Dosage-sensitive regulation of Cofilin-1 and LIMK signaling controls myoblast differentiation and fusion

Abstract Skeletal muscle differentiation requires coordinated cytoskeletal remodeling and transcriptional reprogramming. Members of the actin-depolymerizing factor/Cofilin family regulate actin filament turnover, yet the isoform-specific functions of Cofilin-1 and Cofilin-2 during myogenesis remain incompletely understood. Murine C2C12 myoblasts were used to investigate the expression and function of Cofilin isoforms during differentiation. CRISPR/Cas9-mediated knockout, shRNA-mediated knockdown, immunofluorescence, gene expression analyses, MRTF reporter assays, and pharmacological inhibition of LIM kinase were used to assess myogenic progression and signaling. Myogenic differentiation was accompanied by a pronounced isoform transition characterized by progressive downregulation of Cofilin-1 and Actin-Depolymerizing Factor together with increased expression of Cofilin-2. Loss of Cofilin-1 resulted in marked morphological abnormalities, impaired cell-cycle exit, elevated MRTF activity, and reduced myotube formation, whereas Cofilin-2 deficiency produced comparatively mild effects during early differentiation. Importantly, partial reduction of Cofilin-1 enhanced myoblast fusion, while stronger depletion impaired differentiation, demonstrating a dosage-sensitive requirement for Cofilin-1. In addition, Cofilin-1 activity was dynamically regulated by transient LIM kinase-mediated phosphorylation during early differentiation, and inhibition of this pathway disrupted myogenic progression. Analyses of mRNA and protein stability indicated that isoform switching is primarily regulated at the level of gene expression. Cofilin-1 functions as a dosage-sensitive regulator of myogenic progression that links cytoskeletal remodeling to signaling pathways controlling proliferation and differentiation. Coordinated regulation of Cofilin-1 expression and activity, together with the transition to Cofilin-2, is required for efficient myoblast fusion and muscle formation.