Mutations in the thick filament associated protein cMyBP-C are a major cause of familial hypertrophic cardiomyopathy. The pathological effects of mutations in cardiac myosin binding protein-C are consistent with increasing evidence that it plays both a structural and a regulatory role in myocardial contraction. Although the evidence suggesting a regulatory role for cMyBP-C in myocardial contraction appears compelling, the mechanism of action is far from clear. Most of the research on the functional role of cMyBPC and the effect of its phosphorylation has been done in rat or mouse myocardium which contains primarily the a-myosin heavy chain, despite evidence that thick filaments containing the (3-myosin heavy chain (such as in humans) may differ in the changes shown upon phosphorylation of cMyBP-C. This suggests the importance of investigating in more detail the effect of phosphorylation of cMyBP-C on the structure of the myocardial thick filament in species other than the rat;including species with the (3-myosin heavy chain such as the rabbit. In addition, increasing evidence suggests that N-terminus fragments of cMyBP-C may be able effect myocardial contractility independent of a tethering of the myosin S2 region to the backbone of the filament by cMyBP-C, and may also be able to directly bind actin. These studies emphasize the need to look at the effects of binding of these N-terminus fragments on the cross bridge arrangement and structure of the isolated cardiac thick filament;and further assess by electron microscopy the binding potential of these Nterminus fragments of cMyBP-C to actin. In addition in order to fully understand how cMyBP-C functions in the thick filament, it is vital to have higher resolution 30-reconstructions of the cardiac thick filaments than are currently available. The specfic aims of the research are to: 1) Determine the effect of phosphorylation of cMyBP-C on the crossbridge arrangement of isolated mouse, rat, and rabbit cardiac thick filaments;2) Determine the effect of the C1-C2 and CO-C1 N-terminus fragments of cMyBP-C on the crossbridge arrangement of the isolated thick filaments from rabbit heart, and both wildtype and cMyBP-C""""""""'"""""""" knockout mouse hearts;3) Assess the of binding of C1-C2 and CO-C2 N-terminus fragments of cMyBP-C to actin;and 4) Compute higher resolution 3D-reconstructions of the cardiac thick filaments from both mouse and rabbit thick filaments by single particle analysis to precisely locate the myosin heads and cMyBP-C.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Enhancement Award (SC1)
Project #
5SC1HL096017-02
Application #
7671368
Study Section
Special Emphasis Panel (ZGM1-MBRS-X (CB))
Program Officer
Liang, Isabella Y
Project Start
2008-08-15
Project End
2012-07-31
Budget Start
2009-08-01
Budget End
2010-07-31
Support Year
2
Fiscal Year
2009
Total Cost
$187,500
Indirect Cost
Name
University of Puerto Rico Med Sciences
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
948108063
City
San Juan
State
PR
Country
United States
Zip Code
00936
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
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
González-Solá, Maryví; Al-Khayat, Hind A; Behra, Martine et al. (2014) Zebrafish cardiac muscle thick filaments: isolation technique and three-dimensional structure. Biophys J 106:1671-80
Al-Khayat, Hind A; Kensler, Robert W; Squire, John M et al. (2013) Atomic model of the human cardiac muscle myosin filament. Proc Natl Acad Sci U S A 110:318-23
Kensler, Robert W; Shaffer, Justin F; Harris, Samantha P (2011) Binding of the N-terminal fragment C0-C2 of cardiac MyBP-C to cardiac F-actin. J Struct Biol 174:44-51
Shaffer, Justin F; Kensler, Robert W; Harris, Samantha P (2009) The myosin-binding protein C motif binds to F-actin in a phosphorylation-sensitive manner. J Biol Chem 284:12318-27