The Role of SIRT3 in Energy Metabolism of the Normal and Diabetic Heart
Final Report Abstract
The mitochondrially localized enzyme sirtuin 3 (SIRT3) is a NAD+-dependent deacetylase that decreases lysine acetylation of mitochondrial energy metabolic proteins, thereby increasing mitochondrial ATP generation. In the current project, I proposed (Aim 1) to investigate the function of SIRT3 in cardiac energy metabolism, and (Aim 2) to test if increased expression of PARP-1 in diabetic hearts impairs mitochondrial energetics by depleting mitochondrial NAD+ and thereby decreasing SIRT3-dependent mitochondrial protein deacetylation. Results generated in the context of Aim 1 show that SIRT3-/- mice developed an age-related decline in cardiac function, accompanied by left ventricular hypertrophy and dilation, suggesting that SIRT3 is required to maintain cardiac contractility and structure. In addition, we found evidence of mitochondrial dysfunction and of myocardial energy depletion. Increased acetylation of enzymes involved in fatty acid oxidation, TCA cycle activity and anaplerosis, and oxidative phosphorylation suggests inhibition of these metabolic pathways by hyperacetylation as underlying mechanism of impaired myocardial energetics in SIRT3-/- mice. Thus, we conclude that SIRT3 is required to maintain mitochondrial and contractile function in the heart and that SIRT3 may serve as redoxsensitive rheostat that regulates ATP-generating metabolic pathways by coordinated changes of lysine acetylation of various energy metabolic enzymes. The experiments yielded the expected data and confirm the hypotheses of Aim 1. In Aim 2, we originally proposed to assess the consequences of myocardial NAD+ depletion on mitochondrial energetics in the diabetic heart, based on the assumption that increased mitochondrial protein acetylation results from impaired SIRT3 activity. However, this objective became much more challenging since published studies showed that impaired activity of SIRT4 and SIRT5 may also contribute to increased protein acetylation, since both enzymes have been shown to regulate mitochondrial function, and since NAD+ depletion should also affect their enzymatic activity, resulting in changes of protein acetylation and of other sirtuin-mediated posttranslational modifications that are capable of regulating mitochondrial function. Thus, we decided that the functional consequences of impaired myocardial SIRT4 and SIRT5 activity under non-pathological conditions need to be examined before proceeding to cardiac disease states such as diabetic cardiomyopathy. Using echocardiography, SIRT4-/- mice showed preserved ejection fraction, normal left ventricular internal diameter, and normal posterior wall thickness. In isolated working hearts, cardiac power and output were even increased compared to wildtypes. Mitochondrial function was unaffected by absence of SIRT4, and the hypertrophic response to pressure overload was similar in SIRT4-/- and wildtype mice. Thus, in contrast to SIRT3 deficiency, lack of SIRT4 does not seem to impair cardiac function, left ventricular morphology, mitochondrial function, or the hypertrophic response to cardiac pressure overload. Based on our preliminary findings in SIRT4-/- mice, we suggest that SIRT3 and SIRT4 may comprise a regulatory system which enables the cell to fine-tune mitochondrial energetics under conditions of NAD+ depletion, thereby adding another level of metabolic flexibility. This may be particularly relevant in cardiac pathologies that are accompanied by NAD+ depletion, such as diabetic cardiomyopathy, myocardial ischemia reperfusion, or septic cardiomyopathy. Our next goal will be to study the role of SIRT4 and SIRT5 in the heart in more detail and to identify their protein targets using appropriate animal models. In addition, we will investigate the relevance of altered sirtuin activity in cardiac disease states, including diabetic cardiomyopathy (as initially proposed as Aim 2). From a therapeutic perspective, pharmacologic agonism of SIRT3 may be a feasible possibility to activate catabolic pathways with which to improve myocardial energetics by influencing just one enzyme in its activity, potentially resulting in beneficial effects for an already energy-starved heart, such as the failing heart. Studies addressing this hypothesis will be another goal for future research.
