延迟钠电流在心肌细胞内钙异常介导的心律失常中的作用及其机制

An increase in intracellular calcium concentration exists in many physiological, pathological and pharmacological conditions and is associated with triggered activities and reentry mechanisms and arrhythmias which may be related to the prolongation of action potential duration and polymorphic ventricular tachycardias. This kind of calcium-related arrhythmias common in clinic and is hard to be treated. Recent data from a few small clinical studies indicates that late sodium current inhibitors is effective in preventing or reversing the calcium-induced arrhythmias, including long QT syndrome 8. Based on the research results in this laboratory, we speculated that the endogenous or enhanced late sodium current may play a role in the calcium related ventricular arrhythmias, In this study, L-type calcium channel activator Bay-K 8644 and isoproterenol will be used to induce the intracellular overload in the absence and presence of low concentration of late sodium current enhancer ATX-II, NCX inhibitor calmoduline kinase II modulator. Tetrodotoxin (TTX) and ranolazine will be used to inhibit the endogenous or enhanced late sodium current and to reverse the changes in proarrhythmic electrophysilogical factor, and to prevent or treat ventricular arrhythmia and mechanical abnormalities. The results from this study is speculated to confirm the contribution of late sodium current in the calcium-related ventricular arrhythmias and give a new direction on the prevention and treatment of calcium overload related ventricular arrhythmias.

细胞内钙浓度的增高与多种心律失常的触发和折返形成有关，可合并动作电位时程延长，诱发室性心动过速，甚至猝死，临床上常见，治疗困难。多种生理、病理和药物可造成细胞内钙浓度增高。临床小样本的报告证实延迟钠电流抑制剂可抑制包括长QT综合征8在内的钙相关心律失常，根据以往的研究结合预实验的结果我们推测内源性或增强的延迟钠电流在钙相关心律失常发生中起作用。本研究拟在新西兰兔整体心脏和分离的心室肌单细胞水平，应用L型钙通道激动剂Bay-K 8644和异丙肾上腺素制作细胞内钙浓度增高的模型，结合延迟钠电流增强剂、钙调蛋白激酶II和钠钙交换抑制剂重复出细胞内钙增高引起的电生理改变及心律失常，并观察延迟钠电流抑制剂对心室肌动作电位时程、心律失常发生率、钙瞬变、心肌细胞收缩功能的影响，预期结果可证明延迟钠电流与细胞内钙浓度增高发生协同作用，共同促进心律失常的发生，为进一步研究钙相关心律失常的新的治疗方法打下基础

研究背景：.心肌细胞内钙浓度增高见于多种生理及病理情况：如遗传性钙离子通道病长QT8、心肌缺血再灌注、应用洋地黄等药物后等，常表现为特定的心电和机械功能障碍，合并恶性心律失常，临床评估困难，目前临床治疗效果差。其机制可能与心室肌细胞内过多的Ca2+能够激活钙调蛋白激酶II（CaMK II）和蛋白激酶C（PKC）通路，进而引起延迟钠电流（INaL）增大相关。增大的INaL不仅可以激活细胞的CaMK II，同时还可通过激活反向钠钙交换体（sodium-calcium exchange，NCX），进一步加重细胞内Ca2+的增高，从而形成正反馈式的恶性循环。然而，INaL是否也能与钙内流增多一起协同促进心律失常的发生尚未有报道。.研究内容：.1..在整体心脏水平，建立新西兰兔心肌细胞内Ca2+浓度增高（使用L型钙通道激动剂Bay K 8644、INaL增强剂ATX-II）导致心电学改变及心律失常的动物模型，观察左心室内、外膜MAPD90对Bay K 8644的浓度及频率反应关系，同时运用INaL抑制剂TTX和eleclazine治疗并观察疗效。.2..在细胞水平，分离成年新西兰兔心室肌细胞，观察Bay K 8644（300 nM）、INaL抑制剂TTX（1 µM）和eleclazine (10 µM)处理后的心室肌细胞钙瞬变等指标的变化。.3..在分子生物学水平，应用Western Blotting技术检测Bay K 8644、ATX-II对正常心脏与复极储备下降的心脏组织的CaMK II、受磷蛋白（phospholamban，PLB）的表达与激活的影响，并观察特异性延迟钠电流抑制剂（TTX）可能的作用。.重要结果：.1..Bay-K 8644以浓度依赖及反向频率依赖的方式延长左心室内、外膜MAPD90；而INaL抑制剂TTX和eleclazine在则能够减弱这种增大效应。.2..当Bay K 8644≤300 nM时，偶有早搏、二联律等心律失常事件发生，且可自行恢复，当Bay K 8644的浓度≥600 nM时，心脏易发生PVT、TdP、VF等恶性心律失常；TTX和eleclazine可以消除上述药物诱发的心律失常。.3..Bay K 8644可以增加ICaL，但不增加INaL，而TTX和eleclazine在减小INaL的同时减弱了Bay K 8644增加的心室肌细胞收