B-type natriuretic peptide expression and cardioprotection is regulated by Akt dependent signaling at early reperfusion

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Peptides

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

B-type natriuretic peptide expression and cardioprotection is regulated by Akt dependent signaling at early reperfusion

L. Breivik

, A. Jensen

, S. Guvåg, E.K. Aarnes, A. Aspevik, E. Helgeland, S. Hovland, T. Brattelid, A.K. Jonassen

DepartmentofBiomedicine,FacultyofMedicineandDentistry,UniversityofBergen,Norway

a r t i c l e i n f o

Articlehistory:

Received28October2014

Receivedinrevisedform9January2015 Accepted13January2015

Availableonline16February2015

Keywords:

BNP

Myocardialinfarction Ischemia

Reperfusion Postconditioning Akt

a b s t r a c t

ExogenouslyadministeredB-typenatriureticpeptide(BNP)hasbeenshowntooffercardioprotection throughactivationofparticulateguanylylcyclase(pGC),proteinkinaseG(PKG)andKATPchannelopening.

ThecurrentstudyexploresifcardioprotectionaffordedbyshortintermittentBNPadministrationinvolves PI3K/Akt/p70s6kdependentsignaling,andwhetherthissignalingpathwaymayparticipateinregulation ofBNPmRNAexpressionatearlyreperfusion.IsolatedLangendorffperfusedratheartsweresubjected to30minofregionalischemiaand120minofreperfusion(IR).Applyingintermittent3×30sinfusionof BNPpeptideinapostconditioninglikemanner(BNPPost)reducedinfarctsizeby>50%comparedtocon- trols(BNPPost17±2%vs.control42±4%,p<0.001).Co-treatmentwithinhibitorsofthePI3K/Akt/p70s6k pathway(wortmannin,SH-6andrapamycin)completelyabolishedtheinfarct-limitingeffectofBNP postconditioning(BNPPost+Wi36±5%,BNPPost+SH-641±4%,BNPPost+Rap37±6%vs.BNPPost17±2%, p<0.001).Inhibitionofnatriureticpeptidereceptors(NPR)byisatinalsoabrogatedBNPPostcardioprotec- tion(BNPPost+isatin46±2%vs.BNPPost17±2%,p<0.001).BNPPostalsosignificantlyphosphorylatedAkt andp70s6katearlyreperfusion,andAktphosphorylationwasinhibitedbySH-6andisatin.Myocardial BNPmRNAlevelsintheareaatrisk(AA)weresignificantlyelevatedatearlyreperfusionascompared tothenon-ischemicarea(ANA)(Ctr(AA)2.7±0.5vs.Ctr(ANA)1.2±0.2,p<0.05)andtheischemiccontrol tissue(Ctr(AA)2.7±0.5vs.ischemia1.0±0.1,p<0.05).Additionalexperimentsalsorevealedasignificant higherBNPmRNAlevelinischemicpostconditioned(IPost)heartsascomparedtoischemiccontrols (IPost6.7±1.3vs.ischemia1.0±0.2,p<0.05),butshowednodifferencefromcontrolsruninparallel (Ctr5.4±0.8).AktinhibitionbySH-6completelyabrogatedthiselevation(IPost6.7±1.3vs.IPost+SH- 61.8±0.7,p<0.05)(Ctr5.4±0.8vs.SH-61.5±0.9,p<0.05).Inconclusion,Aktdependentsignalingis involvedinmediatingthecardioprotectionaffordedbyintermittentBNPinfusionatearlyreperfusion, andmayalsoparticipateinregulationofreperfusioninducedBNPexpression.

©2015TheAuthors.PublishedbyElsevierInc.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

The polypeptidehormone brain natriuretic peptide(BNP) is part of the cardiac natriuretic peptide (NP) family, comprising atrial(ANP),BNPandC-typenatriureticpeptide(CNP)[1–6].Until recently,BNPreleasewassolelybelievedbecausedbyventricu- larwalldistension,butaccumulatingevidencesupportischemia [7] and hypoxia[8] to beindependent triggers ofBNP release.

Furthermore, exogenous administered BNP can reduce injuries associatedwithischemiareperfusionintheisolatedheart[9–13].

∗Correspondingauthor.

E-mailaddress:lars.breivik@biomed.uib.no(L.Breivik).

1 Theseauthorscontributedequallytothisstudy.

ThisBNPmediatedcardioprotectionappearstobemediatedviathe natriureticpeptidereceptortypeA(NPR-A)[14],activatingacGMP- dependent protein kinaseG (PKG) [15] that furthermodulates openingofmitochondrialandsarcolemmalKATPchannels(mKATP

andsK_ATP)andendogenousnitricoxidesynthase(eNOS)[9].How- ever,activationofothersignalingpathwaysalsoappearspivotalin NPinducedcardioprotection,asANPadministrationjustpriorto reperfusionlimitedinfarctsizeinrabbithearts,aprotectionthat waslostwheninhibitingeitherNPR-A,PI3K(phosphatidylinositol 3-kinase),ERK (extracellularsignal-regulated kinase), ormK_ATP channels[16].PI3Kispartofthecytoprotectivereperfusioninjury salvagekinasessignalingpathway(RISK)thatdownstreamphos- phorylatesAktandp70S6k[17].Currently,however,itisunknown whetherexogenouslyadministeredBNPmayactivateAktdepend- entRISKsignalingintheisolatedratheartatreperfusion.

http://dx.doi.org/10.1016/j.peptides.2015.01.011

0196-9781/©2015TheAuthors.PublishedbyElsevierInc.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

BNPisconsideredtobeanimmediate-earlygenethathasproven tobebothrapidlysynthesizedandreleased(within1h)inresponse toischemiainvivo[18]andafterreoxygenationintheexvivoper- fusedratheart [8,11].Recently, it hasalsobeensuggested that ischemia-reperfusion(IR)mayleadtoincreasedmyocardialBNP mRNAindependentlyofchangesinventricularfunction[19].Sci- entificevidencealsosuggeststhatnatriureticpeptides(NPs)may serveasautacoidmediatorsofischemicpostconditioning(IPost), sincethenatriureticpeptidereceptorinhibitorisatinabolishedthe IPostinducedprotection[9].ItiswellknownthatIPostmediatesit cardioprotectiveeffectviaAkt-dependentRISKsignaling.Aktpar- ticipatesingene transcription regulation[20–23] andactivated nucleuslocalizedAkthasbeenshowntoenhanceANPexpression [24].Therelationshipbetweenmyocardialreperfusion andBNP synthesishasyettobefullyestablished,anditisunknownwhether AktmayparticipateinregulationofmyocardialBNPexpression levelatearlyreperfusion.

Theobjectivesofthisstudywereto:(1)studyifintermittent exogenousBNPadministrationmaymimicIPostinducedcardio- protection,(2)investigateifthiscardioprotectionismediatedvia AktandNPRdependentsignaling,(3)explorehowearlyBNPmRNA expressionaltersatreperfusionafteranacutemyocardialischemic event,and(4)delineatewhetherAktdependentsignalingmayreg- ulatereperfusioninducedBNPexpression.

Materialsandmethods Langendorffperfusion

AllexperimentswereapprovedbytheNorwegianStateCom- missionfor Laboratory Animals,and carried out in accordance withtheEuropeanCommunitiesCouncilDirective(2010/63/EU).

FemaleWistarrats(n=114,200–300g)wereheparinized(200IU) andanesthetizedwithsodiumpentobarbital(50mg/kg)ip.After excision, theheart was immediately placed in Krebs Henseleit buffer(KHB,4^◦C),andrapidlymountedinLangendorffperfusion modusatconstantpressure(80mmHg).TheKH-buffercontained:

NaCl(118mM),NaHCO₃ (25mM),KCl(4.7mM),MgSO₄×7H₂O (1.22mM),KH₂PO₄(1.21mM),d-glucose(11mM),CaCl₂×2H₂O (1.8mM)(pH7.4,oxygenatedwith95%O2and5%CO2).Coronary flow(CFinml/min)wasmeasuredbytimedcollectionofcoronary effluent,andathermo-probeplacedinthepulmonaryarterymon- itoredthecardiactemperature(keptat∼37^◦C).A3-0silksuture waspassedaroundthemainbranchoftheleftcoronaryartery, andthreadedthroughasmallvinyltubetoformasnare.Regional ischemia(RI)wasachievedbypullingthesnare,andconfirmedby asubstantialfallinleftventriculardevelopedpressure(LVDP)and CF.Reperfusionwasachievedbyreleasingthesnare.

Measurementofischemicriskzoneandinfarctsize

Thesilksuturewassecurelytightenedattheendofeachexperi- mentanda0.2%(w/v)EvansBluesuspensioninfusedtodemarcate theriskzone(DukeScientificCorp.,PaloAlto,CA,USA).Thereafter, theheartswerefrozen(−20^◦C)andlatercutinto2-mmthickslices fromapextobasisoftheheart,andstainedwith1%triphenylte- trazoliumchloride(TTC)inphosphatebuffer(pH7.4)at37^◦Cfor 20min,beforefixationin4%formalintoenhancethecontrastofthe stain.Theareaoftheleftventricle,theinfarctedarea(TTCnegative) andtheriskzone(notblue)weredeterminedusingacomputerized planimetryprogram(Planimetry⁺v2.0;ENK,Norway)andinfarct sizeexpressedastheinfarct/riskratio(%).Thecoefficientofvari- ation(CV)fortheareaatrisk/leftventriclevolumeratio(AAR/LV) were28%.

Experimentalprotocolfortheexvivoratheart

Threesetsofperfusionexperimentswereperformed:(1)infarct sizedeterminationafterintermittentBNPinfusion,(2)heartperfu- sionsfortissueisolationtodeterminetheeffectofBNPtreatment onphosphorylationofAktandp70s6kand(3)heartperfusionsfor tissueisolationforinvestigationofBNPmRNAexpressionintheleft ventricle(LV).Infarctsizedetermination:allheartswerestabilized for20minbeforebeingsubjectedto30minregionalischemiaand 120minreperfusion(Fig.1).RatBNP(10nM)(Sigma–Aldrich)[11]

wasadministeredin apostconditioninglikemannerfor3×30s atimmediate ischemia-reperfusion(BNPPost).Toinvestigatethe roleofpro-survivalRISKsignalinginBNP_Postinducedcardioprotec- tion,thefollowinginhibitors±BNPtreatmentwereadministered atischemicreperfusion:PI3Kinhibitorwortmannin(Wi;1␮M) [25],AktinhibitorSH-6(SH-6;10␮M)[26]andthemTOR/p70s6k inhibitorrapamycin(Rap;1nM)[25](allfromCalbiochem).The effect of inhibiting thenatriuretic peptide receptor (NPR) with isatin(100␮M)[9](Sigma)duringBNPpostconditioningwasalso investigated.Tissueisolationforimmunoblotting:tissuefordeter- minationofthephosphorylationstatusofAktandp70s6kinthe LVwereisolatedatbaselineandat15minofreperfusion.Tissue isolation forBNPmRNAexpressionanalysis: tissue samplesfor mRNAanalysiswerecollectedatendischemia,at15and120min intotheischemic-reperfusionperiod.Furthermore,inadditional experiments,tissuewasisolatedfromheartsexposedtoischemic postconditioning(IPost;3×30sof globalischemia)andparallel runcontrolsatearlyreperfusion±theAktinhibitorSH-6.

Immunoblotanalysis

MyocardialAkt(Phospho-Akt,Ser⁴⁷³)andp70s6kinasephos- phorylation (Phospho-p70s6k, Thr³⁸⁹) in the area at risk was determinedbySDS-PAGEelectrophoresis(allantibodiesfromCell SignalingTechnology,USA),andphosphorylationwasexpressedas theratiobetweenphosphorylatedandtotalproteinlevels.Hearts perfused with BNP for 10min (BNPB) or KH-buffer for 10min servedasbaselinecontrols(CtrB).Tissuewascollectedat15min ofischemia-reperfusion,snapfrozeninliquidnitrogenandstored at−80^◦C.Homogenization,proteinquantification,sampleprepara- tion(40␮g/lane)andelectrophoresiswereperformedaspreviously described[26].PonceauSstaining(Sigma,St.Louis,USA)confirmed successfultransferandGAPDHconfirmequalloading(SantaCruz Biotechnology,CA,USA).Densitometric analysiswasperformed usingQuantityOnesoftware(Bio-Rad,CA,USA).

Real-timequantitativereversetranscriptasepolymerasechain reaction(qRT-PCR)

Areaatrisk(AA,ischemictissue)andareanonatrisk(ANA, non-ischemictissue)werecollectedimmediatelyafteridentifica- tionofareaatriskbyEvansBluestainingandstoredinRNAlater (Ambion) accordingtothemanufacturer’sprotocol.Thecardiac tissue washomogenized in TRIzol (Invitrogen, 1ml/100mg tis- sue) ina Precellys 24 homogenizer(BertinTechnologies) using ceramicbeadsCK28(BertinTechnologies;twotimesat6000rpm for15sseparatedwith30s).AftertreatmentwithRecombinant DNaseI(Takara)andammoniumacetateprecipitation(Ambion), total RNAwasfurtherpurified using RNeasyMiniKit (Qiagen).

FirststrandcDNAwassynthesizedfrom3␮gtotalRNAina40␮L reactionprimedwith600ngoligo(dT_12–18;Invitrogen)using2␮L AffinityScript(Stratagene).Astandardcurvewith0.1–10␮gtotal RNAwasmadetocontrolforreversetranscriptionandPCRquan- tification.Reactionswithoutreversetranscriptaseenzymerunin parallelwerenegativewhentestedforpresenceofgenomicDNA.

ThesynthesizedcDNAwasdilutedbyadding160␮LRNasefree

0

20’ 30’

120’

Stabilisa tion Isc hemi a Reperfusion

20 170

Control Control + Wi/SH-6/Rap/Isatin

BNPPost+ Wi/SH-6/Rap/Isatin BNP_Post

Tissue collection

Fig.1. Experimentalprotocol.IsolatedfemaleratheartsweremountedandretrogradelyperfusedwithKrebsHeinsleitBuffer(KHB,solidlines)inaLangendorffperfusion setup.Allheartsweresubjectedto20minstabilization,30minregionalischemia(RI)and120minreperfusion.Solidlines=bufferperfusion;BNPPost=BNPpostconditioning, 3×30sofexogenousBNPinfusion(10nM).ThePI3Kinhibitorwortmannin(Wi,1␮M),theAkt-inhibitorSH-6(10␮M),themTOR/p70s6kinhibitorrapamycin(Rap,1nM), andthenatriureticpeptidereceptor-inhibitorisatin(100␮M)wereadministeredfor15minstarting1minpriortoreperfusionTissueisolationforimmunoblotting:tissue sampleswerecollectedatstabilizationandat15minofreperfusion.TissueisolationforBNPmRNAexpressionanalysis:tissuesamplesformRNAanalysiswerecollectedat endischemia,at15and120minintotheischemic-reperfusionperiod(indicatedbyarrows).

water.BNPwasquantifiedusingMessagreen(Eurogentech)with 2␮lcDNA astemplatewith300nMof eachprimerpairforthe natriureticpeptideprecursorB(preproBNP,Nppb)[27]orGAPDH [28].TheqPCRwasperformedonaHTS7900(AppliedBiosystem) withinitialdenaturationat95^◦Cfor10minandthePCRcycled at95^◦Cfor1sand60^◦Cfor30s(45cycles)followedbyamelt- ingpointanalysisconfirmingtheprimerspecificitybydetection ofonePCRproductonly.Amplificationefficiencyforeach qPCR reactionwascalculatedfromtheslopeofthestandardcurve[effi- ciency=^{10(−1/slope)}−1]andthestandardcurvemethodwasused tocalculateindividuallevelforeachmRNAanalysed.Thelevelof NppbmRNAwasanalysedand expressedrelative tothehouse- keepinggeneGAPDH.Themeanofischemictissueprioratstartof reperfusionwereassignedthevalue1tosimplifycomparisonof mRNA.

Statisticalanalysis

Values are presented as mean±standard errorof the mean (S.E.M.).Infarctsize, AAR/LV,LVDP, HR,CFand SDS-PAGEelec- trophoresisresultsweretestedforgroupdifferencesbyoneway analysisofvariance(ANOVA)combinedwiththeBonferronipost

hoctest.TheBNPmRNAdataweretestedfor groupdifferences byapplyingaStudentst-test.Avalueofp<0.05wasconsidered statisticallysignificant.

Results

BNPpostconditioninglimitsinfarctsizeviaPI3K/Akt/p70s6k dependentsignalingandnatriureticpeptidereceptorinhibition

Intermittentadministrationof BNPfor3×30sattheimme- diateonsetofischemic-reperfusion(BNPPost),tomimicischemic postconditioning, resulted in a >50% reduction in infarct size as compared to the control group (BNP_Post 17±2% vs. control 42±4%, p<0.001) (Fig. 2). In order to determine the involve- ment of PI3K/Akt/p70s6k dependentsignaling afforded by BNP postconditioning,three inhibitorsofthis pathwaywereutilized (experimental protocol, Fig. 1). The PI3K-inhibitor Wi (1␮M), Aktinhibitor SH-6 (10␮M) and themTor/p70s6k-inhibitor Rap (1nM)allcompletelyabolishedtheeffectofBNPpostconditioning (BNP_Post+Wi36±5%,BNP_Post+SH-641±4%,BNP_Post+Rap37±6%

vs. BNPPost 17±2%, p<0.001) (Fig. 2). Inhibitor administration alonehadnoeffectoninfarctsize(Wi47±2%,SH-639±4%,Rap

10 20 30 40 50 60

+Rap +SH-6 +Wi

BNP

Ctr

*

+Rap +SH-6 +Wi

Infarct/Ris k ratio (% )

Fig.2.AdministrationofBNPasamimeticofpostconditioninglimitsinfarctsizeviaPI3K/Akt/p70s6kdependentsignaling.Co-administrationofinhibitorsofRISKdependent signalingsuchasthePI3KinhibitorWortmannin(Wi,1␮M),theAktinhibitorSH-6(10␮M)andthemTOR/p70s6kinhibitorRapamycin(Rap,1nM)abrogatedtheinfarct sizesparingeffectofBNPPost.Infarctsizeisexpressedasapercentageoftheareaatrisk.Barsrepresentmeans±S.E.M.N≥7ineachgroup.*P<0.001vs.Ctrgroup.

10 20 30 40 50 60

BNP_Post Ctr

+ Isatin + Isatin

Infarct/R isk ratio (% )

*

Fig.3. AdministrationofBNPasamimeticofpostconditioninglimitsinfarctsizevia NPR.Blockingthenatriureticpeptidereceptor(NPR)withisatin(100␮M)revoked theinfarct-limitingeffectofBNPPost.Infarctsizeisexpressedaspercentageofthe areaatrisk.Barsrepresentmeans±S.E.M.N≥7ineachgroup.*P<0.001vs.Ctr group.

Table1

Ratioofareaatrisk(AAR)andleftventricle(LV)volumes.

Group Inhibitor AAR/LV(%)

Ctr

Noinhib 41.2±3.4

Wi 43.7±5.1

SH-6 48.6±6.2

Rap 40.7±5.8

Isatin 41.3±2.6

BNPPost

Noinhib 46.0±3.8

Wi 35.4±4.3

SH-6 45.2±6.4

Rap 37.1±1.9

Isatin 50.2±3.7

Valuesaremean±S.E.M.

41±2% vs. control 42±4%, ns) (Fig. 2). In order to reveal the involvementofcGMP/PKGsignalinginBNP_Postinducedcardiopro- tection,thenon-specificNPRinhibitorisatinwasco-administered atimmediatereperfusion.Isatindidnotaffectinfarctsizeunder control conditions (isatin 40±3% vs. control 42±4%, ns), but completelyabrogatedthe cardioprotection provided byBNP_Post (BNPPost17±2%vs.BNPPost+isatin46±2%,p<0.001)(Fig.3).The ischemicriskzoneconstitutedapproximately40–50%oftheleft ventricularmusclevolume,andtherewerenosignificantgroup differences in therelative sizesof the AAR (volumeof area at risk/volumeofleftventricle)(Table1).

Baselinefunctionalparameterswereobtainedafter18minof stabilization.AsignificantreductioninLVDPandCFafter5minof regionalischemiaconfirmedthatallgroupsobtainedsimilarand expecteddegreesofischemiarelativetocorrespondingstabiliza- tionvalues(Table2).Furthermore,therewasnodifferencebetween groupswithregardstoLVDP,CFandHRat120minofreperfusion.

BNPpostconditioningphosphorylatesAktandp70s6katearly reperfusion

AdministrationofBNPasamimeticofischemicpostcondition- ing(BNP_Post)atearlyreperfusionresultedinasignificantincreasein Aktphosphorylationcomparedtothecontrolgroup(Fig.4).Apply- ingtheAktinhibitorSH-6andtheNPRinhibitorisatinabrogated theBNP_PostinducedAktphosphorylation(Fig.4).Thedownstream Akttargetp70s6kwasalsosignificantlyphosphorylatedafterBNP postconditioningcomparedtothecontrolgroup(Fig.5).BNPinfu- sionatbaselinedidnotalterthephosphorylationstatusofAktor p70s6kcomparedtocorrespondingbaselinecontrols(Figs.4and5).

Fig.4. PhosphorylationstatusofmyocardialAktinisolatedheartsexposedtoBNP postconditioning.Representativeimmunoblots(top)anddensinometricanalysis (bottom)ofphosphorylatedAkt(Ser⁴⁷³)inexvivoratheartsdemonstratesthat BNPPosttreatmentresultedinasignificantincreaseinAkt(Ser⁴⁷³)phosphoryla- tionascomparedtothecontrolgroup,whiletheAktinhibitorSH-6andtheNPR inhibitorisatincompletelyabolishedit.BaselineBNPinfusion(BNPB)didnotsig- nificantlyalterthedegreeofAktphosphorylationascomparedtocontrolbaseline (CB).DensitometricanalysisoftotalandphosphorylatedAktexpressedinarbitrary units(AU)wherep-AktareexpressedasaratiooftotalAktwithCB=1.Barsrepresent means±S.E.M.N≥3ineachgroup.*P<0.001vs.BNPPost,#P<0.05vs.BNPB,¤P<0.05 vs.Ctr.RepresentativeimmunoblotsoftotalandphosphorylatedAkt(Ser⁴⁷³)(bot- tom).GAPDHimmunoblotsindicateequalloading.

Fig.5.Phosphorylationstatusofmyocardialp70s6kinexvivoheartsexposedto BNPpostconditioning.Representativeimmunoblots(top)anddensinometricanal- ysis(bottom)ofphosphorylatedp70s6k(Thr³⁸⁹)inexvivoratheartsindicatethat BNPPosttreatmentsignificantlyincreasedp70s6kphosphorylationstatusatThr³⁸⁹ ascomparedtothecontrolgroup(Ctr).BNPinfusionatbaseline(BNPB)didnot alterp70s6kphosphorylationascomparedtocontrolbaseline(CB).Densitomet- ricanalysisoftotalandphosphorylatedp70s6kexpressedinarbitraryunits(AU) wherep-p70s6kareexpressedasaratiooftotalp70s6kwithCB=1.Barsrepre- sentmeans±S.E.M.N≥3ineachgroup.*P<0.001vs.BNPPost.GAPDHimmunoblots indicateequalloading.

Table2

Functionalparametersrecordedduringtheexperimentalprotocol.

Group 18stab 5RI 30rep 120rep

LVDP (mmHg)

Ctr 137±7 70±7^* 84±6^* 59±6^*

Wi 134±10 65±7^* 82±6^* 57±5^*

SH6 141±9 71±9^* 61±2^* 44±5^*

Rap 126±19 68±7^* 71±3^* 47±5^*

Isatin 148±16 78±9^* 86±11^* 62±6^*

BNPPost 141±10 84±10^* 85±6^* 63±6^*

BNPPost+Wi 122±12 85±13^* 85±15 56±15^*

BNPPost+SH6 142±8 84±12^* 52±4^*,# 42±4^*

BNPPost+Rap 125±12 87±18^* 90±9^* 61±8^*

BNPPost+Isatin 135±6 70±12^* 76±6^* 47±7^*

CF (ml/min)

Ctr 12±2 7±1^* 10±1 7±1^*

Wi 10±1 6±1^* 8±2 6±1^*

SH6 13±1 8±1^* 9±1^* 7±1^*

Rap 11±1 8±1^* 11±1 8±1^*

Isatin 11±1 7±1^* 8±1^* 7±1^*

BNPPost 12±1 6±1^* 9±1^* 7±1^*

BNPPost+Wi 13±1 8±1^* 12±2 9±2^*

BNPPost+SH6 12±1 7±1^* 7±1^* 7±2^*

BNPPost+Rap 11±3 7±1^* 10±3 8±2^*

BNPPost+Isatin 12±1 7±1^* 8±2 6±1^*

HR (beats/min)

Ctr 259±9 278±12 294±9 277±10

Wi 265±15 261±16 243±18 242±22

SH6 294±11 286±14 253±14 239±17^*

Rap 274±17 257±21 295±8 271±9

Isatin 282±17 248±14 248±18 249±17

BNPPost 267±15 228±22 238±21 227±19

BNPPost+Wi 285±20 291±24 278±38 279±22

BNPPost+SH6 289±5 243±50 235±34^# 199±39

BNPPost+Rap 297±27 266±28 259±48 275±38

BNPPost+Isatin 280±18 250±22 229±27 231±25

Valuesrepresentmean±S.E.M.

Ctr–ischemiareperfusioncontrol;BNPPost–BNPpostconditioning;Wi–15minWortmannin(1␮M)atreperfusion;SH6–15minSH-6(10␮M)atreperfusion;Rap–15min ofrapamycin(1nM)atreperfusion;isatin–15minofisatin(100␮M)atreperfusion.

*P<0.05vs.correspondingstab.

#P<0.05vs.Ctr.

TheleveloftotalAkt,totalp70s6kandloadingcontrolGAPDHdid notalterbetweenthegroups.

BNPmRNAlevelsatearlyreperfusionintheareaatriskof infarctionisregulatedbyAktdependentsignaling

MyocardialBNPmRNAlevelsinareaatrisk(AA)weresignif- icantlyelevatedatearlyreperfusion(15min)ascomparedtothe non-ischemicmyocardium(ANA,areanotatrisk)(Ctr_(AA)2.7±0.5 vs.Ctr_(ANA)1.2±0.2,p<0.05)(Fig.6A).Furthermore,theventricu- larBNPexpressioninAAat15minofreperfusionwassignificantly up-regulatedascomparedtotheischemiccontrol(0minreper- fusion)(Ctr_(AA)2.7±0.5vs.ischemia1.0±0.1,p<0.05),whilethere werenodifferencesbetweenANAandtheischemiccontrol(Ctr_(ANA) 1.2±0.2vs.ischemia1.0±0.1,ns)(Fig.6A).TheventricularBNP expressionlevelsinAAreturnedtoischemiclevelsafter120min ofreperfusion(Ctr_(AA)1.2±0.3vs.ischemia1.0±0.1,ns),andwith nodifferencesinexpressionlevelsbetweenAAandANA(Ctr_(AA) 1.2±0.3 vs. Ctr_(ANA) 0.9±0.3, ns). These resultsimply that the reperfusionprocessamplifiesBNPexpressionintheareaatrisk ascomparedtotheischemicgroupandtheremotenon-ischemic partofthemyocardiumatearlyreperfusion.

In separate experiments, we also investigated the effect of ischemicpostconditioning(IPost)andAktinhibitiononBNPmRNA expressionatearlyreperfusion intheareaatrisk ofinfarction.

MyocardialBNPmRNAwassignificantlyup-regulatedinischemic postconditioned(IPost)heartsascomparedtoischemiccontrols atearlyreperfusion(IPost6.7±1.3vs.ischemia1.0±0.2,p<0.05) (Fig.6B).BNPexpressionintheparallelruncontrolswerealsoele- vated(Ctr5.4±0.8vs.ischemia1.0±0.2,p<0.05).Furthermore, inhibitingAktwithSH-6completelyabrogatedtheelevatedBNP

mRNAexpressionlevelsbothintheIPostandcontrolgroup(IPost 6.7±1.3vs.IPost+SH-61.8±0.7,p<0.05)(Ctr5.4±0.8vs.SH-6 1.5±0.9,p<0.05)(Fig.6B),indicatingthatAktmaymodulateBNP expressionlevelsatearlyreperfusion.

Discussion

Thefindingscanbesummarizedasfollows:(1)whenexogenous B-type natriureticpeptide (BNP)wasintermittently infused for 3×30s,inapostconditioninglikefashion(BNP_Post),itsignificantly reducedinfarctsizeintheexvivoratheart.(2)BNP_Postcardiopro- tectionwasabolished byinhibitingPI3K/Akt/p70s6k dependent signalingandbyinhibitionofnatriureticpeptidereceptorsusing isatin.(3)PharmacologicalpostconditioningwithBNPpeptidesele- vatedAktandp70s6kphosphorylationatearlyreperfusion.(4)The myocardialBNPmRNAlevelsintheareaatrisk(AA)weresignifi- cantlyelevatedatearlyreperfusioncomparedtoischemiccontrol tissueandremoteANA(areanotatrisk).(5)Ischemicpostcondi- tioning(IPost)didnotelevatetheBNPmRNAlevelsbeyondthe controlgroupatearlyreperfusion.(6)InhibitionofAktabrogated theearlyreperfusioninducedBNPmRNAexpressionlevels.

Ithaspreviouslybeenshownthatexogenousadministrationof BNPpriorto,duringandimmediatelyaftercoronaryocclusionpro- tectstheheartagainstischemiareperfusioninducedinjury[9–13]

viaacGMP/PKGdrivenmechanism[11],regulatingtheopeningof intracellularK_ATP-channels[9,29].ItiswellknownthattheRISK memberAktservesasafocalpointforsignaltransductionpath- wayscodingforcellsurvival[30]andAktmediatedsignalingevents hasbeenassociatedwithactivationofcGMPandPKG[31].Inthe currentstudy,weshowthatintermittentpharmacologicalmanip- ulationoftheheartfor3×30swithBNPatearlyreperfusion,in

0 1 2 3 4 5

15 120

0

Time of reperfusion (min)

BN P m R N A (A U )

AA ANA

A

*

0 2 4 6 8 10

Ctr IPost

BN P m R N A (A U )

B

+ SH-6 + SH-6

* *

Fig.6. RelativeexpressionofleftventricularBNPmRNAatischemicreperfusion.(A)BNPmessengerRNAwasquantifiedbyrealtimequantitativeqRT-PCRatvarioustime pointsduringreperfusion(0=endischemia,15and120min)intheareaatrisk(AA,blackbar)andtheareanotatrisk(ANA,graybar)ofinfarction.*P<0.05vs.CtrR+15(AA). (B)Inadditionalexperiments,BNPmRNAexpressionanalysisintheAAofheartsexposedtoischemicpostconditioning(IPost;3×30sofglobalischemia),withorwith outtheAktinhibitorSH-6,wascomparedtoparallelruncontrolhearts.ThemRNAdatawasnormalizedtoGAPDHandexpressedinarbitraryunits(AU).Barsrepresent means±S.E.M.N≥3ineachgroup.*P<0.05vs.correspondingcontrolgroup.

apostconditioninglikefashion(BNP_Post),reducedinfarctsizein isolatedrathearts.ThisBNPPostinducedcardioprotectionwaslost wheninhibitingtheclassicalRISK-kinases;PI3Kwithwortman- nin,AktwithSH-6andp70s6kwithrapamycin.BNP_Posttreatment alsoenhancedthephosphorylationofAktandp70s6k.Inaddition, inhibitionofAktwithSH-6atreperfusioncompletelybluntedBNP inducedAktphosphorylation.Ourfindingsareinaccordancewith previousstudieswhere BNPtreatmentduringreperfusion have beenfoundtoactivatebothAktandPKCinisolatedmousehearts [10],whenadministratedathypoxia-reoxygenationtoneonatalrat cardiomyocytestopreventedopeningofthemitochondrialperme- abilitypore(mPTP),partlyviaPI3K/Aktdependentsignaling[32].

AdministrationofANPinfusionjustpriortoreperfusionalsoacti- vatedtheRISKmembersPI3KandERKinisolatedperfusedrabbit hearts[16].

Natriureticpeptides(NPs)interactwiththreetypesofmem- branenatriureticpeptidereceptors(NPR)A,BandC.BNPhasbeen showntoactivatebothNPR-AandC[15].ThedensityofNPR-Cin mosttissueishigherthanthatofNPR-A/B,andNPR-Chasahigh affinityforBNP[33].SeveralstudieshavereportedthatNPsrecruit thePI3/AktsignalingpathwayviaNPR-C[33,34].Thus,cGMP/PKG andRISKsignalingcouldbetheresultofBNPbindingtotwodiffer- entreceptors;cGMPisproducedwhenBNPbindsNPR-AandRISK signalingactivatedwhenBNPbindstheNPR-C[9,33].Inthisstudy, theBNPPostinducedcardioprotectionandAktphosphorylationwas completelyabolishedbythenon-selectiveNPreceptorinhibitor isatin.Attheconcentrationappliedinthisstudy(100␮M),isatin hasbeenshowntoinhibitbothparticulateguanylylcyclaseactiva- tionviatheNPR-Areceptor,andalsotheNPR-Creceptor(reviewed in[35]).Unfortunately,othernon-peptidespecificantagonistsof NPRsarecurrentlynotavailable,andwearethereforeunableto concludewhichNPRthatconveystheRISKmediatedcardioprotec- tionbyBNP_Post.

Acute myocardial ischemia (AMI) up-regulates BNP gene expressionincardiacventricles[7,8],butlatelyithasbeensug- gestedthatischemia-reperfusion(IR)mayserveasanindependent triggerofincreasedBNPmRNAmyocardialcontent[19].Ourresults indicatethatreperfusionpersestimulatesBNPmRNAexpression beyondtheischemiainducedexpressionlevelintheareaatrisk (AA).Noregulationwasevidentintheremotenon-ischemicarea (ANA)atthesametimepoint,andwithin120minofreperfusion

theBNPexpressionlevelshadreturnedtoischemiclevels,withno differencebetweentheAA andANA.Theseresultsarein accor- dancewiththeresultsofRamoset al.[19],whofoundthatthe BNPmRNAlevelwasincreasedintheIRregion(AA)ascompared tothenon-ischemicarea(ANA)oftheisolatedratheartindepen- dentofchangesintheventricularfunction.Seeninconjunction withourstudy,thisimpliesthatearlyreperfusioninducedmod- ulationofBNPgeneexpressionisrestrictedtotheischemicarea, reinforcingtheconceptoftemporalmolecularchangesintheIR myocardium(AA)atvery earlyreperfusionas comparedtothe remotemyocardium(ANA).

InadditiontobeingamarkerofAMIandheartfailure,Burley etal.speculatedthatNPsmaybeautacoidmediatorsofischemic postconditioning(IPost),sincetheunspecificNPR-A/-Cinhibitor isatinabolishedtheIPostinducedprotection[9].Ourresultsshow thatIPostdoesnotinfluenceBNPmRNAexpressionlevelsinthe areaatriskbeyondthecontrolgroupatearlyreperfusion.Despite comparablemRNAexpressionlevelsinIPostandcontrol,itmay notberepresentativefortheBNPcontent/activityortheamountof BNPreleasedfromthemyocardium.However,Goetzeetal.found thatplasmaBNPandproBNPconcentrationsmostlikelyreflected anincrease inBNPgeneexpressionin theischemichumanleft ventricle,sincetheconcentrationwerecloselyassociatedwithven- tricularmRNAexpression[36].

Accumulation of nucleus activated Akt promote cardiomy- ocytes survival [37] via phosphorylation of multiple protein substratesandgenetranscriptionregulation[20–23].Nucleustar- getedAkthasalsobeenshown topromoteANP expressionvia anautocrine/paracrinestimulatedPI3K-dependentsignalingcas- cade[38].OurresultsindicatethatinhibitionofAktusingSH-6has theabilitytodepressleftventricularBNPmRNAexpression.This impliesthatAkt-dependentsignalingmaybeinvolvedinregulat- ingcardiacBNPexpressionatearlyreperfusion.Furtherstudiesare warrantedtofullydelineatetheroleofAktinIRinducedregula- tionofcardiacBNPmRNAlevels,andtofurtherevaluatewhether theshiftingeneexpressionistransitionalorifithaslong-term functionalimplications.

Severalstudies have documenteda correlationbetweenthe plasmaBNPconcentration andinfarctsize inpatientssuffering fromacutemyocardialinfarction(AMI)[7,39,40].Normally,plasma BNPconcentrationsliebetween0and10pmol/L,andcirculating