Abstract 160: Atg7-Dependent Activation of Mitochondrial Autophagy in Cardiomyocytes

Abstract

Introduction: Autophagy-related protein 7 (Atg7) has been studied for its role in cellular macroautophagy induction and regulation in different cell systems. We have previously reported that Atg7 regulates cardiac macroautophagy. However, whether Atg7 plays any role in the initiation and regulation of mitochondrial autophagy (mitophagy) in cardiomyocytes remains elusive. Objective: We investigated subcellular localization and molecular function of Atg7 in the regulation of mitophagy through a series of in vitro and in vivo experiments. Methods and Results: We isolated mitochondria from mouse hearts by subcellular fractionation and conducted biochemical experiments which revealed localization of Atg7 on the mitochondrial membrane. Next, we co-stained for Atg7 in isolated heart mitochondria stained with Mitotracker dye and found Atg7 localizes to mitochondrial membrane. To elucidate molecular function of Atg7 on mitophagy, adenovirus-mediated Atg7 overexpressed cultured neonatal rat cardiomyocytes (NRCs) were treated with mitophagy inducer carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in vitro and stained with organelle-specific mitochondria and lysosome dyes to count mitophagy fluorescent dots by confocal microscopy. Atg7 overexpressed NRCs exhibited an increase in mitophagy dots than β-gal treated NRCs following CCCP induction. Further, we have treated cardiac specific Atg7 overexpressed (Atg7 Tg) mice and littermate non-transgenic (Ntg) mice with CCCP to assess mitophagy induction in vivo . Immunoblotting confirmed enhanced accumulation of mature autophagolysosomal marker LC3-II protein in mitochondrial fraction from Atg7 Tg hearts. Conclusions: Our in vitro and in vivo findings indicate that Atg7 localizes to the mitochondrial membrane, regulates recruitment and initiation of mitophagy machinery, and facilitates mitophagy in cardiomyocytes.