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.
Bibliographical noteFunding Information:
This work was supported by grants from the National Institutes of Health, the American Heart Association (National and Michigan Affiliate), and The Whitaker Foundation. JMM is an Established Investigator of the American Heart Association.