Mechanism of Preconditioning and Cardiocyte Apoptosis

在许多与缺血再灌注(IR)损伤相关的心脏疾病中都发现了心肌细胞的凋亡。预适应，在冠状动脉长时间闭塞前单次或多次短暂的IR，可减少IR的心肌梗死范围和细胞凋亡。这项提议的目的是阐明细胞内信号通路，通过这些信号通路，预适应可以阻止心肌细胞的凋亡。在IR中，预适应产生与心脏保护相关的氧自由基。蛋白激酶C(PKC)和KATP通道是氧自由基的下游信号。然而，在完整的动物中，预适应产生了许多与IR相关的效应，并激活了心肌细胞中的一系列信号转导通路。内源性心脏保护的哪种作用尚不清楚。我们推测，在预适应过程中，氧自由基通过激活PKCE开放线粒体KATP通道是阻断IR细胞凋亡的主要信号转导途径。为了验证这一假设，我们将直接测量体外培养的新生大鼠心肌细胞中氧自由基的产生。我们将使用重组腺病毒载体在心肌细胞中过表达携带其全长cDNA的野生型PKCE(AdCMVPKCe-FL)或显性负性PKCE突变体(AdCMVPKCe-Dn)。这些结构对心肌细胞功能、细胞凋亡和心肌梗死的影响将在体外和体内的IR大鼠模型中进行检验。了解预适应阻断心肌细胞凋亡的特定信号通路的作用，并通过体细胞基因转移发展对这些通路的局部调控，可能为许多临床疾病的治疗提供新的治疗方法。

Cardiocyte apoptosis has been identified in many cardiac conditions associated with ischemia-reperfusion (IR) injury. Preconditioning, single or multiple brief periods of IR before a prolonged coronary artery occlusion, reduced myocardial infarct size and apoptosis in IR. The goal of this proposal is to elucidate intracellular signaling pathways by which preconditioning prevents cardiocyte apoptosis. Preconditioning generates oxygen radicals that correlate with cardioprotection in IR. Protein kinase C (PKC) and KATP channels are downstream signals of oxygen radicals. However, preconditioning produces many effects relevant to IR in intact animals and activates a series of signal transduction cascades in cardiocytes. Which effect accounts for endogenous cardioprotection remains unclear. We hypothesize that during preconditioning oxygen radicals open mitochodrial KATP channels through PKCe activation is the primary signal transduction pathway that blocks apoptosis in IR. To test this hypothesis, we will measure oxygen radical production directly in in vitro neonatal rat cardiomyocytes. We will use recombinant adenoviral vectors to overexpress in cardiocytes a wild type PKCe carrying its full length of cDNA (AdCMVPKCe-FL) or a dominant negative PKCe mutant (AdCMVPKCe-DN). The effects of these constructs on cardiocyte function, apoptosis, and infarction will be examined in both in vitro and in vivo rat models of IR. Understanding the role of specific signaling pathways by which preconditioning blocks cardiocyte apoptosis and developing local modulation of the pathways through somatic gene transfer may provide novel therapies for the management of many clinical disorders.