Analyzing the Role of Dysferlin in Skeletal Muscle in Vitro and in Vivo
We have been studying Ca++ signaling in myofibers in culture that are wild type or dysferlin-null, or that express pathogenic mutants or fragments of dysferlin introduced by electroporation. We have also been examining the effects on Ca++ signaling of different drugs that target the L-type Ca++ channels and ryanodine receptors of skeletal muscle. Our experiments should reveal how dysferlin promotes protection against the loss of Ca++ signaling following injury, as well as how it can promote subsequent repair, and how drugs targeting the calcium channel proteins of the triad junction can help prevent damage to dysferlin-null muscle. Our previous work showed clearly that most of the dysferlin in adult skeletal muscle concentrates in the transverse tubules (TT), and that the TT and Ca++ signaling are more easily damaged when dysferlin is absent. In the course of our studies, we identified a set of dysferlin mutants that do not target the TT, but that may show activity in different assays. By contrast, a few dysferlin mutations target the TT normally but remain inactive in our assays. In our current studies, we continue to examine the physiology of muscle expressing different pathogenic mutants and fragments of dysferlin, before and after injury, with a major focus on the C2A domain, which has a unique sequence and binding activities. We are also continuing to test two alternative hypotheses for the role of dysferlin: 1. Dysferlin must be present in the TT to be active in protecting muscle against damage, but its presence there is not sufficient for protection; or 2. Different parts of the dysferlin molecule, and different locations within the cell where it can concentrate, can mediate different activities of the protein in mature myofibers. Our experiments involve assays of Ca++ transients in control and dysferlin-null myofibers injured by hypo-osmotic shock and, in collaboration with Dr. Noah Weisleder (Ohio State University), by laser wounding. They also include studies of the binding of mutant dysferlins to two key proteins in muscle, caveolin 3, a TT protein, and MG53, involved in membrane repair.