STEADY-STATE [CA2+](I)-FORCE RELATIONSHIP IN INTACT TWITCHING CARDIAC-MUSCLE - DIRECT EVIDENCE FOR MODULATION BY ISOPROTERENOL AND EMD-53998

We have developed a novel method for measuring steady-state force-[Ca2+](i) relations in isolated, membrane-intact rat trabeculae that are microinjected with Fura-2 salt. Twitches are markedly slowed after inhibition of phasic Ca2+ release and uptake from the sarcoplasmic reticulum by addition of cyclopiazonic acid and ryanodine. During relaxation of slowed twitches, force and [Ca2+](i) trace a common trajectory in plots of force versus [Ca2+](i), despite very different histories of contraction. The common trajectory thereby provides a high resolution determination of the steady-state relation between force and [Ca2+](i). Using this method, we show that 1 mu M isoproterenol, a beta-adrenergic agonist, causes a rightward shift (Hill function K-1/2 increased from 0.39 +/- 0.07 mu M to 0.82 +/- 0.23 mu M, p < 0.02, n = 6) and a decreased slope (n(H) decreased from 5.4 +/- 1.1 to 4.0 +/- 1.4, p < 0.02) of the steady-state force-[Ca2+](i) curve, with no change in maximal force (F-max = 99.2 +/- 2.2% of control). In contrast, 2 mu M EMD 53998, a racemic thiadiazinone derivative, causes a leftward shift (K-1/2 decreased from 0.42 +/- 0.02 mu M to 0.30 +/- 0.06 mu M, p < 0.02, n = 4) with no change in slope of the steady-state force-[Ca2+](i) curve, accompanied by a modest increase in maximal force (F-max = 107.1 +/- 4.6% of control, p < 0.02), To gain mechanistic insight into these modulatory events, we developed a simple model of cooperative thin filament activation that predicts steady-state force-[Ca2+](i) relationships. Model analysis suggests that isoproterenol decreases cooperativity arising from nearest-neighbor interactions between regulatory units on the thin filament, without change in the equilibrium constant for Ca2+ binding. In contrast, the effects of EMD 53998 are consistent with an increase in the affinity of strong-binding cross-bridges, without change in either the affinity of troponin C for Ca2+ or cooperative interactions.