Molecular Mechanisms of Muscular Dystrophies
A major focus of many of our studies has been to learn how changes in the composition and organization of the membrane systems of skeletal muscle can lead to muscular dystrophy. We have learned that the organization of structural proteins at the sarcolemma, and especially at costameres, is affected in several forms of muscular dystrophy. The changes at costameres are likely to account in part for the increased susceptibility to injury and to muscle weakness, both of which are typical of dystrophic muscle.
Although our earlier studies to date have focused on an animal model for Duchenne Muscular Dystrophy and for a congenital muscular dystrophy, we are now applying these findings to Facioscapulohumeral Muscular Dystrophy (FSHD), an autosomal dominant disease that affects 1 in 8,000 Americans. We are using our xenografting methods to create grafts of FSHD muscle tissue, to study the pathogenesis of the disease and to determine the physiological, morphological and biochemical changes that underlie it.
Our previous studies have shown that costameres are altered in muscles from patients with FSHD. We have been using large format 2D gel electrophoresis in a custom-built chamber (see figure) to characterize the proteome of muscles from patients with FSHD, to identify the proteins responsible for the altered costameres. The gels, which we typically analyze in quadrants (see figure) resolve ~2000 spots reliably.
To date, we have identified several proteins that change in FSHD muscle, and characterized one– µ-crystallin. µ-Crystallin is a thyroid-hormone binding protein present in the cytoplasm of many mammalian cells and so is unlikely to affect costameres directly. Instead, it probably regulates gene expression by controlling the amount of free thyroid hormone, needed for the extression of genes regulated by the thyroid hormone receptor. Work in progress suggests that μ-crystallin inhibits myogenesis by altering the transcription of muscle-specific thyroid hormone-dependent genes. High levels of µ-crystallin expression also block muscle development in culture (figure). Future studies will address the relationship of hormone binding to anti-myogenic activity, and the variation of protein levels by family and state of health.
We have also been examining the effects on muscle of mutations in the gene encoding dysferlin, to learn how these lead to Limb Girdle Muscular Dystrophy Type 2B and Miyoshi Myopathy. As mentioned above, the absence of dysferlin results in unstable transverse tubules and misregulation of Ca2+. (The figure below shows a section of Miyoshi Myopathy muscle, labeled with antibodies to the Na pump, with regions lacking t-tubules marked with yellow asterisks.) Mutant dysferlins are likely to have additional effects, however, including targeting to the wrong membrane compartments and activation of ERAD (endoplasmic reticulum activated degradation), which may contribute to pathogenesis. In addition to deletion mutants, we are studying individual point mutations associated with dysferlinopathies in man, to learn how the modified dysferlins affect muscle health.