Peroxynitrite in myocardial ischemia-reperfusion injury

Peroxynitrite is a highly reactive oxidant which is produced during reperfusion of the ischemic heart. The role that this molecule plays in reperfusion injury has been controversial. Many investigations have demonstrated toxic effects of peroxynitrite, whereas others have found it to be protective during reperfusion. This review surveys evidence supporting both sides and proposes that peroxynitrite is a dichotomous molecule with beneficial and detrimental effects on the reperfused heart. Its toxic effects are mediated by modification and activation of a variety of targets (including poly (ADP) ribose synthetase and matrix metalloproteinases) while its beneficial effects are primarily mediated through its reaction with thiols, resulting in the formation of NO donor compounds (S-nitrosothiols).     

Protease-activated receptor-2 modulates myocardial ischemia-reperfusion injury in the rat heart

Protease-activated receptor-2 (PAR-2) is a member of seven transmembrane domain G protein-coupled receptors activated by proteolytic cleavage whose better known member is the thrombin receptor. The pathophysiological role of PAR-2 remains poorly understood. Because PAR-2 is involved in inflammatory and injury response events, we investigated the role of PAR-2 in experimental myocardial ischemia-reperfusion injury. We show for the first time that PAR-2 activation protects against reperfusion-injury. After PAR-2-activating peptide (2AP) infusion, we found a significant recovery of myocardial function and decrease in oxidation at reflow. Indeed, the glutathione cycle (glutathione and oxidized glutathione) and lipid peroxidation analysis showed a reduced oxidative reperfusion-injury. Moreover, ischemic risk zone and creatine kinase release were decreased after PAR-2AP treatment. These events were coupled to elevation of PAR-2 and tumor necrosis factor alpha (TNFalpha) expression in both nuclear extracts and whole heart homogenates. The recovery of coronary flow was not reverted by L-nitroarginine methylester, indicating a NO-independent pathway for this effect. Genistein, a tyrosine kinase inhibitor, did not revert the PAR-2AP effect. During early reperfusion injury in vivo not only oxygen radicals are produced but also numerous proinflammatory mediators promoting neutrophil and monocyte targeting. In this context, we show that TNFalpha and PAR-2 are involved in signaling in pathophysiological conditions, such as myocardial ischemia-reperfusion. At the same time, because TNFalpha may exert pro-inflammatory actions and PAR-2 may constitute one of the first protective mechanisms that signals a primary inflammatory response, our data support the concept that this network may regulate body responses to tissue injury.     

150-kDa oxygen-regulated protein attenuates myocardial ischemia-reperfusion injury in rat heart

Early contractile dysfunction and the later death of cardiomyocytes are two major problems that can follow myocardial infarction or major cardiovascular surgery that demands ischemic arrest of the heart. Here, we found that 24 h of hypoxia and 1 h of reoxygenation induced the expression of the chaperone ORP150 in cultured rat cardiomyocytes. Inhibition of its induction using an adenovirus to express anti-sense ORP150 significantly enhanced the hypoxia-reoxygenation-induced cardiomyocyte death; cell death was reduced by overexpressing ORP150. Decreased levels of ORP150 expression also enhanced caspase-3 and -8 activation, cytochrome-c release, and DNA fragmentation, suggesting that this chaperone regulates apoptotic cell death. In contrast, increasing the expression of ORP150 in the cardiomyocytes had the opposite effect on the expression of these molecules. Moreover, apoptotic cell death initiated by myocardial ischemia-reperfusion (I/R) was significantly inhibited in vivo by transfecting an ORP150 expression plasmid into whole rat heart using the hemagglutinating virus of Japan (HVJ)-liposome method. Interestingly, ORP150 seemed to preserve calcium homeostasis in cardiomyocytes that underwent ischemia-reoxygenation in vitro. Calpain activity in the cardiomyocytes was enhanced by anti-sense ORP150 and suppressed by sense ORP150. Finally, we examined the functional recovery of rat hearts that overexpressed ORP150 or GFP protein and were subjected to I/R; we found that ORP150 preserved early contractile function after transient ischemia. Our results indicated cytoprotective roles for ORP150 in rat heart and suggested a therapeutic role for the protein both in preventing cardiomyocyte death and in preserving contractile function after ischemic damage.     

多胺代谢与大鼠心肌缺血再灌注损伤关系的实验研究

目的 观察缺血再灌注不同时期大鼠心肌多胺代谢变化规律,探讨多胺代谢与心肌缺血再灌注损伤的关系.方法 采用结扎冠状动脉方法复制大鼠在体心肌缺血再灌注损伤模型,应用逆转录聚合酶链反应(RT-PCR)、Western免疫印迹(Western blot)方法分别测定正常、缺血再灌注2 h、6 h、12 h和24 h时心肌鸟氨酸脱羧酶(ODC)和精胺/精脒乙酰转移酶(SSAT)mRNA的转录和蛋白表达水平,并用高效液相色谱仪测定多胺含量变化.结果 心肌缺血再灌注后ODC和SSAT mRNA的转录和蛋白表达均上调,至再灌注24 h时,与假手术组比,ODC mRNA和SSAT mRNA转录分别增加了3.1倍和3.8倍(P＜0.01),ODC和SSAT的蛋白表达分别增加了3.1倍和2.9倍(P＜0.01).精胺、精脒和多胺总代谢池含量减少,至再灌注24 h时,分别比假手术组少了33.6%、35.3%和32.9%,而腐胺多了58.9%(P＜0.01).结论 心肌缺血再灌注损伤可导致多胺代谢失衡,二者密切相关.     

大鼠心肌缺血再灌注损伤后无复流模型的制备

目的制备大鼠心肌缺血再灌注损伤后无复流模型,探讨建立模型过程中的关键点,为该疾病模型的成功复制提供参考。方法选取20只SD大鼠,随机分成无复流组(n=15)和假手术组(n=5)。无复流组结扎冠状动脉左前降支45 min后再灌注120 min;假手术组只穿线不结扎。在再灌注末予硫磺素S活体染色确定无复流区,同时监测大鼠呼吸及心电图变化。结果 20只大鼠全部存活,且心电图在心肌缺血期均出现心率加快、Ⅱ导联ST段抬高;恢复心肌灌注后,ST段下降30%;硫磺素S染色显示出无复流区域。结论该方法可成功复制无复流动物模型,且模型制作方法成功率高,动物存活率也高。     

MicroRNAs与心肌缺血再灌注损伤

MicroRNAs（miRNAs）是一类高度保守的非编码小分子RNA,经转录后调节细胞的增殖、迁移、分化、凋亡和免疫应答等。miRNAs与心血管疾病发生发展密切相关。心肌缺血后多种miRNA。异常表达,它们在介导心肌缺血再灌注损伤和调控心肌缺血保护环节中起重要作用,可作为诊断缺血再灌注损伤的标志物和潜在治疗靶点。     

Status of myocardial antioxidants in ischemia-reperfusion injury

BACKGROUND: Myocardial ischemia-reperfusion represents a clinically relevant problem associated with thrombolysis, angioplasty and coronary bypass surgery. Injury of myocardium due to ischemia-reperfusion includes cardiac contractile dysfunction, arrhythmias as well as irreversible myocyte damage. These changes are considered to be the consequence of imbalance between the formation of oxidants and the availability of endogenous antioxidants in the heart. OBSERVATIONS: An increase in the formation of reactive oxygen species during ischemia-reperfusion and the adverse effects of oxyradicals on myocardium have now been well established by both direct and indirect measurements. Although several experimental studies as well as clinical trials have demonstrated the cardioprotective effects of antioxidants, some studies have failed to substantiate the results. Nonetheless, it is becoming evident that some of the endogenous antioxidants such as glutathione peroxidase, superoxide dismutase, and catalase act as a primary defense mechanism whereas the others including vitamin E may play a secondary role for attenuating the ischemia-reperfusion injury. The importance of various endogenous antioxidants in suppressing oxidative stress is evident from the depression in their activities and the inhibition of cardiac alterations which they produce during ischemia-reperfusion injury. The effects of an antioxidant thiol containing compound, N-acetylcysteine, and ischemic preconditioning were shown to be similar in preventing changes in the ischemic-reperfused hearts. CONCLUSIONS: The available evidence support the role of oxidative stress in ischemia-reperfusion injury and emphasize the importance of antioxidant mechanisms in cardioprotection.     

不同β受体阻滞剂对大鼠心肌间隙连接结构作用的对比研究

目的比较三代β受体阻滞剂的代表药物卡维地洛、美托洛尔及普萘洛尔对大鼠心肌缺血再灌注损伤心肌间隙连接（GJ）结构的不同作用。方法将大鼠随机分为假手术组、缺血再灌注组、卡维地洛组、美托洛尔组及普萘洛尔组。除假手术组只穿线不结扎外,其余各组均结扎左冠状动脉前降支30min,然后松开结扎线复灌4h,建立心肌缺血再灌注损伤模型。于再灌4h末用免疫荧光和激光扫描共聚焦显微镜技术观察心肌间隙连接蛋白43（CX43）的分布及组成变化,用激光扫描共聚焦显微镜对CX43进行定量。结果与假手术组相比,缺血再灌注组CX43-GJ结构明显异常。与缺血再灌注组比较,卡维地洛组、美托洛尔组和普萘洛尔组CX43-GJ损伤减轻。各药物治疗组间比较,卡维地洛组CX43-GJ结构损伤最轻。结论各种β受体阻滞剂均具有保护心肌GJ结构的作用,以卡维地洛的作用最明显。     

氧化应激与心肌细胞缺血再灌注损伤

冠状动脉粥样硬化性心脏病血运重建治疗是挽救缺血心肌的重要治疗方式。但血流恢复本身会引起缺血再灌注损伤,氧化还原失衡是缺血再灌注损伤发生的重要起始因素。心肌细胞各结构参与氧化应激反应的发生。细胞膜结构中富集Caveolin-1的质膜微囊影响NADPH氧化酶的组装、移位和ROS介导的信号转导参与缺血再灌注中氧化还原反应的发生。由复合物I、II、III、IV及电子载体组成的线粒体电子传递链在缺氧-复氧过程中遭到破坏导致电子传递受阻,线粒体膜通透性转换孔持续开放,是细胞中氧自由基过量产生的重要来源。细胞核及线粒体中核酸分子损伤影响结构蛋白正常合成,细胞氧化还原平衡代谢受损,此外位于细胞核中的肌球蛋白还可以作为一种核转录因子调控NOX2的表达,增加氧自由基的合成。JNK、p38MAPK、Egr-1、NF-κB作为氧化应激导致心肌缺血再灌注损伤的通路蛋白参与其中。     

The role of neutrophils in myocardial ischemia-reperfusion injury

Reperfusion of ischemic myocardium is necessary to salvage tissue from eventual death. However, reperfusion after even brief periods of ischemia is associated with pathologic changes that represent either an acceleration of processes initiated during ischemia per se, or new pathophysiological changes that were initiated after reperfusion. This 'reperfusion injury' shares many characteristics with inflammatory responses in the myocardium. Neutrophils feature prominently in this inflammatory component of postischemic injury. Ischemia-reperfusion prompts a release of oxygen free radicals, cytokines and other proinflammatory mediators that activate both the neutrophils and the coronary vascular endothelium. Activation of these cell types promotes the expression of adhesion molecules on both the neutrophils and endothelium, which recruits neutrophils to the surface of the endothelium and initiate a specific cascade of cell-cell interactions, leading first to adherence of neutrophils to the vascular endothelium, followed later by transendothelial migration and direct interaction with myocytes. This specific series of events is a prerequisite to the phenotypic expression of reperfusion injury, including endothelial dysfunction, microvascular collapse and blood flow defects, myocardial infarction and apoptosis. Pharmacologic therapy can target the various components in this critical series of events. Effective targets for these pharmacologic agents include: (a) inhibiting the release or accumulation of proinflammatory mediators, (b) altering neutrophil or endothelial cell activation and (c) attenuating adhesion molecule expression on endothelium, neutrophils and myocytes. Monoclonal antibodies to adhesion molecules (P-selectin, L-selectin, CD11, CD18), complement fragments and receptors attenuate neutrophil-mediated injury (vascular injury, infarction), but clinical application may encounter limitations due to antigen-antibody reactions with the peptides. Humanized antibodies and non-peptide agents, such as oligosaccharide analogs to sialyl Lewis, may prove effective in this regard. Both nitric oxide and adenosine exhibit broad spectrum effects against neutrophil-mediated events and, therefore, can intervene at several critical points in the ischemic-reperfusion response, and may offer greater benefit than agents that interdict at a single point in the cascade. The understanding of the molecular processes regulating actions of neutrophils in ischemic-reperfusion injury may be applicable to other clinical situations, such as trauma, shock and organ or tissue (i.e. vascular conduits) transplantation.     

腺苷酸活化蛋白激酶与心肌缺血再灌注损伤

心肌缺血再灌注损伤（MIRI）是指缺血期处于可逆损伤的心肌细胞恢复血液供应后产生更为严重的损伤,主要包括炎症反应、内皮细胞损伤、血流障碍、心肌细胞坏死和凋亡所致心肌梗死面积的扩大、再灌注心律失常、心肌顿抑及冠状微循环障碍等病理生理变化.腺苷酸活化蛋白激酶（5-adenosine monophosphate activated kinase,AMPK）通过调节多种代谢途径控制着心脏能量的供求平衡.AMPK不仅控制葡萄糖和脂类的摄入、储存和利用,还能调节多种代谢酶的活性以及离子通道的开放和相关基因的表达[1].AMPK还能够调节缺血再灌注过程中心肌能量代谢,降低缺血性损伤和心肌凋亡.因此,AMPK被认为是能量应激下心肌细胞代谢调节的关键激酶.     

Glutamine improves myocardial function following ischemia-reperfusion injury

Myocardial ischemia-reperfusion injury is common during cardiac procedures. Glutamine may protect the myocardium by preserving metabolic substrates. Glutamine (0.52 g x kg(-1)) or Ringer's lactate solution (control group) was administered intraperitoneally to 63 Sprague-Dawley rats at 4 or 18 hours prior to experimental ischemia and reperfusion. The hearts were excised and perfused on an isolated working heart model, exposed to global ischemia for 15 min and reperfusion for 1 hour. Left atrial pressure, mean aortic pressure, cardiac flow, coronary flow, and aortic output were measured 15 min before ischemia and every 15 min during reperfusion. There was significantly better cardiac output in the glutamine pretreated groups. Pretreatment at 4 hours before the experiment was superior to pretreatment at 18 hours, with better maintenance of cardiac output and coronary flow. The enhanced protective effect of pretreatment at 4 hours highlights the importance of timing, and suggests a potential clinical benefit.     

LOX-1 abrogation reduces myocardial ischemia-reperfusion injury in mice

LOX-1 is a newly described lectin-like receptor for oxidized-LDL (ox-LDL), which is over-expressed in the ischemic myocardium. To examine the pathogenic role of LOX-1 in the determination of ischemia-reperfusion (I-R) injury to the heart, we developed LOX-1 knockout (KO) mice, and subjected these mice to 60 min of left coronary artery occlusion followed by 60 min of reperfusion. I-R in the LOX-1 KO mice resulted in a significant reduction in myocardial injury as well as in accumulation of inflammatory cells in the I-R myocardium and lipid peroxidation (P < 0.01 vs. wild-type mice). Concomitantly, there was significant preservation of cardiac function in the LOX-1 KO mice despite I-R (P < 0.01 vs. the wild-type mice). The phosphorylation of oxidative stress-sensitive mitogen-activated protein kinase (p38MAPK) and protein kinase B/Akt-1, expression of nitrotyrosine and inducible nitric oxide synthase (iNOS), and superoxide dismutase activity were enhanced during I-R in the wild-type mice. These alterations in p38MAPK, Akt-1 and iNOS were much less pronounced in the LOX-1 KO mice. The superoxide dismutase activity increased further in the LOX-1 KO mice. These observations provide compelling evidence that LOX-1 may be a key modulator of myocardial I-R injury, and its effect is mediated by pro-oxidant signals. LOX-1 may be a potential target for therapy of myocardial ischemic injury.     

柚皮素预处理对大鼠心肌缺血再灌注损伤的保护作用

目的观察柚皮素（naringenin,NAR）对大鼠心肌缺血再灌注损伤（IRI）的保护作用。方法32只雄性SD大鼠（220 g~250 g）随机分为假手术组（control,n=8）、IRI组（n=8）、NAR 50 mg/kg组（n=8）和NAR 100 mg/kg组（n=8）。NAR 50 mg/kg和NAR 100mg/kg组均在IRI前2 h予相应NAR腹腔注射。大鼠心肌IRI模型制备方法：结扎左冠状动脉前降支30 min,再灌注180 min,而后180 min检测各组大鼠血清白介素1β（IL-1β）、肿瘤坏死因子α（TNF-α）、乳酸脱氢酶（LDH）、肌酸激酶（CK）、超氧化物歧化酶（SOD）和丙二醛（MDA）含量以及再灌后24 h检测心肌梗死面积。结果NAR可显著降低大鼠心肌IRI后心肌梗死面积（与IRI相比,P＜0.05）,同时还可以显著减轻IRI后血清中IL-1β、TNF-α、LDH、CK和MDA的水平,增加SOD水平（与IRI相比,P＜0.05）,并呈浓度依赖性。结论NAR对心肌IRI具有明显的保护作用,其保护机制与抑制IRI后炎症反应和清除氧化应激损伤有关。     

Vitamin E attenuates myocardial ischemia-reperfusion injury in murine AIDS

The incidence of myocardial infarction in patients who have the aquired immunodeficiency syndrome (AIDS) is increasing. However, no effective therapeutic agents have been discovered to reduce myocardial ischemia-reperfusion (I/R) injury in pathologies associated with AIDS. The aim of this study was to determine if infarct size is increased in murine AIDS after I/R injury and if I/R injury could be attenuated with vitamin E supplementation. Three groups of mice were studied: control, murine AIDS, and murine AIDS with vitamin E supplementation. Anesthetized mice were subjected to 30 min of left anterior descending coronary artery occlusion and 120 min of reperfusion. The hearts in mice that had murine AIDS had a larger infarct size compared to controls after I/R injury. Vitamin E supplementation significantly reduced infarct size and inhibited polymorphonuclear neutrophil (PMN) CD11b expression (p<0.05). However, vitamin E supplementation did not affect PMN reactive oxygen species (ROS) production and platelet CD62p expression. These results suggest that the reduction of myocardial I/R injury with vitamin E supplementation may be the result of the inhibition of PMN CD11b expression. Vitamin E may be a promising prophylactic agent for the reduction of the severity of myocardial I/R injury in patients who have AIDS.     

褪黑激素对心肌缺血再灌注损伤的保护作用

目的 :探讨褪黑激素增补于停搏液中对缺血再灌注离体鼠心的保护作用。方法 :将 2 4只Wistar大鼠随机分为褪黑激素组 ,对照组。离体鼠心在改良的Langendorff Neely灌注模型上 30min预灌注 ,12 0min停搏 ,30min再灌注。缺血前及再灌注期间测定血流动力学指标 ,心肌酶 (CPK ,LDH)、心肌超氧化物歧化酶(SOD)、过氧化脂质 (LPO)含量。电镜观察心肌超微结构。结果 :再灌注后 ,褪黑激素组心功能、心肌超微结构的改善明显优于对照组 ;CPK ,LDH ,LPO含量显著低于对照组 (P <0 .0 1) ;SOD含量显著高于对照组 (P <0 .0 1)。结论 :褪黑激素增补于停搏液中可显著减轻心肌缺血再灌注损伤 ,具有良好的心肌保护作用     

促红细胞生成素对大鼠心肌缺血再灌注损伤的影响

目的:观察促红细胞生成素(erythropoietin,EPO)对大鼠心肌缺血再灌损伤的影响,并探讨机制.方法:以左冠状动脉前降支(LAD)穿线结扎法制备心肌缺血模型,松开结扎线造成再灌注.45只SD大鼠随机分成5组:①假手术组,②缺血再灌注组,③磷脂酰肌醇-3-激酶(PI3K)的高选择性阻断剂LY294002组,④EPO 组和⑤EPO+LY294002组.观察心电图Ⅱ导联心律失常发生情况,进行室性心律失常评分;电镜观察心肌细胞超微结构;以TUNEL法检测细胞凋亡;检测血清肌酸磷酸激酶同工酶(CK-MB)和肌钙蛋白I(cTnI)水平.结果:EPO能降低心律失常评分(P<0.01),减轻心肌细胞超微结构损伤,减少细胞凋亡(P<0.01),降低血清CK-MB和cTnI水平(P<0.01),但这些作用可被预先给予的LY294002所减弱.结论:EPO能减轻心肌缺血再灌注损伤, PI3K参与其信号转导.     

The role of natural IgM in myocardial ischemia-reperfusion injury

Myocardial ischemia-reperfusion injury represents a combination of factors, namely the intrinsic cellular response to ischemia and the extrinsic acute inflammatory response. Recent studies in mesenteric and skeletal muscle reperfusion models identified natural IgM as a major initiator of pathology through the activation of the complement system and inflammatory cells. To determine whether a similar mechanism is involved in myocardial tissues, mice bearing an altered natural IgM repertoire (Cr2-/-) were examined in a murine model of coronary artery ischemia. Notably, these mice were significantly protected based on the reduced infarct size, limited apoptosis of cardiomyocytes, and decreased neutrophil infiltration. Protection was IgM-dependent as reconstitution of these mice with wild-type IgM restored myocardial reperfusion injury. These results support a model in which natural IgM initiates the acute inflammatory response in the myocardium following ischemia and reperfusion.     

Effects of amlodipine on myocardial ischemia-reperfusion injury in dogs

The effects of the dihydropyridine calcium channel blocker amlodipine on subendocardial segment shortening (%SS), regional myocardial blood flow, myocardial high-energy phosphate levels and tissue water content were compared with those of a saline-treated group of barbital-anesthetized dogs subjected to a 45-minute coronary artery occlusion followed by 60 minutes of reperfusion. Saline or amlodipine (200 micrograms/kg administered intravenously) was given 15 minutes before coronary occlusion. There were no significant differences between groups in ischemic bed size or hemodynamics although dP/dt was higher after amlodipine administration. Subepicardial collateral blood flow was higher in the amlodipine group during coronary occlusion. After occlusion, %SS in the ischemic region was markedly decreased in both series and passive systolic lengthening resulted. Despite similar decreases in %SS during occlusion, the amlodipine-treated dogs showed a marked improvement in myocardial segment function of the ischemic reperfused region throughout 60 minutes of reperfusion compared with saline-treated dogs. In addition, amlodipine prevented the rebound increase in phosphocreatine and attenuated the loss of adenine nucleotides and increase in tissue water in the ischemic reperfused area at 60 minutes of reperfusion. These results suggest that amlodipine has a favorable effect on the functional and metabolic recovery of the ischemic reperfused myocardium and may have potential as a therapeutic agent for the treatment of coronary artery disease. The mechanism of action of amlodipine in this model is unknown but may be partially related to a drug-induced increase in coronary collateral blood flow or a decrease in afterload.