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Of all the myosin filaments in muscle, the most important in terms of human health, and so far the least studied, are those in the human heart. Here we report a 3D single-particle analysis of electron micrograph images of negatively stained myosin filaments isolated from human cardiac muscle in the normal (undiseased) relaxed state. The resulting 28-Å resolution 3D reconstruction shows axial and azimuthal (no radial) myosin head perturbations within the 429-Å axial repeat, with rotations between successive 132 Å-, 148 Å-, and 149 Å-spaced crowns of heads close to 60°, 35°, and 25°(all would be 40°in an unperturbed three-stranded helix). We have defined the myosin head atomic arrangements within the three crown levels and have modeled the organization of myosin subfragment 2 and the possible locations of the 39 Å-spaced domains of titin and the cardiac isoform of myosin-binding protein-C on the surface of the myosin filament backbone. Best fits were obtained with head conformations on all crowns close to the structure of the two-headed myosin molecule of vertebrate chicken smooth muscle in the dephosphorylated relaxed state. Individual crowns show differences in head-pair tilts and subfragment 2 orientations, which, together with the observed perturbations, result in different intercrown head interactions, including one not reported before. Analysis of the interactions between the myosin heads, the cardiac isoform of myosin-binding protein-C, and titin will aid in understanding of the structural effects of mutations in these proteins known to be associated with human cardiomyopathies.thick filaments 3D structure | human heart muscle | electron microscopy | image processing M uscle contraction involves the interaction between actin and myosin filaments that leads to force production mediated by the hydrolysis of ATP. Myosin filaments are assemblies of myosin molecules and accessory proteins. Myosin molecules consist of two heavy chains arranged to form two globular domains [two subfragment-1s (S1s)] at the N-terminal end and a long rod-like α-helical coiled-coil domain (the myosin tail). Each of the globular domains with its associated essential and regulatory light chains forms a myosin head containing the ATPase activity necessary for contraction (1). This is linked to the N-terminal part of the tail known as the subfragment-2 (S2) region. Myosin filaments are generally bipolar, with the myosin tails packing together in an almost cylindrical backbone and the heads projecting out laterally in a helical or quasihelical fashion at intervals of ∼143 Å. In the myosin filaments of vertebrate striated muscles, the myosin heads are arranged in a threestranded quasihelical array on the filament surface (2) with the accessory proteins titin (3, 4), myosin-binding protein-C (MyBP-C) (5), and possibly the MyBP-C analog, X protein (6), located on the surface of the backbone. MyBP-C is located within the C zones of the myosin filament, which are regions ∼3,500 Å long centrally located in the two halves of the bipolar filament...

Myosin-binding protein C (MyBP-C) is a thick filament protein playing an essential role in muscle contraction, and MyBP-C mutations cause heart and skeletal muscle disease in millions worldwide. Despite its discovery 40 y ago, the mechanism of MyBP-C function remains unknown. In vitro studies suggest that MyBP-C could regulate contraction in a unique way—by bridging thick and thin filaments—but there has been no evidence for this in vivo. Here we use electron tomography of exceptionally well preserved muscle to demonstrate that MyBP-C does indeed bind to actin in intact muscle. This binding implies a physical mechanism for communicating the relative sliding between thick and thin filaments that does not involve myosin and which could modulate the contractile process.

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