Mechanisms of Oxidant Signaling in Post-MI Remodeling

描述（由申请人提供）：活性氧（ROS）在衰竭心肌中增加，并模拟体外心肌细胞病理性重塑的许多分子和细胞特征。在前期研究中，我们检测了过量一氧化氮（NO）和超氧化物在心肌梗死后晚期（MI后）介导远端心肌LV重构中的作用。我们发现，NOS 2衍生的NO有助于心肌梗死后重塑，而超氧化物本身，不是一个主要的效应物种。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种类，B）NADPH氧化酶是肥大刺激的ROS来源，以及c）线粒体在ROS依赖性凋亡中起中心作用。在目的1中，我们将测试NADPH氧化酶介导的作用，在培养的心肌细胞重塑刺激通过抑制NADPH氧化酶的活性和表达，使用显性负突变体和小干扰RNA（siRNA）针对特定的酶亚基。在目标2中，我们将通过测试以下假设来检查线粒体在ROS依赖性调节肌细胞凋亡中的作用：凋亡刺激增加线粒体呼吸，导致ROS产生增加，从而激活JNK和bcl-2家族的促凋亡成员，其单独或共同作用以诱导线粒体细胞色素c释放和凋亡级联的激活。在目标3中，我们将通过测试以下假设来检查过氧化氢在体内介导心肌肥大和细胞凋亡中的作用：通过胞质或胞浆定向过氧化氢酶的肌细胞特异性过表达来清除过氧化氢将减少氧化应激，从而减弱小鼠MI后的病理性重塑。在目标4中，我们将使用来自目标1和2的体外范例来检查Ras的氧化还原介导的巯基修饰在介导重塑刺激的肥大效应中的作用，并鉴定具有氧化还原依赖性巯基修饰的另外的蛋白质，其由肥大与凋亡重塑刺激差异介导。这些研究将为氧化剂信号传导在MI后心肌重塑中的机制提供新的理解，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.