Exploring The Effect Of Carbon Monoxide-releasing Molecule-2 On Cardiac Function After Resuscitation Via Mitochondrial Pathway

Objective To explore the protection and mechanism of CORM-2 against cardiac injury after resuscitation,so as to provide a new therapeutic strategy for clinical practice.Methods We established the model of post-cardiac arrest myocardial dysfunction by intravenous injection potassium chloride(4℃)combined with asphyxiation for 4 min and then followed by artificial chest compression for 3 min.40 rats were randomly divided into five groups:sham operated group,cardiopulmonary resuscitation group(CPR),DMSO group,inactivated CORM-2 group(i CORM-2 group)and CORM-2 group,each of which contained 8 rats.Animals in the Sham group were instrumented with catheter without experiencing cardiac arrest and resuscitation.The other groups were inserted with catheters and underwent CPR.CORM-2 and i CORM-2(4 mg/kg,dissolved in 2% DMSO and diluted in normal saline)was intraperitoneally injected at 12 hours before beginning CPR.In the Sham group,CPR group and DMSO group,animals were administrated an equivalent content of vehicle.Hemodynamic data(MAP,dp/dtmax,-dp/dt)were continuously monitored and recorded for 4h after resuscitation(or catheterization)in each group.The ultrastructure of myocardium was observed by transmission electron microscope.Myocardial tissue specimen and blood samples were taken at 4h after resuscitation(or catheterization).Lactate dehydrogenase(LDH)activity was measured by lactate-pyruvate method.Serum creatine kinase isoenzyme(CK-MB)concentration was measured by ELISA.Western blot was used to detect the levels of Caspase-3,Caspase-9,Cyt-C,Drp1,and Mfn2 protein in myocardial tissue.Detect the activity of mitochondrial respiratory complex IV,the ROS accumulation in myocardial tissue.Results1.CORM-2 improved hemodynamics and cardiac function after resuscitation Compared with Sham group,CPR group,DMSO group and i CORM-2 group exhibited that MAP was significantly decreased at each time point after resuscitation,and +dp/dtmax and-dp/dt decreased at 0.5h,1h and 2h after resuscitation(all P<0.05).There was no difference in MAP,+dp/dtmax,-dp/dt between the CORM-2 group and the Sham group(all P>0.05),and at0.5h,1h,2h after resuscitation,+dp/dtmax,-dp/dt in CORM-2 group were higher than those in other resuscitated groups(all P<0.05).No difference in hemodynamic data were seen among the CPR group,DMSO group and i CORM-2 group(all P>0.05).2.CORM-2 protects myocardial mitochondrial ultrastructure after resuscitation Under transmission electron microscope,the myocardial fibers in the Sham group were compact and orderly,with clear mitochondrial structure and neat mitochondrial crest.In the CPR group,myocardial fibrils were sparsely arranged,sarcomere was disordered,mitochondria were swollen,and mitochondrial ridge was partially disappeared.The myocardium of CORM-2 group was well arranged with clear sarcomere and clear mitochondrial ridge.Mitochondria of the i CORM-2 group and the DMSO group were swollen,and the crista was fractured and sparse,with light transmission areas visible.Some mitochondria were almost completely destroyed,with multiple vacuoles visible.3.CORM-2 significantly decreased serum myocardial enzyme levels induced by CA-CPR Compared with sham group,serum LDH activity and CK-MB concentration were significantly elevated in the CPR group,DMSO group and i CORM-2 group(all P<0.05);CORM-2 group displayed only increased CK-MB concentration(P>0.05).the CORM-2group had lower serum LDH activity and CK-MB concentrations than the other resuscitation groups(all P <0.01).4.Effect of CORM-2 on mitochondrial-dependent apoptotic pathways Compared with the Sham group,the expression of Caspase-3,Caspase-9 and Cyt-C in the CPR group,DMSO group and i CORM-2 group were significantly increased;CORM-2 group had only increased levels of Caspase-3 and Caspase-9(all P<0.05).The expression of the above three proteins in the CORM-2 group was lower than that in the other resuscitation groups(all P<0.05).5.Effect of CORM-2 on accumulation of ROS Compared with Sham group,ROS levels in CPR group,i CORM-2 group and DMSO group increased(P<0.05).The ROS production of CORM-2 group was slightly higher than that of Sham group,while the ROS content after CORM-2 intervention was significantly lower than that of CPR group,i CORM-2 group and DMSO group(P<0.05).6.Effect of CORM-2 on mitochondrial respiratory complex IV Compared with the Sham group,the activity of mitochondrial respiratory complex IV enzyme in the CPR group,i CORM-2 group and DMSO group were significantly decreased(P<0.05).In contrast,the enzyme activity was significantly increased in the CORM-2intervention group(P<0.05).There was no significant difference among CPR group,i CORM-2 and DMSO group(P>0.05).7.Effect of CORM-2 on mitochondrial dynamics Compared with Sham group,the expressions of drp1 in CRR group,i CORM-2 group and DMSO group were significantly increased(P<0.05).Compared with Sham group,the expression level of Mfn2 protein decreased(P<0.05).Compared with CPR group,CORM-2intervention group can significantly inhibit the increase of Drp1 and increase the expression of Mfn2.There was no significant difference in the expression of Drp1 and Mfn-2 between the CPR group,the i CORM-2 group and the DMSO group.Conclusion CORM-2 can effectively alleviate myocardial injury after cardiopulmonary resuscitation in rats via reducing mitochondrial pathway-mediated cardiomyocyte apoptosis,and improving rat recovery cardiac function.At the same time,CORM-2 reduces myocardial mitochondrial division,promotes fusion,and improves mitochondrial kinetic balance may also be one of the mechanisms to protect cardiac function after resuscitation.