Muscle growth occurs during embryonic development and continues in adult life as regeneration. During embryonic muscle growth and regeneration in mature muscle, singly nucleated myoblasts fuse to each other to form myotubes. In muscle growth, singly nucleated myoblasts can also fuse to existing large,syncytial myofibers as a mechanism of increasing muscle mass without increasing myofiber number. Myoblast fusion requires the alignment and fusion of two apposed lipid bilayers. The repair of muscle plasma membrane disruptions also relies on the fusion of two apposed lipid bilayers. The protein dysferlin, the product of the Limb Girdle Muscular Dystrophy type 2 locus, has been shown to be necessary for efficient, calcium-sensitive,membrane resealing. We now show that the related protein myoferlin is highly expressed in myoblasts undergoing fusion, and is expressed at the site of myoblasts fusing to myotubes. Like dysferlin, we found that myoferlin binds phospholipids in a calcium-sensitive manner that requires the first C2A domain. We generated mice with a null allele of myoferlin. Myoferlin null myoblasts undergo initial fusion events, but they form large myotubes less efficiently in vitro, consistent with a defect in a later stage of myogenesis. In vivo, myoferlin null mice have smaller muscles than controls do, and myoferlin null muscle lacks large diameter myofibers. Additionally, myoferlin null muscle does not regenerate as well as wild-type muscle does, and instead displays a dystrophic phenotype. These data support a role for myoferlin in the maturation of myotubes and the formation of large myotubes that arise from the fusion of myoblasts to multinucleate myotubes.

Mutations in dysferlin, a novel membrane protein of unknown function, lead to muscular dystrophy. Myoferlin is highly homologous to dysferlin and like dysferlin is a plasma membrane protein with six C2 domains highly expressed in muscle. C2 domains are found in a variety of membrane-associated proteins where they have been implicated in calcium, phospholipid, and protein-binding. We investigated the pattern of dysferlin and myoferlin expression in a cell culture model of muscle development and found that dysferlin is expressed in mature myotubes. In contrast, myoferlin is highly expressed in elongated "prefusion" myoblasts and is decreased in mature myotubes where dysferlin expression is greatest. We tested ferlin C2 domains for their ability to bind phospholipid in a calcium-sensitive manner. We found that C2A, the first C2 domain of dysferlin and myoferlin, bound 50% phosphatidylserine and that phospholipid binding was regulated by calcium concentration. A dysferlin point mutation responsible for muscular dystrophy was engineered into the dysferlin C2A domain and demonstrated reduced calcium-sensitive phospholipid binding. Based on these data, we propose a mechanism for muscular dystrophy in which calcium-regulated phospholipid binding is abnormal, leading to defective maintenance and repair of muscle membranes.

Skeletal muscle is a multinucleated syncytium that develops and is maintained by the fusion of myoblasts to the syncytium. Myoblast fusion involves the regulated coalescence of two apposed membranes. Myoferlin is a membrane-anchored, multiple C2 domain-containing protein that is highly expressed in fusing myoblasts and required for efficient myoblast fusion to myotubes. We found that myoferlin binds directly to the eps15 homology domain protein, EHD2. Members of the EHD family have been previously implicated in endocytosis as well as endocytic recycling, a process where membrane proteins internalized by endocytosis are returned to the plasma membrane. EHD2 binds directly to the second C2 domain of myoferlin, and EHD2 is reduced in myoferlin null myoblasts. In contrast to normal myoblasts, myoferlin null myoblasts accumulate labeled transferrin and have delayed recycling. Introduction of dominant negative EHD2 into myoblasts leads to the sequestration of myoferlin and inhibition of myoblast fusion. The interaction of myoferlin with EHD2 identifies molecular overlap between the endocytic recycling pathway and the machinery that regulates myoblast membrane fusion.

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The genetic understanding of the muscular dystrophies has advanced considerably in the last two decades. Over 25 different individual genes are now known to produce muscular dystrophy, and many different "private" mutations have been described for each individual muscular dystrophy gene. For the more common forms of muscular dystrophy, phenotypic variability can be explained by precise mutations. However, for many genetic mutations, the presence of the identical mutation is associated with marked phenotypic range that affects muscle function as well as cardiac function. The explanation for phenotype variability in the muscular dystrophies is only now being explored. The availability of genetically engineered animal models has allowed the generation of single mutations on the background of highly inbred strain. Phenotypic variation that is altered by genetic background argues for the presence of genetic modifier loci that can ameliorate or enhance aspects of the dystrophic phenotype. A number of individual genes have been implicated as modifiers of muscular dystrophy by studies in genetically engineered mouse models of muscular dystrophy. The value of these genes and products is that the pathways identified through these experiments may be exploited for therapy.