Sirtuin-1 (SIRT1) is an NAD+-dependent deacetylase implicated in autophagosome formation; however, whether SIRT1 also regulates autophagosome clearance during late-stage autophagy remains unclear. Here, we investigated the role of SIRT1 in autophagosome clearance during autophagy and mitophagy in cardiomyocytes. Mitochondrial stress induced by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) decreased mitochondrial protein levels and increased phosphorylation of ubiquitin, a PINK1 target, in H9c2 cardiomyocytes. These CCCP-induced decreases in mitochondrial proteins were prevented by co-treatment with chloroquine, an inhibitor of lysosomal degradation, supporting the induction of CCCP-triggered mitophagy. SIRT1 knockdown similarly prevented the CCCP-induced reduction in mitochondrial proteins and led to the accumulation of autophagosomes containing fragmented mitochondria without attenuating ubiquitin phosphorylation, suggesting that SIRT1 acts downstream of mitochondrial tagging. Tandem GFP-RFP LC3 assay and LC3-LAMP1 colocalization analysis demonstrated impaired autophagosome-lysosome fusion following SIRT1 knockdown. In vivo, cardiomyocyte-specific SIRT1 knockout mice exhibited elevated basal LC3-II levels and a blunted LC3-II response to chloroquine, consistent with impaired autophagic flux. In a doxorubicin (DOX)-treated model, SIRT1 deficiency attenuated autophagosome degradation during the early period after DOX administration. Mechanistically, SIRT1 interacted with Rab7, a key regulator of autophagosome-lysosome fusion, raising the possibility that SIRT1 might regulate fusion through post-translational modification of Rab7 or related components. Collectively, these findings identify SIRT1 as a regulator of autophagosome-lysosome fusion that promotes autophagosome degradation during autophagy and mitophagy in cardiomyocytes.
