SS-31 Attenuates Doxorubicin-induced Cardiomyoblast H9C2 Cell Senescence

Abstract ID 92620 Poster Board 463 Background: Doxorubicin (DOX), an effective drug for many types of cancer, is associated with substantial cardiotoxicity. A 3-hour treatment of cardiomyoblast H9C2 cells with a low concentration of DOX (100 nM) can induce senescence-associated β-galactosidase (SA β-gal) staining, the gold standard of cell senescence. Here, we comprehensively characterized the phenotype of the DOX-induced senescent cardiomyocytes for the first time. We plan to use this cell model to search for effective treatments for DOX-induced cell senescence. Methods and Results: 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 hours 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 at S phase and G2 phase, 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), which is in clinical trials for heart failure and other diseases associated with mitochondrial dysfunction, partially attenuated 50 nM DOX-induced SA β-gal staining from 51.4% to 35.8%. SS-31 also prevented increases of the p16, p21, and SASP markers and mitigated mitochondrial ROS production. SS-31 also reversed the 50 nM DOX-induced elevation of mitochondrial respiration. However, 1 μM SS-31 failed to prevent the cell cycle arrest and cell growth retardation induced by 50 nM DOX. Conclusion: We determined that a 3-hour treatment with 50 nM DOX establishes a H9C2 cell senescence model. Treatment with mitochondrial-targeted SS-31 reverses the 50 nM 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. Keywords: Doxorubicin; H9C2 cell; Senescence; Mitochondria; SS-31 H.Z. received support from NIH (R35GM151226) and UAMS (Development Enhancement Awards for Proposals).