Role of Caspase-9 in Heart

描述（由申请人提供） 本研究项目的长期目标是描述 caspase-9在心脏 细胞凋亡是一种受调控的细胞缺失过程， 与多种心血管疾病有关。 因此，具体 针对减弱心脏细胞凋亡的治疗最有可能 潜在的治疗益处。 半胱氨酸蛋白酶家族成员Caspase-9 蛋白酶，在细胞凋亡的调节中起关键作用。 在我们 初步研究，我们表明，-复氧是一个强大的诱导剂， 成年心肌细胞凋亡和再氧诱导的凋亡 涉及与激活 半胱天冬酶-9。 这一结果表明，caspase-9是一个重要的调节剂， Caspase-9的抑制能有效抑制细胞凋亡， 心肌细胞凋亡 然而，细胞凋亡的分子机制， 对心肌细胞的了解很少。此外，我们观察到高caspase-9， 蛋白质表达的成年心肌细胞，这表明可能的 caspase-9在成人心脏中重要作用 然而，人们对 caspase-9在心脏中的作用及其激活机制。 我们的假设是，caspase-9是一个重要的复氧诱导的 抑制caspase-9的表达，将是一个新的研究方向。 在体内外减轻心肌细胞凋亡有效策略。 为了验证这一假设，在具体目标1中，我们将研究 caspase-9及其相互作用在诱导心肌细胞凋亡中的作用 我们 将研究再氧诱导的成年心肌细胞凋亡， 心肌细胞凋亡模型。 探讨caspase-9在肝癌细胞凋亡中的作用及调控机制 我们将产生和分析重组腺病毒， 成人中过表达caspase-9和各种突变形式的caspase-9 心肌细胞 在具体目标2中，我们将研究几种策略， 抑制胱天蛋白酶-9活性，包括药理学胱天蛋白酶-9抑制， caspase-9显性负性形式的过表达， 内源性半胱天冬酶-9抑制剂和抗凋亡Akt的过表达。 最后，在具体目标3中，我们将定义caspase-9在心脏中的作用， 通过产生心脏特异性caspase-9敲除小鼠体内。 由于caspase-9 同源重组敲除导致围产期死亡， 不能研究半胱天冬酶-9在成体组织中的作用。 因此，心脏 使用Cre-loxP技术特异性敲除caspase-9使我们能够检查 capase-9在成年动物中组织特异性作用。

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.