Impact of C4BPA on Muscle progenitor cell differentiation: insights for Duchenne muscular dystrophy treatment

Fibroadipogenic precursor cells (FAPs) are key contributors to the fibrotic and adipogenic remodeling observed in Duchenne muscular dystrophy (DMD), yet their precise role in muscle degeneration remains unclear. In this study, we investigated how FAPs from DMD muscle influence myogenesis by conducting co-culture experiments using healthy myoblasts with FAPs derived from either healthy or DMD biopsies, in both direct and indirect contact conditions. We observed that healthy FAPs enhance myogenic differentiation, increasing myotube area, while DMD FAPs significantly inhibit it. To identify underlying molecular mechanisms, we performed mass spectrometry of the FAP secretome and found that 368 of 760 detected proteins were upregulated in DMD FAPs, including C4b-binding protein alpha chain (C4BPA), which showed a notable increase. In vitro treatment of myoblasts with recombinant C4BPA led to severe impairment of myotube formation, evidenced by reduced myotube area, fewer nuclei per myotube, and smaller myotube size. C4BPA exposure also downregulated myogenic markers and upregulated genes associated with muscle atrophy. These findings were further validated in a 3D engineered muscle model, where C4BPA significantly reduced contractile function. Importantly, silencing C4BPA in DMD-derived cultures partially restored myogenic capacity, improving both the differentiation index and nuclear content per myotube. Together, our data demonstrate that DMD FAPs exert anti-myogenic effects, at least in part, through elevated secretion of C4BPA, which interferes with muscle differentiation and function. This highlights C4BPA as a novel effector of muscle degeneration and a promising therapeutic target for modulating the fibrotic environment in DMD. Targeting specific secreted factors from pathological FAPs may help preserve muscle regeneration and mitigate disease progression in dystrophic muscles.