Type of Document Dissertation Author Brunet, Nicolas Author's Email Address firstname.lastname@example.org URN etd-11092006-113520 Title Mutations in the Human Cardiac CA2+- Regulatory Proteins Affect the Function of the Thin Filament – Lesson for Inherited Cardiomyopathies. Degree Doctor of Philosophy Department Molecular Biophysics, Institute of Advisory Committee
Advisor Name Title Thomas C. S. Keller III Committee Chair P. Bryant Chase Committee Member Peng Xiong Committee Member Peter G. Fajer Committee Member Timothy S. Moerland Committee Member Keywords
- Calciun Sensitivity
- Familial Hypertrophic Cardiomyopathy
- Thermal Stability
- Cross-Bridge Cycle
Date of Defense 2006-10-25 Availability unrestricted AbstractFamilial hypertrophic cardiomyopathy (FHC) is the leading cause of sudden cardiac death in both preadolescents and adolescents. The hallmark of the disorder is myocardial hypertrophy of the left ventricle, which results in an obstruction of blood flow through the left ventricular outflow tract. FHC has been associated with well over 100 mutations, primarily in proteins of the contractile apparatus of the heart. The molecular mechanisms involved in the pathogenesis of FHC are not well understood. For this study, in vitro motility assays (IVM) were conducted to assess the relationship between structure and function of cardiac thin filaments and to elucidate the molecular basis for sequelae of FHC caused by point mutations in the human cardiac regulatory proteins. Our research aims to contribute to the study of FHC by the accomplishment of the following innovations:
1) Development of a simple strategy to express recombinant human cardiac regulatory proteins in E. coli for molecular assays of human cardiac contractility.
2) Fabrication of a thermo-electric controller that allows rapid and reversible characterization over a broad temperature range of the effects of FHC mutations in troponin and tropomyosin using IVM assays.
My research yielded the following novel physiological findings
1) Ca2+-sensitivity of human cardiac thin filament sliding is affected by some of the FHC mutations in the cardiac regulatory proteins, but not by changes in myosin isoform, indicating that Ca2+-sensitivity does not depend upon the kinetics of cross-bridge cycling. This finding implies that the hypertrophic response is communicated through different pathways depending whether the mutation is in troponin, tropomyosin, or myosin
2) Troponin and tropomyosin affect the temperature sensitivity as well as the maximum speed of unloaded filament sliding by reducing the myosin cross-bridge duty ratio. Our results suggest that the duty ratio also might be affected by clinically relevant mutations in troponin and tropomyosin.
Finally, functional characterization of troponin, bearing FHC mutations in subunit I, subunit T, or both subunits, predicts structural relationships of the thin filament.
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