growth agonists hormone-releasing of with hormone Protection rat cardiac of neonatal myocytes against radiation-induceddamage Pharmacological Research

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ContentslistsavailableatScienceDirect

Pharmacological Research

jo u r n al ho me p a g e :ww w . e l s e v i e r . c o m / l o c a t e / y p h r s

Protection of neonatal rat cardiac myocytes against radiation-induced damage with agonists of growth hormone-releasing hormone

Laura Kiscsatári

^a

, Zoltán Varga

^a

, Andrew V. Schally

^b

, Renáta Gáspár

^c,d

, Csilla Terézia Nagy

^e

, Zoltán Giricz

^e

, Péter Ferdinandy

^d,e

, Gabriella Fábián

^a

, Zsuzsanna Kahán

^a,1

, Anikó Görbe (MD PhD)

^c,d,e,∗,1

aDepartmentofOncotherapy,UniversityofSzeged,Szeged,Hungary

bVeteransAffairsMedicalCenter,MiamiFL,USAandDepartmentsofPathologyandMedicine,DivisionsofHematology/Oncology,UniversityofMiami MillerSchoolofMedicine,Miami,FL,USA

cCardiovascularResearchGroup,DepartmentofBiochemistry,UniversityofSzeged,Szeged,Hungary

dPharmahungaryGroup,Szeged,Hungary

eDepartmentofPharmacologyandPharmacotherapy,SemmelweisUniversity,Budapest,Hungary

a r t i c l e i n f o

Articlehistory:

Received12January2016 Receivedinrevisedform5July2016 Accepted25July2016

Availableonline30July2016

Keywords:

Cardiacmyocytes Cardioprotection GHRHagonists GHRH/SV1receptors Radiationdamage

a b s t r a c t

Despitethegreatclinicalsigniﬁcanceofradiation-inducedcardiacdamage,experimentalinvestigationof itsmechanismsisanunmetneedinmedicine.Beneﬁcialeffectsofgrowthhormone-releasinghormone (GHRH)agonistsinregenerationofthehearthavebeendemonstrated.Theaimofthisstudywasthe evaluationofthepotentialofmodernGHRHagonisticanalogsinpreventionofradiationdamageinanin vitrocardiacmyocyte-basedmodel.

Culturesofcardiacmyocytesisolatedfromnewbornrats(NRVM)wereexposedtoaradiationdoseof 10Gy.Theeffectsoftheagonisticanalogs,JI-34andMR-356,ofhumanGHRHoncellviability,proliferation,theirmechanismofactionandtheproteinexpressionoftheGHRH/SV1receptorswerestudied.

JI-34andMR-356,hadnoeffectoncellviabilityorproliferationinunirradiatedcultures.However,in irradiatedcellsJI-34showedprotectiveeffectsoncellviabilityatconcentrationsof10and100nM,and MR-356at500nM;butnosuchprotectiveeffectwasdetectedoncellproliferation.Bothagonisticanalogs decreasedradiation-inducedROSlevelandJI-34interferedwiththeactivationofSAFE/RISKpathways.

UsingWesternblotanalysis,a52kDaproteinisoformofGHRHRwasdetectedinthesamplesinboth irradiatedandunirradiatedcells.

Since GHRHagonistic analogs,JI-34 andMR-356 alleviatedradiation-induceddamageof cardiac myocytes,theyshouldbetestedinvivoaspotentialprotectiveagentsagainstradiogenicheartdamage.

1. Introduction

Radiation-inducedheartdiseasesincludingischemicheartdis- ease,congestiveheartfailure,electricalconductdefectsandvalve abnormalities are typically late consequences of radiotherapy, occurringmanyyearsordecadesaftertreatment[1,2].Suchadverse effectsmaydevelopafterchestirradiationinchildhoodcancers, lung,esophagealorbreastcancerorHodgkin’slymphoma[3,4].

These often unattributed complications cause deterioration in qualityoflifeoflong-survivingpatients,extrahealthcarecosts,

∗ Correspondingauthorat:CardiovascularResearchGroup,DepartmentofBio- chemistry,UniversityofSzegedDómtér9,Szeged,H-6720,Hungary.

E-mailaddress:aniko.gorbe@pharmahungary.com(A.Görbe).

1 Bothauthorscontributedequally.

andleadoccasionallytofataloutcomes.Althoughtheapplicationof modernradiotherapyplanninganddeliverysigniﬁcantlyimproves theradiationprotectionoftheheart,inmanycases,theentireheart ora partofit stillreceivesadosesufﬁcienttocauselong-term adverseeffects.Notably,theevolvinguseofintensity-modulated radiotherapy(IMRT)willincreasethevolumeofnon-targettissues irradiatedwithlowdose.

Thedevelopmentofradiation-inducedheartdiseasesisdose- dependent, and is more likely if radiation exposure occurs at a younger age[5,6].Radiogenic ischemic heart disease,due to atheroscleroticchangesofthecoronaryarteries,isindistinguish- ablefromotheretiologiesthatcauseanginaormyocardialinfarct.

Robustepidemiologicaldataonsuchcardiovasculardiseaseorigi- natesfrombreastcancerstudies.Darbyetal.,analyzingtheSEER databaseofmorethan300,000patients,foundthatamongthose http://dx.doi.org/10.1016/j.phrs.2016.07.036

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860 L.Kiscsatárietal./PharmacologicalResearch111(2016)859–866

whoreceivedadjuvantradiotherapy,thepatientswithleftsided tumors had a significantly increased risk of cardiac death due tocardiovasculardiseasesormyocardialinfarction,appearing10 yearsaftertheirradiation,andincreasingfurtherthereafter[7,8].It seemslikely,thattheradiogenicdamageofthemacrovasculature oftheheartoccursaftertheexposureofthesestructurestorela- tivelyhighdoses.Animalexperimentsrevealedinflammatoryand oxidativedamageleadingtotheformationoftypicalatheroscle- roticplaques[4,9].Radiation-induceddiffusemyocardiumchanges includemicrovasculatureabnormalities,degenerative cardiomy- ocyte and interstitial fibrotic changes. Such abnormalities may developafter evenlow doseradiationexposure, and canresult in thedeterioration of theheart’s pumpfunction or in cardiac conductionabnormalities[10].Cilliersetal.,investigatedradiation- inducedtime-dependent changes of themyocardium in Wistar rats[11].Interestingly,theradiationinducedstructuralchanges oftheendothelialcells and myocytesdevelopedearlyafterthe irradiation,andwereaccompaniedbyatransitorydepressionof themechanicalfunctionoftheheart;100–180daysaftertheirra- diation,onlyinterstitialinflammationandfibrosiswerepresent.

Radiationinducesinﬂammatory responses,andactivatessenes- cencepathways;there is a needtotest new agents thatcould preventthesecellularresponses.

Growthhormone-releasing hormone (GHRH) wasﬁrst iden- tiﬁed in humans as a hypothalamic peptide hormone which primarilyregulatestheproductionandreleaseofpituitarygrowth hormone(GH)[12].Recently,theexpressionofGHRHhasalsobeen demonstratedinaseriesofnormalandcanceroustissues[13].The pituitaryreceptorofGHRH(pGHRHR)belongstotheclasstype-IIG protein-coupledreceptors[14]thebindinginducestheproduction ofcAMPthattriggersaseriesofintracellularsignalingcascades.

Thepresenceofitssplicevariants(mainlySV1,lessimportantly SV2,SV3)hasbeendemonstrated ina seriesofperipheralnor- maltissuesand avarietyofcancers[15–17].Stimulationofthe GHRHRresultsintheactivationoftheERK1/2,AKT,JAK2/STAT3, PI3K/AKTpathways [18]. Numerous experimentaldata support theautocrine-paracrineroleoftheGHRHsysteminregeneration oftissue.GHRHhasbeenshowntopromotesurvivalandinhibit apoptosisincardiomyocytesinvitrodespiteserumstarvationor isoproterenol treatment [19]. The expression and activation of pGHRHRafterGHRHadministrationweredemonstratedin both adultratventricularmyocytesandinH9c2embryonic ratheart cells,whichexpressedpGHRHR[20].

GHRHpreventeddeathofadultratcardiacmyocytesinduced by serum starvation or by isoproterenol treatment which was antagonizedbyantagonisticanalogofGHRH,JV-1-36.Thesepro- tectiveeffectsweremediatedbytheactivationoftheextracellular signal−regulatedkinase(ERK)1/2andphosphoinositide-3kinase (PI3K)/Akt signaling pathways. Additionally, isolated rat hearts subjectedtoischemia-reperfusion(I/R)injurywereprotectedby theadministrationofGHRHbeforeischemia,whichwasabolished ifco-administeredwithJV-1-36[19].Pennaatal.demonstrated similarresultsintheisolatedratheartmodel;theadministrationof GHRHattheonsetofreperfusionreducedthesizeoftheinfarct,and thiseffectwasabrogatedifantagonisticanalogofGHRH,JV-1-36 wasco-administered[21].

Kanashiro-Takeuchidemonstrated thattheadministration of the GHRH agonist, JI-38, reversed ventricular remodeling and enhancedfunctional recoveryaftermyocardial infarction,while reducingexperimentalinfarctsizeinrats[22].Theseeffectswere absentifaGHRHantagonistwasco-administered,orbytreatment withGHonly.TheseeffectspointtotheroleofaGHRHR-mediated mechanism.

FollowingthedemonstrationofbeneﬁcialresultsoftheJ.I.class ofagonisticanalogsofGHRHinmultipleﬁelds,newclassesofGHRH agonistshavebeendevelopedwiththeultimategoalofclinicaluse

Table1

Cellsurvivalinfunctionofradiationdose.The24-hcultureswereexposedtodiffer- entradiationdoses(5,10,15,20Gy),andcelllosswasdetectedatdifferentlatency times(0,24,48,72,96,120h).Dataareexpressedinpercentofunirradiatedcontrol (mean±SEM).

Cellsurvivalinratiotocontrol(%)

Timeafterirradiation 5Gy 10Gy 15Gy 20Gy

0h 100±2 98±2 95±2 96±3

24h 76±2 75±3 79±4 89±6

48h 63±2 50±2 43±1 44±1

72h 58±3 49±3 43±3 42±2

96h 71±7 78±8 53±3 49±3

120h 50±2 38±1 37±1 39±2

[23].Asnotedsomeofthemhavebeentestedintheexperimental myocardialinfarctratmodel,whichconﬁrmedthecardioprotective effectsofGHRHagonistsbymeasurementofinfarctsizeandheart function[23,24].

Sinceradiationtriggers similarmechanismsas othercellular stresses,such asstarvation and hypoxia,we hypothesized that GHRHanditsreceptor(s)mighthavearoleintherecoveryafter irradiation.WethereforesetouttostudytheeffectsofGHRHandits agonisticanalogsinvitro,innewbornratcardiomyocytes(NRVM), underdifferentconditions.

2. Materialsandmethods

TheseexperimentsconformtheNationalInstitutesofHealth GuidefortheCareandUseofLaboratoryAnimals(NIHPub.No.85- 23,Revised1996)andwasapprovedbythelocalethicscommittee attheUniversityofSzeged.

2.1. Preparationofculturesofprimaryneonatalratcardiac myocytes

Neonatalratcardiacmyocytes(NRVM)wereisolatedasprevi- ouslydescribed[25].Brieﬂy,newbornWistarratsweredisinfected with70%ethanol andthenkilledbycervicaldislocation.Hearts wereexcisedand transferredtocold phosphate-bufferedsaline (PBS),pH7.2.Theventricleswereseparated,mincedgentlybyﬁne forcepsanddigestedin0.25%trypsinsolution(5ml/heart)at37^◦C for25min.Thecellsuspensionwasthencentrifugedat2000RPMat 4^◦Cfor15min.Pelletswereresuspendedingrowthmedium(Dul- becco’sMEMsupplementedwith10%fetalbovineserum[FBS],0.1%

glutamine,0.1%Antibiotic/Antimycoticsolution)andplatedonto6- wellplatestoeliminatefibroblastsat37^◦Cfor90min(pre-plating step).Cellsofthesupernatantwerethenre-platedontofresh6-and 96-wellplatesandflasks(5×10⁵,15×10⁴and4×10⁶cells/well, respectively).NRVMweresuppliedwith2ml,150␮land20mlper wellgrowthmediumwithorwithout10%FBSin6-well,96-well platesandflasks,respectively,andwereincubatedat37^◦Cand5%

CO₂.Theexperimentswerestarted24hafterplating.

2.2. Irradiationofcellcultures

Thecellcultureswereirradiated24hafterplating,usingalinear accelerator(SiemensPrimus,SiemensMedicalSolutions,USA).The plateswereirradiatedwith6MVenergyphotonbeamswithoppos- ingﬁeldtechnique.Toachieveahomogeneousdosedistributionin thesample,2cmthickPMMAsheetswereapplied.

2.3. TestingofGHRHagonisttreatment

First, theeffects ofsynthetic humanGHRH(hGHRH) (Bertin Pharma,MontignyleBretonneux,France)wastestedatconcen- trationsof1,10,50,and100nMinunirradiatedandirradiatedcell

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Fig.1.Viabilityofunirradiated(A)andirradiated(B)NRVMafterhGHRHtreat- ment(48h).TheeffectsofhGHRHontheproliferationofunirradiatedandirradiated NRVMareshowninpanelsCandD,respectively.Dataareexpressedasmean±SEM incomparisontocontrol(100%);*p<0.05,n=8–16ineachgroup.

cultures.Next,GHRHagonists,JI-34andMR-356,werestudiedboth inunirradiatedandirradiatedcellculturesatconcentrationsof1, 10,50,100and500nMincultureswithorwithout1%FBSsupple- mentation(forJI-34).Cellviabilityandproliferationassayswere performed48hthereafter.

NRVMculturesmaintainedinFBS-supplementedmediumwere alsostudiedforGHRHRexpressionandpossibleinvolvedsignaling pathwaysviaWesternblotanalysesasfollows:1.non-irradiated, non-treated, 2. non-irradiated, JI-34-treated,3. irradiated, non- treated,4.irradiatedandJI-34-treated;intheseexperiments,JI-34 wasappliedatthemosteffectiveconcentrationof10nM.Cellswere collected1and48haftertheirradiation.

2.4. Cellviabilityandproliferationassays

Bothtestswereperformedfollowingtherespectiveexperimen- talprocedure(irradiationand/ortreatment).Fortheassessment of cell viability,NRVM were incubated with1␮Mcalcein ace- toxymethyl ester (calcein-AM, Sigma, St Louis, MO) at room temperaturefor30min.Fluorescenceintensitywasmeasuredwith aﬂuorescenceplatereader(FluostarOptima,BMGLabtech,Orten-

Fig.2.Viabilityofunirradiated(A)andirradiated(B)NRVMafterJI-34treatment (48h).Bromodeoxyuridine(BrdU)proliferationassayperformedinunirradiated(C) andirradiatedNRVM(D),48hafterthetreatmentwithJI-34.Dataareexpressed asmean±SEM;incomparisontounirradiateduntreatedcontrol(100%).*p<0.05, n=8–16ineachgroup.

berg,Germany)[26].Cellviabilitywascomparedtothatofvehicle control.Eachplatecontainednon-irradiatedcolumnsservingas technicalcontrols.

FortheassessmentofcellproliferationtheNRVMcultureswere incubatedandlabeledwith10␮l/well5-bromo-2-deoxyuridine (BrdU) (Cell Proliferation ELISA, BrdU (colorimetric), Roche, Germany) labelingsolution at 37^◦C, 5% CO₂ for 20h. Then the labelingmediumwasremoved,thecellsﬁxed,andtheDNAwas denaturedinonestep withﬁx/denaturesolution.Thenananti- BrdUmousemonoclonalantibodywasaddedfollowedbyanHRP conjugatedsecondaryantibodytodetecttheincorporatedBrdU.

Theabsorbanceintensitywasmeasuredwithaplatereader.

2.5. Westernblot

Aftertheindicatedtimeofincubation,cellcultureswerewashed withD-PBSandincubatedfor5mininhomogenizationbuffer(1x RIPA supplemented with a protease and phosphatase inhibitor cocktail).Thencellswerescraped, collectedand sonicatedwith an ultrasound homogenizer for 10s on ice. The homogenate wascentrifugedat 11800RPM, 4^◦Cfor 10min(HettichUniver- sal320RCentrifuge,AndreasHettichGmbHandCo.,Tuttlingen,

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Fig.3. NRVMcellviabilityculturedwithorwithout1%FBS.Unirradiatedandirra- diatedcultureswerecomparedafter48hoflatencytime(A).Viabilityofirradiated nrcmNRVMcellsafterJI-34treatment,culturedwithoutFBSsupplementation;data areexpressedasmean±SEM;incomparisontocontrol(100%).*p<0.05,n=8–16 ineachgroup(B).

Germany);thesupernatantwastransferredintoaconcentrating tube(Amicon^® Ultra-4Centrifugal FilterUnits, 10kDaMWCO), and was centrifuged again at 6300 RPM, 4^◦C for 20min. The concentratedsamplewascollectedandstoredat−80^◦C.Protein concentrationwasmeasuredwithbicinchoninicacid(BCA)assay (Pierce^TM BCA Protein Assay Kit, Thermo Fisher Scientiﬁc Inc., Waltham,MAUSA)byreadingtheopticaldensityatUVmaxof 560nm,andcalculatingautomaticallybytheAnthossoftware.

Foranalysis ofGHRHRexpression 20-␮gsamplesof protein wereloadedon10%SDS-PAGEfollowed bythetransferofpro- teins onto a nitrocellulose membrane (90V, 1h). Membranes werethen blocked overnightat 4^◦C in 1%bovine serum albu- min(BSA).MembraneswereincubatedbothwithGHRHRantibody (1:1000,ab76263,AbcamInc.,Cambridge,MA)andGAPDHanti- body(1:5000,Cell SignalingTechnology,Danvers,MA) for1.5h atroomtemperaturein1%BSA(CellSignalingTechnology,Dan- vers,MA)andthenanti-rabbitHRPsecondaryantibody(1:2000;

1:10000,Dako,Glostrup,Denmark)for40min.Membraneswere then developed with an enhanced chemiluminescence kit (GE HealthCare,Little Chalfont, UK), exposed to X-ray ﬁlm (Kodak, Rochester,NY,US),andscanned[27].

For the analysis of ERK and Akt, equal amounts of protein (20␮g) were mixed with reducing 5×Laemmli buffer, loaded andseparatedin 4–20%precastTris-glycineSDSpolyacrilamide gels(Bio-Rad Hungary Ltd., Budapest, Hungary). Proteins were transferredontoapolyvinylidenediﬂuoridemembrane(Bio-Rad HungaryLtd.,Budapest,Hungary)at350mAfor2h.Transferwas visualizedwith Ponceau staining (Sigma, St Louis, MO). Mem- branes were blocked with 5% BSA (Santa Cruz Biotechnology, Inc.,Heidelberg,Germany)inTris-bufferedsalinecontaining0.05%

Tween-20(0.05%TBS-T;Sigma,StLouis,MO)atroomtemperature for2h.Membraneswereprobedwithprimaryantibodies(dilution 1:1000)overnightat4^◦C(phospho-Akt[Ser473]–#9271;Akt–

#9272;phospho-Erk1/2[Thr202/Tyr204]–#9106;Erk1/2–#9107;

Fig.4. Viabilityofunirradiated(A)andirradiated(B)NRVMafterMR-356treatment.

Bromodeoxyuridine(BrdU)proliferationassayperformedinunirradiated(C)and irradiatedNRVM(D)48hafterthetreatmentwithMR-356.Dataareexpressedas mean±SEM;incomparisontocontrol(100%).*p<0.05,n=8–16ineachgroup.

GAPDH– #5174),andwithcorrespondingHRP-conjugatedsec- ondaryantibodies(CellSignalingTechnology,Danvers,MA)for2h atroomtemperature.Signalsweredetectedwiththechemilumi- nescencekit.QuantiﬁcationwasmadebyintensityratiobyImage Lab4.1 (ImageLab^TM Software,Bio-Rad Laboratories,Inc., Cali- fornia,USA).Antibodiesdetectingphosphorylatedepitopeswere removedwithPiercestrippingbuffer(ThermoScientiﬁcLaborKft., Budapest,Hungary)beforeincubationwithantibodiesdetecting thetotalprotein.

2.6. Oxidativestressdetection

Thepresenceofsuperoxidewasdetectedwithaplatereader using the oxidative ﬂuorescent dye dihydroethidium (DHE) (SigmaSt.Louis,MO;D7008).Cellmembranesarefreelyperme- abletoDHEthatﬂuorescesredwhenoxidizedtoethidiuminthe presenceofsuperoxide.ThepresenceofgeneralROSproduction wasdetectedbyDCFH-DA(Sigma;D6883).Cardiacmyocyteswere rinsedwithPBS,thenincubatedwith10uMDHEorDCFH-DAat roomtemperaturefor60mininadarkchamber,Afterremoving

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Fig.5. Westernblotanalysis:pGHRHRandGAPDHhousekeepinggeneexpression (A).TheexpressionofGHRHRnormalizedtotheGAPDHhousekeepinggenein unirradiated(B)andirradiated(C)NRVMcultures.Westernblotswereperformed bothonuntreatedcellsandcellstreatedwith10nMJI-34.Resultsaremean±SEM (expressedin%ascomparedtonon-treatedcells);n=6sample/group,*p<0.05.

extradyewithD-PBS(pH7.4),theﬂuorescenceintensitywasmea- sured[28].

2.7. Statisticalanalysis

Resultsarepresentedasamean±SE.One-wayanalysisofvari- ance(ANOVA)followedbyFisher’sleastsigniﬁcantdifference(LSD) post-hoctestsorDunnet’spost-hoctestswereusedtoevaluatedif- ferencesbetweengroups.Twoexperimentalgroupswereanalyzed withthet-test.Westernblotresultswereanalyzedwithtwo-way analysisofvariance(ANOVA).Differenceswereconsideredsigniﬁ- cantifp<0.05.

3. Results

To determineoptimal experimental conditions, NRVMwere exposedtovariousradiationdoses,andviabilitywasmeasuredfol-

Fig.6.TheexpressionofpERK/ERKandtheexpressionofGAPDHhousekeeping geneinunirradiatedandirradiatedNRVMcultures.Westernblotswereperformed bothonuntreatedcellsandcellstreatedwith10nMJI-34.Resultsaremean±SEM;

n=6sample/group*p<0.05.

Fig.7.TheexpressionofpAkt/AktandtheexpressionofGAPDHhousekeepinggene inunirradiatedand10GyirradiatedNRVMcultures.Westernblotswereperformed bothonuntreatedcellsandcellstreatedwith10nMJI-34.Resultsaremean±SEM;

n=6sample/group*p<0.05.

lowingvaryinglatencytimes(Table1).Asigniﬁcantcelllossof50%

wasobtainedafter10Gydoseofradiationandlatencytimeof48h.

Thissetofconditionswaschosenandappliedthroughoutallthe experiments.

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Fig.8.ROSproductioninratioofviability(A)detected48hpostirradiation.TheeffectofJI-34onROSformationinunirradiated(B)andirradiatedNRVM(C);theeffect ofMR-356onROSproductioninunirradiated(D)andirradiatedcells(E).Thesuperoxidelevelinratioofviabilitydetected48hafterirradiation(F).TheeffectofJI-34on superoxidelevelsinunirradiated(G)andirradiatedNRVM(H);theeffectofMR-356onsuperoxidelevelinunirradiated(J)andirradiatedNRVM(I).Resultsaremean±SEM;

n=8–16ineachgroup*p<0.05.

First,theeffectofhGHRHwastestedoncellviabilityandcell proliferationinunirradiatedandirradiatedNRVMcultures.hGHRH didnotsigniﬁcantlyinﬂuencecellsurvivalascomparedtocontrol (Fig.1AandB).hGHRHwastestedforitseffectoncellproliferation viatheBrdUincorporationassay.Cellproliferationofbothirradi- atedandnon-irradiatedcellswasslightlystimulatedbyhGHRHat aconcentrationof50nM(Fig.1C).

TheeffectsoftheGHRHagonists,JI-34andMR-356,wereﬁrst testedinunirradiatedNRVM.TheadministrationofJI-34hadno effecton cell viabilityat concentrations of 1–500nM (Fig.2A).

However,inirradiatedcells,JI-34 showedaprotective effectat

concentrationsof10and100nM(Fig.2B).Anti-proliferativeeffect ofJI-34wasdetectedat50nMinunirradiatedandat1–50nMin irradiatedcultures(Fig.2CandD).Wehavetestedthesameparam- etersunderserum-deprivedconditionstotestthepossibleeffects oftheabsenceof1%FBSincludingtheavoidanceofthebindingof theanalogstotheplasmaproteins.NRVMculturesmaintainedin serum-freemediumfor2dayscontainedroughly50%lesscellsboth intheirradiatedandunirradiatedplates(Fig.3A).Again,astrong protectiveeffectofJI-34wasdetectedafterirradiation,atconcen- trationsof10and100nM(Fig.3B).TheGHRHagonist,MR-356,had nosigniﬁcanteffectoncellsurvivalinunirradiatedNRVMcultures

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(Fig.4A),butpreventedcelllossataconcentrationof500nMafter irradiation(Fig.4B).NoproliferativeeffectofMR-356wasdetected ineithertheunirradiatedortheirradiatedcultures(Fig.3CandD).

InordertoinvestigatetheexpressionofGHRHRsinNRVMatthe proteinlevel,Westernblotanalysiswasperformedusingananti- bodyabletodetectpGHRHRs.A52kDaproteinisoformwasreadily detectedinthesamplestogetherwithGAPDHusedasinternalcon- trol(Fig.5A).This52kDaglycosylatedGHRHRwasexpressedin bothirradiatedandunirradiatedcells.Thetreatmentofcellswith JI-34,didnotinﬂuencetheexpressionofGHRHRs(probablydueto lowdoseofagonist)inirradiated(Fig.5B)orunirradiatedNRVM (Fig.5C).Irradiationcausedsigniﬁcantdeclineinreceptorexpres- sionafter48h

ForfurtherinvestigationofintracellularactionofGHRHago- nists,theactivationofRISK/SAFEpro-survivalkinaseswastested.

ThephosphorylationratioofERKsignificantlyincreasedafterthe irradiationwith10Gy,whichwassignificantlyattenuatedbyJI- 34treatment(Fig.6).Likewise,thephosphorylationratioofAKT significantlyincreasedafterthe10-Gyirradiationwhichwassig- nificantlyattenuatedafterJI-34treatment(Fig.7).

Next,JI-34agonistwasadditionallytestedforitspossibleeffect on ROS production. Both the overall level of ROS and that of superoxidesignificantlyincreased48haftertheirradiation.JI-34 treatmentsignificantlydecreasedoverallROSproductionafterirra- diation,whileMR-356decreaseditslevelinbothirradiatedand unirradiatedcultures.Bothagonistsdecreasedsuperoxidelevelsig- nificantlyinirradiatedcultures(Fig.8).

4. Discussion

InthisinvitroNRVMmodel,theagonisticanalogsofGHRH,JI- 34andMR-356,reducedradiation-inducedcellloss.Thisistheﬁrst demonstrationthatGHRHagonistsexertprotectiveeffectinirra- diatedcardiacmyocytesviatheattenuationofROSformationand othermechanismspresentindifferentkindsofstresssituations.

ThesedataandtheexpressionofGHRHRinNRVMcontributetothe previousﬁndingsontheroleoftheGHRHsystemincardiomyocyte regeneration.Radiogeniclatecardiaccytotoxicityiswidely-studied ininvivomodels,however,limitedexperienceisavailablewiththe useofinvitromodelsappropriateforthestudyofacuteradiogenic effects.Thepresentstudyseemstoprovidereproducibleconditions forfurtherstudies.

ThemechanismofcardiocytoprotectionofGHRHagonistsisnot fullyclarified,andprobablydependsonthesystemexamined.The antiapoptoticeffectsofGHRHagonistshavebeendemonstratedin invivoexperimentalinfarctsintherat.Thechronicadministra- tionoftheGHRH-agonistJI-38upregulatedtheexpressionofthe anti-apoptoticBCL2, whiledown-regulated thatoftheproapop- toticBAXgene,andincreasedtheproliferationofcardiacprecursor cellsin experimentalheartinfarcts[22,24].Ina similarsystem, GHRHagonistsreducedtheexpressionofinflammatoryandpro- fibrotic markers [20]. In H9c2 cardiomyoblasts cultured under serum-deprivedcondition,agonisticanalogs ofGHRHrepressed theexpressionofaseriesofgenesrelatedtocardiacremodelling [20].SomestudiesconcludetothatGHRHoritsagonisticanalogs modulatethe kinases includedin theRISK and SAFE pathways [19,21].GHRHtreatmentpreventedapoptosisinducedbyserum starvationorisoproterenoltreatmentincardiacmyocytesthrough theinteractionwithmultiplesignallingmechanismsinvolvingthe cAMP/proteinkinaseA,ERK½andPI3K/Aktpathways[19].GHRH protected the rat heart fromI/R-caused injury administered at reperfusionviaactivationoftheRISKandSAFEpathways,asmea- sured20minafterthestartofreperfusion[21].Theearlyactivation ofERK1/2andAktwasdetectedbyGranata,inserum-deprived isolatedcardiacmyocytes[19].Inourstudy,thepost-irradiation

administrationofGHRHagonistsalsostronglyaffectedtheactiva- tionoftheSAFE/RISKsignalingpathways.BothJI-34andMR-356 treatmentsigniﬁcantlydecreasedphosphorylationofAktandERK after48hoflatencytime.Theirprotectiveeffectmaybeexplained bythisphenomenonsinceirradiationinducestheMAPKpathway andthephosphorylationofERKviaROS formation[29].Infact, ourstudyindicatesthattheagonistic analogs ofGHRH directly inﬂuenceoxidativestress.ROSformationreadilydetectable48h aftertheirradiationwaseffectivelyreducedbythepost-irradiation administrationofJI-34andMR-356.ThepretreatmentofC3Hmice withtheGHRHantagonist,JMR-132,causedaresponsedependent ontheradiationdoseofwholebodyradiation[30,31].

TheeffectsofGHRHortheagonisticanalogsaremediatedby theGHRHR.TheadministrationofGHRHantagoniststogetherwith GHRHoritsagonisticanalogsabolisheditsprotectiveeffectsoncar- diomyocytes[19,21].WebelievethatGHRHanditsreceptorshave physiologicalrolesintherecoveryfromcellinjury,andthatthe explanationforourﬁndingsinNRVMisthepresenceandfunction oftheGHRHR.Granataetal.havedemonstratedboththemRNAand proteinexpressionofthepGHRHRincardiaccells[19].ThepGHRHR hasbeen detectedin rat hearts bymeans of Westernblotting, immunohistochemistryand ligandbindingassay[24].Inconsis- tencewiththeseﬁndings,we showedthepresence ofa 52kDa proteinisoformofGHRHR,withWesternblotting;theexpression ofthisproteindeclinedbytimeinirradiatedNRVM,irrespective ofwhetherJI-34treatmentwasapplied.Thedifferenceirradiation made,maypointtotheroleoftheGHRHsystemincellrecovery afterradiationinjury.

Inconclusion,thepresentstudyprovidesawell-reproducible in vitro cell culture model for the investigation of irradiation- inducedcardiomyocytedamage,inwhichGHRHanalogshavebeen tested.GHRHagonisticanalogs,JI-34andMR-356exertedprotec- tiveeffectsonradiation-inducedcelldamage,henceGHRHagonists shouldbetestedinvivoaspotentialprotectiveagentsagainstradio- genicheartdamage.

Declarationofinterest

Theauthorsreportnoconﬂictofinterest.Theauthorsaloneare responsibleforthecontentandwritingofthepaper.

Acknowledgements

TheauthorswouldliketothankDr.MiklósJászberényi(Depart- ment of Pathophysiology,University of Szeged)for his support andguidancethroughoutthestudy.Thisstudywassupportedby NationalDevelopmentAgency–NewHungaryDevelopmentPlan TAMOP-4.2.2.A-11/1/KONV-2012-0035andTAMOP-4.2.2/B-10/1- 2010-0012andNationalResearch,DevelopmentandInnovation Ofﬁce–NKFIHPD106001(NKFI).Dr.GörbeheldBolyaiJánosfel- lowshipfromtheHungarianAcademyofSciences.

ProfessorFerdinandywasaSzentágothaiFellowoftheNational ProgramofExcellence(TAMOP4.2.4.A/2-11-1-2012-0001).

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,athttp://dx.doi.org/10.1016/j.phrs.2016.07.

036.

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