Mechanism of Preconditioning and Cardiocyte Apoptosis

心肌细胞凋亡在许多与缺血再灌注（IR）损伤相关的心脏疾病中已被证实。预处理，单或多个短暂的IR期之前，长时间的冠状动脉闭塞，减少心肌梗死面积和细胞凋亡的IR。本建议的目标是阐明细胞内信号通路预处理防止心肌细胞凋亡。预处理产生的氧自由基与IR中的心肌保护相关。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，预处理产生了许多与IR相关的效应，并激活了心肌细胞中的一系列信号转导级联。内源性心脏保护的作用机制尚不清楚。我们假设，在预处理过程中，氧自由基开放线粒体KATP通道通过PKCe激活是主要的信号转导途径，阻止IR中的细胞凋亡。为了验证这一假设，我们将直接在体外新生大鼠心肌细胞中测量氧自由基的产生。我们将使用重组腺病毒载体在心脏细胞中过表达携带全长cDNA的野生型PKCe（AdCMVPKCe-FL）或显性负性PKCe突变体（AdCMVPKCe-DN）。这些结构对心脏细胞功能，细胞凋亡和梗死的影响将在体外和体内IR大鼠模型中进行研究。了解预处理阻断心脏细胞凋亡的特定信号通路的作用，并通过体细胞基因转移对通路进行局部调节，可能为许多临床疾病的管理提供新的治疗方法。

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.