L型钙通道在LQTl发病机制中作用的实验研究

目的 分析L型钙电流(IcaL)在犬三层心室肌细胞中的特点,探讨其在LQTl发病机制中的作用.方法 成年杂种犬14只,体重13～15 kg,雌雄不拘.分离犬三层心室肌细胞,采用全细胞膜片钳技术记录动作电位(AP)和ICaL,依次用Chromanol 293B(50ìμmoL/L)阻断慢激活延迟整流性钾电流(IKs)模拟LQTl,用异丙肾上腺素(100 nmo/L)激活13肾上腺素受体(β-AR),观察AP和,ICaL的变化.分三层取少量心室肌组织,采用实时定量逆转录聚合酶链反应(RT-PCR)技术,检测各层L型钙通道a1C亚单位的mRNA含量.结果 正常情况下,犬三层心室肌细胞ICaL电流密度差异无统计学意义外层(4.253±0.782)pA/pF,中层(4.392±0.714)pA/pF,内层(4.182±0.665)pA/pF,P＞0.05],而中层心室肌细胞动作电位时限(APD)较内层和外层的长外层(721.48±26.59)ms,中层(911.80±31.24)ms,内层(783.52±25.27)ms,P＜0.05];阻断IKs后ICaL电流密度没有变化,而APD均明显延长(外层(835.21±27.34)ms,中层(1089.21±30.55)ms,内层(830.64±27.12)ms,与阻断IKs前相比,P＜0.05)];β-AR兴奋使三层心室肌细胞ICaL显著增加,且三者变化差异无统计学意义(外层(5.654±0.756)pA/pF,中层(5.458±0.702)pA/pF,内层(5.600±0.819)pAZpF,P＞0.05].但β-AR兴奋使外层和内层心室肌细胞APD缩短,中层心室肌细胞APD延长,三者变化差异有统计学意义外层(792.63±26.71)ms,中层(1127.85±32.10)ms,内层(811.32±27.52)ms,P＜0.05].实时定量RT-PCR结果显示,三层心室肌细胞中alC亚单位的mRNA含量差异无统计学意义(外层0.112±0.019,中层0.077±0.018,内层0.109±0.012,P＞0.05).结论 L型钙通道在犬三层心室肌中的分布没有差异,在LQTl模型中,Iso使三层心室肌细胞,ICaL均匀增加,推测ICsL本身没有引起LQTl复极不稳定.

The experimental research of L-type calcium current in the mechanism of ventricular arrhythmia of the LQT1 model

Objective To explore the characteristics of L-type calcium current in canine ventricular myocytes of three different layers and its effect on LQTl.Methods Whole-cell patch clamp technique was used to record action potential(AP)and ICaL in canine epicardial,midmyocardial,and endocardial ventricular myocytes,when the cells were under baseline conditions;or perfused with chromanol 293 B(50μmol/L)-IKs blocker alone;or isoproterenol(100 nmol/L)-β-adrenergic receptor with the present of chromanol 293B.Realtime RT-PCR was used to detect the mRNA levels of ICaL in three layers of canine ventricular muscle.Results The peak ICaL density showed no significant difference among the epicardial,midmyoeardial and endocardial ventricular myocytesEpi(4.253±0.782)pA/pF;M(4.392±0.714)pA/pF,Endo(4.182±0.665)pA/pF,P＞0.05],and did not change after the cells were perfused with chromanol 293 B.But chromanol 293 B significantly prolonged the action potential duration(APD)of three types of cells(Epi(721.48±26.59)ms vs(835.21±27.34)ms;M(911.80±31.24)ms vs(1089.21±30.55)ms;Endo(783.52±25.27)ms vs(830.64±27.12)ms,compared with the baseline conditions,P＜0.05]without increasing transmural dispersion of repolarization(TDR).Iso activated ICaL markedly in all the cells,but the increase in the peak ICaL density was identical throughout three layers(Epi(5.654±0.756)pA/pF,M(5.458±0.702)pA/pF,Endo(5.600±0.819)pA/pF,P＞0.05].In contrast,the APDs were prolonged in midmyocardial myocytes,but abbreviated in epicardial and endocardial myocytes when perfused with IsoEpi(792.63±26.71)ms;M(1127.85±32.10)ms;Endo(81 1.32±27.52)ms,P＜0.05].The changes of APDs resulted in TDR increasing.The mRNA levels of ion conducting subunit of ICaL(alC)was similar among three layers of canine ventricular muscle(Epi:0.112±0.019,M:0.077±0.018,Endo:0.109±0.012,P＞0.05).Conclusion These results indicate that the distribution of ICaL is not different among three layers of canine ventricular muscle with regard to genetic and electrophysiological expression.In the model of LQTl ICaL increased homogeneously by β-adrenergic receptor stimulmion.It may suggest that ICaL is not involved in the repolarization instability to cause LQTl.