reperfusion was 31.0+/-2.6% of the risk zone in controls and was 10.3+/-2.2% in PC hearts (p<0.001). Neither the CK2 inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) nor the Mek1/2 inhibitor PD98059 infused during ischemia blocked protection by PC. The activation of CK2 and Erk1 in ischemic preconditioned hearts appear to be epiphenomena and not required for the reduction of infarction from myocardial ischemia.     

Ethyl pyruvate reduces myocardial ischemia and reperfusion injury by inhibiting high mobility group box 1 protein in rats

High mobility group box 1 protein (HMGB1) plays an important role in myocardial ischemia and reperfusion (I/R) injury. Ethyl pyruvate (EP), a potent reactive oxygen species scavenger, has been reported to inhibit myocardial apoptosis and reduce myocardial I/R injury. The aim of this study was to investigate the mechanism by which EP reduces myocardial I/R injury in rats. Anesthetized male rats were once treated with EP (50 mg/kg, i.p.) before ischemia, and then subjected to ischemia for 30 min followed by reperfusion for 4 h. Lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD) activity and infarct size were measured. HMGB1 expression was assessed by immunoblotting. The results showed that pretreatment of EP (50 mg/kg) could significantly reduce the infarct size and the levels of LDH and CK after 4 h reperfusion (all P < 0.05). EP could also significantly inhibit the increase of the MDA level, the decrease of the SOD level (both P < 0.05). Meanwhile, EP could significantly inhibit the expression of HMGB1 induced by I/R. The present study suggested that ethyl pyruvate could attenuate myocardial I/R injury by inhibiting HMGB1 expression.     

下肢骨骼肌缺血后处理对兔缺血/再灌注心肌坏死和凋亡的影响

目的:探讨在体情况下,骨骼肌缺血后处理对兔缺血/再灌注心肌坏死和凋亡的影响。方法:新西兰大白兔36只,随机分成3组(每组随机选取6只进行梗死范围的测定,另外6只进行凋亡测定):①假手术组(Sham组);②缺血/再灌注组(I/R组);③远端后处理组(RPostC组)。在缺血前、后及再灌注60 min、120 min分别抽血测定肌酸激酶(CK),乳酸脱氢酶(LDH)的活性。采用伊文思兰(evans blue)和三苯基氯化四氮唑(TTC)染色方法确定心肌缺血区范围以及心肌坏死区范围。用Tunel法检测兔心肌缺血区细胞凋亡情况,免疫组织化学方法检测心肌缺血区蛋白caspase-3、Bcl-2及Bax的表达。结果:RPostC组心肌坏死程度、再灌注末CK活性较I/R组明显减低。RPostC组缺血区心肌Tunel阳性指数显著低于I/R组(21.79%±1.07%vs35.81%±1.10%,P<0.05)。而RPostC组缺血区心肌细胞caspase-3阳性指数显著低于I/R组(25.03%±1.16%vs39%±2.43%,P<0.05)。与Sham组比较,I/R组及RPostC组Bax蛋白表达指数、Bcl-2蛋白表达指数均升高;但RPostC组的Bax/Bcl-2比值降低,而I/R组的Bax/Bcl-2比值升高。与I/R组相比较,RPostC组Bax蛋白表达指数及Bax/Bcl-2比值显著降低,Bcl-2表达指数显著升高,差异均有统计学意义。结论:远端后处理能够明显的减少缺血/再灌注心肌细胞的坏死和凋亡,其减轻心肌细胞凋亡的机制可能与抑制促凋亡基因caspase-3的活化及Bcl-2表达的上调有关。     

AMPK mediates autophagy during myocardial ischemia in vivo

We have recently shown that autophagy is induced by ischemia and reperfusion in the mouse heart in vivo. Ischemia stimulates autophagy through an AMP activated protein kinase (AMPK)-dependent mechanism, whereas reperfusion after ischemia stimulates autophagy through a Beclin 1-dependent, but AMPK-independent, mechanism. Autophagy plays distinct roles during ischemia and reperfusion: autophagy may be protective during ischemia, whereas it may be detrimental during reperfusion. We will discuss the role of AMPK in mediating autophagy during myocardial ischemia in vivo.     

不同强度及时间窗骨骼肌缺血后处理对兔心肌的保护作用

目的:通过比较不同强度及时间窗骨骼肌缺血后处理对兔缺血/再灌注心肌的保护效能,试图寻找最佳强度和时间窗的骨骼肌缺血后处理方案。方法:健康新西兰大白兔42只(雄性)随机分为7组(n=6):①假手术组(Sham)、②缺血对照组(CON)、③标准骨骼肌后处理组(SP)、④延迟6min骨骼肌后处理组(6M-DSP)、⑤延迟1 min骨骼肌后处理组(1M-DSP)、⑥骨骼肌后处理加强组(SSP)、⑦骨骼肌后处理减弱组(WSP)。以开胸结扎冠状动脉左室支固定部位方法制作缺血/再灌注模型,以游离并夹闭双侧腹股沟髂外动脉固定位置方法造成骨骼肌缺血,再灌注末以TTC法确定心肌梗死范围,并分别于心肌缺血前、后及再灌注1 h、2 h,以生化法测定血清肌酸激酶(CK)及乳酸脱氢酶(LDH)水平。结果:和CON组相比,1M-DSP组心肌梗死重量比及面积比分别下降了42.32%及42.68%、SP组分别下降了49.97%及43.78%、SSP组分别下降了48.36%及48.86%,(P均<0.05),但三组之间相比,心梗范围未见明显差异;而6M-DSP、WSP组与CON组相比未见心肌保护作用;肌酸激酶(CK)的水平和梗死范围变化趋势一致。结论:兔在心肌缺血/再灌注之前完成骨骼肌5 min缺血/1 min再灌注1次循环的缺血后处理,可以起到明显的心肌保护作用。     

Reduction of asymmetric dimethylarginine involved in the cardioprotective effect of losartan in spontaneously hypertensive rats

Previous studies have indicated that nitric oxide synthase (NOS) inhibitors can induce an increase of blood pressure and exacerbate myocardial injury induced by ischemia and reperfusion, whereas angiotensin II receptor antagonists protect the myocardium against injury induced by ischemia and reperfusion. Isolated hearts from male spontaneously hypertensive rats (SHR) or male Wistar-Kyoto rats (WKY) were subjected to 20 min global ischemia and 30 min reperfusion. Heart rate, coronary flow, left ventricular pressure, and its first derivatives (+/-dP/dt(max)) were recorded, and serum concentrations of asymmetric dimethylarginine (ADMA) and NO and the release of creatine kinase in coronary effluent were measured. The level of ADMA was significantly increased and the concentration of NO was decreased in SHR. Ischemia and reperfusion significantly inhibited the recovery of cardiac function and increased the release of creatine kinase, and ischemia and reperfusion-induced myocardial injury in SHR was aggravated compared with WKY. Vasodilation responses to acetylcholine of aortic rings were decreased in SHR. Treatment with losartan (30 mg/kg) for 14 days significantly lowered blood pressure, elevated the plasma level of NO, and decreased the plasma concentration of ADMA in SHR. Treatment with losartan significantly improved endothelium-dependent relaxation and cardiac function during ischemia and reperfusion in SHR. Exogenous ADMA also aggravated myocardial injury induced by ischemia and reperfusion in isolated perfused heart of WKY, as shown by increasing creatine kinase release and decreasing cardiac function. The present results suggest that the protective effect of losartan on myocardial injury induced by ischemia and reperfusion is related to the reduction of ADMA levels.     

The Protective Role of Interleukin-33 in Myocardial Ischemia and Reperfusion Is Associated with Decreased HMGB1 Expression and Up-Regulation of the P38 MAPK Signaling Pathway

Interleukin-33 (IL-33) plays a protective role in myocardial ischemia and reperfusion (I/R) injury, but the underlying mechanism was not fully elucidated. The present study was designed to investigate whether IL-33 protects against myocardial I/R injury by regulating both P38 mitogen-activated-protein kinase (P38 MAPK), which is involved in one of the downstream signaling pathways of IL-33, and high mobility group box protein 1 (HMGB1), a late pro-inflammatory cytokine. Myocardial I/R injury increased the level of IL-33 and its induced receptor (sST) in myocardial tissue. Compared with the I/R group, the IL-33 group had significantly lower cardiac injury (lower serum creatine kinase (CK), lactate dehydrogenase (LDH), and cTnI levels and myocardial infarct size), a suppressed inflammatory response in myocardial tissue (lower expression of HMGB1, IL-6, TNF-α and INF-γ) and less myocardial apoptosis (much higher Bcl-2/Bax ratio and lower cleaved caspase-3 expression). Moreover, IL-33 activated the P38 MAPK signaling pathway (up-regulating P-P38 expression) in myocardial tissue, and SB230580 partially attenuated the anti-inflammatory and anti-apoptosis effects of IL-33. These findings indicated that IL-33 protects against myocardial I/R injury by inhibiting inflammatory responses and myocardial apoptosis, which may be associated with the HMGB1 and P38 MAPK signaling pathways.     

Dynamics of Ca(2+)/calmodulin-dependent protein kinase II following acute myocardial ischemia-translocation and autophosphorylation

Ca(2+)/calmodulin-dependent protein kinase (CaMK) family is responsive to changes in the intracellular Ca(2+) concentration. However, their functions have not been well established in the ischemia/reperfusion heart. The effects of myocardial ischemia on CaMKII, the most strongly expressed form, were investigated using isolated rat hearts. Rat hearts were rendered globally ischemic by stopping perfusion for 15 min, and then reperfused, heart ventricles being analyzed in each phase. Western blotting detected a decrease in the cytosolic and concomitant increase in the particulate fraction of CaMKII following transient ischemia. Redistribution to the cytosol was revealed on reperfusion. Northern blot showed CaMKII gene expression decreased by ischemia. Furthermore, autoradiography and confocal immunohistochemical findings provided autophosphorylation of CaMKII in the cytosol, ischemia causing decrease, with gradual recovery on reperfusion. These results indicate a transient partial translocation of CaMKII accompanied by kinase activity, with residual myocardial CaMKII undergoing autophosphorylation during ischemia and reperfusion, demonstrating two different characteristic dynamics of CaMKII.     

Apelin-13 limits infarct size and improves cardiac postischemic mechanical recovery only if given after ischemia

We studied whether apelin-13 is cardioprotective against ischemia/reperfusion injury if given as either a pre- or postconditioning mimetic and whether the improved postischemic mechanical recovery induced by apelin-13 depends only on the reduced infarct size or also on a recovery of function of the viable myocardium. We also studied whether nitric oxide (NO) is involved in apelin-induced protection and whether the reported ischemia-induced overexpression of the apelin receptor (APJ) plays a role in cardioprotection. Langendorff-perfused rat hearts underwent 30 min of global ischemia and 120 min of reperfusion. Left ventricular pressure was recorded. Infarct size and lactate dehydrogenase release were determined to evaluate the severity of myocardial injury. Apelin-13 was infused at 0.5 μM concentration for 20 min either before ischemia or in early reperfusion, without and with NO synthase inhibition by N(G)-nitro-l-arginine (l-NNA). In additional experiments, before ischemia also 1 μM apelin-13 was tested. APJ protein level was measured before and after ischemia. Whereas before ischemia apelin-13 (0.5 and 1.0 μM) was ineffective, after ischemia it reduced infarct size from 54 ± 2% to 26 ± 4% of risk area (P < 0.001) and limited the postischemic myocardial contracture (P < 0.001). l-NNA alone increased postischemic myocardial contracture. This increase was attenuated by apelin-13, which, however, was unable to reduce infarct size. Ischemia increased APJ protein level after 15-min perfusion, i.e., after most of reperfusion injury has occurred. Apelin-13 protects the heart only if given after ischemia. In this protection NO plays an important role. Apelin-13 efficiency as postconditioning mimetic cannot be explained by the increased APJ level.     

Two-dimensional analysis of myocardial protein expression following myocardial ischemia and reperfusion in rabbits

Myocardial ischemia and reperfusion injury (MI/R) can be related to leukocyte activation with subsequent release of cytokines and oxygen derived free radicals. Activation of the complement system has been implicated in the pathogenesis of myocardial ischemia and reperfusion injury. Inflammatory injury will subsequently result in cellular activation and protein synthesis. In the present study we analyzed the myocardial protein expression and its pattern following myocardial ischemia and reperfusion, with and without complement inhibition with the synthetic serine protease inhibitor Futhan/nafamstat mesilate (FUT-175) known to inhibit classical and alternative complement pathway in a rabbit model of myocardial ischemia and reperfusion (60 min I+180 min R). FUT-175 significantly reduced myocardial necrosis, i.e. creatine kinase release which were analyzed for the three groups (p<0.05). Similarly, histological analysis demonstrated preservation of myocardial tissue injury and reduced leukocyte accumulation following FUT-175 treatment. Further, the myocardial protein expression was analyzed by two-dimensional gel electrophoresis following MI/R in the different groups. The protein patterns were evaluated by means of MELANIE III, a computer assisted gel analysis system. The biochemical identification of the proteins of interest was, achieved using nanohigh-performance liquid chromatography/electrospray ionization-tandem mass spectrometry. On average, 509 +/- 25 protein spots were found on the gels. A pattern of 480 spots with identical positions was found on every gel of five animals of each group. We analyzed ten spots which were significantly altered (i.e., in eight spots we observed decreased protein expression and in two spots we observed increased expression, vehicle vs. sham), by using mass spectrometry. Superoxide dismutase precursor and alphaB-crystallin were identified. We compared sham group vs. vehicle group and vehicle group vs. FUT-175 treated animals. Expression of the two identified proteins decreased by half the amount in the vehicle group when compared to sham treated animals. Treatment with FUT-175 preserved significantly superoxide dismutase precursor and alphaB-crystallin protein expression when compared to vehicle animals. The results present marked differences in myocardial protein expression after ischemia and reperfusion and following treatment with the complement inhibitor FUT-175. Our results illustrate the application of proteomics to discover possible new therapeutic targets or to detect unexpected effects of pharmacological inhibitors.     

Heme oxygenase-1-derived bilirubin ameliorates postischemic myocardial dysfunction

Bilirubin is a potent antioxidant generated intracellularly during the degradation of heme by the enzyme heme oxygenase. The purpose of this study was to determine the role of increased cardiac bilirubin in protection against postischemic myocardial dysfunction. Rat hearts were isolated and perfused according to the Langendorff technique to evaluate the recovery of myocardial function after 30 min of global ischemia and 60 min of reperfusion. We found that upregulation of the inducible isoform of heme oxygenase (HO-1) by treatment of animals with hemin 24 h before ischemia ameliorated myocardial function and reduced infarct size (tetrazolium staining) on reperfusion of isolated hearts. Tin protoporphyrin IX, an inhibitor of heme oxygenase activity, completely abolished the improved postischemic myocardial performance observed after hemin-mediated HO-1 induction. Likewise, cardiac tissue injury was exacerbated by treatment with tin protoporphyrin IX. Increased cardiac HO-1 expression and heme oxygenase activity were associated with enhanced tissue bilirubin content and an increased rate of bilirubin release into the perfusion buffer. Furthermore, exogenously administered bilirubin at concentrations as low as 100 nanomolar significantly restored myocardial function and minimized both infarct size and mitochondrial damage on reperfusion. Our data provide strong evidence for a primary role of HO-1-derived bilirubin in cardioprotection against reperfusion injury.     

Activation of p38 mitogen-activated protein kinase abolishes insulin-mediated myocardial protection against ischemia-reperfusion injury

Myocardial ischemia-reperfusion injury contributes significantly to morbidity and mortality in patients with diabetes. Insulin decreases myocardial infarct size in animals and the rate of apoptosis in cultured cells. Ischemia-reperfusion activates p38 mitogen-activated protein kinase (MAPK), which regulates cellular apoptosis. To examine whether p38 MAPK affects insulin's cardioprotection against ischemia-reperfusion injury, we studied overnight-fasted adult male rats by use of an in vivo rat model of myocardial ischemia-reperfusion. A euglycemic clamp (3 mU.min(-1).kg(-1)) was begun either 10 min before ischemia (InsulinBI), 5 min before reperfusion (InsulinBR), or 30 min after the onset of reperfusion (InsulinAR), and continued until the end of the study. Compared with saline control, insulin decreased the infarct size in both InsulinBI (P < 0.001) and InsulinBR (P < 0.02) rats but not in InsulinAR rats. The ischemic area showed markedly increased phosphorylation of p38 MAPK compared with the nonischemic area in saline animals. Acute activation of p38 MAPK with anisomycin (2 mg/kg iv 10 min before ischemia) had no effect on infarct size in saline rats. However, it completely abolished insulin's protective effect in InsulinBI and InsulinBR rats. Activation of p38 MAPK by anisomycin was associated with marked and persistent elevation in IRS-1 serine phosphorylation. Treatment of animals with SB-239063, a potent and specific inhibitor of p38 MAPK, 10 min before reperfusion enabled insulin-mediated myocardial protection in InsulinAR rats. We conclude that insulin protects myocardium against ischemia-reperfusion injury when given prior to ischemia or reperfusion, and activation of p38 MAPK abolishes insulin's cardioprotective effect.     

Adenosine-enhanced ischemic preconditioning: adenosine receptor involvement during ischemia and reperfusion

Adenosine-enhanced ischemic preconditioning (APC) extends the cardioprotection of ischemic preconditioning (IPC) by both significantly decreasing myocardial infarct size and significantly enhancing postischemic functional recovery. In this study, the role of adenosine receptors during ischemia-reperfusion was determined. Rabbit hearts (n = 92) were used for Langendorff perfusion. Control hearts were perfused for 180 min, global ischemia hearts received 30-min ischemia and 120-min reperfusion, and IPC hearts received 5-min ischemia and 5-min reperfusion before ischemia. APC hearts received a bolus injection of adenosine coincident with IPC. Adenosine receptor (A(1), A(2), and A(3)) antagonists were used with APC before ischemia and/or during reperfusion. GR-69019X (A(1)/A(3)) and MRS-1191/MRS-1220 (A(3)) significantly increased infarct size in APC hearts when administered before ischemia and significantly decreased functional recovery when administered during both ischemia and reperfusion (P < 0.05 vs. APC). DPCPX (A(1)) administered either before ischemia and/or during reperfusion had no effect on APC cardioprotection. APC-enhanced infarct size reduction is modulated by adenosine receptors primarily during ischemia, whereas APC-enhanced postischemic functional recovery is modulated by adenosine receptors during both ischemia and reperfusion.