The Role of SIRT5 and Protein Succinylation in Regulating Cardiac Function and Metabolism

Sirtuin 5 (SIRT5) is one of three mitochondrial proteins that belongs to the sirtuin family of NAD+-dependent deacylases. Mitochondrial sirtuins (SIRT3-5) control metabolism in physiological and pathophysiological conditions by their deacylation activity. SIRT5 possesses demalonylase, desuccinylase, and deglutarylase activity. While the enzymatic activity of SIRT5 has been well characterized, the physiological role of SIRT5 is less understood. Recent evidence suggests that SIRT5 may have a role in responding to cardiac stress. Given that succinylation is abundant in the SIRT5KO heart, it is important to understand the role of SIRT5 mediated desuccinylation in the heart. Since it appears that there are no defects in cardiac function in SIRT5KO mice under basal conditions, I hypothesized that a stress would be required to determine a protective effect of SIRT5 in the heart. Additionally, I hypothesized that multiple sites of lysine succinylation would contribute to the overall phenotype observed. To address this hypothesis, genetic mouse models were exposed to a well characterized model of pressure overload induced cardiac hypertrophy—transverse aortic constriction (TAC). Two main mouse models were used: 1) a whole body SIRT5KO mouse and 2) a cardiomyocyte tamoxifen-inducible heart specific SIRT5KO mouse. In order to characterize cardiac structure and function following TAC, the methods of serial echocardiogram and pressure volume loops with inferior vena cava suppression were used. Mechanistic studies included metabolomic and succinyl- proteomic profiling of heart samples. Additionally, Western Blot and RT- qPCR were used to further gain mechanistic insight. Chapter 3 of this dissertation characterizes the response of the whole body SIRT5KO mouse to pressure overload induced hypertrophy compared to littermate controls. We found that exposure to chronic TAC significantly increases mortality in SIRT5KO mice. Analysis of cardiac morphology and function after 4 weeks of TAC shows that SIRT5KO that have survived to this point have similar cardiac morphology and function compared to WT TAC mice. We predict that impaired oxidative metabolism is a major contributor to accelerated death in SIRT5KO mice. To specifically investigate the role of SIRT5 in cardiomyocytes, a heart-specific, inducible SIRT5KO mouse was generated and exposed pressure overload via TAC surgery. We find that the phenotype of increased mortality in whole-body SIRT5KO mice is not recapitulated under the conditions tested in heart-specific SIRT5KO mice. However, the two genetic mouse models show differences in protein succinylation, leading us to perform succinyl-proteomics in this model. The results of this investigation are presented in Chapter 4. Collectively, the results of these studies provide new insight into the role of SIRT5 mediated desuccinylation in regulating cardiac function and metabolism.