Troponin C regulates the rate constant for the dissociation of force-generating myosin cross-bridges in cardiac muscle

It is well known that cardiac troponin C (cTnC) regulates the association o f force-generating myosin cross-bridges. We report here evidence for an add itional role for cTnC. This hypothesis states that Ca2+ binds more strongly to cTnC when force-generating myosin cross-bridges are attached to actin a nd that removal of this bound Ca2+ accelerates the dissociation of force-ge nerating myosin cross-bridges. Intact Fura-2-loaded rat papillary muscles a nd skinned (permeabilized) ventricular preparations were used. The preparat ions were mounted in the Guth Muscle Research System which is capable of me asuring simultaneously fluorescence and force in response to length perturb ations. All mechanical perturbations of muscle length (isotonic shortening, quick stretches and releases, and length vibrations) which cause dissociat ion of force-generating myosin cross-bridges during a twitch resulted in Ca 2+ being released from troponin as judged from changes in the Ca2+ transien ts (Fura-2 (340/380) fluorescence ratio). Thus dissociation of force-genera ting myosin cross-bridges cause Ca2+ to be released from cTnC. Conversely, it would be expected that removal of strongly bound Ca2+ from cTnC would re sult in an increase in the rate of dissociation of force-generating myosin cross-bridges. To test this hypothesis actomyosin ATPase (NADH fluorescence change) and isometric force were measured in skinned cardiac preparations. The ratio of the ATPase/Force is proportional to the rate constant (g(a)pp ) for the dissociation of force-generating myosin cross-bridges. The data s howed that decreasing the amount of Ca2+ bound to cTnC in skinned cardiac f ibers caused an increase in the ratio of ATPase/Force, the rate of dissocia tion (g(app)) of force-generating myosin cross-bridges.