Cardiovascular disease is the leading cause of death in the United States and its occurrence dramatically increases with age. Preventing or delaying cardiac aging can therefore have a significant effect on longevity and healthspan. A central goal of this proposal is to explore a novel mechanism by which mitochondria decline with age and contribute to loss of cardiac function. We are focusing on the post-translational modification, acetylation, because it increases with age and the enzyme that reverses this modification, sirtuin-3 (SIRT3), is a known longevity factor that decreases with age. However, if mitochondrial acetylation accelerates cardiac aging remains unresolved and controversial. Our overarching hypothesis is that an increase in acetylation has two effects in the aged heart. First, it causes metabolic inflexibility by directly affecting the activity of key regulatory enzymes. Second, the increase in acetylation affects proteostasis (protein synthesis and degradation homeostasis). Dysfunctional proteostasis in turn contributes to metabolic inflexibility by causing improper synthesis and degradation of modified mitochondrial metabolic enzymes. For this proposal, we generated a cardiomyocyte (CM) specific SIRT3 knockout mice (SIRT3CM-/-). Our preliminary data show that SIRT3CM-/- mice exhibit dramatic hypertrophy, loss of contractile function, fibrosis, metabolic abnormalities, and dysfunctional proteostasis by 10- months. This accelerated model of aging and hyperacetylation will be used to test our hypothesis through the following aims. Aim 1 will determine if the increase in acetylation caused by the loss of SIRT3 affects metabolic flexibility with age. We hypothesize that the loss of SIRT3 causes metabolic inflexibility by affecting the activity of discrete mitochondrial regulatory enzymes that result in the increased reliance on glucose. This aim will determine if the loss of SIRT3 affects metabolic flexibility by measuring cardiac and mitochondrial functions, enzymatic activities, and metabolic flexibility longitudinally in SIRT3CM-/- mice and isolated adult cardiomyocytes. Global acetylation and the acetylation of specific metabolic enzymes will be measured by mass spectrometry. The results of these studies will determine if there is a direct consequence of hyperacetylation on cardiac pathology in the absence of SIRT3. Aim 2 will determine how the loss of SIRT3 affects mitochondrial proteostasis. Acetylation of proteins can affect structure and function, yet little is known regarding its role in global changes in mitochondrial protein quality and turnover. This aim will use deuterium oxide (D2O) labeling and proteomics to determine if the loss of SIRT3 affects mitochondrial protein synthesis and the relative turnover rates of specific metabolic regulatory enzymes. Rescue experiments will be performed by AAV delivery of SIRT3. Mechanistic studies in cell culture will be performed to demonstrate how acetylation affects proteostasis. T...