The Loss Of Cardiac SIRT3 Decreases Metabolic Flexibility And Proteostasis In An Age Dependent Manner

Backgrounds&Objectives:SIRT3 is a longevity factor that acts as the primary deacetylase in mitochondria.Whole-body SIRT3knockout mice develop age-related pathologies in the heart,including hypertrophy and fibrosis.Here,we sought to determine how specifically knocking out SIRT3 in cardiomyocytes（SIRT3CM-KOmice）temporally affects cardiac function and metabolism.Methods:1)Cardiomyocyte-specific SIRT3 knockout mice:SIRT3^CM-KOmice was generated by crossing homozygous SIRT3 flox/flox mice withαMHC-Cre mice.Whole hearts from 3 months and 10-12months mice were weighed and imaged after collection.The protein levels of SIRT3 and lysine acetylated protein in heart tissue were determined by Western Blot.Myocardial cross-sectional area and fibrosis were measured by H&E and Masson staining.Besides,the systolic function was evaluated by echocardiography.2)Mitochondrial respiration assay:Freshly isolated mitochondria were added with pyruvate,palmitoyl-carnitine,or glutamate.And respirations were measured with a dissolved oxygen system.3)Effect on glucose metabolism pathway:The proteomic analysis of170 target proteins in 10-month-old heart tissue through LC-MS and pathways analysis over KEGG database.The increase of glycolysis flux of primary myocardial cells stimulated by insulin was detected by isotope labeling method.Besides,the enzyme activity,protein level and phosphorylation level of mitochondrial PDH were measured.4)Explore the BCAA metabolic pathway:The content of BCAA in mouse serum and cardiac tissue homogenate was determined by GC-MS.BCKDH and its phosphorylation level,m TOR pathway related protein and phosphorylation level and LC3I/II protein expression level were determined by Western Blot.5)In vivo labeling to determine heart proteostasis:The mice were labeled in vivo by injecting a large dose of 99%heavy water and feeding them with 8%heavy water for 13 days The fractional synthesis rate of DNA and proteins in cytoplasm and mitochondria fraction were determined by GC-MS.Results:1)Compared with WT,the increase of cardiac tissue lysine acetylation level,cardiac hypertrophy,systolic dysfunction,and myocardial tissue and perivascular fibrosis in the SIRT3^CM-KOmice with cardiomyocyte-specific knockout gradually worsened with age,showing an obvious age-dependent.2)Compared with WT mice,the heart mitochondria of 10-month-old SIRT3^CM-KOmice are less dependent on the energy provided by fatty acid oxidation pathway,and more dependent on pyruvate.With the increase of age,the utilization efficiency of Pyr in mouse heart mitochondria increased significantly;Moreover,the utilization efficiency of Pyr in mitochondria of 3-month-old SIRT3^CM-KOmice also increased significantly.3)After specific knockout of SIRT3 in cardiac myocytes,the abundance of proteins involved in glycolysis,TCA cycle,pyruvate and other metabolic pathways changed significantly.Compared with SIRT3^CM-KOcardiomyocytes,the glycolytic flux of WT cardiomyocytes increased more significantly.In addition,compared with WT mice,the abundance of GLUT4 protein in heart tissue of 10-month-old SIRT3^CM-KOmice was significantly reduced,and the phosphorylation level of AKT was also reduced.Compared with WT mice,the activity of PDH in heart mitochondria of SIRT3^CM-KOmice was significantly increased at the age of 10 months;However,there was no significant change at the age of 3months.The total protein level of PDH decreased,but its phosphorylation level did not change significantly.While the abundance of PDK4 protein decreased significantly.4)The protein level of BCKDH in heart tissue of 10-month-old SIRT3^CM-KOmice decreased significantly,but its phosphorylation level（playing an inhibitory role）did not change significantly.Compared with WT mice,the contents of valine,leucine,and isoleucine in the serum of SIRT3^CM-KOmice did not change significantly.However,the levels of valine and leucine in the heart tissue of SIRT3^CM-KOmice were significantly increased.There was no significant difference in the levels of m TOR and its phosphorylated form,p70 S6K and its phosphorylated form,and 4E-BP1 phosphorylated protein among the groups;Only the content of 4E-BP1 protein in the heart tissue of 10-month-old SIRT3^CM-KOmice decreased,accompanied with a significant decrease in the ratio of LC3-II/LC3-I.5)The FSR of DNA,cytoplasmic protein,and mitochondrial protein in heart tissue of 10-month-old SIRT3^CM-KOmice were significantly higher than those of wild-type mice.On the contrary,the ratio of cytoplasmic protein to DNA synthesis rate and the ratio of mitochondrial protein to DNA synthesis rate of 10-month-old SIRT3^CM-KOmice decreased significantly.Conclusions:In conclusion,cardiomyocyte-specific deletion of SIRT3results in age-dependent hyperacetylation and cardiac hypertrophy through decreasing metabolic flexibility and proteostasis in heart.