species and expression of matrix metalloproteinases generation by suppressing apoptosis of reactiveoxygen/nitrogen Activation protects of PPAR cardiac ␤ / ␦ myocytes from oxidativestress-induced Pharmacological Research

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ContentslistsavailableatScienceDirect

Pharmacological Research

jo u r n al hom e p ag e :w w w . e l s e v i e r . c o m / lo c a t e / y p h r s

Activation of PPAR ␤ / ␦ protects cardiac myocytes from oxidative stress-induced apoptosis by suppressing generation of reactive

oxygen/nitrogen species and expression of matrix metalloproteinases

Eleftheria Barlaka

^a

, Anikó Görbe

^b,d

, Renáta Gáspár

^b

, János Pálóczi

^b

, Péter Ferdinandy

^c,d

, Antigone Lazou

^a,∗

aLaboratoryofAnimalPhysiology,SchoolofBiology,AristotleUniversityofThessaloniki,Greece

bCardiovascularResearchGroup,DepartmentofBiochemistry,UniversityofSzeged,Hungary

cDepartmentofPharmacologyandPharmacotherapy,SemmelweisUniversity,Budapest,Hungary

dPharmahungaryGroup,Szeged,Hungary

a r t i c l e i n f o

Articlehistory:

Received2November2014

Receivedinrevisedform15March2015 Accepted15March2015

Availableonline28March2015

Keywords:

Cardiacmyocytes Oxidativestress Apoptosis

Matrixmetalloproteinases PPAR␤/␦

Caspase3 Bcl-2

Chemicalcompoundsstudiedinthisarticle:

Hydrogenperoxide(PubChemCID:784) GW0742(PubChemCID:9934458) GSK0660(PubChemCID:46233311)

a b s t r a c t

Heartfailurestillremainsoneoftheleadingcausesofmorbidityandmortalityworldwide.Amajor contributingfactorisreactiveoxygen/nitrogenspecies(RONS)overproductionwhichisassociatedwith cardiacremodelingpartlythroughcardiomyocyteapoptosis.Peroxisomeproliferator-activatedreceptors (PPARs)areligand-activatedtranscriptionfactorsthatbelongtothenuclearreceptorsuperfamilyand havebeenimplicatedincardioprotection.However,themolecularmechanismsarelargelyunexplored.In thisstudywesoughttoinvestigatethepotentialbeneﬁcialeffectsevokedbyactivationofPPAR␤/␦under thesettingofoxidativestressinducedbyH2O2inadultratcardiacmyocytes.TheselectivePPAR␤/␦ago- nistGW0742inhibitedtheH2O2-inducedapoptosisandincreasedcellviability.Inaddition,generation ofRONSwasattenuatedincardiacmyocytesinthepresenceofPPAR␤/␦agonist.Theseeffectswereabol- ishedinthepresenceofthePPAR␤/␦antagonistindicatingthattheeffectwasthroughPPAR␤/␦receptor activation.TreatmentwithPPAR␤/␦agonistwasalsoassociatedwithattenuationofcaspase-3andPARP cleavage,upregulationofanti-apoptoticBcl-2andconcomitantdownregulationofpro-apoptoticBax.

Inaddition,activationofPPAR␤/␦inhibitedtheoxidative-stress-inducedMMP-2andMMP-9mRNA upregulation.ItisconcludedthatPPAR␤/␦activationexertsacytoprotectiveeffectinadultratcardiac myocytessubjectedtooxidativestressviainhibitionofoxidativestress,MMPexpression,andapoptosis.OurdatasuggestthatthenovelconnectionbetweenPPARsignalingandMMPdown-regulationin cardiacmyocytesmightrepresentanewtargetforthemanagementofoxidativestress-inducedcardiac dysfunction.

1. Introduction

Cardiovasculardiseasesstillremainoneoftheleadingcauses ofmortalityandmorbidityworldwide.Overthepastyears,clinical andexperimentalstudieshaveprovidedsubstantialevidencethat oxidativestress,deﬁnedasanimbalancebetweenfreeradicalpro- ductionandtheirdetoxiﬁcationbyantioxidants,isanimportant contributingfactorinthepathogenesisofavarietyofcardiovas- culardiseasesincludingischemicheartdiseaseandheartfailure

∗ Correspondingauthorat:LaboratoryofAnimalPhysiology,SchoolofBiology, AristotleUniversityofThessaloniki,Thessaloniki54124,Greece.

Tel.:+302310998381;fax:+302310998269.

E-mailaddress:lazou@bio.auth.gr(A.Lazou).

[1,2]. Excessivegeneration of reactive oxygen/nitrogen species (RONS) causescellular dysfunction,protein and lipidperoxida- tion,DNAdamage,activationofmatrixmetalloproteinases(MMPs) andcanleadtoirreversiblecelldamageandcelldeath.Thecon- trolofmyocytelossthroughsuppressionofcelldeathpathways representsapromisingstrategytopreventcardiacdiseases.Ter- minally differentiated adult cardiac myocytes die primarily by apoptosis or necrosis[3]. Mitochondrial damage, cytochrome c releaseandactivationofcysteineproteasescaspase9and3have beenimplicatedinapoptoticpathways.TheBcl-2familyproteins, which containboth pro-apoptotic and anti-apoptoticmembers, havebeenrecognizedasimportantmodulatorsofcardiacmyocyte apoptosisasin othercells [4,5].Bcl-2familyproteinsactatthe mitochondriaeitherasheterodimersorashomodimerstoregu- latecytochromecwhereasthedynamicequilibriumbetweensuch http://dx.doi.org/10.1016/j.phrs.2015.03.008

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E.Barlakaetal./PharmacologicalResearch95–96(2015)102–110 103

complexesappearstodeterminethepredispositiontoapoptosis [6].

Thezinc-dependentendopeptidases,matrixmetalloproteinases (MMPs),which havebeenknownfor theirability toproteolyse extracellular matrix proteins, play an important role in tissue remodelingassociatedwithvariousphysiologicalandpathological processesincludingchronicheartfailure[7].Itisnowrecognized thatMMPscanalsohavenon-matrixrelatedintracellularprotein targetsandcanregulatediversecellularfunctionscontributingto cardiacdysfunction,suchasoxidative stress-inducedcell death [8–10].Thus,MMPsareconsideredtobeviabledrugtargetsinthe therapyofcardiacpathologies.

Peroxisomeproliferatoractivatedreceptors(PPARs)areligand activatedtranscription factorsthat belongtothesuperfamilyof nuclearhormonereceptorsandinclude3members(␣,␤/␦,␥)with tissuespecificbutoverlappingexpressionpatterns.Uponactivation bytheirnaturalorpharmaceuticalspecificligands,PPARsregulate theexpressionofgenesmostlyinvolvedinlipidandcarbohydrate metabolism,energyproductionandinflammation[11].Ithasbeen alsoshownthatPPARsmayplayaroleinregulatingtranscriptional expressionofantioxidants,suchasCu/Zn-superoxidedismutase (SOD1),manganese superoxide dismutase (SOD2),and catalase [12,13].Furthermore,emergingevidenceindicatesthatactivation ofPPARs,inparticularPPAR␣orPPAR␥,mayexertcardioprotec- tiveeffects beyondtheirmetaboliceffects,decreasinginfarction afterI/Randpreventingcardiacremodelingandfailure[14–17].

However,themechanismsunderlyingtheseprotectiveeffectsare largelyunexplored,anditisstillamatterofdebatewhethercar- dioprotectionisattributabletothemodulationofcardiacenergy metabolismortoantioxidativeandanti-inﬂammatoryeffectsof PPARs[18,19].AlthoughPPAR␤/␦isthepredominantsubtypein theheart,itistheleaststudiedmemberofthePPARfamily.Car- diacPPAR␤/␦deletioninmiceresultedindepressedbioenergetics, cardiachypertrophy,andcongestiveheartfailure[20]whileithas beenshownthatPPAR␤/␦isessentialfortheadulthearttomaintain mitochondrialcapacityandoxidativemetabolism[21].

GiventheemergingroleofPPAR␤/␦inregulatingmitochondrial biogenesisandmetabolism,wehypothesizedthatPPAR␤/␦acti- vationmayameliorateoxidativestress-inducedcardiacmyocyte damage.WeusedthehighlyselectiveandpotentPPAR␤/␦agonist GW0742[22]andaspeciﬁcPPAR␤/␦antagonist,GSK0660[23]as pharmacologicaltoolstoevaluatetheeffectsofreceptoractivation inH₂O₂-inducedcardiacmyocyteapoptosisandexplorethecon- tributingmechanisms.WedemonstratethatPPAR␤/␦activationin cardiacmyocytesleadstotheinhibitionofRONSgeneration,down- regulationofMMP-2andMMP-9gene expressionandprevents apoptosis.

2. Materialsandmethods 2.1. Animals

MaleWistarratsweighingbetween250and300gwereused.

Animalsreceivedpropercareincompliancewiththe“Guidelines for the Care and Use of Laboratory Animals” publishedby US NationalInstitutesofHealth(NIHpublicationNo85-23,revised 1996) and the “Principlesof laboratoryanimal care” published by theGreek Government (160/1991)based on EU regulations (86/609).Allsurgerywasperformedundersodiumpentobarbital anesthesia,andalleffortsweremadetominimizesuffering.

2.2. Isolationofadultratcardiacmyocytesandtreatments

Ventricularmyocyteswereisolatedbycardiacretrogradeaor- ticperfusionandcollagenase treatmentas describedpreviously

[24]. Cells were ﬁnally resuspended in incubation medium, in which added Ca²⁺ was gradually increased to 1mM. Prepara- tionswereconsideredsatisfactoryonlyiftheyieldofrod-shaped cells was more than 70%. Experiments were performed30min afterdissociation.Cardiacmyocyteswereeitherleftuntreated,or theywerepre-treatedwiththePPAR␤/␦agonistGW0742(50nM) (Sigma–AldrichChemieGmbH,Taufkirchen,Germany)for10min beforeexposuretoH2O2 (0.1mM).Whenthe PPAR␤/␦antago- nistGSK0660(0.5␮M)(TocrisBioscience,Bristol,UK)wasused, itwasadded10minbeforetheadditionoftheagonistGW0742.

TheconcentrationofH2O2toinduceapoptosiswaschosenaccord- ingtoourpreviousstudies[24–26].Theoptimalconcentrationof PPAR␤/␦agonistandantagonistwerechosenaccordingtoprelim- inaryexperiments.Preliminaryexperimentsalsoestablishedthat theconcentrationsoftheagonistandantagonistuseddidnotaffect theviabilityofcardiacmyocytes.Theexperimentalprotocolsare summarizedinFig.1.

2.3. Determinationofcellviability

Cellviabilitywasdeterminedusingthe3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay which assesses mitochondrialactivityin livingcells. Cardiacmyocytes (10⁵/sample)wereeitherleftuntreatedortheywerepretreated with PPAR␤/␦ agonist and/or antagonist (10min), followed by exposuretoH₂O₂for3h.Tetrazoliumbromideataconcentration 0.1mg/mlwasadded10minbeforetheendofincubation.Atthe endofincubation,1mlofethanolwasaddedfor10mintodissolve formazanprecipitate.Absorbancewasmeasuredat570nmusinga microplateautoreader.Cellviabilitywascalculatedaspercentage of control OD. Negativecontrols wereused to ensure that the drugsusedforthecelltreatmentsdidnotinterfereontheirown withtheformationofformazan.

2.4. Measurementoflactatedehydrogenase(LDH)

LDH release into the cell culture medium was used as an indicatorofcelldeath. LDHactivitywasdeterminedbyspectro- photometric analysis measuringthe decrease in absorbance at 340nmresultingfromtheoxidationofNADH.Attheendoftreat- ments,cellculturemediumwascollectedand LDHactivitywas measuredafteradditionofNADH(0.17mM)andsodiumpyruvate (1mM).LDHactivitywasexpressedasmunits/mgafterdetermi- nationofproteincontentpersamplebyBradfordassay.

2.5. Evaluationofapoptosis

Apoptosiswasdeterminedbystainingcells withAnnexinV- FITCandpropidiumiodideforflowcytometricanalysis(Biolegend Europe).Briefly,aftertreatments,cells werewashedtwicewith coldphosphate-bufferedsalineandresuspended.Thecells were incubatedwithAnnexinV-FITCandpropidiumiodidefor15min inthedark.Thesampleswerethen subjectedtoflowcytomet- ricanalysistodetectthepercentage ofapoptotic (FITC-stained) and necrotic (PI-stained) cells in a given population. Fluores- cence was measured on a CyFlow ML flow cytometer (Partec GmbH,Germany).Preliminary experimentsestablishedthat the agonistGW0742 ortheantagonist GSK0660usedfor thetreat- mentofcellsdidnotinterfereontheirowninthemeasurementof fluorescence.

2.6. RONSdetection

Intracellular RONS production was evaluated using the 5- (and-6)-chloromethyl-2,7-dichlorodihydroﬂuorescein diacetate (CM-H2DCFDA) (Sigma–Aldrich Chemie GmbH, Taufkirchen,

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Fig.1.Diagramofexperimentalprotocolfordrugadministrationandinductionofoxidativestressincardiacmyocytes.Cardiacmyocyteswereeitherleftuntreated(control) ortheywerestimulatedwithH2O2(0.1mM).Whenused,PPAR␤/␦agonist(GW,50nM)wasaddedfor10minbeforestimulationwithH2O2.WhenthePPAR␤/␦antagonist wasused(GSK,0.5␮M),itwasadded10minbeforetheadditionofPPAR␤/␦agonistGW0742.

Germany). In brief, cardiac myocytes were loaded with CM- H₂DCFDA (5␮g/ml) and then, they were either left untreated ortheywerepretreatedwiththePPAR␤/␦agonistGW0742 for 10minand/ortheantagonistGSK0660(0.5␮M),inthepresence orintheabsenceofH₂O₂(0.1mM)for3h.RONS-inducedfluores- cencewasdetectedbyflowcytometry(CyFlowML,PartecGmbH, Germany).PreliminaryexperimentsestablishedthatGW0742or GSK0660didnotinterfereontheirownin themeasurement of fluorescence.

2.7. IsolationofRNAandquantitativePCR(qPCR)

Total RNA was extracted using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manu- facturer’sinstructions. TheRNA was resuspended in 0.1% (v/v) diethylpyrocarbamate-treated water and its concentration was determined by absorbance at 260nm. The integrity of isolated RNAwasveriﬁedona 1%agarosegel.For cDNAsynthesis, l␮g oftotal RNA wasdenaturedin thepresence ofrandom 9-mers (Takara,Japan) ina reactionvolumeof10␮lat65^◦C for5min.

RNAwasreverse-transcribedwithPrimeScriptReverseTranscrip- tase enzyme (Takara, Japan), dNTP’s (HyTest Ltd., Finland) and HumanPlacentalRibonuclease inhibitor(HTBiotechnologyLtd., Cambridge,UK). Thereaction,intotalvolumeof20␮l,wascar- riedoutfor1hat37^◦C,andwasterminatedbyheatinactivation ofthereversetranscriptaseat95^◦Cfor5min.qPCRanalysiswas performedusing a Real-TimePCR System(Applied Biosystems) withaspeciﬁcsetofprimersforeachgene(VBCGenomics).The forwardandreverseprimersusedforgeneexpressionwerethe following:

Catalase: For:CCTGTGAACTGTCCCTACCG

Rev:ACCCAGTCCCATGCTCTCTC

MMP-2: For:TTGATGACGATGAGCTGTGG

Rev:CTGCTGTATTCCCGACCATT

MMP-9: For:CCGTGGTCCCCACTTACTTT

Rev:CCGTGGTGCAGGACAAATAG

␤-actin: For:GCCCTGAGGCACTCTTCCA Rev:CGGATGTCCACGTCACACTTC

Eachreaction mixcontained 5␮l KAPASYBR FAST qPCR Kit (KapaBiosystems),0.3␮loligonucleotides(10pmoleach offor- wardand reverse primers)and 2␮lcDNA (diluted1:10). qPCR analysisof␤-actinwasperformedasanendogenouscontrol.PCR conditionswere95^◦Cfor20min,followedby40cyclesof95^◦C for 3sand60^◦C for 30min.FollowingqPCR, dissociationcurve analysiswasroutinelyperformedtocheckforaberrantampliﬁca- tionproducts(e.g.primers-dimers).Relativechangesinexpression werecalculatedusingthe2⁽⁻^ct)method.

2.8. Preparationofwholecellextracts

Cardiac myocytes were lysed at 4^◦C in a buffer containing 20mM ␤-glycerophosphate, 20mM HEPES pH 7.5, 50mM NaF, 2mM EDTA, 0.2mM Na₃VO₄, 10mM benzamidine, 5mM DTT, 0.3mMPMSF, 0.2mMleupeptin,0.01mM E64and1%(v/v)Tri- ton X-100. Lysateswere incubated onice for 10min and then centrifugedat4^◦C,for15minat10,000×g.Supernatantswerecol- lected,andtotalproteinwasmeasuredusingtheBradfordassay (BioRad,Hercules,CA,USA).

2.9. Immunoblotting

Total protein extracts were boiled with 0.33vol of SDS- polyacrylamidegelelectrophoresissamplebuffer(10%SDS(w/v),

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13%glycerol(v/v),300mMTris–HCl,pH6.8,130mMdithiothre- itoland0.2%bromophenolblue(w/v).Proteinswereseparatedby SDS-PAGEon10%acrylamide,0.275%(w/v)bis-acrylamideslabgels andtransferredelectrophoreticallyontonitrocellulosemembranes (0.45␮m).Nonspeciﬁcbindingsiteswereblocked(30minatroom temperature)with5% (w/v)nonfatmilk powderinTBST buffer [20mMTris–HClpH7.5,137mMNaCl,0.1%(v/v)Tween20].Mem- braneswereincubated(overnight,4^◦C)withprimaryantibodies dilutedinTBSTbuffercontaining5%(w/v)bovineserumalbumin, thenwashedinTBSTbuffer(3×5min,roomtemperature).Primary antibodiesagainstBcl-2(#2876),cleavedcaspase-3(#8698),PARP (#9542),␤-actin(#8457),␣-tubulin(#2125)(CellSignaling,Bev- erly,MA,USA)andBax(sc-7480,SantaCruzBiotechnology,Texas, USA)wereusedatvariousdilutions.Membraneswereincubated (60min,roomtemperature) withhorseradishperoxidase(HRP) conjugatedsecondaryantibodies(1/5000)inTBSTbuffercontain- ing1%(w/v)non-fatmilkpowder,andtheywerethenwashedin TBSTbuffer(3×5min,roomtemperature).Proteinsweredetected byenhancedchemiluminescence(CellSignaling,Beverly,MA,USA) andquantiﬁedbyscanningdensitometry.

2.10. Statisticalanalysis

Data are presented as mean±SEM of n independent experiments using different cell isolations from different animals.

Statisticalanalyses(ANOVAwithDunnett’scomparisontest)were performedwithsigniﬁcancetakenasbeingestablishedatp<0.05.

3. Results

3.1. ActivationofPPARˇ/ıwiththeagonistGW0742increases cellviabilityandattenuatesLDHreleaseduringexposureof cardiacmyocytestoH2O2

WehavepreviouslyshownthatH₂O₂,atconcentrationshigher than0.05mM,decreasesviabilityandinducesapoptosisincardiac myocytes[24–26].Consistentwiththesestudies,exposureofcar- diacmyocytesto0.1mMH₂O₂for3hdecreasedtheviabilityof cardiacmyocytesbyalmost40% andcaused analmostfourfold increaseinLDHactivitymeasuredintheculturemedium(Fig.2A andB).Pre-treatmentofcardiacmyocyteswiththePPAR␤/␦agonist, GW0742, signiﬁcantly enhanced survival while attenuated LDH release. Inorder todeterminewhetherthis is a PPAR␤/␦- mediatedeffect,thespeciﬁcantagonistofPPAR␤/␦,GSK0660,was used. As shown in Fig. 2A, the PPAR␤/␦ agonist GW0742 was unabletoincreasecellviabilityin thepresence ofthePPAR␤/␦

antagonistGSK0660indicatingthatthiseffectoncardiacmyocyte survivalisdependentofPPAR␤/␦activation.Similarly,theantag- onistabolishedtheprotectiveeffectofPPAR␤/␦agonistonLDH release(Fig.2B).NeithertheagonistGW0742northeantagonist GSK0660hadanyeffectoncellsurvivalorLDHactivitywhenused alone.

3.2. ActivationofPPARˇ/ıwiththeagonistGW0742protects cardiacmyocytesfromH₂O₂-inducedapoptosis

Consistent with previous data [24–26] exposure of cardiac myocytesto0.1mMofH₂O₂ resultedinalmost50%increasein therate ofapoptosis (Fig. 3A)whereas pre-treatment withthe PPAR␤/␦agonist,GW0742,signiﬁcantlyattenuatedapoptosis.In thepresenceofthePPAR␤/␦antagonistGSK0660,thePPAR␤/␦ago- nistGW0742wasunabletoattenuatecardiacmyocyteapoptosis indicatingthattheprotectiveeffectoftheagonistisdependenton PPAR␤/␦activation.Tocorroboratefurthertheseresults,caspase- 3 activation and poly ADP-ribose polymerase (PARP) cleavage were alsoevaluated under thesame conditions. Theactivation of caspase-3, the primary executioner of cell death byproteo- lytic processing of pro-caspase, serves as an early marker of apoptosisinvariouscelltypes.Theactivationofcaspasesresults in proteolyticcleavage of numerous substratesincluding PARP, a nuclear enzyme involved in DNA repair that is considered to be a hallmark of apoptosis [27]. As shown in Fig. 3B and C, pre-treatmentof cardiacmyocyteswiththePPAR␤/␦ agonist GW0742inhibitedH₂O₂-inducedcaspase-3andPARPcleavage,an effectthatisreversedinthepresenceofthePPAR␤/␦antagonist GSK0660.

3.3. ActivationofPPARˇ/ıwiththeagonistGW0742protects cardiacmyocytesfromH2O2-inducedoxidativestress

Given the protective effect of PPAR␤/␦ activation on car- diacmyocyteviability,andapoptosis,wesoughttoexaminethe potentialof PPAR␤/␦ agonistGW0742 toameliorateintracellu- lar generation of RONS in cardiac myocytes.Cells were loaded withthefluorophoreCM-H2DCFDAbeforetreatmentwithH2O2, and/orPPAR␤/␦agonistGW0742andfluorescencewasdetermined withflowcytometry.Asexpected,exposureofcardiacmyocytes toH2O2 resulted inincreased generationof RONSindicative of oxidativestress(Fig.4).However,additionofthePPAR␤/␦agonist completelyabolishedtheincreaseofRONSinducedbyH₂O₂.The inhibitoryeffectofPPAR␤/␦agonistGW0742onintracellularRONS productionwasreversedwhentheantagonistGSK0660wasadded,

Fig.2.Effectofpre-treatmentofcardiacmyocyteswithPPAR␤/␦agonistGW0742onviabilityandH2O2-inducedLDHrelease.Cardiacmyocyteswereeitherleftuntreatedor theywerepretreatedwithPPAR␤/␦agonistGW0742(GW,50nM)and/orPPAR␤/␦antagonistGSK0660(GSK,0.5␮M).Thentheywereincubatedintheabsenceorpresenceof H2O2(0.1mM)for3h.(A)CellviabilitywasmeasuredusingtheMTTmethod.(B)LDHactivitywasevaluatedbyspectrophotometricmeasurement.Resultsaremean±SEM of4–5independentexperiments.*p<0.05comparedwithcontrol,^#p<0.05comparedwithH2O2,^ˆp<0.05comparedwithcellstreatedwithH2O2andPPAR␤/␦agonist GW0742.

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Fig.3. Effectofpre-treatmentofcardiacmyocyteswithPPAR␤/␦agonistGW0742onH2O2-inducedapoptosislevelsandcleavedcaspase-3andPARP.Cardiacmyocyteswere eitherleftuntreatedortheywerepretreatedwithPPAR␤/␦agonistGW0742(GW,50nM)and/orPPAR␤/␦antagonistGSK0660(GSK,0.5␮M).Thentheywereincubatedin theabsenceorpresenceofH2O2(0.1mM)for3h.(A)Fortheevaluationofapoptosis,cellswereloadedwithannexin/PIandﬂuorescencewasdeterminedbyﬂowcytometry.

(B,C)Cellextractswereimmunoblottedforcleavedcaspase-3andPARP.Equalloadingwasveriﬁedbyimmunoblottingfor␣-tubulin.(B)Representativeblotsareshown.

(C)Blotswerequantiﬁedusingscanningdensitometryandplotted.Resultsaremean±SEMof3experimentswithindependentcellpreparations.*p<0.05comparedwith control,^#p<0.05comparedwithcellstreatedonlywithH2O2,^ˆp<0.05comparedwithcellstreatedwithH2O2andPPAR␤/␦agonistGW0742.

verifyingthatthiseffectismediatedthroughPPAR␤/␦activation.

Furthermore,themRNAlevelsofcatalase,whichisknowntobe aPPAR␤/␦targetgene[28],werealsomeasuredunderthesame conditions(Fig.5).Catalasewasup-regulatedincardiacmyocytes thatwerepre-treatedwithPPAR␤/␦agonistGW0742ascompared withthenon-treatedcellswhereasadditionofthePPAR␤/␦antag- onistabolishedthiseffectindicatingtheactivationofthereceptor.

Takentogethertheseexperimentspointtowardtheantioxidant roleofPPAR␤/␦activation.

3.4. PPARˇ/ıactivationmodulatesBcl-2familyprotein expressionduringcardiacmyocyteoxidativestress

WenextdeterminedwhethertheprotectiveeffectofPPAR␤/␦ activationcanbeattributabletoupregulationofBcl-2protein.The Bcl-2family proteinsarekeycomponentsoftheapoptoticpro- cessin cardiacmyocytesand theratioBcl-2/Baxiswidelyused asamarkerofapoptosis[24,25].We,thus,determinedthepro- teinlevelsoftheanti-apoptoticBcl-2andthepro-apoptoticBax.

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Fig.4.Effectofpre-treatmentofcardiacmyocyteswithPPAR␤/␦agonistGW0742onRONSgeneration.Cardiacmyocyteswerepre-loadedwith5-(and-6)-chloromethyl- 2,7-dichlorodihydrofluoresceindiacetate(CM-H2DCFDA)andthentheywereeitherleftuntreatedortheywerepretreatedwithPPAR␤/␦agonistGW0742(GW,50nM) and/orPPAR␤/␦antagonistGSK0660(GSK,0.5␮M).ThentheywereincubatedintheabsenceorpresenceofH2O2(0.1mM)for3h.ChangesinDCFfluorescencewere determinedbyflowcytometry.Resultsaremean±SEMof5independentexperiments.*p<0.05comparedwithcontrol,^#p<0.05comparedwithtreatmentwithH2O2,

ˆp<0.05comparedwithcellstreatedwithH2O2andPPAR␤/␦agonistGW0742.

Fig.5. Effectofpre-treatmentofcardiacmyocyteswithPPAR␤/␦agonistGW0742 oncatalasemRNAexpression.Cardiacmyocyteswereeitherleftuntreatedorthey werepretreatedwithPPAR␤/␦GW0742(GW,50nM)and/orPPAR␤/␦antagonist GSK0660(GSK,0.5␮M).Thentheywereincubatedintheabsenceorpresenceof H2O2(0.1mM)for3h.CatalasemRNAlevelsweredeterminedbyreal-timeRT-PCR andwerenormalizedtothemRNAlevelsofthehousekeepinggene␤-actin.Results aremean±SEMof5independentexperiments.*p<0.05comparedwithcontrol,

#p<0.05comparedwithtreatmentwithH2O2,^ˆp<0.05comparedwithcellstreated withH2O2andPPAR␤/␦agonistGW0742.

AsshowninFig.6,treatmentofcardiacmyocyteswithPPAR␤/␦ agonistfollowedbyH2O2resultedinincreasedexpressionlevels ofanti-apoptoticBcl-2comparedwithcellsthatwereexposedto H₂O₂alone.Inasimilarfashion,theH₂O₂-inducedincreaseinBax wasattenuatedbythePPAR␤/␦agonist.Theeffectoftheagonist GW0742ontheexpressionofBcl-2familyproteinswasreversed inthepresenceoftheantagonistGSK0660,indicatingthatthisis aPPAR␤/␦-mediatedeffect.Takentogethertheseresultssuggest thatactivationofPPAR␤/␦leadstoincreasedresistancetoH₂O₂- inducedcardiacmyocyteapoptosisthroughupregulationofBcl-2 andconcomitantdownregulationofBax.

3.5. PPARˇ/ıactivationleadstoinhibitionofMMP-2andMMP-9 mRNAexpressionincardiacmyocytesexposedtoH2O2

Severalstudieshavedocumentedtheroleofmatrixmetallo- proteinases(MMPs)incardiacremodelingandthedevelopment

ofcardiacdisease[29].Furthermore,recentevidenceimplicates MMPsintheproteolyticregulationofseveralintracellularproteins inmyocardialoxidativestressinjuryleadingtocardiacdysfunc- tionandcelldeath[30].Thus,wedeterminedtheeffectofPPAR␤/␦ activationonMMP-2andMMP-9mRNAexpression.Asexpected, exposureofcardiacmyocytestoH₂O₂resultedinincreasedmRNA expression of both MMP-2 and MMP-9 (Fig. 7A and B). Pre- treatment of cardiac myocytes with PPAR␤/␦ agonist GW0742 resultedinattenuationofMMP-2andMMP-9mRNAlevels(Fig.7A andB).ThiseffectwasreversedbytheadditionofPPAR␤/␦antagonist.

4. Discussion

Inthisstudy,wefocusonthepotentialbeneﬁcialroleofPPAR␤/␦ againstoxidativestressinduceddysfunctionincardiacmyocytes.

WeprovideevidencethatPPAR␤/␦activationbyitsspeciﬁcagonist GW0742protectscardiacmyocytesfromapoptosisviasuppression ofRONSgenerationandMMPgeneexpression.

TheroleofPPARsinthepathogenesisofvariouscardiovascular diseaseshasnotbeenfullyelucidatedandremainsamatterofcon- troversy.AlthoughPPAR␤/␦isthelessstudiedisoform,especially intheheart,thereismountingevidencesupportingitscardiopro- tectiverole.PPAR␤/␦isdownregulatedinratheartafterI/R[14]

whereasconditionalcardiac-speciﬁcdeletionofPPAR␤/␦results inincreasedcardiaclipidaccumulationandcardiomyopathy[20].

Furthermore, PPAR␤/␦ agonists arecardioprotective in the set- tingofI/Rasmanifestedbyareducedinfarctsizeandimproved postischaemicrecoveryofcontractilefunctionindifferentanimal models[31,32,Lazouetal.unpublisheddata].Inaddition,activa- tionofPPAR␤/␦hasbeenrelatedtoinhibitionofhypertrophyand inﬂammationthroughthesuppressionofNF-␬Bandinﬂammatory cytokines[33–35].

H₂O₂, as a physiologically relevant form of oxidative stress, induces cardiac myocyte apoptosis [24–26]. In line with a cardioprotectiveroleofPPAR␤/␦,wedemonstratethatpretreat- ment of cardiac myocytes with the PPAR␤/␦ selective agonist GW0742increasescellviabilityandinhibitsH₂O₂-inducedapoptosis(Figs.2and3)Furthermore,pretreatmentwiththeselective

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Fig.6.PPAR␤/␦activationmodulatesBcl-2familyproteinexpressionduringoxidativestress.Cardiacmyocyteswereeitherleftuntreatedortheywerepretreatedwith PPAR␤/␦agonistGW0742(GW,50nM)and/orantagonistGSK0660(GSK,0.5␮M).ThentheywereincubatedintheabsenceorpresenceofH2O2(0.1mM)for3h.Cellextracts wereimmunoblottedforBcl-2andBax.Equalloadingwasveriﬁedbyimmunoblottingfor␤-actin.Representativeblotsareshown(upperpanels).Blotswerequantiﬁed usingscanningdensitometryandplotted(lowerpanels).Resultsaremean±SEMof3–4experimentswithindependentcellpreparations.*p<0.05comparedwithcontrol,

#p<0.05comparedwithcellstreatedonlywithH2O2,^ˆp<0.05comparedwithcellstreatedwithH2O2andPPAR␤/␦agonistGW0742.

PPAR␤/␦ antagonist GSK0660 abolishes theprotection afforded byGW0742,andthisﬁndingimpliesthattheobservedbeneﬁcial effectsofGW0742areindeedsecondarytotheselectiveactivation ofthePPAR␤/␦receptor.Thereareseveralpotentialmechanisms bywhichthePPAR␤/␦agonistmayexertitsanti-apoptoticeffect.

Our results showthat additionof thePPAR␤/␦ agonist in cardiacmyocytes amelioratesoxidative stressasevidenced bythe inhibitionofRONSgeneration(Fig.4).Manyimportantendoge- nousanti-oxidantssuchasCu/Zn-superoxidedismutase(SOD1), manganesesuperoxidedismutases (SOD2) and catalasecontain functionalPPARresponseelements(PPREs)intheirpromoteranda

roleofPPAR␤/␦inthetranscriptionalregulationoftheseenzymes hasbeenpreviouslyreported[21,28,36].Upregulationofthecata- lase gene wasobserved in the presence of PPAR␤/␦agonist in cardiacmyocytes(Fig.5).Althoughcatalaseactivitywasnotmea- suredinthisstudy,increasedcatalasegeneexpressionmayindicate anincreasedanti-oxidantcapacity.PPAR␤/␦hasbeensuggestedto inhibitROSgenerationthroughtheinhibitionofNADPHoxidasesin vascularsmoothmusclecellsexposedtoangiotensinII[37].How- ever,astheH₂O₂-inducedRONSgenerationincardiacmyocytes cannotbefullyrecoveredinthepresenceofbothPPAR␤/␦ago- nistandantagonist(Fig.4),wecannotexcludethepossibilitythat

Fig.7. PPAR␤/␦activationinhibitsMMP-2andMMP-9geneexpression.Cardiacmyocyteswereeitherleftuntreatedortheywerepre-treatedwithPPAR␤/␦agonistGW0742 (GW,50nM)and/ortheantagonistGSK0660(GSK,0.5␮M)for10min.ThentheywereincubatedintheabsenceorpresenceofH2O2(0.1mM)for3h.(A,B)TotalRNAwas extractedandMMP-2andMMP-9weredeterminedbyreal-timeRT-PCR.LevelsofmRNAwerenormalizedtothemRNAlevelsofthehousekeepinggene␤-actin.Results aremean±SEMof4–5experimentswithindependentcellpreparations.*p<0.05comparedwithcontrol,^#p<0.05comparedwithcellstreatedonlywithH2O2,^ˆp<0.05 comparedwithcellstreatedwithH2O2andPPAR␤/␦agonistGW0742.

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theagonistGW0742hasanadditionalanti-oxidanteffectindepen- dentofPPAR␤/␦activation.Infact,wehavepreviouslyshownthat PPAR␤/␦agonistGW0742caninhibitthephenylephrine-induced RONS generation in cardiac myocytes through a non-genomic redoxmechanism[38].

Bcl-2 family proteins are key components of the apoptotic processincardiacmyocytes[5].Bcl-2proteinscanberegulated eitherbytranscription, heterodimerizationor proteolyticcleavage,whereastheirphosphorylationstatusplaysimportantrolein thoseevents.TreatmentwithPPAR␤/␦agonistGW0742reversed thedetrimentaleffectofH2O2onBcl-2expressionwhileitledtoa signiﬁcantdecreaseoftheproapoptoticBaxproteinlevels(Fig.6).

These resultsare consistentwith previousstudies demonstrat- ingcardioprotectionassociatedwithmodulationofcardiaclevels ofantiapoptotic(Bcl-2andBcl-xl)orproapoptotic(BaxandBad) proteins[17,31,39].Thepresent datado not documenta direct causallinkbetweenPPAR␤/␦activationandmodulationofBcl-2 orBaxexpression.ItispossiblethattheobservedBcl-2upregula- tioninourexperimentalsettingresultsfromthePPAR␤/␦-induced ameliorationofoxidativestressorPPAR␤/␦crosstalkwithother signalingpathways. Furthermore,wecannot excludethepossi- bility that besides Bcl-2 and Bax, other proteins acting in the mitochondrial-dependentapoptoticpathwayarealsoaffectedby PPAR␤/␦activationandmightcontributetotheobservedcardio- protectiveeffects.

Increased RONS promote MMP activities. MMP activation is associatedwithdegradationandremodelingofextracellularmatrix underpathologicalconditions,thusleadingtoheartfailure[40].

It is now becoming clear that, beyond their effects on matrix metabolism,MMPs(inparticularMMP-2)arecapableofmodulat- inginﬂammatorypathwaysbyprocessingcytokines,chemokines, and growth factors [41] as well as affecting many other rele- vantmediatorsinvolvedinavarietyoffunctionsandpathogenic mechanismsleadingtocardiacdysfunction[30,42].Infact,MMP- 2hasbeenshowntobeinvolvedintheintracellulardegradation of troponinI [43], myosinlight chain 1 [44] and nuclearPARP [45,46].Inhibition of MMP activityhas been shown to protect themyocardiumformoxidative/nitrosativestress-inducedinjury suggestingnoveltargetsforcardiacfailuremanagement[10,47].

ThedataofthisstudyclearlydemonstratethatPPAR␤/␦agonist GW0742 downregulates the H2O2-induced MMP-2and MMP-9 geneoverexpression(Fig.7)incardiacmyocytes.Moreoverthis effect is reversed by administration of the PPAR␤/␦ antagonist GSK0660.ItisnotclearwhetherthereisadirectlinkbetweenPPAR activationandMMPinhibition.TheinhibitoryeffectofPPARson NF-␬Biswelldocumented[19],whereastherearereportsdemon- stratingaroleofNF-␬BintheactivationofMMPs[48,49].Thus, the observed MMP dowregulation in cardiac myocytes (Fig.7) maybetheresultoftheanti-inﬂammatoryactionofPPAR␤/␦.In thiscontext,ithasbeenshownthataPPAR␤/␦agonistmarkedly suppressestheIL-1␤-inducedMMP-2and MMP-9expressionin vascular smooth muscle cells [50]. This issue warrants further investigation.Furthermore,wecannotruleoutthepossibilitythat MMP suppressionby the agonistGW0742 in our experimental model might be a secondary effect through themodulation of oxidativestress.

Inconclusion,ourresultsdemonstratethatthePPAR␤/␦ago- nistGW0742inhibitscardiacmyocyteinjurybyreducingoxidative stressandinhibitingMMPexpressionand apoptosis.Itsprotec- tiveeffectsaredependentontheactivationofPPAR␤/␦because theyareabolishedbypretreatmentwiththePPAR␤/␦antagonist GSK0660.Althoughadditionalexperimentsareneededtoclarify theroleofPPAR␤/␦incardiacmyocytesapoptosis,theresultspre- sentedheresuggestthattheconnectionbetweenPPARsignaling andMMPsuppressionmightrepresentanewtargetfortheman- agementofoxidativestress-inducedcardiacdysfunction.

Acknowledgements

Theworkhasbeenco-ﬁnancedbytheEuropeanUnion(Euro- peanSocialFund)andGreekNationalFunds(GSRT-HUN57),and the Hungarian Scientiﬁc Research Fund: OTKA PD 106001. G.A holdsa “JánosBolyai Fellowship”fromtheHungarian Academy of Sciences, P.J.holds a “Apáczai-Csere János Fellowship” from TÁMOP-4.2.4.A/2-11/1-2012-0001(NationalExcellenceProgram).

P.F.isaSzentágothaiFellowoftheNationalProgramofExcellence (TAMOP4.2.4.A/2-11-1-2012-0001).

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