Metformin Regulates Autophagy to Attenuate Mitochondrial Dysfunction in High Glucose-stimulated H9C2 Cardiomyocytes via the AMPKα/Sirt1/Parkin Signaling Axis

Abstract

Abstract Purpose: Mitophagy reduces mitochondrial dysfunction and accumulation of reactive oxygen species (ROS) to prevent myocardial injury in diabetic cardiomyopathy (DCM). Accumulated studies have confirmed that metformin enhances autophagy to maintain mitochondrial homeostasis and scavenge ROS. However, whether and how metformin regulates mitophagy in cardiomyocytes remain unclear. Methods: Diabetic cardiomyopathy was modeled in H9c2 Cardiomyocytes treated with high glucose (30 mM) . Then high Glucose-stimulated H9C2 cells were exposed to metformin, AMPKα inhibitor and Sirt1 inhibitor for 24 h. Mitochondrial dysfunction and mitophagy were detected by fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), mitochondrial membrane potential(MMP), flow cytometry and western blot. Resluts: We found that the protein expression of Parkin, ROS level, and mitochondrial membrane potential showed dynamic changes in H9C2 cells under the stimulation of high glucose. Importantly, metformin enhanced mitophagy, scavenged ROS, improved mitochondrial function and inhibited apoptosis in H9C2 cells treated with high glucose. Mechanistically, metformin increased the protein expression of p-AMPKα, Sirt1, Parkin, and LC3-II in H9C2 cells after a high glucose challenge. Depletion of AMPKα and Sirt1 abolished the increase of protein levels of Parkin and LC3-II and mitophagy levels induced by metformin. Conclusion: Our data indicated that metformin improves mitochondrial dysfunction of H9C2 cells under hyperglycemia by activating AMPKα/Sirt1/Parkin-mediated mitophagy, which provides novel evidence for the treatment of DCM.