| BackgroundMyocardial necrosis,apoptosis and ventricular remodeling after ischemia/reperfusion injury(IRI)is the most important pathophysiological basis of cardiac insufficiency after thrombolytic or surgical therapy for acute myocardial infarction,and about 50%myocardial infarction area is ultimately attributable to IRI.When combined with diabetes,it is more likely to induce myocardial IRI,and the extent of myocardial injury and mortality can be further increased.This issue is a serious threat to human life and health,but its mechanism is unclear.Therefore,both exploring potential protective measures to prevent or reduce myocardial IRI and providing prevention and treatment basis for diabetic patients with ischemic cardiomyopathy have good clinical application prospects.Ischemia preconditioning(IPC)refers to improve the myocardial tolerance for the followed long time ischemia by multiple transient coronary ischemia,and thus to reduce the myocardial IRI.The strong myocardial protective effects of IPC are to reduce myocardial infarction area after IRI,the incidence of arrhythmia,myocardial apoptosis and promote cardiac functional recovery.The role of IPC in IRI has attracted much attentions,nevertheless,the mechanism of its endogenous myocardial protection has not been fully elucidated.Autophagy is an intracellular defense and stress regulation mechanism,and meanwhile is also involved in myocardial IRI.As a kind of important material metabolism way,the occurrence and development of autophagy are strictly regulated.Autophagy is regulated by autophagy related genes and several key protein complexes are involved in the regulation,including mammalian target of rapamycin(mTOR),phosphatidylinositol 3 kinase(PI3K),Atg8(LC3)and Atg6(Beclin-1),et al.At present,multiple regulation pathways of autophagy are known,while PI3K-protein kinase B-mTOR signal transduction pathway(PI3K-Akt-mTOR pathway)is the only inhibitory pathway of autophagy process.The role of autophagy in IRI process is two-sided,but whether its effect is protective or harmful is still controversial.Diabetes is a serious threat to human life and health,and is an independent risk factor for ischemic cardiomyopathy.Diabetes could aggravate myocardial IRI,and weaken the protective effect of IPC against myocardial IRI.It hs been shown that the expression of autophagy and PI3K-Akt-mTOR signaling transduction pathway in diabetic myocardium changes significantly,and plays different roles in the IRI processes of the normal and diabetic myocardium.The diabetic myocardial IRI is closely related to autophagy and PI3K-Akt-mTOR signaling transduction pathway,but the specific mechanisms and their interactions need to be clarified and studied.Based on the above analysis,this basic research was designed.This study assessed the expression of PI3K-Akt-mTOR signaling transduction pathway in normal and diabetic myocardial IRI from the in vitro and in vivo levels,in order to explore the role of PI3K-Akt-mTOR signaling transduction pathway and autophagy in diabetes myocardialIRI.Meanwhile,IPC was applied on diabetic myocardial IRI when autophagy activator and inhibitor interventions were used,and then the protective effect of IPC against myocardiac IRI was observed.By the above measures,the protective mechanisms of autophagy regulation in diabetes myocardial IRI were further explored.This study was divided into four parts:Part 1:The establishment of cardiomyocyte anoxia/reoxygenation injury model and hypoxic preconditioning modelThe aim of this part was to build a stable and high qualitied cells culture method of the primary cardiomyocyts.Further,constructing the neonatal rat cardiomyocyt anoxia/reoxygenation(AR)injury model by a three air incubator,and exploring the best hypoxia preconditioning by observing cardiomyocyt apoptosis.The neonatal rat cardiomyocyte in vitro hypoxia preconditioning model was built by a three gas incubator.The cardiomyocyts were cultured for 72 h,and were randomly divided into 5 groups:(1)control group(group S),without any treatment for normal cultured cardiomyocyts;(2)3h/6h group:treatments were a 3-h anoxia and a 6-h reoxygenation for cardiomyocyts;(3)30min*3h/6h group,treatments were a 30-min anoxia and a 30-min reoxygenation for cardiomyocyts,and then a 3-h anoxia and a 6-h reoxygenation;(4)6h/6h group,treatments were a 6-h anoxia and a 6-h reoxygenation for cardiomyocyts;(5)30min*6h/6h group,treatments were a 30-min anoxia and a 30-min reoxygenation for cardiomyocyts,and then a 6-h anoxia and a 6-h reoxygenation.The cardiomyocyt purity was identified by combined cTnT immunofluorescence method with nucleus DAPI staining.Lactatedehydrogenase(LDH)leakage rate was tested by LDH detection kit.Myocardial cells were stained by annexin V/and propidium iodide(PI)(Annexin V/PI),and apoptosis was observed by flow cytometry.The results demonstrated myocardial cells showing synchronous beating and syncytium after 48-72 h under inverted phase contrast microscope.Cardiomyocyts contracted strongly and had a high activity.The cardiomyocyt purity determined by the method of combined cTnT immunofluorescence with nuclear DAPI staining was up to 94.4±1.2%.As compared with the S group,LDH leakage rate,early and late apoptotic percentages of cardiomyocyts were significantly increased(P<0.05),and percentage of normal cells was significantly decreased in 30min*3h/6h group and 30min*6h/6h group(P<0.05).Compared with the 3h/6h group,LDH leakage rate,early and late apoptotic percentage of cardiomyocyts were significantly increased(P<0.05),and percentage of normal cells was significantly decreased(P<0.05)in the 6h/6h group;LDH leakage rate was obviously decreased(P<0.05),and percentage of normal cells was significantly increased(P<0.05)in the 30min*3h/6h group.Compared with the 6h/6h group,LDH leakage rate,early and late apoptotic percentage of cardiomyocyts were significantly decreased(P<0.05),and the percentage of normal cells was increased significantly(P<0.05)in the 30min*6h/6h group.Part 2:Experimental study on roles of autophagy and PI3K-Akt-mTOR signaling transduction pathway in protection of hypoxia preconditioning against anoxia/reoxygenation injury when cardiomyocyts were cultured in high glucose conditionIn this part,expressions of autophagy and PI3K-Akt-mTOR signaling transduction pathway in hypoxia preconditioning cardiomyocyts cultured in normol and high glucose conditions were observed.The study aims were to explore the protection of hypoxia preconditioning against cardiomyocyte anoxia/reoxygenation(AR)injury and its mechanisms.After cardiomyocytes were cultured for 72h,they were randomly divided into 6 groups:(1)Normal cardiomyocyte control group(S group);(2)High glucose cardiomyocyte control group(HS group);(3)Normal cardiomyocyte AR group(C group),cardiomyocytes were treated with a 6-h anoxia followed by a 6-h reoxygenation in a normal culture condition;(4)High glucose cardiomyocyte AR group(HC group):cardiomyocytes were treated with a 6-h anoxia followed by a 6-h reoxygenation in a high glucose culture condition;(5)Normal cardiomyocyte anoxia preconditioning group(HPCgroup):cardiomyocytes were treated with a 3 0-min anoxia followed by a 30-min reoxygenation,and then a 3-h anoxia followed by a 6-h reoxygenation in a normal culture condition;(6)High glucose cardiomyocytanoxia preconditioning group(HHPCgroup):cardiomyocytes were treated with a 30-min anoxia followed by a 30-min reoxygenation,and then a 3-h anoxia followed by a 6-h reoxygenation in a high glucose culture condition.Lactatedehydrogenase(LDH)leakage was tested by LDH detection kit.Cardiomyocytes were stained by Annexin V/PI,and apoptosis were observed by flow cytometry.Cardiomyocyte expressions of LC3-Ⅱ,Beclin-1,PI3K,mTOR and P-Akt Akt ratio in all samples were assessed by Western-blotting analysis.The results showed that compared with the C group,LDH leakage rate was significantly decreased in the HPC group(P<0.01).Compared with the HC group,LDH leakage rate was not significantly different in the HHPC group(P>0.05).Compared with the HPC group,LDH rate leakage was significantly increased in the HHPC group(P<0.01).Compared with C group,early and late apoptotic percentages of cardiomyocytes were significantly decreased in the HPC group,percentage of normal cells was significantly increased(P<0.05).Compared with the HC group,the percentages of early and late apoptosis and normal cells were not significantly different in the HHPC group(P>0.05).Compared with the HPC group,the percentages of early and late apoptosis were significantly increased in the HHPC group(P<0.05).The Western-blotting analysis showed that compared with the S group,cardiomyocyte expression of LC3-Ⅱ was increased significantly in the other groups(P<0.05),cardiomyocyte expressions of mTOR and PI3K were significantly decreased(P<0.05);cardiomyocyte expression of Beclin-1 was significantly increased(P<0.05),and the P-Akt/Akt ratio was significantly decreased in the HS,C,HC and HHPC groups(P<0.05).Compared with the C group,cardiomyocyte expressions of LC3-Ⅱ and Beclin-1 were significantly decreased(P<0.05),and cardiomyocyte expression of mTOR was significantly increased in the HPC group(P<0.05);cardiomyocyte expression of Beclin-1 was significantly increased(P<0.05),cardiomyocyte expression of mTOR was significantly decreased(P<0.05)and cardiomyocyte expression of LC3-Ⅱ was increased but no significant statistical difference was achieved in the HC group(P>0.05).Compared with the HC group,cardiomyocyte expression of mTOR was significantly increased(P<0.05),cardiomyocyte expression of PI3K was significantly decreased in HHPC group(P<0.05).Compared with the HPC group,cardiomyocyte expressions of LC3-Ⅱ and Beclin-1 were significantly increased(P<0.05)and cardiomyocyte expressions of mTOR and PI3K,and P-Akt/Akt ratio were significantly decreased in the HHPC group(P<0.05).Part 3:Protection and mechanisms of ischemia preconditioning against myocardial ischemia-reperfusion injury in normal and diabetic ratsThe aims of this part study were to observe the expression of autophagy in normal and diabetic myocardial ischemia-reperfusion injury,compare the protective effects of ischemia preconditioning against normal and diabetic myocardial ischemia-reperfusion injury,and investigate the mechanism of autophagy in the process.Thirty normal and thirty diabetic male Sprague Dawley rats(weighed 350 to 450 g)were randomly divided equally into six groups(n=10 in each group):Normal blank control group(S group);Diabetic blank control group(DS group);Normal IRI group(C group);Diabetic IRI group(DC group);Normal IPC group(IPC group);Diabetic IPC group(DIPC group).After left thoracotomy in all rats,a 5-0 silk ligature was placed around the left anterior descending coronary artery(LAD)and encircled with a suture.In C and DC groups,LAD was ligated for 30 min followed by a 120 min reperfusion.In IPC and DIPC groups,rats underwent three 3 circles of a 5-min LAD ligature followed by a 5-min reperfusion,which was performed before a 30 min LAD ligation,and then LAD was again ligated for 30 min followed by a 120 min reperfusion.Throughout all the experiment,a lead II electrocardiogram,mean artery pressure(MAP),and the heart rate(HR)were continuously monitored.The rectal temperature was maintained at 36.5-37.5℃.Blood samples were taken at 120 min of reperfusion for measuring serum troponin I(TnI)and myocardial-bound creatine kinase(CK-MB)concentrations by the kits specifically for rats.At the end of experiment,the infarct size(IS%)was assessed by Evans blue and triphenyltetrazolium chloride(TTC)staining.The cardiomyocyte expressions of LC3-Ⅱ,Beclin-1,PI3K,mTOR and P-Akt/Akt ratio in all groups were assessed by Western-blotting technique.The results showed that rat’s body weight,body temperature and baselines of HR,MAP and RPP were not significantly different among six groups(P>0.05).As compared to the C group,number of rats with reperfusion ventricular arrhythmia and arrhythmia score were significantly decreased in the IPC group,number of rats with ischemic arrhythmia and arrhythmia score were significantly increased in the DC group.Compared to the DC group,number of rats with reperfusion ventricular arrhythmia and arrhythmia score were not significantly different in the DIPC group.As compared to S group,the IS%was significantly higher in the C and DC groups(P<0.05);Compared to the C group,serum cTnI and CK-MB concentrations,and IS%were significantly lower in the IPC group(P<0.05),serum cTnI and CK-MB concentrations,and IS%tended to rise but did not achieve a statistical difference(P>0.05);Compared to the DC group,serum cTnI and CK-MB concentrations,and IS%were not significantly different DIPC group(P>0.05);Compared to the IPC group,serum cTnI and CK-MB concentrations,and IS%were significantly increased in the DIPC group(P<0.05).The Western-blotting analysis showed that compared with the S group,myocardial expression of Beclin-1 in the ischemic area were significantly increased(P<0.05)and myocardial expression of mTOR was significantly decreased(P<0.05)in the other groups.Compared with the C group,myocardial expressions of LC3-Ⅱ and Beclin-1 were significantly increased in the DC group(P<0.05),myocardial expressions of LC3-II and Beclin-1 were significantly decreased(P<0.05),and myocardial expressions of mTOR and PI3K,and P-Akt/Akt ration were significantly increased in the IPC group(P<0.05).As compared with the DC group,myocardial expressions of LC3-Ⅱ,Beclin-1,PI3K,mTOR and Akt were not significantly different in the DIPC group(P>0.05).Compared with the IPC group,myocardial expression of LC3-Ⅱ was significantly increased(P<0.05),and myocardial expression of mTOR,and P-Akt/Akt ratio were significantly decreased(P<0.05)in the DIPC group.Part 4:Experimental study on roles of autophagy and PI3K-Akt-mTOR signaling transduction pathway in protection of ischemia preconditioning against myocardial ischemia-reperfusion injury in diabetic ratsBased on the results of above third part experiment,ischemia preconditioning produced a protective effect against myocardial IRI in normal rats,but not in diabetic rats.Thus,this part experiment was designed.Both autophagy activater(rapamycin)and inhibitor(wortmannin)were used before IPC,myocardial expressions of PI3K-Akt-mTOR signaling transduction pathway and autophagy were assessed and protective effect of IPC on diabetic myocardial IRI was detected.Sixty anesthetized diabetic male Sprague Dawley rats(weighed 350 to 450 g)were randomly divided into six groups(n=10 in each group):(1)Diabetic rat blank control group(DS group):1.5 ml saline was injected intraperitoneally 30 min before modeling,a 5-0 silk ligature was placed around the left anterior descending coronary artery(LAD)and encircled with a suture,and stay for 150 min;(2)Diabetic rat IRI group(DC group):1.5 ml saline was injected intraperitoneally 30 min before modeling,then LAD was ligated for 30 min followed by a 120 min reperfusion;(3)Rapamycin+diabetic IRI group(Ra+DIRI group):rapamycin 0.25 mg/kg(1.5 ml)was given by intraperitoneal injection 30 min before modeling.After in vivo IRI model was successfully established and stabilized for10 min,LAD was ligated for 30 min followed by a 120-min reperfusion;(4)Wortmannin+diabetic IRI group(Wo+DIRI group),Wortmannin 0.6 mg/kg(1.5 ml)was given by intraperitoneal injection 30 min before modeling and other treatments were same as the Ra+DIRI group;(5)Rapamycin+diabetic IPC group(Ra+DIPC group):rapamycin 0.25mg/kg was given by intraperitoneal injection 30 min before modeling,IPC including 3 circles of a 5-min LAD ligature followed by a 5-min reperfusion was performed before a 3 0-min LAD ligature followed by a 120-min reperfusion;(6)Wo+diabetic IPC group(Wo+DIPC):Wortmannin 0.6mg/kg was given by intraperitoneal injection 30 min before modeling and other treatments were same as the Ra+DIPC group.The results showed that rat’s body weight,body temperature and baselines of HR,MAP and RPP did not differ among six groups(P>0.05).As compared to the DS group,serum cTnI and CK-MB concentrations,and IS%were significantly increased in the other groups.Compared to the DC group,serum cTnI and CK-MB concentrations,and IS%did not statistically differ in the Ra+DIRI and Ra+DIPC groups(P>0.05),tended to reduce but did not achieve statistical difference in the Wo+DIRI group(P>0.05),and were significantly lower in the Wo+DIPC group.Compared to the Ra+DIRI group,serum cTnI and CK-MB concentrations,and IS%did not significantly different in the Wo+DIRI and Ra+DIPC groups,but were significantly lower in the Wo+DIPC group(P<0.05).Compared to the Wo+DIRI group,serum cTnI and CK-MB concentrations,and IS%were significantly lower in the Wo+DIPC group(P<0.05).Compared to the Ra+DIPC group,serum cTnI and CK-MB concentrations,and IS%were significantly lower in the Wo+DIPC group(P<0.05).The Western-blotting analysis showed that compared with the DS group,myocardial expressions of LC3-Ⅱ and Beclin-1 were significantly increased,and myocardial expressions of P-Akt/Akt,mTOR and PI3K were significantly lower in the DC and Ra+DIRI groups(P<0.05);in the Wo+DIRI group,only myocardial expression of LC3-Ⅱ was significantly increased,and P-Akt/Akt ratio was significantly decreased(P<0.05).Compared with the DC group,myocardial expression of LC3-Ⅱ was significantly increased(P<0.05),and myocardial expression of PI3K was significantly decreased(P<0.05)in the Ra+DIRI group;myocardial expression of PI3K and P-Akt/Akt ratio were significantly increased in the Wo+DIRI group(P<0.05).Compared with the DC group,myocardial expression of LC3-Ⅱ was increased significantly in the Ra+DIPC group(P<0.05),myocardial expression of Beclin-1 was significantly lower in the Wo+DIPC group(P<0.05),and myocardial expressions of mTOR and PI3K,and P-Akt/Akt ratio were significantly increased in the Wo+DIPC group(P<0.05).Compared with the Ra+DIPC group,myocardial expressions of LC3-Ⅱ and Beclin-1 were significantly lower(P<0.05),and myocardial expressions of mTOR and PI3K,and P-Akt/Akt ratio were significantly increased in the Wo+DIPC group(P<0.05).Conclusions1.AR injury model of neonatal rat cardiomyocytes can be successfully constructed by anoxia 6 h/reoxygenation 6 h.Anoxia 30 min/reoxygenation 30 min before anoxia 6 h/reoxygenation 6 h can be used to construct hypoxia preconditioning model of neonatal rat cardiomyocytes.2.High glucose culture and AR injury can decrease the cardiomyocyte expression of PI3K-Akt-mTOR signal transduction pathway,but increase the cardiomyocyte expression of autophagy.3.Hypoxia preconditioning including a 30-min anoxia/a 30-min reoxygenation can produce protection against AR injury of cardiomyocytes cultured in a normal condition,and increase cardiomyocyte expression of PI3K-Akt-mTOR signaling transduction pathway.However,hypoxia preconditioning has no obvious protection against AR injury of cardiomyocytes cultured in a high glucose condition and does not improve cardiomyocyte expression of PI3K-Akt-mTOR signaling transduction pathway.4.Ischemia/reperfusion injury and diabetes can reduce the myocardial expression of PI3K-Akt-mTOR signaling transduction pathway,but enhance myocardial expression of autophagy.5.Ischemia preconditioning can produce protection against myocardial ischemia/reperfusion injury in normal rats,but can not result in anobvious cardioprotection in diabetic rats,which may be associated with the increased myocardial expression of autophagy in diabetic rats.6.After the intervention with Wortmannin,ischemia preconditioning can produce protection against myocardial ischemia/reperfusion injury in diabetic rats,which may be associated with activation of PI3K-Akt-mTOR signaling pathway. |
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