Mitochondrial pathways in NO induced cardioprotection

描述(由申请人提供)： 过去几十年的研究极大地促进了我们对心肌缺血损伤保护和发病机制的理解。越来越多的人认识到线粒体功能的保护在抗缺血再灌注损伤(I/R)中的关键作用。然而，对于线粒体中谁是心肌保护的分子靶点；哪些特定的分子事件导致线粒体的保护；以及线粒体上是否存在心脏保护信号的系统整合以支持受保护表型的表现，仍然是未知的。使用一氧化氮(NO)诱导的心肌保护晚期阶段的小鼠模型，我们选择使用一种新的实验策略来研究线粒体可信的内在信号特性，该策略能够通过计算模型并行检查线粒体信号、线粒体蛋白质组和线粒体行为。所提出的研究是基于他人和我们自己的初步证据，证明在NO供体处理的小鼠中存在PKC？-Src模块的激活，并且两者都定位于线粒体膜。在这项提案中，我们将检验这一创新假设，即PKC？-Src模块与短暂的MPTP开放相互作用，以保护心肌细胞免受钙超载损伤。工作假说是，NO激活PKC？-Src模块，导致短暂的MPTP开放，导致一过性钙释放和活性氧(ROS)爆发，从而抑制钙再摄取，进而激活PKC？-Src形成前馈环路。当动态平衡被破坏时(例如，钙超载或ROS升高)，短暂的MPTP开放转变为不可逆转的、持久的MPTP开放，这反而会导致心脏损伤。在这一应用中，我们建议描述MPTP短暂开放对钙处理和ROS产生的功能影响，并阐明线粒体PKC？-Src模块在NO诱导的心脏保护晚期阶段对MPTP的调节(目标1)。在特定的目标2中，我们将最终确定激活线粒体中的PKC？-Src信号模块作为NO诱导的心肌缺血损伤保护心肌的强制性信号元件。最后，我们将系统地确定线粒体Src-Kinase在NO诱导的心肌保护晚期的分子靶点(目标3)。这些研究将促进我们对心脏生物学的理解，为短暂的MPTP开放与线粒体PKC？-Src模块的相互作用如何在介导NO诱导的心脏保护的晚期阶段提供新的机制见解。(摘要结束)

DESCRIPTION (provided by applicant): Investigations in the past decades have significantly advanced our understanding of signaling mechanisms underlying the protection and pathogenesis of myocardial ischemic injury. It is increasingly recognized that preservation of mitochondrial function plays a pivotal role in cardioprotection against ischemia reperfusion injury (I/R). However, it remains virtually unknown as to who the molecular targets of cardioprotection are in the mitochondria; what specific molecular events led to the protection of mitochondria; and whether there is a systems integration of cardioprotective signaling at the mitochondria to support the manifestation of a protected phenotype. Using a murine model of nitric oxide (NO) induced late phase of cardioprotection, we elect to examine the plausible intrinsic signaling properties of mitochondria using a novel experimental strategy enabling a parallel examination of mitochondrial signaling, mitochondrial proteomes, and mitochondrial behavior by computational modeling. The proposed studies are based upon preliminary evidence by others and our own demonstrating that activation of PKC?-Src module occurs in the NO donor treated mice and that both are localized to mitochondrial membranes. In this proposal we will test the innovative hypothesis that the PKC?-Src module interacts with the brief mPTP openings to protect cardiomyocytes from Ca++ overload induced jury. The working hypothesis is that NO activates PKC?-Src module, leading to brief mPTP openings which results in transients Ca++ releases and reactive oxygen species (ROS) bursts, and consequently inactivates Ca++ reuptake and further activates PKC?-Src to form a feed- forward loop. When the homeostasis is interrupted (e.g., calcium overload or elevated ROS), brief mPTP openings transits into irreversible, long-lasting mPTP openings, which instead induce cardiac injury. In this application we propose to delineate the functional effects of brief openings of mPTP on Ca++ handling and ROS production; and to elucidate mPTP regulation by the mitochondrial PKC?-Src module in the setting of NO-induced late phase of cardioprotection (Aim 1). In the specific Aim 2 we will conclusively establish the activation of a PKC?-Src signaling module in the mitochondria as a mandatory signaling element of NO-induced cardioprotection against myocardial ischemic injury. At last we will systematically define the molecular targets of mitochondrial Src-kinase in NO- induced late phase of cardioprotection (Aim 3). The proposed studies will advance our understanding of cardiac biology by providing novel mechanistic insights into how interactions of brief mPTP openings with mitochondrial PKC?-Src module can be beneficial in mediating NO-induced late phase of cardioprotection. (End of Abstract)