Orientation of Myosin Binding Protein C in the Cardiac Muscle Sarcomere Determined by Domain-Specific Immuno-EM. Issue 2 (30th January 2015)

• Record Type: Journal Article
• Authors: Lee, Kyounghwan; Harris, Samantha P.; Sadayappan, Sakthivel; Craig, Roger
• Is Part Of: Journal of molecular biology. Volume 427:Issue 2(2015:Jan. 15)
• Page Start: 274
• Page End: 286
• Publication Date: 2015-01-30
• Subjects: MyBP-C myosin binding protein C -- cMyBP-C cardiac myosin binding protein C -- EM electron microscopy -- RLC regulatory light chain -- S2 subfragment 2 -- LMM light meromyosin -- 3D three-dimensional -- BSA bovine serum albumin; cMyBP-C -- cardiac muscle contraction -- cardiac muscle disease -- cardiac muscle structure -- cardiac muscle regulation; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2014.10.023
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: 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. Graphical abstract: Highlights: The organization of cMyBP-C in the cardiac muscle sarcomere is controversial. Cardiac muscle myofibrils were labeled with domain-specific antibodies to cMyBP-C. The antibodies revealed the axial positions of different domains of cMyBP-C. Different domains were located at the same axial positions along the thick filament. We conclude that most of cMyBP-C is oriented perpendicular to the thick filament.