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 Ca(2+) 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 Ca(2+) dependence of the rate constant of force redevelopment (k(tr)) in skinned single fibers in which cross-bridge and Ca(2+) binding were also perturbed. Ca(2+) sensitivity of tension, the steepness of the force-pCa relationship, and Ca(2+) 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 Ca(2+), 3 microM NEM-S1 increased k(tr) to maximal values, and higher concentrations of NEM-S1 (6 or 10 microM) 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 microM 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 [Ca(2+)], and suggest that activation dependence of k(tr) is due to the combined activating effects of Ca(2+) binding to TnC and cross-bridge binding to actin.
Fitzsimons
D P
DP
Department of Physiology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706, USA. fitzsimons@physiology.wisc.edu
Patel
J R
JR
Campbell
K S
KS
Moss
R L
RL
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D066298:In Vitro Techniques; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D018656:Muscle Fibers, Fast-Twitch / Q000502:physiology*; D015879:Myosin Subfragments / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D016658:Psoas Muscles / Q000166:cytology; D011817:Rabbits; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D019209:Troponin C / Q000302:isolation & purification
D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; D013439:Sulfhydryl Reagents; D019209:Troponin C; D005033:Ethylmaleimide; D002118:Calcium