Mechanisms of Oxidant Signaling in Post-MI Remodeling

描述(由申请人提供)：在衰竭的心肌中，活性氧物种(ROS)增加，并模仿体外培养的心肌细胞病理性重构的许多分子和细胞特征。在之前的资助阶段，我们测试了过量的一氧化氮(NO)和超氧化物歧化在心肌梗死后晚期(后MI)远端心肌中介导左室重构的作用。我们发现NOS2衍生的NO参与了心肌梗死后的重塑，而超氧化物本身并不是主要的效应物质。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种，b)NADPH氧化酶是肥大刺激的ROS的来源，c)线粒体在ROS依赖的细胞凋亡中发挥核心作用。在目标1中，我们将通过针对特定酶亚基的显性负性突变体和小干扰RNA(SiRNA)来抑制NADPH氧化酶的活性和表达，以测试NADPH氧化酶在心肌细胞重构刺激反应中的作用。在目标2中，我们将通过检验以下假设来研究线粒体在ROS依赖的心肌细胞凋亡调控中的作用：凋亡刺激增加线粒体呼吸，导致ROS生成增加，从而激活JNK和bcl2家族的促凋亡成员，它们单独或共同作用，诱导线粒体细胞色素c释放，激活凋亡级联反应。在目标3中，我们将通过检验这样一种假设，即通过胞浆或线粒体导向的过氧化氢酶的特异性表达来清除过氧化氢将减少氧化应激，从而减轻小鼠心肌梗死后的病理重塑，从而检验过氧化氢在体内介导心肌肥大和细胞凋亡中的作用。在目标4中，我们将使用AIMS 1和AIMS 2的体外范例来研究氧化还原介导的RAS的硫醇修饰在介导重塑刺激的肥大效应中的作用，并识别其他具有氧化还原依赖的硫醇修饰的蛋白质，这些蛋白质是由肥大的重塑刺激和凋亡的重塑刺激差异介导的。这些研究将对氧化信号在心肌梗死后心肌重构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.