二附心胸外科中文评估有9.29mary vincy

non-canonicalautophagyincardiacmyocytesHaoYan*†,Jian-JunXu*,Wen-linLi†,Xiao-YuShi†,Yong-BingWu,Shu-QiangZhu*，XiangLong*andXiao-QiangZhang**Departmentofcardiacsurgery,TheSecondAffiliatedHospital,NanchangUniversity,Nanchang,jiangxi,330006,PRChina.†JiangxiProvincialKeyLaboratoryofMedicalbiologicalhightechnology,Nanchang,NanchangUniversity,Jiangxi,330006,PRChina.Correspondingauthors:Jian-JunXu: Departmentofcardiacsurgery,TheSecondAffiliatedHospital,nanchangUniversity,nanchang,jiangxi,330006,PRChina.: Fax: :Autophagy,DopamineReceptor,Raclopride,non-canonicalautophagyShorttitle:RacloprideInducedatypicalautophagyHeartdiseasestillistheleadingcauseofdeathininduatrializeworld[1],andadultcardiacmyocyteislong-livedandterminallydifferentiated，whichcannotbefullyreplacedbyitsproliferation，socatabolicpathwaysplayimportantroletomaintaincardiacmyocytecellularhomeostasis.Whilemostcellularproteindegradationismediatedthroughthreemajorpathways,includingcalpains,autophagyandubiquitin.Since1963deDuveintroducedthetermautophagy,autophagy(generallyreferredtomacroautophagy)befoundtodeliversthelong-livedproteinsanddamagedorganellestolysosomesfordegradation,andreplenishdepletedenergystoretomaintainingcellularhomeostasis,butexcesslevelsofautophagycanalsobetoxicandmayeveninducecelldeath,termedtypeIIprogrammedcelldeath.Cellkeepbasiclevelsautophagy,Autophagyalsocanbeinducedbycellstress,andmorethan30ATGgenescontroltheconservingdynamicprocessesofautophagy,however,recentlymanyreaserchreportatypicalautophagyoralternativemacroautophagyisindependentofULK1/2[2],LC3[3],l1[4]ormTOR[5].Thenon-canonicalautophagymechanismisstillmultiplicity,suchasbyCa2+-calpain-GsεpathwayorcAMP-Epac-PLC-ε-IP3pathway[5].DuringmanycardiacPathophysiologyofischemiaandreperfusion(I/R),hypertrophyandheartfailure,autophagyisexaminedtoinvolveinall.ButduringI/Rcardicautophagyeffectiscontroversial,duringischemiasomereaserchreportthatautophagyviaAMPKisbeneficial,butduringreperfusionautophagyviaJNKisdetrimental[6].sotheforms,levelorphaseofautophagymaydeterminewhetherautophagyisprotectiveordetrimentalinresponsetoheartDopamine(DA)isapotentcardiovascularneurotransmitter.Dopaminereceptorsplayanimportantroleincontrollingtheprincipalfunctionsofthecardiovascularcirculation,dopaminereceptorsweretraditionallydividedintotwofunctionalgroups,D1-like(D1andD5subtypes)andD2-like(D2,D3,andD4subtypes)families[6]D1-likeandD2-likereceptorshavebeenbothidentifiedinhumanandrathearts[7].D2-likereceptorsmayantagonizesD1-likereceptors,suchascoupledtotheadenylylcyclaseandL-typecalciumchannel.AlthoughDopamineinduceautophagyinneuroblastomacell[8]hasbeenreported，andDopamineReceptorAntagonistinvolveinindependentofmTORautophagy[9],theexactautophagymechanismsviaCa2+fluxordopaminereceptorsubtypesremaininconclisive,maybedependingondrugconcentration,timeofexposure,differentcelltypes.theaimofthepresentstudywastodeterminewhethertheD1--likeorD2-likeparticipateininduceautophagybydifferentmechanism.Inthisstudy,wehaveidentifiedanovelfunctionofracloprideasanautophagyinducerincardiacmyocytes.Wepresumedthatraclopride–inducedautophagyisnotincanonicalform.TheinvestigationconformstotheGuidefortheCareandUseofLaboratoryAnimals,publishedbytheNationalInstitutesofHealth(NIHPublicationNo.85-23,Revised1996).TheprocedureforcardiomyocyteisolationwasapprovedbytheSecondAffiliatedHospitalofUniversityofNanchang’sInstitutionalAnimalCareandUseCommittee.Primaryculturesofventricularcardiacmyocyteswerepreparedfrom1-2day-oldSprague-Dawleyrats(Animalsciencecenter,NanchangUniversity)byenzymaticdigestionwith0.25%trypsin(Invitrogen).NeonatalRatCardiomyocyteswerepreplatedfor2hrstoreducenon-myocytecontamination，thenwashingtoremovalerythrocytes，plated(2.0x106cells)incultureflasksandincubatedat37℃and5%CO2inhumidifiedatmosphere.CellswereculturedinDulbecco’smodifiedEagle’smedium(DMEM,Gibco,USA)with10%fetalbovineserum(FBS,SIGMA,USA)containingappropriateantibiotics，changedper24h.MostPrimaryculturesofventricularcardiacmyocytesbeatspontaneouslyinaconfluentmonolayer48–72hafterplating.Followingtreatment,NeonatalRatCardiomyocyteswerelysedinRIPAlysisbuffer(BeyotimeInstituteofBiotechnology,China).ProteinconcentrationsweredeterminedusingtheBCAproteinassayreagent(BeyotimeInstituteofBiotechnology,China).Equalproteinamounts(40-60μg)wereelectrophoresedinansodiumdodecylsulfatepolyacrylamidegel(8-15%)andtransferredtoapolyvinylidenefluoridemembrane,whichwasblockedwith2%albuminbovineⅤandincubatedwithrabbitanti-LC3antibody( ,USA)，Atg5，Atg7,P62(SIGMA,antibody(SANTACRUZ,USA)overnightat4ºC.MembraneswerethenincubatedwithHRP-conjugatedanti-rabbitIgG(Sigma,USA)for1hatroomtemperature.Allprimaryantibodieswereusedatadilutionof1:500-1000andthesecondaryantibodywasusedatadilutionof1:5000.BlotsweredevelopedwithECLreagent(Thermo,USA)andexposedtofilm(Kodak,USA).Cardiacmyocyteswerecentrifugedforcollectionafterdrugincubated24h,fixedwith4%glutaraldehyde,post-fixedwith1%osmiumtetroxide,wereimmersedin0.1Mofsodiumcacodylatebuffer(pH7.3)containing2.5%glutaraldehydefor4h,andthenrinsedwiththecacodylatebufferalone,followedwithpost-fixationby1%osmiumtetroxideinthecacodylatebufferfor1h.Thecellwereembeddedineponresinandcutinto600-nm-thickslicesbyanultramicrotome.Thesliceswerestainedwithuranylacetateandleadcitrate,thenexaminedwithaelectronmicroscope(Hitachi,H7500,Japan)at75kV.Afterdrugsincubated24h,Cardiacmyocyteswerefixedwith4.0%paraformaldehydefor30minandacetonefor10min，and0.2%triton100forpermeation,thenblockedin2%albuminbovineⅤimmunostainingblockingbufferfor1hatroomtemperature.Forduallabelingstudies,thefixedcellswereincubatedwithrabbitanti-LC3antibody(1:50)( ,USA)andmouseanti-α-actinantibody( ,USA)overnightat4ºCovernight.CellularproteinwasstainedbyincubatingwithFITCconjugatedanti-rabbitIgG(1:50)andTRITCconjugatedanti-mouseIgG(1:50)(Jackson,USA)for1hatroomtemperature.NucleiweredyedwithDAPI(Roche).Immunofluorescenceofcellswasvisualizedusingaconfocalmicroscopy(LeicaSP5,Mannheim,Freeintracellularcalciumconcentrations([Ca+2])inmyocardialcellswasdeterminedbyusingtheFluo-3/AM(sigma,USA)probe.Aftertreatedwithdrugs,NeonatalRatCardiomyocytewereincubatedwith5mol/LFluo-3/AMfor40minat37℃and5%CO2inhumidifiedatmosphere,washedthreetimeswithphosphate-bufferedsalineandfurtherincubatedfor20mininDMEM.allproceduresprotectfromlight.Dynamicchangesin[Ca+2]inmyocardialcellsweremeasuredafterstimulationwithdrugfor30minbymeasuringFluo-3fluorescence.Theimageswerecapturedusing40×oilimmersionobjective(488nmexcitation,522nmemission).TThechangeoffluorescentintensityinthecellscouldrepresentthechangeofintracellularCa2+concentration.Thefluorescenceintensitywasobservedineightrandomlychosencellsusingalaserscanningconfocalmicroscope(LeicaSP5,Mannheim,Germany)tocalculateaveragefluorescenceintensityforallcellsbyLASAFLite（LeicaSP5,Mannheim,Germany）.GeneSilencingwithsiRNA-rab7andsiRNA-ThesilencerselectsiRNAstargetingratrab9(Cat#: )andasilencerAccuTarget™Negativecontrol(Cat#:SN-1004)wereobtainedfromBioRP(Daejeon,SouthKorea).Fortransfection,cellswereplatedin35-mmtissueculturedishesat0.7x106cellsperdish,grownfor24h,thentransfectedwithsiRNAusingLipofectamineRNAiMAXandOpti-MEMinserum-freeandantibiotics-freemedium,Atg7siRNAandrab9siRNAatadoseof180pmolforperdish，accordingtothemanufacturer’sinstructions(Invitrogen).Threedaysaftertransfection,Atg7andRab9proteinsinthecellswerereducedtoalevelundetectablebyWesternblotysis(Fig.3A).indicatedbestrab9siRNAandAtg7siRNAinhibitautophagybyracloprideinduced.WeFoundtheDopamineDopaminedrugsinvolveinregulationofcardicPathophysiology,p62isanautophagysubstrate,andconversionofLC3-ⅠtoLC3-Ⅱisaimportantpartofautophagy，wetestedSKF-38393,Bromocriptine,SCH2390,racloprideandrapamycin(autophagyinducer0.1µM).neonatalratventricularmyocytesweretreatedwith40μMofeachDopamineDopamineReceptordrugsfor24hbeforeharvested.thelysateswereananlyzedbywesternblottingusinganti-LC3antbodytop62clearandanti-LC3antbodytodetecttheconversionofLC3-ⅠtoLC3-Ⅱ.Amongthe5drug,tworacloprideandrapamycinmoderayactivetop62clear,threeSKF-38393,BromocriptineandSCH2390wereshowednop62clear.similarlyintheconversionofLC3-ⅠtoLC3-Ⅱ,racloprideandrapamycintheconversionofLC3-ⅠtoLC3-Ⅱ,butSKF-38393,BromocriptineandSCH2390wereshowednotheconversionofLC3-ⅠtoLC3-ⅡweusedEMtoevaluatetheautophagyprocessesbyexaminingcharacteristicstructures.AsshowninFig.3,differenttypesofautophagosomes,whichcontainedheterogeneousorganellesrangingfrommitochondriatomultivesicularbodiessurroundedbyasequesteringmembrane,wereobservedinmyocytesunderraclopride(40μM)for24h(Fig.3c),rapamycin(0.1µM)for6h(Fig.3b)andcontrol(Fig.3a).BafilomycinA1wasoriginallyreportedtoinhibitautophagosome-lysosomefusionandlysosomalactivitybyraisinglysosomalpH,WeusedBafilomycinA1toinhibitlysosomalfunctiontomeasureautophagicfluxinneonatalratventricularmyocytescellculture,byimmunoblottingyses,asignificantincreaseinthenumberofLC3wasobservedinthecytosolafterthetreatmentwithraclopride,BafilomycinA1onlytraceactivitiesofLC3-Ⅱconversion,butBafilomycinA1reducedegradeofautolysosome,increaseLC3-Ⅱrecruit.Inthepresenceof200nMBafilomycinA1(2h),endogenousLC3-IIdramaticallyincreasedinracloprideinducedcells.TheamountofLC3-IIincellstreatedwithBafilomycinA1was0.8-foldhigherthaninuntreatedcontrolcellsat3h(Fig.4A).Theautophagyformationwasyzedbyanconfocalmicroscopyusinganti-LC3antibodyforLC3andanti-α-actinantibodytoidentificationforPrimarycardiacmyocytes，asignificantincreaseinthenumberofLC3wasobservedinthecytosolafterthetreatmentwithraclopride,recruitfromperipheraltoperinuclear,similarly,BafilomycinA1enchanceLC3plaqueaccumulation(Fig.4B).WeFoundtheDopamineDopamineReceptordrugsinvolveinregulationofcardicthatwefoundthatSKF-38393andbromocriptinestronglyincreased[Ca+2],SCH23390hadtenuityeffectinincreased[Ca+2].However,raclopridehadonlytraceactivitiesofdecreased[Ca+2](Fig.24E-BP1andp70S6KinasearedownstreamofmTOR,levelofPhospho-4E-BP1andPhospho-p70S6KinasearemTORactive,rapamycindown-regalationPhospho-4E-BP1andPhospho-p70S6Kinase,andinducedautophagydependentofmTOR,butDopamineDopamineReceptordrugsdoesnotchanglevelofPhospho-4E-BP1andPhospho-p70S6Kinase(Fig.1A).racloprideinducibleautophagyfluxwithoutdown-regalationPhospho-4E-BP1andPhospho-p70S6Kinase,itsautophagyisindependentofmTOR.racloprideenchanceautophagyInatime-dependentmanner(Fig.5).increasedconversionofLC3-ⅠtoLC3-Ⅱandp62clearfrom12h-24h，butATG5-ATG12didslightincrease.andrab9increaseduring0h-24h，butrab7increasein12h.Atg7IsRequiredforEfficientconventionalmacroautophagyFormationofAggresomes,Rab9IsRequiredforalternativemacroautophagy,butAtg7cannotchangeduring0h-24h，butrab9increasedat8h.weusesiRNAdown-reglateAtg7,0.4-foldlowerthanincontrolcells;wealsousesiRNArab9todown-reglaterab9,0.9-foldlowerthanincontrolcells,usedindicatedbestrab9siRNA(GUGCAUUUUAACCAACCAA)andAtg7siRNA(CAGACAAGAAGCUCCUUCU),wefoundthatsiRNAAtg7reduceracloprideconversionofLC3-ⅠtoLC3-Ⅱ,p62clearandincreasingrab9,butsiRNArab9didnotchangeracloprideconversionofLC3-ⅠtoLC3-Ⅱandp62clear(Fig.及教育水平有关[10]SNP[111-13],并与内质网应激产生的自噬等胞内转运也相关，敲除D2多巴胺受体小鼠表现出帕金森病特征[14]，在大鼠模型中发现D2多巴胺受体参与代谢调节15]，使D2多巴胺受体阻断剂raclopride通过增加血糖浓度来减少肌老化过程。多巴胺受体2激动剂bromocriptine可能参与了促进瓣膜病变[18,19]，还有对抗血自噬[10，被认为与受体对L-type钙离子通道功能有关。我们研究发现对于心肌，明显多于IP3Rs，发挥主导作用[21]。在缺血心肌病中“钙超载”被认为激活各种信号，而在缺血再灌注中[22DR1CDRThapsigargin，ionomycin,ATP,vitaminD3[Ca2+]升高增加细胞自也认为[25]Thapsigargin被认为是升高钙浓度引起内质网应急，阻断吞噬体与溶酶体结合L-typeCa2+通道，这也可能导致异常低钙离子转移到线粒体[5]。一般认为细胞内Ca2+水平升高通过calpain依赖mTOR，包括无所不在的表达的calpain1和calpain2[28]。L-typeCa2+通道开发激活Calpain或过表达激活calpain2阻断自噬和延迟清除体蛋白[5]，calpain激活剪切Atg5研究认为自噬并不完全依赖LC3-IconversiontoLC3-II[30]，这可能是其激活经典自噬的机制之一。yuyanishida等，在通常经典自噬之外还存在替代自噬途径macroautophagyAtg7缺乏细胞。然而，这些过程仍可以被PI3kinase阻断剂阻断，同时依赖于Ulk1和运调控子Vps34的激活也可以不依赖于钙离子[32]，其他方式的自噬，如Microautophagic于外界环境，脂[34]、等都影响自噬过程。多巴胺受体D2阻断剂作为阻断L-typeLloyd-JonesD,AdamsRJ,BrownTM,etal.Heartdiseaseandstrokestatistics--2010update:areportfromtheAmericanHeartAssociation.Circulation.2010.121(7):e46-e215.CheongH,LindstenT,WuJ,LuC,ThompsonCB.Ammonia-inducedautophagyisindependentofULK1/ULK2kinases.ProcNatlAcadSciUSA.2011.108(27):11121-6.NishidaY,ArakawaS,FujitaniK,etal.DiscoveryofAtg5/Atg7-independentalternativemacroautophagy.Nature.2009.461(7264):654-8.ZhuJH,HorbinskiC,GuoF,WatkinsS,UchiyamaY,ChuCT.Regulationofautophagybyextracellularsignal-regulatedproteinkinasesduring1-methyl-4-phenylpyridinium-inducedcelldeath.AmJPathol.2007.170(1):75-86.WilliamsA,SarkarS,CuddonP,etal.NoveltargetsforHuntington'sdiseaseinanmTOR-independentautophagypathway.NatChemBiol.2008.4(5):295-305.MatsuiY,TakagiH,QuX,etal.Distinctrolesofautophagyintheheartduringischemiaandreperfusion:rolesofAMP-activatedproteinkinaseandBeclin1inmediatingautophagy.CircRes.2007.100(6):CavallottiC,NutiF,BruzzoneP,ManconeM.Age-relatedchangesindopamineD2receptorsinratheartandcoronaryvessels.ClinExpPharmacolPhysiol.2002.29(5-6):412-8.Gimenez-XavierP,FranciscoR,SantidrianAF,GilJ,AmbrosioS.EffectsofdopamineonLC3-IIactivationasamarkerofautophagyinaneuroblastomacellmodel.Neurotoxicology.2009.30(4):658-65.ZhangL,YuJ,PanH,etal.Smallmoleculeregulatorsofautophagyidentifiedbyanimage-basedhigh-throughputscreen.ProcNatlAcadSciUSA.2007.104(48):19023-8.Keltikangas-JarvinenL,ElovainioM,KivimakiM,RaitakariOT,ViikariJS,LehtimakiT.DopaminereceptorD2geneTaq1A(C32806T)polymorphismmodifiestherelationshipbetweenbirthweightandeducationalattainmentinadulthood:21-yearfollow-upoftheCardiovascularRiskinYoungFinnsstudy.Pediatrics.2007.120(4):756-61.SangrajrangS,SatoY,SakamotoH,OhnamiS,KhuhapremaT,YoshidaT.Geneticpolymorphismsinfolateandalcoholmetabolismandbreastcancerrisk:acase-controlstudyinThaiwomen.BreastCancerResTreat.2010.123(3):885-93.GemignaniF,LandiS,MorenoV,etal.PolymorphismsofthedopaminereceptorgeneDRD2andcolorectalcancerrisk.CancerEpidemiolBiomarkersPrev.2005.14(7):1633-8.MurphyG,CrossAJ,SansburyLS,etal.DopamineD2receptorpolymorphismsandadenomarecurrenceinthePolypPreventionTrial.IntJCancer.2009.124(9):2148-51.TinsleyRB,ByeCR,ParishCL,etal.DopamineD2receptorknockoutmicedevelopfeaturesofParkinsondisease.AnnNeurol.2009.66(4):472-84.WongKJ,WojnickiFH,CorwinRL.Baclofen,raclopride,andnaltrexonedifferentiallyaffectintakeoffat/sucrosemixturesunderlimitedaccessconditions.PharmacolBiochemBehav.2009.92(3):528-36.BalthazarCH,RibeiroOAJr,MarubayashiU,dosRAM,CoimbraCC.Chronictreatmentwithbromocriptinemodifiesmetabolicadjustmentsinresponsetorestraintstressinrats.AutonAutacoidPharmacol.2007.27(2):GomezMJ,RousseauG,NadeauR,BerraR,FloresG,SuarezJ.FunctionalandautoradiographiccharacterizationofdopamineD2-likereceptorsintheguineapigheart.CanJPhysiolPharmacol.2002.80(6):TanLC,NgKK,AuWL,LeeRK,ChanYH,TanNC.Bromocriptineuseandtheriskofvalvularheartdisease.MovDisord.2009.24(3):344-9.StephensJW,PriceDE,IonescuA.Dopamineagonistsandvalvularheartdisease.NEnglJMed.2007.356(16):1676;authorreply1678-80.LiH,ShiS,SunYH,etal.DopamineD2receptorstimulationinhibitsangiotensinII-inducedhypertrophyinculturedneonatalratventricularmyocytes.ClinExpPharmacolPhysiol.2009.36(3):312-8.HarrMW,DishorstCW.Apoptosisandautophagy:decodingcalciumsignalsthatmediatelifeordeath.ColdSpringHarbPerspectBiol.2010.2(10):a005579.LiHZ,HanLP,JiangCM,etal.Effectofdopaminereceptor1onapoptosisofculturedneonatalratcardiomyocytesinsimulatedischaemia/reperfusion.BasicClinPharmacolToxicol.2008.102(3):329-36.XiaoJF,WangCY,HuangYP,etal.N-n-butylhaloperidoliodidepreservescardiomyocytecalciumhomeostasisduringhypoxia/ischemia.CellPhysiolBiochem.2011.27(5):433-42.Hoyer-HansenM,BastholmL,SzyniarowskiP,etal.Controlofmacroautophagybycalcium,calmodulin-dependentkinasekinase-beta,andBcl-2.MolCell.2007.25(2):193-205.GanleyIG,WongPM,GammohN,JiangX.Distinctautophagosomal-lysosomalfusionmechanismrevealedbythapsigargin-inducedautophagyarrest.MolCell.2011.42(6):731-43.KhanMT,JosephSK.RoleofinositoltrisphosphatereceptorsinautophagyinDT40cells.JBiolChem.2010.285(22):16912-20.CardenasC,MillerRA,SmithI,etal.EssentialregulationofcellbioenergeticsbyconstitutiveInsP3receptorCa2+transfertomitochondria.Cell.2010.142(2):270-83.GollDE,ThompsonVF,LiH,WeiW,CongJ.Thecalpainsystem.PhysiolRev.2003.83(3):731-DemarchiF,BertoliC,CopettiT,etal.Calpainisrequiredformacroautophagyinmammaliancells.JCellBiol.2006.175(4):595-605.Gimenez-XavierP,FranciscoR,PlatiniF,PerezR,AmbrosioS.LC3-IconversiontoLC3-IIdoesnotnecessarilyresultincompleteautophagy.IntJMolMed.2008.22(6):781-5.ZafarS,vonAN,OellerichM,etal.ProteomicsapproachtoidentifytheinteractingpartnersofcellularprionproteinandcharacterizationofRab7ainteractioninneuronalcells.JProteomeRes.2011.10(7):3123-35.YanY,FlinnRJ,WuH,SchnurRS,BackerJM.hVps15,butnotCa2+/CaM,isrequiredfortheactivityandregulationofhVps34inmammaliancells.BiochemJ.2009.417(3):747-55. A,SchwarzH,MayerA.MicroautophagicvacuoleinvaginationrequirescalmodulininaCa2+-independentfunction.JBiolChem.2005.280(39):33289-97.MoRH,ZaroJL,OuJH,ShenWC.EffectsofLipofectamine2000/siRNAComplexesonAutophagyinHepatomaCells.MolBiotechnol.2011.Fig.1.DopamineDopaminedrugsinduceDopamineDopaminedrugswerescreenedforautophagyinducibleactivity.cardiacmyocyteswereincubatedwithindicatedDopamineDopaminedrugs(40.mM)for24hand（0.1μM）rapamycinfor8h,andconversionofLC3