The effect of a thiadiazinone derived Ca2+ sensitizer on the responsiveness of Mg2+-ATPase to Ca2+ in myofibrils isolated from stunned and nonstunned porcine and human myocardium

Previously, we showed, in an in situ porcine model, that the thiadiazinone derivative [+]EMD 60263, a putative Ca2+ sensitizer with minimal phosphodiesterase III inhibitory properties, increased contractility more profoundly in stunned than in nonstunned myocardium. The aim of the present investigation was to study the mechanism of action by determining the in vitro effects of [+]EMD 60263 on the Ca2+ responsiveness of the Mg2+-dependent ATPases of myofibrils and sarcoplasmic reticulum membrane vesicles, isolated from normal ventricle of swine and hypertrophic septum of cardiomyopathic patients. Contamination of the myofibrils with sarcoplasmic reticulum membranes was excluded by testing the effect of the sarcoplasmic reticulum Ca2+-pumping ATPase inhibitor thapsigargin. The plasma concentrations at which [+]EMD 60263 exerted its inotropic effect in the in situ porcine model were found to be submicromolar. [+]EMD 60263 stimulated concentration-dependently (1-10 mu M) the submaximally activated Mg2+-ATPase (at pCa 6.1) of pig heart myofibrils. [+]EMD 60263 (10 mu M) shifted the pCa(50) of porcine myofibrillar Ca2+-stimulated, Mg2+-dependent ATPase from 6.00 +/- 0.05 to 6.67 +/- 0.05, whereas the [-]enantiomer EMD 60264 had no significant effect. Although the effect was much less at 1 and 3 mu M, [+]EMD 60263 (10 mu M) also stimulated maximal myofibrillar Mg2+-ATPase activity. The Hill coefficient, reflecting the steepness of the fitted pCa/Mg2+-ATPase curve at half-maximal activation, was not affected by [+]EMD 60263 (10 mu M) [+]EMD 60263 (10 mu M) had no effect on sarcoplasmic reticulum Ca2+-stimulated, Mg2+-dependent ATPase from swine heart. The thiadiazinone derivative [+]EMD 57033 (10 mu M), but not its [-]enantiomer EMD 57439, had similar, although less potent, effects on pig heart myofibrillar Mg2+-ATPase activity as compared to [+]EMD 60263. [+]EMD 60263 (3 mu M) produced a significantly larger leftward shift of the pCa(2+)/Mg2+-ATPase activity curve of myofibrils isolated from the stunned compared to the adjacent nonstunned myocardium (Delta pCa(50)s caused by the presence of [+]EMD 60263 amounted to +0.57 +/- 0.04 and +0.42 +/- 0.05, respectively) in the in situ porcine model. The effects of [+]EMD 60263 on myofibrillar Mg2+-ATPase of hypertrophic human heart were identical to those observed with porcine heart myofibrils. The results indicate that the positive inotropic action of [+]EMD 60263 observed in the in situ porcine model of stunned myocardium may be primarily due to myofilament sensitization to Ca2+, and that this compound may have a similar action on diseased human myocardium.