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 steadystate isometric contraction; force generation in the actin-attached head is fast compared with the attachment step.C ontraction of skeletal muscles is driven by a cyclical interaction between myosin and actin, fueled by the hydrolysis of ATP. The myosin and actin are polymerized into parallel thick and thin filaments, which themselves are organized into a hexagonal array in the muscle cell. The head domains of myosin lie on the surface of the thick filaments and bind to actin in the thin filaments. Filament sliding is driven by a change in conformation of the actin-bound myosin head: its working stroke (1-3). A detailed molecular model for the working stroke has been derived from biochemical and structural studies of isolated myosin head domains and their interaction with actin and ATP (3-6), and the quasi-crystalline organization of myosin and actin in muscle has allowed this model to be tested and elaborated by mechanical and structural studies on muscle cells (1, 2, 7-11).Many of these cell-based studies used rapid perturbations to synchronize the actions of the myosin heads in a muscle cell. Typically, the length of an active muscle fiber was rapidly decreased, displacing each set of myosin filaments by a few nanometers with respect to the opposing actin filaments (2). Such a shortening step produces an elastic force decrease during the step, followed in the next few milliseconds by rapid force regeneration driven by the working stroke in actin-attached myosin heads (2,7,8). This and related protocols have revealed fundamental properties of the working stroke, including its size, speed, and load dependence, and shown how ...

Myosin filament activation in the heart is tuned to the mechanical task

“…This work, by combining sarcomere-level mechanics with nanometermicrometer-scale X-ray diffraction in intact trabeculae from the rat cardiac ventricle, describes the changes in the thick filament and the myosin motors after force development in the cardiac twitch and their relation to SL, with the subnanometer precision achieved by using the X-ray interference between the two halves of the thick filament. We find that, in diastole, all of the myosinbased reflections mark the quasihelical three-stranded symmetry followed by the myosin molecules when they are in their off state on the surface of the thick filament with a short periodicity (2,4,7). At the peak of twitch force, the intensities of all of the meridional reflections and that of the ML1 decrease because of the myosin motors leaving their helical tracks as the thick filament switches on.…”

“…2A and Fig. S2A) shows the first-order layer line reflection (ML1) at an axial spacing of 43 nm and up to the sixth order of the meridional reflections (M1-M6) indexing on the quasihelical three-stranded symmetry with 43-nm periodicity followed by the myosin molecules when they are on the surface of the thick filament in their resting (off) state (2,4,7). The spatial resolution achieved along the meridian (parallel to the trabecula axis) with vertically mounted trabeculae is adequate to record the reflection fine structure (Fig.…”

“…In diastole, the motors are on the surface of the thick filament folded back toward the center of the sarcomere (blue in the cartoon in Fig. 3A), and the backbone has a short periodicity (2,4,7). The corresponding axial mass density is represented by a Gaussian with the center at an axial position (z) displaced by −7.97 nm from the head-rod junction (negative direction is toward the center of the thick filament) and σ = 3.5 nm (Fig.…”

“…The corresponding axial mass density is represented by a Gaussian with the center at an axial position (z) displaced by −7.97 nm from the head-rod junction (negative direction is toward the center of the thick filament) and σ = 3.5 nm (Fig. 3B, blue) (4). The calculated intensity profile, convoluted with the point spread function of the recording system (beam and detector) (dashed line in Fig.…”

Structural dynamics of troponin during activation of skeletal muscle

“…Thus, the original concept of the steric blocking model of muscle regulation, in which azimuthal movement of tropomyosin allows myosin head binding (1, 34) must be modified to the extent that the rate of myosin head binding is not limited by the rate of activation of the thin filament; myosin head binding is relatively slow, and this limits the rate of force generation in physiological conditions. The physiological rate of myosin head binding may be limited by structural changes in the thick rather than in the thin filaments (35).…”