Duchenne muscular dystrophy (DMD) is a common untreatable inherited disease with an incidence of approximately 1:3,500 male births. Patients generally present at three to five years of age with proximal muscle weakness and succumb to diaphragmatic respiratory insufficiency in the late teens to early twenties. The disease preferentially affects fast twitch muscle fibers, which undergo pathologic necrosis, degeneration, regeneration and fibrosis. At the genetic level DMD is caused by mutations of a gene on the X-chromosome named dystrophin, which encodes a large protein with overall structural similarity to cytoskeletal proteins. Dystrophin is found beneath the plasma membrane of all skeletal muscle cells where it functions as a bridge linking a family of sarcolemmal membrane proteins to the actin based cytoskeleton. This dystrophin associated complex is believed to serve both as receptor for extracellular ligands and as a structural element which protects the muscle from stress of contraction. The absence of dystrophin in DMD disrupts this associated complex; however, the specific cause of muscular dystrophy remains uncertain. Our recent studies show that the nitric oxide synthase (NOS) and dystrophin physically associate in a complex that is selectively present at the sarcolemma of fast twitch skeletal muscle fibers. Furthermore, we find that NOS is inappropriately displaced from this complex in dystrophic muscle. These unexpected results suggest novel mechanisms for signaling and disease associated with dystrophin. Nitric oxide (NO), the product of NOS, functions as an endogenous mediator in diverse processes throughout the body. In skeletal muscle NO participates in myoblast fusion and regulates contractile force in active muscle. On the other hand, because NO is responsible for tissue damage in certain diseases including autoimmune and neurodegenerative processes, the derangement of NOS function in dystrophic muscle may contribute to disease. We now propose to define the role of the NOS dystrophin complex in neuromuscular development and disease. To achieve a detailed understanding of the molecular interaction between these proteins, we will use mutagenesis to precisely define the domains near the N-terminus of NOS and C-terminus of dystrophin which mediate binding. Using cultured myoblasts we will elucidate mechanisms for regulation of NO mediated signaling by the dystrophin associated complex. Developmental studies will clarify the role for NO in dystrophin associated processes at the neuromuscular junction. Finally, we will determine whether the selective displacement of NOS from fast twitch muscle fibers contributes to the selective pathology of this fiber type on muscular dystrophy.
