Role of Caspase-9 in Heart

描述(由申请人提供) 这项研究项目的长期目标是确定 心中的caspase-9。细胞凋亡是一个受调控的细胞删除过程，它具有 与各种心血管疾病有关。因此，具体来说 以减少心脏细胞凋亡为目标的治疗很可能会有 潜在的治疗益处。半胱氨酸家族成员caspase-9 蛋白水解酶在细胞凋亡的调控中起着至关重要的作用。在我们的 初步研究表明，复氧是一种强烈的诱导剂。 成人心肌细胞的凋亡及复氧诱导的细胞凋亡 涉及线粒体介导的途径，与激活有关 Caspase-9。这一结果表明caspase-9是一种重要的调节因子。 抑制caspase-9可有效抑制细胞凋亡。 心肌细胞凋亡。然而，细胞凋亡的分子机制目前尚不清楚。 人们对心肌细胞知之甚少。此外，我们观察到高水平的caspase-9 在成年心肌细胞中的蛋白表达，提示可能存在 Caspase-9在成人心脏中的重要作用。然而，人们对此知之甚少。 Caspase-9在心脏中的作用及其激活机制。 我们的假设是caspase-9是一种重要的复氧诱导的 成人心肌细胞的凋亡和对caspase-9的抑制将是一种 体外和体内抑制心肌细胞凋亡的有效策略。 为了检验这一假设，在具体目标1中，我们将调查 Caspase-9及其在诱导心肌细胞凋亡过程中的相互作用。我们 将作为我们的研究对象研究复氧诱导的成年心肌细胞凋亡 心肌细胞凋亡模型。研究caspase-9及其调控的作用 对于其相互作用，我们将产生并分析重组腺病毒以 Caspase-9在成人中的过表达及其各种突变型 心肌细胞。在具体目标2中，我们将研究几种策略，以 抑制caspase-9活性，包括药理上的caspase-9抑制， Caspase-9的主要负性形式过表达，过度表达 内源性caspase-9抑制物和抗凋亡Akt的过表达。 最后，在特定的目标3中，我们将定义caspase-9在心脏中的作用 通过产生心脏特异的caspase-9基因敲除小鼠。自从caspase-9 同源重组基因敲除导致围产儿死亡 Caspase-9在成人组织中的作用还无法研究。因此，心脏 使用Cre-loxP技术对caspase-9进行特定的敲除使我们能够检查 CAPase-9在成年动物中的组织特异性作用。

DESCRIPTION (provided by applicant) The long-term goal of this research project is to characterize the roles of caspase-9 in heart. Apoptosis is a regulated cell deletion process that has been implicated in various cardiovascular diseases. Therefore, specific therapies targeted toward attenuating cardiac apoptosis most likely will have potential therapeutic benefit. Caspase-9, a member of a family of cysteine proteases, plays a critical role in the regulation of apoptosis. In our preliminary study, we showed that-reoxygenation is a strong inducer of apoptosis in adult cardiomyocytes, and reoxygenation-induced apoptosis involves the mitochondria-mediated pathway associated with the activation of caspase-9. This result suggests that caspase-9 is an important regulartor of apoptosis, and that the inhibition of caspase-9 could effectively inhibit cardiomyocyte apoptosis. However, the molecular mechanism of apoptosis in cardiomyocyte is poorly understood. Furthermore, we observed high caspase-9 protein expression in the adult cardiomyocytes, suggesting a possible important role of caspase-9 in adult heart. Yet, very little is known about the role of caspase-9 in heart and the mechanism involved in its activation. Our hypothesis is that caspase-9 is an important reoxygenation-induced apoptosis in adult cardiomvocytes, and the inhibition of caspase-9 will be an effective strategy to attenuate cardiac apoptosis in vitro and in vivo. To test this hypothesis, in Specific Aim 1, we will investigate the role of caspase-9 and its interactions during the induction of cardiac apoptosis. We will study reoxygenation-induced apoptosis in adult cardiomyocytes as our model of cardiac apoptosis. To study the effect of caspase-9 and modulation of its interaction, we will generate and analyze recombinant adenoviruses to overexpress caspase-9 and various mutant forms of caspase-9 in adult cardiomyocytes. In Specific Aim 2, we will examine several strategies to inhibit caspase-9 activity including, pharmacological caspase-9 inhibition, overexpression of the dominant negative form of caspase-9, overexpression of endogenous caspase-9 inhibitor, and overexpression of anti-apoptotic Akt. Finally, in Specific Aim 3, we will define the role of caspase-9 in heart in vivo by generating cardiac-specific caspase-9 knockout mice. Since caspase-9 knock-out by homologous recombination results in perinatal lethality, the effect of caspase-9 in adult tissue can not be studied. Thus, the cardiac specific knockout of caspase-9 using Cre-loxP technology allows us to examine the tissue-specific role of capase-9 in adult animal.