兔左室肌三层细胞动作电位和L型钙离子流异质性以及胺碘酮急性灌流作用的研究

摘要 目的 本实验用膜片钳技术研究正常兔心室内、外膜层及M细胞的动作电位时程（APD）跨壁离散度（TDR）和三层细胞的L-型钙通道电流的异质性,以及胺碘酮急性灌流对上述生理指标的影响。方法 以混合气-二型胶原酶消化法分离兔左室游离壁内、中、外三层心肌，继而对其采用全细胞膜片钳记录生理台氏液和10μmol/L浓度胺碘酮急性灌流测量AP和Ica-L离子电流。结果：(1)APD以M细胞为最长，内膜细胞最短，外膜细胞居中，APD 90分别为488.13±22.88 ms、326.36±14.51 ms和391.1±23 ms，P<0.05；TDR为185 ms。存在显著的l相切迹和2相驼峰。(2)应用胺碘酮后的三层细胞动作电位的APD 20，50和90在M细胞显著缩短（P<0.05），Edno显著延长（P<0.05），TDR明显减小。胺碘酮对动作电位的形态无影响。(3)正常兔三层心肌细胞的ICa-L电流密度存在明显差异，外膜细胞和内膜细胞的ICa-L电流密度分别为：6.21±1.35 和 7.20±0.83 pA／pF。M细胞的ICa-L电流密度较大为11.59±3.71pA／pF， P <0.05。三层细胞的ICa-L 电压-电流曲线形态和电压依赖性无差异。(4)10μmol/L胺碘酮急性灌流对ICa-L的影响，在内、外细胞和M均有所抑制，但只有M细胞有统计学意义11.59±3.71 vs 7.28±2.01，（P<0.05）。胺碘酮对三层细胞的ICa-L的电压-电流（I-V )曲线形态和电压依赖性无差异。结论 (1)兔三层心肌细胞的AP存在明显的异质性，M细胞拥有明显大的APD，Endo细胞的APD最短，三层细胞的TDR较大。外膜细胞的AP形态有大的2相穹窿，M细胞拥有小的穹窿和尖锐的0时相上升支，呈尖峰圆顶型。M细胞明显大的ICa-L，外膜细胞和内膜细胞的ICa-L大小相仿，三层细胞的ICa-L的形态无差异。(2)胺碘酮延长Endo细胞的APD，缩短M细胞的APD，使得TDR减小。胺碘酮能显著抑制M细胞的ICa-L，但不影响三层细胞的ICa-L的形态。

Heterogeneity of action potential and Ica-L in the left ventricular myocytes of rabbit and the acute electrophysiological effects of amiodarone

Abstract Objective The heterogeneity of the action potential(AP)，transmural dispersion of repolarization (TDR) and Ica-L ion current in left ventricular three layers myocytes were investigated in rabbit by using patch-clamp technique. And the acute electrophysiological effects of amiodarone on both AP and TDR were also studied. Methods Rabbits myocytes were isolated by O2 & CO2 mixed-gas combined with type –II collagenase enzymatic method．And the sub-endocardial (Endo) and sub-epicardium (Epi) tissues were separated from the other regions (midmyocardium，M)．Separated ventricular myocytes were perfused with Tyrod’s solution and 10 umol/L amiodarone. And then the whole cell patch clamp technique was used to study the AP and Ica-L. Result 1. The action potential (AP) in the three myocardial layers were significantly different．The action potential duration (APD) in M cells was the longest, while the Endo cells were the shortest. The APD 90 of M cells was 488.13±22.88ms, and that of Endo and Epi cells were 326.36±14.51ms and 391.1±23ms, respectively (P<0.001). TDR was 185ms. The M cells had more pronounced spike-and-dome configuration，with significantly larger phase 1 magnitude and plateau voltage．2) After acute amiodarone perfusion, the APD 20, 50 and 90 in M cells were sharply shortened (P<0.003). On the contrary， Endo cells have a remarkably prolonged period of AP (P <0.0003). So the TDR obviously decreased. Amiodarone has no effect on AP configuration. 3) ICa-L of three myocardial layers was significance different in the current density. The current density of Epi and Endo cells were 6.21±1.35 and 7.20±0.83 pA/pF, respectively. The M cells had a larger current density, the value was 1.59±3.71pA/pF, P<0.05. The three layers myocytes had no significance change in the I-V curved and voltage-dependence. 4) Compared with the normal ventricular myocytes，the current density of ICa-L were all decreased in three layers myocytes perfused by 10μmol/L amiodarone，but the M cells were the significant ones, 11.59±3.71pA/pF vs 7.28±2.01 pA/pF, (P<0.05). And amiodarone had no effect the I-V curved and voltage-dependence of ICa-L. Conclusion 1) The action potential and the L-Ca ion currents in the three myocytes layers were significantly different．Action potential duration (APD) in M cells was longer than that in Epi or Endo cells．M cells had a more pronounced spike—and—dome configuration，with a significantly larger phase 1 magnitude an d plateau voltage．2) ICa-L in M cells were higher than those of Epi and Endo．The ICa-L in Epi and Endo were similar with each other. And they were no difference in I-V curved and voltage-dependence of ICa-L. 3) Amiodarone could prolong APD in Endo cells and decrease that in M cells, so the TDR was decreased. Amiodarone could remarkably inhibited ICa-L in M cells, but had no influence on configuration of ICa-L．