REGULATION OF MITOCHONDRIAL PERMEABILITY TRANSITION IN ISCHEMIC INJURY

主要研究者/项目负责人（最后、第一、中间）：Ping，Peipei（韦斯，项目1） 本计划项目申请的主题是了解信号转导通路介导的 使用结合生物物理学、生理学、蛋白质组学和遗传学的多学科方法进行心脏保护。 项目1聚焦于这一主题的中心原则，即心脏保护信号汇聚于保护心脏。 通过阻止线粒体通透性转换（MPT）来抑制线粒体膜电位。项目1将突出 MPT的两种不同组分使线粒体在缺氧/复氧期间易于损伤。的MPT 启动成分与缺氧或缺血期最相关，并启动线粒体进行缺氧或缺血。 再灌注期间的MPT。MPT启动成分表现为进行性MPT非依赖性内源性启动成分， 线粒体膜（IMM）质子泄漏、基质浓缩和重塑以及细胞色素c动员 并通过长链脂肪酸（FA）和活性氧（ROS）的积累而释放。的 MPT触发成分与复氧或再灌注阶段最相关。MPT是否发生在 再灌注由复氧期间MPT诱导剂和抑制剂之间的相互作用决定 （特别是基质游离Ca水平）和再生线粒体膜的电子传递能力 电位（Av|/m），这又取决于细胞色素c含量和IMM质子泄漏。 基于先前的研究，证明了mitoKATp通道和PKCe在调节这些细胞中的作用， 项目1将进一步探索保护线粒体免受MPT的信号转导途径 在通常与缺血/再灌注相关的条件下，通过整合功能性 蛋白质组学分析。与心脏生物学核心合作，功能研究将使用 荧光分光光度法、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，原位 透化的肌细胞和分离的肌细胞中的线粒体。与项目2、项目3合作， 蛋白质组学核心和心脏生物学核心，蛋白质组学分析将剖析线粒体蛋白质复合物 与电压依赖性阴离子通道（VDAC，MPT孔组分）、PKCe和新的 受保护和未受保护的完整心脏中的线粒体蛋白磷酸酶PP 2CK。使用该集成 方法，项目1将（1）进一步表征缺血/再灌注元素促进 MPT启动和触发组件，以及mitoKATp通道激动剂在这种情况下如何保护;（2） 确定关键线粒体亚蛋白质组在保护免受MPT引发和触发组分的作用; (3)表征缺氧/复氧诱导的Avym去极化波，以确定其与 细胞色素c释放和MPT及其对mitoKATp激活和心脏保护信号的响应 途径。

Principal Investigator/Program Director (Last, First, Middle): Ping, Peipei (Weiss, Project 1) The theme of this Program Project application is to understand the signal transduction pathways mediating cardioprotection using a multidisciplinary approach that combines biophysics, physiology, proteomics and genetics. Project 1 focuses on a central tenet of this theme, that cardioprotective signaling converges on protection of mitohchondria by preventing the mitochondrial permeability transition (MPT). Project 1 will characterize the role of two distinct components of MPT to predispose mitochondria to injury during anoxia/reoxygenation. The MPT priming component is most relevant to the anoxic, or ischemic, period and primes the mitochondria to undergo MPT during reperfusion. The MPT priming component manifests as progressive MPT-independent inner mitochondrial membrane (IMM) proton leak, matrix condensation and remodeling, and cytochrome c mobilization and release and is promoted by accumulation of long chain fatty acids (FA) and reactive oxygen species (ROS). The MPT trigger component is most relevant to the reoxygenation, or reperfusion, phase. Whether MPT occurs during reperfusion is determined by the interplay between MPT inducers and inhibitors present during rexoygenation (particularly matrix free Ca levels) and electron transport capacity for regenerating mitochondrial membrane potential (Av|/m), which in turn depends on cytochrome c content and IMM proton leak. Building upon previous studies demonstrating a role of mitoKATp channel and PKCe in modulating these components, Project 1 will further explore the signal transduction pathways protecting mitochondria from the MPT priming and trigger components under conditions generally relevant to ischemia/reperfusion by integrating functional studies with proteomic analyses. In collaboration with the Heart Biology Core, the 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. In collaboration with Project 2, Project 3, the Proteomic Core, and the Heart Biology Core, the proteomic analyses will dissect mitochondrial protein complexes associated with the voltage-dependent anion channel (VDAC, an MPT pore component), PKCe and the novel mitochondrial protein phosphatase PP2CK in protected and unprotected intact hearts. Using this integrated approach, Project 1 will (1) further characterize the mechanisms by which ischemic/reperfusion elements promote the MPT priming and trigger components, and how mitoKATp channel agonists are protective in this setting; (2) define the roles of key mitochondrial subproteomes in protection against the MPT priming and trigger components; (3) characterize Avym depolarization waves induced by anoxia/reoxygenation to define their association with cytochrome c release and MPT and their responsiveness to mitoKATp activation and cardioprotective signaling pathways.