Motion of myosin head domains during activation and force development in skeletal muscle

Abstract

Muscle contraction is driven by a change in the structure of the head domain of myosin, the "working stroke" that pulls the actin filaments toward the midpoint of the myosin filaments. This movement of the myosin heads can be measured very precisely in intact muscle cells by X-ray interference, but until now this technique has not been applied to physiological activation and force generation following electrical stimulation of muscle cells. By using this approach, we show that the long axes of the myosin head domains are roughly parallel to the filaments in resting muscle, with their center of mass offset by approximately 7 nm from the C terminus of the head domain. The observed mass distribution matches that seen in electron micrographs of isolated myosin filaments in which the heads are folded back toward the filament midpoint. Following electrical stimulation, the heads move by approximately 10 nm away from the filament midpoint, in the opposite direction to the working stroke. The time course of this motion matches that of force generation, but is slower than the other structural changes in the myosin filaments on activation, including the loss of helical and axial order of the myosin heads and the change in periodicity of the filament backbone. The rate of force development is limited by that of attachment of myosin heads to actin in a conformation that is the same as that during steady-state isometric contraction; force generation in the actin-attached head is fast compared with the attachment step.

Fig. 1.

Changes in the M3 X-ray reflection during isometric force development. (A) Arrangement of thick (black) and thin (white) filaments in the sarcomere showing axial periodicity of myosin heads (d), and interference distance (L) between the two arrays of heads in each thick filament. (B) Axial profiles of the M3 X-ray reflection at different times during force development; colors correspond to symbols in C and D. (C) Intensity of the M3 X-ray reflection (I_M3) after width correction, normalized by tetanus plateau value. (D) Spacing of the M3 X-ray reflection (S_M3). Thicker line in C and D is force normalized by its plateau value (T₀); thinner line is change in half-sarcomere (hs) length. Error bars denote ± SE for five fibers. Dashed lines in C and D were calculated from the model in Fig. 3.

Fig. 2.

Analysis of the component peaks of the M3 reflection. (A and B) Black lines are axial profiles of the M3 reflection at rest (A) and at the tetanus plateau (B). Red, blue, and green lines are Gaussian functions fitted to the lower, middle, and higher angle peaks, respectively. (C) Fractional intensity and (D) axial spacing of the three peaks (mean ± SE, n = 5 fibers); continuous lines show mean calculated from the model described in the text.

Fig. 3.

Three populations of myosin heads during isometric force development: (A) Force-generating (Top, red), active detached (Top, orange), and two possible resting conformations (Middle and Lower, green). The globular catalytic domain of the head binds to actin, and the more elongated light chain domain connects it to the myosin tail in the filament backbone. (B) Axial mass distributions of these three conformations, and from isolated thick filaments (22) (black). (C) Comparison of experimental and model M3 axial profiles at rest (black and thick red continuous lines, respectively) and at the tetanus plateau (blue and thin red line). (D) Fraction of heads in the resting (f_R, green), active detached (f_D, orange), and force-generating (f_A, red) conformations, and mean center of mass (CoM) of the heads (violet).

Fig. 4.

Changes in the intensities of other X-ray reflections during isometric force development. (A) Meridional associated with troponin (T1). (B) First myosin layer line (ML1). (C) First myosin-based meridional (M1). (D) Second myosin-based meridional (M2). Line is force normalized by its plateau value. Error bars denote ± SE for the five fibers in Fig. 1.