DESCRIPTION (provided by applicant): Reactive oxygen and nitrogen species (ROS) are implicated in the pathophysiology of myocardial failure. Our laboratory and many others have shown that remodeling stimuli, including mechanical strain and Gq activation, cause myocyte hypertrophy by using ROS to activate the small GTPase, Ras, via the oxidative S-glutathiolation of a specific cysteine; and further, that Ras activation and myocyte hypertrophy can be prevented by glutaredoxin. A hallmark of myocardial failure is cardiac myocyte contractile dysfunction, which is due in part to reduced activity of sarcoplasmic reticulum calcium ATPase 2 (SERCA2). Our preliminary data show that Gq- and pressure overload-induced myocardial failure are associated with oxidative post-translational modifications (OPTM) of thiols and tyrosines in SERCA2, and that OPTM can regulate SERCA2 activity in vitro in myocytes. Thus, work during the prior grant period has led to our working hypothesis that remodeling stimuli cause OPTM of SERCA2 leading to calcium dysregulation that contributes to myocardial failure. Aim 1 examines the hypothesis that ROS cause OPTM that regulate SERCA2, calcium and contractile function, by testing whether a) specific SERCA2 OPTM are determined by the ROS species, concentration and duration of exposure; and b) specific ROS-induced OPTM up- or down- regulate SERCA2 activity. Using cardiac myocytes in primary culture we will measure the effects of graded a) concentrations, b) durations and c) types of defined reactive oxygen / nitrogen species on myocyte SERCA2 activity and OPTM, calcium handling and contractile function. The role of specific OPTM will be tested using site-directed mutations of SERCA2. Aim 2 extends the role of the OPTM delineated in Aim 1 to heart failure disease models by testing the hypothesis that remodeling stimuli, acting via oxidants, cause calcium and contractile dysfunction by causing SERCA2 OPTM. We will test this thesis in myocytes exposed to remodeling stimuli in vitro in cultured myocytes or in vivo in myocytes from mice with Gq- and pressure overload-induced myocardial failure. We will test the ability of pharmacologic and genetic manipulations that modify or prevent the occurrence of SERCA2 OPTM, including site-directed mutation of suspected OPTM targets, to alleviate calcium and contractile dysfunction. To test the clinical relevance of our hypothesis, we will assess SERCA2 OPTM in myocardium from dogs and humans with heart failure. Aim 3 tests the hypothesis that SERCA2 OPTM causes myocyte calcium and contractile dysfunction in vivo in mice with pressure overload-induced myocardial failure. We will examine the ability of transgenic manipulations that, a) reduce the level of offending ROS (myocyte-specific catalase, NOS2 knockout), b) maintain reactive thiols in a reduced state (glutaredoxin 1), or c) modify suspected OPTM targets (SERCA2 C674S knock-in) to inhibit or prevent the occurrence of specific SERCA2 OPTM, improve myocardial function and ameliorate pathological remodeling in vivo. PUBLIC HEALTH RELEVANCE Heart failure, which affects over 5 million Americans, is most often caused by high blood pressure or heart attack leading to overworking of the heart muscle. There is now evidence that overwork of the heart muscle leads to oxidative stress. This grant seeks to understand the ways in which oxidant stress leads to heart failure, and has the potential to discover new treatments for this condition.