Determination of MyBP-C Orientation in the Cardiac Sarcomere by Immuno-EM

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Myosin binding protein-C (MyBP-C) is a thick filament accessory protein of vertebrate striated muscle. It is essential for normal cardiac function, and mutations in MyBP-C cause cardiac and skeletal muscle disease. The 40 nm long molecule is composed of 10 or 11 immunoglobulin- or fibronectin-like domains and is located in up to 9 axial stripes 43 nm apart in each half of the A-band. To understand MyBP-C function it is important to know its structural organization in the sarcomere. Several models have been proposed, in which MyBP-C either wraps around the thick filament, or extends radially from or longitudinally along the filament (or some combination of these). To distinguish between these models, we have used immuno-EM of mouse cardiac myofibrils labeled with antibodies to different domains of MyBP-C to determine the relative axial positions of the domains. Myofibrils were obtained from mouse cardiac muscle by homogenizing chemically skinned hearts under rigor conditions. They were labeled with antibodies specific for the N-terminal domain, the middle of the molecule, and the C-terminal domain, and observed by negative stain EM. Labeled myofibrils showed nine stain-excluding stripes in each half of the A-band. The average distance of each stripe from the middle of the M-line was measured. All antibodies labeled axially within 10 nm of each other, at the same axial positions as the unlabeled stripes, suggesting that the bulk of MyBP-C runs radially or circumferentially. A small axial difference between the C-terminal and the central and N-terminal antigenic sites suggests that a short portion of the C-terminus runs longitudinally. Electron tomography of muscle sections (Luther et al., PNAS 2011) and 3D reconstruction of isolated thick filaments (Zoghbi et al., PNAS 2008) support the radial arrangement.