We will use presteady state kinetic methods and electron microscopy to determine the mechanism of thin filament regulation of cardiac actomyosin ATP hydrolysis. Most biochemistry and physiology textbooks depict a """"""""steric blocking"""""""" mechanism of thin filament regulation in which tropomyosin blocks the binding of myosin to actin at low calcium. However, there is now compelling experimental data that are inconsistent with this mechanism. Calcium and rigor myosin binding to fast skeletal muscle thin filaments accelerate the maximum rate of the product dissociation from myosin-ADP-Pi approximately 200 times but produce at most a 3 fold increase in the affinity of myosin-ADP-Pi binding to the thin filament. This indicates that steric blocking is a very small component of the regulatory mechanism. There are significant differences in the amino acid sequences between skeletal and cardiac actin, myosin, troponin, and tropomyosin and the mechanism of calcium regulation of cardiac actomyosin ATP hydrolysis is much less well characterized. Cardiac troponin- C has only a single regulatory calcium binding site and cardiac troponin-l has a 26 amino acid extension that contains a physiologically significant phosphorylation site. In addition to providing key information in the study of the mechanism of thin filament regulation, the proposed work is directly relevant to a number of diseases involving control of the regulation of cardiac muscle contraction, such as dilated cardiomyopathy, in which the force of the contraction is inadequate. Multi-mixing stopped-flow will be used to measure the dependence of the acceleration of product dissociation (Pi and ADP) from cardiac myosin-ADP-Pi by native cardiac thin filaments upon calcium and rigor myosin binding to the thin filament, and from phosphorylation of troponin-l. Parallel electron microscopy using negative stain and cryo-EM methods will be used to determine the structure of cardiac thin filaments (in various states of activation by calcium and bound myosin) and the distribution of the myosin bound to thin filaments. The structural data will be analyzed by single particle methods that have been adapted to filament structures by the laboratory of the CoPI (John Trinick). The combined data will be used to determine the mechanism of regulation by cardiac thin filaments and increase our understanding of mechanism of regulation of cardiac contractility. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL084604-01
Application #
7086753
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Przywara, Dennis
Project Start
2006-07-01
Project End
2010-06-30
Budget Start
2006-07-01
Budget End
2007-06-30
Support Year
1
Fiscal Year
2006
Total Cost
$331,400
Indirect Cost
Name
Eastern Virginia Medical School
Department
Physiology
Type
Schools of Medicine
DUNS #
058625146
City
Norfolk
State
VA
Country
United States
Zip Code
23501
Belknap, Betty; Harris, Samantha P; White, Howard D (2014) Modulation of thin filament activation of myosin ATP hydrolysis by N-terminal domains of cardiac myosin binding protein-C. Biochemistry 53:6717-24
Yang, Shixin; Barbu-Tudoran, Lucian; Orzechowski, Marek et al. (2014) Three-dimensional organization of troponin on cardiac muscle thin filaments in the relaxed state. Biophys J 106:855-64
Houmeida, Ahmed; Heeley, David H; Belknap, Betty et al. (2010) Mechanism of regulation of native cardiac muscle thin filaments by rigor cardiac myosin-S1 and calcium. J Biol Chem 285:32760-9