Dynamics of Structural Changes in Myosin-Containing Filaments of Intact Mouse EDL During Twitch and Tetanic Contractions

Regulation of contraction in skeletal muscle is mediated by structural changes in both the actin-containing thin filaments and the myosin-containing thick filaments, and contractile dynamics are determined by interactions between motor and regulatory mechanisms. However, those interactions have not been characterised in the physiological context of the twitch response to single action potential stimulation. To do so, we recorded X-ray diffraction patterns using a Pilatus-2M detector at the I22 beamline of the Diamond Light Source in 5ms-frames during the twitch response of isolated intact mouse EDL muscles (starting sarcomere length 2.4μm, 28°C) and compared the results to full activation and relaxation in a 100-ms tetanus. Although peak force in the twitch was only 25% of that in the tetanus, X-ray signals associated with thick filament activation, like the decrease in the intensity of the myosin layer line (ML1) associated with the loss of the folded helical state of myosin motors in the OFF state of the thick filaments, were largely complete in the twitch. The difference reflects the substantial lag between myosin filament activation and motor attachment to actin and force development. Switching off the myosin filament tracked mechanical relaxation in the twitch, but did not start until the end of the 20-ms sarcomere-isometric phase of slow force relaxation following the tetanus, although other X-ray signals showed that myosin motors were detaching from actin during that phase. Moreover, the OFF state of the myosin filament had still not fully recovered at the end of mechanical relaxation, likely reflecting phosphorylation of the regulatory light chain of myosin associated with post-tetanic potentiation. Supported by Medical Research Council and Diamond Light Source, UK.