Mechanisms of Oxidant Signaling in Post-MI Remodeling

描述（由申请人提供）：衰竭心肌中的活性氧（ROS）增加，并模仿体外心肌细胞病理重塑的许多分子和细胞特征。在之前的资助期内，我们测试了过量一氧化氮（NO）和超氧化物在心肌梗死后晚期远端心肌中介导左室重构的作用。我们发现nos2衍生的NO有助于心肌梗死后的重塑，而超氧化物本身并不是主要的影响物质。我们的体外研究进一步表明，a)过氧化氢或其衍生物是重塑刺激的主要效应物质，b) NADPH氧化酶是增生性刺激ROS的来源，c)线粒体在ROS依赖性细胞凋亡中起核心作用。在Aim 1中，我们将通过使用显性阴性突变体和针对特定酶亚基的小干扰RNA （siRNA）抑制NADPH氧化酶的活性和表达，来测试NADPH氧化酶在培养心肌细胞中介导心肌细胞肥大以响应重塑刺激中的作用。在Aim 2中，我们将检验线粒体在ROS依赖性肌细胞凋亡调节中的作用，通过验证凋亡刺激增加线粒体呼吸导致ROS生成增加的假设，从而激活JNK和bcl-2家族的促凋亡成员，它们单独或协同作用诱导线粒体细胞色素c释放和凋亡级联激活。在Aim 3中，我们将检验过氧化氢在体内介导心肌肥大和凋亡中的作用，通过验证一个假设，即通过心肌细胞特异性过表达细胞质或线粒体定向过氧化氢酶来清除过氧化氢，将减少小鼠心肌梗死后的氧化应激，从而减轻病理性重构。在目标4中，我们将使用目标1和目标2中的体外范式来检查氧化还原介导的Ras硫醇修饰在介导重塑刺激的肥厚效应中的作用，并确定氧化还原依赖性硫醇修饰的其他蛋白质，这些蛋白质在肥厚和凋亡重塑刺激的差异介导下。这些研究将为心肌梗死后心肌重构中氧化信号的机制提供新的认识，因此将与患者心力衰竭的常见原因直接相关。

DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) are increased in failing myocardium and mimic many of the molecular and cellular features of pathologic remodeling in cardiac myocytes in vitro. In the prior funding period we tested the roles of excessive nitric oxide (NO) and superoxide in mediating LV remodeling in remote myocardium late after myocardial infarction (post-MI). We found that NOS2-derived NO contributes to post-MI remodeling, whereas superoxide, per se, was not a primary effector species. Our in vitro studies further suggest that a) hydrogen peroxide, or a derivative, is the primary effector species for remodeling stimuli, b) NADPH oxidase is the source of ROS for hypertrophic stimuli, and c) mitochondria play a central role in ROS-dependent apoptosis. In Aim 1 we will test the role of NADPH oxidase in mediating myocyte hypertrophy in response to remodeling stimuli in cultured cardiac myocytes by inhibiting NADPH oxidase activity and expression using dominant negative mutants and small interference RNA (siRNA) directed at specific enzyme subunits. In Aim 2 we will examine the role of mitochondria in the ROS-dependent regulation of myocyte apoptosis by testing the hypothesis that apoptotic stimuli increase mitochondrial respiration leading to increased ROS generation and thereby activate JNK and pro-apoptotic members of the bcl-2 family, which act alone or in concert to induce mitochondrial cytochrome c release and activation of the apoptotic cascade. In Aim 3 we will examine the role of hydrogen peroxide in mediating myocardial hypertrophy and apoptosis in vivo by testing the hypothesis that scavenging hydrogen peroxide by the myocyte-specific overexpression of cytosolic or mitochondrially-directed catalase will reduce oxidative stress and thereby attenuate pathologic remodeling post-MI in the mouse. In Aim 4 we will use the in vitro paradigms from Aims 1 and 2 to examine the role of redox-mediated thiol modifications of Ras in mediating the hypertrophic effects of remodeling stimuli, and to identify additional proteins with redox-dependent thiol modifications that are differentially-mediated by hypertrophic vs. apoptotic remodeling stimuli. These studies will provide new understanding of the mechanisms of oxidant signaling in myocardial remodeling post-MI, and will therefore have direct relevance to a common cause of heart failure in patients.