Liguzinediol的正性肌力作用机制及心脏安全性

Positive inotropic mechanism of liguzinediol and heart safety evaluation

目的探索Liguzinediol(LZDO)对正常大鼠离体心脏正性肌力作用的机制,并评价其心脏安全性。方法①大鼠离体心脏实验:按照灌流液(空白对照)→LZDO 100μmol.L-1→洗脱的顺序灌流,持续5 min并于灌流5 min末记录大鼠离体心脏左心室收缩压(LVSP)、左心室舒张末期压(LVEDP)、左室内压最大上升/降速率(±dp/dtmax)及心率(HR)。②豚鼠在体和离体实验:在体实验按照生理盐水→LZDO 1.7 g.kg-1的顺序经颈外静脉缓慢推注,持续5 min,于每次处理后5 min记录5只豚鼠心电图。离体实验按照灌流液(空白对照)→LZDO 300μmol.L-1的顺序灌流,持续5 min,于灌流5 min末记录豚鼠离体心脏心电图。分析P-R间期和心率校正QT间期(QTc间期)。③膜片钳全细胞法记录细胞膜离子通道电流:按照灌流液(空白对照)→尼莫地平2μmol.L-1顺序灌流左心室肌细胞,记录电流。灌流液(空白对照)→LZDO100μmol.L-1的顺序灌流,于灌流5 min末记录其5个细胞的L型钙电流。另两组实验按照灌流液(空白对照)→LZDO 1→10→100→300μmol.L-1的顺序灌流,于灌流5 min末记录hNav1.5和hERG电流。④激光共聚焦方法测定左心室心肌细胞的钙释放量:按照灌流液(空白对照))→LZDO 100μmol.L-1的顺序灌流,于灌流2 min和30 min末记录心肌细胞钙释放量。结果①大鼠离体心脏实验:尼莫地平1μmol.L-1和rethenium red 5μmol.L-1均能完全阻断LZDO 100μmol.L-1的正性肌力作用。②豚鼠在体和离体心电图实验:豚鼠在体给予LZDO 1.7 g.kg-1或豚鼠离体心脏灌流LZDO 300μmol.L-1后,P-R及QTc间期并没有显著性改变。③细胞膜离子通道电流实验:LZDO 100μmol.L-1未能显著地增加大鼠左心室肌细胞的L型钙电流;LZDO 1,10,100和300μmol.L-1未能显著地改变hNav1.5和hERG电流。④激光共聚焦测定左心室心肌细胞钙释放实验:LZDO 100μmol.L-1显著增加左心室心肌细胞钙释放,于2 min末钙释放量达到最大值,并能维持到30 min(P<0.05)。结论 LZDO对L型钙通道无直接作用,LZDO是通过作用肌浆网钙释放来起到正性肌力作用的;LZDO在体1.7 g.kg-1或离体300μmol.L-1无致心律失常的作用。

OBJECTIVE To explore the mechanism underlying positive inotropic effect in rat isolated hearts and evaluate cardiac safety of liguzinediol.METHODS ① In vitro isolated rat hearts were used to analyze the effects of tested compounds on the peak isovolumic left ventricular systolic pressure（LVSP）,left ventricular end diastolic pressure（LVEDP）,heart rate（HR）,peak rate of rise/decrease of LVSP（±dp/dtmax）.The isolated rat hearts were exposed to blank control→100 μmol·L-1 or in presence of L-type Ca2＋ channel or ryanodine receptor blocker by Langendorff perfusion.② In vivo ECG recordings were made to analyze effects of jugular intravenous（iv） injection of LZDO 1.7 g·kg-1 on P-R and QTc intervals in guinea pigs.In vitro ECG recordings were made to analyze effects LZDO 300 μmol·L-1 on P-R and QTc intervals in isolated guinea pig hearts.③ Patch clamp technique was used to analyze the effects of LZDO on L-type Ca2＋ current which underlies mechanism of LZDO′s positive inotropic effect and hNav1.5 and hERG currents which serve for cardiac safety evaluation of LZDO.④ Intracellular Ca2＋ imaging was used to investigate the effect of LZDO on Ca2＋ transient.RESULTS ① LZDO 100 μmol·L-1 significantly enhanced the isolated rat left ventricular contractility.Meanwhile,nimodipine 1 μmol·L-1 and rethenium red 5 μmol·L-1 completely blocked the positive inotropic effect of LZDO 100 μmol·L-1.LZDO 100 μmol·L-1 failed to increase the L-type Ca2＋ current in rat left ventricular myocyte.Also,LZDO 100 μmol·L-1 significantly enhanced intracellular Ca2＋ transient from normalized control（100±4）% to（138.4±2）%（P0.05,n=5）.② LZDO 1.7 g·kg-1 in vivo and LZDO 300 μmol·L-1 in vitro could not prolong P-R and QTc intervals.LZDO 1,10,100 and 300 μmol·L-1 failed to change hNav1.5 and hERG currents.CONCLUSION The positive inotropy effect of LZDO is mediated by sarcoplasmic reticulum Ca2＋ release and LZDO has no direct effect on L-type Ca2＋ current.LZDO 1.7 g·kg-1 in vivo and LZDO 300 μmol·L-1 in vitro could have no proarrhythmic effect.