Researches Of Activating The Reperfusion Injury Salvage Kinases By Sevoflurane Postconditioning In Rats

ObjectiveBrief episodes of ischemia during early reperfusion after myocardial ischemia would reduce the extent of myocardial infarction.This deliberate use is termed "ischemic postconditioning".Pharmacological postconditioning,the administration of a volatile agent during early reperfusion after global ischemia,could produce an effect of myocardial protection similar to ischemic postconditioning.Some data showed pharmacological postconditioning can decrease myocardial damage and activate reperfusion injury salvage kinases(RISKs),which consist of phosphatidylinositol-3-kinase(PI3K)and extracellular signal-regulating kinases (ERK1/2).Sevoflurane,a new type of volatile agent,is prevalent in clinical practice because of its better characteristics.However,the mechanisms underlying sevoflurane postconditioning are still unclear.The experiment tested the hypotheses thatⅰ)sevoflurane postconditioning produces cardioprotection not only by activating phosphatidylinositol-3-kinase pathway,but also activating extracellular signal-regulating kinases pathway at the early reperfusion,ⅱ)Combining sevoflurane and ischemic postconditioning may offer an additional benefit against reperfusion injury.Meanwhile,the experiment investigated influences of sevoflurane postconditioning on myocardial mitochondrial membrane potential(△Ψm)and cell apoptosis in isolated rat heart model,and discovered the possible mechanisms of sevoflurane postconditioning protecting ischemic myocardium. Methods1.Research of activating PI3K pathway by sevoflurane postconditioningIsolated,perfused rat hearts were exposed to 25 min of ischemia followed by 90 min of reperfusion.Sevoflurane postconditioning was induced by 15 min of 3.0 vol% sevoflurane administered at the onset of reperfusion.Ischemic postconditioning was induced by 3 cycles of 30s of reperfusion followed by 30s ischemia immediately after the global ischemia.After equilibration for 20 min,the hearts were randomly allocated into 10 groups as follows:1.Sham group:Perfusion for 115 min in a KH buffer.2.Control group:Global ischemia for 25 min followed by reperfusion for 90 min.3.Ischemic postconditioning group(Post group):Three cycles of ischemic postconditioning(30 s of ischemia followed by 30 s of reperfusion)during early reperfusion after 25 min of ischemia.4.Sevoflurane postconditioning group(Sevo group):Administration of 3.0 vol%sevoflurane for 15 min during early reperfusion.5.LY294002 group(LY group):Administration of the selective PI3K inhibitor LY294002(15μmol/L)with its drug vehicle dimethyl sulfoxide (DMSO)for 15 min during early reperfusion.6.Ischemic postconditioning together with LY294002 group(LY+Post group): Administration of LY294002 in DMSO(15μmol/L)for 15 min during early reperfusion followed by three cycles of ischemic postconditioning simultaneously.7.Sevoflurane postconditioning together with LY294002 group(LY+Sevo group):Administration of both LY294002 in DMSO(15μmol/L)and 3.0 vol% sevoflurane for 15 min during early reperfusion.8.Combining ischemic postconditioning and sevoflurane postconditioning group(Post+Sevo group):Administration of 3.0 vol%sevoflurane for 15 min during early reperfusion followed by three cycles of ischemic postconditioning simultaneously.9.Combining ischemic postconditioning and sevoflurane postconditioning together with LY294002 group(LY+Post+Sevo group):Administration of both LY294002 in DMSO(15μmol/L)and 3.0 vol%sevoflurane for 15 min during early reperfusion,followed by three cycles of ischemic postconditioning simultaneously.10.Dimethyl sulfoxide group(DMSO group):Administration DMSO(15μmol/L)for 15 min during early reperfusion.In each group,separate experiments were performed to determine coronary flow (CF)and hemodynamic variables including left ventricular systolic pressure(LVSP), left ventricular developed pressure(LVDP:end-systolic minus end-diastolic),positive and negative LV dp/dt(dp/dt max,dp/dt min),heart rate(HR)after equilibration for 20 min and after reperfusion for 5 min,15 min,30 min,60 min,90 min. Western blot analysis was used to determine Akt and GSK3βactivity after 15 min of reperfusion(n=6).Infarct size was determined with 2,3,5-triphenyltetrazolium chloride(TTC)staining at the end of reperfusion(n=6).2.Research of activating extracellular signal-regulating kinases (ERK1/2)pathway by sevoflurane postconditioning.Group 1-4 were treated in the same way to those in part one.5.PD98059 group(PD group):Administration of the selective PI3K inhibitor PD98059(20μmol/L)with its drug vehicle dimethyl sulfoxide(DMSO) for 15 min during early reperfusion.6.Sevoflurane postconditioning together with PD98059 group(PD+Sevo group):Administration of both PD98059 in DMSO(20μmol/L)and 3.0 vol% sevoflurane for 15 min during early reperfusion.7.DMSO group:Administration DMSO(15μmol/L)for 15 min during early reperfusion.The determination of CF and hemodynamic variables and infarct size in this part and part one were alike.Western blot analysis was used to determine ERK1/2 activity after 15 min of reperfusion(n=6).Then,the subsarcolemmal mitochondria (SSM)was isolated and its morphology was observed and quantified under the electron microscope at the end of reperfusion(n=6).3.Influences of sevoflurane postconditioning on ischemic myocardium cell apoptosis and mitochondrial membrane potentialGroup 1-4 were treated in the same way to those in part one.At the reperfusion of 15 min,cardiac myocytes were isolated acutely from heart,and observed mitochondrial membrane potential(△Ψm)under laser scanning confocal microscope with JC-1 (5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide)probe((n=6), in addition,apoptosis cells were detected with TUNEL and apoptotic index (AI)was calculated simultaneously(n=6).At the reperfusion of 90 min,used western blot analysis to determine contents of cytosolic and mitochondrial cytochrome C and the activity of caspase-3(n=6).The subsarcolemmal mitochondria (SSM)was isolated and its morphology was observed and quantified under the electron microscope at the same time(n=6).Results1.Effects on coronary flow and systemic hemodynamicsExcept the Sham group,CF of hearts in all experimental groups decreased, compared with the preischemic state throughout reperfusion(P＜0.05).Overall,CF in all experimental groups showed a tendency to improve during reperfusion.Between 60 and 90 minutes from the onset of reperfusion,CF in Sevo group,Post group and Sevo+Post group increased significantly,compared to the control group(P＜0.05), but CF did not differ among the sevoflurane-treated groups and Post groups. LY294002 and PD98059 with its vehicle,DMSO,did not affect CF but prevented the beneficial effect vascular of sevoflurane or ischemic postconditioning or sevoflurane and ischemic postconditioning.Therefore,CF in LY294002 treated groups,PD98059 treated groups and DMSO group were comparable to control group during reperfusion.HR did not vary before Preischemia and after the reperfusion of 60 min in any of the experimental groups.No significant differences were observed between or within groups after the 20 min stabilization period for the parameters examined(HR,CF,LVSP,LVDP,dp/dt max,dp/dt min).There was a gradual recovery in the haemodynamic parameters of all groups throughout reperfusion.At 15 min of reperfusion,LVSP,LVDP,dp/dt max, and dp/dt min in the sevoflurane group was significantly lower than Post group(P＜0.05).After 15 min of reperfusion,hemodynamic parameters in Sevo,Post,and Sevo+Post groups were higher than control and other groups(P＜0.05).However, hemodynamic parameters in the three groups were not statistically different. Hemodynamic parameters in LY294002 treated groups,PD98059 treated groups and DMSO group showed no differences compared to those in control group during reperfusion.2.Effects on myocardial infarct sizeInfarct size in Post and Sevo groups significantly reduced(28±3%and 31±2%, respectively,P＜0.05)at the end of reperfusion,compared to the control group(42±3%).Similarly,there was significantly decreased infarct size in Post+Sevo group(26±4%,P＜0.05).However,the differences among the three experimental groups were not statistically significant.LY294002 with its vehicle,DMSO,did not affect myocardial infarct size,but they abolished the protection of sevoflurane or ischemic postconditioning or sevoflurane and ischemic postconditioning to ischemic myocardial.Infarct size in the LY+Post,LY+Sevo,and LY+Post+Sevo groups was 41±2%,43±3%,and 42±2%,respectively.Similarly that of LY and DMSO group was 40±3%and 43±2%,respectively.They were no statistical differences compared to control group.The data in PD98059 treated groups were similar to those in LY294002 treated groups. 3.Effects on phosphorylation of Akt and GSK3βCompared with control group,a significant increase in phosphorylation of Akt and GSK3βwas observed in Sevo group at the early reperfusion(P＜0.05). Similar increases were also observed in Post and Post+Sevo groups.However,there were no statistical differences between the three experimental groups.In all three groups,phosphorylation of Akt and GSK3βwas strongly suppressed by administration of LY294002.4.Effects on phosphorylation of ERK1/2(p-ERK1/2)Compared with Sham group,the expression of the phosphorylation of ERK1/2 in Control,Post,Sevo,DMSO groups was reinforced,however,that of PD and PD+Sevo was not statistically significant.Compared with control group,the expression of p-ERK1/2 was strengthened further in Post and Sevo group(P＜0.05).Nonetheless, difference between two groups was not statistically significant.The expression of p-ERK1/2 in PD and PD+Sevo was significantly lower than that of control group (P＜0.05).5.Effects on SSM morphologyThe micrographs showed there were electron dense mitochondria with fewer blebs and the majority of SSM was intact in Sham group.SSM in Post and Sevo group were moderately swollen,with reduced matrix density and altered cristal patterns.However,micrographs from the other groups revealed a decreased number of mitochondria,accompanied by swelling,disorganized cristae,vacuolation,and even rupture.Semiquantitative morphometry also showed that myocardial mitochondria injury in Sevo(1.45±0.24)and Post(1.40±0.21)group was less than that of control group(2.23±0.28)(P＜0.05),but comparisons between two groups revealed no significant differences.Nonetheless,those in PD group and DMSO group were equivalent with respect to the control group during reperfusion.6.Effects on myocardial cell apoptosisAfter reperfusion of 15 min,the apoptotic cells were not observed obviously in Sham group,and its AI was only(4.1±1.1)%.AI of Post and Sevo groups were (12.6±2.4)%and(13.8±2.7)%,respectively,and were significantly lower than that of control group(38.2±9.0)%(P＜0.05),However,AI in Post and Sevo groups were not statistically different.7.Effects on mitoehondrial membrane potential(MMP)After the reperfusion of 15 min,the mitochondrial membrane potential of normal cardiocytes in Sham group was in a high level(1.875±0.375),and the number of cardiocytes was also more than control group.On the contrary,MMP was eventually dissipated(1.149±0.287),and the number of cardiocytes decreased in control group (P＜0.05).Compared to the control group,MMP in Post group(1.534±0.318)and Sevo group(1.507±0.321)were maintained in higher level(P＜0.05).However,there was not significant statistically between these two groups.8.Effects on eytoehrome C(CytC)and Caspase-3Compared with Sham group,a significant decrease in the CytC content of mitochondria and a significant increase in the CytC content of cytosol in other groups were observed(P＜0.05).At the end of reperfusion,the content of CytC in Sevo and Post group's mitochondria was higher,and the content of CytC in two groups' cytosol was lower than of control,group(P＜0.05).Moreover, comparisons between two groups revealed no significant difference.Compared with Sham group,at the end of reperfusion,the expression of caspase-3 was significantly strengthened in other groups(P＜0.05).However,the expression of caspase-3 was weakened in two groups(P＜0.05),and there was not significant statistically between two groups.Conclusion1.Sevoflurane postconditioning can increase coronary flow,salvage myocardium from infarction and improve functional recovery,elevate the level of phosphorylation of Akt,GSK313,ERK1/2,and diminish the degree of mitochondria lesions,which showing a comparable protective effect in ischemic postconditioning.Some selective inhibitor,LY294002 for PI3K and PD98059 for ERK1/2,can abrogate these protections of the two ways about postconditioning.It indicates that sevoflurane postconditioning activates not only phosphatidylinositol-3-kinase signal transduction but also extracellular signal-regulating kinases signal transduction to protect ischemic myocardium against myocardial reperfusion injury.2.The combination of sevoflurane and ischemic postconditioning play an important protective role to ischemic myocardium.The cardioprotection effects were equivalent with ischemic postconditioning or sevoflurane postconditioning.It shows that the combination of sevoflurane postconditioning and ischemic postconditioning does not offer an additional benefit against ischemia reperfusion injury.3.During reperfusion,sevoflurane postconditioning inhibit the dissipation of mitochondrial membrane potential to ischemic myocardium,prevent the release of cytochrome C from mitochondria into cytosol,depress the activity of ischemic myocardial caspase-3,and reduce myocardial cell apoptosis.So,it is one of important mechanisms of sevoflurane postconditioning to reduce cell apoptosis via many sites.