Heart failure can be a result of impaired cardiac contraction or relaxation or both, which are regulated by Ca2+induced thin filament activation and deactivation. Therefore, understanding the mechanism of thin filament regulation is of great importance in prevention and treatment of heart failure. The molecular basis of the thin filament regulation involves Ca2+induced alterations in the structure and structural kinetics and dynamics associated with the protein-protein interactions at the interface between troponin and actin. The objective of this proposal is to define the detailed kinetic mechanism that underlies Ca2+induced thin filament activation/deactivation and how it is affected by troponin phosphorylation and cardiomyopathy mutations found in cTnI. To achieve the goal, this proposal will address the following questions: 1) What is the kinetic role of different functional regions of cTnI in regulating crossbridge kinetics? 2) How do the structural dynamics of the cTnI C-domain play roles in thin filament regulation? and 3) How are the structural changes and structural kinetics of the thin filament modified in the skinned muscle fibers and myofibrils? The underlying hypothesis of this proposal is that the Ca2+induced structural transitions involving the different regions of troponin I at the interface between troponin and actin play different roles in regulating kinetics of the interaction between actin and mysoin, and the structural transitions can be affected by strong crossbridges, cardiomyopathy mutations, and phosphorylation of troponin proteins, as well as cellular lattice environment found in intact muscle cells. This project capitalizes on our FRET and fluorescence anisotropy expertise in determining protein structural kinetics and dynamics to exploit a new approach by implementing FRET methodologies into muscle fibers and myofibril measurements. This will enable us to acquire the urgently needed kinetic and dynamic information associated with the Ca2+induced thin filament regulation on muscle function. The results from this study will have a positive impact by advancing our understanding of the regulatory role of cardiac thin filament in cardiac muscle contraction and relaxation in health and disease.

Public Health Relevance

The objective of this proposal is to define the detailed kinetic mechanism that underlies Ca2+induced thin filament activation/deactivation and how it is affected by troponin phosphorylation and cardiomyopathy mutations found in cTnI. This application is related to research to improve depressed cardiac contractility under pathological conditions.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL080186-08
Application #
8242740
Study Section
Special Emphasis Panel (ZRG1-CVRS-F (03))
Program Officer
Evans, Frank
Project Start
2005-04-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
8
Fiscal Year
2012
Total Cost
$365,119
Indirect Cost
$117,619
Name
Washington State University
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
041485301
City
Pullman
State
WA
Country
United States
Zip Code
99164
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 107:682-93
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
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
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
Jacroux, Thomas; Rieck, Daniel C; Cui, Rong et al. (2013) Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection. Anal Biochem 432:106-14
Rieck, Daniel C; Li, King-Lun; Ouyang, Yexin et al. (2013) Structural basis for the in situ Ca(2+) sensitization of cardiac troponin C by positive feedback from force-generating myosin cross-bridges. Arch Biochem Biophys 537:198-209
Zhou, Zhiqun; Rieck, Daniel; Li, King-Lun et al. (2013) Structural and kinetic effects of hypertrophic cardiomyopathy related mutations R146G/Q and R163W on the regulatory switching activity of rat cardiac troponin I. Arch Biochem Biophys 535:56-67
Zhou, Zhiqun; Li, King-Lun; Rieck, Daniel et al. (2012) Structural dynamics of C-domain of cardiac troponin I protein in reconstituted thin filament. J Biol Chem 287:7661-74
Qiao, Yanling; Tang, Hongmin; Munske, Gerhard R et al. (2011) Enhanced fluorescence anisotropy assay for human cardiac troponin I and T detection. J Fluoresc 21:2101-10
Bottenus, Danny; Hossan, Mohammad Robiul; Ouyang, Yexin et al. (2011) Preconcentration and detection of the phosphorylated forms of cardiac troponin I in a cascade microchip by cationic isotachophoresis. Lab Chip 11:3793-801

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