Muscle contraction is triggered by an increase in intracellular free calcium concentration leading to a structural change in the actin-containing thin filament that allows myosin motors to bind and generate force. The number of motors available for binding to actin is determined by the structure of the myosin-containing thick filaments, so that more motors are available at higher load  (Linari et al., 2015; Hill et al., 2021, 2022). Previous studies have focused on the activation of the thick filament at the start of contraction; here we used time-resolved X-ray diffraction to characterise the inactivation of the thick filament when the load is rapidly decreased by imposing rapid shortening during maximal calcium activation.

Small-angle X-ray diffraction patterns were recorded from extensor digitorum longus muscles of the mouse at 27°C using an Eiger 2X-4M detector at the ID02 beamline at the ESRF, Grenoble, France, at a camera length of 3.2m or 2.0m for the low-angle X-ray reflections, and at 31m for the sarcomere reflections. Initial sarcomere length (SL) was set to 2.87±0.006μm (mean ± S.E.M) in the resting muscle. Muscles were stimulated continuously for 120ms to produce a fused tetanus, and X-ray data were collected in 2-ms time frames. 60ms after the first stimulus, when SL was 2.67±0.007μm, rapid shortening was imposed for 15ms (SL 2.33±0.04μm), then force redeveloped at fixed muscle length (SL 2.13±0.008μm). Data were collected from approximately 30 tetani in each muscle (n=7). All procedures accorded with current national legislation.

The first order myosin layer line (ML1), associated with the folded helical array of myosin motors in the OFF state of the thick filament, decreased at the start of stimulation but recovered partially towards its resting level during unloaded shortening, indicating partial recovery of the thick filament OFF state. The axial periodicity of the thick filament backbone, signalled by the spacing of the M6 reflection, which increases at the start of stimulation, also partly recovered during unloaded shortening, as did the second actin-based layer line, associated with the azimuthal position of tropomyosin. Thus, both the thin and thick filament are partially inactivated during unloaded shortening, indicating positive coupling between the regulatory states of the two filaments. Unexpectedly, the spacing of the M3 reflection, associated with the axial repeat of myosin motors, which increases during contraction, decreased below the resting level during unloaded shortening. Finally, the sarcomere-based X-ray reflections revealed two distinct phases of relaxation following electrical stimulation: a ~20ms sarcomere-isometric phase followed by ~60ms of chaotic relaxation associated with two distinct sarcomere populations.

These results indicate that activation of both the thick and thin filaments decreases during unloaded shortening at the tetanus plateau. These results are consistent with the mechano-sensing paradigm in the thick filaments, activation of the thin filaments by myosin motors, and positive coupling between the regulatory states of the thick and thin filaments.