We have developed two complementary in vivo approaches to investigate fundamental aspects of troponin T (TnT) protein interactions, the function of TnT in muscle contraction, and the effects of TnT mutations that cause human hypertrophic cardiomyopathy (HCM). A vertebrate myotube transfection/protein replacement assay will be used to study the cell biological and physiological consequences of different TnT mutations and functions of different TnT isoforms, and 2) C. elegans suppressor genetics will be used to study the in vivo interactions of specific TnT domains with other sarcomeric proteins. Qual fast TnT isoform cDNAs with variable N-terminal domains, human cardiac TnT cDNAs bearing HCM mutations, and site-directed mutations in quail fast TnT cDNA, will be introduced into the vertebrate myotube transfection/protein replacement assay. We will analyze the impact of incorporation of isoform and mutant proteins of myotube structure and function, including force-velocity relationships at maximal and submaximal calcium concentrations, and isometric ATPase activity of myotubes. These studies test specific hypotheses concerning the roles of Tnt as a regulator of the calcium responsiveness of muscles and of actin-myosin crossbridge mechanics. In addition, we will characterize site-directed HCM mutations duplicated in the C. elegans TnT- 1 gene, and site-directed mutations of the C-terminal domain of TnT that is hypothesized to interact to Tm, TnI and Tn, to define mutations that disrupt C. elegans muscle function in stable transgenic lines. Genetic suppressor analyses will test specific hypotheses of TnT domain interactions, will define specific residues of interaction between TnT and interacting muscle proteins, and likely will reveal unexpected TnT domain functions and interactions not measurable by physiological assays in the proposed protein replacement studies. The combined application of these in vivo assays provides a new approach to investigate TnT function, which is poorly understood. Our findings will provide fundamental knowledge of the mechanisms of muscle contraction and a basis for development of gene and drug therapies for the treatment of HCM.
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