胺碘酮对人心房肌细胞钙电流和钙离子浓度影响的研究

Influences of angiotensin Ⅱ on calcium channel current and intracellular free calcium concentration of human atrial myocytes and the antagonistic effect of amiodarone

目的 采用血管紧张素(Ang)Ⅱ刺激人的心房肌细胞,观察细胞膜钙通道电流及胞内游离钙浓度的变化,了解AngⅡ参与房性心律失常的电生理机制,同时观察胺碘酮的干预作用,为临床应用胺碘酮提供实验依据。方法 急性分离单个人心房肌细胞,采用全细胞膜片钳方法记录L 型钙电流(ICa L),同时应用共聚焦显微镜技术测定细胞内游离钙的浓度。实验分为对照组、AngⅡ组(0.1μmol/L AngⅡ)、胺碘酮组(30 nmol/L胺碘酮)及AngⅡ+胺碘酮组。结果 与对照组相比,0.1μmol/L AngⅡ能使人心房肌细胞膜ICa L峰值电流密度明显增加[(-12.77±1.61) pA/pF比(-5.78±0.81) pA/pF,P<0.05];30 nmol/L胺碘酮对人心房肌细胞膜ICa L无明显影响[(-5.58±0.62) pA/pF];但胺碘酮可拮抗AngⅡ的作用,AngⅡ+胺碘酮组的ICa L峰值电流密度[(-7.16±0.82) pA/pF]与AngⅡ组的差异有显著性(P<0.05)。共聚焦显微技术发现,对照组和胺碘酮组人心房肌细胞内荧光强度和荧光吸光度值较低;加入AngⅡ后即刻,细胞内荧光吸光度值开始增加,15 min后细胞内荧光强度和荧光吸光度值明显增高,与对照组比较,差异有显著性(P<0.05);而AngⅡ+胺碘酮组细胞内荧光吸光度值显著低于AngⅡ组(P<0.05)。结论 AngⅡ使人心房肌细胞膜ICa L峰值电流密度明显增加。

Objective To observe the influences of angiotensin Ⅱ (AngⅡ) on calcium channel current and intracellular free calcium concentration ([Ca 2+]i) of human atrial myocytes, so as to reveal the electrophysiological mechanisms of atrial arrhythmia induced by AngⅡ, and to investigate the antagonistic effect of amiodarone for providing the theoretical basis in treating atrial arrhythmias.Methods Single human atrial myocyte was isolated. The conventional whole-cell configuration of the patch-clamp technique was used to detect membrane I Ca-L, and confocal microscope was used with Fluo-3/AM as indicator to detect changes of [Ca 2+]i. The experiment comprised of four groups, ie, control group, AngⅡ group, amiodarone group and AngⅡ+amiodarone group.Results Comparing with the control group, AngⅡ of 0.1 μmol/L significantly increased the peak density of I Ca-L in human atrial myocyte [(-12.77±1.61) pA/pF vs (-5.78±0.81) pA/pF, P<0.05). Amiodarone of 30 nmol/L had no significant effect on I Ca-L of human atrial myocyte [(-5.58±0.76) pA/pF], but it could antagonize the effects of AngⅡ. In AngⅡ+amiodarone group, the peak density of I Ca-L was [(-7.15±0.82)] pA/pF, which had a significant difference in comparing with the AngⅡ group (P<0.05). The intracellular fluoresence intensity of single cardiomyocyte in both control and amiodarone groups was low. The intracellular fluoresence intensity of single cardiomyocyte in AngⅡ group was significantly higher than that in the control group after 15 minutes intervention (P<0.05). When amiodarone was added simultaneously with AngⅡ intervention, the values were much lower comparing with AngⅡ group (P<0.05).Conclusion AngⅡ of 0.1 μmol/L can significantly increase the peak density of I Ca-L and induce intracellular calcium overload in human atrial myocytes, which may participate in human atrial electrical remodeling and relevant to occurrence and progression of atrial fibrillation. Amiodarone can antagonize the above effects of AngⅡ in human atrial myocytes.