Mitochondrial-Targeted SS-31 Attenuates the Doxorubicin-Induced Cardiomyoblast H9C2 Cell Senescence

Doxorubicin (DOX), an effective chemotherapeutic agent for many types of cancer, is known for significant cardiotoxic side effects, which largely limit its clinical usage. A 3 h treatment of cardiomyoblast H9C2 cells with a low concentration of DOX (100 nM) can induce senescence-associated β-galactosidase (SA β-gal) staining, a gold standard of cell senescence. In the current study, we comprehensively characterized the phenotype of the DOX-induced senescent cardiomyocytes for the first time. Establishing this in vitro model will facilitate an expanded capacity for searching for effective treatments for DOX-induced cell senescence. Using SA β-gal staining and cell growth rate as readouts, we assessed the concentration-dependent effect of DOX on H9C2 cell senescence. The cells were treated with DOX for 3 h and subsequently cultured for 3 days. We found that a 50 nM concentration of DOX induced ~50% SA β-gal staining and completely inhibited cell growth. The DOX-induced H9C2 cell senescence was further confirmed by several well-accepted senescence markers, including cell hypertrophy, increased p16 and p21 expression, increased Senescence Associated Secretory Phenotype (SASP) markers, arrested cell cycle, and increased ROS production. Interestingly, we found that 50 nM DOX increased mitochondrial respiration. Translationally, we found that mitochondrial-targeted tetrapeptide SS-31 (elamipretide, 1 µM) partially attenuated 50 nM DOX-induced SA β-gal staining from 51.4% to 35.8%. SS-31 also prevented increases in the p16, p21, and SASP markers and mitigated mitochondrial ROS production. Additionally, SS-31 reversed the 50 nM DOX-induced elevation of mitochondrial respiration. However, 1 µM SS-31 failed to prevent the cell cycle arrest induced by 50 nM DOX. Using a 3 h treatment of 50 nM DOX, we established an H9C2 cell senescence model. Treatment with SS-31 attenuates this DOX-induced cell senescence but not the cell cycle arrest. These data suggest that SS-31 is a promising drug to treat DOX-induced cardiomyocyte senescence.