No abstract

FilaminC (FLNc) is the muscle-specific member of a family of actin binding proteins. Although it interacts with many proteins involved in muscular dystrophies, its unique role in muscle is poorly understood. To address this, two models were developed. First, FLNc expression was stably reduced in C2C12 myoblasts by RNA interference. While these cells start differentiation normally, they display defects in differentiation and fusion ability and ultimately form multinucleated "myoballs" rather than maintain elongated morphology. Second, a mouse model carrying a deletion of last 8 exons of Flnc was developed. FLNc-deficient mice die shortly after birth, due to respiratory failure, and have severely reduced birth weights, with fewer muscle fibers and primary myotubes, indicating defects in primary myogenesis. They exhibit variation in fiber size, fibers with centrally located nuclei, and some rounded fibers resembling the in vitro phenotype. The similarity of the phenotype of FLNc-deficient mice to the filamin-interacting TRIO null mice was further confirmed by comparing FLNc-deficient C2C12 cells to TRIO-deficient cells. These data provide the first evidence that FLNc has a crucial role in muscle development and maintenance of muscle structural integrity and suggest the presence of a TRIO-FLNcdependent pathway in maintaining proper myotube structure.

Limb-girdle muscular dystrophy, type 2A (LGMD 2A), is an autosomal recessive disorder that causes late-onset muscle-wasting, and is due to mutations in the muscle-specific protease calpain 3 (C3). Although LGMD 2A would be a feasible candidate for gene therapy, the reported instability of C3 in vitro raised questions about the potential of obtaining a stable, high-level expression of C3 from a transgene in vivo. We have generated transgenic (Tg) mice with muscle-specific overexpression of full-length C3 or C3 isoforms, which arise from alternative splicing, to test whether stable expression of C3 transgenes could occur in vivo. Unexpectedly, we found that full-length C3 can be overexpressed at high levels in vivo, without toxicity. In addition, we found that Tg expressing C3 lacking exon 6, an isoform expressed embryonically, have muscles that resemble regenerating or developing muscle. Tg expressing C3 lacking exon 15 shared this morphology in the soleus, but not other muscles. Assays of inflammation or muscle membrane damage indicated that the Tg muscles were not degenerative, suggesting that the immature muscle resulted from a developmental block rather than degeneration and regeneration. These studies show that C3 can be expressed stably in vivo from a transgene, and indicate that alternatively spliced C3 isoforms should not be used in gene-therapy applications because they impair proper muscle development.