Role of Ca²⁺ and cross-bridges in skeletal muscle thin filament activation probed with Ca²⁺ sensitizers

Thin filament regulation of contraction is thought to involve the binding of two activating ligands: Ca²⁺ and strongly bound cross-bridges. The specific cross-bridge states required to promote thin filament activation have not been identified. This study examines the relationship between cross- bridge cycling and thin filament activation by comparing the results of kinetic experiments using the Ca²⁺ sensitizers caffeine and bepridil. In single skinned rat soleus fibers, 30 mM caffeine produced a leftward shift in the tension-pCa relation from 6.03 ± 0.03 to 6.51 ± 0.03 pCa units and lowered the maximum tension to 0.60 ± 0.01 of the control tension. In addition, the rate of tension redevelopment (k(tr)) was decreased from 3.51 ± 0.12 s^-1 to 2.70 ± 0.19 s^-1, and V(max) decreased from 1.24 ± 0.07 to 0.64 ± 0.02 M.L./s. Bepridil produced a similar shift in the tension-pCa curves but had no effect on the kinetics. Thus bepridil increases the Ca²⁺ sensitivity through direct effects on TnC, whereas caffeine has significant effects on the cross-bridge interaction. Interestingly, caffeine also produced a significant increase in stiffness under relaxing conditions (pCa 9.0), indicating that caffeine induces some strongly bound cross-bridges, even in the absence of Ca²⁺. The results are interpreted in terms of a model integrating cross-bridge cycling with a three-state thin-filament activation model. Significantly, strongly bound, non-tension-producing cross- bridges were essential to modeling of complete activation of the thin filament.