Endurance exercise preconditioning alleviates ferroptosis induced by doxorubicin-induced cardiotoxicity through mitochondrial superoxide-dependent AMPK2 activation

Doxorubicin-induced cardiotoxicity (DIC) adversely impacts patients' long-term health and quality of life. Its underlying mechanism is complex, involving regulatory cell death mechanisms, such as ferroptosis and autophagy. Moreover, it is a challenge faced by patients undergoing cardiac rehabilitation. Endurance exercise (EExe) preconditioning effectively counters DIC injury, potentially through the adenosine monophosphateactivated protein kinase (AMPK) pathway. However, detailed studies on this process's mechanisms are scarce. Here, E-Exe preconditioning and DIC models were established using mice and primary cultured adult mouse cardiomyocytes (PAMCs). Akin to ferrostatin-1 (ferroptosis inhibitor), rapamycin (autophagic inducer), and MitoTEMPO (mitochondrial free-radical scavenger), E-Exe preconditioning effectively alleviated Fe2+ accumulation and oxidative stress and improved energy metabolism and mitochondrial dysfunction in DIC injury, as demonstrated by multifunctional, enzymatic, and morphological indices. However, erastin (ferroptosis inducer), 3-methyladenine (autophagic inhibitor), adenovirus-mediated AMPK alpha 2 downregulation, and AMPK alpha 2 inhibition by compound C significantly diminished these effects, both in vivo and in vitro. The results suggest a nontraditional mechanism where E-Exe preconditioning, under mild mitochondrial reactive oxygen species generation, upregulates and phosphorylates AMPK alpha 2, thereby enhancing mitochondrial complex I activity, activating adaptive autophagy, and improving myocardial tolerance to DIC injury. Overall, this study highlighted the pivotal role of mitochondria in myocardial DIC-induced ferroptosis and shows how E-Exe preconditioning activated AMPK alpha 2 against myocardial DIC injury. This suggests that E-Exe preconditioning could be a viable strategy for patients undergoing cardiac rehabilitation.