Novel Regulatory Role of SIRT3 on Cardiomyocyte Mitochondrial Frataxin and Ferroptosis
Authors: Cantrell Aubrey , Su Han , Zeng Heng , Chen Jian‐Xiong 
Published: May 2022

AIMSSirtuin 3 (SIRT3) has been shown to contribute to the mitochondrial cardiomyopathy of Friedreich’s Ataxia (FRDA), which is characterized by a deficiency in mitochondrial frataxin along with mitochondrial hyperacetylation. Using a cardiomyocyte‐specific SIRT3 knockout (SIRT3cKO) mouse model, we addressed two key questions: (1) whether SIRT3‐induced acetylation of proteins is specific to the mitochondria; and (2) if mitochondrial SIRT3 is a key regulator of mitochondrial frataxin which impacts mitochondrial iron homeostasis and ferroptosis.METHODS AND RESULTSThe mitochondrial and cytosolic fractions were isolated from the ventricles of the hearts of SIRT3cKO mice. The mitochondrial and cytosolic proteins and mitochondrial iron levels were analyzed by comparison to SIRT3‐Loxp wild‐type (WT) control mice. Cardiac function study showed that SIRT3cKO mice developed heart failure as evidenced by reduction of ejection fraction (EF) and fraction shortening (FS) and increased isovolumic relaxation time (IVRT) and myocardial performance index (MPI) when compared to WT controls. Comparison of the mitochondrial and cytosolic fractions of the SIRT3cKO model to those of the WT control shows that, upon loss of SIRT3, mitochondrial, but not cytosolic, total lysine acetylation was significantly increased in the heart. Similarly, acetylated p53 (p53ace) was significantly upregulated only in the mitochondria, while levels of p53 were not altered in either compartment. These data demonstrate that SIRT3 is the primary mitochondrial deacetylase, while acetylation in the cytosol is independent of SIRT3 in cardiomyocytes.Most importantly, loss of SIRT3 in the mitochondria resulted in significant reduction of the protein frataxin and the Iron (II) export protein ferroportin (FPN). Furthermore, levels of glutathione peroxidase 4 (GPX4) were also downregulated in the mitochondria. This was accompanied by a significant increase in levels of 4‐hydroxynonenal (4‐HNE), an indicator of lipid peroxidation, and suggestive of upregulated mitochondrial ferroptosis in SIRT3cKO mouse hearts. Additionally, mitophagy marker beclin‐1 expression was significantly reduced in the mitochondria of SIRT3cKO mice. Levels of glucose transporter‐1 (GLUT1), which functions to deliver the antioxidant Vitamin C to the mitochondria and reduce ROS production, were also diminished in the mitochondria. Mechanistically, mitochondrial levels of hypoxia inducible factor‐2α (HIF‐2α) were downregulated, while those of HIF‐1α remained unchanged in SIRT3cKO mouse hearts. Treatment with ferroptosis inhibitor ferrostatin‐1 for 14 days significantly reduced 4‐HNE levels and rescued preexisting impaired cardiac function in SIRT3cKO mice.CONCLUSIONSFor the first time, we have demonstrated that cardiomyocyte SIRT3 deficiency causes mitochondrion‐specific acetylation and impairment of frataxin and ferroportin potentially via downregulation of HIF‐2α. Our results suggest that the SIRT3‐ferroptosis pathway may be a novel target for the mitochondrial cardiomyopathy of FRDA.