Force development during striated muscle contraction is initiated by the binding of Ca2+ to the specific sites in troponin C (TnC), triggering a series of functional structural changes within the thin filament, including opening of the N-domain of TnC, conformational change of the inhibitory region of troponin I (Tnl), and switching interaction between Tnl and actin to Tnl and TnC, which ultimately lead to a cyclic interaction between actin and myosin to form strong force-generating cross-bridges. Full muscle activation requires both Ca2+ binding and feed back modulation of cross-bridge cycling. In cardiac muscle it is also modulated by protein phosphorylation which plays important roles in heart failing/hypertrophic process. To fully understand muscle regulatory mechanism requires structural, thermodynamic and kinetic information on each of these structural transitions during force development. My long-term research goal is to elucidate the kinetics of movements of the thin finlament betweem extremes of contraction/relaxation, and understand how they are modified by cross-bridge cycling and phosphorylation. To achieve the goal, this proposal addresses the following three issues: (1) What is the kinetics of each individual activation/deactivation process of the thin filament? (2) How does the cross-bridge cycling affect these kinetic processes? And (3) what is the role of phsophorylation in modulating these transitions? Newly designed conformational markers based on Forster resonance energy transfer to monitor these structural transitions will be used for stopped-flow kinetic and Ca2+ titration measurements at different activation conditions to acquire the desired information. These markers will be incorporated into reconstituted thin filament, myofibrils and skinned fibers along with/without phosphotylated proteins to specify the time-dependent changes of specific domain movements of the thin filament in response to Ca2+. Results of this study will enhance our understanding of molecular mechanisms of thin filament activation in response of Ca2+ and the role of protein phosphorylation in heart failure.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL080186-05
Application #
7597232
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Evans, Frank
Project Start
2005-04-01
Project End
2010-03-31
Budget Start
2009-04-01
Budget End
2010-03-31
Support Year
5
Fiscal Year
2009
Total Cost
$347,982
Indirect Cost
Name
Washington State University
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
041485301
City
Pullman
State
WA
Country
United States
Zip Code
99164
Bohlooli Ghashghaee, Nazanin; Li, King-Lun; Solaro, R John et al. (2018) Role of the C-terminus mobile domain of cardiac troponin I in the regulation of thin filament activation in skinned papillary muscle strips. Arch Biochem Biophys 648:27-35
Schlecht, William; Dong, Wen-Ji (2017) Dynamic Equilibrium of Cardiac Troponin C's Hydrophobic Cleft and Its Modulation by Ca2+ Sensitizers and a Ca2+ Sensitivity Blunting Phosphomimic, cTnT(T204E). Bioconjug Chem 28:2581-2590
Bohlooli Ghashghaee, Nazanin; Tanner, Bertrand C W; Dong, Wen-Ji (2017) Functional significance of C-terminal mobile domain of cardiac troponin I. Arch Biochem Biophys 634:38-46
Li, King-Lun; Ghashghaee, Nazanin Bohlooli; Solaro, R John et al. (2016) Sarcomere length dependent effects on the interaction between cTnC and cTnI in skinned papillary muscle strips. Arch Biochem Biophys 601:69-79
Pulcastro, Hannah C; Awinda, Peter O; Methawasin, Mei et al. (2016) Increased Titin Compliance Reduced Length-Dependent Contraction and Slowed Cross-Bridge Kinetics in Skinned Myocardial Strips from Rbm (20?RRM) Mice. Front Physiol 7:322
Schlecht, William; Li, King-Lun; Hu, Dehong et al. (2016) Fluorescence Based Characterization of Calcium Sensitizer Action on the Troponin Complex. Chem Biol Drug Des 87:171-81
Schlecht, William; Zhou, Zhiqun; Li, King-Lun et al. (2014) FRET study of the structural and kinetic effects of PKC phosphomimetic cardiac troponin T mutants on thin filament regulation. Arch Biochem Biophys 550-551:1-11
Jayasundar, Jayant James; Xing, Jun; Robinson, John M et al. (2014) Molecular dynamics simulations of the cardiac troponin complex performed with FRET distances as restraints. PLoS One 9:e87135
Li, King-Lun; Rieck, Daniel; Solaro, R John et al. (2014) In situ time-resolved FRET reveals effects of sarcomere length on cardiac thin-filament activation. Biophys J 107:682-693
Jacroux, Thomas; Bottenus, Danny; Rieck, Bennett et al. (2014) Cationic isotachophoresis separation of the biomarker cardiac troponin I from a high-abundance contaminant, serum albumin. Electrophoresis 35:2029-38

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