胰岛素保护缺血再灌注心脏：PI3-KAkt和JNKs信号通路间的交互作用.pdf

651Acta Physiologica Sinica,October 25,2007,59(5):651-659http:/ Received 2007-07-20 Accepted 2007-08-29 This work was supported by the National Science Fundation of China for Outstanding Young Investigators(No.30625033),theNational Natural Science Foundation of China(No.30471923,30500577),and the National Basic Research Development Program ofChina(No.2007CB512106).*Corresponding author.GAO Feng:Tel:[phone];Fax:[phone];E-mail:;WANG Hai-Chang:Tel:[phone];Fax:[phone];E-mail:Insulin protects isolated hearts from ischemia/reperfusion injury:cross-talk between PI3-K/Akt and JNKsLIU Hai-Tao1,ZHANG Hai-Feng2,SI Rui1,ZHANG Quan-Jiang2,ZHANG Kun-Ru2,GUO Wen-Yi1,WANG Hai-Chang1,*,GAO Feng2,*1Department of Cardiology,Xijing Hospital;2Department of Physiology,the Fourth Military Medical University,Xian 710032,ChinaAbstract:Our previous results have demonstrated that insulin reduces myocardial ischemia/reperfusion(MI/R)injury and increasesthe postischemic myocardial functions via activating the cellular survival signaling,i.e.,phosphatidylinositol 3-kinase(PI3-K)-Akt-endothelial nitric oxide synthase(eNOS)-nitric oxide(NO)cascade.However,it remains largely controversial whether c-Jun NH2-terminal kinase(JNK)is involved in the effects of insulin on MI/R injury.Therefore,the aims of the present study were to investigatethe role of JNK,especially the cross-talk between JNK and previously expatiated Akt signaling,in the protective effect of insulin onI/R myocardium.Isolated hearts from adult Sprague-Dawley rats were subjected to 30 min of regional ischemia and followed by 2 or4 h of reperfusion(n=6).The hearts were pretreated with PI3-K inhibitor LY294002,or phosphorylated-JNK inhibitor SP600125,respectively,then perfused retrogradely with insulin,and the mechanical functions of hearts,including the heart rate(HR),leftventricular developed pressure(LVDP)and instantaneous first derivation of left ventricular pressure(LVdp/dtmax)were measured.Atthe end of reperfusion,the infarct size(IS)and apoptotic index(AI)were examined.MI/R caused significant cardiac dysfunction andmyocardial apoptosis(strong TUNEL-positive staining).Compared with the control group,insulin treatment in MI/R rats exertedprotective effects as evidenced by reduced myocardial IS(28.92.0)%vs(45.04.0)%,n=6,P0.01,inhibited cardiomyocyteapoptosis decreased AI:(16.00.7)%vs(27.61.3)%,n6,P0.01 and improved recovery of cardiac systolic/diastolic function(including LVDP and LVdp/dtmax)at the end of reperfusion.Moreover,insulin resulted in 1.7-fold and 1.5-fold increases in Akt andJNK phosphorylation in I/R myocardium,respectively(n=6,P0.05).Inhibition of Akt activation with LY294002 abolished,andinhibition of JNK activation with SP600125 enhanced the cardioprotection by insulin,respectively.And the abolishment by LY294002could be partly converted by SP600125 pretreatment.In addition,SP600125 also decreased the Akt phosphorylation(n=6,P0.05).These results demonstrate that insulin simultaneously activates both Akt and JNK,and the latter further increases the phosphorylationof Akt which attenuates MI/R injury and improves heart function;this cross-talk between Akt and JNK in the insulin signaling isinvolved in insulin-induced cardioprotective effect.Key words:ischemia/reperfusion injury;insulin;apoptosis;Akt;JNK;cross-talk胰岛素保护缺血胰岛素保护缺血/再灌注心脏：再灌注心脏：PI3-K/Akt 和和 JNKs 信号通路间的交互作用信号通路间的交互作用刘海涛1，张海锋2，司 瑞 1，张全江2，张昆茹 2，郭文仪1，王海昌1,*，高 峰2,*第四军医大学1西京医院心血管内科；2生理学教研室，西安 710032摘 要：摘 要：我们前期研究表明胰岛素可激活细胞内信号转导机制如磷脂酰肌醇3-激酶-蛋白激酶B-内皮型一氧化氮合酶-一氧化氮(PI3-K-Akt-eNOS-NO)信号通路，减轻心肌缺血/再灌注(ischemia/reperfusion,I/R)损伤，改善缺血后心肌功能恢复。然而c-Jun 氨基末端激酶(c-Jun NH2-terminal kinase,JNK)信号通路在胰岛素保护I/R 心肌中的作用尚不清楚，本研究旨在探讨JNK Research PaperActa Physiologica Sinica,October 25,2007,59(5):651-659652信号通路在胰岛素保护I/R 心肌中的作用及其与PI3-K/Akt 信号通路间的相互关系。离体Sprague-Dawley大鼠心脏缺血30 min后施行2 h 或4 h 的再灌注，缺血前用LY294002(15 mmol/L)和SP600125(10 mmol/L)灌注15 min，分别阻断PI3-K/Akt和磷酸化JNK(phosphorylated-JNK,p-JNK)活化，观测心脏功能、心肌梗死、细胞凋亡和蛋白磷酸化水平。与对照组相比，胰岛素再灌注 2 h 后，心率、左心室发展压和左心室收缩/舒张最大速率均明显增加，梗死面积减少约 16.1%(28.92.0)%vs(45.04.0)%,n=6,P0.01，细胞凋亡指数从(27.61.3)%减少到(16.00.7)%(n=6,P0.01)，Akt 的活性增加1.7 倍(n=6,P0.05)，同时JNK活性增加1.5 倍(n=6,P0.05)。用LY294002 处理后胰岛素对I/R 心肌的保护作用消失；而用SP600125 处理可增强胰岛素的保护作用，且可部分逆转LY294002 的抑制作用。进一步观察发现SP600125 减弱了Akt 的磷酸化(n=6,P0.05)。上述结果表明，在I/R 心肌中，胰岛素可同时激活PI3-K/Akt 及 JNK 信号通路，且通过后者进一步增加Akt 活化，从而减轻I/R 损伤，改善心肌功能。这种PI3-K/Akt 与 JNK 信号通路交互机制对胰岛素保护I/R 心肌有重要意义。关键词：关键词：缺血/再灌注损伤；胰岛素；凋亡；蛋白激酶 B；c-Jun 氨基末端激酶；交互机制中图分类号中图分类号：R331.3Reperfusion therapy has become a practical and effectivestrategy in the salvage of ischemic myocardium.The di-rect enhancement of cardiac cellular tolerance againstmyocardial ischemia/reperfusion(MI/R)injury should fur-ther improve patient outcome in acute coronary syndromes(ACS).Our previous studies demonstrated that insulin mightplay an important role in attenuating MI/R injury in vivowhen it was administered during reperfusion and activa-tion of the cellular survival signaling,that is,phosphatidyl-inositol 3-kinase(PI3-K)-Akt-endothelial nitric oxide syn-thase(eNOS)-nitric oxide(NO)cascade,is one of the cen-tral mechanisms underlying the protective effect of insulinagainst MI/R injury1-5.Besides the PI3-K/Akt pathway,the mitogen-activated protein kinase(MAPK)cascades alsoare the downstream molecules of insulin in the heart6,7,which have been shown to play an important regulatoryrole in a variety of cellular processes8,9.MAPKs phospho-rylate selected intracellular proteins,including transcrip-tion factors,which subsequently regulate gene expressionby transcriptional and post-transcriptional mechanisms10.MAPKs are,in turn,activated by phosphorylation at con-served threonine and tyrosine residues by upstream dual-specific MAPK kinases(MAPKKs),which themselves areactivated by MAPKK kinases(MAPKKKs)11.In particular,the c-Jun NH2-terminal kinases(JNKs)are a group ofMAPKs that play a role in apoptosis,proliferation,andembryonic morphogenesis in both transcription-dependentand-independent mechanisms10,12,13.However,regardingthe MAPK signaling cascades are one of the main signalingpathways of insulin,it remains largely elusive whether JNKis also involved in insulin-induced anti-apoptotic effectson I/R myocardium.In addition,JNK activities were shown to be antago-nized by Akt kinase activity in numerous cell systems,andthis cross-talk may underlie many of the prosurvivaleffects of Akt14.It has been demonstrated that Akt regu-lates several proteins to suppress the JNK pathway,suchas extracellular regulated kinases(ERKs),JNK-interactingprotein(JIP)115-18,and MAPK/ERK kinase(MEK)15,19.Indeed,the role of Akt-JNK cross-talk in the cardiopro-tective effects of insulin against MI/R is unclear so far.Therefore,the aims of the present study were:(1)toinvestigate whether the JNK signaling pathway participatesin the protective effect of insulin,and if so,(2)to furtherexplore the possible role of Akt-JNK cross-talk in insulin-induced anti-apoptosis and subsequent attenuation of MI/Rinjury.1 MATERIALS AND METHODS1.1 Experimental protocolsThe experiments performed were in adherence with Guide-lines on the Use of Laboratory Animals in the NationalInstitutes of Health and were approved by the Fourth Mili-tary Medical University Committee on Animal Care.The experimental protocol was shown in Fig.1.Isolatedhearts from adult male Sprague-Dawley rats(20050)gwere randomly assigned to the following groups.(1)Shamgroup:the hearts were subjected to the MI/R procedure(30 min of regional ischemia and followed by 2 or 4 h ofreperfusion)except that the suture that was passed underthe left coronary artery was left untied(n=6);(2)Control(Vehicle)group:the hearts were subjected to 30 min ofregional ischemia and followed by 2 or 4 h of reperfusionwith vehicle(n=6);(3)Insulin group:insulin(Novo Nordisk,60 U/L)was administered 15 min before MI/R procedure(n=6);(4)Insulin+LY294002(a specific inhibitor for PI3-K)group:the hearts were pretreated with LY294002(15mmol/L)19 for 15 min before insulin administration(n=6);(5)Insulin+SP600125(a specific inhibitor for JNK)group:LIU Hai-Tao et al:Cross-talk between Akt and JNK653the hearts were pretreated with SP600125(10 mmol/L)12,20,21for 15 min before insulin administration(n=6);(6)Insulin+LY294002+SP600125 group:the hearts were pretreatedwith SP600125 and LY294002 for 15 min before insulinadministration(n=6).The doses of the reagents were basedon our previous experiments in rat hearts.1.2 Isolated heart preparation and MI/RAfter exteriorized through a left thoracic incision,the heartswere placed rapidly in ice-cold Krebs-Henseleit buffer toarrest and prevent them from ischemic preconditioning.The hearts were then perfused retrogradely through theascending aorta by a peristaltic pump at a constant flowrate of 10 mL/min,with perfusion medium containing(inmmol/L):NaCl 11.9,NaHCO3 24.9,KCl 4.74,KH2PO4 1.19,MgSO4 1.2,CaCl2 1.8 and glucose 11,oxygenated and keptat pH 7.4 by gassing with the mixture of 95%O2 and 5%CO221,22.The heart was kept throughout at constant tem-perature of 36.5 oC by being placed in a recirculation glassjacket.The initial perfusion pressure was 60 mmHg.Whenthe heart began contracting spontaneously,a latex balloon,connected to a pressure transducer,was inserted into theleft ventricle via the left atrium.The balloon was then filledwith saline buffer at a steady diastolic pressure of 4-8 mmHgto measure left ventricular pressure.Two silver electrodeswere fixed,one at the apex and another at the atria,forelectrocardiogram recording of a bipolar derivative.Theefficiency of the peristaltic pump in maintaining a stablecoronary flow was determined by collecting the effluentFig.1.Protocol chart.Isolated hearts from adult male Sprague-Dawley rats(20050)g were randomly assigned to the followinggroups.(1)Sham group:the hearts were subjected to the MI/R procedure(30 min of regional ischemia and followed by 2 or 4 h ofreperfusion)except that the suture that was passed under the left coronary artery was left untied(n=6);(2)Control(Vehicle)group:the hearts were subjected to 30 min of regional ischemia and followed by 2 or 4 h of reperfusion with vehicle(n=6);(3)Insulin group:insulin(Novo Nordisk,60 U/L)was administered 15 min before MI/R procedure(n=6);(4)Insulin+LY294002(a specific inhibitor forPI3-K)group:the hearts were pretreated with LY294002(15 mmol/L)for 15 min before insulin administration(n=6);(5)Insulin+SP600125(a specific inhibitor for JNK)group:the hearts were pretreated with SP600125(10 mmol/L)for 15 min before insulinadministration(n=6);(6)Insulin+LY294002+SP600125 group:the hearts were pretreated with SP600125 and LY294002 for 15 minbefore insulin administration(n=6).Acta Physiologica Sinica,October 25,2007,59(5):651-659654from the heart in a graduated glass container at fixedintervals.All variables were recorded using a computeracquisition data system23.Making a slipknot with a 4-0 silk around the left anteriordescending(LAD)coronary artery produced myocardialischemia.After 30 min of ischemia,the slipknot wasreleased and the heart was reperfused for 2 h to measureinfarct size(IS)or 4 h for terminal deoxynucleotidyl nick-end labeling(TUNEL)and Western blot assays24.1.3 Determination of myocardial infarctionTo determine myocardial infarct,the LAD coronary arterywas ligated again after 2 h of reperfusion,and perfusedwith 3 mL/kg of 1%Evans blue to stain the area at risk(AAR).The heart was then removed and divided into 4-5slices of 1-2 mm width transversally.The Evans blue so-lution stained the perfused myocardium leaving the oc-cluded vascular bed uncolored.All the colored non-ischemictissue and the non-colored AAR were weighed to calculatethe percentage of AAR with respect to the whole leftventricle.To distinguish between viable ischemic and in-farcted tissue,the slices were incubated with TTC(10mg/mL,20 min,at 37 oC).In the presence of intact dehy-drogenase enzyme systems(normal myocardium),TTCformed dark red formation,while areas of necrosis lack-ing dehydrogenase activity were not stained,and TTC-stained tissue was weighed.IS was then calculated andexpressed as percentage of AAR25.1.4 Western blot analysisHeart tissue samples were lysed with lysis buffer.Aftersonication,the lysates were centrifuged;proteins were sepa-rated by electrophoresis on SDS-PAGE and transferredonto a polyvinylidene difluoride-plus membrane.After be-ing blocked with 5%fat-free milk,the immunoblots wereprobed with anti-p-JNK and anti-p-Akt antibodies over-night at 4 oC followed by incubation with the correspond-ing secondary antibodies at room temperature for 1 h.Theblots were visualized with ECL-plus reagent.p-JNK andp-Akt immunoblots were then stripped with strip buffer at50 oC for 30 min and reblotted for total JNK and Akt26.1.5 TUNEL assayHearts were washed by perfusion with ice-cold PBS for 1min followed by a fixation step with ice-cold 4%parafor-maldehyde in phosphate buffer(peristaltic pump flow:50mL/min for 10 min).After 24 h in fixation solution,heartswere cryosectioned at a thickness of 10 m and serialsections were stained with TUNEL reagents(Roche Co.,USA),according to the manufacturers instructions.Thedigoxigenin-conjugated dUTP was incorporated to the endsof DNA fragments by terminal deoxynucleotidyl transferase(TdT).The signal of TdT-mediated dUTP nick-end label-ing was then detected by an anti-fluorescein antibody con-jugation with alkaline phosphatase,a reporter enzyme thatcatalytically generates a red-colored product from vectorred substrate.For each slide,10 fields were randomly chosenand a defined rectangular field area(40 objective)wasused,a total of 200 cells per field were counted.Theapoptotic index(AI)was determined(i.e.,number of posi-tively stained apoptotic myocytes/total number of myocytescounted 100%)from a total of 80 fields per heart.Theassays were performed in a blinded manner26.1.6 Statistical analysisAll values were presented as meansSEM.Differences werecompared by ANOVA or Students t-test,when appropriate.P0.05 was considered to be statistically significant.All ofthe statistical tests were performed with the GraphPad Prismsoftware version 4.0(GraphPad Software,Inc.,San Diego,CA).2 RESULTS2.1 Cardiac hemodynamics in isolated hearts sub-jected to MI/RBaseline values for functional parameters were obtainedafter stabilization.In sham group,the left ventricular deve-loped pressure(LVDP),heart rate(HR)and instantaneousfirst derivation of left ventricular pressure(LVdp/dtmax)remained at a relatively stable level throughout the wholeMI/R procedure.In the control group,the left ventricularpressure showed a large scatter,which was frequentlyfound despite a large sample.Coronary occlusion caused asignificant decline in LVDP,HR and LVdp/dtmax.However,compared with the control,administration of insulin at 60 U/Linduced a significant increase in LVDP,HR and LVdp/dtmaxby 26%,25%,29%and 28%(n=6,all P0.05)(Fig.2),respectively.In insulin+LY294002 group,the values ofLVDP,HR,LVdp/dtmax decreased by 34