TY - JOUR
T1 - Orientation of myosin binding protein C in the cardiac muscle sarcomere determined by domain-specific immuno-EM
AU - Lee, Kyounghwan
AU - Harris, Samantha P.
AU - Sadayappan, Sakthivel
AU - Craig, Roger
N1 - Funding Information:
We thank Drs. Christine E. Seidman and Jonathan G. Seidman for their generosity in providing the cMyBP-C (t/t) mouse model; Elaine Hoye for assistance in the development of the C5-C7 antibody; and Drs. John Woodhead, Michael Previs, and David Warshaw for comments on the manuscript. We are also grateful to Drs. George Witman and Branch Craige for training on and use of their immunofluorescence microscope. This work was supported by National Institutes of Health grants R01 AR034711 (R.C.) and P01 HL059408 (to D. Warshaw), grant R01 HL080367 (S.P.H.), and grants R01 HL105826 and K02 HL114749 (S.S.) and S10 RR027897 (for the purchase of the electron microscope).
Publisher Copyright:
© 2014 Elsevier Ltd. All rights reserved.
PY - 2015/1/30
Y1 - 2015/1/30
N2 - Myosin binding protein C is a thick filament protein of vertebrate striated muscle. The cardiac isoform [cardiac myosin binding protein C (cMyBP-C)] is essential for normal cardiac function, and mutations in cMyBP-C cause cardiac muscle disease. The rod-shaped molecule is composed primarily of 11 immunoglobulin- or fibronectin-like domains and is located at nine sites, 43 nm apart, in each half of the A-band. To understand how cMyBP-C functions, it is important to know its structural organization in the sarcomere, as this will affect its ability to interact with other sarcomeric proteins. Several models, in which cMyBP-C wraps around, extends radially from, or runs axially along the thick filament, have been proposed. Our goal was to define cMyBP-C orientation by determining the relative axial positions of different cMyBP-C domains. Immuno-electron microscopy was performed using mouse cardiac myofibrils labeled with antibodies specific to the N- and C-terminal domains and to the middle of cMyBP-C. Antibodies to all regions of the molecule, except the C-terminus, labeled at the same nine axial positions in each half A-band, consistent with a circumferential and/or radial rather than an axial orientation of the bulk of the molecule. The C-terminal antibody stripes were slightly displaced axially, demonstrating an axial orientation of the C-terminal three domains, with the C-terminus closer to the M-line. These results, combined with previous studies, suggest that the C-terminal domains of cMyBP-C run along the thick filament surface, while the N-terminus extends toward neighboring thin filaments. This organization provides a structural framework for understanding cMyBP-C's modulation of cardiac muscle contraction.
AB - Myosin binding protein C is a thick filament protein of vertebrate striated muscle. The cardiac isoform [cardiac myosin binding protein C (cMyBP-C)] is essential for normal cardiac function, and mutations in cMyBP-C cause cardiac muscle disease. The rod-shaped molecule is composed primarily of 11 immunoglobulin- or fibronectin-like domains and is located at nine sites, 43 nm apart, in each half of the A-band. To understand how cMyBP-C functions, it is important to know its structural organization in the sarcomere, as this will affect its ability to interact with other sarcomeric proteins. Several models, in which cMyBP-C wraps around, extends radially from, or runs axially along the thick filament, have been proposed. Our goal was to define cMyBP-C orientation by determining the relative axial positions of different cMyBP-C domains. Immuno-electron microscopy was performed using mouse cardiac myofibrils labeled with antibodies specific to the N- and C-terminal domains and to the middle of cMyBP-C. Antibodies to all regions of the molecule, except the C-terminus, labeled at the same nine axial positions in each half A-band, consistent with a circumferential and/or radial rather than an axial orientation of the bulk of the molecule. The C-terminal antibody stripes were slightly displaced axially, demonstrating an axial orientation of the C-terminal three domains, with the C-terminus closer to the M-line. These results, combined with previous studies, suggest that the C-terminal domains of cMyBP-C run along the thick filament surface, while the N-terminus extends toward neighboring thin filaments. This organization provides a structural framework for understanding cMyBP-C's modulation of cardiac muscle contraction.
KW - Cardiac muscle contraction
KW - Cardiac muscle disease
KW - Cardiac muscle regulation
KW - Cardiac muscle structure
KW - cMyBP-C
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U2 - 10.1016/j.jmb.2014.10.023
DO - 10.1016/j.jmb.2014.10.023
M3 - Article
C2 - 25451032
AN - SCOPUS:84920784546
VL - 427
SP - 274
EP - 286
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 2
ER -