Mitochondrial Mechanism Of SDH Protective Effect Of Ischemic Postconditioning On Diabetic Myocardium During Cardiopulmonary Bypass

Objective:To observe the myocardial mitochondrial respiratory function,ROS,membrane potential,mitochondrial permeability transition pores（mPTP）,SDH activity,succinic acid and fumaric acid content in normal and diabetic rats during the whole heart ischemia/reperfusion under cardiopulmonary bypass（CPB）.From the perspective of mitochondria,to explore the role of SDH in IPO to alleviate normal heart I/RI,and to determine whether the reduction of MI/RI effect in diabetic myocardium by IPO is related to SDH,and further clarify whether the use of SDH inhibitor can restore the myocardial protective effect of IPO in diabetic state.Methods:Established a rat model of whole heart ischemia-reperfusion in normal and diabetic rats（DM）under cardiopulmonary bypass（CPB）.36 healthy SPF male SD rats were randomly divided into 3 groups（n=12）:sham operation group（Con+Sham group）,ischemia/reperfusion group（Con+I/R group）,and ischemic postconditioning group（Con+IPO group）;72 DM rats were randomly divided into 6 groups（n=12）:sham operation group（DM+Sham group）,ischemia/reperfusion group（DM+I/R group）,ischemia Post-treatment group（DM+IPO group）,sham operation group+dimethyl malonate（DM+Sham+dme group）,ischemia/reperfusion group+dimethyl malonate（DM+I/R+dme group）,ischemic postconditioning group+dimethyl malonate（DM+IPO+dme group）.All groups except Sham group were treated with I/R.In the Sham group,after balancing for 10min,the aircraft was transferred under CPB for 90min（maintain a flow rate of100ml/kg·min-1 during CPB.）.I/R group:after 10min of balance,the aorta was ligated under CPB,the whole heart was subjected to ischemia 30 minutes,and then the aortic recanal was transferred for 60 minutes（maintain a flow rate of 100ml/kg·min-1 during CPB.）.IPO group:on the basis of the I/R group,the aorta was opened for 30s reperfusion and clamping for 30s ischemia after the aorta was closed for a total of 3 cycles,the aorta was opened and transferred for 57min（maintain a flow rate of 100ml/kg·min-1 during CPB.）.In the DM+I/R+dme group and DM+IPO+dme group,the SDH-specific inhibitor dme 4mg/kg was continuously pumped from the caudal vein after the establishment of CPB to the end of total cardiac arrest（40 min）,while DM+Sham group applied the same dose of dme at the first 40min after the establishment of CPB.The measurement was performed at 5 minbefore CPB（T0）,at the beginning of CPB（T1）,at cardiac arrest（T2）,cardiac arrest at the end（T3）,5min after reperfusion（T4）,and at the end of reperfusion（T5）.Arterial blood pressure were measured and mean arterial pressure（MAP）was calculated to identify the success of the CPB model.At the end of reperfusion:（1）extract mitochondria from left ventricular tissue,mitochondrial 3-state respiration（State3）and respiratory control rate（RCR）were measured by Hansa oxygen electrode method,mitochondrial membrane potential were detected by JC-1 method,the open level of mPTP was examined by absorb spectrophotometry.（2）The changes of ROS in the left ventricle were observed by fluorescence microscopy.（3）The left ventricular tissue was taken and the changes of SDH activity,succinic acid and fumaric acid content were detected by spectrophotometry.Results:1.OGTT and IPGTT results:DM normarats and DM rats showed an increase in blood glucose after GS use and a decrease in blood glucose after 30 minutes.Compared with the blood glucose of normal rats,the blood glucose of DM rats was maintained at a high level（P<0.05）,suggesting that the glucose tolerance of DM rats was impaired.2.Changes in MAP:The MAP of rats in each group decreased significantly at the beginning of CPB（P<0.05）,and returned to the original level when the heart stopped,and MAP>60mmHg throughout the CPB period.3.Arterial blood gas analysis results:RT,PaO₂ and PaCO₂ remained stable throughout CPB,and there was no significant difference between each time point compared with T0time point（P>0.05）;pH and blood K+levels remained stable after CPB（P>0.05）;Hct decreased significantly after CPB began due to hemodilution（P<0.05）.4.Changes in mitochondrial respiratory function and membrane potential:mitochondrial State3,RCR and membrane potential in Con+I/R group were lower than those in Con+Sham group（P<0.05）;Con+IPO group was higher than Con+I/R group（P<0.05）.The DM+I/R group was lower than the Con+I/R group（P<0.05）;the DM+I/R group and the DM+I/R+dme group were lower than the DM+Sham group and the DM+Sham+dme group,respectively.P<0.05);there was no statistically significant difference between DM+IPO group and DM+I/R group（P>0.05）,DM+IPO+dme group was higher than DM+I/R+dme group and DM+IPO group（P<0.05）.5.Changes in the open levels of ROS and mPTP:ROS and the open of mPTPin the Con+I/R group were more than the Con+Sham group（P<0.05）;the Con+IPO group was less than the Con+I/R group（P<0.05）;DM+I/R group was more than the Con+I/R group（P<0.05）;the DM+I/R group and the DM+I/R+dme group were more than the DM+Sham group and the DM+Sham+dme group,respectively（P<0.05）.there was no statistically significant difference between DM+IPO group and DM+I/R group（P>0.05）,DM+IPO+dme group was more than DM+I/R+Dme group and DM+IPO group（P<0.05）.6.Changes in SDH activity:SDH activity in Con+I/R group was higher than Con+Sham group（P<0.05）;Con+IPO group was lower than Con+I/R group（P<0.05）;DM+I/R group was higher In the Con+I/R group（P<0.05）;the DM+I/R group and the DM+I/R+dme group were higher than the DM+Sham group and the DM+Sham+dme group,respectively（P<0.05）;there was no statistically significant difference between DM+IPO group and DM+I/R group（P>0.05）,and the DM+IPO+dme group was lower than the DM+I/R+dme group and DM+IPO.Group（P<0.05）.7.Changes in succinic acid content:succinic acid content in Con+I/R group was less than Con+Sham group（P<0.05）;Con+IPO group was more than Con+I/R group（P<0.05）;DM+I/R group was less than Con+I/R group（P<0.05）;DM+I/R group and DM+I/R+dme group were less than DM+Sham group and DM+Sham+dme group,respectively（P<0.05）;there was no statistically significant difference between DM+IPO group and DM+I/R group（P>0.05）,The DM+IPO+dme group was more than the DM+I/R+dme group and DM+IPO group（P<0.05）.8.Changes of fumaric acid content:the content of fum（P>0.05）.aric acid in the Con+I/R group was higher than that in the Con+Sham group（P<0.05）;the Con+IPO group was less than the Con+I/R group（P<0.05）;the DM+I/R group was more in the Con+I/R group（P<0.05）;the DM+I/R group and the DM+I/R+dme group were more than the DM+Sham group and the DM+Sham+dme group,respectively（P<0.05）;there was no statistically significant difference between DM+IPO group and DM+I/R group（P>0.05）.the DM+IPO+dme group was less than the DM+I/R+dme group and DM+IPO group（P<0.05）.Conclusion:1.When normal rats whole heart are subjected to I/R under CPB,IPO can improve myocardial mitochondrial function and reduce MI/RI.The mitochondrial mechanism may be related to the inhibition of SDH activity.2.When diabetic rats whole heart are subjected to I/R under CPB,the effect of IPO on reducing MI/RI decreased,Which may be related to the fact that the SDH activity of diabetic myocardial mitochondria cannot be inhibited by IPO.3.Application of SDH inhibitor can restore the protective effect of IPO on I/R myocardium in diabetic rats under CPB.