The goal of this project is to understand myofibrillar factors that control striated muscle relaxation. There is controversy regarding the relative contribution of the Ca2+ dissociation rate from the regulatory site(s) of troponin C (TnC), the number of force generating cross-bridges and the kinetics of cross-bridge detachment to the process of relaxation. The effect of altering these factors on the relaxation rate in skinned rabbit skeletal muscle and rat cardiac trabeculae will be determined.
Aim #1 : Develop a biochemical model that mimics Ca2+ exchange with TnC during muscle relaxation. Working hypotheses: A) For skeletal muscle, TnC-Tnl, but not TnC alone, is an adequate model system. B) For cardiac muscle, the effects of crossbridges on Ca2+ dissociation from TnC must be considered and thus the minimally sufficient model is the reconstituted thin filament or isolated myofibril.
Aim #2 : Elucidate the molecular communication pathway between TnC and Tnl. Working hypothesis: The hydrophobic interactions between the regulatory domain of TnC and Tnl are critical for binding to Tnl, transferring the high Ca2+ affinity and slow Ca2+ dissociation rates to the complex and sustaining maximal force in muscle.
Aim #3 : Determine the effects of modifying Ca2+ exchange with TnC, cross-bridge number and cross-bridge kinetics on the rates of muscle contraction and relaxation. Working hypotheses for skeletal muscle: A) Ca2+ dissociation from TnC and cross-bridge detachment rates are similar, such that decreasing either rate slows relaxation but accelerating either rate has limited effect on relaxation. B) Relaxation rate is independent of the number of force generating crossbridges. C) The rates of Ca2+ exchange with TnC do not affect the kinetics of contraction or Vmax. Working hypothesis for cardiac muscle: Relaxation rate is modulated by the Ca2+ dissociation rate from TnC, cross-bridge number and cross-bridge kinetics. Fluorescently labeled mutants of TnC and Tnl will be utilized to alter Ca2+ dissociation rate from TnC. Cross-bridge number will be altered by [Ca2+], crossbridge inhibitors and NEM myosin S1. Cross-bridge kinetics will be modified with inorganic phosphate. Contraction and relaxation will be induced by flash photolysis of NP-EGTA and diazo-2, respectively. Elucidating the fundamental factors that modulate relaxation will improve understanding of muscle diseases that exhibit defects in relaxation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR020792-24
Application #
7190589
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Nuckolls, Glen H
Project Start
1978-01-01
Project End
2009-12-31
Budget Start
2007-01-01
Budget End
2007-12-31
Support Year
24
Fiscal Year
2007
Total Cost
$280,671
Indirect Cost
Name
Ohio State University
Department
Physiology
Type
Schools of Medicine
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
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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