AMPK Enhances Autophagy and Inhibits Ferroptosis by Upregulating ULK1 to Ameliorate Acute Skeletal Muscle Ischemia-Reperfusion Injury

Skeletal muscle ischemia-reperfusion (I/R) injury is a critical clinical condition. AMP-activated protein kinase (AMPK) during IR injury remains unclear. A hindlimb I/R model was established in Sprague-Dawley rats using vascular occlusion. AMPK Activator Metformin and AMPK Inhibitor Compound C were applied to investigate the effects of AMPK on hindlimb I/R injury. Differentiated C2C12 myotubes were subjected to hypoxia/reoxygenation to mimic I/R injury in vitro. Histopathology, Apoptosis, and angiogenesis were observed by Hematoxylin-Eosin, TUNEL staining, and immunohistochemistry. Western blotting and Enzyme-Linked Immunosorbent Assay were performed to assess Autophagy and ferroptosis-related Indicators. Co-immunoprecipitation validated AMPK-ULK1 interactions. Furthermore, Unc-51-like kinase 1 (ULK1) was inhibited to explore its effects in vivo and in vitro. AMPK activation via Metformin reduced I/R-induced muscle damage, Apoptosis, and endothelial dysfunction, while Compound C exacerbated these effects. Metformin upregulated p-AMPK and suppressed Ferroptosis in vivo. In C2C12 cells, AMPK overexpression attenuated hypoxia/reoxygenation-induced Ferroptosis (increased GPX4 and SLC7A11) and activated Autophagy (increased LC3-II/LC3-I and decreased p62). Mechanistically, AMPK phosphorylated ULK1 at Ser555, promoting Atg7/ATG5-dependent Autophagy. ULK1 inhibition suppressed Autophagy and abolished AMPK's protective effects, exacerbating muscle injury and Ferroptosis. AMPK activation protects against skeletal muscle I/R injury by enhancing ULK1/ATG-mediated Autophagy and inhibiting Ferroptosis.