Skinned muscle fiber bundles were attached to the arm of a displacement motor (model 308B; Aurora Scientific Inc.) on one end and to a force transducer (AE 801; Sensor One Technologies Corporation) on the other end using aluminum T-clips. Mounted bundles were placed in a 15-µl well, where they were bathed in either activating or relaxing solutions. Sarcomere length, SL, was measured by laser light diffraction methods. Visual display of the first-order laser light diffraction allowed judgment as to the accuracy of the SL assessment and the quality of the preparation. SL in all preparations was set to 2.2 µm before Ca2+ activation. Fiber bundle length at SL 2.2 µm was recorded as baseline muscle length (ML). Fiber bundle cross-sectional area was calculated from optical measurements of major and minor axes using an assumption of an elliptical cross section.
Ca2+-activated force was measured at maximal level of activation (pCa 4.3). The amount of free [Ca2+] in solution and corresponding pCa value was determined using methods described previously (Fabiato and Fabiato, 1979 (link)). Once steady-state isometric force was achieved, the length of the constantly activated muscle was commanded to change according to the following step length change protocol. A command was given to the motor for the muscle to increase its length by 0.5% of ML in a step-like fashion. This length was maintained for 5 s at which time the muscle was commanded to rapidly return to ML. After another 5 s, a command was given to increase muscle length to 1.0% of ML, which was held for 5 s before rapidly returning to ML. This procedure was repeated for additional step increases in muscle length of 1.5 and 2.0%. All step-like changes in muscle length were essentially completed in 1–2 ms. Measurements of force and muscle length were digitally sampled every 1 ms during the entire procedure. In all cases, force stabilized to a steady-state value within ∼1.5 s after a change in length. The first 1.5 s of the force response to the increase in muscle length was taken as a response to quick stretch, and the first 1.5 s of force response after the return of muscle length to ML was taken as a response to quick release. All force and muscle length records were normalized to their respective values just before length change.
Ca2+-activated force was measured at maximal level of activation (pCa 4.3). The amount of free [Ca2+] in solution and corresponding pCa value was determined using methods described previously (Fabiato and Fabiato, 1979 (link)). Once steady-state isometric force was achieved, the length of the constantly activated muscle was commanded to change according to the following step length change protocol. A command was given to the motor for the muscle to increase its length by 0.5% of ML in a step-like fashion. This length was maintained for 5 s at which time the muscle was commanded to rapidly return to ML. After another 5 s, a command was given to increase muscle length to 1.0% of ML, which was held for 5 s before rapidly returning to ML. This procedure was repeated for additional step increases in muscle length of 1.5 and 2.0%. All step-like changes in muscle length were essentially completed in 1–2 ms. Measurements of force and muscle length were digitally sampled every 1 ms during the entire procedure. In all cases, force stabilized to a steady-state value within ∼1.5 s after a change in length. The first 1.5 s of the force response to the increase in muscle length was taken as a response to quick stretch, and the first 1.5 s of force response after the return of muscle length to ML was taken as a response to quick release. All force and muscle length records were normalized to their respective values just before length change.
