Point mutations or deletions in the T or I subunits of troponin may result in disorders such as Familial Hypertrophic Cardiomyopathy and Nemaline myopathy. Our data indicate that mutations of troponin tend to alter the distribution between states of actin-tropomyosin-troponin having different activities. We propose to test this hypothesis by examining several mutations of these troponin subunits. We will avoid the difficulty of obtaining human tissues by using expressed human troponin subunits in solution and animal muscle fiber models. Biochemical studies in solution will determine changes in the key parameters that define the regulatory response. We will test the hypothesis that mutations tend to alter the distribution of actin states or the rate of transition among these states. Emphasis will be placed on the cooperative activation of steady-state ATP hydrolysis by myosin S1, the cooperative equilibrium binding of S1 to actin-tropomyosin-troponin (regulated actin), and on the rate of S1 binding to regulated actin. We will examine several mutations of the T and I subunits of troponin and make detailed measurements on a set of mutants with varying degrees of effect on regulation. If there is evidence of changes in multiple steps we will confirm those changes. The effect of the troponin mutations will be examined in both skeletal and cardiac muscle fiber preparations. The native troponin in the fibers will be replaced with mutant troponin and/or fluorescently labeled troponin using our exchange protocol. Our method of exchange is an important part of this proposal because the procedure itself does not affect the mechanical properties of the fiber. We will observe changes in the mechanical properties of fibers caused by the mutations. By measuring the fluorescence of a probe on the troponin we can correlate changes in performance with biochemical changes that are measured in solution. We will utilize mathematical modeling to test models of regulation, to compare the solution results with the results from mechanical studies and to predict other behaviors that can be tested. Most importantly, the detailed mathematical analyses may allow us to predict specific interventions to limit the effects of these mutations in afflicted individuals.
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