title: GRAF1 deficiency blunts sarcolemmal injury repair and exacerbates cardiac and skeletal muscle pathology in dystrophin-deficient mice.
abstract: BACKGROUND
  The plasma membranes of striated muscle cells are particularly susceptible to rupture as they endure significant mechanical stress and strain during muscle contraction, and studies have shown that defects in membrane repair can contribute to the progression of muscular dystrophy. The synaptotagmin-related protein, dysferlin, has been implicated in mediating rapid membrane repair through its ability to direct intracellular vesicles to sites of membrane injury. However, further work is required to identify the precise molecular mechanisms that govern dysferlin targeting and membrane repair. We previously showed that the bin-amphiphysin-Rvs (BAR)-pleckstrin homology (PH) domain containing Rho-GAP GTPase regulator associated with focal adhesion kinase-1 (GRAF1) was dynamically recruited to the tips of fusing myoblasts wherein it promoted membrane merging by facilitating ferlin-dependent capturing of intracellular vesicles. Because acute membrane repair responses involve similar vesicle trafficking complexes/events and because our prior studies in GRAF1-deficient tadpoles revealed a putative role for GRAF1 in maintaining muscle membrane integrity, we postulated that GRAF1 might also play an important role in facilitating dysferlin-dependent plasma membrane repair.
  
  
  METHODS
  We used an in vitro laser-injury model to test whether GRAF1 was necessary for efficient muscle membrane repair. We also generated dystrophin/GRAF1 doubledeficient mice by breeding mdx mice with GRAF1 hypomorphic mice. Evans blue dye uptake and extensive morphometric analyses were used to assess sarcolemmal integrity and related pathologies in cardiac and skeletal muscles isolated from these mice.
  
  
  RESULTS
  Herein, we show that GRAF1 is dynamically recruited to damaged skeletal and cardiac muscle plasma membranes and that GRAF1-depleted muscle cells have reduced membrane healing abilities. Moreover, we show that dystrophin depletion exacerbated muscle damage in GRAF1-deficient mice and that mice with dystrophin/GRAF1 double deficiency phenocopied the severe muscle pathologies observed in dystrophin/dysferlin-double null mice. Consistent with a model that GRAF1 facilitates dysferlin-dependent membrane patching, we found that GRAF1 associates with and regulates plasma membrane deposition of dysferlin.
  
  
  CONCLUSIONS
  Overall, our work indicates that GRAF1 facilitates dysferlin-dependent membrane repair following acute muscle injury. These findings indicate that GRAF1 might play a role in the phenotypic variation and pathological progression of cardiac and skeletal muscle degeneration in muscular dystrophy patients.
authors: Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 USA.
authors: Kaitlin C
authors: KC
authors: Lenhart
authors: Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 USA.
authors: Thomas J
authors: TJ
authors: O'Neill
authors: Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 USA.
authors: Zhaokang
authors: Z
authors: Cheng
authors: Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 USA.
authors: Rachel
authors: R
authors: Dee
authors: Center for Genetic Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611 USA.
authors: Alexis R
authors: AR
authors: Demonbreun
authors: Department of Gene Therapy Molecular Pharmaceutics, Northwestern University Feinberg School of Medicine, Chicago, IL USA.
authors: Jianbin
authors: J
authors: Li
authors: Department of Gene Therapy Molecular Pharmaceutics, Northwestern University Feinberg School of Medicine, Chicago, IL USA.
authors: Xiao
authors: X
authors: Xiao
authors: Center for Genetic Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611 USA.
authors: Elizabeth M
authors: EM
authors: McNally
authors: Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 USA ; McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 USA.
authors: Christopher P
authors: CP
authors: Mack
authors: Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 USA ; McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 USA.
authors: Joan M
authors: JM
authors: Taylor
keywords: Dysferlin; Muscle repair; Muscular dystrophy; Rho-GAP