内向整流钾通道（IK1）激动剂抗缺血性心律失常的作用及机制

Life-threatening arrhythmias resulting from ischemia and infarction is important for risk stratification of patients and for the development of interventions to decrease mortality caused by coronary artery disease. However, antiarrhythmic drugs, with the exception of β-adrenergic receptor blocking drugs and amiodarone，are largely ineffective or even harmful. Arrhythmogenesis in acute myocardial ischemia and chronic infarction are associated with the decrease of resting potential (RP) and inward rectifier potassium current (IK1). However, there are no clinical antiarrhythmic agents mainly acted on resting potential or IK1 up to date. Recently, we reported firstly a selective agonist of IK1, zacopride. By this pharmacological tool, we hypothesized that moderate enhancing IK1, accordingly enhancing or restoring resting potential might exhibit protection against ischemic arrhythmias. Therefore, the antiarrhythmic effect of zacopride would be tested first in acute ischemic and chronic myocardial infarcted(MI) rats subjected to coronary artery ligation. Then in hypoxic atmosphere, the effects of zacopride on IK1 in native rat ventricular myocytes or cloned Kir2.1, Kir2.2 and Kir2.3 channels in HEK 293 cells were to be observed using whole-cell patch-clamp technique.The latter was designed to identify the role of those different Kir2.x channel subunits in this regulation. Finally, the change of mRNA or expression of IK1 / Kir2.x channels in presence and absence of zacopride in acute simulated ischemic /chronic infarcted myocardium were to be compared by Real Time-PCR and Western blot analysis. If the hypothesis is tested to be true, it will bring new ideas to antiarrhythmic theories and shed light on the development of new antiarrhythmic drugs, especially the ones against ischemic arrhythmias.

心肌缺血和心肌梗死时发生的室性心律失常与静息电位(RP)减小和内向整流钾电流（IK1）下降有关，但目前临床应用的抗心律失常药物没有一个是以激动IK1和（或）增大RP而发挥作用的。由此设想适度增强IK1，进而增大或恢复RP，可以预防或治疗缺血性心律失常。我们发现并报道了首个IK1选择性激动剂zacopride。本课题将通过结扎大鼠冠状动脉致急性缺血和慢性心梗模型，检验zacopride抗缺血性心律失常的效应；利用膜片钳技术观察缺氧条件下zacopride对大鼠心室肌细胞IK1及RP的影响；观察它对HEK293细胞转染Kir2.x通道的作用，明确介导zacopride调节IK1的Kir2.x通道类型；利用Real Time PCR和免疫印迹法分析其对急性模拟缺血和慢性缺血心肌IK1/Kir2.x 通道mRNA和蛋白表达的影响。该设想如被证实，将是抗心律失常理论上的创新，并为药物研发提供新方向。

心肌缺血和心肌梗死时发生的室性心律失常与静息电位（RP）减小和内向整流钾电流（IK1）下降有关，但目前临床应用的抗心律失常药物没有一个是以激动IK1和（或）增大RP而发挥作用的。课题组利用IK1选择性激动剂zacopride观察其对大鼠冠状动脉结扎致急慢性缺血性心律失常的效应。在麻醉大鼠，zacopride可剂量依赖性抑制离体及在体急性缺血性心律失常的发生，期前收缩（VPB）数目、室速（VT）和室颤（VF）持续时间和发生率均显著降低（P<0.01），低浓度BaCl2可以逆转其保护作用。在清醒大鼠，采用心电血压遥测记录系统，观察zacopride对心梗致慢性缺血性心律失常的效应。zacopride能够显著减少室性心律失常和房室传导阻滞的发生，但对室上性心律失常无明显作用。利用全细胞膜片钳和免疫蛋白印迹技术进一步分析其细胞和分子机制。缺氧细胞RP降低，动作电位时程（APD）延长，zacopride可通过激动IK1而恢复RP和APD，但对IKATP无影响；在HEK293细胞建立大鼠心肌Kir2.x通道基因表达系统，发现zacopride仅激动Kir2.1通道，而对Kir2.2和Kir2.3通道无明显影响。Western blot结果示大鼠心梗边缘区心肌IK1表达较伪手术组明显降低，zacopride可以恢复IK1表达，而低剂量氯喹选择性阻断IK1可逆转其效应。这些实验结果充分证明我们的假说，即适度增强IK1，进而增大或恢复RP，可以预防或治疗缺血性心律失常。此外，人心室肌IK1通道主要以Kir2.1和Kir2.2亚型为主，心房肌以Kir2.3亚型为主，而zacopride通过激动心肌Kir2.1亚型发挥抗缺血性心律失常效应，这就有力排除了其对心房电活动的潜在影响（致房颤风险）。同时，课题组还发现zacopride可通过激动心肌IK1发挥抗心室重构效应。与单纯心梗大鼠相比，zacopride干预组全心肥厚指数、左心室肥厚指数显著降低，心脏收缩和舒张功能全面改善，心肌细胞肥大、超微结构损伤和间质纤维化显著减轻，血浆BNP水平降低。低剂量氯喹选择性阻断IK1可明显逆转zacopride的抗重构作用。本课题的实施有望在抗心律失常理论上取得突破；同时，有望为心血管疾病治疗提供一条兼具抗心室重构与抗心律失常双重效应的药物干预途径，具有重要的理论意义和潜在的临床价值。