The nemaline myopathies are neuromuscular disorders characterized by muscle weakness and rod-shaped """"""""nemaline"""""""" inclusions in skeletal muscle fibers. Recent studies have identified a recessively inherited form of nemaline myopathy, the Amish nemaline myopathy (ANM), among the Old Order Amish at the rate of 1 of 500 births. The molecular cause of ANM is a nonsense mutation in the gene encoding slow skeletal muscle troponin T (TnT), a muscle- specific Ca2+ -regulatory protein, truncating the protein at amino acid 179. We have shown that the truncated slow TnT is degraded and not incorporated into the myofibrils, consistent with the recessive inheritance of the disease. The ANM phenotypes include tremors, contractures and hypotonia. A rapid postnatal progress results in death from respiratory insufficiency, usually in the second year. No effective treatment is available. The discovery of ANM gene has given the affected families the first hope for a cure of this fatal disease. Three homologous genes are present in the vertebrate genome encoding the slow, fast and cardiac TnT isoforms. Despite the loss of slow TnT in ANM muscle, only mild phenotypes are shown at birth. The postnatal worsening of myopathy is concurrent with the developmental down-regulation of cardiac TnT in skeletal muscle and the embryonic to adult isoform transition of fast skeletal muscle TnT. Therefore, cardiac and embryonic fast TnT, but not adult fast TnT, may compensate for the lost function of slow TnT. To understand the pathology of ANM in which the loss of only one isoform of TnT causes severe myopathy, we shall investigate the functional relationship between TnT isoforms. In addition to a better understanding of the Ca2+-regulation of muscle contraction, this study aims at the development of therapies for ANM.
Four specific aims are proposed to bridge the basic studies of TnT isoform gene regulation and structure-function relationships to the clinical management of this lethal disease.I. Biochemical characterization of TnT isoforms to investigate their functional difference and whether the cardiac TnT and embryonic fast TnT, both of which are acidic isoforms, are functionally similar to slow TnT which is also acidic.II. Functional characterization of the truncated slow TnT to determine whether it may have a dominant negative effect in ANM.III. Protein interaction and transgenic expression experiments to investigate whether disorganization of muscle thin filament due to change in one regulatory protein forms the basis of nemaline myopathy.IV. Production and characterization of the slow TnT mutation in an animal model of ANM to study muscle pathophysiology, fatigue tolerance, and therapeutic approaches.
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