Metformin Inhibits Autophagy, Mitophagy and Antagonizes Doxorubicin-Induced Cardiomyocyte Death

Article Metformin Inhibits Autophagy, Mitophagy and Antagonizes Doxorubicin-Induced Cardiomyocyte Death Jennifer Van 1, Younghee Hahn 1, Brett Silverstein 1, Cairong Li 2, Fei Cai 2, Jia Wei 3, Lokesh Katiki 1, Puja Mehta 1, Katherine Livatova 1, Jaclyn DelPozzo 1, Tamayo Kobayashi 1, Yuan Huang 1, Satoru Kobayashi 1, and Qiangrong Liang 1, * 1 Department of Biomedical Sciences, New York Institute of Technology, College of Osteopathic Medicine, Old Westbury, New York 10001, United States 2 Clinical Medical College, Hubei University of Science and Technology, Xianning 332306, China 3 Department of Cardiology, the Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an 710000 , China * Correspondence: qliang03@nyit.edu Received: 28 November 2022 Accepted: 12 January 2023 Published: 17 February 2023 Abstract: The antidiabetic drug metformin has been shown to reduce cardiac injury under various pathological conditions, including anticancer drug doxorubicin (DOX)-induced cardiotoxicity, which makes metformin a prime candidate for repurposing. However, the mechanisms that mediate the cardioprotective effects of metformin remain highly controversial. In this study, we tested a prevailing hypothesis that metformin activates autophagy/mitophagy to reduce DOX cardiotoxicity. FVB/N mice and H9C2 cardiac myoblasts were treated with metformin, respectively. Autophagy/mitophagy was determined by Western blot analysis of microtubule-associated protein light chain 3, form-II (LC3-II), a well-established marker of autophagic vesicles. Although metformin had minimal effects on basal LC3-II levels, it significantly inhibited the accumulation of LC3-II levels by the lysosomal protease inhibitors pepstatin A and E64d in both total cell lysates and mitochondrial fractions. Also, dual fluorescent autophagy/mitophagy reporters demonstrated that metformin slowed the degradation rate of autophagic cargos or mitochondrial fragments in the lysosomes. These surprising results suggest that metformin inhibits rather than stimulates autophagy/mitophagy, sharply contrasting the popular belief. In addition, metformin diminished DOX-induced autophagy/mitophagy as well as cardiomyocyte death. Together, these results suggest that the cardioprotective effects of metformin against DOX cardiotoxicity may be mediated by its ability to inhibit autophagy and mitophagy, although the underlying molecular mechanisms remain to be determined.