Mitochondria and Cardiac Cell Death

描述(申请人提供)：在缺血/再灌流期间导致坏死和细胞凋亡的一个中心机制被认为是线粒体通透性转换(MPT)，这是由于线粒体内膜上的通透性转换孔(PTP)开放。根据我们最近的工作，我们假设。在这个修订的应用中，两个可分离的成分使线粒体在缺氧/复氧过程中容易受到损伤。MPT阈值分量与缺氧期或缺血期最相关，并设置再灌注期间MPT的阈值。它表现为进行性的MPT非依赖性细胞色素c丢失和内膜渗漏，这可归因于长链脂肪酸(FA)和活性氧(ROS)的积累。MPT触发成分与再灌流的相关性最大。再灌注期间是否发生MPT取决于复氧过程中存在的MPT诱导剂/抑制物(特别是基质游离钙水平)和线粒体膜电位再生的电子传递能力(Delapsim)之间的相互作用，后者反过来又取决于细胞色素c含量和内膜泄漏。与其已知的心脏保护作用一致，我们发现mitoKATP通道激动剂二氮嗪对MPT阈值和MPT触发成分都有保护作用，这种保护作用可被mitoKATP拮抗剂5-HD阻断。此外，PKC epsilon是心脏保护中的关键信号成分，对MPT触发成分具有保护作用。本研究的目的是进一步探讨在与缺血/再灌注相关的条件下保护线粒体免受MPT阈值和MPT触发成分影响的信号转导途径。我们的战略是将功能研究与蛋白质组学分析结合起来。功能研究将使用荧光光谱、成像(荧光、共聚焦和高压电子显微镜)和腺病毒基因转移技术在三个水平上研究线粒体和心脏保护：分离的线粒体、通透性心肌细胞中的原位线粒体和分离的心肌细胞。蛋白质组学分析将剖析受保护和未受保护的完整心脏中与PKCepsilon和PTP成分相关的线粒体蛋白质复合体。使用这一综合方法，我们将1)进一步表征缺血/再灌流元件促进MPT阈值和触发成分的机制，以及mitoKATP通道激动剂如何起到保护作用；2)确定异构体特异性PKC信号在保护MPT阈值和触发成分中的作用；3)研究与心脏保护有关的其他信号通路是否调节对MPT阈值和MPT触发成分的敏感性。4)利用功能蛋白质组学方法，在未保护和保护的心脏中鉴定与PKCepsilon和已知的PTP组分形成多蛋白信号复合体的蛋白质，以及5)表征缺氧/复氧诱导的潜伏期去极化波，以确定它们与细胞色素c释放和MPT的关系，以及它们对mitoKATP激活和心脏保护信号通路的反应。

DESCRIPTION (provided by applicant): A central mechanism leading to necrosis and apoptosis during ischemia/reperfusion is believed to be the mitochondrial permeability transition (MPT), due to permeability transition pore (PTP) opening in the inner mitochondrial membrane. Based on our recent work, we hypothesize. In this revised application that two separable components predispose mitochondria to injury during anoxia/reoxygenation. The MPT threshold component is most relevant to the anoxic or ischemic period, and sets the threshold for MPT during reperfusion. It is manifested as progressive MPT-independent cytochrome c loss and inner membrane leakiness, which can be attributed to accumulation of long chain fatty acids (FA) and reactive oxygen species (ROS). The MPT trigger component is most relevant to reperfusion. Whether MPT occurs during reperfusion is determined by the interplay between MPT inducers/inhibitors present during rexoygenation (particularly matrix free Ca levels) and electron transport capacity for regenerating mitochondrial membrane potential (deltapsim),which in turn depends on cytochrome c content and inner membrane leak. Consistent with its known cardioprotective role, we find that mitoKATP channel agonist diazoxide protects against both the MPT threshold and MPT trigger components, and that this protection is blocked by mitoKATP antagonist 5-HD. In addition, PKC epsilon, a key signaling component in cardioprotection, protects against the MPT trigger component. The objective of this proposal is to further explore the signal transduction pathways protecting mitochondria from the MPT threshold and MPT trigger components under conditions generally relevant to ischemia/reperfusion. Our strategy is to integrate functional studies with proteomics analysis. Functional studies will use spectrofluorometric, imaging (fluorescent, confocal and high voltage electron microscopy), and adenoviral gene transfer techniques to study mitochondria and cardioprotection at three levels: isolated mitochondria, in situ mitochondria in permeabilized myocytes, and isolated myocytes. Proteomic analysis will dissect mitochondrial protein complexes associated with PKCepsilon and PTP components in protected and unprotected intact hearts. Using this integrated approach, we will 1) further characterize the mechanisms by which ischemic/reperfusion elements promote the MPT threshold and trigger components, and how mitoKATP channel agonists are protective; 2) define the roles of isoform-specific PKC signaling in protection against the MPT threshold and trigger components; 3) examine whether other signaling pathways implicated in cardioprotection modulate susceptibility to the MPT threshold and MPT trigger components. 4) identify, using functional proteomics, the proteins forming multiprotein signaling complexes with PKCepsilon and known PTP components in unprotected and protected hearts, and 5) characterize deltapsim depolarization waves induced by anoxia/reoxygenation to define their association with cytochrome c release and MPT and their responsive to mitoKATP activation and cardioprotective signaling pathways.