Muscle contraction is due to myosin motors that transiently attach with their globular head to an actin filament and generate force. After a sudden reduction of the load below the maximum isometric force ( T ₀ ), the attached myosin heads execute an axial movement (the working stroke) that drives the sliding of the actin filament toward the center of the sarcomere by an amount that is larger at lower load and is 11 nm near zero load. Here, we show that an increase in temperature from 2 to 17°C, which increases the average isometric force per attached myosin head by 60%, does not affect the amount of filament sliding promoted by a reduction in force from T ₀ to 0.7 T ₀ , whereas it reduces the sliding under low load by 2.5 nm. These results exclude the possibility that the myosin working stroke is due to the release of the mechanical energy stored in the initial endothermic force-generating process and show that, at higher temperatures, the working stroke energy is greater because of higher force, although the stroke length is smaller at low load. We conclude the following: ( i ) the working stroke is made by a series of state transitions in the attached myosin head; ( ii ) the temperature increases the probability for the first transition, competent for isometric force generation; and ( iii ) the temperature-dependent rise in work at high load can be accounted for by the larger free energy drop that explains the rise in isometric force.

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.