Abstract Body: Backgroud: Skeletal muscle regeneration requires coordinated satellite cell activation. Gasdermin D (GSDMD) classically mediates pyroptosis, while its non-canonical roles in muscle repair remain unclear.
Hypothesis: GSDMD undergoes caspase-3-dependent, non-inflammatory processing during myogenesis to regulate muscle cell state transitions.
Aims: To determine the impact of GSDMD deficiency on muscle regeneration and characterize the functional roles of specific GSDMD cleavage products.
Mehods: Regeneration was assessed in GSDMD^-/- and WT mice following cardiotoxin injury. Mechanistic studies utilized primary satellite cells and C2C12 myoblasts via histology, force assessment, subcellular localization, GSDMD-N13 overexpression, and transcriptomic/in silico analysis.
Results: GSDMD deficiency significantly impaired muscle regeneration. Functional assessment showed reduced force generation (P=0.04), while RT-PCR revealed delayed Myod and Myh3 expression. Histological analysis confirmed reduced myofiber cross section area (P=0.021) and decreased eMyHC⁺ fibers. In vitro, GSDMD knockout increased cell doubling time (40.6h vs 22.2h), induced G1 stage arrest, impaired differentiation, and increased senescence (β-galactosidase staining+). Mechanistically, a non-canonical ~13kDa N-terminal fragment (GSDMD-N13) was detected concomitant with caspase-3 activation. Subcellular localization revealed marked nuclear enrichment of GSDMD-N13, while the C-terminal remained cytoplasmic. GSDMD-N13 overexpression increased proliferation (P=0.029), up-regulated Ccna2 (P=0.027), and reduced senescence. Furthermore, GSDMD-N13 overexpression partially rescued the GSDMD^-/- phenotype and improved in vivo force output. In silico perturbation identified GSDMD as a master regulator of myogenic gene programs. Transcriptomic analysis confirmed enrichment of cell cycle pathways (Myc/E2f targets; NES>1.5) and suppression of senescence/inflammatory pathways.
Conclusions: Caspase-3-dependent GSDMD processing generates a nuclear-translocating N13 fragment that promotes the myogenic cell cycle and prevents senescence. These findings highlight a novel, non-pyroptotic GSDMD function essential for effective muscle repair.