Role of Sirtuin6 in the maintenance of cardiac lysosomal homeostasis

Abstract

The response to cardiac stress can be divided into two phases- an initial adaptive compensatory phase followed by a maladaptive decompensatory phase. Protein homeostasis in cardiomyocytes has been observed to behave dynamically as the heart goes from compensation to decompensation before it undergoes failure. Autophagy helps in the degradation of damaged organelles and protein aggregates through the involvement of lysosomes. However, during the maladaptive stage of hypertrophy, autophagy becomes dysregulated causing the cell to die. Degradation of malfunctioning organelles and proteins becomes equally important in the context of decompensation. Sirtuin 6 has been studied extensively in the context of cardiac dysfunction, including protein synthesis. The present work attempts to unravel the role of SIRT6 in the maintenance of lysosomal homeostasis in the heart. Part I: Developing a quantitative method to study lysosomes in cardiomyocytes- Most traditional methods involve the quantification of lysosomes after imaging them using fluorescence or electron microscopy. In this section, we illustrate the use of flow cytometry to assess the acidity of organelles and the activity of cathepsin B in cardiomyocytes using LysoTracker Red and MagicRed respectively. We validated the assay using three models of induction of lysosomes. Part II: Role of SIRT6 in the maintenance of cardiac lysosomal homeostasis- In the current study, we validated two mouse models of cardiac stress including doxorubicin-induced cardiomyopathy and isoproterenol-induced hypertrophy. Doxo- and ISO-treated mice hearts show an increase in lysosome levels in addition to a decrease in SIRT6 levels. Using loss- and gain-of-function experiments, we observed that SIRT6 negatively regulates lysosomal biogenesis in primary cardiomyocytes. This effect was dependent on the activity of SIRT6. Further, in SIRT6 knockout mice hearts, we found an increased expression of lysosomal proteins in different organs. To study the specific role of SIRT6 in cardiac lysosomes, we generated cardiomyocyte-specific SIRT6- depleted mice which showed cardiac atrophy at 4 months of age with a concomitant increase in lysosomal levels. Our transcriptomic analysis supports the phenotype of the csSIRT6 KO mice hearts observed. Mechanistically, SIRT6 interacts with TFEB, the master regulator of lysosomal biogenesis, and causes its deacetylation. Under normal conditions, SIRT6 binds to the TFEB target promoters, causes the deacetylation of H3K56, and inhibits transcriptional activity at the said promoters. It also binds to the promoters of the TFEB-activating phosphatases including calcineurin and PP2A and inhibits TFEB. Additionally, pharmacological inhibition of Calcineurin restored the lysosomal levels and activity to basal levels. Our study has therefore unraveled a critical link between SIRT6 and lysosomes in the maintenance of cardiac health. Modulating lysosomes by targeting SIRT6 could be a potential therapeutic intervention in managing heart conditions.