超极化停搏对大鼠离体缺血再灌注心脏功能和心肌细胞凋亡的影响

Effect of hyperpolarized cardioplegic arrest on cardiac function and cardiomyocyte apoptosis of isolated heart following ischemia-reperfusion in rats

目的研究ATP敏感性钾通道(KATP)开放剂心脏超极化停搏对大鼠缺血再灌注时离体心脏功能及心肌细胞凋亡的影响。方法SD大鼠108只,随机分为对照组(CON组)、去极化停搏组(D组)、超极化停搏组(H组)、5-羟葵酸(选择性线粒体ATP敏感性钾通道阻滞剂)+超极化停搏组(5-HD+H组)、HMR-1098(选择性膜ATP敏感性钾通道阻滞剂)+超极化停搏组(HMR-1098+H组)、5-羟葵酸+HMR-1098+超极化停搏组(5-HD+HMR-1098+H组)。每组18只。每组再分为缺血前、缺血40min、再灌注60min3个亚组(n=6)。取心脏建立Langendorff灌注模型,平衡15min。除对照组心脏不停搏持续灌注外,其余各组分别灌注各自的停搏液,心脏停搏缺血40min,再灌注60min。持续测定心功能指标[心率(HR)、左心室发展压(LVDP)、左心室舒张末压(LVEDP)、冠脉流量(CF)],计算率压双乘积(DP);各组于缺血前、缺血40min、再灌注60min时分别取心肌组织,运用原位末端TUNEL法检测心肌细胞凋亡指数。结果与各停搏组比较,CON组心功能指标最好,心肌细胞凋亡指数最少(P<0.01);与其余停搏组比较,超极化停搏组能明显提高再灌注期间LVDP及DP,降低LVEDP,减少心肌细胞凋亡率(P<0.01)。5-羟葵酸及HMR-1098能拮抗超极化停搏对缺血再灌注损伤的保护作用。结论超极化停搏对心肌缺血再灌注损伤的保护作用与保护心功能及抑制心肌细胞凋亡有关。这也是通过共同开放两类ATP敏感性钾通道实现的。

[Objective] To study the effect of hyperpolarized cardioplegic arrest on cardiac function and cardiomyocyte apoptosis of isolated heart following ischemia-reperfusion in rats. [Methods] One hundred and eight Sprague-Dawley （SD） rats weighing 250-300g were randomly divided into six groups（18 rats in each group）： control group （group CON）, depolarized arrest group（group D）, hyperpolarized arrest group（group H）, hyperpolarized arrest with 5-HD （mito-KATP channel blocker） group（group 5-HD＋H）, hyperpolarized cardioplegia with HMR-1098 group （group HMR-1098＋H）, hyperpolarized arrest with both 5-HD and HMR-1098 group （group 5-HD＋HMR- 1098＋H）.The animals were anaesthetized and heparinized. Chests were opened and hearts were quickly removed to a Langendorff apparatus. The hearts were perfused with Krebs-Henseleit buffer （37℃） balanced with gas mixture （O2：CO2=95%：5%） at 5.8 kPa（perfusion pressure） for 15 rain and then the heart were arrested with different cardioplegic solutions. Hearts in each groups, except group CON, were subjected to global ischemia at 37℃ for 40 min followed by 60 rain reperfusion after a 10-rninute equilibration period. HR, LVDP, LVEDP and coronary flow. （CF）were measured consistently,and DP（DP=HR×LVDP） was calculated. Cardiomyocyte apoptosis was determined before and after ischemia and at the end-reperfusion by terminal deoxynucleotidy 1 transferase-mediated dUTP-fluorescent nick end labeling （TUNEL） method. [Results] ①Compared with other groups, significant differences were found in group CON with regard to LVDP, LVEDP, DP, HR; CF （P 〈0.01） was improved and apoptotic index decreased remarkably（P 〈0.01）. ②In group H LVDP, DP, HR, CF significantly increased and LVEDP and apoptosis index decreased after ischemia for 40 min and reperfusion for 60 min. ③In the other 3 groups with KATP blocker,the cardiac function was lower and the apoptosis index was significently increased after ischemia for 40 min and reperfusion for 60 min（P 〈0.01）. [Conclusion] Hyperpolarized cardioplegia protects cardiomyocyte from ischemia- reperfusion injury by better preserved cadiac function and inhibiting apoptosis of cardiomyocyte. Both mito-KATP and sacoKATP openings may play an important role in protecting cardiomyocytes.