Myocardial Infarction Induces Cardiac Fibroblast Transformation within Injured and Noninjured Regions of the Mouse Heart - Shah et al. -

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pubs.acs.org/jprArticleMyocardialInfarctionInducesCardiacFibroblastTransformationwithinInjuredandNoninjuredRegionsoftheMouseHeartHaisamShah,*AlisonHacker,DylanLangburt,MichaelDewar,MeghanJ.McFadden,HangjunZhang,UrosKuzmanov,Yu-QingZhou,BilalHussain,FahadEhsan,BorisHinz,AnthonyO.Gramolini,*andScottP.Heximer*CiteThis:J.ProteomeRes.2021,20,2867−2881ReadOnlineACCESSMetrics&MoreArticleRecommendations*sıSupportingInformationABSTRACT:Heartfailure(HF)isassociatedwithpathologicalremodelingofthemyocardium,includingtheinitiationoffibrosisandscarformationbyactivatedcardiacfibroblasts(CFs).AlthoughearlyCF-dependentscarformationhelpspreventcardiacrupturebymaintainingtheheart’sstructuralintegrity,ongoingdepositionoftheextracellularmatrixintheremoteandinfarctregionscanreducetissuecompliance,impaircardiacfunction,andaccelerateprogressiontoHF.Inourstudy,weconductedmassspectrometry(MS)analysistoidentifydifferentiallyalteredproteinsandsignalingpathwaysbetweenCFsisolatedfrom7dayshamandinfarctedmurinehearts.Surprisingly,CFsfromboththeremoteandinfarctregionsofinjuredheartshadawidenumberofsimilarlyalteredproteinsandsignalingpathwaysthatwereconsistentwithfibrosisandactivationintopathologicalmyofibroblasts.Specifically,proteinsenrichedinCFsisolatedfromMIheartswereinvolvedinpathwayspertainingtocell−cellandcell−matrixadhesion,chaperone-mediatedproteinfolding,andcollagenfibrilorganization.Theseresults,togetherwithprincipalcomponentanalyses,providedevidenceofglobalCFactivationpostinjury.Interestingly,however,directcomparisonsbetweenCFsfromtheremoteandinfarctregionsofinjuredheartsidentified15differentiallyexpressedproteinsbetweenMIremoteandMIinfarctCFs.Elevenoftheseproteins(Gpc1,Cthrc1,Vmac,Nexn,Znf185,Sprr1a,Specc1,Emb,Limd2,Pawr,andMcam)werehigherinMIinfarctCFs,whereasfourproteins(Gstt1,Gstm1,Tceal3,andInmt)werehigherinMIremoteCFs.Collectively,ourstudyshowsthatMIinjuryinducedglobalchangestotheCFproteome,withthemagnitudeofchangereflectingtheirrelativeproximitytothesiteofinjury.KEYWORDS:cardiovasculardisease,cardiacfibroblasts,myofibroblasts,cardiacfibrosisandremodeling,myocardialinfarction,massspectrometryDownloadedviaBUTLERUNIVonMay16,2021at16:38:17(UTC).■INTRODUCTIONFollowingMI,theinitialmyofibroblast-dependentformationoffibroticscartissuepreservesthestructuralintegrityoftheHeartdiseaseisoftenassociatedwithpathologicalremodelingmyocardium,preventstheriskofcardiacrupture,andisaofthemyocardium,reductionofhearttissuecompliance,and181−3largelycardioprotectiveprocess.ChronicdepositionandSeehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.acceleratedprogressiontoheartfailure(HF).ThesematurationofECMcomponentscancontributetoprogressiveprocessesaremediated,inpart,bycardiacfibroblasts(CFs).4,5Inthehealthyheart,theCFisanabundantandtissuestiffening,noncompliantscarformation,andcardiac7,19dysfunction.Thisprocess,knownascardiacfibrosis,essentialcelltypethatplaysanimportantroleincardiacdevelopmentandthemaintenanceofextracellularmatrixprovokesfurtherpathologicalchangesthatculminateinleft(ECM)andelectrophysiologicalhomeostasis.6Duringische-ventricularstrain,chamberdilatation,cardiomyocytehyper-micinjurieslikeamyocardialinfarction(MI),however,trophy,cellularapoptosis,andultimately,thedevelopmentofresidentCFsdifferentiateintoanactivemyofibroblastHF.Despitetheirknownpathophysiologicalimportanceinstate.7−10WhilequiescentCFsarecharacterizedbytheheartdisease,theCFremainsrelativelyuncharacterized.expressionofseveralmarkers,includingvimentinandtype1Likewise,veryfewstudieshavecharacterizedtherolesofcollagen,themyofibroblastexhibitsincreasedlevelsofα-11Received:February3,2021smoothmuscleactin(α-SMA)-containingstressfibers.Additionally,inthisactivatedstate,myofibroblastsbegintoPublished:March31,2021secreteanddepositexcessiveamountsofECMproteins,12−14includingcollagenandfibronectin.ThesechangesintheCFphenotypefollowinganinjurycanultimatelycontributeto15−17thedevelopmentofscartissueintheheart.©2021AmericanChemicalSocietyhttps://doi.org/10.1021/acs.jproteome.1c000982867J.ProteomeRes.2021,20,2867−2881

1JournalofProteomeResearchpubs.acs.org/jprArticledifferentCFpopulationsafteracuteischemicinjuryandmLdispaseII(SigmaCat#:D4693)inDulbecco’sphosphate-throughoutthedevelopmentofHF.Lastly,therearefewbufferedsaline(dPBS)supplementedwith0.9mmol/LCaCl2clinicalinterventionsthatspecificallytargetmyofibroblastfor45minat37°Cwithperiodicmixingevery15min.Thefunctiontopromoteimprovedcardiacfunctionfollowingandigestwasthenfilteredthrougha70μmmeshinto25mLofischemicinsult.1×dPBS.Next,thedigestwascentrifugedfor7minat500gatExperimentalanimalmodelsofMIareaneffectivetoolfor4°C.Resultingpelletswereresuspendedinfibroblastmedia,accuratelystudyingthespatiotemporalchangesofCFcontaining1%L-glutamine(ThermoCat#:25030-081),1%populationsduringcardiacrepair.Specifically,bothMIinjurypenicillin−streptomycin(ThermoCat#:15140-122),and10%andsurgicalligationoftheleftanteriordescendingcoronaryfetalbovineserum(ThermoCat#10437-028)inDulbecco’sartery(LAD)leadstotherapiddeathofcardiomyocytesinthemodifiedEagle’smedia(ThermoCat#:11965-092),andischemicleftventricle(LV)andtheprogressiveformationofplatedfor2honastandardtissueculture-treatedPetridish.distinctmyocardialzones,namely,theinfarctandremoteFollowingthe2hpreplate,oldmediawasaspiratedand20−22regions.Theinfarctzone,definedastheregiondirectlyreplacedwithfreshfibroblastmedia.Themediawasaffectedbytheischemicinsult,mainlyundergoesreplacementsubsequentlyreplacedeveryotherdayuntiltheendofthefibrosis,whichoccursinresponsetothelossofcardiomyocytesexperiment.Allexperimentswereconducted1,3,or5days23,24andviabletissue.Thisreplacementfibrosisinitiallyaftertheinitialpreplate.preventsventricularrupture,butifleftunmanaged,itcanImmunoblottingcontributetothedevelopmentofanECM-rich,noncompliantCFswerelysedwithRIPAlysisbuffer,containing50mMTris-scarthatworsenscardiacfunctionovertime.Incontrast,theHCl(pH8.0),150mMNaCl,1%NP-40(BioChemikaCat#remoteregion,whichisdefinedasthenonischemicregion,74385),0.5%sodiumdeoxycholate(SigmaCat#:D6750),visuallyresemblesthehealthymyocardium,butstilldevelops25−280.5%sodiumdodecylsulfate(SDS),1%sigmaproteasereactivefibrosis.Interestingly,studiessuggestthatinhibitor,andgentlesonication.Proteinconcentrationwasreplacementfibrosisintheinfarctzoneandreactivefibrosisdeterminedusingthebicinchoninicacidassay(BCA)intheremote,nonischemicareaarebothcriticalregulatorsof(ThermoCat#:23227).Equalamountsofproteinwerechronic,pathologicalcardiacremodeling,progressivecardiac29−31separatedbythemolecularweightviagelelectrophoresis.dysfunction,andthedevelopmentofHFpost-MI.ProteinswerethentransferredtoanitrocellulosemembraneTherefore,webelieveitisimportanttostudythemolecular(BioRadCat#1620115)andblockedfor1hwithTris-characteristicsandphenotypicdifferencesbetweenCFsinthebufferedsaline0.1%Tween20(TBS-T)in5%skimmilkremoteandinfarctregionstoidentifyhowbothpopulations(BioShopCat#:SKI400).Primaryantibodiesweredilutedcontributetopost-MIcardiacremodeling.withTBS-Tcontaining5%bovineserumalbumin(BioShop■Cat#ALB001)andincubatedwithmembranesovernightat4MATERIALSANDMETHODS°C.Theprimaryantibodiesusedincludeanti-vimentin(CellExperimentalAnimalssignalingCat#:5741S,1:1000dilution)andanti-α-SMA(AbcamCat#:ab5694,1:1000dilution).MembraneswereAllanimalworkwasconductedunderguidelinesestablishedbywashedinTBS-TfollowedbyincubationwiththehorseradishtheCanadianCouncilonAnimalCare(CCAC).Allperoxidase-linkedsecondaryantibody(CellsignalingCat#:experimentalprotocolswereapprovedbytheInstitutional7074S),diluted1:3000withTBS-Tin5%skimmilk,for1hatAnimalCareandUseCommitteeattheUniversityofTorontoroomtemperature.ThesignalwasdetectedusingtheandconductedinaccordancewithCanadiananimalprotectionSuperSignalWestPicochemiluminescentsubstrate(Thermolaws.Allanimalswerehousedunderstandard14:10hlight−Cat#:32106).darkcycleswithadlibitumaccesstowaterandchow.Amixture(roughly1:1)ofmaleandfemale10−16weekoldImmunofluorescenceadultC57Bl/6micewereusedforthestudiesdescribedbelow.CFswereisolatedfromC57Bl/6miceandinitiallyplacedonaLeftAnteriorDescending(LAD)CoronaryArteryLigationstandardtissueculture-treatedPetridishfor2h.Followingthe(SurgicallyInducedMI)2hpreplate,cellsweretransferredtoacollagen-coated,glass-bottomMatTekPetridish(MatTekCat#:P35GCol-1.5-14C)MIwasinducedusingsurgicalligation,aspreviously32andculturedfor1,3,or5days.Sampleswerefixedin4%described.Briefly,a7-0Prolenesuturewaspassedthroughparaformaldehyde(PFA)(SigmaCat#:P6148)in1×calciumthemyocardiumbeneaththeleftanteriordescendingcoronaryandmagnesium-freephosphate-bufferedsaline(PBS)for10arteryapproximately2mmbelowtheleftatriumandtheminatroomtemperatureandpermeabilizedonicewith0.1%suturecutandtiedofftocompletethearteryligation.Sham-TritonX-100(BioShopCat#:TRX777)in1×PBSfor10min.operatedanimalsunderwentthefullsurgicalprocedureexceptNext,thesampleswereblockedfor1hatroomtemperaturethe7-0suturewasnottiedoffafterpassingundertheLAD.Allwith5%bovineserumalbuminin1×PBS,andincubatedwithmicewereallowedtorecoverfor1week(7days)beforetheprimaryantibodyovernightat4°C.Theprimaryphenotypingandfibroblastisolationprocedureswerecarriedantibodiesusedincludeanti-vimentin(CellsignalingCat#:out.5741S,1:100dilution)andanti-α-SMA-Cy3(SigmaCat#:AdultMurineCFIsolationandCultureC6178,1:100dilution).Thefollowingday,sampleswereOurCFisolationprotocolwasadaptedfromastrategyusedbyincubatedinthedarkwithasecondaryfluorophore-conjugated33Pintoetal.(2016).Specifically,thewholeheartofanadultantibodyfor1hatroomtemperature(ifrequired).TheC57Bl/6mousewasexcised,andtheatriaandatrioventricularsecondaryantibodiesusedincludegoatantirabbitIgGAlexavalvesweresurgicallyremoved.TheventriclesandseptumFluorophore488(ThermoCat#:A11034,1:1000dilution).weremincedinto1mmpiecesanddigestedwith2mg/mLCellsweresubsequentlytreatedwith2μg/mLHoechst33342collagenasetypeII(WorthingtonCat#:LS004176)and2mg/for10minatroomtemperaturetovisualizenuclei.Imaging2868https://doi.org/10.1021/acs.jproteome.1c00098J.ProteomeRes.2021,20,2867−2881

2JournalofProteomeResearchpubs.acs.org/jprArticlewasperformedusinganOlympusconfocalmicroscoperesuspendedin120μLof100mMABC.AfractionoftheFV3000.sampleswasusedforproteinquantificationusingBCA.Approximately6μgofproteinfromeachsamplewasEchocardiographyDataAcquisitiontransferredtoanewEppendorftubeandtoppedupto100Ahigh-frequencyultrasoundimagingsystem(Vevo770orμLwith100mMABC.Proteinswerereducedwith5mMVevo3100)(FUJIFILMVisualSonicsInc.)witha25or30dithiothreitol(DTT)at55°Cfor30minandalkylatedintheMHztransducerwasusedtoevaluatethestructureandcardiacdarkwith15mMiodoacetamideatroomtemperaturefor30functionof7dayshamandpost-MImice.Theprocedureusedmin.Calciumchloride(2mM)wasaddedwith1μgofthe34hasbeenpreviouslydescribedindetail.One-dimensionalMS-gradetrypsin/Lys-Cmix(Promega).Digestionwas(M-mode),two-dimensional(B-mode),andpulse-waveallowedtoproceedovernightat37°Cbeforeanother0.5μgDopplerwererecorded.TheLVwallthicknessatthemiddleoftrypsin/Lys-Cwasaddedandallowedtoproceedforansegment,LVend-diastolicdiameter(LVEDD),andLVend-additional3h.Digestionwasquenchedwiththeadditionofsystolicdiameter(LVESD)weremeasuredinmm.TheLV0.1%formicacid,andtheresultingpeptideswereisolatedandfractionalshortening(LVFS),fractionalareachange(FAC),desaltedusingOMIXC18solid-phaseextractiontips(Agilent)strokevolume(SV),heartrate(HR),andcardiacoutput(CO)asperthemanufacturer’sinstruction.Samplesweredriedinawerealsomeasured.vacuumconcentratorandstoredat−80°CuntilliquidEchocardiographyDataAnalysischromatography−massspectrometry(LC-MS)analysis.Throughtheleftparasternalacousticalwindow,theLVinitsLiquidChromatography−MassSpectrometryAnalysislongaxisviewwasvisualizedusingtwo-dimensional(B-mode)CFswereanalyzedintechnicalduplicateonanEASY1200-imagingtoobservetheoverallmorphologyoftheLV.UsinganLCcoupledtoaQExactivePlusmassspectrometerwithafunctionmodalitycalledECG-basedkilohertzvisualizationNanosprayFlexIonSource(ThermoScientific).Dried(EKV),acineloopofLVwithaframerateupto1KHzwaspeptideswerereconstitutedin24μLof5%(v/v)formiccreatedforonecardiaccycle,andtheend-diastolicandpeak35acidinhigh-performanceliquidchromatography(HPLC)-systolicframeswereidentified.TheendocardiumoftheLVgradewaterand5μLwasloadedontoa10cm,75μmi.dwastracedtomeasuretheend-diastolicareaandpeaksystolicanalyticalcolumn(Reprosil-PurBasicC18,3μm,100Å;Dr.area.TheFACwascalculatedasthedifferencebetweentheMaischHPLC,Ammerbuch,Baden-Württemberg,Germany)end-diastolicareaandpeaksystolicarea,dividedbytheend-andseparatedusingalinear90mingradientfrom2−43%Batdiastolicarea,andexpressedasapercentage.Furthermore,theaflowrateof250nL/min,whereAwas0.1%formicacidinLVwasdividedintothreeequalsegmentsbetweentheapexHPLC-gradewaterandBwas80%acetonitrilewith0.1%andmitralorifice(e.g.,theapical,middle,andbasalsegments)formicacid.DatawasacquiredinTop10data-dependentmodeinthelongitudinalaxisview.UndertheguidanceofB-modewithonefullMS1scanatR=70000from400−1500m/zimaging,M-moderecordingwasmadefromthecenterofeachwithanautomaticgaincontrol(AGC)targetof1E6andasegment.TheM-modetracefromthemiddlesegmentwasmaximuminjectiontimeof100ms.TheMS1surveyscanwasusedtomeasuretheleftventricularanteriorandposteriorwallfollowedby10data-dependentMS2scansatR=17500,anthicknessesandchamberdimensionsatpeaksystoleandend-isolationwindowof1.3m/z,AGCof5E5,andamaximumdiastole.Fortheapicalandbasalsegments,onlytheLVESDinjectiontimeof55ms,usinganormalizedhigh-energyandLVEDDweremeasured.TheLVFSwascalculatedasthecollision-induceddissociation(HCD)settingof28.Only2+,differencebetweenLVEDDandLVESD,dividedbythe3+,4+,and5+precursorchargestateswereincluded,withtheLVEDD,foreachofthethreesegments.Finally,thediameterdynamicexclusionsetto30s.TechnicalreplicateswereoftheaorticannulusandtheDopplerflowvelocityatthecombinedandRAWfilessearchedusingtheMaxQuantmiddleoftheaorticorificeweremeasuredtocalculatetheLV,version1.6.0.1againsttheUniProtmouseproteomesequenceSV,andCOforevaluatingtheglobalfunctionoftheLV.database(updatedAugust22,2017)withtheprecursormassSamplePreparationforMassSpectrometrytoleranceat6ppmandMSMStoleranceat20ppm.ToRemoteandinfarcttissuefromshamcontrolandMI-operatedminimizemissingquantitativevalues,the“matchbetweenC57Bl/6micewereisolated7daysaftertheinitialsurgeryandruns”featurewasenabled.Carbamidomethylationofcysteinedigested,asdescribedearlier.TogeneratesufficientCFproteinwasincludedasafixedmodificationandvariablemodificationslysatesamplesforanalysis,eachbiologicalreplicatecontainedwereselectedformethionineoxidation,proteinamino-pooledsamplesoftheremoteorinfarctregiontissuefrom4toterminalacetylation,andasparagineandglutaminedeamida-5mice/samples.CD31+endothelialcellsandCD45+tion.Thefalsediscoveryrate(FDR)wassetto1%usingaleukocyteswereremovedusingamagneticcellsorter(Miltenyireversetarget-decoystrategy,andthemaximummissedBiotecCat#:130-097-418andCat#:130-052-301)beforecleavagesallowedwere2.AllRAWMSdataandsearchresultspreplatingthenegativefractionsofthetissuedigest.AfterhavebeenuploadedtoMASSIVEandareavailableundertheovernightculture,plateswerewashedthreetimeswith10mLaccessioncode:MSV000085454.Proteinswerefilteredusingof1×PBSfor5mineach.About1mLof50%(v/v)2,2,2-thePerseussoftwareversion1.6.1.3toexcludecontaminants,trifluoroethanol(TFE)(SigmaCat#:T63002)in100mMdecoys,andproteinsidentifiedonlybythesite.Label-freeammoniumbicarbonate(ABC)(SigmaCat#:A6141)wasquantification(LFQ)valuesgeneratedbyMaxQuantwerelog2addedtotheplatesandcellswerescrapedoff.Tolysecellstransformed,andidentificationswerefilteredforaminimumofeffectively,samplesweresonicatedforsix15scyclesata20%threevalidvaluesforatleastoneoftheexperimentalgroups.maximumsetting.Lysateswereincubatedat60°Cfor2htoMissingvalueswereimputedfromanormaldistributionusingdenature,extract,andsolubilizeproteins,andcentrifugedatawidthof0.3anddownshiftof1.8standarddeviations.All13000gfor5minat4°Ctoremoveaggregatesandcelldebris.heatmapsweregeneratedusingnormalizedlog2(LFQ)valuesSamplesweredriedusingaspeedvacuumovernightandacrossallsamplesforeachprotein.Differentialprotein2869https://doi.org/10.1021/acs.jproteome.1c00098J.ProteomeRes.2021,20,2867−2881

3JournalofProteomeResearchpubs.acs.org/jprArticleexpressionwasdeterminedusingthemultisampleanalysisofvariance(ANOVA)test,withapermutation-basedFDR<0.05andafoldchangeparameter(S0)of1.0.Binarycomparisonswerealsomadeusingthesignificanceofmicroarrayanalysis,35withanFDRthresholdof<0.05andS0of1.0.Thegeneontology(GO)termpathwayanalysiswasperformedusingtheDatabaseforAnnotation,Visualization,andIntegratedDiscovery(DAVID)tool(version6.8).StatisticalAnalysisAllexperimentswereperformedinatleastbiologicaltriplicate.Forallnon-MSbinarycomparisons,statisticalanalysiswasconductedusinganunpaired,two-tailedt-testwithap-valueof<0.05beingconsideredassignificant.Allnon-MScomparisonswithmorethanthreeconditionswereconductedusingaone-wayANOVAandTukey’smultiplecomparisontestwithap-valuecutoffof<0.05beingconsideredsignificant.Alldatawereexpressedasmean±standarddeviation.ForMSdata,differentiallyexpressedproteinsbetweenatleasttwoofthefoursampleswereidentifiedusingamultisampleANOVA,withapermutation-basedFDRcutoffof<0.05andafoldchangeparameter(S0)of1.0.ForMSbinarycomparisons(volcanoplots),weusedthesignificanceanalysisofmicro-arraysdevelopedbyTusheretal.(2001)withthesignificancedeterminedusingapermutation-basedFDRcutoffof<0.0536andanS0=1.0.FortheGOtermanalysis,thesignificanceFigure1.Immunofluorescenceimagingrevealedthatthemajorityof(EASEscore<0.05)wasdeterminedusingaFischerExacttest.ourcellswerevimentin+after1,3,and5daysinculture.Specifically,TheEASEscoreisaFisherExacttest-modifiedp-value.Dataourdatashowedthatapproximately96%oftheisolatedcellswerevimentin+onday1,98%werevimentin+positiveafter3days,andwereanalyzedusingGraphPadPrism(version6.01).Volcano100%werevimentin+after5daysinculture.Threeindependentplots,heatmaps,andPCAplotswereproducedinPerseus(version1.6.2.1).biologicalreplicateswereusedfortheexperiment(n=3).Foreachbiologicalreplicate,wetookfiveimageswithapproximately10−30■cellsperfield.Imagingwasperformedusinga40×objectiveonaRESULTSLeicaTCSSP8confocalmicroscope.Thescalebarrepresents50μm.OptimizationofCFIsolationandCultureThegraphdepictsmean±standarddeviation.SinceCFsaremoreadhesivethanothercardiaccelltypes,theypreferentiallyadheretotheplateduringthepreplatingstep.SMA,asensitivemarkerofthefibroblasttomyofibroblastThepurityofourpreplatedCFcultureswasassessedbytransitioninculture,increasedfromapproximately6%ondaydeterminingtheproportionofisolatedcellsthatexpressed1,to53%onday3,and71%onday5followingpreplatingandvimentin,atypeIIIintermediatefilamentproteinthatiswidelyCFculture(Figure2A).Immunoblottingalsorevealedthatα-usedasafibroblastmarker.ImmunofluorescenceimagingSMAexpressionlevelsweremuchhigherin3and5day-performedonCFsculturedfor1,3,and5daypostisolationculturedCFs,comparedto1day(overnight)-culturedCFsshowedthatthemajorityofcellsfromourpreplatedcultures(Figure2B;SupplementalFigure1).Thesefindingsindicatewerevimentin+(Figure1).Moreprecisely,96%ofcellswerethatalowbaselinelevelofα-SMAexpressionexistsinCFsvimentin+onday1,98%werepositiveonday3,and100%fromhealthyheartsafter1dayofculture,butthatextendedwerepositiveonday5,indicativeofanisolationprotocolthattimeincultureincreasesitsexpressionlevelsdramatically.producedhighlypurifiedpreparationofCFsfromintactAccordingly,the1daytimepointwasselectedastheCFcardiactissue.isolationintervalwiththeleasteffectonmyofibroblastCharacterizationofCFChangesOverTimeinCulturedifferentiationasdefinedbyα-SMAinduction.PreviousstudieshaveshownthatCFsaremechanosensitiveCardiacFunction7daysFollowingShamorLAD-Ligationandwilldifferentiateintoamoreactivated(myofibroblast)(MI)SurgerystatewhentheyareculturedonartificiallystifftissuecultureBeforeproceedingtomassspectrometryexperiments,37,38substrates.Forexample,normalplastictissueculturePetriechocardiographywasusedtoverifyifLAD-ligationsurgeriesdishesareseveralordersofmagnitudestifferthanthehealthyinducedcardiacinjurythatwasconsistentwithanacuteMI.39,40humanheart.Thus,weperformedaseriesofpreplatingEchocardiographyofsham-operatedandLAD-ligatedmicewasandculturetime-coursestudiestocharacterizeisolationperformed7daysaftertheinitialsurgery.One-dimensionalM-conditionsthatprovidedsufficientmaterialformassmodeanalysisrevealedthattheleftventricleanteriorwallwasspectrometryanalysisbutretainedaCFphenotypethatmostsignificantlythinnerintheMIhearts,relativetoshamcontrols,closelyresembledtheirinvivostate.whereastheposteriorwallthicknessremainedunaffectedAccordingly,CFswereculturedfor1,3,and5days(SupplementalFigure2A).Furthermore,theLVEDDandfollowingisolation.Cellswereeitherfixedforimmunofluor-LVESDweresignificantlyelevatedattheapical,middle,andescenceorlysedforimmunoblotting.ImmunofluorescencebasalsegmentsoftheheartinmicethatunderwentLADexperimentsshowedthattheproportionofcellsexpressingα-ligation(SupplementalFigure2B).Bothwallthinningand2870https://doi.org/10.1021/acs.jproteome.1c00098J.ProteomeRes.2021,20,2867−2881

4JournalofProteomeResearchpubs.acs.org/jprArticleFigure2.Proportionofα-SMA+CFsandtheproteinexpressionofα-SMAinCFsincreasessignificantlyovertimeinculture.Specifically,wefoundthattheproportionofα-SMA+cellsincreasedovertimeinculture(A).Threeindependentbiologicalreplicateswereusedfortheexperiment(n=3).Foreachbiologicalreplicate,wetookfourimageswithapproximately50−100cellsperfield.Imagingwasperformedona20×objectiveonanOlympusconfocalmicroscopeFV3000.Thescalebarrepresents50μm.Immunoblottingdemonstratedthattheexpressionofα-SMAincreasessignificantlyfromday1todays3and5inculture(p<0.05)(B).Vimentinwasusedasaloadingcontrol.Notably,weobservedadoublebandfor41vimentin,whichcanbeattributedtopost-translationalmodifications.Theexperimentwasrepeatedforatotaloffivetimes(n=5).BandswerequantifiedusingFIJI.Inbrief,meangrayvaluesforα-SMAwerenormalizedtovimentinforeachsample.Aone-wayANOVAusingTukey’smultiplecomparisontestwasperformedforstatisticalanalysis,withsignificance(*)atp<0.05.Graphsdepictmean±standarddeviation.chamberdilationarecharacteristicfeaturesofpost-MIcardiacMassSpectrometryAnalysisofCFsIsolatedfrom7Dayremodeling.ShamorMIHeartsExperimentalOverviewandGlobalNext,LVEDDandLVESDmeasurementswereusedtoAnalysiscalculateLVFSattheapical,middle,andbasalsegmentsoftheAworkflowchartoutliningtheprotocolforthemassheart.Atallthreelevelsoftheheart,theLVFSwassignificantlyspectrometry-basedanalysisofculturedCFsisshowninFigure4.Briefly,LAD-ligationorshamcontrolsurgerywasimpairedininjuredhearts,relativetoshamcontrols(FigureconductedonadultC57Bl/6mice,andheartswereisolatedfor3A).Moreover,two-dimensional,B-modeanalysisrevealed7daysfollowingtheprocedure.AtriaandatrioventricularthatFACwasalsosignificantlyreducedininjuredhearts,valveswereremovedfromthehearts,andtheremainderofthecomparedtoshamcontrols(Figure3B).ThesedatasuggestcardiactissuewasdividedintotheremoteandinfarctzonesthatLADligationsignificantlyimpairedleftventricularusingtissuepalingtodefinetheboundarybetweeninfarctedcontractilityatalllevels,whichistypicalofaninfarctedheart.andnoninfarctedtissue.NotethatwhileshamheartsdidnotCardiacfunctioninshamandinfarctedheartswasalsohavevisuallydistinctremoteandinfarctzones,roughlydeterminedbymeasuringSV,HR,andCO.Thedatashowedequivalentregionsweretakenascontrolsforregion-specificdifferencesintheCFphenotype.Next,thedissectedsamplesthatLAD-ligationresultedinsignificantlyreducedSVat7daysweredigestedseparatelyforCFisolation,asdescribedabove.postsurgerycomparedtoshamcontrols;however,noTissuedigestswereincubatedwithantibody-conjugatedsignificantchangesinHRandCOweredetected(Figure++microbeadstoremoveCD31endothelialcellsandCD453C−E).Interestingly,however,thereisatrendtowardleukocytesinamagneticcellsorter.TheremainingtissueincreasedHRintheinjuredmice,whichmayhelptodigestwasplatedonastandardtissueculture-treatedPetridishcompensatefortheSVreductiontomaintainadequateCO.for2handnonadherentcellsanddebriswerewashedoffwithThisisconsistentwiththepreviousliterature,whichprovidesfibroblastmedia.CFswereculturedovernightandpreparedforsimilarevidenceforsuchcompensatorymechanisms,includingMSthefollowingday,asdescribedintheMethodssection.PriortoMSpreparation,abicinchoninicacidassaywasusedtoincreasedsympatheticinnervationoftheheartfollowingMI,toquantifyandnormalizeeachsampletoapproximately6μgofmaintainadequatebloodflowtothebody.Together,theprotein.EachsamplewasrunasatechnicalduplicateonanechocardiographydatademonstratedthatLAD-ligationnLCcoupledinlinetoaQExactivemassspectrometer.inducedsignificantcardiacdysfunctionintheinfarctedAcrossallexperiments,2148uniqueproteinswereidentifiedcomparedtothesham-operatedcohort.acrossallMSruns(SupplementalTables1and2;2871https://doi.org/10.1021/acs.jproteome.1c00098J.ProteomeRes.2021,20,2867−2881

5JournalofProteomeResearchpubs.acs.org/jprArticleFigure3.EchocardiographyrevealedthatMImiceexhibitedsignificantcardiacdysfunction7daysaftertheinitialsurgerywhencomparedtosham-operatedcontrolmice.Specifically,one-dimensionalM-modeechocardiographywasperformedattheapical,middle,andbasalsegments.TheM-modetracingwasusedtoassessanteriorandposteriorwallthicknesses,aswellastheLVEDDandLVESD(SupplementalFigure2).ThelatterwasusedtocalculateLVFSattheapical,middle,andbasalsegments.Ourdatashowedthatthefractionalshorteningwassignificantlyimpairedatallthreesegmentsoftheinfarctedheartswhencomparedtosham-operatedcontrolmiceat7days(A).Furthermore,ECG-basedkilohertzvisualization(EKV)wasusedtocreateacineloopoftheLVfor1cardiaccycle.Framescorrespondingtoend-diastoleandpeaksystolewereidentifiedandtracedtomeasuretheend-diastolicareaandpeaksystolicarea,whichwerethenusedtocalculateFAC.OurdatarevealedthattheinfarctedheartshadasignificantlylowerFACwhencomparedtoshammice(B).Additionally,measurementsofthepulse-waveDopplerflowvelocityattheaorticorificeandthediameteroftheaorticannuluswereusedtocalculateSVandCO.Wealsomeasuredeachmouse’sHR.OurdatademonstratedsignificantlylowerSVinMImicecomparedtoshamcontrols;however,nodifferenceswereobservedinHRandCObetweenthetwogroups(C−E).Significance(*)atp<0.05usingunpaired,two-tailed,student’st-testcomparingshamandMImice.Thegraphdepictsmean±standarddeviation.SupplementalFigures3and4).Specifically,therewere1957,Figure4).MultisampleANOVAwasusedtoidentify10251854,2131,and2133uniqueproteinsinshamremote,shamproteinsthatweredifferentiallyexpressedbetweenatleasttwoinfarct,MIremote,andMIinfarctregions,respectively(Figureofthesamples(permutation-basedFDRcutoffof<0.05andan5A).Furthermore,1802ofthe2148proteinswereidentifiedinS0of1.0)(SupplementalTable3).Hierarchicalclusteringofallfoursamples.Principalcomponentanalysis(PCA)ofthethe1025differentiallyexpressedproteinsrevealedthatCF1170proteinswithvalid,nonzeroLFQvaluesforall12samplesfromsham-operatedheartsweremoresimilartoeachsamples,showedpreferentialclusteringsham-operatedversusotherthantotheCFsamplesfromtheinfarcted(MI)miceinjured(MI)CFsamplesalongwithcomponent1,with(Figure6).CFsamplesfrominjured(MI)heartspreferentiallyminimalregion-specificdifferencesinclusteringforsamplesclusteredintoinfarctandremoteregion-derivedgroups.Byisolatedfromsham-operatedanimals(Figure5B).Althoughcontrast,CFsamplesfromsham-operatedmiceshowednothedistinctionbetweenCFsamplesfromtheremoteandregion-specificdifferencesintheirexpressionprofiles(seealsoSupplementalFigure5),andassuch,thesesampleswereusedinfarctregionsofinfarctedheartswashardertovisualize,assixindependentshamcontrolsinsubsequentbinaryandrightwardmigrationalongprincipalcomponent1appearstogeneontologyanalyses.reflectagradientoffibroblastactivationfromthelowest(nonactiveCFsinshamhearts),totheintermediate(activatedMassSpectrometryAnalysisofCFsIsolatedfrom7DayCFsintheremoteregionofinjuredhearts),tothehighestShamorMIheartsBinaryComparisonsandGene(highlyactivatedmyofibroblastsintheinfarctedregionofOntologyAnalysisinjuredhearts).SimilarobservationswereinferredfromtheFinally,volcanoplotsweregeneratedforbinarycomparisonsheatmapproducedbyplottingPearson’scorrelationvaluesforbetweendifferentsamples,withsignificancelinesrepresentingeachbinarycomparisonbetweensamplesasanintensityapermutation-basedFDRcutoffof<0.05andanS0=1.042markerinthePerseussoftwarepackage(Supplemental(Figure7).Reddotsrepresentproteinsthatweremorehighly2872https://doi.org/10.1021/acs.jproteome.1c00098J.ProteomeRes.2021,20,2867−2881

6JournalofProteomeResearchpubs.acs.org/jprArticleFigure4.Overviewofthemassspectrometryworkflow.Briefly,heartswereexcisedfrom7dayshamandMI-operatedmice,andtheatriaandatrioventricularvalveswereremoved.TheremainderofthetissuewasdividedintotheinfarctandremotezoneforbothshamandMIsamples.Notethatwhileshamheartsdidnothaveavisuallydistinctremoteandinfarctzone,wetookroughlyequivalentregionsascontrolstoaccountforanypotentialregion-specificdifferences.Eachtissuewasdigested,aspreviouslydescribed.CD31+andCD45+cellswereremovedusingamagneticcellsorter,andtheresultingdigestwasplatedfor2htoallowCFstopreferentiallyattach.Plateswerewashedafter2handtheadherentcellswereculturedovernight.Sampleswerelysedthefollowingdayandproteinswerequantifiedusingabicinchoninicacidassay.6μgofproteinfromeachsamplewaspreparedforMSandrunonananoliquidchromatography(nanoLC)coupledtoaQExactivePlus.RawfilesweresearchedinMaxQuantagainstamouseproteomicsequencedatabaseandadownstreamanalysiswasconductedusingthePerseussoftwareversion1.6.1.3.expressed,whilethegreendotsrepresentproteinswithtothesiteofischemia;rather,CFsinremoteregionsalsoreducedexpressioninthereferencecondition.Insummary,underwentproteinexpressionchangesthatappeartobeweidentified1052proteinsdifferentiallyexpressedbetweenrelevanttoongoingcardiacremodelingandHFprogression.MIinfarctandsham(SupplementalTable4),579betweenMIThisdataprovidesfurtherevidenceofaCFactivationgradientremoteandsham(SupplementalTable5),and15betweenMIbetweencellsinnoninfarctedtissuesandthoselocatedintheinfarctandMIremoteCFs(SupplementalTable6;Figure7).differentmyocardialzonespost-MI.ThesenumberssuggestedthatwhilethemajorityoftheMI-Asexpected,severalofthedifferentiallyexpressedproteinsdependentchangesinCFproteinexpressionoccurredattheinMIcomparedtoshamCFshavebeenpreviouslyimplicatedsiteofinfarct,asubstantialnumberofproteinswereinpost-MIremodelingandfibrosis.Specifically,severalECMdifferentiallyexpressedinCFsfromremoteareasofthecomponentswereupregulatedinMICFs,includingstructuralheartaswell.Moreprecisely,theMIinjurywasnotexclusiveproteinslikecollagenfamilymembers(Col1a1,Col3a1,2873https://doi.org/10.1021/acs.jproteome.1c00098J.ProteomeRes.2021,20,2867−2881

7JournalofProteomeResearchpubs.acs.org/jprArticleFigure5.ExploratoryanalysisoftheMSexperimentusing1day-culturedCFsfrom7dayshamandMImice.AcrossallMSruns,2148proteinswereidentified.Specifically,weidentified1957,1854,2131,and2133proteinsintheshamremote(rem),shaminfarct(inf),MIremote,andMIinfarctCFs,respectively(A).WeconductedPCAusingthe1170proteinsthathadvalid,nonzerovaluesforall12samples(B).TherewerecleardistinctionsbetweentheshamandMIsamplesacrosscomponent1.Noregion-specificclusteringwasobservedbetweentheshamandinfarctsamples,suggestingthatCFsfromtheseregionsweresimilar.Figure6.GlobalanalysisoftheMSexperimentusing1day-culturedCFsfrom7dayshamandMImice.StatisticalanalysisusingmultisampleANOVA(permutation-basedFDR<0.05andanS0=1.0)revealed1025differentiallyexpressedproteinsbetweenatleasttwoofthefourexperimentalgroups.Hierarchicalclusteringofthe1025proteinsrevealeddifferentexpressionprofilesbetweenshamandMICFs.Therewerethreebiologicalreplicatespercondition.AllheatmapsweregeneratedusingnormalizedLFQvaluesona1to1000intensityscaleforeachprotein.Azoomed-inviewoftheregionoftheheatmaphighlightsproteinsappearingtodifferbetweenCFsamplesfromtheinfarctandremoteregionsofinfarctedmice.2874https://doi.org/10.1021/acs.jproteome.1c00098J.ProteomeRes.2021,20,2867−2881

8JournalofProteomeResearchpubs.acs.org/jprArticleFigure7.Volcanoplotoftherelevantbinarycomparisons.Thecurvedsolidblacklinesareknownasthe“lineofsignificance”,whicharedeterminedbasedonapermutation-basedFDRcutoffof<0.05andanS0=1.0.Thereddotsrepresentproteinsenrichedinthefirstcondition,whilethegreendotsrepresentproteinsrelativelyenrichedinthesecondcondition.WhencomparingMIinfarcttosham,weidentified867proteinsenrichedintheMIinfarctand185proteinsenrichedinshamCFs.WhencomparingMIremotetosham,weidentified505proteinsenrichedinMIremoteand74proteinsenrichedinshamCFs.Finally,whencomparingMIinfarctversusMIremote,weidentified15differentiallyexpressedproteins,where11wereenrichedintheMIinfarctand4wereenrichedinMIremoteCFs.AfewofthedifferentiallyexpressedproteinsandenrichedGOtermsaredepictedforeachcondition.NoGOtermsareshownforMIinfarctversusMIremotecomparisonsbecausetherewereonly15differentiallyexpressedproteins,whicharenotenoughforGOtermenrichmentanalysis.Col4a2,Col5a1,Col1a2,andCol5a2)andfibronectin,aswellasexperiencingadversecardiaceventsandtheobservationthatnonstructuralcomponentslikeSparc(alsoknownasmanyofthesesamegenes(GstandMtfamilies)arealso43−4550−53osteonectin).Moreover,follistatin-relatedprotein1,decreasedinpathologicversusphysiologichypertrophy.whichlabelspathologicalCFspost-MI,wasshowntobeTakentogether,thesefindingsindicatedthatMIinjurydid,asupregulatedinbothMIinfarctandremoteCFswhenexpected,induceCFactivationtowardamoremyofibroblast-46comparedtoshamcontrols.Thedataalsoshowedthatlikephenotype,especiallywithintheinfarctregion.Itwasoffocaladhesionproteins,includingpaxillin(Pxn),numerousparticularinterest,however,thatmanyofthesamechangesintegrins(Itgb3,Itgb1,Itgav,Itga1,andItgb6),andfocalcouldbeobserved,albeittoagenerallylesserextentinCFsadhesionkinase1(Ptk2)wereallincreasedinMIconditionsfromtheremote(noninfarcted)areasoftheheart.Directcomparedtosham.Indeed,focaladhesionprocessesconnectbinarycomparisonidentified15proteinsthatwerediffer-cellstotheECMandhavebeenshowntofacilitatecellularentiallyexpressedbetweentheremoteandinfarctregions.Of47−49migrationtowardthesourceofinjury.Incontrast,these,11proteinsweremorehighlyexpressedinCFsfromthegelsolin,whichisfoundtobeamarkerofadulthomeostaticinfarctregion(Gpc1,Cthrc1,Vmac,Nexn,Znf185,Sprr1a,CFs,wasdecreasedinMIsamples,comparedtoitslevelinSpecc1,Emb,Limd2,Pawr,andMcam),while4proteins46shamcontrols.Moreover,proteinsinvolvedinglutathioneshowedlowerexpressioninCFsfromtheinfarctzone(Gstt1,metabolismandxenobioticclearance,includingGstt1andGstm1,Tceal3,andInmt).TofurtherinvestigatepotentialGstm1,weredownregulatedinCFspost-MI.ThesedataaredifferencesbetweentheinfarctandremoteregionsfollowingconsistentwithstudiesshowingthatpatientswithGstt1andMI,weidentified285proteinsthatweredifferentiallyGstm1-nullmutationsorpolymorphismsareatahigherriskofexpressedbetweenthesesampleswhenalowerstringency2875https://doi.org/10.1021/acs.jproteome.1c00098J.ProteomeRes.2021,20,2867−2881

9JournalofProteomeResearchpubs.acs.org/jprArticleFigure8.Proteinexpressionofα-SMAandCthrc1inCFsisolatedfromdifferentregionsofsham-operatedandLAD-ligatedmurinehearts.Notably,althoughα-SMAistheexpressioninallsamples,itwasfoundtobehigherinCFsfromtheMIinfarctregion(A).Moreover,therewasahigherCthrc1expressionintheMIinfarctCFscomparedtoshamcontrolandMIremoteCFs(B).Vimentinwasusedasaloadingcontrol.Eachsamplewasobtainedbypoolingtherespectiveregionsfromfivemurinehearts.cutoff(p<0.05)wasused(SupplementalFigure6andTablelevelswerepresentatdetectablelevelsinsham-operatedhearts7).andincreasedonlyslightlyintheinfarctedanimals(FigureGeneontologyenrichmentsweregeneratedusingthe8A).Whilethisresultwassurprisingatfirst,itwasconsistentDAVIDfunctionalannotationtooltoidentifyGOterms(pwithourproteomicresultsandmayindicatethatα-SMAisa<0.05)associatedwiththedifferentiallyexpressedproteinsformuchmoresensitiveindexofplating/cultureonastiffeachbinarycomparison.Ofthe1052differentiallyexpressedsubstratethanitisofmyofibroblastactivationfollowingproteinsbetweenMIinfarctandsham,867wereupregulatedmyocardialinfarction.Asdiscussedabove,Cthrc1wasoneofand185weredownregulatedinMIInfarctCFs,relativetothemostdifferentiallyexpressedproteinsintheinfarctzoneshamcontrols(SupplementalTable4).TheproteinsenrichedcomparedtoinfarctedremoteandidentifiedasbeinginMIinfarctwerefoundtobeassociatedwithGOtermssignificantlydifferentinthefollowingbinarycomparisons:pertainingto“cell−cellandcell−matrixadhesion”,“ribosomesMI-remoteversussham,MI-infarctversussham,andMI-andtranslation”,“chaperone-mediatedproteinfolding”,andinfarctversusMI-remote(Figure7).Consistentwiththese“collagenfibrilorganization”(SupplementalTable8).Inobservations,postproteomicimmunoblottingforCthrc1contrast,proteinsthatweredownregulatedinMIinfarctshowedthatitwasexpressedatlowbasallevelsinshamrelativetoshamCFswereassociatedwithtermsrelevanttosamples,increasedmodestlyinremoteregions,andincreased“glycolyticprocesses”,“lipidmetabolism”,and“insulin-likedramaticallyintheinfarctzone(Figure8B).growthfactor-binding”(SupplementalTable9).Ofthe579proteinsdifferentiallyexpressedbetweenMIremoteandsham■DISCUSSIONCFs,505wereenrichedand74weredownregulatedinMIToourknowledge,thisisthefirststudytousemassremoteCFs,relativetoshamcontrols(SupplementalTable5).spectrometrytocharacterizechangesinproteinexpressionTheproteinsenrichedinMIremotewereassociatedwithGOforregion-specificCFsisolatedacutelyfollowingshamorMI-termsrelatedto“cell−cellandcell−matrixadhesion”,surgery.ArecentpaperfromtheLundbygroupexaminedthe“ribosomesandtranslation”,“proteinfolding”,and“collagendeepproteomeofdifferentcardiaccelltypesthatwerefreshlyfibrilorganization”(SupplementalTable10).Incontrast,the54isolatedfromhealthyrathearts.CFsintheirstudywereproteinsdownregulatedinMIremoteCFswereassociatedisolatedusingasimilardigestionandpreplatingmethodwithtermspertainingto“oxidation−reductionprocesses”,consistentwiththeapproachhere,albeitonlyinhealthycells;“lipidmetabolism”,and“insulin-likegrowthfactor-binding”however,theydedicatedasignificantlygreatermassspectrom-(SupplementalTable11).Whileonly15proteinsdifferedetrytimetotheirstudyresultinginamuchdeeperproteomicbetweenremoteandinfarctregionsofMI-injuredheartswhendataset.However,whenourmouseCFproteomedatawerethepermutation-basedFDRcutoffof<0.05andanS0=1.0comparedinintensity/rankplotstoLundby’sratdatawereused,ifwecomparetheseregionsusingthe285(SupplementalFigure8),thedatashowaveryconsistentdifferentiallyexpressedproteinsbasedonthep<0.05cutoff,dispersion.DespitetheproportionatelysmalleramountofCFweseeanumberofthesameGOterm-relatedfibrosis-linkedproteinavailablepermouseheart,ourprotocolsprovidedgoodpathwaysthatwereidentifiedaboveincludingcell−cellandcoverageacrosstherangeofproteinabundancesseeninthecell−matrixadhesion,andglutathionemetabolism(Supple-Lundbydataset.Asexpected,someofthecoverageatverylowmentalTable12).Thesedataareconsistentwiththenotionabundanceswasmissingfromourmousedata.thatfibroblastsarebeingactivatedonaglobalscale,butthatItisgenerallyacceptedthatCFswithintheinfarctedthosewithintheinfarctzonearethemostaffectedbythemyocardiumexperiencethegreatestextentofmechanical,injury.Notably,afewadditionalGOtermssuchasthebiochemical,andpathophysiologicstressorsasaresultoftheextracellularexosomes,cytoplasm,andmyosincomplexareinjury.Consistentwiththisnotion,wefoundthatMIinducedhighlightedaspathwaysthatappeartodifferbetweenCFsinthemostsignificantproteinexpressionchangesinCFslocatedtheremoteandinfarctzonesofMI-injuredmice.atthesiteofischemia,however,weweresurprisedtofindthatPostproteomicAnalysisConfirmsInfarct-SpecificCFsfromlocationsdistaltotheinfarctunderwentasimilarsetInductionofCthrc1ofchangesasthoseintheinfarct.AnalysisofvolcanoplotdataFinally,weperformedthewesternblottinganalysisonlysatesforbinarycomparisonsbetweensham,remote-andinfarct-fromthevarioussamplesusedfortheproteomiccomparisonresidentCFsidentifiedanumberofregion-specificdifferences.above(Figure8;SupplementalFigure7).Importantly,α-SMACthcr1,Nexn,andSprr1awereinducedinCFsthroughoutthe2876https://doi.org/10.1021/acs.jproteome.1c00098J.ProteomeRes.2021,20,2867−2881

10JournalofProteomeResearchpubs.acs.org/jprArticleheartbuttoamuchgreaterextentintheinfarctregion.NotTheheartisknowntoundergoprofoundmetabolicchanges68surprisingly,allthreeoftheseproteinshavebeenpreviouslyfollowingmyocardialinfarction.Toourknowledge,however,55−60implicatedinthefibroblastfunctionandfibrosis.therehasbeennocomprehensiveproteomicmeasureofthePostproteomicimmunoblottinganalysisconfirmedthatCFmetabolicstatefollowingischemicinjury.TheGOtermCthrc1expressionwasincreasedspecificallyintheinfarctanalysisofproteinchangesinCFsfrominfarctedheartsregioncomparedtoremoteandshamCFsamples.ThisfindingshowedclearevidenceofmetabolicreorganizationfollowingwascorroboratedbytherecentdemonstrationthatCthrc1MI.Indeed,areductionofproteinsassociatedwithβ-oxidationmarksapreviouslyunappreciatedpopulationofprofibroticandlipidmetabolismwasobservedaswellasareductioninCFsthatlocalizespecificallytothescar/infarctregionandmitochondrialproteinsthatwereassociatedwithcarbohydratehighlightsthepotentialbiologicalimportanceoftheothermetabolism,specificallythoserequiredforpyruvatetoacetyl-proteinsweidentifiedasselectivelyinducedintheinfarctCoAconversion.Thelatterobservationisconsistentwiththeregion(Gpc1,Vmac,Znf185,Specc1,Emb,Limd2,Pawr,andnotionthathypoxicandischemicinjuriescaninduceMcam)duringthescar-formingprocess.61InadditiontoGstt1significantmitochondrialdamageanddysfunctionincells.andGstm1(discussedabove),Tceal3,atranscriptionIndeed,apreviousstudyfromourgroupfoundthattheelongationfactorA-likeprotein,andInmt,anindolethylaminesecretomeofCFsinhypoxicconditionswasenrichedin69N-methyltransferasethatisalsoknowntoparticipateinmitochondria-associatedproteins.Together,thesefindingsxenobioticclearancewerefoundtobedecreasedinCFssuggestthathypoxiaandLADligationpromotemitochondrialfromtheinfarctzonesuggestingthatthereductionofthesedysfunctionandreducedβ-oxidationinCFs.Furtherresearchfactorsmaycontributetoscarformationintheinfarctedheart.isnecessarytofullycharacterizethemetabolismofCFsinInadditiontounderstandingthese15proteins,interrogationhealthyanddiseasedstates.WhiletheCFswithintheinfarctzoneshowaprofoundofthefunctionandbiologicpathwayaffiliationofall285injury-inducedchangeintheirproteome,themostsurprisingproteinsidentifiedasdifferent(p<0.05)willlikelyprovideresultfromourstudywastheextentofchangethatalsoimportantcluestounderstandingthesignalingconditionsthatoccurredinCFsthatresidedinmoreremote(noninfarcted)drivereactive-typeandreplacement-typefibrosisintheremoteregionsoftheventricles.Thesefindingsareconsistentwithandinfarctregions,respectively.othersimilarobservations,includingthosebyNagarajuetal.AsindicatedbyGOtermanalyses,MIinducedasimilarset(2017),thatreportedaglobalactivationoffibroblastsinofCF-specificproteomicchangesinboththeremoteandinfarctedpighearts,withthemajorityofmeasurableinterstitialinfarctregionsthatreflectedcellsurfaceandsignalingchangesandperivascularreplacementtypeoffibrosislocalizedtotheincludingincreasedcell−cellandcell−matrixadhesion20scar(infarct)zone.TheseauthorsreasonthatglobalCFmolecules,andcollagenfibrilorganization.Forinstance,itactivationmaybecausedbyanincreasedmechanicalloadonhasbeenshownthatcoordinatedECMremodelingisachievedtheheart,includingattheremotezone,anotionthatisthroughthecrosstalkbetweenmyofibroblastsatcell−cellconsistentwiththeBogaertetal.(2000)study,thatshowedjunctions,inadditiontothecell−matrixcommunication.Moresignificantcardiacdysfunctionandleftventriclestrainintheprecisely,myofibroblastsformadhesionstructureswiththe31remoteandborderregionsofaninfarct.ThereareseveralECM,whichhelpsdistributetheircontractileforcethroughoutpotentialexplanationsforthesefindings.Forinstance,MIthematrix.Likewise,chaperone-mediatedproteinfoldinghasinjurycaninducearobustimmuneresponse,whichincludesbeenshowntobeimportantintheprocessingandassemblyofinfiltrationofcirculatingneutrophilsandmonocytes,aswellascollagenandotherECMproteins.Forinstance,astudyanincreasedexpressionofinflammatorymediatorsinbothshowedthattheFK506-bindingprotein,whichisimportantfor70−72remoteandinfarctregions.Onestudyfoundthatcollagenassembly,waselevatedinbleomycin-inducedlunginflammatorymarkers,includinginterleukin-1βandinterfer-62fibrosisandinpatientswithidiopathicpulmonaryfibrosis.on-γ,wereelevatedintheremoteandinfarctregionofpigTogether,thesedatademonstratethatMIinjurysignificantlyheartsfollowingMI.20GiventherecentappreciationfortheinducedsimilarprofibroticchangesinCFslocatedatthesiteofinteractionbetweenfibroblastsanddifferentimmunecellischemiaandthosefoundmoreremotelyintheheart.types,itisreasonabletopostulatethatatleastsomeoftheWealsoidentifiedGOtermsthatareassociatedwithchangesintheremoteCFproteomemaybelinkedtothesignalingpathwayswhoseroleislesswellappreciatedintheproinflammatoryenvironmentcreatedbytheinfarction.contextofcardiacinjuryandrepair.Forexample,proteinsLikewise,impairedcardiacfunctionfollowingMIisexpectedassociatedwiththeSNAREcomplexesandvesicle-mediatedtohavesystem-wideeffectstoincreasetheactivitiesofthetransportwerealsoshowntobeenrichedinMI,relativetosympatheticnervoussystemandtherenin−angiotensin−shamandremoteCFs.Althoughmanyofthespecificproteinsaldosteronesystem(RAAS).Itistobeexpectedthatsuchandsignalingmechanismsremaintobecharacterizedinpost-neurohormonalresponsestodecreasedLVfunctionfromanMICFs,numerousstudieshavedemonstratedtheimportanceinfarctedheartaffectremoteregionsofthemyocardiumandofexosomesecretionandvesicle-mediatedcellularcommuni-similarlyalterthephysiologyofCFswithinremoteareasofthe63,64cationinvariousdiseases.Forinstance,TGF-β-containingmyocardiumaswell.exosomescantriggerbothdifferentiationandprofibroticWhencombinedwithothernext-generationsequencingresponsesinfibroblasts.Moreover,miRNAsfromfibroblasttechniquessuchastranscriptomicsandnewimagingtoolssuchexosomepreparationshavebeenshowntostimulatehyper-asRNAscopeourdatawillbeusefulfordefiningthenature65trophyincardiomyocytes.InadditiontotheirroleinandregionalityofCFactivationfollowingmyocardialexocytosis,SNAREproteinchangesmightalsobeimportant73infarction.Indeed,therecentRNA-seqstudybyFarbehietforalteringcell-surfacemarkerexpressionandturnoveroffocalal.(2019)showedthereareanumberofdistinctfibroblastadhesioncomplexesinthecontextofcardiacremodelingandpopulationsinbothuninjuredandinjuredheartsandthat66,67fibrosis.thesepopulationschangetheirtranscriptionprofilesdramat-2877https://doi.org/10.1021/acs.jproteome.1c00098J.ProteomeRes.2021,20,2867−2881

11JournalofProteomeResearchpubs.acs.org/jprArticle74icallypost-MI.ItwouldbeinterestingtoinvestigatewhetherAnthonyO.Gramolini−TranslationalBiologyandanyofthesepopulationsareregion-specificandtoidentifytheEngineeringProgram,TedRogersCentreforHeartResearch,CFpopulationsthatregulateremodelingineithertheremoteToronto,Ontario,CanadaM5G1M1;Departmentoforinfarctzones.Physiology,FacultyofMedicine,UniversityofToronto,Toronto,Ontario,CanadaM5S1A8;orcid.org/0000-■ASSOCIATEDCONTENT0003-1109-2070;Phone:416-946-8257;*sıSupportingInformationEmail:Anthony.gramolini@utoronto.caScottP.Heximer−TranslationalBiologyandEngineeringTheSupportingInformationisavailablefreeofchargeatProgram,TedRogersCentreforHeartResearch,Toronto,https://pubs.acs.org/doi/10.1021/acs.jproteome.1c00098.Ontario,CanadaM5G1M1;DepartmentofPhysiology,AlistofallunfilteredidentificationsfortheMSFacultyofMedicine,UniversityofToronto,Toronto,Ontario,experiments(SupplementalTable1);filteredlistofallCanadaM5S1A8;Phone:416-978-6048;2148proteinswithimputationfortheMSexperimentEmail:scott.heximer@utoronto.ca(SupplementalTable2);listof1025proteinsthatweredifferentiallyexpressedbetweenatleasttwooftheAuthorsexperimentalgroupsbymulticomparisonANOVA(FDRAlisonHacker−TranslationalBiologyandEngineering<0.05,S0=1.0)(SupplementalTable3);listofProgram,TedRogersCentreforHeartResearch,Toronto,proteinsfromthebinarycomparisonofMIinfarctandOntario,CanadaM5G1M1shamCFs(FDR<0.05,S0=1.0)(SupplementalTableDylanLangburt−TranslationalBiologyandEngineering4);listofproteinsfromthebinarycomparisonofMIProgram,TedRogersCentreforHeartResearch,Toronto,remoteandshamCFs(FDR<0.05,S0=1.0)Ontario,CanadaM5G1M1;DepartmentofPhysiology,(SupplementalTable5);listofproteinsfromthebinaryFacultyofMedicine,UniversityofToronto,Toronto,Ontario,comparisonofMIinfarctandMIremoteCFs(FDRCanadaM5S1A8<0.05,S0=1.0)(SupplementalTable6);listofproteinsMichaelDewar−TranslationalBiologyandEngineeringfromthebinarycomparisonofMIinfarctandMIProgram,TedRogersCentreforHeartResearch,Toronto,remoteCFswithalessstringentcutoff(FDR<0.05)Ontario,CanadaM5G1M1;DepartmentofPhysiology,(SupplementalTable7)(XLSX)FacultyofMedicine,UniversityofToronto,Toronto,Ontario,GOtermsassociatedwithproteinsenrichedinMICanadaM5S1A8infarctCFs,relativetoshamcontrol(n=867protein)MeghanJ.McFadden−TranslationalBiologyand(SupplementalTable8);GOtermsassociatedwithEngineeringProgram,TedRogersCentreforHeartResearch,proteinsdownregulatedinMIinfarctCFs,relativetoToronto,Ontario,CanadaM5G1M1shamcontrols(n=185protein)(SupplementalTableHangjunZhang−TranslationalBiologyandEngineering9);GOtermsassociatedwithproteinsenrichedinMIProgram,TedRogersCentreforHeartResearch,Toronto,remoteCFs,relativetoshamcontrols(n=505protein)Ontario,CanadaM5G1M1(SupplementalTable10);GOtermsassociatedwithUrosKuzmanov−TranslationalBiologyandEngineeringproteinsdownregulatedinMIremoteCFs,relativetoProgram,TedRogersCentreforHeartResearch,Toronto,shamcontrols(n=74protein)(SupplementalTableOntario,CanadaM5G1M1;DepartmentofPhysiology,11);GOtermsassociatedwithproteinsdifferentiallyFacultyofMedicine,UniversityofToronto,Toronto,Ontario,expressedbetweenMIinfarctversusMIremoteCFs(nCanadaM5S1A8=285protein)(SupplementalTable12)(PDF)Yu-QingZhou−TranslationalBiologyandEngineeringRawimmunoblotsofα-SMAandvimentin(Supple-Program,TedRogersCentreforHeartResearch,Toronto,mentalFigure1);echocardiographyofsham-operatedOntario,CanadaM5G1M1andLAD-ligatedmice(SupplementalFigure2);histo-BilalHussain−TranslationalBiologyandEngineeringgramoftransformedandimputedLFQvaluesProgram,TedRogersCentreforHeartResearch,Toronto,(SupplementalFigure3);theglobalcorrelationbetweenOntario,CanadaM5G1M1individualsamples(SupplementalFigure4);volcanoFahadEhsan−TranslationalBiologyandEngineeringplotcomparingshaminfarctandshamremoteCFsProgram,TedRogersCentreforHeartResearch,Toronto,(SupplementalFigure5);volcanoplotcomparingMIOntario,CanadaM5G1M1;DepartmentofPhysiology,infarctandMIremoteCFs(SupplementalFigure6);FacultyofMedicine,UniversityofToronto,Toronto,Ontario,rawimmunoblotsofα-SMA,Cthrc1,andvimentinCanadaM5S1A8(SupplementalFigure7);andthewaterfallplotofCFBorisHinz−LaboratoryofTissueRepairandRegeneration,proteinsidentifiedbyPoulsenetal.(SupplementalFacultyofDentistry,UniversityofToronto,Toronto,Ontario,Figure8)(PDF)CanadaM5G1G6■Completecontactinformationisavailableat:AUTHORINFORMATIONhttps://pubs.acs.org/10.1021/acs.jproteome.1c00098CorrespondingAuthorsHaisamShah−TranslationalBiologyandEngineeringNotesProgram,TedRogersCentreforHeartResearch,Toronto,Theauthorsdeclarenocompetingfinancialinterest.Ontario,CanadaM5G1M1;DepartmentofPhysiology,FacultyofMedicine,UniversityofToronto,Toronto,Ontario,CanadaM5S1A8;orcid.org/0000-0001-6740-5465;■ACKNOWLEDGMENTSPhone:+61-416-070-074;Email:haisam.shah@ThisprojectwasfundedbyaTeamGrantfromtheTedmail.utoronto.caRogersCentreforHeartResearchTranslationalBiologyand2878https://doi.org/10.1021/acs.jproteome.1c00098J.ProteomeRes.2021,20,2867−2881

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