The molecular mechanism by which cMyBP-C exerts its effect on actomyosin force power generating system remains largely undefined. With its low ratio relative to myosin and it being located in distinct regions of the thick filament, we will determine in this Project how cMyBP-C's modulates actomyosin's power generation by either interacting with a limited population of crossbridges or whether it cooperatively affects all crossbridges within the thick filament. This project serves as a physiological bridge between the animal (Project #3, Robbins), whole heart (Core B, Kass) and fiber (Core B, Palmer, Maughan) studies.
In Aim #1, we will use state-of-the-art single molecule biophysical techniques (e.g. laser trap assay) to probe the effect that cMyBP-C exerts on actomyosin function along the length a single native thick filament isolated from transgenic mice designed in Project #3 (Robbins) and produced in Core C (Robbins).
In Aim #2, we will use expressed N-terminal fragments of cMyBP-C produced in Core C (Robbins) to probe the binding affinity of these fragments for actin and/or myosin. Thus, these data and that obtained in Project #1 (Craig), Project #3 (Robbins) and Core B (Palmer) will help define cMyBP-C's specific binding partners (i.e. myosin and/or actin) and thus its physiological site of action. In combination with motility and laser trap assays, we will determine if the N-terminus of CMyBP-C limits myosin's attachment rate to actin or if it directly affects myosin's inherent molecular mechanics and kinetics. Finally, in Aim #3 we will characterize how phosphorylation regulates cMyBP-C action. Using transgenic mouse models (Core C, Robbins) expressing cMyBP C mutants having alanine or aspartic acid substitutions for one or more of the three phosphorylatable serines, we will determine the functional importance of phosphorylation and the hierarchical importance of each site using native thick filaments containing mutant cMyBP-C as well as N-terminal fragments having the same mutations. Once the molecular mechanism of cMyBP-C is defined, the potential for novel therapeutics or clinical intervention may be possible in cases of heart failure associated with genetic mutations in cMyBP-C.

Public Health Relevance

cMyBP-C is critical to normal cardiac performance. Using single molecule biophysical techniques to characterize the molecular mechanism of cMyBP-C function, the results may provide the potential for novel therapies in cases of heart failure associated with genetic mutations in cMyBP-C.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL059408-12
Application #
8215309
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
2011-02-01
Project End
2015-01-31
Budget Start
2011-02-01
Budget End
2012-01-31
Support Year
12
Fiscal Year
2011
Total Cost
$396,215
Indirect Cost
Name
University of Vermont & St Agric College
Department
Type
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Kensler, Robert W; Craig, Roger; Moss, Richard L (2017) Phosphorylation of cardiac myosin binding protein C releases myosin heads from the surface of cardiac thick filaments. Proc Natl Acad Sci U S A 114:E1355-E1364
McLendon, Patrick M; Davis, Gregory; Gulick, James et al. (2017) An Unbiased High-Throughput Screen to Identify Novel Effectors That Impact on Cardiomyocyte Aggregate Levels. Circ Res 121:604-616
Gupta, Manish K; McLendon, Patrick M; Gulick, James et al. (2016) UBC9-Mediated Sumoylation Favorably Impacts Cardiac Function in Compromised Hearts. Circ Res 118:1894-905
Mun, Ji Young; Kensler, Robert W; Harris, Samantha P et al. (2016) The cMyBP-C HCM variant L348P enhances thin filament activation through an increased shift in tropomyosin position. J Mol Cell Cardiol 91:141-7
Tewari, Shivendra G; Bugenhagen, Scott M; Palmer, Bradley M et al. (2016) Dynamics of cross-bridge cycling, ATP hydrolysis, force generation, and deformation in cardiac muscle. J Mol Cell Cardiol 96:11-25
Yang, Shixin; Woodhead, John L; Zhao, Fa-Qing et al. (2016) An approach to improve the resolution of helical filaments with a large axial rise and flexible subunits. J Struct Biol 193:45-54
Previs, Michael J; Mun, Ji Young; Michalek, Arthur J et al. (2016) Phosphorylation and calcium antagonistically tune myosin-binding protein C's structure and function. Proc Natl Acad Sci U S A 113:3239-44
Bhuiyan, Md Shenuarin; McLendon, Patrick; James, Jeanne et al. (2016) In vivo definition of cardiac myosin-binding protein C's critical interactions with myosin. Pflugers Arch 468:1685-95
Warshaw, David M (2016) HEART DISEASE. Throttling back the heart's molecular motor. Science 351:556-7
James, Jeanne; Robbins, Jeffrey (2016) Healing a Heart Through Genetic Intervention. Circ Res 118:920-2

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