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Updated: May 14, 2026

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells
Published on: June 12, 2017
AMPK Enhances Autophagy and Inhibits Ferroptosis by Upregulating ULK1 to Ameliorate Acute Skeletal Muscle
Wanghai Li1, Jiaoqing Zhou1, Jiandong Guo1
1Department of Interventional Radiology and Vascular Surgery, The First Affiliated Hospital of Jinan University, Guangzhou, Guangdong, China.
Abstract:
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.
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