曲美他嗪对大鼠缺血再灌注心肌线粒体的保护作用

目的探讨曲美他嗪对缺血再灌注损伤心肌线粒体的保护作用及其机制.方法将50只SD雄性大鼠随机分为假手术组、生理盐水组和曲美他嗪组(5mg/kg组及10mg/kg组)4组,假手术组只开胸,不结扎冠状动脉.余3组复制缺血再灌注损伤模型,缺血前分别静脉注射曲美他嗪(5或10mg/kg)及等量生理盐水,在缺血30min及再灌注40min时测定缺血再灌注损伤区心肌线粒体丙二醛、超氧化物歧化酶、谷胱甘肽、谷胱甘肽过氧化物酶及总钙浓度,并通过电镜观察心肌超微结构的改变.结果与假手术组比较,生理盐水组及曲美他嗪组线粒体中的丙二醛及总钙显著增高,超氧化物歧化酶、谷胱甘肽及谷胱甘肽过氧化物酶显著降低.与生理盐水组比较,曲美他嗪组的丙二醛及总钙水平显著降低,超氧化物歧化酶、谷胱甘肽及谷胱甘肽过氧化物酶显著增高.电镜观察显示曲美他嗪组线粒体损伤较生理盐水组明显减轻.结论以上提示曲美他嗪能减轻缺血再灌注心肌线粒体的脂质过氧化损伤,其机制可能是通过提高线粒体内谷胱甘肽含量及超氧化物歧化酶和谷胱甘肽过氧化物醇活性,以增强其抗氧化能力,并通过减轻线粒体内钙聚积在细胞水平提供心肌保护作用.     

Activation of HtrA2, a mitochondrial serine protease mediates apoptosis: current knowledge on HtrA2 mediated myocardial ischemia/reperfusion injury

A plethora of apoptotic stimuli converge on the mitochondria and affect their membrane integrity, thereby eliciting release of multiple death-promoting factors residing in the mitochondrial intermembrane space into the cytosol. Among the death-promoting factors, a serine protease, high temperature requirement A2 (HtrA2) has drawn attention as a key player in the apoptosis pathways in different pathological conditions including myocardial ischemia/reperfusion injury. Heart ischemia/reperfusion results in HtrA2 translocation from the mitochondria to the cytosol, where it promotes cardiomyocyte apoptosis via a protease activity-dependent and caspase-mediated pathway. Once released, cytosolic HtrA2 causes X-chromosome-linked inhibitor of apoptosis protein (XIAP) degradation, caspase activation, and subsequent apoptosis. Consistent with the hypothesis, inhibition of HtrA2 improved postischemic myocardial contractile functions along with reduction of myocardial infarct size. The precise mechanism underlying HtrA2-induced apoptosis in mammalian cells has been studied through biochemical, structural, and genetic studies, in which HtrA2 promotes proteolytic activation of caspases through multiple pathways in heart ischemia. Therapeutic interventions that inhibit HtrA2 expression, translocation, or protease activity (such as by using the ucf-101 inhibitor) may provide an attractive therapeutics in the treatment of cardiovascular diseases.     

心肌缺血再灌注损伤亚细胞Ca~(2 )反常与ATP酶泵功能抑制

目的　研究心肌缺血再灌注损伤亚细胞Ca2 分布、含量及ATPase活性变化 ,探讨Ca2 反常与ATP酶泵功能抑制的关系。方法　采用离体心脏灌注模型 ,电子探针显微分析测定心肌缺血再灌注原位亚细胞Ca2 变化 ;电镜酶细胞化学及生化方法测定ATPase分布及活性变化。结果　心肌缺血 30min再灌注 30、6 0min肌浆网及肌膜Ca2 明显减少 (P <0 .0 1) ,而胞浆及线粒体内Ca2 明显增加 (P <0 .0 1)。再灌注肌浆网及肌膜Ca2 减少与胞浆及线粒体Ca2 增加呈负线性相关 ,r分别为- 0 .96 4和 - 0 .994,胞浆与线粒体Ca2 变化呈正线性相关 ,r为 0 .997。心肌缺血 30minATPase活性明显降低 ,缺血 30min再灌注 30及 6 0min进一步加重。结论　心肌缺血再灌注Ca2 超载发生在亚细胞水平 ,即亚细胞Ca2 的重新分布。ATPase泵功能抑制是心肌缺血再灌注亚细胞Ca2 紊乱的直接原因之一。     

Ischemia-reperfusion induces myocardial infarction through mitochondrial Ca²⁺ overload

Both mitochondria and the sarcoplasmic reticulum (SR) are essential for myocardial homeostasis and control of cardiac function. Uptake of Ca(2+) from the cytosol into SR is mediated by the Ca(2+)-dependent ATPase SERCA2a, which is reversibly inhibited by phospholamban (PLN). We previously showed that removal of PLN inhibition of SERCA2a with an antibody to (anti-) PLN reduces cytosolic Ca(2+) overload, thereby attenuating the spread of contraction bands and fodrin proteolysis, during reperfusion after cardiac ischemia. We have now examined the effects of anti-PLN injection into the heart on the development of myocardial infarction (MI) after ischemia-reperfusion in rats. Whereas anti-PLN injection attenuated cytosolic Ca(2+) overload, it did not affect MI size 6h after the onset of reperfusion and actually increased it at 30 min. The antibody also increased the release of apoptosis-inducing factor (AIF) from mitochondria into the cytosol, indicative of enhanced opening of the mitochondrial permeability transition pore (mPTP). Administration of an mPTP blocker at the time of reperfusion or of a blocker of the mitochondrial Ca(2+) uniporter significantly suppressed the release of AIF and the development of MI. These results indicate that the enhancement of SR Ca(2+) loading by anti-PLN injection facilitated Ca(2+) uniporter-dependent mitochondrial Ca(2+) uptake and thereby induced mPTP opening and MI development during early reperfusion. The enhancement of SR Ca(2+) loading thus aggravates MI in a manner independent of cytosolic Ca(2+) overload. Given that cytosolic Ca(2+) overload induces contraction bands, our findings are inconsistent with a causal relation between contraction bands and MI.     

Effects of amiloride and an analogue on ventricular arrhythmias, contracture and cellular injury during reperfusion in isolated and perfused guinea pig heart

The present study was designed to examine whether activation of Na+/H+ exchange and subsequent massive Ca2+ influx via Na+/Ca2+ exchange are involved in the pathogenesis of myocardial reperfusion injury. We tested the effects of 1 mM amiloride, which is known to inhibit both Na+/H+ and Na+/Ca2+ exchange, and 3 microM 5-(N-ethyl-N-isopropyl) amiloride (EIPA), which is known to act as a specific inhibitor against Na+/H+ exchange, on the incidence of ventricular arrhythmias, isovolumic left ventricular function and creatine kinase (CK) release during reperfusion after 15 or 30 min of global ischemia in the isolated and perfused guinea pig heart. Treatment of a normally perfused heart with amiloride decreased heart rate significantly and tended to increase coronary flow and left ventricular developed pressure (LVDP), whereas treatment with EIPA decreased all of these 3 measurements significantly. Treatment with amiloride or EIPA for 15 min before ischemia, and during reperfusion after 15 min of ischemia, under electrical pacing at 240 rpm to eliminate a negative chronotropic effect abolished ventricular tachycardia (VT) and ventricular fibrillation (VF) during reperfusion associated with highly significant inhibition of increases in left ventricular end-diastolic pressure (LVEDP) and CK release. Amiloride or EIPA pretreatment also inhibited the incidence of VF and increases in LVEDP and CK release significantly during reperfusion after 30 min of ischemia. However, amiloride was more effective in preventing these events than EIPA. The treatment with amiloride or EIPA only during reperfusion after 15 or 30 min of ischemia also decreased the incidence of VF and inhibited the increases in LVEDP and CK release significantly, though less effectively than the pretreatment modality. These results suggest that EIPA prevents ventricular arrhythmias, contracture and myocardial cellular injury during reperfusion after 15 min of ischemia by inhibiting Na+/H+ exchange, while amiloride exerts more powerful protection against these events than EIPA during reperfusion after 30 min of ischemia by inhibiting both Na+/H+ and Na+/Ca2+ exchange.     

再灌注致大鼠心肌线粒体损伤及益心康胶囊的保护作用

目的　观察再灌注对心肌线粒体的损伤 ,评价中药复方———益心康胶囊 (H30 3)的保护作用。方法　利用离体大鼠全心停灌 /再灌 (I/R)模型 ,测定心肌线粒体结构和功能的变化。结果　I/R可致线粒体结构和功能损伤 ,H30 3预先灌注给药可明显减轻心肌脂质过氧化程度 ,抑制磷脂酶A2 活性 ,抑制线粒体磷脂降解和游离脂肪酸生成 ,改善线粒体膜脂流动性。此外对呼吸功能及Ca2 + ATPase活性也具有明显的保护作用。结论　H30 3对再灌注所致的线粒体的损伤具有明显的保护作用     

Oxygen free radicals and cardiac reperfusion abnormalities.

Oxygen free radicals are highly reactive compounds causing peroxidation of lipids and proteins and are thought to play an important role in the pathogenesis of reperfusion abnormalities including myocardial stunning, irreversible injury, and reperfusion arrhythmias. Free radical accumulation has been measured in ischemic and reperfused myocardium directly using techniques such as electron paramagnetic resonance spectroscopy and tissue chemiluminescence and indirectly using biochemical assays of lipid peroxidation products. Potential sources of free radicals during ischemia and reperfusion have been identified in myocytes, vascular endothelium, and leukocytes. In several different experimental models exogenous free radical-generating systems have been shown to produce alterations in cardiac function that resemble the various reperfusion abnormalities described above. Injury to processes involved in regulation of the intracellular Ca2+ concentration may be a common mechanism underlying both free radical-induced and reperfusion abnormalities. Direct effects of free radicals on each of the known Ca(2+)-regulating mechanisms of the cell as well as the contractile proteins and various ionic membrane currents have been described. Free radicals also inhibit critical enzymes in anaerobic and aerobic metabolic pathways, which may limit the metabolic reserve of reperfused myocardium and contribute to intracellular Ca2+ overload. Inhibiting free radical accumulation during myocardial ischemia/reperfusion with free radical scavengers and inhibitors has been demonstrated to reduce the severity of myocardial stunning, irreversible injury, and reperfusion arrhythmias in many, but not all, studies. This evidence strongly implicates free radical accumulation during myocardial ischemia/reperfusion as an important pathophysiological mechanism of reperfusion abnormalities, although many issues remain unresolved.     

Quantification of [Ca2+]i in perfused hearts. Critical evaluation of the 5F-BAPTA and nuclear magnetic resonance method as applied to the study of ischemia and reperfusion

Calcium has been implicated as a mediator of cell injury in ischemia and reperfusion, but direct measurements of Ca2+ are required to refine this idea. We used nuclear magnetic resonance spectroscopy and the Ca2+ indicator 5F-BAPTA to measure [Ca2+]i in perfused ferret hearts. Several lines of evidence are presented to show that loading with the acetoxymethyl ester of 5F-BAPTA is not significantly complicated by accumulation of partially de-esterified metabolites, compartmentalization into mitochondria, or disproportionate uptake into endothelial cells. During 20 minutes of total global ischemia at 30 degrees C, time-averaged [Ca2+]i increased significantly, reaching peak values roughly three times control at 15-20 minutes. Reperfusion resulted in a persistent elevation of [Ca2+]i during the first 5 minutes, but not afterward. Although the nonlinear response of 5F-BAPTA to [Ca2+] leads to underestimation of the true time-averaged [Ca2+]i, the measured alterations of intracellular Ca2+ homeostasis during ischemia are large compared with the likely errors in quantification. Phosphorus nuclear magnetic resonance spectroscopy of 5F-BAPTA-loaded hearts reveals changes during ischemia similar to those recorded previously in hearts not containing a Ca2+ indicator. Developed pressure recovers to only 50% of control values during reflow, indicating that the presence of 5F-BAPTA in the cytosol does not protect against stunning, at least when the extracellular calcium concentration has been raised to 8 mM. We conclude that 5F-BAPTA provides useful measurements that reveal that time-averaged [Ca2+]i rises during ischemia and returns to control levels soon after reperfusion.     

瞬时受体电位通道亚族M7样电流在小鼠心肌梗死后的变化及意义

目的 观察小鼠心肌成纤维细胞(CFs)在心肌梗死后瞬时受体电位通道亚族M7(TRPM7)样电流的变化及其对胶原生成的影响,探讨TRPM7离子通道在心脏纤维化形成中的潜在病理生理作用. 方法 (1)制备小鼠心肌梗死模型并分离CFs细胞;(2)培养传代CFs及小分子干扰RNA(SiRNA)技术感染;(3)应用膜片钳技术观察CFs缺血后TRPM7通道内外向电流特征;(4)钙荧光显像技术观察缺血心肌对CFs的钙离子内流影响;(5)测定缺血对CFs总胶原含量的影响.结果 (1)心肌缺血能使CFs的含钙离子内向电流较对照组显著增加,分别为(7.4±0.7)pA/pF和(16.25±1.7)pA/pF(P<0.05);(2)SiRNA使TRPM7样电流的mRNA水平明显下降,且电流幅值减少约90%;(3)心肌缺血组CFs的TRPM的mRNA丰度及总胶原含量较基础值增加2.3倍左右.结论 致纤维化因子心肌缺血可通过TRPM7样电流介导的钙离子信号机制调节CFs功能,在心肌组织纤维化的病理生理过程中发挥重要作用.     

再灌注损伤抢救激酶对小鼠缺血后适应心肌再灌注损伤中的减轻作用

目的 研究缺血后适应(IPC)对离体小鼠心肌缺血再灌注(I/R)损伤的作用及其影响因素,探讨再灌注损伤抢救激酶在IPC心肌保护中的作用.方法 建立Langendofff小鼠心肌I/R模型,全心缺血30 min后分为6组[(1)对照组,(2)3次IPC组(采取缺血10 s及再灌注10 s的3次IPC周期),(3)6次IPC组(采取缺血10 s及再灌注10 s的6次IPC周期),(4)延迟IPC组(恢复再灌注1 min后进行IPC),(5)IPC+PD98059组,(6)I/R+PD98059组],随后再灌注2 h;观察IPC对心脏血流动力学、心肌酶的释放、心肌超氧化物歧化酶活性和丙二醛的含量、梗死心肌范围的影响以及与细胞外信号调节激酶(ERK1/2)、磷脂酰肌醇3激酶-蛋白激酶B表达水平的关系.结果 与对照组比较,3次IPC组和6次IPC组小鼠心脏血流动力学显著改善,心肌酶释放减少,心肌丙二醛减少、超氧化物歧化酶活性增加,心肌梗死范围减小.6次与3次IPC周期的保护作用相似.而IPC作用在恢复再灌注1 min后消失.3次IPC组和6次iPC组心肌的ERK1/2磷酸化水平显著增高,蛋白激酶B磷酸化水平无明显变化.PD98059显著抑制IPC所致的ERK1/2的磷酸化,并能消除IPC对心肌的保护作用.结论 IPC能有效地减轻离体小鼠心肌缺血再灌注损伤,增加IPC的周期数并没有扩大保护作用,延迟IPC没有产生类似的保护作用.ERK1/2细胞信号途径参与介导IPC对离体心脏缺血再灌注心肌的保护作用.     

再灌注损伤挽救激酶和生存活化因子增强信号通路在心肌缺血再灌注损伤中的研究进展

经皮冠状动脉介入治疗、溶栓治疗可以使心肌梗死患者明显获益并且改善预后,但是由此引起的心肌缺血再灌注损伤问题也凸显出来,心肌缺血再灌注损伤涉及到多个靶点通路,不同信号通路之间的关系错综复杂。近几年医学家提出的再灌注损伤挽救激酶和生存活化因子增强两个促存活激酶信号通路成为了再灌注干预治疗的新靶点,从而成为心血管疾病甚至其他血管性疾病的新的切入点。本文拟阐述这两条信号通路对缺血再灌注后心肌保护的作用机制,为心肌梗死新药研发提供新思路。     

辛伐他汀保护缺血-再灌注损伤心肌的线粒体蛋白质组学研究

目的建立大鼠心肌缺血-再灌注损伤模型,通过蛋白质组学的方法研究辛伐他汀对大鼠缺血-再灌注损伤心肌线粒体代谢的保护作用。方法将大鼠随机分为辛伐他汀干预组(n=14)和生理盐水对照组(n=14),建立缺血-再灌注损伤模型,通过伊文思蓝和TTC染色评估梗死面积,提取大鼠左心室心肌线粒体蛋白行双向凝胶电泳,应用质谱分析鉴定差异蛋白点。结果辛伐他汀组和对照组相比,梗死区与危险区(梗死区+缺血区)的比值有统计学差异(29.4%±8.4%vs57.7%±6.5%,P0.0001);梗死面积与左室面积的比值有统计学差异(15.9%±5.6%vs29.0%±8.9%,P=0.012)。双向凝胶电泳图谱分析有19个蛋白点的表达有差异,质谱鉴定了9种差异蛋白,相比对照组,辛伐他汀组4个蛋白表达上调:三功能酶亚基α(线粒体前体)、电子转移黄素蛋白脱氢酶、肌动蛋白α(心肌)、细胞色素c氧化酶亚基5A亚单位(线粒体前体);5个蛋白表达下调:L-乳酸脱氢酶B链、异柠檬酸脱氢酶[NAD]α亚基(线粒体前体)、α晶状体蛋白B链、内膜蛋白(线粒体)、肌动蛋白类似物(细胞质)。结论辛伐他汀组大鼠心肌在缺血-再灌注损伤后,心肌梗死面积显著减少,辛伐他汀改变线粒体呼吸链、能量代谢等途径上的蛋白,为阐明辛伐他汀保护缺血再灌注损伤提供了理论依据。     

Role of cardiac renin angiotensin system in ischemia reperfusion injury and preconditioning of heart

Cardio-vascular diseases are the leading cause of morbidity and mortality. Ischemia is a state of oxygen deprivation in tissues, whereas reperfusion is restoration of blood flow in ischemic tissues. Myocardial damage of tissue during reperfusion after ischemic insult is known as myocardial ischemia–reperfusion (I/R) injury. It induces damage to cardiac muscle via increasing expression of oxygen, sodium and calcium ions which are responsible in the activation of proteases and cell death. Heart renin angiotensin system (RAS) plays an important role in the myocardial ischemia and reperfusion injury. Angiotensin (1–7) is responsible for vasodilation and angiotensin II for vasoconstriction. Here-in we reviewed how myocardial I/R injury sets in by up-regulation of angiotensin II that leads to increased infarct size, which can be reduced by the use of ACE inhibitors, ACE2 activators and angiotensin II antagonist.     

Na+/Ca++ exchanger and myocardial ischemia/reperfusion

Myocardial hypoxia and ischemia are characterized by the depletion of ATP and the development of intracellular acidosis, which alter cellular ionic homeostasis. Specifically, elevated cytosolic free Ca++ concentrations cause cellular injury during hypoxia/ischemia and lead to irreversible myocardial damage during reoxygenation/reperfusion. An increase in the intracellular Na+ concentration has been shown to correlate with Ca++ overload. Although inhibition of Na+/K+ exchange because of decreased ATP production may be involved, it is more likely that intracellular acidosis drives Na+ into the cells via Na+/H+ exchange. Experimental evidence supports the notion that Na+/H+ exchange is primarily responsible for Na+ influx during hypoxia/ischemia. The accumulation of intracellular Na+ may then activate the Na+/Ca++ exchanger causing Ca++ overload. Therefore, the Na+/Ca++ exchanger plays a crucial role in cellular injury during hypoxia/ischemia and in cell death during reoxygenation/reperfusion. In the past few years, the Na+/Ca++ exchanger has been cloned and the structure/function relationship studied intensively. Agents which inhibit the Na+/Ca++ exchanger may have therapeutic potential for the treatment of ischemic heart disease. These advances will greatly accelerate the understanding of the cellular and molecular mechanisms underlying the role of the Na+/Ca++ exchanger in the development of myocardial damage during hypoxia/ischemia and reoxygenation/reperfusion.     

基因治疗在心肌缺血再灌注损伤中的研究进展

通过溶栓和经皮冠状动脉血管成形术达到再灌注是即将发生急性心肌梗死的标准处理措施。尽管缺血区的血流复灌对心肌的挽救具有重要意义,再灌注本身可能导致缺血损伤基础上的进一步心肌损伤。目前对心肌缺血再灌注损伤分子和细胞机制的更深理解以及心血管系统基因操作工具的利用,为基因治疗提供了可能性。为了提高人类血管基因治疗的有效性和安全性,对基因转导载体,基因递送技术和有效治疗基因的鉴定等方面的基础研究是非常必要的。     

Alterations in phospholipid metabolism in the globally ischemic rat heart: emphasis on phosphoinositide specific phospholipase C activity

The effect of global ischemia on myocardial ventricular membrane phospholipids was evaluated using a modified Langendorff preparation. Isolated rat hearts were perfused at 37 degrees C with oxygenated Krebs Ringer solution or rendered ischemic by cessation of perfusion (10 min to 3 h). Longer periods of ischemia were assessed by incubating preperfused (10 min) intact hearts in non-oxygenated Krebs (37 degrees C) for 6 to 18 h. Ischemia-induced alterations in phosphatidylinositol levels and phosphoinositide-specific phospholipase C (PI PLC) activity were assessed in detail, since inositol phospholipids and PI-PLC play putative roles in the regulation of cell function and Ca2+ homeostasis. Decreases in major membrane phospholipids (phosphatidylcholine, phosphatidylserine, cardiolipin and sphingomyelin) were demonstrated after long ischemic periods (6 to 18 h). While periods of ischemia (3 h or less) induced no change in structural phospholipids, an elevation in lysophosphatidylcholine and free fatty acids was found by 1 h. Notably a significant increase in phosphatidylinositol content and an accompanying decrease in cytosolic PI PLC activity was detected by 30 mins of ischemia. Reduced enzymic activity was not due to altered in vitro activation or deactivation of PI-PLC, to a change in the Ca2+ requirement of the enzyme, or to translocation of the enzyme from the cytosol to a membrane fraction. The isolated rat heart made globally ischemic for 30 mins under conditions described for this investigation shows signs of irreversible injury i.e. increased cell Ca2+ content and inability to initiate and maintain rhythmic contraction upon reperfusion. Therefore, it is possible that altered phosphoinositide metabolism may contribute to the evolution of ischemia-elicited irreversible cell injury.     

Postconditioning: a mechanical maneuver that triggers biological and molecular cardioprotective responses to reperfusion

Infarct size is determined not only by the duration and severity of ischemia, but also by pathological processes initiated at reperfusion (reperfusion injury). Numerous pharmacological strategies have been reported which administer drugs at or just before the onset of reperfusion, with subsequent salubrious effects, notably a reduction in infarct size. However, few if any of these strategies have become standard of care in the catheterization laboratory setting. Postconditioning, defined as repeated brief cycles of reperfusion interrupted by ischemia (or hypoxia) applied at the onset of reperfusion, was recently introduced as a mechanical strategy to attenuate reperfusion injury. Postconditioning intervenes only during the first few minutes of reperfusion. However, it reduces endothelial activation and dysfunction, the inflammatory response to reperfusion, necrosis, and apoptosis both acutely and long-term. Cardioprotection has been demonstrated by multiple independent laboratories and in multiple species. Postconditioning stimulates G-protein coupled receptors by their cognate endogenously released ligands and surprisingly activates survival kinases that may converge on mitochondrial K_ATP channels and the permeability transition pore. Postconditioning has been shown in two clinical studies to reduce infarct size in patients undergoing percutaneous coronary intervention in the catheterization laboratory, and at least five other studies are in some phase of implementation. This significant reduction in infarct size has implications for reduction in heart failure as a consequence of myocardial infarction, but this link has yet to be demonstrated. The salubrious effects of postconditioning are an indirect validation of the experimental and clinical importance of reperfusion injury in the setting of coronary artery occlusion.     

Specific inhibition of the mitochondrial permeability transition prevents lethal reperfusion injury

The aim of the present study was to determine whether specific inhibition of mitochondrial permeability transition (MPT) by NIM811 at the time of reperfusion following acute myocardial infarction may protect the heart. MPT pore opening appears to be a pivotal event in cell death following acute myocardial infarction. Recently, MPT pore opening has been involved in ischemic preconditioning. In protocol 1, NZW rabbits underwent either no intervention (sham) or 10 min of ischemia followed by 5 min of reperfusion, preceded (preconditioned, PC) or not (control, C) by 5 min of ischemia and 5 min of reperfusion. Additional rabbits were treated by cyclosporin A (CsA) or its non-immunosuppressive and more specific derivative (NIM811) (10 mg kg(-1), IV bolus), either 10 min before ischemia or 1 min before reperfusion. Hearts were excised and mitochondria isolated for further assessment of Ca(2+)-induced MPT. In protocol 2, animals were randomly assigned into similar experimental groups and underwent 30 min of ischemia and 4 h of reperfusion. Infarct size was assessed by TTC staining, and apoptosis by TUNEL assay. The Ca2+ overload required to induce MPT pore opening was significantly higher in NIM811, CsA and PC groups than in controls. Both necrotic and apoptotic cardiomyocyte death were significantly reduced by NIM811, CsA and PC. In both protocols, administration of NIM811 at reperfusion provided full protection. These data indicate that specific inhibition of MPT pore opening at reperfusion following acute myocardial infarction provides a powerful antinecrotic and antiapoptotic protection.     

Protection by verapamil of mitochondrial glutathione equilibrium and phospholipid changes during reperfusion of ischemic canine myocardium

Pretreatment of the ischemic myocardium with verapamil protects against mitochondrial respiratory depression observed during ischemic arrest as well as during reperfusion. Since ischemic mitochondrial function appears not to be altered further by reperfusion, the purpose of this study is to identify a biochemical event affecting mitochondria that is specifically associated with reperfusion injury. It has been proposed that increased cellular Ca2+ influx and oxygen toxicity may result from reintroduction of coronary flow. Increased cytosolic Ca2+ is transmitted to the mitochondria with subsequent activation of Ca2+-dependent events, including phospholipase A2. Net production of lysophospholipids (and loss of total diacylphospholipids from the mitochondria) will proceed when reacylation mechanisms are inhibited. Since acyl-CoA:lysophospholipid acyltransferase is a sulfhydryl-sensitive enzyme and since increased activity of glutathione peroxidase shifts the levels of the mitochondrial sulfhydryl buffer, glutathione, towards oxidation, levels of glutathione and its oxidation state were measured during reperfusion in the absence or presence of verapamil pretreatment. Ischemia lowers total glutathione and reduces the redox ratio (reduced glutathione: oxidized glutathione) by 85%. Reperfusion partially returns the redox ratio to control by causing oxidized glutathione to disappear from the matrix. Verapamil maintains both the concentration and the redox potential of glutathione at control levels. Concomitant with alterations in reduced glutathione:oxidized glutathione is a decrease in ischemic mitochondrial phospholipid content. During reperfusion, phosphatidylethanolamine and its major constituent fatty acids (C 18:0 and C 20:4) are specifically lost from the mitochondrial membrane. Accompanying the significant loss of arachidonic acid during reperfusion is the decreased content of 11-OH, 12-OH, and 15-OH arachidonate. These lipid peroxidation products are not increased in ischemia. It is proposed that oxidation of matrix glutathione to glutathione disulfide during ischemia results in formation of glutathione-protein mixed disulfides and inhibition of sulfhydryl-sensitive proteins, including acyl-CoA lysophosphatide acyltransferase. Thus, metabolic events occurring within the ischemic period set the stage for prolonged dysfunction during reperfusion.     

Effects of cardioplegic arrest and reperfusion on rabbit cardiac ryanodine receptors

Calcium overload is considered to be a primary contributor to ischemia-reperfusion injury. Cardiac sarcoplasmic reticulum (SR), the main regulator of intracellular Ca2+ concentration under normal conditions, is a target for ischemic myocardial injury. The ryanodine receptor (RyR) is the SR Ca2+ release channel. Previous reports have shown that a reduction in RyR activity during global myocardial ischemia correlates with concomitant myocardial dysfunction. Crystalloid cardioplegia, a technique for myocardial protection during heart operations, reduces Ca2+ accumulation during global ischemia. Hence, the effects of cardioplegia on RyR in isolated rabbit hearts was investigated. The study also compared [3H] ryanodine binding before ischemia (control group), after 30 min of ischemia (either global ischemia (GI group) or cardioplegic arrest (CA group)), and after 20 min of reperfusion. The GI group, but not the CA group, showed a significant reduction in the maximum number of binding sites (Bmax) for RyR compared with the control group (Control vs GI group: after ischemia, 1.33+/-0.27 vs 0.83+/-0.12 pmol/mg protein, p<0.05; after reperfusion, 1.33+/-0.27 vs 0.80+/-0.08 pmol/mg protein; p<0.05). CA group: after ischemia, 1.22+/-0.20 pmol/mg protein; after reperfusion, 1.15+/-0.28 pmol/mg protein). The affinity (Kd) values for [3H] ryanodine binding were not different among the 3 groups at any point. The preservation of RyR numbers during cardioplegia correlated with the concomitant preservation of cardiac functions. The results indicate that number of functional RyR was much better preserved during cardioplegia than during global ischemia. It is postulated that cardioplegia-induced protection of cardiac RyR may result in the protection of SR function during ischemia-reperfusion.