Research Of Succinate Dehydrogenase And Mitochondrial Adenosine Triphosphate-sensitive Potassium Channel In Ischemic Postconditioning Of Rats

Objective:In this study,we observed the variation of succinate dehydrogenase?SDH?in myocardial ischemia-reperfusion injury?MIRI?during cardiopulmonary bypass?CPB?of rats.We try to identify the role that SDH and mitochondrial adenosine triphosphate-sensitive potassium channel(mitoK_ATPC)play in ischemic postconditioning?IPO?while treating MIRI.We also want to find out the interaction between SDH and mitoK_ATPC.Methods:160 adult male Spragye-Dawley?SD?rats,weighting 300-350g,CPB models were created.Rats were randomly divided in to 8 groups,20 rats in each group:normal group?Nor?,SDH competitive inhibitor,dimethyl malonate?dm?control group?dm+Nor?,ischemia reperfusion group?I/R?,dm+I/R group?dm+I/R?,IPO group?IPO?,dm+IPO group?dm+IPO?,mitoK_ATPC specific inhibitor,5-hydroxydecanoate?5-HD?+IPO group?5-HD+IPO?and dm+5-HD+IPO group?dm+5-HD+IPO?.In Nor group,rats were perfused for 140 minutes till the end.In I/R group,rats went through 30 minutes'ischemia after 10 minutes'perfusion,then we complemented the perfusion up to 140 minutes.On the basis of I/R group,IPO group were treated with three cycles of 30s'reperfusion and 30s'ischemia after 30minutes'ischemia,henceforth,117 minutes'reperfusion was carried on.Importantly,dm+I/R group,dm+IPO group and dm+5-HD+IPO group were all pumped into vein with dm(4mg/kg·min^-1)10 minutes before I/R for 40 minutes,dm+Nor group were treat with dm(4mg/kg·min^-1),and other groups were pumped into vein with isopycnic normal saline in the corresponding period.5-HD+IPO group and dm+5-HD+IPO group were injected with 5-HD?5mg/kg?5 minutes before IPO.Other groups were injected with isopycnic normal saline at the corresponding time.During the surgery,electrocardiogram and hemodynamic index were monitored.At the end of reperfusion,we drew blood and removed the whole heart for detection as follows:1.Measuring myocardial infarct size?IS%?by 2,3,5-triphenyltetrazolium chloride?TTC?staining.2.Assessing ultrastructure of myocardial tissue and calcula-ting mitochondrial Flameng score by electronic dyeing and electron microscope.3.Detecting the concentration of creatine kinase-MB?CK-MB?and cardiac troponin I?cTnI?in plasma by ELISA.4.Assessing the generation of reactive oxygen species?ROS?in myocardium by immune fluorescent staining and laser scanning confocal microscope.5.Detecting SDH activity,concentration of succinic acid?SA?and fumaric acid?FA?in myocardium by absorptiometry.6.Detecting succinate dehydrogenase flavoprotein?SDHA?mRNA and protein expression in myocardium by RT-qPCR and Western blotting.Results:1.IS%:I/R group got higher IS%than Nor group?P<0.05?;compared with the I/R group,dm+I/R group,IPO group decreased?P<0.05?;compared with the IPO group,dm+IPO group decreased?P<0.05?while the 5-HD+IPO group increased?P<0.05?;difference between 5-HD+IPO group and dm+5-HD+IPO group was not statistically significant?P>0.05?.2.Myocardial ultrastructure and mitochondrial Flameng score.2.1 Myocardial ultrastructure:Nor group and dm+Nor group were mostly normal;myocardial ultrastructure in I/R group which was worst,5-HD+IPO group and dm+5-HD+IPO group showed serious damage;IPO group which was worse than dm+IPO group,and dm+I/R group showed less damage than I/R group.2.2 Mitochondrial Flameng score:compared with the Nor group,I/R group got higher?P<0.05?;difference between Nor group and dm+Nor group was not statistically significant?P>0.05?;compared with the I/R group,dm+I/R group and IPO group got lower?P<0.05?;compared with the IPO group,dm+IPO group got lower?P<0.05?while the 5-HD+IPO group got higher?P<0.05?;difference between 5-HD+IPO group and dm+5-HD+IPO group was not statistically significant?P>0.05?.3.Concentration of CK-MB&cTnI in plasma:compared with the Nor group,I/R group increased?P<0.05?;difference between Nor group and dm+Nor group was not statistically significant?P>0.05?;compared with the I/R group,dm+I/R group and IPO group decreased?P<0.05?;compared with the IPO group,dm+IPO group decreased?P<0.05?while the 5-HD+IPO group increased?P<0.05?;difference between 5-HD+IPO group and dm+5-HD+IPO group was not statistically significant?P>0.05?.4.ROS in myocardium:compared with the Nor group,I/R group increased?P<0.05?;difference between Nor group and dm+Nor group was not statistically significant?P>0.05?;compared with the I/R group,dm+I/R group and IPO group decreased?P<0.05?;compared with the IPO group,dm+IPO group decreased?P<0.05?while the 5-HD+IPO group increased?P<0.05?;difference between 5-HD+IPO group and dm+5-HD+IPO group was not statistically significant?P>0.05?.5.SDH activity,SA and FA in myocardium.5.1 SDH activity in myocardium:compared with the Nor group,I/R group increased?P<0.05?,while dm+Nor group decreased?P<0.05?;compared with the I/R group,dm+I/R group and IPO group decreased?P<0.05?;compared with the IPO group,dm+IPO group decreased?P<0.05?while the 5-HD+IPO group increased?P<0.05?;difference between 5-HD+IPO group and dm+5-HD+IPO group was not statistically significant?P>0.05?.5.2 Concentration of SA in myocardium:Nor group got higher SA than dm+Nor group?P<0.05?,but obviously lower than I/R group?P<0.05?;compared with the I/R group,dm+I/R group and IPO group decreased?P<0.05?;compared with the IPO group,dm+IPO group decreased?P<0.05?while the 5-HD+IPO group increased?P<0.05?;There was no statistical difference between 5-HD+IPO group and dm+5-HD+IPO group?P>0.05?.5.3 Concentration of FA in myocardium:compared with the Nor group,I/R group decreased?P<0.05?,while dm+Nor group increased?P<0.05?;compared with the I/R group,dm+I/R group and IPO group increased?P<0.05?;compared with the IPO group,dm+IPO group increased?P<0.05?while the 5-HD+IPO group decreased?P<0.05?;difference between 5-HD+IPO group and dm+5-HD+IPO group was not statistically significant?P>0.05?.6.SDHA mRNA&protein expression in myocardium:compared with the Nor group,I/R group increased?P<0.05?,while dm+Nor group decreased?P<0.05?;compared with the I/R group,dm+I/R group and IPO group decreased?P<0.05?;compared with the IPO group,dm+IPO group decreased?P<0.05?while the 5-HD+IPO group increased?P<0.05?;difference between 5-HD+IPO group and dm+5-HD+IPO group was not statistically significant?P>0.05?.Conclusion:1.Myocardial ischemia-reperfusion promotes the expression of succinate dehydrogenase flavoprotein and activates succinate dehydrogenase,which lead to myocardial ischemia-reperfusion injury.2.Inhibiting succinate dehydrogenase by dimethyl malonate,an inhibitor of succinate dehydrogenase,effectively alleviates myocardial ischemia-reperfusion injury.3.Ischemic postconditioning could protect heart from myocardial ischemia-reperfusion injury in cardiopulmonary bypass model,by opening mitochondrial adenosine triphosphate-sensitive potassium channel and inactivating succinate dehydrogenase which is based on the activation of mitochondrial adenosine triphosphate-sensitive potassium channel.