Abstract

The goals of the proposed studies are to identify and characterize the rate-limiting step for relaxation in rabbit skeletal muscle fibers and rat cardiac trabeculae by selectively altering each event of relaxation: 1.) lowering of myoplasmic (Ca2+ by the sarcoplasmic reticulum (SR) Ca- ATPase, 2.) dissociation of Ca2+ from the regulatory sites(s) of troponin C (TnC) and 3.) cross-bridge detachment and examining the effect of the alterations on relaxation rate.
Aim 1 : Directly assess the role of TnC in relaxation induced by flash photolysis of a caged Ca2+ chelator, diazo-2, by determining the effect on relaxation rates of: a) functional TnC mutants which exhibit a 10-fold difference in Ca2+ affinities and off-rates, and b) phosphorylation of cardiac TnI.
Aim 2 : Determine the effect of increasing (with intracellular Ca2+ chelators) and decreasing (with selective inhibitors of the SR Ca-ATPase) the rate of Ca2+ sequestration on the relaxation rate of intact cardiac trabeculae.
Aim 3 : Evaluate the contribution of cross-bridge cycling rate and number of cross-bridges to relaxation kinetics by determining the rates of diazo-2 induced relaxation in: a) fast-twitch psoas and slow-twitch soleus fibers, and b) in cardiac muscle from rats which have been treated to produce 100 % V1 or V3 myosin isoforms and by altering the number of force generating cross-bridges by varying Ca2+ concentration or by vanadate.
Aim 4 : Separate the effect of phosphorylation of phospholamban from phosphorylation of TnI on relaxation rate in cardiac muscle. Twitch-like contractions will be induced in skinned trabeculae with functionally intact SR by flash photolysis of a caged Ca2+. Relaxation rate after phosphorylation of phospholamban and TnI will be compared to relaxation rate when Ca2+ off- rate from TnC is accelerated by a TnC mutant, thus mimicking the effect of TnI phosphorylation. Selectively altering rates of Ca2+ sequestration, Ca2+ dissociation from TnC and cross-bridge kinetics and number will allow determination of the rate-limiting step for relaxation under various conditions.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR020792-18
Application #
6124120
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Program Officer
Lymn, Richard W
Project Start
1978-01-01
Project End
2003-11-30
Budget Start
1999-12-01
Budget End
2000-11-30
Support Year
18
Fiscal Year
2000
Total Cost
$187,990
Indirect Cost

  Institution

Name
Ohio State University
Department
Physiology
Type
Schools of Medicine
DUNS #
098987217
City
Columbus
State
OH
Country
United States
Zip Code
43210

  Publications

Liu, Bin; Lee, Ryan S; Biesiadecki, Brandon J et al. (2012) Engineered troponin C constructs correct disease-related cardiac myofilament calcium sensitivity. J Biol Chem 287:20027-36
Lee, Ryan S; Tikunova, Svetlana B; Kline, Kristopher P et al. (2010) Effect of Ca2+ binding properties of troponin C on rate of skeletal muscle force redevelopment. Am J Physiol Cell Physiol 299:C1091-9
Norman, Catalina; Rall, Jack A; Tikunova, Svetlana B et al. (2007) Modulation of the rate of cardiac muscle contraction by troponin C constructs with various calcium binding affinities. Am J Physiol Heart Circ Physiol 293:H2580-7
Davis, Jonathan P; Norman, Catalina; Kobayashi, Tomoyoshi et al. (2007) Effects of thin and thick filament proteins on calcium binding and exchange with cardiac troponin C. Biophys J 92:3195-206
Swartz, Darl R; Yang, Zhenyun; Sen, Asok et al. (2006) Myofibrillar troponin exists in three states and there is signal transduction along skeletal myofibrillar thin filaments. J Mol Biol 361:420-35
Luo, Ye; Rall, Jack A (2006) Regulation of contraction kinetics in skinned skeletal muscle fibers by calcium and troponin C. Arch Biochem Biophys 456:119-26
Tikunova, Svetlana B; Davis, Jonathan P (2004) Designing calcium-sensitizing mutations in the regulatory domain of cardiac troponin C. J Biol Chem 279:35341-52
Davis, Jonathan P; Rall, Jack A; Alionte, Catalina et al. (2004) Mutations of hydrophobic residues in the N-terminal domain of troponin C affect calcium binding and exchange with the troponin C-troponin I96-148 complex and muscle force production. J Biol Chem 279:17348-60
Gomes, Aldrin V; Venkatraman, Gayathri; Davis, Jonathan P et al. (2004) Cardiac troponin T isoforms affect the Ca(2+) sensitivity of force development in the presence of slow skeletal troponin I: insights into the role of troponin T isoforms in the fetal heart. J Biol Chem 279:49579-87
Davis, Jonathan P; Rall, Jack A; Reiser, Peter J et al. (2002) Engineering competitive magnesium binding into the first EF-hand of skeletal troponin C. J Biol Chem 277:49716-26

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