含吡那地尔的心脏保存液对大鼠离体心脏线粒体呼吸功能的影响

Effect of heart preservation solution containing pinacidil on mitochondrial function in isolated rat hearts

目的探讨含吡那地尔的心脏保存液（HTK液）对大鼠离体心脏线粒体呼吸功能的影响。方法健康清洁级SD大鼠120只，建立离体心脏Langendorff灌注模型，随机分为5组，HTK组（I组）、吡那地尔＋HTK液组（II组）、吡那地尔＋HTK液＋5-羟葵酸（5-HD）组（Ⅲ组）、吡那地尔＋HTK液＋HMR-1098组（Ⅳ组）、吡那地尔＋HTK液＋HMR-1098＋5-HD组（V组）。K—H液平衡灌注10min后灌注心脏停搏液：I组灌注HTK液；Ⅱ组灌注含吡那地尔0．5mmol／L的HTK液；Ⅲ组灌注含吡那地尔0．5mmol／L和5-HD100umol／L的HTK液；IV组灌注含吡那地尔0．5mmol／L和HMR-1098 100umol／L的HTK液；V组灌注含吡那地尔0．5mmol／L、5-HD100umol／L和HMR-1098 100umol／L的HTK液。停搏后取心脏保存于4℃各组相应液体中8h，然后用37℃含氧K—H液再灌注60min。于平衡灌注10min（T1）、保存8h（T2）、复灌60min（T3）时测定线粒体呼吸功能[3态呼吸（S3）和4态呼吸（S4）的耗氧速率、呼吸控制率（RCR）及磷氧比（P／O）]。于T1和T3时收集冠脉流出液测定心肌肌钙蛋白I（cTnI）浓度、肌酸激酶同功酶（CK—MB）及乳酸脱氢酶（LDH）的活性。电镜下观察心肌细胞超微结构。结果与I组比较，Ⅱ组。Ⅳ组RCR、P／O、S3耗氧速率升高，cTnl、CK-MB和LDH的水平降低（P〈0．05）。与Ⅱ组比较，Ⅲ组～V组RCR、P／O、S3耗氧速率降低，cTnI、CK—MB和LDH的水平升高（P〈0．05）。与Ⅲ组比较，Ⅳ组RCR、P／O、S3耗氧速率升高，cTnI、CK—MB和LDH的水平降低，V组RCR、P／O、S3耗氧速率降低，cTnI、CK—MB和LDH的水平升高（P〈0．05）。与Ⅳ组比较，V组RCR、P／O、S3耗氧速率降低，cTnI、CK-MB和LDH水平升高（P〈0．05）。各时点S4耗氧速率组间比较差异无统计学意义（P〉0．05）。Ⅱ组心肌细胞损伤较轻，Ⅲ组和Ⅳ组损伤程度相近但重于Ⅱ组，I组和V组损伤最重。结论含吡那地尔的HTK液可改善心脏线粒体呼吸功能，减轻心肌损伤，提高心脏保存效果，线粒体ATP敏感性钾通道和细胞膜ATP敏感性钾通道均参与了吡那地尔的心肌保护效应，且线粒体ATP敏感性钾通道发挥主导作用。

Objective To investigate the effect of mitochondrial function in isolated rat hearts. Methods One heart preservation solution containing pinacidil on hundred and twenty pathogen-free SD rats of both sexes weighing 250-350 g were used in this study. The animals were anesthetized with intraperitoneal pentobarbital 65 mg/kg. Their hearts were immediately removed and perfused in a Langendorff apparatus. Left ventricular enddiastolic pressure was measured from a fluid-filled latex balloon inserted in the left ventricle. The isolated hearts were randomized into 5 groups （ n = 24 each） ： group I was perfused with cardioplegic solution HTK; group II with HTK containing pinacidil （a non-specific sarcKATP and mitoKATP channel opener） 0.5 mmol/L; group III with HTK containing pinacidil 0.5 mmol/L ＋ 5-HD （a selective mitoKATP channel blocker） 100 umol/L; group IV with HTK containing pinacidil 0.5 mmol/L ＋ HMR-1098 100 umol/L （a selective sarcKATP channel blocker） and group V with HTK containing pinacidil 0.5 mmol/L ＋ 5-HD 100 umol/L ＋ HMR-1098 100 umol/L. The isolated hearts were perfused with simple HTK or HTK containing pinacidil or pinacidil ＋ 5-HD and/or HMR 20 ml/kg at 10 ml/min and then removed from Langendorff apparatus and dipped into the same HTK solution for 8 h at 4℃ followed by 60 rain reperfusion. The respiratory function of mitoebondria （respiratory control rate （RCR）, the rate of oxygen consumption in state 3/state 4 and P/O） was measured at the end of equilibration （T1 ） after 8 h preservation （T2） and at the end of 60 min reperfusion （T3 ）. The CK-MB and LDH activities and cTnI expression in myocardium was detected at T2 and T3 . The ultrastructure of myocardimn was examined at T3 . Results Perfusion suspension-reperfusion （PS/R） significantly decreased mitochondfial respiratory function （RCR, P/O and rate of O2 consumption in state 3） and increased myocardial cTnI concentration and CK-MB and LDH activities at T3 compared with baseline at T1 in group I. Pinacidil significantly increased mitochondrial respiratory function （RCR, P/O and rate of O2 consumption in state 3） and decreased myocardial cTnI concentration and CK-MB and LDH activities in group II as compared with group I indicative of protective effect of pinacidil on mitochondfia against PS/R injury. The protective effect of pinacidil against PS/R injury was attenuated by 5-HD and/or HMR- 1098. The myocardial damage was slightest in group II. Conclusion Both sarcolemmal and mitochondrial KATe channel are involved in the protective effect of pinacidil against PS/R-induced myocardial damage during heart preservation.