Cross-bridge kinetics studied with staircase shortening in single fibres from frog skeletal muscle.

The kinetics of actin-myosin interaction has been studied in single active muscle fibres by repetitively eliciting tension transients with staircase shortening, consisting in a sequence of step releases of identical size (1-5 nm per half-sarcomere) imposed at regular time intervals (3-11 ms). Under sarcomere length-clamp conditions, the quick phase of tension recovery following each step in the staircase is the manifestation of the working stroke by synchronized cross-bridges. Different average shortening velocities are obtained by varying both the size of the step and the time interval between steps. Ti, the tension just before each step in the sequence, T2, the tension attained at the end of the quick phase of tension recovery, decrease with the number of steps, reaching a steady state value, which is lower the larger the shortening velocity. In agreement with previous results on tension response to steady shortening, the overall shortening necessary to approach the steady state values of Ti and T2 is about 15 nm. The normalized amplitude of quick tension recovery (T2r), which is measured by the ratio of the amount of tension recovered at the end of the quick phase (T2-T1) over the tension drop simultaneous with the step (Ti-T1), has been used to measure the extent of the working stroke elicited by each step in the staircase. The steady state value of T2r decreases progressively with the increase of shortening velocity. At velocities higher than 0.5 microns s-1 per half-sarcomere the steady state value of T2r is attained after a transitory depression, which reaches a maximum for an amount of overall shortening increasing from about 8 nm up to about 13 nm with increase in shortening velocity from 0.5 to 1.4 microns s-1 per half-sarcomere. The velocity-dependent transitory depression of T2r can be explained with the mechanical-kinetic model described previously. In the model cross-bridges cycle through two pathway distinct for the kinetics of the detachment/reattachment process. Shortening promotes a redistribution of cross-bridges interacting in the isometric conditions among the various states of the force-generating process. Shortening at high speed, preventing most of cross-bridges from undergoing the relatively fast (100 s-1) detachment/reattachment process, uncovers a rate limiting step in the cycle at the end of the 12 nm working stroke. Under these conditions, the finding that the fraction of the working stroke elicited by each step is transitory depressed with respect to the steady state value reveals that in the original isometric state a large fraction of interacting cross-bridges was accumulated near the beginning of the working stroke.