Ubiquitin Specific Peptidase 51 Exacerbates Skeletal Muscle Injury by Enhancing APAF1 ‐Mediated Apoptosis and Pyroptosis

ABSTRACT Skeletal muscle injury (SMI) involves complex cellular events, including oxidative stress, inflammation, and programmed cell death. However, the molecular mechanisms governing the cell death pathways in SMI remain incompletely understood. In this study, we identified the deubiquitinating enzyme USP51 as a novel modulator of SMI. Ubiquitin Specific Peptidase 51 (USP51) expression is significantly upregulated in the tBHP‐induced oxidative stress C2C12 myotubes model. Functional assays demonstrated that USP51 knockdown enhances cell viability and suppresses both apoptosis and pyroptosis. Bioinformatic screening and protein–protein interaction analysis identified APAF1, a key regulator of intrinsic apoptosis, as a potential USP51‐interacting partner. Co‐immunoprecipitation and ubiquitination assays confirmed that USP51 interacts with and deubiquitinates APAF1, thereby increasing its protein stability in C2C12 cells. Notably, APAF1 overexpression in USP51‐deficient C2C12 myotubes mitigated the protective effects of USP51 knockdown, restoring apoptosis and pyroptosis. AAV‐mediated knockdown of USP51 alleviated muscle damage, preserved myofiber integrity, reduced inflammation, and decreased apoptotic and pyroptotic cell death in the SMI mouse model. However, concurrent APAF1 overexpression abolished these beneficial effects, underscoring the critical role of the USP51‐APAF1 axis in amplifying SMI. Collectively, our study suggests that targeting the USP51‐APAF1 axis may represent a promising therapeutic strategy for mitigating muscle damage.