REGULATION OF MITOCHONDRIAL PERMEABILITY TRANSITION IN ISCHEMIC INJURY

首席调查员/项目主任(最后、第一、中间)：平，佩佩(魏斯，项目1) 本计划项目申请的主题是了解信号转导途径 利用结合生物物理学、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侧重于这一主题的一个中心原则，即心脏保护信号集中在对 防止线粒体通透性转变(MPT)。项目1将描述 MPT有两种截然不同的成分，使线粒体在缺氧/复氧过程中容易受到损伤。MPT 启动成分与线粒体将要经历的缺氧期和启动最相关 再灌注期MPT。MPT启动组件表现为渐进的MPT非依赖内部 线粒体膜质子泄漏、基质凝聚和重塑及细胞色素c动员 和释放，并通过积累长链脂肪酸(FA)和活性氧物种(ROS)来促进。这个 MPT触发成分与复氧期或再灌注期最为相关。MPT是否发生在 再灌注是由复氧过程中MPT诱导物和抑制物之间的相互作用决定的。 (特别是基质游离钙水平)和线粒体膜再生的电子传递能力 电势(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在调节这些过程中的作用 组件，项目1将进一步探索保护线粒体免受MPT影响的信号转导途径 在一般与缺血/再灌流相关的条件下通过整合功能启动和触发组件 用蛋白质组学分析进行研究。在与心脏生物学核心的合作下，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 线粒体移植技术在三个水平上研究线粒体和心脏保护：分离线粒体，原位 通透性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，以及心脏生物学核心，蛋白质组学分析将剖析线粒体蛋白质复合体 与电压依赖的阴离子通道(VDAC，MPT的孔成分)、PKCE和新型 保护和未保护的完整心脏中的线粒体蛋白磷酸酶PP2CK。使用此集成 方法，项目1将(1)进一步描述缺血/再灌注素促进 MPT启动和触发组件，以及mitoKATp通道激动剂在这种设置下如何保护；(2) 确定关键的线粒体亚蛋白质组在防御MPT启动和触发成分中的作用； (3)表征缺氧/复氧引起的Avym除极波，以确定它们与 细胞色素c释放和MPT及其对mitoKATp激活和心肌保护信号的反应性 小路。

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.