The Role of Autophagy in Hydrogen Peroxide‐ or Methylglyoxal‐induced Cellular Damage in H9c2 Cells

Abstract

Oxidative stress (e.g., increased hydrogen peroxide [H2O2] levels) and dicarbonyl stress (e.g. increased methylglyoxal [MG]) serves as an initiator for the pathogenesis of cardiovascular diseases and diabetic complications. Increased H2O2‐derived free radicals and MG can be highly reactive and attack cellular components resulting in cell damage and even cell death. Normally, endogenous antioxidant mechanisms and glyoxalase degrade H2O2 and MG, respectively. Additionally, autophagy facilitates the break‐down of damaged cellular components allowing the components to be recycled by the cells. Researchers are still unclear about whether autophagy exacerbates cell damage or protects cells under higher doses of H2O2 and MG. This study investigated cell damage and autophagy changes when rat cardiac H9c2 myoblasts were treated with H2O2 or MG. Moreover, autophagy enhancing or inhibiting drugs were tested to evaluate their effects on H2O2‐ or MG‐induced cell damage. We found that H2O2 (300–900 mM, n=4) dose‐dependently decreased cell viability via measuring live cell dehydrogenase activity. H2O2 (600 mM, n=4) decreased cell viability to 16 ± 1% of the control. Similarly, MG (400–1400 mM, n=5) decreased cell viability in a dose‐dependent manner. MG (1200 mM, n=5) decreased cell viability to 29 ± 6% of the control. These results were further confirmed with the use of double staining, which contained calcein‐AM for labeling live cells and propodium iodide for labeling dead cells. By using Cyto ID autophagy detection dye, we found that H9c2 cells exhibited higher autophagy fluorescence after incubation of H2O2 (300–600 mM, n=2) or MG (400–1200 mM, n=2). Furthermore, trehalose (0.1–100 mM, n=3), an autophagy enhancer, reduced MG (1200 mM) induced cell damage. Trehalose (100 mM, n=3) increased cell viability by 69 ± 27% when compared to MG (1200 mM). Our preliminary results suggest that higher doses of H2O2 and MG can cause cell damage accompanied with increased autophagy. The autophagy enhancer trehalose provided cell protection against MG. Further studies are needed to confirm the effects of autophagy modulation on H2O2 and MG‐induced cell damage by using different autophagy modulators.Support or Funding InformationThis research was supported by the Division of Research, Department of Biomedical Sciences, and the Center for Chronic Disorders of Aging at Philadelphia College of Osteopathic Medicine.