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. ? ?
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