Does cross-bridge activation determine the time course of myofibrillar relaxation?

The ability of force-generating cross-bridges to activate the thin filament in cardiac muscle was tested by studying the effects of initial force and [MgADP] on force relaxation kinetics in subcellular myofibrillar bundles prepared from left ventricles of the guinea pig. Relaxation was initiated by rapidly reducing the [Ca(2+)] from pCa 4.5 to 7.5. Initiating relaxation from lower force levels during pre-steady-state force development did not significantly accelerate the kinetics of the force decay compared to relaxations initiated from steady-state force development. This suggests that the force-generating cross-bridges which become formed during maximally Ca(2+)-activated steady-state contractions do not maintain thin filament activation for significant enough times after Ca(2+)-removal to exert a rate-limiting influence on force relaxation kinetics. Adding 2 mM MgADP to solutions slowed down relaxation kinetics approximately 4-fold. To differentiate whether these slower kinetics result from either (1) MgADP favoring accumulation of cross-bridges during the preceding contraction in a state of activating capability or (2) slow-down of cross-bridge turnover by the presence of the product MgADP during relaxation, the [MgADP] was either increased or removed at the time of Ca(2+)-removal. The addition of 2 mM MgADP to activating solutions (subsequent relaxation in the absence of MgADP) slowed-down the kinetics of the initial, slow, linear force decay following Ca(2+)-removal approximately 1.5-fold, suggesting that the high [MgADP] during contraction favors formation of cross-bridges which contribute in rate-limiting early relaxation kinetics by transiently sustaining thin filament activation. On the other hand, the addition of 2 mM MgADP to the relaxing solution (preceding Ca(2+)-activation in absence of MgADP) slowed-down the kinetics of the initial force decay approximately 3-fold, more similar to the kinetics observed in the continuous presence of 2 mM MgADP both before and after Ca(2+)-removal. This suggest that, despite some influence of cross-bridge activation, the main effect of MgADP on relaxation kinetics results from product inhibition of cross-bridge turnover. In summary, whereas under certain conditions (high [MgADP]) cross-bridge activation of the thin filament can weakly take part in rate-limiting relaxation kinetics induced by complete Ca(2+)-removal, cross-bridge activation does not influence relaxation kinetics under more physiologically normal conditions.