Chapter1

GeneralIntroduc2on

andOutlineoftheThesis

INTRODUCTION

BackgroundHeartFailure(HF)isacomplexsyndromecausedbyanabnormalityofcardiacstructureorfunc8onleadingtotheinabilityofthehearttodeliveroxygenataratecommensuratewiththerequirementsofthemetabolizing8ssues,oriftheheartisonlyfeasibletoachievethisattheexpenseofincreasedfillingpressuresoftheleAventricle(LV).1ThediagnosisofHFrequirestypicalsymptoms(suchasbreathlessness,ankleswellingandfa8gue)incombina8onwithsigns(suchaselevatedjugularvenouspressure,displacedapexbeatandpulmonarycrackles).2Sincethesesignsandsymptomscanhavemanydifferentcausesoraredifficulttodetectinanelderlyorobesepopula8onofHFpa8ents,furtherevidenceofcardiacdysfunc8oniswarrantedforthediagnosisofHF.

HeartFailurewithReducedandPreservedEjec2onFrac2onInthebeginningofthe1980s,theimportanceofLVejec8onfrac8on(LVEF)originatedfromindustry-drivenclinicaltrialsandtheemphasisonsta8s8csinthelightofthenovelprincipleofevidence-basedmedicine3.Inthesetrials,onlyHFpa8entswithaLVEF<40-45%wereincludedbecausetheywereexpectedtohaveagrimprognosisandthisselec8onbiaswasaimedatincreasingthesta8s8calpowerwithareasonablenumberofpa8ents.3Thisapproachledtoatremendousimprovementintherapeu8cop8onsinpa8entswithHFandareducedLVEF(HFrEForsystolicheartfailure)asdescribedinthecurrentguidelinesonthetreatmentofHFfromtheHeartFailureAssocia8onoftheEuropeanSocietyofCardiologyandtheAmericanCollegeofCardiologyFounda8on/AmericanHeartAssocia8on.2,4However,duringthepastdecadeitbecamemoreandmoreevidentthatthepopula8onofHFpa8entswithapreservedLVEF(HFpEF)increasesgradually.5,6SinceHFpEFischaracterizedbydiastolicLVdysfunc8on,thistypeofHFisalsoreferredtoasdiastolicheartfailure.Currently,HFpEFandHFrEFaccountforroughlyequalpropor8onsofHFpa8ents,7

butintheelderlyHFpEFhasalreadybecomethemostcommonformofHF.8Moreover,sincemoderntherapeu8cstrategieshaveproventopreventdeteriora8onofLVEF,ithasbeensuggestedthatmanypa8entshaveshiAedfromaHFrEFtoaHFpEFpopula8on.7Also,mortalityinHFpEFpa8entsisonlyslightlylowerthaninHFrEFpa8ents,9indica8veoftheburdenofthistypeofHFwithits

increasingprevalence.UnfortunatelyandinstarkcontrasttoHFrEF,notreatmentstrategystudiedtodateinlargeHFpEFtrialshasproventoimprovediseaseprogressionandsurvival,includingbetablockers,10angiotensin-conver8ngenzymeinhibitors,11angiotensin2receptorblockers,12,13anddigoxin.14Probablythemostimportantreasonforthesedisappoin8ngtrialsandthelackofevidence-basedtreatmentop8onsistheabsenceofaprofoundknowledgeaboutHFpEFpathophysiology.Indeed,justoveradecadeago,knowledgeaboutmyocardialstructureandfunc8oninHFpEFwasverypoor.15Thefollowingyearsmanystudiesaddressedepidemiological,clinicalandfundamentalaspectsinHFpEF,graduallyelucida8ngitspathophysiology.

Clinicalcharacteris2csofHFpEFpa2entsHFpEFischaracterisedbyahighincidenceofnon-cardiaccomorbidi8es.Althoughwaistcircumferenceandoverweightpa8entsareoAennotreportedinstudies,onethirdofpa8entshasabodymassindex≥30kg/m2.6,16Diabetesmellitusprevalencerangesfrom37to45%invariousregistries,whilearterialhypertensionshowsanevenhigherprevalence,rangingfrom76to96%.6,16–18Furthermore,around1/3ofHFpEFpa8entssufferfromchronicobstruc8vepulmonarydisease(COPD)and26-52%ofpa8entshavechronickidneydisease.6,18Moreover,manypa8entshavemorethanonecomorbidityandthenumberofcomorbidi8escorrelateswithprognosis.19,20Thesecomorbidi8essharethecapacitytoinduceachronic,low-gradeinflammatorystateandoxida8vestress,aswillbediscussedindetaillater.

Cardiaccharacteris2csofHFpEFTheLVinHFpEFischaracterizedbyconcentricremodeling,whereasHFrEFischaracterizedbyeccentricLVremodelingasillustratedinFigure1.21 BesidesmorphologicaldifferencesbetweenHFrEFandHFpEF,pressure-volume(PV)loopanalysesrevealtwodis8ncthemodynamicalprofiles(Figure2).22Thedominantfunc8onalabnormali8esinHFrEFaredecreasedLVcontrac8lity,asevidencedbyadecreaseintheslopeoftheend-systolicPVrela8onship(systolicelastance)andaglobaldown-andrightwardshiAofthePV-curve.Incontrast,inHFpEFthePV-loopisshiAedupwardandtotheleA,indica8veofincreasedLVdiastolics8ffnessandhigherfillingpressuresinasmallerLVcavityvolume.2ManystudiesthereaAerfocusedonthemyocardialmechanismsunderlyingtheincreasedLVdiastolics8ffnessobservedinHFpEF.

Myocardialcharacteris2csofHFpEFThemyocardiumconsistsof2majorcompartmentscontribu8ngtoLVdiastolics8ffness:theextracellularmatrix(ECM)andthecardiomyocytes.23StudiesonLVendomyocardialbiopsysamplesexpandedourknowledgeonHFpEFpathophysiologyenormously.Thefirstexperimentalstudiesshowedmyocardialfibrosiswithanincreasedcollagenvolumefrac8on(CVF)inHFpEFpa8entscomparedtocontrols.24Associa8onsofmyocardialCVFwithparametersrelatedtodiastolicdysfunc8onsuchasLVend-diastolicpressure24ortheE:E’ra8o(thera8ooftransmitralEvelocitytoearlydiastolicmitralannularvelocity)25havebeenfoundinHFpEFpa8ents.However,quan8fica8onoftotalcollagencontentwithCVFseemstohavelessfunc8onalimplica8onsthantherela8veamountofthes8ffercollagentypeIoverthemorecompliantcollagentypeIII,ortheamountofcross-linkedcollagenbylysyloxidase(LOX).26,27Forexample,humanHFpEFmyocardialbiopsysamplescontainedincreasedlevelsofcollagentypeI,enhancedcollagencross-linkingandLOXexpressionandthesefindingswereassociatedwithparametersofdiastolicdysfunc8onon8ssueDopplerechocardiography.25MyocardialfibrosisinHFpEFismainlyinters88al,whereasHFrEFischaracterizedbyreplacementfibrosisfollowingcardiomyocytecelldeath,sugges8ngdifferentpathophysiologicalmechanismsinbothHFen88es.28

Figure1:ComparedtothenormalheartintheleApanel,theHFpEFheartischaracterizedbyanormal-sizedLVcavitywiththickenedventricularwalls(concentricLVhypertrophy)andpreservedsystolicfunc8on.Incontrast,inHFrEF(rightpanel),theLVwallsarethinnerwitheccentricremodelingandanoveralldecreaseinsystolicfunc8on.Reproducedwithpermission,CopyrightMassachusejsMedicalSociety.21

However,besidesaltera8onsintheECM,cardiomyocytesundergotypicalchangesinHFpEF.Forexample,irrespec8veofCVF,cardiomyocytediameterwaslargerinbiopsysamplesfromHFpEFpa8entscomparedtoHFrEFpa8ents.29Also,whenmountedbetweenaforcetransducerandpiezzolectricmotor(Figure3),HFpEFcardiomyocytesappearedtobes8fferthancontrolorHFrEFcardiomyocyteswhenstretchedoverawiderangeofsarcomerelengths(SL).24,29Moreover,thisincreasedpassives8ffness(Fpassive)uponstretchcorrelatedwithLVend-diastolicpressuresandLVdiastolics8ffness.24 Also,cardiomyocytes8ffnesswashigherinHFpEFthaninHFrEFandhighestinHFpEFpa8entswithDM.29,30Uponadministra8onofproteinkinaseA(PKA),cardiomyocytes8ffnessdeclinedmoreinHFpEFthaninHFrEFcardio-myocytes,sugges8ngthataphosphoryla8ondeficitcontributestomyocardialanddiastolics8ffnessinHFpEF.Finally,infailinghearts,Ca2+reuptakeintothesarcoplasmicre8culumbytheSERCA(sarcoplasmic/endoplasmicre8culumcalcium-ATPase)pumpisdelayed.31ThisleadstoincreasedcytoplasmicCa2+concentra8onsandsubsequentincreaseddiastolics8ffness. Ithasbeenhypothesizedthatwhentheheartfillsandthemyocardiumisstretchedwithinaphysiologicalrange,Fpassiveisprimarilycausedby88ninthecardiomyocytes.32Inpathologicalsemngswherethemyocardiumisacutely

Figure2:Schema8cLVpressure-volumerela8onshipthrough1cardiaccycleinnormalhearts(green),HFpEF(red)andHFrEF(blue).HFrEFischaracterizedbyincreasedLVvolume,decreasedstrokevolume(SV,bluearrow)comparedtoanormalheart(greenarrow),buttheend-diastolicpressure-volume(PV)rela8onshipiscomparable(dojedgreyline).InHFpEF,theSVisdecreased(redarrow)andtheend-diastolicPV-rela8onshipisshiAedupwardandtotheleA(reddojedline),indica8ngincreaseddiastolics8ffness.

stretchedorinthecaseofmyocardialremodeling,ithasbeensuggestedthattheECMplaysamoreimportantrole,probablytoprotectthecardiomyocytesfromoverstretching.32

Molecularcharacteris2csofHFpEF-Ti2nSinceadministra8onofPKAcouldlowerFpassiveinHFpEFcardiomyocytesandthistoalargerextentthaninHFrEFcardiomyocytes,molecularchangesinHFpEFcardiomyocytesweretobeexpected.29Thegiantprotein88nisthemaindeterminantofmyocardials8ffnessincardiomyocytessuppor8ngdiastolicdistensibility.Ti8nmodulatescardiomyocytes8ffness,viaeitherphosphoryla8onoroxida8on.32Ti8ncan,forexample,bephosphorylatedbyproteinkinaseA(PKA)24,PKG33,byPKC34,calcium/calmodulin-dependentkinaseII(CaMKII)35andextracellularsignal-regulatedkinase(ERK).36PKGiss8mulatedbycyclicguanosinemonophosphate(cGMP),whichonitsturnissynthesizedfromguanylylcyclase.37Thelajerhas2isoformsandiseithers8mulatedbynitricoxide(NO)viasolubleGC(sGC)ornatriure8cpep8des(NP)viapar8culateGC(pGC).38LowPKGac8vityandcGMPconcentra8onwereobservedinhumanHFpEFLVmyocardiumandFpassiveofisolatedcardiomyocytesdecreaseduponinvitroPKG-administra8on.39 Itwasrecentlyproposedthatthelowgradeinflammatorystatethatisinducedbymetaboliccomorbidi8es,leadstomicrovascularendothelialinflamma8onandoxida8vestress.28Theseinflammatoryandoxida8veprocessesmaydecreaseNObioavailabilityandsubsequentlyNO-sGC-cGMP-PKGsignalling,eventuallyleadingto88nhypophosphoryla8onandincreasedmyocardials8ffness.However,besides88nhypophosphoryla8on,othermechanismsinside

Figure3:Amyocardialmusclestripmountedbetweenaforcetransducerandpiezoelectricmotoraboveaninvertedmicroscope.

thecardiomyocytesareexpectedtoincreaseFpassive.Recentstudiessuggestedaroleforproteotoxicityincardiacdysfunc8on,aspreviouslydemonstratedinneurologicaldiseasessuchasAlzheimer’sandHun8ngton’sdisease.40Thebasisforthisproteotoxicityresidesinfailingquality-controland/orrepairmechanismstocorrectforaggregatedordamagedproteinsasaconsequenceofinflamma8onandoxida8vestress,butitisalsopartofnormal(cardiac)aginganditcanaffectmitochondrialdysfunc8on.40,41Knowledgeabouttheroleforproteotoxicityandmitochondrialdysfunc8oninrela8ontoaging,inflamma8onandoxida8vestressinHFpEFiss8llpremature,butmightexplaintheabsenceofbenefitfromdifferenttreatmentstrategiessofar.42 Thegoalofthecurrentthesisistoinves8gatethepathophysiologicalmechanismsleadingtoincreasedmyocardials8ffnessinHFpEF.Moreover,amorethoroughknowledgeaboutthepathophysiologymighthelptoimprovediagnos8cstrategiesandiden8fypoten8altherapeu8ctargets.

1.1 AIM,OBJECTIVESANDOUTLINE

Theaimofthisthesiswastogainmoreinsightinthepathophysiology(chapter2,3,4),diagnos8c(chapter5,6)andtherapeu8cop8ons(chapter7,8)ofHFpEF.Forthis,weusedatransla8onalapproach,rangingfromaratmodeltohumanendomyocardialbiopsysamples(Figure4). InChapter2,thefocusisonmyocardials8ffnessinHFpEFanditscontributors.Inthisstudy,anovel,metabolicallyinducedZSF1-HFpEFratmodelwascharacterizedwithechocardiography,invasivehemodynamics,metaboliccagestudiesandon8ssueandproteinlevelaAersacrifice.Forcemeasurementswereperformedonsmallmusclestripsandisolatedcardiomyocytestodiscernthedifferentfactorscontribu8ngtomyocardials8ffness. Chapter3inves8gatesifsystemic,low-gradeinflamma8onofmetabolicriskcontributestoHFpEFthroughcoronarymicrovascularendothelialac8va8on.Inflammatoryendothelialac8va8on,myocardialoxida8vestress,NObioavailabilityandcGMP-PKGsignallingwereinves8gatedinhumanHFpEFmyocardialbiopsiesandvalidatedinZSF1-HFpEFrats. InChapter4,theroleof88nandECMremodelinginHFpEFarereviewed.DifferenttechniquestoassessECMquality,quan8tyanditsrelevancearediscussed,followedbychangesincardiomyocytesobservedinHFpEF.Also,cardiomyocyteandECMcross-talkisdiscussed. InChapter5,thefocuschangestodiagnos8caspectsofHFpEF.Weaknessesofthecurrentdiagnos8calgorithmarediscussed,followedbythepoten8alstrengthsofseveralbiomarkersreflec8ngmyocardialremodelling. Chapter6highlightsthedifficul8escliniciansareconfrontedwithinpa8entswithpulmonaryhypertension(PH),whenitsae8ologyisunclear.Pulmonarycapillarywedgepressure(PCWP)aidsinthisdiagnosis.IfPCWPishigh,PHislikelytobecausedbyleAsidedheartfailure.However,manypa8entswithHFpEFhaveanormalPCWPatrestandthischapterfocusesonhowtocopewiththisdiagnos8ctrap. Chapter7focusesoncardiomyocytebaseds8ffnessandapoten8altherapeu8cop8on(α-Βcrystallin,aheatshockprotein)isexaminedinvitro.Thisstudyuseshumanendomyocardialbiopsyspecimensonwhichforcemeasurementsareperformedonmusclestripsandsinglecardiomyocytes.

Chapter8concludeswithareviewonthecurrentknowledgeonHFpEF,includingdiagnosis,pathophysiologyandcurrentandpoten8alfuturetherapeu8cstrategies.

Figure 4. Outline of the thesis.

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