MOLECULAR MECHANISMS OF APOPTOSIS IN MYOCARDIAL ISCHEMIA

这项建议的长期目标是阐明 缺血/再灌流后心肌细胞凋亡及其与血管内皮生长因子的关系 心脏重塑和心力衰竭的进展。直到 最近，心肌梗死(MI)时的心肌细胞死亡被认为是 完全是由于细胞坏死。然而，随着证据的积累 提示细胞的凋亡性死亡对心肌细胞有重要作用。 ML后急性期和慢性期的丢失。特别是， 细胞凋亡在再灌注损伤中可能起重要作用。然而，在 目前，几乎没有实验证据可以证明 心肌细胞因缺血/再灌流而发生细胞凋亡。AS 为此，我们建立了成人心肌细胞的体外模型。 缺氧/复氧诱导的细胞凋亡与心肌梗死相关的小鼠模型 伴随着心脏重塑。我们的初步结果表明， 从线粒体释放细胞色素c是早期的一步 心肌细胞凋亡。此外，丝裂原激活蛋白的成员 (MAP)激酶，特别是JNK，迅速被激活以响应 缺血/再灌流。来自其他细胞类型的证据表明 应激激活激酶JNK的持续激活导致 细胞凋亡，可能是通过磷酸化线粒体蛋白Bc l-2。论 另一方面，磷脂酰肌醇-3激酶(PI-3K)/Akt的激活 生长因子刺激途径，如胰岛素样生长因子-1 (IGF-1)，已被证明可促进细胞存活，推测是通过 促凋亡线粒体蛋白Bad的磷酸化。 因此，我们假设线粒体膜外膜的丧失 缺血/再灌流在关键决策中的完整性 心肌细胞因细胞凋亡和潜在的坏死而不可逆转的死亡。 我们还假设，有额外的抗细胞凋亡和促进- 除已知的Bcl-2蛋白家族外，其他凋亡分子表达 在调节线粒体反应的心肌细胞中 缺血/再灌流。我们对这项建议的具体目标是：具体 目的1：进一步描述我们的心肌细胞凋亡模型 低氧/复氧诱导的体外和心肌 体内缺血/再灌流。具体目标2：确定 线粒体功能障碍、细胞色素c释放及Bcl2、Bclx/L 体外低氧/复氧诱导的细胞凋亡及其机制 体内缺血/再灌流。具体目标3：阐明 应激激活蛋白激酶在心肌细胞凋亡调控中的作用 在缺血/再灌注期间，并鉴定新的线粒体和非线粒体 促凋亡蛋白激酶JNK的线粒体靶标。具体目标4： 阐明抗细胞凋亡的PI-3K/Akt信号转导通路在细胞周期调控中的作用 缺血/再灌流诱导的心肌细胞凋亡及调控 鉴定新的抗线粒体和非线粒体靶点 细胞凋亡蛋白Akt.我们期待着对其机制的阐明 缺血/再灌流引起的心肌细胞凋亡及其鉴定 新的心肌支持细胞凋亡和抗凋亡分子将导致 设计更具体的心肌细胞保护策略。

The long term goal of this proposal is to elucidate the mechanisms of cardiomyocyte apoptosis following ischemia/reperfusion and its relation to the progression of ventricular remodeling and heart failure. Until recently, myocyte death during myocardial infraction (MI) was considered to be exclusively due to cell necrosis. However, accumulating evidence suggests that apoptotic cell death contributes significantly to myocyte loss during both the acute and chronic phases after ML. In particular, apoptosis may play a major role in reperfusion injury. However, at present, little experimental evidence is available as to how cardiomyocytes undergo apoptosis in response to ischemia/reperfusion. As such, we have established an in vitro model of adult cardiomyocyte apoptosis by hypoxia/reoxygenation and a murine model of MI associated with ventricular remodeling. Our preliminary results suggest that the release of cytochrome c from mitochondria is an early step in cardiomyocyte apoptosis. In addition, members of mitogen activated protein (MAP) kinases, particularly JNK, are rapidly activated in response to ischemia/reperfusion. Evidence from other cell types suggest that persistent activation of the stress-activate kinase JNK leads to apoptosis, possibly by phosphorylating mitochondrial protein Bcl-2. On the other hand, activation of the phosphatidylinositol-3 kinase (PI-3K)/Akt pathway by growth factor stimulation, such as insulin-like growth factor-1 (IGF-1), has been shown to promote cell survival, presumably by phosphorylation of the pro-apoptotic mitochondrial protein Bad. Accordingly, we hypothesize that the loss of outer mitochondrial membrane integrity by ischemia/reperfusion in the critical decision making for irreversible myocyte death by apoptosis, and potentially necrosis as well. We also hypothesize that there are additional anti-apoptotic and pro- apoptotic molecules, other than known Bcl-2 family of proteins, expressed in cardiomyocytes that modulate the mitochondrial response to ischemia/reperfusion. Our Specific Aims of this proposals are: Specific Aim 1: To further characterize our models of cardiomyocyte apoptosis induced by hypoxia/reoxygenation in vitro and myocardia ischemia/reperfusion in vivo. Specific Aim 2: To define the roles of mitochondrial dysfunction, cytochrome c release and Bcl-2 and Bcl-x/L in apoptosis induced by hypoxia/reoxygenation in vitro and ischemia/reperfusion in vivo. Specific Aim 3: To elucidate the roles of stress-activated kinases in the regulation of cardiomyocyte apoptosis during ischemia/reperfusion and identify novel mitochondrial and non- mitochondrial targets of the pro-apoptotic kinase JNK. Specific Aim 4: To elucidate the roles of anti-apoptotic PI-3K/Akt signaling in the regulation of ischemia/reperfusion-induced cardiomyocyte apoptosis and identify novel mitochondrial and non-mitochondrial targets of the anti- apoptotic kinase Akt. We anticipate that elucidation of the mechanisms of cardiomyocyte apoptosis by ischemia/reperfusion and identification of novel myocardial prop-apoptotic and anti-apoptotic molecules will lead to the design of more specific cardiomyocyte protective strategies.