Di Francescantonio, S. | Search bioRxiv

bioRxiv2026

Myofibers drive postnatal myonuclear accretion through Arp2/3-dependent membrane protrusions

Skeletal muscle postnatal growth depends on myoblast fusion with pre-existing myofibers, but whether myofibers actively regulate this process remains unclear. The Arp2/3 complex, which nucleates branched actin networks, is required for myoblast fusion during embryogenesis, but its role in postnatal myofibers is unknown. Here, we investigated the role of the Arp2/3 complex in myofibers using an inducible skeletal muscle-specific Arpc4 knockout model. We find that loss of Arp2/3 in myofibers impairs myonuclear accretion, resulting in smaller myofibers and reduced muscle strength. Satellite cells are still activated and able to differentiate but accumulate around Arpc4-deficient myofibers rather than fusing, pointing to a myofiber-intrinsic defect. In co-culture, myofibers form long-lived membrane protrusions enriched in Arp2/3 at myoblast contact sites, and optogenetic activation of protrusion in myofibers induced myoblast fusion. Genetic depletion of Arpc4 in myofibers markedly reduces membrane protrusions both in vitro and in vivo. These findings unveil a fundamental role for the Arp2/3 complex in postnatal muscle growth, and reveal a mechanism by which myofibers actively extend membrane protrusions to drive myoblast fusion. This repositions myofibers as active fusogenic partners and cell-autonomous drivers of their own nuclear accretion.

cell biology↗

bioRxiv2024

Gene-edited primary muscle stem cells rescue dysferlin-deficient muscular dystrophy

Dystrophy-associated fer-1-like protein (dysferlin) conducts plasma membrane repair. Mutations in the DYSF gene cause a panoply of genetic muscular dystrophies. We targeted a frequent loss-of-function, DYSF exon 44, founder frameshift mutation with mRNA-mediated delivery of SpCas9 in combination with a mutation-specific sgRNA to primary muscle stem cells from two homozygous patients. We observed a consistent >60% exon 44 re-framing, rescuing a full-length and functional dysferlin protein. A new mouse model harboring a humanized Dysf exon 44 with the founder mutation, hEx44mut, recapitulated the patients phenotype and an identical re-framing outcome in primary muscle stem cells. Finally, gene-edited murine primary muscle stem-cells were able to regenerate muscle and rescued dysferlin when transplanted back into hEx44mut hosts. These findings are the first to show that a CRISPR-mediated therapy can ameliorate dysferlin deficiency. We suggest that gene-edited primary muscle stem cells could exhibit utility, not only in treating dysferlin deficiency syndromes, but also perhaps other forms of muscular dystrophy.

genetics↗