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目的运用膜片钳全细胞技术和实时定量聚合酶链式反应(real-time quantitativepolymerase chain reaction,real-time PCR),研究胺碘酮对乳鼠心室肌细胞起搏电流(funny current,I_f)及其基因表达的影响。方法分离培养乳鼠心室肌细胞,记录 I_f 并测定超极化激活的环核苷酸门控通道(hyperpolarization-activated cation channel,HCN)亚型1、HCN2、HCN3和 HCN4;分别用0.01、0.1、1、10、100 μmol/L 的胺碘酮刺激细胞3 h 和 10μmol/L胺碘酮分别刺激细胞0、0.5、1、3、6 h,观察胺碘酮对 I_f 和 HCN 的影响。结果 (1)心室肌细胞中,HCN1～4在总 HCN 的表达中所占比例分别为(0.23±0.01)%、(83.58±0.04)%、(0.79±0.01)%和(15.44±0.01)%。(2)不同浓度胺碘酮使I_f 电流密度分别降低(3.1±0.9)%、(9.7±2.4)%、(36.7±5.8)%、(80.3±1.8)%和(85.9±3.1)%;HCN2表达减少(2.1±0.8)%、(8.9±3.6)%、(30.1±4.2)%、(78.3±3.6)%和(81.1±1.9)%;HCN4减少(0.5±0.2)%、(2.1±2.6)%、(8.8±3.2)%、(60.1±4.6)%和(59.6±6.5)%。(3)刺激不同时间后,I_f 分别降低(1.1±0.1)%、(12.6±2.3)%、(80.6±2.2)%和(80.1±2.1)%;HCN2减少(1.0±0.1)%、(9.8±3.9)%、(82.9±4.6)%和(83.9±1.7)%;HCN4减少(0.1±0.1)%、(1.9±1.1)%、(59.4±7.8)%和(60.9±3.1)%;对 HCN1和 HCN3的影响差异无统计学意义。结论胺碘酮呈浓度和时间依赖性降低乳鼠心室肌细胞 HCN2和 HCN4的表达并抑制 I_f,这有利于其抗室性心律失常的治疗。
Objective To investigate the effects of amiodarone on the expression of funny current (I_f) and the effect of amiodarone on myocardial cells in neonatal rat ventricular myocytes by using whole-cell patch clamp technique and real-time quantitative polymerase chain reaction (real-time PCR) Its gene expression. Methods The neonatal rat ventricular myocytes were isolated and cultured. The I_f was recorded and the hyperpolarization-activated cation channel (HCN) subtypes 1, HCN2, HCN3 and HCN4 were assayed. Amiodarone stimulation of 1,10,100 μmol / L cells for 3 h and 10 μmol / L of amiodarone stimulated cells at 0, 0.5, 1, 3, and 6 h, and observed the effect of amiodarone on I_f and HCN. Results (1) The percentage of HCN1 ~ 4 in total HCN expression in ventricular myocytes was (0.23 ± 0.01)%, (83.58 ± 0.04)%, (0.79 ± 0.01)% and (15.44 ± 0.01)% . (2) Amiodarone decreased (3.1 ± 0.9)%, (9.7 ± 2.4)%, (36.7 ± 5.8)%, (80.3 ± 1.8)% and (85.9 ± 3.1)%, respectively, (2.1 ± 0.8)%, (8.9 ± 3.6)%, (30.1 ± 4.2)%, (78.3 ± 3.6)% and (81.1 ± 1.9)%; )%, (8.8 ± 3.2)%, (60.1 ± 4.6)% and (59.6 ± 6.5)%, respectively. (3) The I_f decreased by (1.1 ± 0.1)%, (12.6 ± 2.3)%, (80.6 ± 2.2)% and (80.1 ± 2.1)% (3.9 ± 1.1)%, (59.4 ± 7.8)%, and (60.9 ± 3.1)%, respectively; (3.9 ± 1.1)%, (82.9 ± 4.6)% and There was no significant difference in the effects of HCN1 and HCN3. Conclusions Amiodarone decreases the expression of HCN2 and HCN4 and inhibits I_f in a concentration-and time-dependent manner in neonatal rat ventricular myocytes, which is beneficial to the treatment of anti-ventricular arrhythmia.