The Role of SIRT3 and Acetylation in the Pathogenesis of Friedreich’s Ataxia Cardiomyopathy
Lysine acetylation and its regulation by the mitochondrial NAD+-dependent protein deacetylase sirtuin 3 (SIRT3) are emerging as important regulators of cardiac pathophysiology. SIRT3 serves as a negative regulator of cardiac hypertrophy, and several studies demonstrate decreased SIRT3 expression and increased mitochondrial acetylation in the setting of heart failure. However, the mechanisms that enable the cardio-protective activities of SIRT3 are not well understood. Furthermore, the mechanisms of how acetylation translate to repression of cardiomyocyte metabolism are under studied. Of interest is the genetic cardiomyopathy model that mimics Friedreich’s Ataxia (FRDA) cardiomyopathy–the frataxin knock-out (Fxn-/-, a.k.a. FXNKO) mouse–a model of primary mitochondrial dysfunction due to complex I-III deficiency and/or inhibition that exhibits progressive mitochondrial protein hyperacetylation. We predicted that complex I deficiency in the FXNKO heart may lead to mitochondrial protein hyperacetylation, altered NAD+ homeostasis, and SIRT3 inactivity. We further hypothesized that the hyperacetylation and SIRT3 inactivity in the FXNKO heart are pathogenic and contribute to maladaptive shifts in carbon and energy metabolism known to occur with disease progression in this model and in FRDA patients. Thus, the goal of my dissertation work was to elucidate the role of SIRT3 and acetylation in the pathogenesis of FXNKO and FRDA heart failure. We developed a multi-pronged strategy to realize this goal.
The bulk of my dissertation work focuses on our first approach which involved examining the cardiac intrinsic role of SIRT3 in a cardiac/skeletal-muscle-specific ablation model, as well as the role of SIRT3 in the context of the FXNKO heart failure (see Chapter 3). For these studies, we genetically manipulated SIRT3 in wild-type and FXNKO mice to evaluate the role of cardiac SIRT3 in normal and pathophysiological states. In these studies, we found a cardiac intrinsic role for SIRT3 in maintenance of normal hypertrophic response and contractile function. These data may explain the increased susceptibility of Sirt3 deficient mouse models to cardiac stress, as observed in the literature. Additionally, we explored NAD+ precursor supplementation as a therapeutic means to modulate SIRT3 activity for FXNKO cardiomyopathy. Remarkably, NMN administered to FXNKO mice restored cardiac function to near-normal levels. These effects of NMN required SIRT3, as the improvement in cardiac function upon NMN treatment in the FXNKO were lost in a SIRT3KO/FXNKO (dKO) knockout model. Coupled with cardio-protection, we found that chronic NMN supplementation resulted in improvements in both cardiac and extra-cardiac metabolic function and energy metabolism, and that many of these improvements required cardiac SIRT3. Taken together, these results serve as important preclinical data for a potential strategy of NMN supplementation or SIRT3 activator therapy in FRDA patients.
As a second approach, we conducted preliminary studies to investigate the development of hyperacetylation in the FXNKO heart, with the aim to assess the metabolic source of acetyl-CoA (i.e., the acetylating agent) throughout disease progression (see Appendix A). With these studies, we found that the contribution of various metabolic substrates to the acetyl-CoA pool led to differential acetylation of cardiac proteins in the FXNKO. Additionally, we sought to manipulate the degree of acetylation and test if reducing global acetylation could improve disease outcome. Preliminary results here suggest that the degree of global acetylation is less contributory to disease progression than acetylation at specific sites on proteins. Together, these studies support the notion that shifts in cardiac metabolism with disease progression result in the accumulation of mitochondrial protein hyperacetylation. Additionally, these findings suggest that key sites of hyperacetylation are most critical to the development of cardiomyopathy in the FXNKO, rather than global hyperacetylation.
Lastly, the above findings could serve as preliminary preclinical data for either SIRT3 activation or inhibition of acetylation as therapeutic approaches to treat FXNKO and perhaps FRDA patients. For the results of these studies to have clinical importance, parallel observations of mitochondrial protein hyperacetylation and/or SIRT3 inactivity in the human FRDA heart would need to be assessed. Therefore, we evaluated the cardiac acetylome of autopsied FRDA patient hearts (see Appendix B). Alongside these samples, we included non-failing hearts as a negative control and two other types of human heart failure (i.e., non-ischemic failing and ischemic failing hearts) as positive controls. We found significant hyperacetylation of cardiac proteins in all three types of human heart failure, as expected based on the literature. Importantly, we found that the FRDA patients, like the preclinical FXNKO mouse, develop marked hyperacetylation of cardiac proteins at end-stage disease. Altogether, these preliminary data provide some clinical relevance of acetylation and/or SIRT3 inactivity in the FRDA heart. Additionally, these findings strongly support furthering efforts to develop therapeutic approaches to manipulate acetylation and/or SIRT3 activity as a prospective treatment for FRDA.
Overall, our work provides insight into the role of SIRT3 and acetylation in the heart under basal and stressed conditions. These data validate an important role for SIRT3 in the heart as a mediator of the hypertrophic response. Furthermore, these data identify a novel role of SIRT3 in mediating contractile function in the heart, a finding that may explain the increased sensitivity of Sirt3 ablation mouse models to cardiac stress. Finally, these data provide early mechanistic insight into the contribution of acetylation at key SIRT3 regulated sites to the pathogenesis of cardiomyopathy in FXNKO mice and FRDA patients. This insight is necessary to fully realize the potential of metabolic therapy via manipulation of SIRT3/acetylation in the FRDA heart and in other types of human heart failure.
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