Project Summary: Heart failure is a debilitating condition in which the heart cannot meet the metabolic demands of the body. Chronic β-adrenergic (β-AR) stimulation causes pathological myocardial remodeling that leads to heart failure. Chronic β-AR stimulation contributes to myocardial remodeling by promoting apoptosis of cardiac myocytes. Work from our laboratory has shown that β-AR stimulated apoptosis is dependent on reactive oxygen species (ROS), but the molecular target(s) by which ROS mediates apoptosis is not known. We have also shown that sarcoplasmic reticulum calcium ATPase (SERCA2), a key regulator of cardiac myocyte calcium, can undergo oxidative post-translational modification (OPTM) of cysteine C674: High levels of ROS inhibit SERCA2 activity through irreversible OPTM of SERCA2 C674. SERCA2 is responsible for moving the large majority of intracellular calcium in the cardiac myocyte, and may thereby affect the level of calcium in mitochondria, where excessive calcium activates the intrinsic mitochondrial apoptosis pathway. We hypothesize that oxidative inhibition of SERCA2 at C674 increases mitochondrial calcium, thereby activating the mitochondrial apoptosis pathway. To address this thesis, we will use of a novel redox-insensitive SERCA2 mutation in which C674 is replaced by serine (C674S) to determine the role of oxidative inhibition in β-AR stimulated apoptosis both in vitro in cardiac myocytes and in vivo in mice. We will elucidate the role of SERCA2 oxidative inhibition in vitro by assessing β-AR stimulated apoptosis in adult rat ventricular myocytes (ARVM) that overexpress wild type or C674S SERCA2. To identify whether oxidative inhibition of SERCA2 at C674 affects mitochondrial calcium we have created ultrasensitive genetically-encoded calcium indicators (GECI) targeted to the mitochondria, mitochondrial-associated membrane (MAM) and cytosol which will be expressed in ARVM via adenovirus infection. These novel GECI will allow us to measure calcium levels in the mitochondria and MAMs, and assess the relationship to apoptosis. To test our hypothesis in vivo, we will generate a cardiac myocyte-specific redox- insensitive SERCA2 mouse in which there is knock-in of the C674S mutant. With this mouse we will assess the role of C674 oxidation in mediating pathological remodeling in response to a chronic β-AR challenge at the cellular and organ levels. Taken together, these experiments will elucidate the role of SERCA2 oxidation in mediating apoptosis in cardiac myocytes, thereby identifying a novel mechanism contributing to heart failure.