心肌内向整流钾通道IRK1激动剂后适应可减轻心肌细胞缺氧/复氧损伤

Postconditioning with agonist of inwardly rectifying potassium channel IRK1 attenuates cardiomyocyte hypoxia/reoxygenation injury

目的:探讨心肌内向整流钾通道IRK1激动剂扎考必利(Zac)后适应减轻心肌细胞缺氧/复氧(H/R)损伤的作用及机制。方法:胶原酶法分离成年雄性SD大鼠心室肌细胞,体外利用矿物油覆盖法建立H/R损伤模型。细胞分别缺氧45和75 min之后去除上层石蜡油密封层并更换新鲜台氏液,复氧30 min;2种损伤模型复氧前分别给予0.1、1和10μmol/L的Zac进行药物后适应;激光共聚焦显微镜观察比较Fluo-4负载细胞内游离钙离子浓度;Westernblot法测定各组心肌细胞IRK1主要亚单位Kir2.1蛋白的表达。此外,在心肌H9c2(2-1)细胞建立H/R模型,分别缺氧3和5 h,之后复氧2 h;2种损伤模型复氧前分别加入0.1、1和10μmol/LZac进行药物后适应;ELISA法检测乳酸脱氢酶和caspase-3含量;CCK-8法检测细胞活力;Westernblot法测定各组心肌细胞PI3K/Akt信号通路相关蛋白的水平。结果:成年大鼠心室肌细胞复氧时预先给予0.1~10μmol/LZac可有效抑制H/R引起的IRK1抑制和细胞内钙超载。在心肌H9c2(2-1)细胞H/R模型中,0.1~10μmol/LZac后适应可激活PI3K/Akt信号通路,减少心肌细胞的坏死和凋亡,其中0.1μmol/LZac为最适效应浓度;IRK1阻断剂BaCl_(2)可有效抑制Zac的效应(P<0.01),表明Zac的后适应保护是IRK1依赖的。结论:通过激动心肌IRK1减轻心肌细胞内钙超载并激活PI3K/Akt信号通路是减轻H/R损伤的有效策略。IRK1可能成为拮抗心肌缺血/再灌注损伤药物作用的新靶点。

AIM:To clarify whether postconditioning with inwardly rectifying potassium channel IRK1 agonist zacopride(Zac)attenuates cardiomyocyte hypoxia/reoxygenation(H/R)injury and the underlying mechanisms.METHODS:Hypoxia for 45 or 75 min/reoxygenation for 30 min injury model was established in primary ventricular myocytes of adult male SD rats by mineral oil covering method in vitro to simulate myocardial ischemia/reperfusion injury.Treatment with Zac at 0.1,1 and 10μmol/L was applied before reoxygenation.The average intensity of Ca2+fluorescence in the cardiomyocytes was recorded by confocal microscopy.The protein expression of Kir2.1(the dominant subunit of IRK1 in the ventricle)in each group was determined by Western blot.The H/R model was also established in H9c2(2-1)cells.After 3 or 5 h of hypoxia,0.1,1 and 10 mol/L Zac were applied before 2 h of reoxygenation.Lactate dehydrogenase release,caspase-3 activation,cell viability and the protein levels of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signaling-related molecules were detected.RESULTS:Postconditioning with 0.1,1 and 10μmol/L Zac in adult rat ventricular myocytes effectively attenuated IRK1 inhibition and intracellular calcium overload caused by H/R.In H9c2(2-1)cell H/R model,0.1,1 and 10 mol/L Zac activated the PI3K/Akt signaling pathway,and reduced necrosis and apoptosis of the cells,among which the superlative effects occurred at 0.1μmol/L.The IRK1 blocker BaCl_(2) reversed the effect of Zac(P<0.01),indicating that the protective effect of Zac postconditioning was mediated by the enhancement of IRK1.CONCLUSION:Activating myocardial IRK1 to reduce intracellular calcium overload and activating PI3K/Akt pathway are effective strategies to reduce H/R injury of myocardial cells,and IRK1 may be a potential new drug target for preventing or alleviating acute myocardial ischemic/reperfusion injury.