A search for novel treatments against ventricular arrhythmias

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Asearchfornoveltreatmentsagainstventriculararrhythmias

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© Jonas Skogestad, 2020

Series of dissertations submitted to the Faculty of Medicine, University of Oslo

ISBN 978-82-8377-613-3

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Print production: Reprosentralen, University of Oslo.

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Acknowledgments

TheworkinthisPhDthesiswascarriedoutattheInstituteforExperimentalMedicalResearch(IEMR) atOsloUniversityHospitalUllevålunderprincipalsupervisionofAssoiciateProfessorJanMagnus AronsenandcoͲsupervisionofProfessorIvarSjaastad.Theresearchwasfinanciallysupportedby AndersJahre´sfoundation,theResearchCurriculumStudentprogramatUiO,theNorwegian ResearchCouncil,SPARKNorwayandUiO:LifeScience.

Ifirststartedattheinstituteatamedicalstudentresearcherin2011,butthebeginningcanbetraced backtotheUnderwaterPubin2010whenNilsTovsrudnotifiedmethatHelsehøyskolenwaslooking fornewstudentteachers.IgotthejobandmetMagnusduringahistologyclass,whorecruitedmeto IEMR.UnderMagnus´sinspiringsupervision,Iwasgivenanacademicfreedomalmostwithout limitations,whichwasbothinspiringandgavemetheopportunitytoexploretherichnessofcardiac basicsciences.Frommyperspective,thisjourneyhasbeenintellectuallyrewardingandwehave accomplishedsomescientificachievementsthatweallcanbeproudof.

Firstandforemost,IwouldliketothankMagnusforthemanyyearsofexcitingscientific

collaboration.Becomingascientistwasachild´sdreamcomingtrue,andIwillalwaysremember theseyearsassomethingunique.Magnusisincrediblyinspiringtoworkwith,alwaysbringingnew ideastothetableandalwaysallowingmetoputmyindividualfootprintonthework.Thankyoufor allyoureffortsandpassion,ithasbeenanhonortoworkwithyou.Healsogatheredatalented groupofresearcherstogether,inwhatwecalledthe“PDEgroup”,whichconsistedofIngrid,Mira, SerenaandGustav.Ihavebeentrulyluckytoworkwithyouall.AspecialthanksstilltoIngrid,forour collaborationandconversationsduringthelastcoupleofyears.

WhenIstartedattheinstitute,Ivaropenedthedoorandgavemethechance.Thankyoualsoforthe highgradeofindependencethatyougavemeandMagnus,whichenabledustoexploredifferent scientificdirections.Finally,Iwouldalsothankyouforbeingmy“privatecardiologist”duringthe yearsof“geneticuncertainty”.

ThesocialenvironmentatIEMRhasalwaysbeenspecial,andIwillmissthediscussionsduringlunch.

IwillmentionMichael,ArneOlav,BillandPerAndreasinparticular,forbeingmylunchpartnersover alltheseyears.

Severalotheralsodeservementioning,althoughyourfullandindispensablecontributionscannotbe accountedforinthisshortsection.FredrikandMathisforteachingmepatchclamp.Karinafor teachingmecellisolations.Maniforallyouraltruism.ProfessorOleSejerstedforintroducingmeto therealmofsodiumandNKA.ProfessorFinnOlavLevyandLiseMoltzauforopeningthedooratthe departmentofpharmacology,OUSRH.Royformakingallthesesmallandcomplicatedtechnical things.Drs.EmmaRobinsonandCathrineCarlssonandMarianneLundeforyourimportantscientific contributions.AndtoIdaforyourpersonalsupport,Itrulyappreciateit.Thankyouall.

Finally,Ineedtoexpressmydeepestaffectiontomyfamily.Wordsareinsufficient.Thisthesisis dedicatedtomyfather,whoismymostimportantrolemodelandwhoalwayshasbelievedinme.

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1Abbreviations

AKAP–AͲkinaseanchoringprotein AnkB–AnkyrinB

AP–Actionpotential

APD–Actionpotentialduration ATP–Adenosinetriphosphate

BAPTA–1,2Ͳbis(oͲaminophenoxy)ethaneͲN,N,Nඁ,NඁͲtetraaceticacid CaMKII–Ca²⁺/CalmodulinͲdependentproteinkinaseII

cAMP–Cyclicadenosinemonophosphate cGMP–Cyclicguanosinemonophosphate

CICR–Ca²⁺ͲinducedCa²⁺release

CPVT–CatecholaminergicPolymorphicventriculartachycardia DAD–Delayedafterdepolarization

EAD–Earlyafterdepolarization

EͲCcoupling–ExcitationͲcontractioncoupling ECG–Electrocardiogram

EDTA–Ethylenediaminetetraaceticacid HF–Heartfailure

ICa–Ca²⁺current

ICD–Implantablecardioversiondefibrillator

IK1–InwardrectifyingK⁺current IKr–RapiddelayedrectifyingK⁺current IKs–SlowdelayedrectifyingK⁺current IKur–UltraͲrapiddelayedrectifyingK⁺current INa–Na⁺current

IP3–Inositoltriphosphate Ito–TransientoutwardK⁺current LTCC–LͲtypeCa²⁺channel MAB–Minimalankyrinbinding MI–Myocardialinfarction

mRNA–Messengerribonucleicacid

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NCX–Na⁺/Ca²⁺exchanger

NHE–Na⁺/H⁺exchanger NKA–Na⁺/K⁺ͲATPase

ORAB–OͲringaortabanding PDE–Phosphodiesterase PKA–ProteinKinaseA PLM–Phospholemman

PVC–Prematureventricularcontraction RMP–Restingmembranepotential RyR–RyanodineReceptor

SA–Sinoatrial

SBFI–SodiumͲbindingbenzofurzanisophtalate SCD–Suddencardiacdeath

Ser–Serine

SERCA2–Sarco(Endo)plasmicReticulumCa²⁺ATPase2 SR–Sarcoplasmicreticulum

Thr–Threonine

VF–Ventricularfibrillation VT–Ventriculartachycardia

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2Summary

Ventriculartachyarrhythmias,suchasventriculartachycardiaandventricularfibrillation,areleading causesofdeathworldwide,andareespeciallyprevalentinpatientswithchronicheartdisease.

Currentantiarrhythmictreatmentslackefficacyandhaveasevereburdenofsideeffects,highlighting theneedfornewandspecificantiarrhythmictreatments.

WehypothesizedthatreducedactivityoftheNa⁺/K⁺ͲATPase(NKA)couldleadtoCa²⁺overloadand arrhythmiasbyalteringtheactivityoftheNa⁺/Ca²⁺exchanger(NCX).Inpaper1,wefoundthat extracellular[K⁺]correspondingtomoderatehypokalemiainducedCa²⁺overloadandCa²⁺wavesin ratcardiomyocytesbyspecificallyreducingtheactivityofNKAɲ2,therebyalteringthe[Na⁺]sensed bytheNCX.Moreover,inpaper2,wefoundthatspecificdisruptionoftheNKAɲ2fromAnkyrinB (AnkB)ledtoalteredlocalregulationof[Na⁺]and[Ca²⁺]andincreasedfrequencyofCa²⁺wavesinrat cardiomyocytes,whichpotentiallyrepresentsanimportantproarrhythmicmechanismintheAnkyrin Bsyndrome.

Thefindingsinpaper1and2suggestthatNKAregulateslocal,NCXͲsensed[Na⁺],allowingthe NKA/NCXcomplextofunctionasanupstreammodulatorofarrhythmias.However,towhatextent [Na⁺]isdifferentiallyregulatedintheNKAcompartmentandwhethersuchlocalizedsubsarcolemmal Na⁺poolsexist,arenotknown.Inpaper3,wefoundthat[Na⁺]washeterogeneouslydistributed betweendifferentcompartmentsinthesubsarcolemmalspace,suggestingthat[Na⁺]canbe

discretelyregulatedinspecificdomains.Thisprovidesabiologicalbasisforthemainfindingsinpaper 1and2,andalsosuggestthattargetingtheNKA/NCXcomplex,duetothecompartmentalized regulationof[Na⁺]and[Ca²⁺],couldrepresentanopportunityforspecificantiarrhythmictreatments.

Inpaper4,wefoundthatinhibitionofphosphodiesterase2(PDE2)increasedNKAactivityand reducedintracellular[Ca²⁺]bymodulatingNCXactivityinratcardiomyocytes.PDE2inhibitors reducedthefrequencyofCa²⁺wavesandafterdepolarizationsindiseasedcardiomyocytes,and preventedventriculartachyarrhythmiasinmicewithheartfailureandingeneticallymodifiedmice resemblingtheAnkyrinBsyndrome.

ThisthesisthusconcludesthatreducedNKAactivityrepresentsanupstreammechanismfor

ventriculartachyarrhythmiasinchronicheartdiseasebylocallyregulatingtheNCXͲsensed[Na⁺],and thatPDE2inhibitorscouldrepresentanovelantiarrhythmictreatmentbyspecificallyactivatingNKA andlowerintracellular[Ca²⁺].

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3Listofpapers

Paper1

HypokalemiainducesCa²⁺overloadandCa²⁺wavesinventricularmyocytesbyreducingNKAɲ2 activity

AronsenJM*,SkogestadJ*,LewalleA,LouchWE,HougenK,StokkeMK,SwiftF,NiedererS,SmithNP, SejerstedOM,SjaastadI(2015).*Equalcontribution

JournalofPhysiology,Mar15.

Paper2

CouplingoftheNa⁺/K⁺ͲATPasetoankyrinBcontrolsNa⁺/Ca²⁺exchangeactivityincardiomyocytes

SkogestadJ,AronsenJM,TovsrudN,WanichawanP,HougenK,StokkeMK,CarlsonCR,SjaastadI, SejerstedOM,SwiftF(2019).

CardiovascularJournal,2019Apr5

Paper3

EvidenceforheterogeneoussubsarcolemmalNa⁺levelsinratventricularmyocytes SkogestadJ,LinesGT,LouchWE,SejerstedOM,SjaastadI,AronsenJM(2019).

AmericanJournalofPhysiology–HeartandCirculatoryphysiology,2019May1

Paper4

Phosphodiesterase2inhibitionactivatestheNa⁺/K⁺ͲATPaseandpreventsventriculartachycardia

SkogestadJ,RobinsonEL*,MellebyAO*,BørstadM,LundeM,MarshallS,AlbertI,SjaastadI,Carlson CR,AronsenJM.*Equalcontribution

Manuscript

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4Introduction

Fromthefirstfeebleheartbeatsinyourmother’swombuntillife’sfinalmoment,theheartdiligently pumpsbloodtoprovidetheentirebodywithnutrientsandoxygen.Cardiovasculardiseaseisa leadingcauseofmorbidityandmortality[1],andcommonheartdiseasesincludemyocardial infarction(MI),heartfailure(HF),andgeneticdiseases(e.g.hypertrophiccardiomyopathyandlong QTsyndromes).Despitethedifferentetiologiesofchroniccardiacdisease,acommoncauseofdeath inthesepatientsislethalrhythmdisorders(ventriculartachyarrhythmias).Currentlyused

antiarrhythmicdrugshavesuboptimalefficacyandahighsideeffectburden,andthereisaclear unmetneedfornewantiarrhythmictreatments.Thus,theoverallaimofthisthesishasbeento identifynovelmoleculartreatmenttargetsagainstventriculartachyarrhythmias.

4.1Cardiacarrhythmias

Eachheartbeat,andhencehumanlife,isdependentonasynchronizedcontractionandrelaxationof 3Ͳ5billioncardiomyocytes[2].Thenormalandsteadyheartrhythm,thesinusrhythm,isinitiatedby aspecializedgroupofmyocytesinthesinoatrial(SA)node,whichgeneratesanelectriccurrentthat rapidlyspreadsthroughtheheartthroughtheconductionsystem.Thiscausesaneffectiveand synchronouscardiaccontractionbyinducingopeningofvoltageͲgatedCa²⁺channels,leadingtoCa²⁺

influxandsarcomereshortening[3].Theprocessthatlinkselectricalexcitationwithcontractionin individualcardiomyocytesiscalledexcitationͲcontractioncoupling(EͲCcoupling),brieflysummarized inFigure1.However,inawidevarietyofcardiacpathologies,theconductionsystembecomes defectiveorpathologicalrhythmgeneratorsintheatriaorventriclesdevelop.Thismayleadtoan abnormalheartrhythm,collectivelycoinedarrhythmias.

Arrhythmiascanbeclassifiedaccordingtotheirorigin(atrialorventricular)andtheirpace(bradyͲor tachyarrhythmiaswhenthearrhythmiaisslowerorfaster,respectively,thanthesinusrhythm).The prevalenceisusuallyinverselyrelatedtotheseverityofthearrhythmia.E.g.prematureatrial contractionsarealmostuniversallypresenteveninthehealthypopulation[4],butrepresentsa clinicalproblemonlyinexceedinglyrareoccasions.Atrialfibrillationisrelativelycommon(e.g.

prevalenceisabout4%inEuropeansaged60Ͳ70)anddoublestheriskofdeath,mainlyduetoafiveͲ foldriskofstroke[5].Finally,themostseveretypearrhythmiasareventriculartachyarrhythmias (ventriculartachycardia(VT)andventricularfibrillation(VF)),whicharelifeͲthreateningunless properlyaddressedimmediately.AlthoughVT/VFarerareinthehealthypopulation,theyrepresenta seriousclinicalproblemforpatientswithcardiacdisease[6].

4.1.1Ventriculartachyarrhythmias

VTisarapid(>100beats/min)rhythmoriginatingfromtheventricles,whichacutelythreatensto compromisethehemodynamicstabilityofthepatient.VTcanalsodeteriorateintoVF,wherethe ventriclesfibrillateandareunabletopumpblood.VTcommonlyandVFinevitablyleadstosudden cardiacarrest,andpreventionofVT/VFiscriticalinpatientsatrisk.MIrepresentsthemostcommon causeofVT/VF,whereVT/VFarepresentin5Ͳ10%ofpatientsadmittedtothehospital,while additional5%ofthepatientswilldevelopVT/VFwithinthenext48hours[6,7].VT/VFarealsoan importantcauseofdeathinHF.E.g.about20Ͳ30%ofHFdeathsarecausedbyVT/VFwithout concomitantworseningofpumpcapacity[8].EarlytrialsshowedanageͲadjustedfiveͲfoldriskof suddencardiacdeath(SCD)amongHFpatients[9,10],althoughtheriskofSCDhasdeclinedinlater trials,probablyduetoimplementationofevidenceͲbasedmedications[11].Nevertheless,50Ͳ80%of

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patientswithHFhavenonͲsustainedVTduring24ͲhourHolterelectrocardiogram(ECG)registration [12,13],emphasizingthehighprevalenceofventriculartachyarrhythmiasinHFpatients.

Figure1:OverviewofEͲCcouplingandcardiaccontraction.(a)Thewallofheart(i)consistsofthreelayers:pericardium, myocardium,andendocardium(ii).Myocardiumisthecontractingelementoftheheart,comprisingbillionsofindividual cardiomyocytesinanadulthumanheart.Theouterboundaryofacardiomyocyte(iii)issarcolemma,whichhasregular invaginations,TͲtubules,tofacilitateefficientpropagationoftheelectricalsignalandcardiaccontraction.(b)Schematic overviewofEͲCcoupling.Na⁺channelopening(1)depolarizesthemembranepotential,leadingtoopeningofLͲtypeCa²⁺

channels(LTCC)andCa²⁺influx(2).Ca²⁺bindingtoRyanodineReceptors(RyR)triggersreleaseoftheintracellular sarcoplasmicreticulum(SR)Ca²⁺stores(3),andthereleasedCa²⁺bindsthemyofilaments,causingcardiaccontraction.

RelaxationisinducedbyCa²⁺extrusion(4),eitherintotheSRthroughSarco(Endo)plasmicReticulumCa²⁺ATPase2 (SERCA2)oracrossthesarcolemmathroughtheNa⁺/Ca²⁺exchanger(NCX),whichexchangesCa²⁺for3Na⁺.NKAremoves theNa⁺thatenteredthecellduringEͲCcoupling(5).(c)Thetimingoftheactionpotential(AP),cellularCa²⁺fluxesand contraction.(d)ThetimingandsizeofthemajorcurrentsconstitutingtheAP.

Other,rarercausesofVT/VFincludecongenitalchannelopathies(longQTsyndrome,

catecholaminergicpolymorphicventriculartachycardia(CPVT),andBrugadasyndrome),acommon causeofSCDinpersonsbelowtheageof35[14],electrolytedisturbances(suchashyperͲand

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hypokalemia),andsideeffectstoQTͲprolongingdrugs(suchastricyclicantidepressantsand antiarrhythmicdrugs)[6,15].

Insummary,VT/VFarebothprevalentandanimportantcauseofSCDincardiacpatients.Inorderto developnewpharmacologicaltreatmentsagainstVT/VF,itisnecessarywithacomprehensive understandingoftheunderlyingcellularmechanismsresponsibleforinducingventricular tachyarrhythmiasincardiacpatients.

4.2Cellularmechanismsofventriculartachyarrhythmias

ThetwomainmechanismsforVTarereͲentryandtriggeredarrhythmias.Althoughregularly describedastwoseparateentities,bothmechanismsareofteninvolvedintheinitiationand

maintenanceofaVT.MostVTsinnonͲischemicHFandabout50%ofVTsinischemicHFareinitiated bytriggeredarrhythmias,andthenoftenmaintainedbyreͲentrymechanisms[16,17].InreͲentry arrhythmias,thereisastructural(fibrosis,scarring)orelectrophysiologicalabnormality(e.g.delayed regionalconduction)thatcausesanabnormalelectricalcircuit.ThetypicalexampleisreͲentry circuitsaroundapostͲMIscar[18].Triggeredarrhythmiasarecausedbyearlyafterdepolarizations (EADs)ordelayedafterdepolarizations(DADs),whicharespontaneousfluctuationsofthemembrane potentialduringanactionpotential(AP).Ifthespontaneousdepolarizationreachesathreshold,a new,abnormalspontaneousAParises.Whenthisoccurssimultaneouslyinasufficientnumberof cardiomyocytes,anectopicbeatisgenerated,potentiallyinducingaVT[19,20].

DADsarecausedbyaseriesofeventsthatstartswithspontaneousCa²⁺releasefromthe

SarcoplasmicReticulum(SR)throughtheRyanodineReceptors(RyR),calledspontaneousCa²⁺sparks.

ThisoccursinsituationwhereSRCa²⁺concentration,theSRCa²⁺load,isincreasedabovethe

thresholdforrelease,orwhenthethresholdforreleaseisreduced,i.e.whenRyRopenprobabilityis increased[21,22].IfthespontaneousCa²⁺sparksaresufficientlylargetoactivatetheneighboring RyRs,thenthespontaneousCa²⁺releasecanspreadalongtheSRasaCa²⁺wave[23](Figure2a).The Ca²⁺releasedduringtheCa²⁺waveisthenextrudedacrossthesarcolemmabytheNa⁺/Ca²⁺

exchanger(NCX),whichgeneratesaninward,depolarizingcurrent,Iti[3].Itiisobservedasa

fluctuationofthemembranepotential,aDAD,duringtherestingphaseoftheAP(Figure2b).EADs occurduringtheplateauphaseoftheAPorearlyintherepolarizingphase,andtheyaremainly causedbyreducedrepolarizationreserve,i.e.increasedinwardCa²⁺currentsorreducedoutwardK⁺ currents,orbyCa²⁺waves[24Ͳ26].DuetotheprotectivesourceͲsinkmismatch,600000Ͳ800000 cardiomyocytesmustdevelopafterdepolarizationsinordertogenerateaPVCinahealthyheart, althoughthisissignificantlyreducedincardiacdisease[27,28].

Previously,digitalisͲderiveddrugswerewidelyusedasinotropicagentsinHF,butdigitalistoxicityhas longbeenrecognizedasacauseoftriggeredarrhythmias[29].Themechanismofbothcardiac inotropyandarrhythmiasisbelievedtobeinhibitionoftheNa⁺/K⁺ͲATPase(NKA)withsubsequent riseinintracellularCa²⁺concentration[Ca²⁺]duetoalteredNCXactivity[30].ReducedNKAactivity hasalsobeenproposedtobeinvolvedinthearrhythmogenesisofhypokalemia[31]andAnkyrinB syndrome[32],butmorestudiesareneededtoconfirmthehypothesisofNKAasanupstream modulatorofarrhythmias.Inthisthesis,weaimedtoinvestigatewhetherreducedNKAactivityis involvedasaproarrhythmicmechanisminhypokalemiaandtheAnkyrinBsyndrome,asintroduced inthefollowingparagraphs.

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Figure2:MechanismforconversionofCa²⁺wavesintoafterdepolarizations(a)SpontaneouslyreleasedCa²⁺ionsfromthe SR,aspontaneousCa²⁺spark,havethreepossiblefates.(I)ReͲpumpingbackintotheSR.(II)ActivationofnearbyRyR, leadingtofurtherreleaseofSRCa²⁺.Ifensuing,thismaytriggeraCa²⁺wave.(III)SomeofthereleasedCa²⁺isexchanged acrossthesarcolemmathroughNCX.SinceNCXmovesanevenamountofcharges(3Na⁺for1Ca²⁺),thisleadstoa depolarizationofthecellmembrane,calledadelayedafterdepolarization(DAD).(b)IftheDADreachesthresholdforNa⁺ channelopening,aspontaneousAPoccur.Reprintedfrom[33].

4.2.1Hypokalemia

HypokalemiaisdefinedasserumK⁺<3.5mMandisawellͲrecognizedriskfactorforVTs,inparticular TorsadesDePointesandVF[34,35].Hypokalemiaispresentinnearly20%ofhospitalizedpatients [36,37],with5%ofpatientshavingserumK⁺<3.0mM[36].Thiscorrespondstomoderate

hypokalemia,withanincreasedriskofcardiacarrhythmias[34].Themostcommoncauseof hypokalemiaisdiuretics,oftenusedbyHFpatientsalreadysusceptibletocardiacarrhythmias[38].

Hypokalemiainducesamultitudeofeffectsontheheartpossiblycontributingtothe

arrhythmogenesis.ThisincludesfunctionalreͲentrycircuits(duetoregionalvariabilityinconduction velocityandactionpotentialduration(APD)),reducedcardiacrepolarizationreserve,andincreased intracellularCa²⁺[31,38].ThelatterwasexploredbyEisneretal.alreadyin1979,wheretheyfound thatPurkinjefibersexposedtoK⁺ͲdepletedsolutionsdevelopedaftercontractionsdependentonNKA [39,40].However,sinceNKAishalfͲactivatedbyextracellularK⁺at1.5Ͳ2.0mM[41],andveryfew patientsdevelopK⁺<2.0mM,theclinicalrelevanceofthesedataisquestionable.Inaddition,others havereportedthatlowextracellular[K⁺]reducesratherthanincreasesintracellular[Ca²⁺][42].Thus, inpaper1,ouraimwastoinvestigatewhetherNKAinhibitioninducedbymoderatehypokalemia(2.7

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mM)isanupstreammechanismfortheensuingCa²⁺overload.Further,sinceNKAɲ2hasalower affinityforextracellular[K⁺]thanNKAɲ1andisknowntopreferentiallyregulateintracellular[Ca²⁺], wehypothesizedthattheeffectsofhypokalemiaonintracellular[Ca²⁺]weredependentonNKAɲ2.

4.2.2ArrhythmiamechanismsinAnkyrinBsyndrome

SpecificmutationsintheAnk2geneoftenleadtoacomplex,inheritablearrhythmiasyndrome referredtoas“AnkyrinBsyndrome”(previouslyLongQTsyndrometype4),characterizedbysinus bradycardia,conductionabnormalities,andVTs[32,43Ͳ45].TheAnk2genecodesforAnkyrinB (AnkB),ananchoringproteinthatassemblesNKA,NCX,andinositoltriphosphate(IP3)receptorsin macromolecularcomplexes[46].ThisfacilitateslocalregulationofNa⁺andCa²⁺,andtheAnkB^+/Ͳmice exhibitsdysfunctionalregulationofCa²⁺,leadingtocardiacarrhythmias[47,48],buttheroleofNKAͲ mediatedregulationofNCXͲsensed[Na⁺]inpromotingarrhythmiasisnotknown.However,the molecularmechanismsarenoteasilyinferredfromstudiesontheAnkB^+/Ͳmice,astheprotein assembly,proteintraffickingandexpressionofNKAandNCXarealteredinthismodel[32,46].Thus, inpaper2,weaimedtospecificallystudytheeffectofdisruptingNKAfromitsbindingsiteonAnkB onNCXͲsensed[Na⁺]andcellulararrhythmias.Tothisend,wesynthesizedadisruptorpeptidebased ontheMinimalAnkyrinͲBinding(MAB)domainonNKA,a25aminoacidresidueontheɲsubunitthat mediatesbindingtoAnkB[49].Wehypothesizedthatdisruptionofthisdomainwouldalterlocal [Na⁺]and[Ca²⁺]andleadtoCa²⁺waves,therebyprovidingevidencefortheNKA/NCXcomplexasan upstreamregulatorofcardiacarrhythmias.

4.3NKAͲmediatedregulationofNCXactivity

NKAhasforlongbeenrecognizedasaregulatorofintracellular[Ca²⁺]andcardiaccontractilityby regulatingintracellular[Na⁺].ThismodulatestheactivityofNCX,whichextrudes1Ca²⁺for3Na⁺ions, providinganinterfaceforNa⁺andCa²⁺regulation.NKAregulationofNCXhastwoaspectsof

particularimportanceforphysiologicalregulationofcardiaccontractility,andpotentiallyalsoforthe developmentofarrhythmias.First,theevidencesuggestthatNKAregulationofNCXisisoformͲ dependent,and,second,NKAregulationofNCXͲsensed[Na⁺]isprobablycompartmentalized,i.e.

dependentonintracellularNa⁺gradients.Thesetwoaspectsarestudiedinthisthesisandare introducedinthefollowingparagraphs.

ϰ͘ϯ͘ϭE<ɲϮƌĞŐƵůĂƚĞƐĐĂƌĚŝĂĐĐŽŶƚƌĂĐƚŝůŝƚǇ

NKAconsistsoftwosubunits,ɲandɴ,andintheheart,therearethreedifferentNKAɲisoforms[50, 51].NKAɲ1isthedominantisoform(70Ͳ95%)inhumanandrodenthearts,whereasNKAɲ2exhibitsa lowerexpression(10Ͳ30%)[52Ͳ55].WhilehumanheartsexpressNKAɲ3[53],thedataismore uncertainregardingNKAɲ3expressioninrodents[56Ͳ58].NKAɲ1isubiquitouslyexpressedinthe membrane,whereasNKAɲ2clustersintheTͲtubules [55].SeveralgroupshaveshownthatNKAɲ2 playsanimportantincontrollingcardiaccontractility[54,59,60],potentiallybyregulatinglocal[Na⁺] closetoNCX[61,62],andtherebymodulatingintracellular[Ca²⁺][59].

Twoaspectsofparticularimportanceforthisthesisare:

1. AnkyrinBisananchoringproteinthatassemblesNKA,NCXandIP3receptorsinamacromolecular complex[46],possiblyfacilitatingcompartmentalizedregulationofNa⁺andCa²⁺.Althoughboth NKAɲ1andNKAɲ2coͲimmunoprecipitatewithAnkyrinB,itisnotknownwhetherthedomainsare functionallysimilar.Inpaper2,weexploredthepossibilitythatNKAɲ1andNKAɲ2bindingto

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AnkyrinBdifferedfunctionally,andhypothesizedthatNKAɲ2bindingtoAnkyrinBwasimportant forregulationoflocal[Na⁺]and[Ca²⁺]andforthedevelopmentofcellulararrhythmias.

2. ThehalfͲmaximalactivationofNKA,k0.5,forintracellular[Na⁺]is8Ͳ25mM[51,63],butthe intracellular[Na⁺]dependencyissimilarbetweenNKAɲ1andNKAɲ2[64].Incontrast,NKAɲ1has alowerextracellular[K⁺]dependency( ๝1.3mM)comparedtoNKAɲ2( ๝2.7mM)[41].This difference makes the NKAɲ2 activity more susceptible to changes in extracellular K⁺ at physiologicallevels(3.5Ͳ5mM),butitiscurrentlyunknownhowNKAactivityisregulatedat pathologicallylow[K⁺]e(i.e.hypokalemia).Inpaper1,wehypothesizedthathypokalemiaͲinduced arrhythmiaswasmediatedbyNKAɲ2.

ϰ͘ϯ͘Ϯ>ŽĐĂůEĂ^нƉŽŽůƐĂŶĚƐƵďƐĂƌĐŽůĞŵŵĂůƐƉĂĐĞ

SeveralpublicationssuggestthatNKAɲ2preferentiallyregulates[Ca²⁺]andcardiaccontractility comparedtoNKAɲ1[54,59Ͳ62,65,66],andthisisexplainedbytheabilityofNKAɲ2inregulating localpoolsof[Na⁺][62,67,68].However,theconceptoflocalNa⁺poolsishighlycontroversial[69Ͳ 71].Basicchemistrysuggeststhationswouldfreelyandrapidlydiffuseandequilibrateinacell, preventinganylocalaccumulationofNa⁺ions.Nevertheless,severallinesofevidenceindicatethat localpoolsofNa⁺doexist,althoughtheexactnatureofthesepoolsremainelusive.Leblancand HumewerethefirsttoshowthatNa⁺influxthroughNa⁺channelswassufficienttoinduceCa²⁺Ͳ inducedCa²⁺release(CICR)byactivatingreverseNCX[72].Thisfindingrequiredrestricteddiffusion ofNa⁺withinacellularcompartmentcoined“fuzzyspace”,anundefinedandsmallspacebeneath themembrane,inwhichNa⁺channels,NCXandRyRreside[73].Sincethen,severalstudieshave shownthatNa⁺couldaccumulateinsubmembranecompartmentsnotdirectlyinvolvedinCICR (includingNKA)[54,60,74Ͳ77],andtheterm“subsarcolemmalspace”referstotheideaofadiffuse submembranelayerwithrestrictedmovementofNa⁺andwithdifferentconcentrationofNa⁺ comparedtobulkcytosol[68].

Adifferentpossibilityisthatthereareseveralsubmembranecompartmentswithdistinctive

regulationof[Na⁺],where[Na⁺]islocallyincreased(“hotspots”)orreduced(“coldspots”)dependent onlocalNa⁺influxorefflux[68].Theideaofaheterogeneoussubsarcolemmalspacecouldexplain whythereapparentlyisnoNa⁺accumulationaroundNHE[78],whilestudiesthathaveusedNKAasa sensorofsubsarcolemmal[Na⁺]concludesdifferently[75Ͳ77].Thehypothesisofaheterogeneous subsarcolemmalspacewith“hotspots”ofNa⁺isintriguingbecauseitcouldprovideabasisforNKAɲ2 asapreferentialregulatorofNCXͲsensed[Na⁺]andtherebyintracellular[Ca²⁺]andcardiac

contractility.Thus,inpaper3,weinvestigatedwhetherNa⁺wasdifferentiallyregulatedbetweenthe Na⁺channelandtheNKAcompartments.

Inthisthesis,amainhypothesisisthatNKAisanupstreammodulatorofventriculararrhythmiasby regulatinglocal,NCXͲsensed[Na⁺].Byextension,wehypothesizedthatincreasingNKAactivitymight holdpromiseasanantiarrhythmicstrategy.Theprospectoftargetingapotentialupstream

mechanismofventriculararrhythmiashaspotentialbenefitsoverthecurrenttreatments,which havelimitedefficacyandunfavorablesideeffects.

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4.4Currentandfutureantiarrhythmictreatmentstrategies

4.4.1Currentantiarrhythmictreatments

CurrentpharmacologicalantiarrhythmictreatmentscanbeclassifiedasclassIͲIVaccordingtothe classicalVaughanWilliamsscheme:ClassIisNa⁺channelblockers,classIIbetaͲblockers,classIIIK⁺ channelblockers,andclassIVCa²⁺channelblockers[6,79].Digitalisisantiarrhythmicdrugthat worksbyinhibitingNa⁺/K⁺ͲATPase(NKA)(andhencefallsoutsidethissimpleschematics),which currentlyisprimarilyusedtotreatatrialfibrillationinHF.Inaddition,althoughamiodaroneis classifiedasaclassIIIantiarrhythmic,ithasabroadclassIͲIVeffect[79].

Betablockersaretheonlyantiarrhythmicdrugsusedforprimaryorsecondarypreventionwitha proveneffectonmortalityinlargerandomizedclinicaltrials[6,64,80].Theexactmortalityreducing effectsofbetablockersaredifficulttoidentify,asbetablockersexertabroadrangeofeffects[81], andonestudyfoundnoreductioninarrhythmicdeathdespiteanoverallreductioninmortality[82].

However,betablockersappeartohaveahighlyprotectiveeffectinpatientswithlongQTsyndrome (withstructurallynormalhearts)[6,83],althoughnorandomized,doubleͲblind,placeboͲcontrolled trialshavebeenperformedduetotheethicalaspectsofrandomizingpatientsatriskofsudden cardiacdeathtoreceiveplacebotreatment.

Besidesbetablockers,severaloftheantiarrhythmicdrugshaveseveresideeffects,e.g.classIantiͲ arrhythmicdrugs(e.g.flecanideandlidocaine)andsomeclassIIIdrugs(e.g.dronedarone)are contraindicatedinstructuralheartdisease(e.g.postͲMIandHF)duetoaproarrhythmicriskand increasedmortality[6,84].Inaddition,Ca²⁺channelblockershavenegativeinotropiceffectswith littleeffectonVT[85,86],andhavenoroleinpreventionofVTinHF.Finally,amiodaronehasalong halfͲlifeinbloodandaccumulatesinorgans,causingorgantoxicity(e.g.thyroidglandandlungs)in about20%ofpatients[87].SeveraltrialshaveshownthatanImplantableCardioverterDefibrillator (ICD)reducesmortalitywhenusedinselectedpatientpopulations[88Ͳ90],butthereducedriskof SCDcouldinsomepatientpopulationsbeoffsetbyincreasedriskofdeviceinfectionsandby inappropriateshocks[91,92].

4.4.2Developmentofnewantiarrhythmictreatments

DespitethedevelopmentofthecurrentevidenceͲbasedtreatmentforHF,includingintroductionof ICDs,ventriculararrhythmiasandsuddencardiacdeathremainaprevalentclinicalproblem.Lackof sufficientantiarrhythmicprotectionisalsorelevantforpostͲMIandthechannelopathies,including longQTsyndromes,CPVT,andAnkyrinBsyndrome.Themainproblemofcurrenttreatmentsis, besideslackofefficacy,ahighburdenofsideeffects,whichlimitsthenumberofantiarrhythmic drugsavailableforuseandalso,inmanycases,preventstheuseofoptimaldosages.E.g.beta

blockershaveabroadrangeofintracellularpharmacologicaleffects,givingrisetoseveralsideeffects (e.g.bradycardia,dizziness,fatigue)thatpreventsmanypatientsfromreachingtheevidenceͲbased targetdoseusedinclinicaltrials[93].Otherantiarrhythmicdrugs,suchasNa⁺channelandK⁺ channelblockers,haveabroadandunfavorableelectrophysiologicalprofile,prolongingtheAPand theQTinterval[94,95],increasingtheriskofTorsadedesPointesandSCDinstructuralheartdisease [96Ͳ98].

Toovercometheselimitations,newantiarrhythmicstrategiescouldfocusontargetingspecific signalingdomainstoreduceundesiredintracellularpharmacologicaleffects.Inaddition,targeting

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compartmentalizedionregulationcouldalsopreventAPprolongationandreducetheriskof proarrhythmicsideeffects.

ϭ͘ϰ͘Ϯ͘ϭdĂƌŐĞƚŝŶŐƐƉĞĐŝĨŝĐƐŝŐŶĂůŝŶŐĂŶĚŝŽŶĐŽŵƉĂƌƚŵĞŶƚƐ͗<WƐĂŶĚWƐ

Oneapproachtoachievespecificnewtherapiesistotargetlocalsignalingdomains,e.g.distinct cAMPcompartments.CatecholaminesandprostaglandinEgiveasimilarintracellularincreaseinthe secondmessengercAMP,butthefunctionalresponsesareverydifferent[99].Thisisdueto

compartmentationofthecAMPsignaling[99Ͳ102],facilitatedbyAͲkinaseanchoringproteins (AKAPs),whichassembledifferentproteinsinvolvedincAMPsignaling,includingadenylylcyclases, phosphodiesterases(PDEs),proteinkinases,andphosphatases,inuniquesignalingdomains[103].

ThismoleculararrangementallowsdifferentialregulationofcAMPanditsdownstreameffectsin differentintracellularcompartments.OfparticularimportanceforthefineͲtuningofthecAMPsignal aredifferentisoformsofPDEs,whichdegradecAMP(andcGMP)withdifferentkineticsandmaximal velocity[104].TargetingisoformsofPDEsinuniquecompartmentsmightbeastrategyfor

developingspecificandefficienttreatmentswithlessundesiredeffects.

OnesuchcompartmentistheNKAcompartment,andacentralhypothesisofthisthesisisthatNKA activationcouldbeanovelantiarrhythmicstrategy.ThiscouldbeachievedbytargetingspecificPDE regulationoftheNKAcompartment,butcompartmentalizedɴͲadrenergicregulationoftheNKA compartmentisscarcelystudied.AprimaryregulatorofNKAactivityisphospholemman(PLM),a smallaccessoryprotein(72aminoacids)thatreversiblyinhibitsNKAactivity,mainlybyreducingthe affinityforintracellular[Na⁺][105].PLMhasthreephosphorylationsites,Serine(Ser)63,Ser68,and Ser69,whereSer68istheProteinKinaseA(PKA)site[106,107].ɴͲadrenergicstimulationincreases thephosphorylationofPLMatSer68,whichrelievestheinhibitoryactionofPLMandincreasesNKA activity[108],andɴͲadrenergicactivationofNKAisimportantissituationswithincreasedNa⁺ loading,e.g.exercise[105].ArecentstudyfoundthatPDE2andPDE3werethemainregulatorsof cyclicadenosinemonophosphate(cAMP)inthePLMcompartment[109],butalthoughAKAP150is showntointeractwithNKAinparotidglands[110,111],PDE/AKAPͲmediatedregulationofNKAhas notbeenshownincardiomyocytes.Inpaper4,westudiedcompartmentalizedregulationofNKAby PDEsandhypothesizedthattargetedactivationofNKAcouldbeanovelantiarrhythmicstrategy.

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5Mainaim

TheoverallaimofthisthesisistoidentifyupstreammechanismsofVT,andusethesetodevelop novel,targetedtreatmentsagainstVT.

5.1Specificaims

1. TostudywhetherreducedNKAactivityoralteredactivityoftheNKA/NCXcomplexrepresent anupstreammechanismforarrhythmiasinhypokalemiaandAnkyrinBsyndrome.

2. ToinvestigatewhetherspecificsubmembraneNa⁺compartmentsarepresentandtheroleof localNa⁺poolsinregulatingtheactivityoftheNKA/NCXcomplex.

3. ToidentifyNKAactivatorsandtotestwhetherNKAactivationpreventsventricular tachyarrhythmiasinpreclinicalmicemodels.

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6Methodsandresults

Themainmethodsandresultsfromthethesisarebrieflysummarizedinthissection.Acomplete overviewofmethodsandresultsisfoundintheattachedmanuscripts.

Paper1

HypokalaemiainducesCa²ЀoverloadandCa²ЀwavesinventricularmyocytesbyreducingNaЀ,KЀͲ ATPaseɲЇactivity

HypokalemiaisawellͲknownriskfactorforventriculararrhythmias,andinpaper2,weinvestigated theunderlyingcellularandelectrophysiologicalmechanismsinisolatedratcardiomyocytes.Low extracellularK⁺([K⁺]e=2.7mM),correspondingtomoderatehypokalemia,increasedCa²⁺transient amplitudeinfieldstimulatedcardiomyocytesthroughabiphasicresponse.Experimentsinvoltage clampedandfieldstimulatedcardiomyocytesshowedthatlow[K⁺]ereducedNKAcurrent,increased intracellular[Na⁺],increasedNCXͲsensed[Na⁺].ThisreducedNCXͲmediatedCa²⁺extrusion,leadingto agraduallyincreasedCa²⁺transientamplitude,Ca²⁺overloadandCa²⁺waves.Mathematical

modelingsuggestedthatthehypokalemiaͲinducedCa²⁺overloadwascausedbyreducedNKAactivity andreducedNCXͲmediatedCa²⁺extrusion,similartotheexperimentallyderivedresults.Specific inhibitionoftheɲ2isoformofNKApreventedhypokalemiaͲinducedCa²⁺overloadandincreased NCXͲsensed[Na⁺],suggestingthatNKAɲ2ismoresensitivetochangesinlow[K⁺]e.Ourdatasuggests thatreducedNKAɲ2activityandsubsequentreducedNCXͲmediatedCa²⁺extrusionmightbean importantmechanismofhypokalemiaͲinducedCa²⁺overloadandventriculararrhythmias.

Paper2

CouplingoftheNa⁺/K⁺ͲATPasetoAnkyrinBcontrolsNa⁺/Ca²⁺exchangeractivityincardiomyocytes

WefoundthatfunctionalinteractionbetweenNKAandNCXwasdisruptedinvoltageclamped AnkB^+/Ͳmicecardiomyocytes,possiblycontributingtoincreasedriskofventriculararrhythmiasthis modelandinhumanAnkBsyndrome.However,duetoarangeofunspecificalterationsintheAnkB^+/Ͳ mice,thismodelisnotoptimalforstudiesaimingtoinvestigatetheNKA/AnkB/NCXinteraction.To thisend,wesynthesizedadisruptorpeptide(MABpeptide)fortheNKA/AnkBinteractionbasedon publishedinteractionsequences.SimilartotheAnkB^+/Ͳmice,dialysiswiththeMABpeptidereduced thefunctionalinteractionbetweenNKAandNCX,andincreasedNCXͲsensed[Na⁺]involtageclamped ratcardiomyocytes.TheMABpeptidedidnotalterglobalCa²⁺amplitudeorextrusioninfield

stimulatedcardiomyocytes,butcaffeineͲinducedCa²⁺extrusionwasslower,suggestingthatNCXͲ mediatedCa²⁺extrusionwasreduced.TheMABpeptidefurtherincreasedthefrequencyofCa²⁺

sparksandCa²⁺waveswithnoeffectonCa²⁺transientamplitude.InhibitionofNKAɲ2prevented increaseinNCXͲsensed[Na⁺]involtageclampedratcardiomyocytesandcellulararrhythmiasinfield stimulatedratcardiomyocytesfollowingNKA/AnkBdisruptionwiththeMABpeptide.Ourresults suggestthatNKAɲ2bindingtoAnkBisnecessaryforlocalregulationof[Na⁺]inaNCXͲsensed compartment,andthatdisturbedNKAɲ2/AnkBinteractioncouldbeaproarrhythmicmechanismin humanAnkyrinBsyndrome.

Paper3

EvidenceforheterogeneoussubsarcolemmalNa⁺levelsinratventricularmyocytes

ToinvestigatethepropertiesofsubsarcolemmalNa⁺,weperformedaseriesofexperimentsin voltageclampedratcardiomyocytes.WeestablishedaconcentrationͲresponserelationshipbetween

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theNKAcurrentandintracellular[Na⁺],acurvethatwouldlaterbeusedtoestimateNKAͲsensed [Na⁺]basedonthemeasuredNKAcurrents.DuringlongͲtermexposuretophysiological[Na⁺](130 mM),therewasagradientbetween[Na⁺]intheNKAcompartmentandbulkcytosol.Shorttrainsof Na⁺currentsdidnotincreasedNKAͲsensed[Na⁺],whereasNa⁺accumulatedintheNKAcompartment followinglongertrainsofNa⁺currents,whichdissipatedveryslowlyfromtheNKAcompartment.We nextaimedtodeterminewhetherNa⁺accumulationinsubsarcolemmalspacewasdistributed homogeneouslyorheterogeneouslybetweentheNa⁺channelandNKAcompartment.Mathematical modelingpredictedthatifhomogeneouslydistributed,NKAͲsensed[Na⁺]wouldincreasemore following20secondsof6HzactivationofNa⁺currentscomparedto240secondsof1Hzactivation.

Whenexperimentallytested,wefoundtheoppositeresult,andmodelpredictingheterogeneity withinthesubsarcolemmalspacelargelyresolvedthediscrepancy.OurresultssuggeststhatNa⁺is differentiallyregulatedindifferentsubmembranecompartments,whichcouldhavegreatinfluence ontheregulationofNKA,cardiaccontractilityanddevelopmentofarrhythmias.

Paper4

Phosphodiesterase2inhibitionactivatestheNa+/K+ͲATPaseandpreventsventriculartachycardia WeinvestigatedPDEregulationofNKAandfoundthatinhibitionofPDE2,butnotPDE3orPDE4, increasedtheNKAcurrentinvoltageclampedratcardiomyocytes.NKAandPDE2coͲlocalizedinrat cardiomyocytes,asevidentbyproximityligationassay,andcoͲtransfectionofNKAandPDE2in HEK293cellsrevealedcoͲimmunoprecipitation.AluminescenceͲbasedcAMPassayrevealedthat therewasnoglobalincreaseincAMPfollowingPDE2inhibitioninratormicecardiomyocytes.Inline withthesefindings,disruptionofPKAͲRIIabolishedtheincreaseinNKAcurrentfollowingPDE2 inhibitioninvoltageclampedratcardiomyocytes.PDE2inhibitioninfieldstimulatedrat cardiomyocytesshowedreducedCa²⁺transientamplitudeandincreasedNCXͲmediatedCa²⁺

extrusionwithoutaglobalincreasein[Na⁺],suggestingthatPDE2functionallyregulatesalocal NKA/NCXdomain.VoltageclampexperimentsshowedthatPDE2inhibitiondidnotaltertheLͲtype Ca²⁺current,K⁺currents,ortheNa⁺current.PDE2inhibitionreducedCa²⁺wavesfrequencyinfield stimulatedandpreventedafterdepolarizationsinvoltageclampedAnkB^+/Ͳcardiomyocytes.Acute inductionofventriculartachycardiawaspreventedinAnkB^+/ͲmicepreͲinjectedwitheitherBay60Ͳ 7550orPF05180999,whichalsopreventedQTprolongation.Similarly,PDE2inhibitionincreasedthe NKAcurrent,reducedCa²⁺wavesandpreventedinvivoventriculartachycardiasinHFmice.CaMKII waslessautophosphorylated,suggestiveoflessactivity,inAnkB^+/ͲandHFmicereceivingPDE2 inhibition.PKAͲRIIdisruptionwithsuperAKAPpreventedtheantiarrhythmiceffectofPDE2inhibition bothinfieldstimulatedcardiomyocytesandinvivoinbothAnkB^+/ͲandHFmice,suggestingthatthe antiarrhythmiceffectofPDE2inhibitionistargetedanddependentuponregulationoflocalcAMP pools.ChronictreatmentwithaPDE2inhibitororthecombinationofPDE2inhibitorandmetoprolol wassuperiortometoprololaloneinpreventingventriculararrhythmiasinAnkB^+/Ͳmice.RealͲtime quantitativePolymeraseChainReaction(RTͲqPCR)showedthatPDE2ͲmRNAwasupregulatedinflow sortedcardiomyocytenucleifromAnkB^+/ͲandHFmiceandinhumancohortsofleftventricular hypertrophyandHF,suggestingthatPDE2couldbeatreatmenttargetinchronicheartdisease.Our findingsidentifyPDE2asamainregulatorofNKAactivity,andweshowthatPDE2inhibitionactivates NKA,preventscellularCa²⁺overload,andpreventsventriculartachycardiasinAnkB^+/ͲandHFmice.

PDE2inhibitorsspecificallytargetingNKAmightbeafutureantiarrhythmictreatmentinhumans.

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7Methodologicalconsiderations

Inthissection,Iaimtocriticallydiscussthecentralexperimentsandmethodsemployedtoreachthe conclusionsinthethesis.Theintentionisnottoprovidedetaileddescriptionsofalltheindividual methods(whicharefoundintheattachedmanuscripts),buttodiscussstrengthsandweaknessesof themethodsandwhetherthemethodsemployedareappropriatetosupporttheconclusions.

7.1Animalmodels

7.1.1Ethicalconsiderations

Ideally,allcellularexperimentswouldhavebeenperformedincellsisolatedfromhumansamples.

However,thisisnotfeasibleduetoethicalandpracticalconcerns,andbasicscientistscommonlyuse animalmodelsintheirstudies,whichofcourseevokesotherethicaldilemmas.Wehaveworked alongtheprinciplesofthe3R’s:Replace,reduceandrefine[112].Inparticular,wehaveemployeda micemodelofHFthatincreasesreproducibility,reducestheinterͲanimalvariationandreducesthe numberofanimalneeded[113].Inaddition,differentpeopleonthelabcollaboratedand

coordinatedexperimentswhenpossible,enablingseveralpeopletousecellsfromthesameanimal.

ExperimentsonmiceandratswereapprovedbytheNorwegianNationalAnimalResearch CommitteeandconformedtotheGuidefortheCareandUseofLaboratoryAnimals.

7.1.2Rodentmodels

Wehavemainlyusedrodents(miceandrats)inourexperiments.Rodentsarerelativelycheap,easy tohandle,andareconsideredarelevantanimalmodelforstudyingcardiacphysiologyand

pathophysiology[114]..However,therearesomeimportantdifferencesbetweentherodentand humanelectrophysiologythatneedtobeconsidered.Theheartrateisapproximately300beats/min and600beats/mininratsandmice,respectively,comparedto60Ͳ80beats/mininhumans.The fasterheartrateinrodentscorrespondstoamuchshorterAPD,andinmiceinparticular,wherethe APDisabout5Ͳ8timesshortercomparedtohumans(40Ͳ50msvs250Ͳ300ms)[115Ͳ117],mainly causedbyhighexpressionandactivityofItoinrodents[118].ThelargerItoleadstoaprominentnotch ontherodentAP,amorenegativeplateauphasepotentialand,hence,ashorterplateauphase comparedtohumansandotherlargeanimals[119,120].Otherdifferencesbetweenrodentsand largeranimalsrelatedtoCa²⁺andNa⁺homeostasisincludealowercontributionofNCXtototalCa²⁺

removalinrodents[3]andhigherintracellular[Na⁺]inrodents(10Ͳ15mMcomparedto6Ͳ8mMin largeranimals)[121,122],thelattercausedbyhigherbaselineNa⁺influx[123].Thesedifferencesare importanttoconsiderwhenextrapolatingexperimentalresultsincardiacelectrophysiologyfrom rodentstohumans.

DuetothebriefAPD,rodentsarenotpertinentmodelsfortypicallongQTarrhythmias,inwhich reducedK⁺currentsincreaseAPDandtheriskofEADs,astheseK⁺currentsareoftennotactivated duringtherodentAP.Incontrast,duetothehighrelativeSarco(Endo)plasmicReticulumCa²⁺ATPase 2(SERCA2)activity,rodentsareeasilyCa²⁺overloadedandconsideredtobeasensitivemodelfor Ca²⁺Ͳinducedarrhythmias[124],whichistheproposedmechanismforthearrhythmiasstudiedinthis thesis.

7.1.3Aorticbanding

Inpaper4,weusedmicewithaorticbanding(AB).Thebandingwasinducedbyinsertingarubber ringintheascendingaorta,anoveltechniquecalledORAB(OͲringaortabanding)recentlydeveloped byoneofthecoͲauthorsinpaper4[113].ThenovelORABmethodallowsforhighlyconsistent hypertrophicphenotypes,andmostmicedevelopsovertHFwithdilatedleftatrium,increasedheart

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weightandpulmonarycongestionwithinweekswiththeringdiameterusedinthisstudy.Inpaper4, weperformedinvivoandcellularexperiments14Ͳ18weekspostͲbanding,andonlyinmicewithHF confirmedbyechocardiography.

7.1.4Transgenicmodelsvsdisruptorpeptides

Inpaper2,weusedthetransgenicAnkB^+/ͲmiceasatooltostudytheeffectofAnkBontheNKA/NCX interactionandregulationofNa⁺andCa²⁺.However,trafficking,assemblingandexpressionofnonͲ AnkBproteins(includingNKAandNCX)arealteredintheAnkB^+/Ͳmice,andmightobscure

straightforwardconclusionsregardingtheNKA/AnkB/NCXinteraction.Tothatend,wesynthesizeda disruptorpeptidebasedontheMinimalAnkyrinBinding(MAB)domainonNKAandperformed experimentsinvoltageclampedratcardiomyocytes.Thisapproachallowedustodirectlystudythe effectofdisruptingNKAfromAnkB.ProximityligationassayverifiedthattheMABpeptidecoupledto NKAintheTͲtubules,andvoltageclampexperimentsshowedthatratcardiomyocytesdialyzedwith theMABpeptideandAnkB^+/ͲcardiomyocyteshadasimilarlackofcorrelationbetweenNKAandNCX currents.WethusconsideredtheMABpeptideapertinenttooltostudythefunctionalconsequences ofdisruptingtheNKA/AnkB/NCXdomain.

ϳ͘ϭ͘ϱVentriculararrhythmiasinvivo

Inpaper4,weaimedtoinvestigatewhetherpharmacologicinhibitionofPDE2preventedventricular arrhythmias,andweusedtwomicemodelsofventriculararrhythmias,HFmiceandAnkB^+/Ͳmice.

TheidealapproachwouldbecontinuouslysurveillanceoftheheartrhythmbyusingatelemetryECG systemthatregistersallarrhythmicevents.However,intheabsenceofproarrhythmicstimuli,such asisoprenaline/adrenalineandcaffeineinjectionsorexercise,miceveryrarelyexhibitVTs.Although itispossibletodetectprematureventricularcontractions(PVC),theseareonlysurrogatemarkersfor moreseriousVTsanddeath[125],withquestionableclinicalrelevance[126,127].Sincea

proarrhythmicstimulusisneededanyway,wechoseonlytomonitortheheartrhythmpriortoand followingtheinjectionoftheproarrhythmicdrug.Comparedtousingacohortofmicewithtelemetry recorders,thissavedusatremendousamountoftimeandresources,allowingustoincludeahigh numberofmiceinthestudies(about100miceinpaper4),muchhigherthanothercomparable papersinthefield[128].Webelievethatthiswouldincreasethereliabilityandreproducibilityofthe resultsinourstudy[129].

Standardprotocolstoacutelyinduceventriculararrhythmiasoftenincludeinjectionofboth isoprenalineandcaffeineinhighdoses(0.2Ͳ20mg/kgand120mg/kg,respectively).Inpaper4,we hypothesizedthatPDE2inhibitionwouldpreventarrhythmiasbylocallyincreasingcAMPandthereby activatingNKA.InjectionoftheseveryhighdosesofisoprenalinewouldlikelysaturatecAMPinall cellularcompartments[130,131],andwealsohaveunpublisheddatashowingthatmaximum contractilityinrodentheartsisreachedatisoprenalineconcentrationsintheμg/kgrange.The proarrhythmicmechanismofcaffeineismainlytoincreaseRyRconductanceandthereby intracellular[Ca²⁺][132,133],butitcouldalsoactasaPDEinhibitorathigherdoses[134].1mM caffeineelicitedventriculartachycardiain100%ofhealthyhearts[133],whereaswefoundthata singleinjectionof120mg/kgcaffeinewasnotsufficienttoinducearrhythmiasinWTmice(paper4).

Assumingthatina25mgmousethevolumeofdistributionforcaffeinewas~80%[135],120mg/kg correspondsto~0.75mM,atwhichtherateofPDEinhibitionislow[134].

SincewehypothesizedthatPDE2inhibitionwouldactantiarrhythmicbyreducingintracellular[Ca²⁺], wechosetousecaffeineasthemainarrhythmogenictrigger.Inunpublishedpilotexperiments,we

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alsotestedlowerconcentrationsofisoprenalineandcaffeineincombination(50ng/kgand60 mg/kg,respectively),whichyieldedsimilarresults.

Themicewererandomizedtoreceiveinterventionalorcontroltreatmentpriortoinitiationof experiment.Thenextmouse,oftenalittermatecontrol,wasthenstratifiedtotheothertreatment group.WealsobenchmarkedtheefficacyofthePDE2inhibitorBay60Ͳ7550againstthecommonly usedbetaͲblockerMetoprolol.Inthistrial,themicereceivedtheassignedtreatmentas

intraperitonealinjectionsfor5days,assomeofthechronicbetaͲblockereffectsstarttomaterialize afterlessthan1weektreatment[136,137].

Intheinvivoexperiments,weusedthePDE2AinhibitorsBay60Ͳ7550andPF05180999indoses(3 mg/kgand1mg/kg,respectively)wheretheydonotinfluencetheactivityofotherPDEs[138Ͳ141].

7.2Cellisolation

Theabundanceofexperimentsincludedinthisthesiswereperformedonisolatedrodent cardiomyocytes(ratsormice).Allelectrophysiologicalexperimentswereperformedonfreshly isolatedcardiomyocytes,6Ͳ8hourswithinexcision,asalterationsincellstructureandfunction appearrapidlyincardiomyocytesinprimarycultures[142].Individualcardiomyocyteswereselected forexperimentsmainlybasedontheirappearance(rodͲshaped,striated,absenceofspontaneous contractions).Forisolationofratcardiomyocytes,weemployedamodifiedLangendorffsetup,in whichtheexcisedratheartwasmountedonacannulaandperfusedwithaconstantpressure retrogradelythroughaorta.Forisolationofmousecardiomyocytes,weslightlymodifiedanovel techniquedescribedbyAckersͲJohnsonetal.[143],whichinvolvesmanualinjectionsofisolation buffer(withcollagenase)throughtheleftventricleoftheexcisedheart.Implementationofthis isolationtechniquedramaticallyimprovedthequality,yieldandconsistencyofourmiceisolations comparedtopreviousapproaches(usingretrogradeperfusion),andallowedrapidprogressthrough criticalstagesoftheprojectsdescribedinpaper4.

Severalmeasuresweretakentoincreasethelikelihoodofasuccessfulisolation.Weintubatedthe ratsandplacedthemiceonfacemask,whichallowedoxygenationoftheheartthroughoutthe surgicalprocedurepriortoexcisionoftheheart.Ratsreceivedanintravenousheparininjectionin theinferiorvenacava,whilethemiceheartswereinjectedwithasolutioncontaining

ethylenediaminetetraaceticacid(EDTA),bothtopreventbloodclotting,whichcompromisesthe perfusionoftheisolationbuffer.Followingexcision,theheartswererapidlytransferredtoiceorice coldbufferwithoutK⁺andCa²⁺(hypothermiccardioplegia)toreducemetabolicdemands.

7.3Electrophysiology

Electrophysiologicalexperiments,i.e.measurementsoftranssarcolemmalorintracellularionfluxes ormembranevoltage,constitutethemainpartofpaper1Ͳ3,andarealsoimportantinpaper4.The intentionofthefollowingparagraphsistocriticallydiscusstherationalefortheexperimentalsetups.

7.3.1Fluorescentdyes

MeasurementofintracellularCa²⁺isakeymethodthroughoutthisthesis.Inallfieldstimulation experiments,thecardiomyocyteswereloadedwith5μMFluo4ͲAM,afluorescentdyethatissimple touse[144],ataconcentrationthatminimizesunspecificeffects[145].Smithandcolleagues

reportedthat1,2Ͳbis(oͲaminophenoxy)ethaneͲN,N,Nඁ,NඁͲtetraaceticacid(BAPTA)Ͳderivedfluorescent dyes(includingFluo4andFura2)hadaprofoundNKAͲinhibitingeffect,especiallyathigher

concentrations.Theauthorsfoundthat10μMFluo4ͲAMsuppressedNKAactivityinculturedmice

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cardiomyocytesby30%,while2μMFluo4ͲAMhadnoeffect.TheNKAsuppressionwasofparticular concernintheCNS,whereswellingandastrocytedeathwasfound[145].Ofnote,thegenetically encodedCa²⁺indicatorGCaMP3didnotinhibitNKA,butthisapproachrequiresatransgenicmice and/orviraltransfectionofcellcultures,andarelessconvenientforexperimentsinfreshlyisolated cardiomyocytes.

7.3.2NKAcurrents

ThereareprincipallytwodifferentwaysofmeasuringtheNKAcurrentinvoltageclamped

cardiomyocytes.OneistousetheNKAinhibitorouabain(orothercardiacglycosides),whilethemain alternativeistoaddorremoveextracellularK⁺.Themaindrawbackofthelatteristhattherecould benanomolartracesofK⁺eveninasupposedlyK⁺Ͳfreesolution,whichcouldkeepsomeNKAsinan inactivatedstate,especiallywhenextracellular[Na⁺]iszero[146].Themainadvantagebymeasuring theNKAcurrentasaK⁺Ͳsensitivecurrent,isthatunliketheslowwashͲoutofouabain,K⁺isreadily washedout,convenientlyallowingsequentialactivationsofNKA.

Inpaper4,wheretheaimsimplywastocompareNKAactivityatdifferentinterventions(e.g.PDE inhibition),theNKAcurrentwasmeasuredinsymmetricalNa⁺solutionsbyremovingextracellularK⁺ fromthesuperfusate,andthecellswerenotexposedtoK⁺Ͳfreesolutionthatmightinfluencethe recordings.Intheseexperiments,wealsoemployedsymmetricalNa⁺solutions,i.e.solutionswhere thesuperfusateandtheinternalsolutionhavethesameNa⁺content,inordertoreduceanyeffects fromlocalNa⁺poolsontheNKArecordings.

Inallpatchclampexperimentsinpaper4,wherePDEfunctionwasstudied,5μMcAMPwasincluded intheinternalsolutiontoinvestigatetheeffecstofPDE2inhibitiononawiderangeofionchannels andpumps.5μMcAMPwaschosenbasedonastudythatmeasuredcAMPconcentrationsincytosol atdifferentdosesofIsoprenaline;5μMcAMPwasatthelowermiddlepartofthedoseͲresponse curve[147].TheideawasthatthecAMPaddedinthepipettebecomesasubstrateforthePDEsand activatesPKA,andthisapproachhasbeenemployedpreviouslybyothers[148Ͳ150].Thelowseries resistanceintheseexperimentsensuredthatpipettecAMPbecamethemaindeterminantof cytosoliccAMP,andthatanyeffectsofPDEinhibitorswereduetolocalregulationofcAMPin specificcompartments.AsaproofͲofͲprincipleoftheseassumptions,wehaveunpublisheddata showingthatPDE3inhibitionincreasesSERCA2activityinvoltageclampedcardiomyocytesonly whendialyzedwithcAMP;intheabsenceofcAMPPDE3inhibitionhasnoeffectonSERCA2activity.

7.3.3Globalandlocal[Na⁺]measurements

ComparedtotheadvancementswithinCa²⁺imaging,thetoolsforNa⁺measurementsare unsatisfactoryandlackthepropertiesnecessarytodetectsubsarcolemmalchangesinthe nanometerscale.Tomeasureglobal[Na⁺]inbeatingcardiomyocytes,weusedsodiumͲbinding benzofurzanisophtalate(SBFI)AMinthesingleexcitation(340nm)anddualemission(410nm/590 nm)mode,asdescribedbyBaartscheer[151],whichcandetectchangesinglobalintracellular[Na⁺] above2mM.Intheabsenceoftechniquestoadequatelyvisualizeandmeasuresubsarcolemmal [Na⁺],wemeasuredNCXorNKAcurrentsincarefullydesignedexperiments,andinterpretedtheseas ameasureofNCXͲorNKAͲsensed[Na⁺].

x MeasurementsofNCXͲsensed[Na⁺]:Inpaper2and3,thereversemodeNCXcurrentwas measuredbyrapidlyswitchingfromaCa²⁺Ͳfreesolutiontoasolutioncontaining2mMCa²⁺. Extracellular[Na⁺]wasconstant,whileintracellular[Ca²⁺]washeavilybufferedwithEGTA, andthecurrentamplitudewasthusinterpretedasNCXͲsensed[Na⁺].Inpaper2,we activatedreverseNCXintheabsenceandpresenceofactiveNKA(e.g.Fig.1A).Upon

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activationofNKA,intracellular[Na⁺]decreases[108],andreverseNCXisreduced

correspondingly[54].Innormalcardiomyocytes,thereisalinearcorrelationbetweenthe reductionsinNKAandreverseNCXcurrentsfollowingNKAactivation[54],acorrelationthat isabolishedinfailingcardiomyocytes[152].Arecentpublicationsuggestedthat

glutathionylationoftheNKAɴsubunitcouldinfluencethecurrentdecline[70].Similarly, anotherpapersuggestedthatthedeclinecouldbeduetoNKAsequestration[69].However, theideathatNKAactivationlowersintracellular[Na⁺],andthatthisinturnreducestheNKA activity,isinlinewithbasicbiologicalconceptsandseveralindependentobservations.Thus inpaper3,weusedthismethodtoinvestigatewhethertheNKA/NCXcorrelationwas presentinAnkB^+/ͲcardiomyocytesandinnormalcardiomyocytesdialyzedwithanNKA/AnkBͲ disruptingpeptide,butweinterpretedtheseresultsinthelightofotherdatausingdifferent methods.

x MeasurementofNKAͲsensed[Na⁺]:Inpaper3,weusedtheNKAcurrentasasensorfor[Na⁺] intheNKAcompartment,wherewecalibratedtheNKAcurrentagainstintracellular[Na⁺] (Fig.1).TominimizetheinfluenceofintracellularNa⁺gradients,weexposedthecellsto symmetricalNa⁺solutions.WethenmadeaconcentrationͲresponsecurvebetween intracellular[Na⁺]andNKAcurrents,whichwouldlaterenableconversionbetweenthe measuredNKAcurrentamplitudeandNKAͲsensed[Na⁺],similartotheapproachby

Verdoncketal.[153].IntheabsenceofCl^Ͳcurrentinhibitors(e.g.niflumicacid),NKAcurrents weremeasuredatͲ20mV,i.e.thecalculatedequilibriumpotentialofCl^Ͳ.However,this theoreticallycreatesaslightinwardNa⁺leak,possiblyleadingtoasmallerrorinour calibrationcurvethatwouldhavebeenavoidedbymeasuringNKAcurrentsat0mVand inclusionofNiflumicacidinthesuperfusate.Althoughthisdoesnotaffectanyofthe conclusionsinthepaper,itcouldleadtoanunderestimationoftheactual[Na⁺].

x ExperimentalapproachtoinvestigateheterogeneoussubsarcolemmalNa⁺levels:Tostudy thecommunicationbetweenNa⁺channelsandNKA,wemeasuredtheNKAcurrentbefore andaftervoltagepulses(Ͳ70mVͲ0mV)thatwouldactivatetheNa⁺current.Weinterpreted increasedNKAcurrentasincreased[Na⁺]intheNKAcompartment.However,ithasbeen showninskeletalmusclethathighfrequencyvoltagepulses(60Hz)activatesNKAandlead toapostͲactivationreductioninintracellular[Na⁺][154,155].Thisisaplausiblemechanism inskeletalmuscle,whereNKAactivationcouldpartlycounteracttheNa⁺loadinginducedby rapid,repetitivecontractions.However,cardiomyocyteshavemuchlowerstimulation frequencies(e.g.1Hz)andtheincreaseinintracellular[Na⁺]issufficienttoreachanew steadystate[156].WealsoconductedNKAmeasurementsbeforeandafterincreasing extracellular[Na⁺](Fig.2)andatdifferentholdingpotentials(Ͳ20mVvsͲ70mV,datanot includedinthethesis).Asallexperimentalinterventionsweredesignedtoincrease

intracellular[Na⁺],andallexperimentsyieldedsimilarresults,ourinterpretationwasthatthe increaseinNKAcurrentfollowingtrainsofdepolarizationswasduetoNa⁺influx.

7.3.4Cellulararrhythmias–Ca²⁺wavesandafterdepolarizations

Ca²⁺overloadinducesCa²⁺wavesandafterdepolarizations(EADsandDADs)andinitiatestriggered arrhythmias.600000Ͳ800000cardiomyocytesmustdevelopafterdepolarizationsinordertogenerate aPVCinahealthyheart[27,28],butitisstillmeaningfultomeasureCa²⁺overloadinindividualcells, asthisrepresentsameasureofthethresholdfortriggeredarrhythmiageneration.However,any findingsonCa²⁺overloadincells,shouldbereplicatedinexvivoorinvivoexperiments.

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TomeasureCa²⁺waves,cardiomyocyteswerefieldstimulatedatdifferentstimulationfrequencies, andCa²⁺wavesweredetectedduringstimulationͲfreeperiods.WavedetectionduringastimulationͲ freeperiodispossibleinrodentcellsduetothelowcontributionofNCXtoCa²⁺extrusion,which preventstherapidreductioninSRCa²⁺loadfollowingcessationofstimulationseeninlargeranimals [124].WealsomeasuredafterdepolarizationsincurrentclampedAnkB^+/Ͳcardiomyocyteswithand withoutPDE2inhibition.Itwasnotnecessarytoprovoketheafterdepolarizationsinthesecellswith isoprenalineorcaffeine,asbothEADsandDADswereprevalent.

7.4Statistics

Appropriatestatisticalanalysisisoftenneglectedinthebasicsciences[157],whichcouldcontribute tothereproducibilitycrisisinpreclinicalresearch[158].Inthisthesis,wehavestrivedtoapplythe correctstatisticaltestsonourdata.Tothisend,allrelevantdatasetswereanalyzedfornormal distribution,andtͲtestswereonlyemployedinthecaseofnormaldistribution;otherwise,nonͲ parametrictestswereused.Whenmultipledatasetsorendpointswerecompared,ANOVAtests withmultiplecomparisonswereused.Whencomparingbinaryoutcomes(e.g.presenceorabsence ofarrhythmias),weusedFisher’sexacttest.

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8Discussion

Theaimsofthisthesiswastoprovidenewinsightinarrhythmiamechanisms,andtousethis knowledgetodevelopnoveltargetedtreatmentsagainstventriculartachyarrhythmias.Ourfindings suggestthattheNKA/NCXcomplexisanupstreammodulatorofarrhythmiasinhypokalemiaand AnkyrinBsyndrome.ThismechanismlikelyinvolvesregulationoflocalNCXͲsensedNa⁺pools,making NKAͲmediatedregulationofNCXapromisingtargetformorespecificantiarrhythmictreatments.By usingPDE2inhibitors,wewereabletoactivateNKA,lowerintracellular[Ca²⁺]andpreventventricular tachyarrhythmiasinmicewithHFandAnkyrinBdeficiency.WesuggestthatactivationofNKA,e.g.

withPDE2inhibitors,couldbeafutureantiarrhythmictreatment.

8.1ReducedNKAactivity–aproarrhythmicmechanism?

8.1.1Hypokalemia

InhibitionofNKA,accumulationofintracellular[Na⁺]andsubsequentincreaseinintracellular[Ca²⁺] byreducinginwardNCXhasforalongtimebeenrecognizedasamechanismofdigitalistoxicity[159, 160],aconditioninwhichtoohighserumlevelsofdigitalisinduceavarietyofdifferentarrhythmias, includingVTandVF[29,161].Similarly,NKAhasbeensuggestedtobeinvolvedinthe

arrhythmogenesisinhypokalemiaandAnkyrinBsyndrome[31,44].Inpaper1and2,weprovide furtherevidencethatNKAisanupstreamregulatorofintracellular[Ca²⁺]andcardiacarrhythmiasin hypokalemiaandintheAnkyrinBsyndrome,andourresultsinparticularsuggestthattheNKAɲ2is importantbylocallyregulatingNCXͲsensed[Na⁺],therebymodulatingtheamountofCa²⁺extruded byNCX.

Inpaper1,wefoundthatextracellular[K⁺]correspondingtomoderatehypokalemia(2.7mM) reducedNKAactivity,increasedNCXͲsensed[Na⁺],reducedNCXͲmediatedCa²⁺extrusion,and inducedCa²⁺overloadandCa²⁺wavesinfieldstimulatedratcardiomyocytes.Followingspecific inhibitionofNKAɲ2,hypokalemianolongerinducedCa²⁺overloadorincreasedNCXͲsensed[Na⁺].A previousstudybyEisnerandLederershowedthatCa²⁺overloadandarrhythmiasinpotassiumͲ depletedsolutionsweremediatedbyNKA[40],buttheyused[K⁺]e=0mM,whichrepresentsan extremeconditionneverencounteredinpatients.Severalothermechanismshavealsobeen proposedtobeinvolvedinhypokalemiaͲinducedarrhythmias,includingreducedrepolarization potentialduetoreducedK⁺currents[31],buttheirrelativeimportanceareexpectedtovarywiththe serumlevelofK⁺,necessitatingstudiesonclinicallymeaningfullevelsofextracellularK⁺.Tothisend, thecellularexperimentsinpaper1wereperformedat[K⁺]e=2.7mM,correspondingtomoderate hypokalemia.

Whiletheresultsinpaper1suggestthatNKAɲ2inhibitioncouldbeanupstreammediatorof hypokalemiaͲinducedCa²⁺overload,wedidnotinvestigatetherelativeimportanceofCa²⁺overload comparedtootherproarrhythmiceffectsofhypokalemia,e.g.reducedK⁺currents.However,this wasinvestigatedinapaperbyPezhoumanandcolleagues,whichshowedthathypokalemiaͲinduced VT/VFinrabbitandratheartswaspreventedbyloweringextracellular[Ca²⁺].Incontrast,theK⁺ channelblockerdofetilideprolongedtheAPD,butdidnotelicitVT/VF[162].Importantly,alater studyalsoshowedthathypokalemiainducedCa²⁺overloadinbeatinghearts[163],whichconfirmed themainresultsofourcellularexperimentsinanintactheart.Mathematicalmodelingpredictedthat hypokalemiaͲinducedVT/VFwasdependentonNKAasanupstreammediatorofCa²⁺overloadin rabbitandrathearts[162].Basedonthis,leadingscientistsinthefieldhavesuggestedreducedNKA activityasakeyproarrhythmicmechanisminhypokalemia[164],inlinewithourmainconclusionsin paper1.

A search for novel treatments against ventricular arrhythmias