钙调节蛋白对七氟烷处理再灌注心律失常的影响

Effects of calcium regulatory protein on sevoflurane conditioning reperfusion arrhythmia

目的:七氟烷对离体大鼠心脏再灌注心律失常的影响的探究。方法:在血流动力学稳定30min后,缺血20min,再灌注60min,建立缺血再灌注损伤模型。参考相关发表大鼠动物模型文献[1-2]将50只大鼠随机分成以下5组,每组10只:时间对照组(TC):用95%氧合KH液恒流在离体心脏内灌流110 min;缺血/再灌注组(I/RI):连续灌流95%氧合KH液30min后停止20min,恢复灌流60min;七氟烷预处理组(sevofluranepreconditioning,Sevo PC):离体心脏用95%氧合KH液灌流20 min,用95%氧合的3%七氟烷饱和KH混合液灌流10 min,停止20 min,恢复灌流60 min;七氟烷后处理组(sevofluranepostconditioning,Sevo Po C):离体心脏用95%氧合KH液灌流30 min,停止20 min,用95%氧合的3%七氟烷饱和KH混合液灌流10 min,恢复灌流50 min;七氟烷联合处理组(sevofluranepreconditioning and postconditioning,PCPO):离体心脏用95%氧合KH液灌流20min,用95%氧合的3%七氟烷饱和KH混合液灌流10min,停止20 min,使95%氧合的3%七氟烷饱和KH混合液灌流10min,恢复灌流50 min。持续监测各组HR,左心室发展压力(left ventricular developed pressure,LVDP),最大LVDP上升率(+dp/dt),最大LVDP下降率(-dp/dt)。监测并统计再灌注心律失常发生率。LTCC、RyR2和NCX1的蛋白和转录水平分别通过Westernblot和RT-PCR进行检测。用荧光分光光度计检测并比较每组细胞中的钙离子水平。结果:七氟烷干预的三组大鼠在再灌注30min、60min后的心功能均有改善,表现为LVDP、+dp/dt、-dp/dt和HR的改善(P<0.05),LVEDP下降(P<0.01)。再灌注心律失常的发生可由SevoPC和SevoPoC改善,表现为室性期前收缩(ventricular premature beat,VPB)总数下降。室性心动过速(ventricular tachycardia,VT)、心室颤动(ventricular fibrillation,VF)发作时程缩短,VF发生率下降,心律失常再灌注评分下降等。SevoPC组RyR2在转录水平的表达明显减少;在SevoPoC组中,转录水平的LTCC、RyR2、NCX1的表达和RyR2、NCX1的翻译水平均有较大幅度的下降。与TC组相比,I/RI、SevoPC、SevoPoC和PCPo组的细胞内钙水平分别增加了145%、88%、79%和65%(P<0.05)。结论:SevoPC和SevoPoc可以改善离体大鼠心脏再灌注心律失常的发生,机制可能与其对RyR2和NCX1表达的抑制作用有关。

Objective:To investigate the effect of sevoflurane on the reperfusion arrhythmias in an isolated rat heart model and explore the related mechanism.Methods:After 30-minute balanced perfusion,isolated hearts were subjected to 20-minute ischemia followed by 60-minute reperfusion,50 isolated hearts were randomly assigned to 5 groups(n=10):Time control(TC)group:continuous perfusion for 110 minutes;Ischemia/reperfusion(I/RI)group:After 30-minute balanced perfusion,ischemia for 20 minutes followed by 60-minute reperfusion;Sevofluranepreconditioning(SevoPC)group:After 20-minute balanced perfusion,perfused with 3%(v/v)sevoflurane-bubbled KHBs oxygenated with 95%oxygen for 10 minutes before 20 minutes of global ischemia and 60 minutes of reperfusion;Sevofluranepostconditioning(SevoPoC)group:After 30-minute balanced perfusion and 20 minutes of global ischemia,perfused with 3%(v/v)sevofluranebubbled KHBs oxygenated with 95%oxygen for 10 minutes at the onset of reperfusion and then with normal KHBs for the remaining 50 minutes;PCPo group:After 20-minute balanced perfusion,perfused with 3%(v/v)sevoflurane-bubbled KHBs oxygenated with 95%oxygen for 10 minutes before 20 minutes of global ischemia and then perfused with 3%(v/v)sevoflurane-bubbled KHBs oxygenated with 95%oxygen for 10 minutes at the onset of reperfusion and then with normal KHBs for the remaining 50 minutes.Heart rate(HR),left ventricular developed pressure(LVDP),and maximum LVDP increase(+dp/dt)and decrease(-dp/dt)rate were continuously collected.The reperfusion arrhythmias were measured.and the protein and messenger RNA(mRNA)levels of LTCCs,RyR2,and NCX1 were determined.Intracellular calcium levels of all groups were compared with the Fluorescence spectrophotometer.Results:Compared with I/RI goup,SevoPC group,SevoPoC group and PCPo group could improve the isolated rat heart function after reperfusion 30 min and 60 min,manifested by improved LVDP,+dp/dt,-dp/dt and HR(P<0.05),reduced LVEDP(P<0.05).SevoPC and SevoPoC could improve the occurring of reperfusion arrhythmias manifested by the decrease of the total number of ventricular premature beat(VPB),incidences of VF and reperfusion arrhythmias score,and the reduction of lasting of ventricular tachycardia(VT)and ventricular fibrillation(VF).In SevoPC group,the expression of RyR2 at the levels of transcription was siginificantly decreased;In SevoPoC group,the expression of LTCCs,RyR2 and NCX1 at the levels of transcription and the expression of RyR2 and NCX1 at the levels of translation were siginificantly decreased.Compared with TC group,intracellular calcium levels in I/RI,SevoPC,SevoPoC and PCPo groups were increased by 145%,88%,79%and 65%,respectively(P<0.05).Conclusions:SevoPC and SevoPoC could improve the occurring of reperfusion arrhythmias and the effects may be due to the inhibition of RyR2 and NCX1.