The Rho-GAP GRAF1 Regulates Skeletal Muscle Maturation and Repair

Lenhart, Kaitlin Christine

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March 20, 2019

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Lenhart, Kaitlin Christine
- Affiliation: School of Medicine, Department of Pathology and Laboratory Medicine

Abstract

Skeletal muscle is a large and highly specialized tissue requiring tightly regulated processes during development and maintenance to prevent the manifestation of debilitating myopathies. The fusion of myoblasts into mature multinucleated syncitia is a critical component of muscle formation and is regulated by the tight coordination of actin- and membrane-based dynamics; however, the spatial/temporal regulation of and interrelationship between these processes is incompletely understood. We recently reported that the BAR domain-containing Rho-GAP, GRAF1, is particularly abundant in perinatal muscle undergoing fusion to form multinucleated muscle fibers and that enforced expression of GRAF1 in cultured myoblasts induced robust fusion by a process that required GAP-dependent actin remodeling and BAR domain-dependent membrane sculpting. Herein we developed a novel GRAF1-deficient mouse line to explore a role for this protein in the formation/maturation of myotubes in vivo. Adult GRAF1-deficient mice exhibited a significant decrease in grip strength with muscle analysis revealing a significant reduction in cross-sectional area and impaired regenerative capacity, indicating a deficiency in myoblast fusion. Indeed, a significant fusion defect was recapitulated in isolated myoblasts depleted of GRAF1. Mechanistically, we show that GRAF1 associates with endocytic recycling vesicles and facilitates myoblast fusion, at least in part, by promoting vesicle-mediated translocation of fusogenic ferlin proteins to the plasma membrane. Muscle plasma membrane is particular vulnerability to contraction-induced rupture and possesses specialized mechanisms for rapid membrane repair so as to preserve the syncytia. Dysferlin has been established as a critical regulator of muscle plasma membrane resealing; however, the mechanism(s) which govern dysferlin trafficking to sites of membrane damage require further investigation. We show that GRAF1 associates with and mediates deposition of dysferlin at plasma membranes of injured muscle, implicating a novel role for GRAF1 in dysferlin-mediated membrane repair. In support of this, GRAF1 depletion enhanced susceptibility to induced membrane injury in isolated myoblasts and as anticipated, exacerbated some hallmarks of muscle degeneration in the mdx mouse. Interestingly, GRAF1-deficient mdx mice exhibited unprecedented myofiber expansion and muscle growth, implicating an additional role for GRAF1 in skeletal muscle–injury response. Together, these data shed new light on the importance of GRAF1 in skeletal muscle formation and maintenance.

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