During the aging process, mammals lose up to a third of their skeletal muscle mass and strength. Although the mechanisms underlying this loss are not entirely understood, we attempted to moderate the loss by increasing the regenerative capacity of muscle. This involved the injection of a recombinant adeno-associated virus directing overexpression of insulin-like growth factor I (IGF-I) in differentiated muscle fibers. We demonstrate that the IGF-I expression promotes an average increase of 15% in muscle mass and a 14% increase in strength in young adult mice, and remarkably, prevents aging-related muscle changes in old adult mice, resulting in a 27% increase in strength as compared with uninjected old muscles. Muscle mass and fiber type distributions were maintained at levels similar to those in young adults. We propose that these effects are primarily due to stimulation of muscle regeneration via the activation of satellite cells by IGF-I. This supports the hypothesis that the primary cause of aging-related impairment of muscle function is a cumulative failure to repair damage sustained during muscle utilization. Our results suggest that gene transfer of IGF-I into muscle could form the basis of a human gene therapy for preventing the loss of muscle function associated with aging and may be of benefit in diseases where the rate of damage to skeletal muscle is accelerated.

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene, leading to the absence of the dystrophin protein in striated muscle. A significant number of these mutations are premature stop codons. On the basis of the observation that aminoglycoside treatment can suppress stop codons in cultured cells, we tested the effect of gentamicin on cultured muscle cells from the mdx mouse -an animal model for DMD that possesses a premature stop codon in the dystrophin gene. Exposure of mdx myotubes to gentamicin led to the expression and localization of dystrophin to the cell membrane. We then evaluated the effects of differing dosages of gentamicin on expression and functional protection of the muscles of mdx mice. We identified a treatment regimen that resulted in the presence of dystrophin in the cell membrane in all striated muscles examined and that provided functional protection against muscular injury. To our knowledge, our results are the first to demonstrate that aminoglycosides can suppress stop codons not only in vitro but also in vivo. Furthermore, these results raise the possibility of a novel treatment regimen for muscular dystrophy and other diseases caused by premature stop codon mutations. This treatment could prove effective in up to 15% of patients with DMD.

Insulin-like growth factor I (IGF-I) is critical in promoting growth of skeletal muscle. When IGF-I is introduced into mouse hindlimb muscles by viral-mediated gene transfer, local overexpression of IGF-I produces significant increases in muscle mass and strength compared with untreated controls (Barton-Davis et al. 1998). We have proposed that this functional hypertrophy is primarily owing to the activation of satellite cells which leads to increased muscle regeneration. In order to test if satellite cells are essential in mediating the hypertrophic effects of IGF-I, we used gamma radiation to destroy the proliferative capacity of satellite cells. The right hindlimbs of adult C57BL/6 male mice were subjected to one of the following treatments: (1) 2,500 rad gamma radiation only, (2) viral-mediated gene transfer of IGF-I only, (3) 2,500 rad gamma radiation plus viral-mediated gene transfer of IGF-I, or (4) no intervention as a control. Approximately 4 months after treatment, the extensor digitorum longus muscles (EDL) from both hindlimbs were removed for mechanical and morphological measurements. Treatment with gamma radiation significantly prevented normal growth of the muscle. When combined with IGF-I treatment, approximately half of the IGF-I effect was prevented by gamma radiation treatment. This suggests that the remaining half of IGF-I induced hypertrophy is owing to paracrine/autocrine effects on the adult myofibres. Thus, these data are consistent with a mechanism by which IGF-I induced muscle hypertrophy via a combination of satellite cell activation and increasing protein synthesis in differentiated myofibres.

In humans, a subset of cases of Limb-girdle muscular dystrophy (LGMD) arise from mutations in the genes encoding one of the sarcoglycan (alpha, beta, gamma, or delta) subunits of the dystrophin-glycoprotein complex. While adeno-associated virus (AAV) is a potential gene therapy vector for these dystrophies, it is unclear if AAV can be used if a diseased muscle is undergoing rapid degeneration and necrosis. The skeletal muscles of mice lacking gamma-sarcoglycan (gsg-/- mice) differ from the animal models that have been evaluated to date in that the severity of the skeletal muscle pathology is much greater and more representative of that of humans with muscular dystrophy. Following direct muscle injection of a recombinant AAV [in which human gamma-sarcoglycan expression is driven by a truncated muscle creatine kinase (MCK) promoter/enhancer], we observed significant numbers of muscle fibers expressing gamma-sarcoglycan and an overall improvement of the histologic pattern of dystrophy. However, these results could be achieved only if injections into the muscle were prior to the development of significant fibrosis in the muscle. The results presented in this report show promise for AAV gene therapy for LGMD, but underscore the need for intervention early in the time course of the disease process.