Myosin binding protein C (MyBP-C) is a component of the thick filament of striated muscle. The importance of this protein is revealed by recent evidence that mutations in the cardiac gene are a major cause of familial hypertrophic cardiomyopathy. Here we investigate the distribution of MyBP-C in the A-bands of cardiac and skeletal muscles and compare this to the A-band structure in cardiac muscle of MyBP-C-deficient mice. We have used a novel averaging technique to obtain the axial density distribution of A-bands in electron micrographs of well-preserved specimens. We show that cardiac and skeletal A-bands are very similar, with a length of 1.58 ± 0.01 μm. In normal cardiac and skeletal muscle, the distributions are very similar, showing clearly the series of 11 prominent accessory protein stripes in each half of the A-band spaced axially at 43-nm intervals and starting at the edge of the bare zone. We show by antibody labelling that in cardiac muscle the distal nine stripes are the location of MyBP-C. These stripes are considerably suppressed in the knockout mouse hearts as expected. Myosin heads on the surface of the thick filament in relaxed muscle are thought to be arranged in a three-stranded quasi-helix with a mean 14.3-nm axial cross bridge spacing and a 43 nm helix repeat. Extra “forbidden” meridional reflections, at orders of 43 nm, in X-ray diffraction patterns of muscle have been interpreted as due to an axial perturbation of some levels of myosin heads. However, in the MyBP-C-deficient hearts these extra meridional reflections are weak or absent, suggesting that they are due to MyBP-C itself or to MyBP-C in combination with a head perturbation brought about by the presence of MyBP-C.

The formation of a high-molecular weight complex between spectrin and F-actin depends on the presence of a third cytoskeletal constituent, protein 4.1. Electron microscopy shows that in this ternary complex the actin filaments are linked by bridges, which have the appearance of spectrin. The spectrin must be in the tetrameric state for such bridges to form: the dimer is evidently univalent, for it binds but forms no cross-links. G-actin also fails to form extended complexes. It is inferred that in the native cytoskeleton the spectrin is tetrameric and associated with 4.1 and probably oligomers of actin.

Purified antibodies to the thick filament accessory proteins, C-protein, X-protein and H-protein, have been used to label fibres of three rabbit muscles, psoas (containing mainly fast white fibres), soleus (containing mainly slow red fibres) and plantaris (a muscle of mixed fibre type) and their location has been examined by electron microscopy. These accessory proteins are present on one or more of a set of eleven transverse stripes about 43 nm apart that have been observed previously in each half A-band. Each protein has a limited set of characteristic distributions. H-protein is present on stripe 3 (counting from the M-line) in the majority of psoas fibres but is absent in soleus and plantaris muscle. C-protein can occur on stripes 4-11 (the commonest pattern seen in psoas); on stripes 5-11 (in psoas and plantaris); on stripes 3 together with stripes 5-11 (in plantaris); or on none (in red fibres of all three muscles). X-protein can occur on stripes 3-11 in the red fibres of all three muscles; on stripe 4 only (in psoas and plantaris); on stripes 3 and 4 (in psoas and plantaris) or on none. Stripes labelled with anti-X are wider than those labelled with anti-C and consist of a doublet with an internal spacing of 16 nm. The patterns for the three accessory proteins, while overlapping, are in no case identical; this suggests the proteins do not simply substitute for one another. The precise axial positions of the anti-C labelled stripes differ from those of the anti-X stripes; the anti-X stripes lie about 8-9 nm further from the M-line than the corresponding anti-C stripes. This implies that the inner member of an X-protein doublet lies in a very similar position to a C-protein stripe. The anti-H labelled stripe seen in most psoas fibres lies 14 nm nearer the M-line than stripe 3 of the anti-X labelled array in psoas red fibres and is staggered from a continuation of the C-protein array by about 4 nm. The labelling patterns were constant within a fibre and suggest a very precise assembly mechanism. The number of classes of fibre, as defined by the accessory proteins present and their arrangement, exceeds the number of fibre types presently recognized.