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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
2R01AR044504-04A2
Application #
6850390
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Nuckolls, Glen H
Project Start
1997-05-01
Project End
2008-05-31
Budget Start
2004-09-29
Budget End
2005-05-31
Support Year
4
Fiscal Year
2004
Total Cost
$259,350
Indirect Cost
Name
East Carolina University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
607579018
City
Greenville
State
NC
Country
United States
Zip Code
27858
Varughese, Jayson F; Baxley, Tamatha; Chalovich, Joseph M et al. (2011) A computational and experimental approach to investigate bepridil binding with cardiac troponin. J Phys Chem B 115:2392-400
Mathur, Mohit C; Chase, P Bryant; Chalovich, Joseph M (2011) Several cardiomyopathy causing mutations on tropomyosin either destabilize the active state of actomyosin or alter the binding properties of tropomyosin. Biochem Biophys Res Commun 406:74-8
Varughese, Jayson F; Chalovich, Joseph M; Li, Yumin (2010) Molecular dynamics studies on troponin (TnI-TnT-TnC) complexes: insight into the regulation of muscle contraction. J Biomol Struct Dyn 28:159-74
Mathur, Mohit C; Kobayashi, Tomoyoshi; Chalovich, Joseph M (2009) Some cardiomyopathy-causing troponin I mutations stabilize a functional intermediate actin state. Biophys J 96:2237-44
Mathur, Mohit C; Kobayashi, Tomoyoshi; Chalovich, Joseph M (2008) Negative charges at protein kinase C sites of troponin I stabilize the inactive state of actin. Biophys J 94:542-9
Wirth, A; Schroeter, M; Kock-Hauser, C et al. (2003) Inhibition of contraction and myosin light chain phosphorylation in guinea-pig smooth muscle by p21-activated kinase 1. J Physiol 549:489-500
She, M; Trimble, D; Yu, L C et al. (2000) Factors contributing to troponin exchange in myofibrils and in solution. J Muscle Res Cell Motil 21:737-45
Brenner, B; Kraft, T; Yu, LC et al. (1999) Thin filament activation probed by fluorescence of N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle Biophys J 77:2677-91
Brenner, B; Chalovich, JM (1999) Kinetics of thin filament activation probed by fluorescence of N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle: implications for regulation of muscle contra Biophys J 77:2692-708
Xu, S; Malinchik, S; Frisbie, S et al. (1998) X-ray diffraction studies of the cross-bridge intermediate states. Adv Exp Med Biol 453:271-8;discussion 278-9

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