description): Familial Hypertrophic Cardiomyopathy (FHC) is an autosomal dominant disorder caused by mutations in any of several genes encoding the proteins of the cardiac contractile apparatus. This project will characterize the effects of FHC-causing mutations on the in vitro function of the various thin filament proteins so far implicated in this disorder: troponin T, troponin 1, and alpha-tropomyosin. By comparing normal and mutant proteins, the project will provide some of the insight required to understand the pathophysiology of cardiac disease in these patients. Also, the applicant will use the mutations to test the mechanism by which cardiac contraction is regulated by troponin and tropomyosin. A multi-faceted study of the mutant proteins is planned, with examination of several protein-protein affinities (including thin filament binding of troponin, of troponin-tropomyosin, and of myosin Sl; troponin binary subunit interactions; effects of calcium and of myosin on these various processes), calcium affinity, myosin MgATPase regulation, folding stability, in vitro motility, in vitro force, and structural effects on the regulatory conformational switching of the thin filament as determined by 3-D reconstructions of electron micrographs. A smaller number of mutations, identified in the cardiac actin gene, have been found causative in a subset of patients with another genetic disorder: dilated cardiomyopathy. Mutant forms of actin will be similarly examined for alterations in interactions with tropomyosin and troponin. (1) FHC mutations occurring in two regions of troponin T will be investigated. Troponin T mutants R92Q, R92W, Al 04V, and Fl 01 I occur in or near a region of troponin T that the applicant recently identified as forming a critical portion of the troponin tail. In a different region, the effects of FHC-linked COOH-terminal truncation of 28 residues will be studied. (2) Six troponin I mutations that occur in FHC will be similarly investigated, all located in the region of troponin I that interacts with the regulatory domain of troponin C. (3) Five FHC-linked tropomyosin mutants will be created and similarly studied, as will an actin mutation that causes inherited dilated cardiomyopathy and that is hypothesized to interact abnormally with tropomyosin.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
7R01HL063774-05
Application #
6826144
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Project Start
2000-01-15
Project End
2004-12-31
Budget Start
2004-01-01
Budget End
2004-12-31
Support Year
5
Fiscal Year
2003
Total Cost
$174,536
Indirect Cost
Name
University of Illinois at Chicago
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Mun, Ji Young; Previs, Michael J; Yu, Hope Y et al. (2014) Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism. Proc Natl Acad Sci U S A 111:2170-5
Sousa, Duncan R; Stagg, Scott M; Stroupe, M Elizabeth (2013) Cryo-EM structures of the actin:tropomyosin filament reveal the mechanism for the transition from C- to M-state. J Mol Biol 425:4544-55
Kowlessur, Devanand; Tobacman, Larry S (2012) Significance of troponin dynamics for Ca2+-mediated regulation of contraction and inherited cardiomyopathy. J Biol Chem 287:42299-311
Li, Xiaochuan Edward; Tobacman, Larry S; Mun, Ji Young et al. (2011) Tropomyosin position on F-actin revealed by EM reconstruction and computational chemistry. Biophys J 100:1005-13
Sousa, Duncan; Cammarato, Anthony; Jang, Ken et al. (2010) Electron microscopy and persistence length analysis of semi-rigid smooth muscle tropomyosin strands. Biophys J 99:862-8
Kowlessur, Devanand; Tobacman, Larry S (2010) Troponin regulatory function and dynamics revealed by H/D exchange-mass spectrometry. J Biol Chem 285:2686-94
Kozaili, Julie Mouannes; Leek, Daniel; Tobacman, Larry S (2010) Dual regulatory functions of the thin filament revealed by replacement of the troponin I inhibitory peptide with a linker. J Biol Chem 285:38034-41
Ali, Laith F; Cohen, Joshua M; Tobacman, Larry S (2010) Push and pull of tropomyosin's opposite effects on myosin attachment to actin. A chimeric tropomyosin host-guest study. Biochemistry 49:10873-80
Kowlessur, Devanand; Tobacman, Larry S (2010) Low temperature dynamic mapping reveals unexpected order and disorder in troponin. J Biol Chem 285:38978-86
Siththanandan, V B; Tobacman, L S; Van Gorder, N et al. (2009) Mechanical and kinetic effects of shortened tropomyosin reconstituted into myofibrils. Pflugers Arch 458:761-76

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