The Experimental Research On The Protective Effect And Mechanism Of Carbon Monoxide Releasing Molecule-3 On Hypoxia Reoxygenation Injury In Rat Cardiomyocytes

ObjectiveTo investigate the effect of carbon monoxide releasing molecule 3?CORM-3?on myocardial apoptosis induced by hypoxia/reoxygenation injury and its role in regulating the expression levels of cytochrome C,Caspase 3 and Caspase 9 protein,mitochondrial respiration and energy generation,providing reference for clinical research.Methods1.Construction of hypoxia/reoxygenation injury model in primary neonatal rat myocytes.1.1 Cardiomyocytes isolated from neonatal rat hearts were cultured for 5 days.Cardiomyocytes being subjected to hypoxia?H?were placed in glucose-free and serum-free DMEM and submitted to hypoxic conditions in tri-gasincubator?1%O₂,5%CO₂,37??for a periods.Cardiomyocytes were exposed to hypoxia for 1h,2h,3h and 4h respectively.Then followed by reoxygenation,the myocytes recieving reoxygenation?R?were incubated in low glucose DMEM medium containing 10%fetal bovine serum for a further 6h.1.2 The cells were divided into five groups:normal control group?group C?,group H1R6,group H2R6,group H3R6 and group H4R6.1.3 After 6h of reoxygenation,morphological features and ultrastructure changes in cardiomyocytes were observed,cell viability was measured by CCK-8 assay,the activity of lactate dehydrogenase?LDH?was determined by lactic acid-pyruvate method and the early cell apoptotic rate was detected by flow cytometry.2.Research on the effect of CORM-3 after hypoxia/reoxygenation injury on neonatal rat cardiomyocytes.2.1 The cardiomyocytes were incubated in glucose-free and serum-free DMEM for 3h and then in low-glucose DMEM for 6h.At the beginning of oxygen-glucose deprivation,the corresponding concentrations of CORM-3 and iCORM-3 were added to the glucose-free DMEM and the the glucose-free DMEM with intervention drug was abandoned immediately after restoring the oxygen and glucose supply.No drugs were added in the C and H/R groups.The index was measured after 6h of reoxygenation.2.2 The cells were divided into six groups:normal control group?group C?,HR group,iCORM-3group,CORM-312.5?mol/L?,25?mol/L?,50?mol/L?group.2.3 The index was measured just the same as the hypoxia/reoxygenation injury model.3.TheeffectofCORM-3onmyocardialapoptosisinducedby hypoxia/reoxygenation injury through the mitochondrial signaling pathway.3.1 The preparation process of cardiomyocyte H/R model and the method of drug administration were the same as previously discribed.The index was measured after6h of reoxygenation.3.2 The cells were divided into four groups:normal control group?group C?,group HR,group iCORM-3,group CORM-312.5?mol/L.3.3 Index detection:flow cytometry was used to detect the change of mitochondrial membrane potential.Western Blot was used to determine protein expression levels of cytochrome C?both in cytoplasm and in mitochondrial?,Caspase3 and Caspase 9.4.The effects of CORM-3 on mitochondrial respiration and energy generation of myocardial cells in hypoxia/reoxygenation injury.4.1 The specimen collection phase was divided into two parts,namely at the end of hypoxia and reoxygenation.The hypoxic cells experienced hypoxia?H_H?only,and the other underwent hypoxia and reoxygenation?HR_R?.The model preperation process and the drug administration method were the same as previously discribed.Hypoxia groups:group C,H_H group,iCORM-3_H group,12.5?mol/L CORM-3_H group.Reoxygenation groups:group C,HR_R group,iCORM-3_R group,12.5?mol/LCORM-3_R group.4.2 Indicators detection:the ROS and ATP levels and the cytochrome C oxidase activity were measured at the end of hypoxia and reoxygenation.Results1.Construction of hypoxia/reoxygenation injury model in primary neonatal rat myocytes.Compared with group C,the morphology and the ultrastructure of cardiomyocytes were obviously damaged in H/R groups,the cell viability decreased and the LDH activity in the culture media increased,the early apoptotic rate of H/R groups were significantly increased?P<0.05?,with the increase of hypoxia time,the damage of cardiomyocytes gradually aggravated.2.Research on the effect of CORM-3 with different concentrations on hypoxia/reoxygenation injury in neonatal rat cardiomyocytes.Compared with group C,the pathological changes of cardiomyocytes were obviously observed,cell proliferation was significantly reduced,and the LDH concentration in the supernatant and the early apoptosis rate were increased in H/Rgroups?P<0.05?.ComparedwithH/RandiCORM-3groups,the pathological changes of cardiomyocytes were significantly attenuated,the cell proliferation increased,the LDH activity in the supernatant and the early apoptosis rate decreased in group CORM-3?,?,?,especially in group CORM-3??P<0.05?,with increasing concentration of CORM-3,the pathological changes were aggravated,the cell proliferation was reduced,and the LDH concentration and the early apoptosis rate were increased.There was no statistically significant difference in early apoptosis rate between group H/R and group iCORM-3?P>0.05?.3.The effect of CORM-3 on the mitochondrial signaling pathway of myocardial apoptosis induced by hypoxia/reoxygenation injury.Compared with group C,the mitochondrial membrane potential level was decreased?and the protein expression levels of cytochrome C,Caspase 3 and Caspase9 were increased?while the protein expression level of cytochrome C in mitochondria was lower in H/R group and iCORM-3 group?P<0.05?.Compared with H/R and iCORM-3 group,the mitochondrial membrane potential level was increased,and the protein expression levels of cytoplasmic cytochrome C,Caspase 3 and Caspace 9 were decreased?the expression of mitochondrial cytochrome C protein was higher in the12.5?mol/LCORM-3group?P<0.05?.There was no statistically significant difference in the above indexs between group H/R and group iCORM-3?P>0.05?.4.The effects of CORM-3 on mitochondrial respiration and energy generation of myocardial cells in hypoxia/reoxygenation injury.At the end of hypoxia:compared with group C,the ROS level were increased?the ATP level and the cytochrome C oxidase activity were decreased in H_H and iCORM-3_H group?P<0.05?.Compared with the H_H and iCORM-3_H group,the ROS level was decreased?the ATP content and the cytochrome C oxidase activity were increased in 12.5?mol/L CORM-3_H group?P<0.05?.There was no statistically significant difference between H_H group and iCORM-3_H group?P>0.05?.At the end of reoxygenation,compared with group C,the ROS level was increased?the ATP content and cytochrome C oxidase activity were decreased in HR_R group and iCORM-3_R group?P<0.05?.Compared with the HR_R group and iCORM-3_R group,the ROS content decreased?the ATP content and cytochrome C oxidase activity were increased in 12.5?mol/L CORM-3_R group?P<0.05?.There was no significant difference between HR_R group and iCORM-3_R group?P>0.05?.Conclusions1.Hypoxia/reoxygenation injury model of cardiac myocytes in rats was established successfully,and the hypoxia time for 3 hours is more appropriate.2.CORM-3 can reduce hypoxia/reoxygenation injury to cardiomyocytes of rats?especially in group CORM-312.5?mol/L?.3.CORM-3 can inhibit apoptosis after hypoxia/reoxygenation cardiomyocytes injury via the mitochondrial pathways.4.CORM-3improvesmitochondrialfunctionofhypoxia/reoxygenation cardiomyocytes,improves cell respiration,as well as increases ATP production.