Time-Dependent Autophagy Flux and Cell Death Responses in Cardiomyocytes Treated with Doxorubicin

Background: Doxorubicin (DOX), an anthracycline-based chemotherapeutic agent, is widely used in cancer treatment. However, its use is limited by cardiotoxicity. While the precise mechanisms underlying DOX-induced cardiotoxicity are not fully understood, they are believed to be multifactorial, involving dysregulation of autophagy, a catabolic process responsible for degrading and recycling cellular components. The impact of DOX on autophagic activity and the role of autophagy in mediating DOX cardiotoxicity remain highly controversial. objective: To determine the optimal duration of DOX treatment that affects autophagic flux in human cardiomyocytes (AC16) and to investigate the correlation between autophagy and DOX-induced cell death. Methods: Cardiomyocytes were seeded three days before DOX treatment. DOX was first administered at 5 p.m., followed by subsequent doses every 2 hours from 9 a.m. to 3 p.m. the next day, with cell harvesting at 5 p.m. Pepstatin A (PepA) and E64d inhibitors were added 8 hours prior to cell harvesting. Western blot analysis was performed using a 10% gel, and the membranes were incubated with antibodies against LC3B, cleaved caspase 3, PARP, and beta-actin. Images were captured, and band intensity was measured to assess autophagic flux and cell death. Results: During autophagy, LC3-I is converted to LC3-II. This correlates with the number of autophagosomes, making it a valuable marker for assessing autophagic activity. DOX increased LC3-II protein levels at all time points throughout the 24-hour period, with further elevation observed in the presence of PepA and E64d, suggesting an accelerated autophagic flux. DOX treatment also resulted in increased levels of cleaved caspase 3 and PARP, indicating enhanced apoptotic cell death. Conclusion: Doxorubicin-treated cells exhibit a gradual increase in autophagy flux and cell death over a 24-hour period. Further investigation is required to clarify the role of autophagy in DOX-induced cardiomyocyte death. This study was supported by NIH grants 1R15HL163707-01A1 (QL) and 1R15HL161737-01 (SK). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.