Inhibition of Calpain-1 ameliorates doxorubicin-induced cardiotoxicity by suppression of p53-mediated ferroptosis.

Doxorubicin (Dox), a widely applied anthracycline antitumor agent, exhibits severe dose-dependent cardiotoxicity that greatly restricts its clinical utilization, yet the molecular mechanism linking Calpain-1 to cardiomyocyte ferroptosis in doxorubicin-induced cardiotoxicity (DIC) remains poorly clarified. This study aimed to explore how Calpain-1 regulates cardiomyocyte ferroptosis in DIC. Differentially expressed proteins from Dox-treated H9c2 cells were screened by proteomics, stable Calpain-1 knockdown cell lines were generated to detect cell viability and ferroptosis indicators through CCK-8, FerroOrange staining, biochemical kits and western blotting, and PPI network, co-IP and cycloheximide chase assays were performed to confirm Calpain-1-p53 binding and p53 protein stability regulation, with p53 rescue tests further verifying their functional relationship. In vivo, Calpain-1 expression was detected in Dox-exposed mouse hearts, and AAV9-driven myocardial Calpain-1 knockdown was established in DIC mice, where myocardial injury and ferroptosis were evaluated by HE staining, serum CK-MB/LDH and protein detection. Calpain-1 was significantly upregulated in Dox-stimulated cardiomyocytes and cardiac tissue. Calpain-1 silencing alleviated Dox-induced ferroptosis in vitro, with decreased Fe2+, MDA, LPO and ACSL4, elevated GSH and SLC7A11; co-IP verified direct Calpain-1-p53 interaction, and cycloheximide assays proved Calpain-1 inhibits p53 degradation to accumulate p53 protein, whereas p53 overexpression completely abolished the protective effect of Calpain-1 knockdown. Consistent with cellular results, cardiac-specific Calpain-1 knockdown mitigated myocardial damage and suppressed ferroptosis in DIC mice. These findings suggest that Calpain-1 exacerbates DIC by stabilizing p53 to initiate p53-dependent cardiomyocyte ferroptosis, offering a novel therapeutic target and theoretical basis for DIC intervention.