Intrinsic Dysfunction in Muscle Stem Cells Lacking Dystrophin Disrupts Secondary Myogenesis and Postnatal Muscle Regeneration

Abstract

Dystrophin deficiency is the underlying cause of Duchenne Muscular Dystrophy (DMD), a severe neuromuscular disorder characterized by progressive muscle degeneration and a significantly diminished lifespan. While DMD is conventionally understood to manifest postnatally due to cumulative muscle damage, the intrinsic dysfunction of muscle stem cells (MuSCs) also contributes to disease progression. Specifically, the absence of dystrophin-MARK2 interactions in activated MuSCs impairs their polarity, resulting in fewer asymmetric divisions and progenitor cells. However, the role of impaired MuSC polarity in muscle development and regeneration has not been extensively studied in vivo. Here, we describe the consequences of MuSC dysfunction during fetal, neonatal, and regenerative myogenesis using the mdx mouse model of DMD. We first characterized disease progression, muscle regeneration, and MuSC function in postnatal muscle. Dystrophin-deficient muscle undergoes progressive changes with age and delayed regeneration in response to acute injury. Moreover, defective MuSC polarity results in fewer progenitor cells at 5 days post-cardiotoxin injury and at neonatal day 7, before chronic inflammation and severe necrosis. We also observe that mdx MuSCs exhibit elevated PAX7⁺ cell engraftment with significantly fewer donor-derived myonuclei in regenerated myofibers. We next explored the developmental onset of mdx MuSC dysfunction. Examination of mdx mouse embryos lacking dystrophin revealed no impairment of the primary myogenic program. By contrast, histological and single cell RNA-sequencing analysis during secondary myogenesis uncovered a reduced proportion of progenitor cells relative to fetal (f) MuSCs, leading to fewer smaller-caliber myofibers. Wild type fMuSCs expressed full-length dystrophin that interacted with MARK2, whereas mdx fMuSCs downregulated MARK2 and NUMB and exhibited markedly reduced polarization. Strikingly, deletion of the Numb Associated Kinase, AAK1, restores NUMB expression in mdx fMuSCs and rescues the proportion of myogenic committed progenitor cells in mdx fetal muscle. Our data provides evidence that dystrophin deficiency in MuSCs and myofibers contributes to the progression of DMD. We also elucidate an acute disease pathology during DMD fetal development and the potential for therapeutic intervention by targeting MuSC.