Abstract Body: Introduction: Autophagy-related protein 7 (Atg7) functions are essential in cellular macro-autophagy. We have previously reported that Atg7 promotes macro-autophagy in the heart; nonetheless, whether Atg7 regulates selective mitochondrial autophagy (mitophagy) remains unknown.
Objectives: We employed complementary biochemical and genetic approaches to decipher the molecular mechanisms of Atg7-mediated mitophagy induction in the heart.
Methods and Results: Atg7 is localized in cardiac mitochondria, as revealed in organelles fractionation and anti-Atg7 quantum dot-labeled electron microscopy. We corroborate these observations in isolated heart mitochondria where Atg7 co-localizes to Mitotracker-labeled mitochondria. Next, we overexpressed Atg7 in cultured primary cardiomyocytes, treated with cyanide 3-chlorophenylhydrazone (CCCP) to induce mitophagy, and stained with pH-sensitive fluorescent mitophagy dye. Atg7 overexpression significantly increased red mitophagy puncta than β-gal expressing control cardiomyocytes. To assess the activation of mitophagy in vivo, we treated the cardiac-specific Atg7 overexpressing transgenic (αMHC-Atg7 Tg) and their littermate non-transgenic (Ntg) mice with CCCP. We found an increased accumulation of mature autophagolysosomal marker protein LC3-II in isolated mitochondria in Atg7 Tg mice hearts. Mito-Keima transgenic (Mt-Keima Tg) mice bear mitochondria-targeted pH-dependent fluorescent lysosomal protease-resistance Keima protein, enabling the assessment of mitophagy extent and activation in vivo. To assess the extent of mitochondria-bearing autophagosome lysosomal delivery (mitophagy flux), we crossed αMHC-Atg7 Tg mice with cardiac-specific Mt-Keima Tg mice. We observed increased Mt-Keima high red-to-green areas in αMHC-Atg7xMt-Keima Tg mice hearts compared to control Mt-Keima Tg mice.
Conclusions: Our in vitro and in vivo experiments corroborate that Atg7 localizes to the mitochondria, promoting mitophagy machinery recruitment and activation. Our data further confirm that genetic activation of Atg7 leads to increased basal mitophagy flux in the heart.