The novel H2S-donor $-carboxyphenylisothiocyanate promotes cardioprotective effects against ischemia/reperfusion injury through activation of mitoKATP channels and reduction of oxidative stress

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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

The

novel

H

2

S-donor

4-carboxyphenyl

isothiocyanate

promotes

cardioprotective

effects

against

ischemia/reperfusion

injury

through

activation

of

mitoK

ATP

channels

and

reduction

of

oxidative

stress

Lara

Testai

a

_,

_Alice

_Marino

b

_,

_Ilaria

_Piano

a

_,

_Vincenzo

_Brancaleone

c

_,

_Kengo

_Tomita

b

_,

Lorenzo

Di

Cesare

Mannelli

d

_,

_Alma

_Martelli

a

_,

_Valentina

_Citi

a

_,

_Maria

_C.

_Breschi

a

_,

Roberto

Levi

b

_,

_Claudia

_Gargini

a

_,

_Mariarosaria

_Bucci

e

_,

_Giuseppe

_Cirino

e

_,

Carla

Ghelardini

d

_,

_Vincenzo

_Calderone

a,∗

a_Department_of_Pharmacy,_University_of_Pisa,_Pisa,_Italy

b_Department_of_{Pharmacology,}_Weill_Cornell_Medicine,_New_York,_USA c_Department_of_Science,_University_of_Basilicata,_Potenza,_Italy

d_Department_of_{Neuroscience,}_Psychology,_Drug_Research_and_Child_Health_{(Neurofarba),}_University_of_Florence,_Florence,_Italy e_Department_of_Pharmacy,_University_of_Naples_Federico_II,_Naples,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received30May2016 Accepted7September2016 Availableonline9September2016 Chemicalcompoundstudiedinthisarticle: 4-hydroxyphenylisothiocyanate(PubChem CID:121230993) Keywords: Hydrogensulphide H2S-donors Isothiocyanate Myocardialischemia/reperfusion Cardioprotection

Mitochondrialpotassiumchannels

a

b

s

t

r

a

c

t

Theendogenousgasotransmitterhydrogensulphide(H2S)isanimportantregulatorofthe

cardiovascu-larsystem,particularlyofmyocardialfunction.Moreover,H2Sexhibitscardioprotectiveactivityagainst

ischemia/reperfusion(I/R)orhypoxicinjury,andisconsideredanimportantmediatorof“ischemic pre-conditioning”,throughactivationofmitochondrialpotassiumchannels,reductionofoxidativestress, activation ofthe endogenous “anti-oxidant machinery”and limitation of inﬂammatoryresponses. Accordingly,H2S-donors,i.e. pro-drugsable togenerate exogenousH2S,are viewed as promising

therapeuticagentsforanumberofcardiovasculardiseases.ThenovelH2S-donor4-carboxy

phenyl-isothiocyanate(4CPI),whosevasorelaxingeffectswererecentlyreported,wastestedhereindifferent experimentalmodelsofmyocardialI/R.

InLangendorff-perfusedratheartssubjectedtoI/R,4CPIsigniﬁcantlyimprovedthepost-ischemic recoveryofmyocardialfunctionalparametersandlimitedtissueinjury.Theseeffectswereantagonized by5-hydroxydecanoicacid(ablockerofmitoKATPchannels).Moreover,4CPIinhibitedtheformation

ofreactiveoxygenspecies.WefoundthewholebatteryofH2S-producingenzymestobepresentin

myocardialtissue:cystathionine␥-lyase(CSE),cystathionine␤-synthase(CBS)and3-mercaptopyruvate sulfurtransferase(MPST).Notably,4CPIdown-regulatedthepost-ischemicexpressionofCSE.

InLangendorff-perfusedmousehearts,4CPIreducedthepost-ischemicreleaseofnorepinephrineand theincidenceofventriculararrhythmias.Inbothratandmousehearts,4CPIdidnotaffectthe degranu-lationofresidentmastcells.

Inisolatedratcardiacmitochondria,4CPIpartiallydepolarizedthemitochondrialmembranepotential; thiseffectwasantagonizedbyATP(i.e.,thephysiologicalinhibitorofKATPchannels).Moreover,4CPI

abrogatedcalciumuptakeinthemitochondrialmatrix.

Finally,inaninvivomodelofacutemyocardialinfarctioninrats,4CPIsigniﬁcantlydecreased I/R-inducedtissueinjury.

Inconclusion,H2S-donors,andinparticularisothiocyanate-basedH2S-releasingdrugslike4CPI,can

actuallybeconsideredasuitablepharmacologicaloptioninanti-ischemictherapy.

∗ Correspondingauthorat:DepartmentofPharmacy,UniversityofPisa,Via Bonanno,6,I-56126Pisa,Italy.

E-mailaddress:calderone@farm.unipi.it(V.Calderone).

1. Introduction

Thegasotransmitterhydrogensulphide(H2S)is apleiotropic

and ubiquitousmediator which inﬂuencesalmost allthe func-tionsofthemammalianbody[1].Amongthese,theregulationof

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thecardiovascularsystemisanimportantroleofthis gasotrans-mitter [2,3].H2Sisbiosynthesized by cystathionine␤-synthase

(CBS), cystathionine ␥-lyase (CSE) [4], and by the cooperation betweencysteineaminotransferase(CAT)and3-mercaptopyruvate sulfurtransferase (MPST) [4–6]. The pattern of distribution and localizationoftheseenzymesisquitecomplexandtheymaycoexist incardiovasculartissues[7–9].

H2S exhibits cardioprotective activity against

ischemia/reperfusion (I/R) or hypoxic injury and is consid-eredanimportantmediatorof“ischemicpreconditioning”(IPC),a self-defencecardioprotectivemechanism againstmyocardialI/R injury.Themechanismsofactionaccountingforthis cardioprotec-tiveactivityareheterogeneousandnotyetcompletelyunderstood. Mitochondrial ATP-sensitivepotassium channels (mitoKATP)are

well-known effectors of ischemic preconditioning [10]. Their activation by H2S is likely to be a relevant cardioprotective

mechanism[11],sincetheanti-ischemiceffectsofH2Sarelargely

antagonized by blockers of mitochondrial potassium channel

[12].Othermechanismshavealsobeenproposedtoexplainthe cardioprotectiveactivity ofH2S. Antiapoptotic responsesplay a

roleincardioprotectionagainstI/Rinjury,andareduetotheH2

S-inducedtriggeringofpathwaysofintracellularsignalling,suchas PI3K/Akt,PKCand ERK1/2,andtheNrf-2-mediatedantioxidant machinery[13].Eventheinhibitionoftype-5phosphodiesterase byH2Splaysapotentialrole.Infact,theintracellularriseofcGMP,

and theconsequentcGMP-dependent proteinkinaseactivation, trigger downstream effectors of ischemic preconditioning [14], upregulateCSElevelsandpromotefurtherH2Sproduction[15].

ExacerbationofI/R-induced tissue injury canbealso dueto anintenseinﬂammatoryresponse,triggeredbythedegranulation ofresidentheartmastcellsandthereleaseofcytokines,growth factors,chemokines,andotherpro-inﬂammatorymediators[16]. Remarkably,cardiacmast cellscontainrenin and therelease of thisproteolyticenzymefromcardiacmastcellscontributestothe I/R-associatedheartinjury[17].

Noteworthy, H2S was reported to inhibit antigen-induced

degranulation of rat basophile leukemic RBL-2H3 cells (a well knownmast-celllikemodel)and murinebone marrow-derived mastcells,suggestinganinhibitoryroleofthisgasotransmitterin mastcell-mediatedinﬂammatoryresponses[18,19].Therefore,a possibleH2S-mediatedinhibitionofthedegranulationofresident

heartmastcellsmaybeafurther(andpoorlyinvestigated) mech-anismcontributing,atleastinpart,totheoverallcardioprotective activity.

Giventhe intriguingbiological activityofH2Sinthe

cardio-vascularfunction,naturalandsyntheticH2S-donors,i.e.pro-drugs

abletogenerateexogenousH2S,areviewedaspromising

cardio-protectiveagents[20–22].4-carboxyphenyl-isothiocyanate(4CPI,

Fig.1)isaH2S-releasingcompound,knowntoevokevasorelaxing

responsesinisolatedrataortaandtoincreasecoronaryﬂowin iso-latedrathearts[23].Moreover,4CPIwasshowntocausemembrane hyperpolarizationinhumanaorticsmoothmusclecellsthrough theactivationofKv7potassiumchannels,whichplayaroleinH2

S-inducedvasodilation[24].Noteworthy,Kv7channels(inparticular,

Fig.1.Chemicalstructureof4CPI.

Kv7.4)havebeenrecentlyrecognizedinheartmitochondria,where theyappeartoplayacardioprotectiverole[25].

AlthoughthevasculareffectsofthenovelH2S-donor4CPIhave

beenelucidated,itspotentialcardioprotectiveactivityisyettobe evaluated.Hence,theaimofthisstudywastoinvestigatetheeffects of4CPIindifferentexperimentalmodelsofI/Rinisolatedheartsand ofacutemyocardialinfarctioninvivo,byevaluatingwell-known markersofI/R-inducedmyocardialinjury(i.e.,reducedmyocardial contractility,ventriculararrhythmias,celldeath,oxidativestress). The involvement of some relevant mechanisms of action, such asactivationof mitochondrialion channelsand/orinhibitionof degranulationofresidentheartmastcells,wasalsotestedin iso-latedcardiacmitochondriaandisolatedhearts,respectively.

2. Materialsandmethods

2.1. Substances

4CPI(Fluorochem Ltd, Hadﬁeld, UK)was dissolved(10−2M) in dimethylsulfoxide(DMSO), andfurtherdilutedin bi-distilled water. 5-hydroxy decanoic acid (5HD; Sigma–Aldrich, Milano, Italy)wasdissolvedinbidistilledwater.Tetraphenylphosphonium chloride(TPP+_Cl−_,_{Sigma–Aldrich,}_Milano,_Italy)_was_dissolved_in

bi-distilled water and 2,3,5-triphenyltetrazolium chloride (TTC, Sigma–Aldrich.Milano,Italy)wasdissolved(1%,p/w)inphosphate buffer (pH 7.4). Olygomicin, 2,4-dinitrophenol (DNP), carbonyl cyanidem-chlorophenylhydrazone(CCCP)andvalinomycinwere purchasedfromSigma–Aldrich(Milano,Italy),dissolvedinpure DMSO(10mM)andfurtherdilutedinbi-distilledwater.

2.2. Pharmacologicalprocedures

Alltheexperimentalprocedureswerecarriedoutfollowingthe guidelinesoftheEuropeanCommunityCouncilLaw2010/63and havebeenapprovedbytheCommitteeforanimalexperimentation oftheUniversityofPisa.Allproceduresonmicewereapprovedby WeillCornellMedicineInstitutionalAnimalCareandUse Commit-tee.

2.3. Langendorff-perfusedrathearts

MaleWistarrats(260–350g) weretreatedwithani.p. injec-tionofdifferentincreasingdosesof4CPI0.072mg/kg,0.24mg/Kg, 0.72mg/kgand2.4mg/kgortheirvehicle(DMSO).After2h,allthe animalswereanaesthetizedwithsodiumpentobarbital(100mg/kg i.p.)andheparinized(100UIi.p.)topreventbloodclotting.When requiredfromexperimentalprocedure,5HD (10mg/kgi.p.)was administrated20minbefore4CPI(0.24mg/kg)-treatment.

Afteropeningthechest,heartswerequicklyexcisedandplaced ina4◦CKrebssolution(compositionmM:NaHCO325.0,NaCl118.1,

KCl4.8,MgSO4 1.2,CaCl2×2H2O1.6,KH2PO41.2,glucose11.5),

equilibrated with95% O2 and 5% CO2,to stop contractionand

reduceoxygenconsumption.Rapidly,theascendingaortawas can-nulatedandtheheartsweremountedonaLangendorffapparatus, andthenperfusedwithKrebssolution(thermostatedat37◦Cand continuouslybubbledwithagasmixtureof95%O2and5%CO2)at

constantpressure(70–80mmHg).Theaboveprocedurewas com-pletedwithin2min.Awater-ﬁlledlatex balloonconnectedtoa pressuretransducer(BentleyTrantec,mod800,UgoBasile, Come-rio,Italy)wasintroducedintotheleftventricleviathemitralvalve andthevolumewasadjustedtoachieveastableleftventricular end-diastolicpressureof5–10mmHgduringinitialequilibration. After30minofequilibration,heartsweresubjectedto30minof globalischemia(noﬂow).Thereafter,heartswerereperfusedfor 120min.Functionalparameterswerecontinuouslyrecorded dur-ingthewholeexperiment.Attheendofreperfusionheartswere

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removedfromtheLangendorffapparatusandleftventricleswere isolatedandsubmittedtomorphometric,histologicaland biochem-icalassays.

2.3.1. Recordingoffunctionalparameters

ReductionofheartcontractilityafterI/Rhasbeenusedasa suit-ableindicatorofI/R-induced injury. Therefore,totest potential cardioprotectiveeffectsof4CPI,werecordedprospective improve-mentsofinotropicparametersinheartssubmittedtoI/R.

Heartrate(HR),leftventriculardevelopedpressure(LVDP)and therateofriseoftheleftventricularpressure(dP/dt)were con-tinuouslymonitoredbyacomputerizedBiopacsystem(California, USA).RPPwascalculatedasRatexPressureProduct.Post-ischemic dP/dtandRPPrecordedduringreperfusionwereexpressedasa% ofthecorrespondingvaluesrecordedinthepre-ischemicperiod. HeartsshowingseverearrhythmiaorunstableLVDPandHRvalues, duringthepre-ischemicphase,werediscarded.

2.3.2. Beta-hexosaminidaseassayincoronaryefﬂuent

Measurement of mast cell-derived beta-hexosaminidase ( ␤-hexo)iswidelyusedasasuitablemarkerofmastcelldegranulation inducedbyI/R.Thus, totestpotentialinhibitoryeffects of4CPI onmastcelldegranulation,weinvestigatedwhetherany reduc-tioninmastcell-derived␤-hexooccurredin4CPI-treatedhearts submittedtoI/R.

Atthebeginningofreperfusion,5mlofcoronaryefﬂuentwere collectedandusedtomeasure␤-hexoconcentration.50␮lof per-fusate were placed into a 96-well plate,then 50_␮l of _␤-hexo substrate(p-nitrophenyl-N-acetyl-d-glucosaminide1.3mg/ml dis-solvedincitratebuffer0.1M,pH4.5)wereaddedandincubatedfor 60minat37◦C.Thereactionwasstoppedwith100␮lofTRISbuffer (pH=9at4◦C).Opticaldensitywasreadat405nmonaplatereader (EnSpire,PerkinElmer).␤-hexoreleasewasexpressedasarbitrary unitsofabsorbance.

2.3.3. Morphometricanalysisoftheischemicarea

ThesizeofventriculartissueexhibitingI/R-inducedcelldeathis widelyusedasasuitableindicatorofI/R-inducedinjury.Therefore, totestpotentialcardioprotectiveeffectsof4CPI,wemeasuredthe possiblereductionofthesizeofinjuredareasin4CPI-treatedhearts submittedtoI/R.

The left ventricle was cut in 2mm-wideslices which were immersedina1%aqueoussolutionofTTCfor20minandthenina 10%aqueoussolutionofformaldehyde.After24h,ventricularslices werephotographedandanalyzedtohighlightnecroticareasdueto theischemicprocess(visibleasawhiteorlightpinkcolor)andthe healthyareas(visibleasastrongredduetotheTTCreaction).

2.3.4. DeterminationofCSE,CBSandMPSTexpression

TheexpressionofCSE,CBSandMPSTwasdeterminedinheart tissue,inordertoevaluatetheinﬂuenceofI/RontheH2S

biosynthe-sizingmachinery.Moreover,theeffectsof4CPIontheexpression oftheseenzymeswasevaluatedinhearts(withandwithoutI/R), inordertoinvestigatethemechanismsofactionofthiscompound. Asabovedescribed,heartswereisolatedfromratspretreatedwith vehicleor4CPIandsubmittedtotheI/Rcycle.Moreover,CSE,CBS andMPSTexpressionwasalsodetectedintheheartsofa“sham group”:inthisgroup,heartswereisolatedfromratspretreatedwith vehicleor4CPI,mountedinaLangendorffapparatusandperfused for150min,butnotsubmittedtoI/R.

Following an I/R cycle or a sham 150min perfusion time, left ventricular tissue from rat hearts were homogenized in lysisbuffer. Denaturedproteins(40_␮g)wereseparated on10% sodium-dodecylsulfate polyacrylamide gels and transferred to polyvinylidene ﬂuoride membrane (PVDF). Membranes were

blockedinphosphate-bufferedsalinecontaining0.1%v/v Tween-20(PBST) and3% w/vnon-fatdry milkfor30min,followedby overnight incubation at 4◦C with rabbit polyclonal anti-MPST (1:500, Novus Biologicals, Italy), mouse monoclonal anti-CSE (1:1000,Proteintech,UK),rabbitpolyclonalanti-CBS(1:1000,Santa Cruz Biotechnology,Germany) or rabbit polyclonal anti-GAPDH (1:5000, Sigma–Aldrich, Italy). Membranes were extensively washedinPBSTpriortoincubationwithhorseradish-peroxidase conjugated secondary antibody for 2h at room temperature. Followingincubation,membraneswerewashedand chemilumi-nescencewasdetectedbyusingImageQuant-400(GE-Healthcare, USA).Targetproteinbandintensitywasnormalizedin considera-tionofhousekeepingproteinGAPDHintensity.

2.3.5. Detectionofoxidativestress

Increasedproductionofreactiveoxygenspecies(ROS)was con-sideredasasuitableindicatorofI/R-inducedoxidativestress,i.e. anadditionalmarkerofmyocardialinjury.Thus,tofurthertestthe potentialcardioprotectiveeffectsof4CPI,werecordedapossible reductionofROSproductionin4CPI-treatedheartssubmittedto I/R.

Forhistologicallabelingandqualitativeimageacquisition,left ventriclesfromratswereisolatedandreadilyincludedin Opti-malCuttingTemperature (OCT,Sakura,Japan) withoutﬁxation. Cryosection20␮mthickwereobtainedusingamanualcryostat (Leica,Germany)andcollectedontoglassslides.Ventriclesections werethenincubatedin10␮Mdihydroethidium(DHE)solutionat 37◦Cfor30min.Sectionswerethenrinsed3×5mininphosphate bufferedsaline(PBS)andmountedforconfocalimagingwith Vec-tashield(Vector,Burlingame).Confocalimageswereobtainedfor eachsectionusinga16×oilobjective(NA0.50)onaTCS-SP2 con-focalmicroscope(Leica,Germany).Eachimagewasacquiredasthe maximumintensityprojectionofa10␮mconfocalstack.Inorder toquantifyROS+areasinthedifferentexperimentalgroup,were acquiredsixﬁeldsofeachsectionoftheleftventricleanalyzed.It wassampledthreesectionsforeachleftventricleofthreeanimals pergroup.

2.4. Langendorff-perfusedmousehearts

Wild-typeC57BL/6Jmice(male,10-to12-weekold,Jackson Lab-oratory,BarHarbor,ME)wereinjectedi.p.with4CPI0.24mg/kg 2hbeforetheexperiment.Micewereinjectedi.p.withheparin (100UI),anaesthetizedwithCO2andeuthanizedbycervical

dis-location.Hearts were isolated and transferred to a Langendorff apparatus(Radnoti,Monrovia,CA,USA).Theaortawascannulated withaﬂanged20-gaugestainless-steelneedle.Theheartwas per-fusedthroughtheaortainaretrogrademodeataconstantpressure of100cmH2OwithmodiﬁedKrebs-Henseleit(KH)buffer

contain-ing(mM):NaCl,120;KCl,4.7;CaCl2,2.5;MgSO4,1.2;KH2PO4,1.2;

NaHCO3,25;glucose,11;Pyruvateacid,2;EDTA,0.5.The

perfu-sionﬂuidwasbubbledwith95%O2 and5%CO2at37◦Ctogivea

pHof7.4.Afterequilibration,allheartsweresubjectedto30-min ischemia(glucose-andpyruvicacid-freeKHbuffer,95%N2+5%CO2

andsodiumdithionite)followedby30-minreoxygenation (reper-fusion) withKH buffer. Coronaryﬂow wasmeasured bytimed collectionsoftheefﬂuentevery5min;sampleswereassayedfor ␤-hexo,reninandnorepinephrine(NE).

2.4.1. Evaluationofventriculararrhythmias

SurfaceECGwasrecordedfromleadsattachedtotheleft ventri-cleandtherightatriumandanalyzedusingPowerLaboratory/8SP (ADInstruments,ColoradoSprings,CO,USA).Reperfusion arrhyth-mias were recorded and quantiﬁed according to the Lambeth Conventions.

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2.4.2. Determinationofnorepinephrinerelease

Increased release of norepinephrine (NE) from heart sym-pathetic nerve endings was considered a suitable indicator of I/R-inducedadrenergicactivation.Thus,tofurthertestpotential cardioprotectiveeffectsof4CPI,weinvestigatedapossible reduc-tionofNEreleasein4CPI-treatedheartssubmittedtoI/R.

Norepinephrine (NE) wasassayed in thecoronary perfusate byhigh-performanceliquid chromatographycoupledto electro-chemicaldetection[26].PerchloricacidandEDTAwereaddedto samplestoachievefinalconcentrationof0.01Nand0.025%, respec-tively.NEpresent intheeffluentwasadsorbedonacid-washed aluminaadjustedatpH8.6withTris2%EDTAbuffer(inagitation for45min)andthenextractedin150␮lof0.1Nperchloricacid. Thesefinalsampleswereinjectedontoa3␮mODSreverse-phase column(3.2×100mm,BioanalyticalSystem,WestLafayette,IN) withanappliedpotentialof0.65V.Themobilephaseconsistedof monochloroaceticacid(75mM),Na2EDTA(0.5mM),sodium

acty-sulfate(0.5mM)and acetonitrile(15%)atpH3.0.Flowratewas 1.0mg/min. Dihydroxybenzylamine (DHBA) was added to each sampleasaninternalstandardbeforealuminaextractionandused forrecoverycalculation.TherecoveryofNEwas77%andthe detec-tionlimitwasapproximately0.2pmol.

2.4.3. Determinationofˇ-hexorelease

Thedeterminationofmastcell-derived␤-hexohasbeenused asa suitable markerofmast celldegranulation inducedbyI/R. Therefore,totestthepotentialinhibitoryeffectsof4CPIonmast celldegranulation, we ascertainedwhethertherelease of mast cell-derived␤-hexowasreducedin4CPI-treatedmousehearts sub-mittedtoI/R.Sampleofcoronaryefﬂuentwereconcentrated 8-to10-foldbycentrifugalﬁltration(EMDMillipore),thenthesame proceduredescribedabovewascarriedout.

2.4.4. Determinationofreninrelease

Determinationofmastcell-derivedreninhasbeenusedasan additionalsuitablemarkerofmastcelldegranulationinducedby I/R.Thus,asafurthertestofpotentialinhibitoryeffectsof4CPIon mastcelldegranulation,weevaluatedapossiblereductionofmast cell-derivedreninin4CPI-treatedheartssubmittedtoI/R.

Reninactivitywasmeasuredinsamplescollectedfrom coro-nary effluent using a GammaCoat Plasma Renin Activity 125I Radioimmunoassaykit(DiaSorin,Stillwater,MN,USA)according tothemanufacturer’sinstructions.Briefly,200␮leachsampleof coronaryeffluentwereincubatedwithporcineangiotensinogen (0.1mg/ml),maleatebufferand PMSF(partof theDiaSorinkit). ThereactionforangiotensinIproductionwasconductedfor1.5hat 37◦C.Resultswerenormalizedtototalheartweight.Thedetection limitwasapproximately0.01pmol.

2.5. Acutemyocardialinfarction

Thecardioprotectiveeffectsof4CPIwereevaluatedinvivo,inan experimentalmodelmorecloselyresemblingtheclinicalcondition ofacutemyocardialinfarction.

Theexperimentalprotocolforcoronaryocclusion-reperfusion wasperformedasdescribedin[27],withminormodiﬁcations.Two hoursbeforetheexperimentalprocedure,ratsreceivedani.p. injec-tion(about0.3ml)of4CPI(0.24mg/kg)orvehicle(DMSO).Then, ratswereanaesthetizedwithsodiumpentobarbital(70mg/kg,i.p.). Thetracheawasintubatedandconnectedtoarodentventilator (mod.7025UgoBasile,Comerio,Italy)forartiﬁcialventilationwith roomair(strokevolume,1ml/100gbodyweight;70strokes/min). Electrocardiogram (ECG) was continuouslymeasuredby leadII (Mindray, PM5000, 2 BiologicalInstruments,Varese, Italy). The chest was opened by a left thoracotomy. A 6-0 surgical nee-dle was passed around the left anterior descending coronary

artery(LAD),locatedbetweenthebaseofthepulmonaryartery and leftatrium. Theends ofthe suturewerepassedthrough a polypropylene tube(PE50)to forma snare, allowingreversible arteryocclusion.Theacuteinfarctionprotocolconsistedof30-min occlusion/120-min reperfusion; successful occlusion was con-ﬁrmedbyobservingregionalcyanosisdownstreamoftheligature, and by ST elevation in theECG recording. A groupof vehicle-pretreatedanimalswassubmittedtoanIPCprocedure,achievedby 2cyclesof5-minocclusion/10minreperfusion,followedby30-min coronaryocclusionand120-minreperfusion.Eachexperimental groupwascomposedof6–10animals.Attheendofreperfusion, rats were euthanized by an overdose of sodium pentobarbital, heartswerequicklyexcised,mountedonaLangendorffapparatus (Radnoti,California,USA)andperfusedfor100withKrebssolution at37◦Ctocleansecoronarybloodvessels.Then,theatriaandright ventriclewereremovedfromthehearts.Theleftventriculartissue wasdried,frozenat−20◦_C,_and_cut_into_4–5_transverse_slices_from

apextobaseofequalthickness(about2mm).Thesliceswerethen incubatedina1%2,3,5-triphenyltetrazoliumchloride(TTC) solu-tioninaphosphatebuffer(pH7.4)at37◦Cfor20min.TTCreacts withNADHinthepresenceofdehydrogenaseenzymes,toform a formazanderivative,whichstaintheviablecells withintense redcolor.Thesliceswerethenﬁxedovernightin10% formalde-hydeandﬁnallyphotographed.Red-stainedviabletissuewaseasily distinguishedfromtheinfarctedwhite-unstainednecrotictissue. 2.6. Isolatedcardiacmitochondria

Toinvestigateapossiblemitochondrialinvolvement,theeffects of4CPIonmembranepotentialandcalciumuptakeweretestedin isolatedheartmitochondria.

Mitochondriawereisolatedbydifferentialcentrifugationfrom heartsexcisedfromadifferentgroupofrats.Theseratswerenot pretreatedwitheither4CPIorvehicle.Theseheartswerenot sub-mitted toany I/R protocol. Male Wistar rats(260–350g) were killedbypentobarbitaloverdose,heartswereremoved immedi-atelyandplacedinanice-coldisolationbuffer(compositionmM: sucrose250,Tris5,EGTA1,pH7.4adjustedwithHCl).Theatria wereremovedandtheventriculartissuewasfinelymincedwith surgicalscissors(about2mmpieces)andhomogenizedusingan Ultra-Turrax homogenizer (20ml of isolation buffer per heart, IKA1-WerkeGmbH&Co.,Staufen-Germany).Three homogeniza-tion cycles(eachof 20s)were performedonice, and then the suspensionwascentrifugedat1075gfor3minat4◦C(EuroClone, SpeedMaster14Rcentrifuge,Milano,Italy).Theresulting super-natantwascentrifugedat11,950gfor 10minat4◦C. Thepellet containingthemitochondrialfractionwasfurtherresuspendedin theisolationbuffer(withoutEGTA)andcentrifugedat11,950gfor 10minat4◦C,thisstepwasrepeatedoncemore.Thefinal mito-chondrialpelletwasresuspendedinaminimalvolumeof400␮lof theisolationbuffer(withoutEGTA)andstoredonicethroughout the experiments,which wereperformed within 2h. Mitochon-drialproteinconcentrationsweredeterminedusingtheBradford reaction.Previous experiments(datanotshown) confirmedthe reliabilityoftheisolationproceduresbymeasurementofthe mito-chondrialrespiratoryfunctionwithanATPbioluminescenceassay, inagreementwiththemethodofDrewandLeeuwenburgh,as pre-viouslydescribed[28].

2.6.1. Mitochondrialmembranepotential

Mitochondrial membrane potential (m) was measured potentiometricallywithtetraphenylphosphonium(TPP+_)-sensitive

mini-electrodes,coupledwithareferenceelectrode(WPI,FL,USA), usingadataacquisitionsoftware(BiopacInc.California,USA),as previouslydescribed[28].Brieﬂy,electrodeswerecalibratedbefore each experimentusing knownconcentrationsofTPP+_Cl−_.

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Mito-chondria(1mgprotein/ml)weresuspendedundergentlystirring intheincubationmedium(compositionmM:KCl120,K2HPO45,

Hepes10,succinicacid10,MgCl22,EGTA1,plusTPP+Cl−10␮M,pH

7.4adjustedwithKOH).Thevalueofthepotentialwascalculatedby thefollowingNernst-derivedexperimentalequation:wheremis themitochondrialmembranepotential(mV),

=60×LogV0·

[TPP+]0

[TPP+]t −Vt−K0P

VmP+KiP

V0isthevolumeoftheincubationmediumbeforethemitochondria

addition,Vtisthevolumeoftheincubationmediumafterthe

mito-chondriaaddition,Vmisthevolumeofthemitochondrialmatrix

(ml/mgprotein),[TPP+_]

0 and[TPP+]t are concentrationsofTPP+

recordedbeforetheadditionofmitochondriaandattimet, respec-tively,Pistheproteinconcentration(mg),K0andKiareapparent

externalandinternalpartitioncoefﬁcientsofTPP+_,_and_were

esti-mated as 14.3ml/mg and 7.9ml/mg, respectively. The volume ofmitochondriawastaken as1ml/mgofprotein.Mitochondria showingabasallevelofmembranepotential>−170mVwere dis-carded.Changesofmwerecontinuouslyrecorded(inmV)before andaftertheaddition intheincubation mediumof cumulative increasingconcentrationsof 4CPI(10–300␮M).Whenrequired, ATP(200␮M),physiologicalblocker ofKATP channels,orXE991

(10␮M,blockerofKv7channels)wereincubatedinthemedium 2minbeforethemitochondriaaddition.Effectsoftheadditionof thecorrespondingvehicleswereevaluated.Each concentration-responsecurvewasobtainedwithmitochondriaisolatedfromthe heartsof6–10differentanimals.

2.6.2. Mitochondrialcalciumuptake

Mitochondrialcalciumuptakewasmeasuredby potentiomet-ric technique, as described [28]. In particular, the changes of calcium concentration in the medium (i.e. extra-mitochondrial calcium) were continuouslymeasuredwith a calcium-selective mini-electrode,coupledwithareferenceelectrode(WPI,FL,USA), usingadataacquisitionsoftware(BiopacInc.California,USA).The selectivityoftheelectrodeforcalciumoverothercations,suchas magnesium,potassiumandsodium,is>105_._In_order_to_correlate

thepotentiometricmeasurements(inmV)withthecorresponding concentrationsofcalciumionsinthesolution,calibrationcurves weregeneratedbeforeeachexperiment,usingknown concentra-tionsofCaCl2.Mitochondria(1mgprotein/ml)wereadded,under

gentlystirring,totheincubationmedium(compositionmM:KCl 120, K2HPO4 5, Hepes10,succinic acid10, MgCl2 2, plus CaCl2

100␮M,pH7.4adjustedwithKOH)containingthevehicle(DMSO 1%)or4CPI(100␮Mor300␮M).Aftertheadditionof mitochon-dria,themaximaldecreaseoftheconcentrationofcalciuminthe medium,relatedtoitsaccumulationinthemitochondrialmatrix, wasmeasured.Eachresultwasobtainedwithmitochondria iso-latedfromtheheartsof6differentanimals.

2.7. Dataanalysis

Data were expressed as means±standard errors and were statisticallyanalyzed byANOVA and Student’st-test (software: GraphPadPrism4.0).Pvalueslowerthan0.05wereconsideredas indicativeofsigniﬁcantdifferences.

3. Results

3.1. EffectsonisolatedratheartsubjectedtoI/Rinjury

I/R caused marked functional damage to isolated hearts of vehicle-treatedrats,withasigniﬁcantreductionofmyocardial con-tractility.Indeed, duringreperfusion,theRPPvaluewasalways

lessthan50%ofthecorrespondingpre-ischemicone.Consistently, dP/dtvaluesweremarkedlyreduced(Fig.2A–B).Thefunctional impairmentinducedbyI/Rwasassociatedwithahighdegreeof tissueinjurydetectedbymorphometricanalysis(Fig.2C).

I/Rcausedlessdamageinisolatedheartsofratstreatedwith 4CPI(0.072-0.72mg/kg), as indicated by an improvedrecovery ofboth functionalandmorphometric parameters(Fig.2A-C). In contrast,atthehighestdosetested(2.4mg/kg),4CPIfailedto pro-ducecardioprotectiveeffects,asanevidentischemicinjury was observedatthisdose(Fig.2A–C).

Finally,pre-treatmentofanimalswith5HD(10mg/kgi.p.), a selectiveblockerofmito-KATPchannels,almostcompletely abol-ished the cardioprotective effects of 4CPI, administered at the selecteddose0.24mg/kg(Fig.2A–C).

DHEstainingofcontrolnon-ischemicsamplesofmyocardial tis-sueshowedaverylowlevelofROSproduction.Incontrast,heartsof vehicle-treatedanimalssubmittedtoI/Rrevealedahighdensityof DHE-stainednuclei,indicatingasigniﬁcantlevelofoxidativestress andROSproduction.Treatmentwith4CPI0.24mg/kgsigniﬁcantly reducedthelevelsofDHEstaininginmyocardialsamplesafterI/R injury(Fig.3A–B).

No release of _␤-hexo, a marker of mast cell degranulation, wasdetectableinthecoronaryperfusateofcontrolnon-ischemic hearts.showedHighlevelsof␤-hexowereinsteaddetectedinthe coronaryperfusateofratheartssubjectedtoI/R.Nochangesin ␤-hexoreleasewereobservedafter4CPItreatment(0.072,0.24and 0.72mg/kg).Incontrast,atthehighestdose(2.4mg/kg)4CPIcaused asigniﬁcantincreasein␤-hexorelease(Fig.3C).

WesternblotanalysisshowedabasalexpressionofCSE,CBS andMPSTinmyocardialtissue(shamgroup).I/Rdidnot signifi-cantlymodifytheexpressionofthethreeenzymes,althoughthere wasaslightnotsignificantincreaseinCSE(Fig.4A).Inhearts sub-jectedtoI/R,treatmentwith4CPIsignificantlyreducedCSEprotein levels,asalsodemonstratedbydensitometryanalysis(Fig.4B).In contrast,nosignificantchangesoccurredineitherCBSorMPST pro-teinexpressionfollowing4CPIadministration(Fig.4C–D).Notably, 4CPIdidnotchangelevelsofCBS,MPSTandCSEin“shamgroup” hearts(datanotshown).

3.2. EffectsonisolatedmouseheartssubjectedtoI/R

WhenLangendorff-perfusedmurineheartsweresubjectedto 30min of ischemia, followed by 30min reoxygenation, a large increaseinrenin,␤-hexoandnorepinephrine(NE)overflowwas observedduringthefirst5minofreperfusion,whichwas accompa-niedbyventriculartachycardia/fibrillation(VT/VF).Pre-treatment with4CPI0.24mg/kgsignificantlydiminishedNErelease,aswell asthedurationofVT/VF,whereasreninand␤-hexoreleasefrom residentmastcellswasnotmodified(Fig.5A-D).

3.3. 3Invivomodelofacutemyocardialinfarction

LADligature led,in vehicle-treatedrats,towideI/R-induced tissue injury (Ai/AVL=39±2%). IPC signiﬁcantly reduced I/R-induced tissue injury (Ai/AVL=17±6%);pretreatmentwith 4CPI 0.24mg/kgalsosigniﬁcantlyreducedthesizeofI/R-injuredareas (Ai/AVL=25±3;Fig.6A–B).

3.4. Effectsonmitochondrialmembranepotential

Membranepotentialofisolatedheartmitochondriawaslower than −170mV. Cumulatively increasing concentrations of 4CPI (10–300␮M)wereaddedtoamitochondrialsuspension(1mg/ml), and only at the highest concentration 4CPI elicited a signiﬁ-cantdepolarizationofmitochondrialmembranepotentialofabout 40mV.The effectof 4CPIwassigniﬁcantly antagonized byATP

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Fig.2. Post-ischemicfunctionalparametersofRPP(A)anddP/dt(B)recordedduringreperfusioninLangendorffratheartspre-treatedwithvehicle(black),withincreasing dosesof4CPI(0.072mg/Kg,magenta;0.24mg/Kg,blue;0.72mg/Kg,red;2.4mg/Kg,green)orwith4CPI0.24mg/kgplus5HD5mg/kg(gray).TwowayANOVAindicatedthat thecurvesobtainedinheartsofratspre-treatedwith4CPI(0.072,0.24and0.72mg/kg)showsigniﬁcantdifferences(P<0.01)vsvehicle.(C)Thecorrespondingdataemerging fromthemorphometricanalysisoftheareasofI/R-inducedtissueinjury(Ai/Avs)arealsoshown;asterisksindicatesigniﬁcantdifferences(*=P<0.05)vsvehicle.Inallthe panels,thedifferentpharmacologicalpre-treatmentsareindicatedbydifferentcolorsoflinesandhistograms:vehicle(black);4CPI0.072mg/Kg,magenta;4CPI0.24mg/Kg, blue;4CPI0.72mg/Kg,red;4CPI2.4mg/Kg,green;4CPI0.24mg/kgplus5HD5mg/Kg,gray.

(200␮M),thephysiologicalmitoKATPblocker(Fig.7A).Incontrast,

XE991,a Kv7 potassium channelblocker, didnot inﬂuencethe effectsof4CPI(datanotshown).

3.5. Effectsonmitochondrialcalciumuptake

Cardiac mitochondria (1mg/ml), exposed to 100_␮M CaCl2,

rapidlyand almostfully accumulatedthis cation,leading toan almostcompletereductionofcalciumconcentrationinthe extra-mitochondrial buffer (82±13␮M). 4CPI 100␮M and 300␮M signiﬁcantly reduced calcium accumulation in a concentration-dependentmode(Fig.7B).

4. Discussion

Heartdiseases,suchasmyocardialischemiaandinfarction,are majorcausesofmortality,and theidentiﬁcationof novel phar-macological tools tolimit I/R-inducedcardiac injury remains a challengingissue.ThediscoveryofH2Sasthethirdgasotransmitter

andtheunderstandingofitspivotalroleinregulating cardiovascu-larfunction,andinmediatingcardioprotection,pavedthewayto newexcitingperspectivesinthisﬁeldofresearch[29].In particu-lar,H2S-releasingdrugs,suchasGYY4137,haveshownsigniﬁcant

protectiveeffects inexperimentalmodelofmyocardialI/R[30], suggestingthatH2S-donorscanactuallybeviewedasapromising

classofanti-ischemicdrugs.

Inthispaper,weevaluatedthecardioprotectiveeffectsof4CPI and investigated the mechanisms of action. The H2S-releasing

propertiesof4CPIwereclearlycharacterizedinpreviousstudiesby usualamperometricandspectrometricmeasurements,andfurther conﬁrmedbygaschromatography,coupledwithmass spectropho-tometry[23].

In an ex-vivo experimental model of myocardial I/R (Langendorff-perfused rat hearts), i.p. pre-administration of 4CPI, at doses of 0.072, 0.24 and 0.72mg/Kg, ledto signiﬁcant recoveryofRPPanddP/dtduringreperfusionandtoanevident limitationoftissueinjury,althoughnocleardose-dependencywas observed.Accordingly, asanH2S-donor,4CPIaffordssigniﬁcant

cardioprotection against I/R-mediated myocardial tissue injury. Notably, at high doses (2.4mg/kg) 4CPI failed to show cardio-protective effects: indeed, both functional and morphometric parameters recordedafterI/Rinheartsofratspre-treated with this dose of 4CPIwere almost equivalentto those recorded in vehicle-treated animals. Such a “bi-modal” effect of 4CPI can be reasonably attributedto an excessiveformation of H2Sand

the consequentinduction of toxic effects, typical of thehigher concentrationofall gasotransmitters[31,32].Thishypothesis is supportedbythedeterminationofmastcell-derived␤-hexo.This markerofmast celldegranulationcouldnotbedetectedin the perfusateofnon-ischemicrathearts,butwasclearlyreleasedin heartsofvehicle-treatedanimals,subjectedtoI/R.Pre-treatment withcardioprotectivedosesof4CPI(0.072,0.24and0.72mg/kg) didnotreducethereleaseof␤-hexo,suggestingthatinhibitionof

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Fig.3. Representativepictures(A)andrelativequantificationoftheROS-positiveareas(B)oftheDHE-positivenucleiobservedbyfluorescencemicroscopyinventricular tissue.Nonischemiccontrol(CTR)heartswereisolatedfromvehicle-treatedratsandwerenotsubjectedtoI/R.HeartssubjectedtoI/Rwereisolatedfromvehicle-treated rats(I/R)orfrom4CPI-treatedrats(0.24mg/kg).InpanelC,␤-hexoreleaseinthereperfusionoverflowofLangendorffheartsofvehicle-(I/R)or4CPI-treatedratsarealso shown.Inthecontrolpre-ischemicoverflow(CTR)noreleaseof␤-hexowasdetected.Asterisksindicatesignificantdifferences(*=P<0.05;**=P<0.01).

mastcelldegranulationdoesnotplayarelevantroleinthe car-dioprotectionaffordedby4CPI.Incontrast,asigniﬁcantincrease of␤-hexo release wasobserved in theperfusate fromheart of animals pre-treated with the highest doseof 4CPI(2.4mg/kg), suggestingapossibletriggeringoftoxicresponses.

Basedontheaboveresults,0.24mg/kgwasselectedasan effec-tiveand safedosefor furthercharacterizing themechanism of actionof4CPI.

Duringmyocardialischemia,excessiveNEreleasefrom sympa-theticnerveendingsisamajorcauseofarrhythmiasandincrease inmetabolicdemand,whichlargelycontributetomorbidityand mortalityfollowingmyocardialinfarction.ThisNEreleaseis rein-forcedbyreninrelease frommastcellsandactivationofalocal renin-angiotensincascade[33].Weevaluatedpossibleeffectsof 4CPIonarrhythmiasandreleaseofNE,reninand␤-hexoinmouse heartssubjected to I/R. In this model, I/R wasassociated with severeventriculararrhythmiasandamassivereleaseofNE,renin and␤-hexo.Asalreadyobservedin rathearts,pre-treatmentof micewith4CPI(0.24mg/kg)ledtoclearcardioprotectiveeffects. Indeed, the H2S-donor caused a signiﬁcant reduction of

post-ischemicarrhythmias,and asigniﬁcantreductionofNE release. Yet,reninand␤-hexoreleasewasnotinﬂuenced.Theseresults sug-gestthatthereductionofNEreleaseandconsequentattenuationof ventriculararrhythmiasmaybeattributedtoanH2S-induced

sta-bilizationofsympatheticnerveendings.Indeed,H2Sdonors,such

asGYY4137,havebeenrecentlyreportedtoinhibit electrically-evokedNEreleasefromisolatedbovineiris-ciliarybodies[34].In contrast,asalreadyobservedintherathearts,inhibitionofthe releaseofmastcellproductsdoesnotseemtoplayasigniﬁcant role.

H2Seasilyreactswithmanyreactivespecies,suchassuperoxide

radical anion, hydrogen peroxide, peroxynitrite and hypochlo-rite, leading to their neutralization and reduction of oxidative stress[35].However,the anti-oxidantactivity ofH2Sis dueto

furtherand more complex mechanisms,such as theactivation ofpowerfulendogenousmechanisms,suchastheNrf2-mediated “anti-oxidant”machinery[36],possiblyinvolvedalsointhe car-dioprotectiveeffectsofH2S[13].Indeed,inexperimentalmodels

ofcardiacI/R,H2S-donorssuchasGYY4137werefoundtostrongly

inhibitoxidativestressinmyocardialtissue[30].Consistently,in ourstudy,I/RcausedadramaticincreaseinDHE-stainednucleiin rathearts,indicatingaveryhighlevelofROSproduction. Impor-tantly,4CPI(0.24mg/kg)completelyabolishedsuchanincrease, suggestingthatareductionofoxidativestressmaybeanadditional protectiveeffectsof4CPIagainstI/Rinjury.

CSE is likely to be the main source of endogenous H2S in

theheart,butconﬂictingresultsconcerningtheinﬂuenceofI/R onCSEexpression have beenreported[37]. Ischemiceventsin

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Fig.4.(A)WesternBlotsshowthebasalexpressionofCSE,CBSandMPSTinventriculartissueofheartsofthe“shamgroup”(sham),notsubjectedtoI/R.Moreover,the expressionofthethreeenzymesinventriculartissueofheartssubjectedtoI/R,isolatedfromvehicle-treatedor4CPI-treatedrats(0.24mg/kg)isalsoshown.Thecorresponding quantitativeanalysisforCSE(B),CBS(C)andMPST(D)expressionisalsoshown.Dataareexpressedasarbitraryunitsofdensitometry.Asterisksindicatesigniﬁcantdifferences vsvehicle(**=P<0.01).TheoriginaluncroppedimagesofWesternBlotexperimentsareavailableasSupplementarymaterial.

Fig.5. PanelAreportstheduration(seconds)ofventriculararrhythmiasrecordedduringreperfusionafterfollowingischemia.ReleaseofNE(B),renin(C)and␤-hexo(D) inthereperfusionoverflowofLangendorffheartsofvehicle-(I/R)or4CPI-treatedrats(0.24mg/kg)arealsoshown.Inthecontrolpre-ischemicoverflow(CTR)noreleaseof NEand␤-hexo,andaverymodestreleaseofreninweredetected.Asterisksindicatesignificantdifferences(*=P<0.05;***=P<0.001).

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Fig.6.MorphometricquantiﬁcationofI/R-inducedtissueinjuryobservedin ven-tricularslicesofrathearts,afteracutemyocardialinfarctioninvivo.Infarctionwas createdinvehicle-treatedrats(vehicle)orin4CPI-treatedrats(0.24mg/kg).Insome vehicle-treatedrats,acutemyocardialinfarctionwasprecededbyischemic pre-conditioning(IPC).Asterisksindicatesigniﬁcantdifferencesvsvehicle(**=P<0.01; ***=P<0.001).Imagesofrepresentativeventricularslicesarealsoshown.Paleareas indicateI/R-injuredtissue,whilehealthytissueiscoloredinred.

myocardiumhave beenassociatedwithanenhancementofCSE expression,suggesting a possible cardiac defensive mechanism directedat increasingH2Slevelsand limiting ischemicdamage

[38].In ourstudy,Westernblottingshowedthebasal presence ofallthreeH2S-biosynthesizingenzymesinratventriculartissue:

CSE,CBSandMPST.I/Rdidnotsigniﬁcantlyaltertheexpression of theseenzymes; however, a slightnot signiﬁcant increase of

CSEexpressionwasobserved,suggestingthetriggeringofa self-defencemechanism.Pre-treatmentwith4CPI(0.24mg/kg)caused adramaticinhibitionofCSEexpressioninheartssubjectedtoI/R, whileitdidnotcausesigniﬁcanteffectsonCBSandMPST.In con-trast,4CPIdidnotinﬂuencetheexpressionofCSEinheartsnot subjectedtoI/R.Thus,wecanspeculatethatsince4CPIdirectly increasesH2Slevel,anincreaseinCSEexpressionasaself-defence

mechanismagainstmyocardialischemicinjurymaybecome super-ﬂuous.

Asreportedabove,theactivationofmitochondrialKATPchannels

islikelytobeinvolvedinthecardioprotectiveeffectsevokedbyH2S

[11,12].Inourstudy,themitoKATP-blocker5HDcompletely

abol-ishedtheeffectsof4CPI,stronglysuggestingthatmitoKATPmaybe

amaintargetoftheanti-ischemiceffectsofthisnovelH2S-donor.

Tosolidifythishypothesis,weinvestigatedtheeffectsof4CPIon isolatedratheartmitochondria.Aspreviouslyobservedwith acti-vatorsofmitochondrialpotassiumchannels,4CPIcausedapartial depolarizationofthemitochondrialmembranepotential[28].This effectwasinhibitedbyATP,thussuggestingtheinvolvementof ATP-sensitivechannels.Incontrast,XE991didnotcauseany sig-nificantinfluence,indicatingthattheactivationofmitochondrial Kv7channeldoesnotcontributesignificantlytocardioprotection. Mitochondrialcalciumaccumulationisakeyeventinpromoting celldeathafterI/R,andtheinhibitionofcalciumuptakeintothe mitochondrialmatrixisconsideredapivotalmechanismofaction oftheanti-ischemiceffectsofmitochondrial potassiumchannel activators[10].Consistently,4CPIstronglypreventedcalcium accu-mulationintothemitochondrialmatrix,furtherindicatingthatthe mitoKATPchannelislikelytobearelevantpharmacologicaltarget

ofthisnovelH2S-donor.

Finally, the cardioprotective effects of 4CPI were tested in vivo,inanexperimentalmodelofacutemyocardialinfarctionin rats,moreclosely resemblingtheclinicalpattern ofmyocardial infarction. Alsoin this model, 4CPI(0.24mg/kg)exhibited car-dioprotective effects, which were comparable to those evoked by IPC, i.e. an endogenous and powerful self-defence mecha-nism. These results strongly suggest that isothiocyanate-based H2S-releasingdrugs,suchas4CPI,cantriggera“pharmacological

pre-conditioning”andcouldrepresentasuitablepharmacological optioninanti-ischemictherapy.

Fig.7.(A)Changeinmitochondrialmembranepotential(yinmV)uponincubationofmitochondriawith4CPI(300␮M),intheabsenceorpresenceofATP(200␮M). Asterisksindicatesigniﬁcantstatisticaldifferences(**=P<0.01).B)ChangesinextramitochondrialCa2+_{concentration}_(in_␮M)_upon_addition_of_mitochondria_to_a_calcium-rich

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AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,athttp://dx.doi.org/10.1016/j.phrs.2016.09. 006.

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