Mechanisms of Hypothermic Protection from Ischemia/Reperfusion Cardiac Injury

描述(申请人提供)：治疗性低温可以显著减轻复苏后的损伤，提高一些患者的存活率。在我们的全脑缺血的细胞和动物模型中，存在显著的复苏后心脏损伤。这一建议的中心假设是，在缺血期间启动的低温通过相互关联的途径保护心肌细胞和保护心功能，涉及一氧化氮(NO)和P53下调的产生，这两个因素作用于线粒体，以防止启动凋亡级联反应。利用已建立的常温和低温细胞和动物模型，我们提出了两个主要的具体目标：1)确定低温诱导的一氧化氮、活性氧自由基(ROS)的调节和线粒体在保护心肌细胞和心脏免受缺血/再灌注(L/R)损伤中的相互作用。我们假设，缺血内低温保护包括产生改变的ROS和反应氮物种(RNS)，允许缺血降低的线粒体膜电位的恢复，并阻止细胞色素c(Cytc)从线粒体释放。在这些研究中，我们将研究低温对特定氧化剂，如超氧化物和一氧化氮的诱导或调节，以及这些对启动线粒体凋亡途径的影响。2)确定线粒体Bcl2家族蛋白(特别是Bax和Bak)与其他以线粒体为靶点的蛋白(特别是P53)之间的相互作用，通过这些蛋白，低温诱导NO的产生提供保护，并阻止促凋亡的线粒体蛋白Cytc的释放和caspase的激活。我们推测，在I/R期间，P53的激活导致Bax和/或Bak的激活，低温阻断P53的激活，与Bax/Bak的相互作用，和/或易位将抑制这些相互作用，阻止Cytc的释放，保护细胞免受凋亡。建议的研究将阐明低温诱导保护心肌细胞的分子机制，包括“适应性”或保护性低温氧化剂的细节。了解低温信号影响线粒体特定的细胞凋亡途径的机制，将有助于开发低温、药物和基因治疗方法的最佳保护方案。

DESCRIPTION (provided by applicant): Therapeutic hypothermia can significantly attenuate post-resuscitation injury and improve survival in some patients. In our cellular and animal models of global ischemia, there is significant post-resuscitation cardiac injury. The central hypothesis of this proposal is that hypothermia initiated during ischemia protects cardiomyocytes and preserves cardiac function through interrelated pathways, involving the production of nitric oxide (NO) and p53 down-regulation, which act at the mitochondria to prevent initiation of the apoptotic cascade. Using a combination of established normothermic and hypothermic cellular and animal models, we propose two major specific aims: 1) Determine the interacting roles of hypothermia-induced nitric oxide, modulation of reactive oxygen species (ROS) and the mitochondrion in protecting cardiomyocytes and the heart from ischemia/reperfusion (l/R)-induced injury. We hypothesize that intra-ischemic hypothermic protection involves the generation of an altered ROS and reactive nitrogen species (RNS) profile, allowing recovery of ischemia-reduced mitochondrial membrane potential and blocking the release of cytochrome c (cyt c) from the mitochondria. In these studies, we will examine the induction or modulation of specific oxidants, such as superoxide and nitric oxide, by hypothermia and these effects on the initiation of the mitochondrial pathway of apoptosis. 2) Determine the interactions between mitochondrial Bcl-2 family proteins, specifically Bax and Bak, and other "mitochondria-targeted" proteins, specifically p53, through which hypothermia-induced NO generation provides protection and prevents release of pro-apoptotic mitochondrial proteins cyt c and activation of caspases. We hypothesize that activation of p53 during I/R leads to activation of Bax and/or Bak and hypothermic blockade of p53 activation, interactions with Bax/Bak, and/or translocation will inhibit these interactions, blocking the release of cyt c and protecting the cells from apoptosis. The proposed studies will elucidate the molecular mechanisms involved in the hypothermic induction of protection of cardiomyocytes, including details of the "adaptive" or protective hypothermic oxidant profile. An understanding of mechanisms by which specific mitochondrial pathways of apoptosis are affected by hypothermic signaling will aid in developing optimal protective protocols in hypothermic, pharmacological and genetic therapeutic approaches.