东亚钳蝎毒素多肽对家兔心室肌细胞钠电流和在体动作电位的影响以及抗心律失常作用研究

目的 探讨基因表达得到重组的东亚钳蝎毒素多肽(BmKXM)对家兔单个心室肌细胞钠电流(INa)和在体动作电位的影响,以及对抗乌头碱诱发的心律失常电活动的作用.方法 用酶解法分离家兔心室肌细胞,全细胞膜片钳技术记录所需INa.采用浮置式玻璃微电极记录在体心肌细胞跨膜动作电位,并监测体表心电图.结果 (1)BmKIM 能明显抑制心肌细胞INa,使INa的电流-电压(I-V)曲线显著上移,但I-V曲线的形态没有发生改变.BmKIM使失活曲线显著左移,50%的INa失活电压分别由用药前的(-70.8±2.6)mV变为(-84.84±3.5)mV(P<0.05).在BmKIM作用下,心肌细胞INa失活后恢复时间明显延长,用药前快、慢恢复时间常数(Tf和Ts)分别是(28.9±6.1)ms和(107±21.6)me,给予BmKIM后,τf和τs分别是(54.2±7.9)ms(P<0.05)和(211.1±34.6)ms(P<0.01).(2)BmKIM 能明显改变记录在体心肌细胞跨膜动作电位时程(APD)和幅度(APA)以及最大上升速率(Vmax),使APD50和APD90都明显缩短,APA和Vmax显著减小.心电图上表现为QT间期缩短,心率明显加快.(3)乌头碱能明显触发后除极,诱发室件心动过速(室速),动作电位上表现为早期后除极和(或)延迟后除极.相对心电图上表现为插入性室性早搏或尖端扭转型室速(发生率为77.8%).BmKIM能明显降低乌头碱诱发的心律失常发生率(22.2%,P<0.01),更不能触发室速.结论 BmKIM对兔的心室肌细胞,INa有明显的抑制作用,这种抑制作用是通过与失活态的钠通道结合而发挥生理学效应,BmKIM 可抑制形成动作电位的钠离子内流,对抗乌头碱所致的心律失常发生,为开发具有选择性直接针对钠通道的阻滞剂BmKIM的应用前景打下基础.

Effects of BmKIM on sodium current of isolated cardiomyocytes, transmembrane action potential and aconitine induced arrhythmia in vivo in rabbits

Objective To investigate the effects of recombinant BmKIM (poly-peptide derived from Asian Scorpion Buthus martensi Karsch) on the sodium current (INa) of isolated ventricular myocytes,transmembrane action potential and aconitine induced arrhythmia in vivo in rabbits. Methods Ventricular myocytes were enzymatically dissociated from adult rabbits. Whole-cell patch-clamp technique was used to record voltage-dependent INa. Standard transmembrane action potentials in rabbit hearts in vivo were recorded by using floating glass microelectrodes. Incidence of arrhythmias, the early afterdepolarization (EAD) and/or delay afterdepelarization (DAD) were measured in vivo in rabbits post aconitine (100 μg/kg, iv) in the absence or presence of BmKIM (50 μg/kg iv). Results (1) BmKIM ignificantly inhibited INa, in a voltage-dependent manner and significantly shifted the I - V curves of INa, upward. BmKIM left shifted the inactivation curve of INa, and voltages at 50% inactivation of INa, were changed from (-70. 8±2. 6 ) mV to (-84. 8±3.5 ) mV ( P < 0.05 ). BmKIM prolonged the recovery of inactivation of INa. In the presence of BmKIM, the time constants of recovery ( both τf and τs ) of INa, were significantly prolonged from ( 28. 9 ±6. 1)ms and (107±21.6)ms in control group to (54.2±7.9) ms(P <0.05) and (211.1±34.6) ms (P <0.01 ), respectively. (2) BmKIM significantly shortened 50% and 90% of action potential duration (APD50 and APD90), and reduced action potential amplitude (APA), declined maximum up stroke velocity of action potential ( Vmax ) in vivo. The Q-T duration was shortened and heart rate significantly increased post BmKIM injection. (3) Incidence of aconitine induced ventrieular arrhythmias (77.8%) was significantly reduced by BmKIM (22.2%, P < 0.01). Conclusions BmKIM significantly blocked INa through affecting the inactivated state of INa in rabbit ventricular myocytes. BmKIM could attenuate the influx of INa, therefore shorten action potential duration and reduce action potential amplitude and reduce the incidence of aconitine induced arrhythmias.