The long-term goal of our research is to document the molecular mechanisms of contraction in the striated muscle system, in particular to characterize the elementary molecular steps of the cross-bridge cycle in the cardiac muscle strips (myocardium). We selectively remove the thin filament from myocardium with a gelsolin treatment, and sequentially reconstitute the thin filament with G-actin, tropomyosin, and troponin complex. The degree of reconstitution is assessed by isometric tension, SDS-PAGE, electron microscopy, and confocal microscopy. At each stage of reconstitution, we assess the cross-bridge kinetics with the """"""""sinusoidal analysis"""""""" method. In this method, the length of the preparation is oscillated in sine waves of varying frequencies, and concomitant tension transients are analyzed in terms of three exponential processes. When the effects of ATP, ADP, and phosphate on the exponential processes are studied together with the ATP hydrolysis rate measurement, the 10 kinetic constants that characterize the elementary steps of the cross-bridge cycle can be deduced. To characterize the preparation at each stage of reconstitution, we also measure the ATP hydrolysis rate, the rate of tension redevelopment (ktr), maximum velocity of shortening (Vmax), and pCa-tension relationship. We propose to test the hypothesis that N-terminal negative charge of actin is essential in force generation in structured muscle system. We further propose to characterize the length of thin filament cooperativity experimentally, and test the hypothesis that the cooperativity is affected by the hydrophobic plug of actin. Our short-term goal is to document the thin filament activation mechanism by using thin-filament extracted and reconstituted models of myocardium, and determine the function of actin domains in the structured muscle system where force can be generated. In the long run, the method developed and the knowledge acquired will help to elucidate the mechanisms of cardiac dysfunction, including ischemia, hypertrophy, and familial hypertrophic cardiomyopathy.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL070041-03
Application #
6718357
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Dunn, Rosalie
Project Start
2002-03-20
Project End
2006-02-28
Budget Start
2004-03-01
Budget End
2005-02-28
Support Year
3
Fiscal Year
2004
Total Cost
$257,250
Indirect Cost
Name
University of Iowa
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Wang, Li; Bai, Fan; Zhang, Qing et al. (2017) Development of apical hypertrophic cardiomyopathy with age in a transgenic mouse model carrying the cardiac actin E99K mutation. J Muscle Res Cell Motil 38:421-435
Wang, Li; Kazmierczak, Katarzyna; Yuan, Chen-Ching et al. (2017) Cardiac contractility, motor function, and cross-bridge kinetics in N47K-RLC mutant mice. FEBS J 284:1897-1913
Bai, Fan; Caster, Hannah M; Dawson, John F et al. (2015) The immediate effect of HCM causing actin mutants E99K and A230V on actin-Tm-myosin interaction in thin-filament reconstituted myocardium. J Mol Cell Cardiol 79:123-32
Wang, Li; Bahadir, Anzel; Kawai, Masataka (2015) High ionic strength depresses muscle contractility by decreasing both force per cross-bridge and the number of strongly attached cross-bridges. J Muscle Res Cell Motil 36:227-41
Wang, Li; Sadayappan, Sakthivel; Kawai, Masakata (2014) Cardiac myosin binding protein C phosphorylation affects cross-bridge cycle's elementary steps in a site-specific manner. PLoS One 9:e113417
Bai, Fan; Caster, Hannah M; Rubenstein, Peter A et al. (2014) Using baculovirus/insect cell expressed recombinant actin to study the molecular pathogenesis of HCM caused by actin mutation A331P. J Mol Cell Cardiol 74:64-75
Wang, Li; Ji, Xiang; Barefield, David et al. (2014) Phosphorylation of cMyBP-C affects contractile mechanisms in a site-specific manner. Biophys J 106:1112-22
Wang, Li; Kawai, Masataka (2013) A re-interpretation of the rate of tension redevelopment (k(TR)) in active muscle. J Muscle Res Cell Motil 34:407-15
Bai, Fan; Wang, Li; Kawai, Masataka (2013) A study of tropomyosin's role in cardiac function and disease using thin-filament reconstituted myocardium. J Muscle Res Cell Motil 34:295-310
Bai, Fan; Caster, Hannah M; Pinto, Jose R et al. (2013) Analysis of the molecular pathogenesis of cardiomyopathy-causing cTnT mutants I79N, ?E96, and ?K210. Biophys J 104:1979-88

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