吡那地尔超极化停搏对大鼠离体心脏缺血再灌时心肌线粒体损伤的影响

Effects of pinacidil-induced hyperpolarized arrest on myocardial mitochondrlal Injury during ischemiareperfusion in rats

目的探讨吡那地尔超极化停搏对大鼠离体心脏缺血再灌注时心肌线粒体损伤的影响。方法健康雄性SD大鼠,成功建立Langendorff再灌注模型的80个心脏随机分为5组：对照组（C组）、去极化停搏组（D组）、吡那地尔超极化停搏组（H组）、线粒体ATP敏感性钾通道阻滞剂5-羟葵酸（5-HD）＋去极化停搏组（5-HD＋D组）和5-HD＋吡那地尔超极化停搏组（5-HD＋H组）。以K-H液平衡灌注20 min后（平衡末）,C组阻断主动脉,不予停搏液灌注,使其自然停搏,D组用37℃ST.ThomasⅡ停搏液灌注,H组用37℃超极化停搏液灌注,5-HD＋D组和5-HD＋H组分别用含有100μmol/L 5-HD的37℃ST.ThomasⅡ停搏液或超极化停搏液20 ml/kg灌注,缺血40 min。分别于平衡末及再灌注30 min时取8个心脏,测定心肌线粒体呼吸功能指标[4态呼吸耗氧速率、3态呼吸耗氧速率、呼吸控制率（PCR）及磷氧比（P/O）]、线粒体酶（NADH氧化酶、琥珀酸氧化酶和细胞色素C氧化酶）活性及线粒体膜电位（MMP）,电镜下观察线粒体的超微结构。结果与平衡末比较,各组再灌注30 min时心肌线粒体呼吸功能指标（3态呼吸耗氧速率、PCR及P/O）、线粒体酶活性及MMP降低（P〈0.05或0.01）;与C组比较,再灌注30 min时其余各组上述指标均升高（P〈0.01）;再灌注30 min时H组线粒体的功能及病理损伤最轻。结论吡那地尔超极化停搏能明显改善大鼠离体心脏缺血再灌注时心肌线粒体功能,减轻线粒体超微结构损伤,其机制与开放线粒体ATP敏感性钾通道有关。

Objective To investigate the effects of hyperpolarized arrest induced with pinacidil on myocardial mitochondrial injury during ischemia-reperfusion（I/R）of the isolated rat hearts.Methods Adult male SD rats weighing 250-300 g were killed after anesthetized and heparinized.The chests were opened and the hearts were immediately removed and perfused with oxygenated（95% O2-5% CO2）balanced K-H buffer for 20 min at 37℃at 5.8 kPa（perfusion pressure）in a Langendorff apparatus.Eighty isolated hearts were randomized into 5 groups（n=16 each）：groupⅠcontrol（C）;groupⅡdepolarized arrest（D）;groupⅢhyperpolarized arrest （H）;groupⅣdepolarized arrest＋5-hydroxydecanoate（5-HD）（5-HD＋D）and groupⅤ5-HD＋H.Cardiac arrest was induced with different cardioplegic solution.The hearts were subjected to ischemia at 37℃for 40 min followed by 30 min reperfusion.The changes of myocardial mitochondrial ultrastructure were determined at the end of post-preparation equilibration（T1）and at the end of reperfusion（T2）and mitochondrial respiratory function, enzyme activities and mitochondrial membrane potential（MMP）were also measured.Results The mitochondrial respiratory function（respiratory control rate,state 3 respiration and P/O）,mitochondrial enzyme activities（NADH oxidase,succinate oxidase and cytochrome C oxidase）and MMP were significantly decreased at the end of 30 min reperfusion（T2）as compared to the baseline values at T1 in all 5 groups and the mitochondrial function in control group was the poorest and that in group H the best among the 5 groups.Electron microscopic examination showed that mitochondrial ultrastructure was damaged in varying degree and the damage was slightest in group H. Conclusion Hyperpolarized cardioplegia can significantly improve mitochondfial function and decrease ultrastructural damage at the end of reperfusion.The opening of mitochondrial ATP sensitive K^＋ channel is involved in the mechanism of the protective effect of hyperpolarized cardiac arrest against I/R injury.