Cooperative mechanisms in the activation dependence of the rate of force development in rabbit skinned skeletal muscle fibers

Regulation of contraction in skeletal muscle is a highly cooperative process involving Ca²⁺ binding to troponin C (TnC) and strong binding of myosin cross-bridges to actin. To further investigate the role(s) of cooperation in activating the kinetics of cross-bridge cycling, we measured the Ca²⁺ dependence of the rate constant of force redevelopment (k_tr) in skinned single fibers in which cross-bridge and Ca²⁺ binding were also perturbed. Ca²⁺ sensitivity of tension, the steepness of the force-pCa relationship, and Ca²⁺ dependence of k_tr were measured in skinned fibers that were (1) treated with NEM-S1, a strong-binding, non-force-generating derivative of myosin subfragment 1, to promote cooperative strong binding of endogenous cross-bridges to actin; (2) subjected to partial extraction of TnC to disrupt the spread of activation along the thin filament; or (3) both, partial extraction of TnC and treatment with NEM-S1. The steepness of the force-pCa relationship was consistently reduced by treatment with NEM-S1, by partial extraction of TnC, or by a combination of TnC extraction and NEM-S1, indicating a decrease in the apparent cooperativity of activation. Partial extraction of TnC or NEM-S1 treatment accelerated the rate of force redevelopment at each submaximal force, but had no effect on kinetics of force development in maximally activated preparations. At low levels of Ca²⁺, 3 μM NEM-S1 increased k_tr to maximal values, and higher concentrations of NEM-S1 (6 or 10 μM) increased k_tr to greater than maximal values. NEM-S1 also accelerated k_tr at intermediate levels of activation, but to values that were submaximal. However, the combination of partial TnC extraction and 6 μM NEM-S1 increased k_tr to virtually identical supramaximal values at all levels of activation, thus, completely eliminating the activation dependence of k_tr. These results show that k_tr is not maximal in control fibers, even at saturating [Ca²⁺], and suggest that activation dependence of k_tr, is due to the combined activating effects of Ca²⁺ binding to TnC and cross-bridge binding to actin.