Inhibition or loss of myostatin activity during development causes excessive muscle growth, but little is known about the consequences of reducing myostatin activity after muscle has stopped growing. The broad goal of this research is to understand what happens to skeletal muscle when myostatin activity is reduced post-developmentally. The proposed experiments will address three specific aims: 1. to test the hypothesis that post-developmental myostatin depletion will increase muscle mass in mice, that this effect will be mediated by muscle fiber hypertrophy with no increase in the number of muscle fibers or the number of myonuclei per muscle, and that the effect will persist long term (tested up to one year after myostatin depletion);2. to examine certain aspects of muscle quality after myostatin depletion, including capacity for force generation, susceptibility to stretch-induced injury, fatigability, proportion of different fiber types as classified by expression of myosin heavy chain isoforms or SDH activity, collagen content, and expression of procollagen genes;and, 3. to test the hypothesis that post-developmental myostatin deficiency will induce muscle hypertrophy via increased myofibrillar protein synthesis rather than inhibition of proteolysis, will upregulate expression of genes involved in mRNA translation, and will stimulate mRNA translation through activation of the Akt-mTOR-4EBP1/S6K1 pathway. Post-developmental disruption of the myostatin gene will be accomplished by activation of a tamoxifen-inducible Cre recombinase, constitutively expressed by a transgene, in mice in which the critical third exon of the myostatin gene is flanked with loxP sequences (the targets of Cre recombinase). Myostatin activity will be inhibited in short-term mechanistic studies by injecting the JA16 anti-myostatin antibody. Myofibrillar protein synthesis will be determined by stable isotope tracer incorporation. Gene expression will be determined by quantitative RT-PCR. Concentrations and phosphorylation status of selected proteins will be determined by immunoblotting. Immunohistochemistry will be used to assess muscle fiber hypertrophy, fiber types, numbers of fibers, and myonuclear domain volume. Force generation, fatigability, and stretch injury will be studied by stimulation of isolated muscles in vitro. Relevance to public health: Muscle atrophy causes weakness in various conditions, including muscular dystrophies, cancer, glucocorticoid administration, inactivity, arthritis, malnutrition, AIDS, and old age. Reducing myostatin activity in these conditions might ameliorate this muscle loss and weakness. Knowledge of the physiological and molecular effects of reducing myostatin activity in mature muscle will reveal potential benefits and limitations of anti-myostatin therapy.
