Quantitation of Proteins Associated with Structural Abnormalities in Dysferlin-Deficient Skeletal Muscle
Dysferlin deficiency syndromes (dysferlinopathies) are late-onset muscular dystrophies characterized by muscle fiber regeneration, infiltration of inflammatory cells and muscle replacement by fatty tissue. Despite intense study, the molecular basis of dysferlin-deficiency disease remains uncertain. We think that dysferlinopathies may be developmental disorders, due the lack of dysferlin protein in development. The dysferlin protein (230 kDa) interacts (directly or indirectly) with many skeletal muscle proteins and may help to organize or stabilize fiber structural organization. The lack of dysferlin may potentiate structural instability in the fiber leading to fiber necrosis.
We are evaluating this hypothesis using array tomography and quantitative western blotting. Array tomography is a high-resolution fluorescence microscopy technique that allows precise localization of specific antigens (i.e. proteins) within the three-dimensional structure of the muscle fiber. Quantitative western blotting is used to determine whether differences in detection of epitopes in array tomography are due to differences in the amount of specific antigen (protein). We will compare the distributions and levels of specific proteins between dysferlin-deficient vs. dysferlin-normal mouse skeletal muscle. We will focus on 1) dysferlin-interacting proteins (i.e. myoferlin, calpain 3), 2) components of the calcium release unit (i.e. ryanodine receptor, dihydropyridine receptor, and associated proteins), 3) structural and organizational proteins of sarcomeres (actin, myosin, nebulin, titin, etc.), t-tubules (BIN1), and sarcoplasmic reticulum (i.e. calsequestrin, SERCA ATP’ase). In addition, we will localize antigens from inflammatory cells (i.e. macrophages) and adipocytes in dysferlin-deficient muscle.