Bezstarosti K, Soei L K, Krams R, Ten Cate F J, Verdouw P D, Lamers J M
Department of Biochemistry, Cardiovascular Research Institute COEUR, Faculty of Medicine and Health Sciences, Erasmus University, Rotterdam, The Netherlands.
Biochem Pharmacol. 1996 May 3;51(9):1211-20. doi: 10.1016/0006-2952(96)00083-4.
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 Mg(2+)-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 Ca(2+)-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 microM) the submaximally activated Mg(2+)-ATPases (at pCa 6.1) of pig heart myofibrils. [+]EMD 60263 (10 microM) shifted the pCa50 of porcine myofibrillar Ca(2+)-stimulated, Mg(2+)-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 microM, [+]EMD 60263 (10 microM) also stimulated maximal myofibrillar Mg(2+)-ATPase activity. The Hill coefficient, reflecting the steepness of the fitted pCa/Mg(2+)-ATPase curve at half-maximal activation, was not affected by [+]EMD 60263 (10 microM). [+]EMD 60263 (10 microM) had no effect on sarcoplasmic reticulum Ca(2+)-stimulated, Mg(2+)-dependent ATPase from swine heart. The thiadiazinone derivative [+]EMD 57033 (10 microM), but not its [-]enantiomer EMD 57439, had similar, although less potent, effects on pig heart myofibrillar Mg(2+)-ATPase activity as compared to [+]EMD 60263. [+]EMD 60263 (3 microM) produced a significantly larger leftward shift of the pCa2+/Mg(2+)-ATPase activity curve of myofibrils isolated from the stunned compared to the adjacent nonstunned myocardium (Delta pCa50s 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 Mg(2+)-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.
此前，我们在一个原位猪模型中发现，噻二嗪酮衍生物[+]EMD 60263是一种假定的Ca2+增敏剂，具有最小的磷酸二酯酶III抑制特性，与非顿抑心肌相比，它在顿抑心肌中更显著地增强了收缩性。本研究的目的是通过测定[+]EMD 60263对从猪正常心室和心肌病患者肥厚间隔中分离出的肌原纤维及肌浆网膜囊泡的Mg(2+)-依赖性ATP酶的Ca2+反应性的体外作用，来研究其作用机制。通过检测肌浆网Ca(2+)-泵ATP酶抑制剂毒胡萝卜素的作用，排除了肌原纤维被肌浆网膜污染的情况。发现在原位猪模型中[+]EMD 60263发挥其正性肌力作用时的血浆浓度为亚微摩尔。[+]EMD 60263浓度依赖性地（1 - 10 microM）刺激猪心肌肌原纤维的亚最大激活Mg(2+)-ATP酶（在pCa 6.1时）。[+]EMD 60263（10 microM）使猪肌原纤维Ca(2+)-刺激的、Mg(2+)-依赖性ATP酶的pCa50从6.00±0.05变为6.67±0.05，而其[-]对映体EMD 60264则无显著作用。尽管在1 microM和3 microM时作用小得多，但[+]EMD 60263（10 microM）也刺激了最大肌原纤维Mg(2+)-ATP酶活性。反映半最大激活时拟合的pCa/Mg(2+)-ATP酶曲线陡度的希尔系数不受[+]EMD 60263（10 microM）影响。[+]EMD 60263（10 microM）对猪心肌肌浆网Ca(2+)-刺激的、Mg(2+)-依赖性ATP酶无作用。噻二嗪酮衍生物[+]EMD 57033（10 microM），而不是其[-]对映体EMD 57439，与[+]EMD 60263相比，对猪心肌肌原纤维Mg(2+)-ATP酶活性有相似但较弱的作用。在原位猪模型中，与相邻的非顿抑心肌相比，[+]EMD 60263（3 microM）使从顿抑心肌分离出的肌原纤维的pCa2+/Mg(2+)-ATP酶活性曲线显著更大程度地向左移位（由[+]EMD 60263引起的Delta pCa50分别为+0.57±0.04和+0.42±0.05）。[+]EMD 60263对肥厚型人心脏肌原纤维Mg(2+)-ATP酶的作用与在猪心肌肌原纤维中观察到的相同。结果表明，在顿抑心肌的原位猪模型中观察到的[+]EMD 60263的正性肌力作用可能主要归因于肌丝对Ca2+的敏感性增加，并且该化合物可能对患病的人心肌有类似作用。
