Thesis
Reference
The cardiac hERG channel: a multiple approach for a better understanding of the Long QT Syndrome
SINTRA GRILO, Liliana
Abstract
Le canal human ether à-gogo related gene (hERG) contrôle le courant potassique qui détermine la durée du potentiel d'action d'un cardiomyocyte. En réduisant ce courant, de nombreuses mutations dans le gène codant pour le canal allongent l'intervalle QT sur l'électrocardiogramme et donnent lieu à une maladie congénitale appelée syndrome du QT long (SQTL). Deux nouvelles mutations ont été caractérisées fonctionnellement et montrent une perte de fonction pouvant expliquer le syndrome observé. La forme acquise de la maladie se distingue de la précédente, car elle ne s'explique pas par des mutations génétiques.
L'étude réalisée sur la régulation physiologique de la densité de canaux hERG à la surface cellulaire constitue une première étape vers la compréhension d'altérations physiopathologiques qui sous-tendraient le SQTL acquis. De nombreux composée réduisent le courant par blocage du canal hERG, aboutissant à la forme induite par le médicament.
L'importance de la stéréosélectivité dans cet effet est abordée en dernière partie de ce mémoire. En conclusion, ce travail de thèse s'est [...]
SINTRA GRILO, Liliana. The cardiac hERG channel: a multiple approach for a better understanding of the Long QT Syndrome. Thèse de doctorat : Univ. Genève, 2010, no. Sc.
4224
URN : urn:nbn:ch:unige-107913
DOI : 10.13097/archive-ouverte/unige:10791
Available at:
http://archive-ouverte.unige.ch/unige:10791
Disclaimer: layout of this document may differ from the published version.
SectiondesSciencesPharmaceutiques ProfesseurH.Abriel
TheCardiachERGChannel:
AMultipleApproachForABetter
UnderstandingOfTheLongQTSyndrome
THÈSE
présentéeàlaFacultédesSciencesdel’UniversitédeGenève
pourobtenirlegradedeDocteurèsSciences,mentionsciencespharmaceutiques
par
LilianaSintraGrilo
de Lausanne(VD)
Thèsen°4224
AtelierReproMailGenève
Août2010
“Cen'estpasdanslasciencequ'estlebonheur, maisdansl'acquisitiondelascience.”
EdgarAllanPoe LePouvoirdesmots
Je tiens à adresser ma plus sincère reconnaissance au Professeur Pierre‐Alain Carrupt pour m’avoir accordé sa confiance et permis d’effectuer ce travail de thèse, partagé entre son groupe de Pharmacochimie et d’autres horizons. Merci pour son soutien, pour les ressources exceptionnelles mises à disposition et pour la liberté d’action qui m’a été accordée durant ces années de thèse.
Je tiens également à remercier tout particulièrement le Professeur Hugues Abriel pour m’avoir accueilli au sein de son groupe et m’avoir permis de profiter d’un environnement de travail très motivant. Merci également pour tous les apports scientifiques de premier plan concernant les canaux ioniques et la recherche scientifique en général, ainsi que pour son énergie inépuisable.
J’aimerais aussi remercier le Docteur Antoine Daïna qui a su me guider dans le monde (pas si) virtuel de la modélisation moléculaire. Merci pour sa disponibilité et sa motivation de tous les instants, et bien évidemment pour sa bonne humeur contagieuse!
J’aimerais également remercier tous mes collègues de Pharmacochimie, en particulier Elisabeth Favre pour sa « joie de vivre » et sa camaraderie lors des TPs, Delphine Cressend, Amandine Guillot, Bénédicte Gross‐Vallotton, les Docteurs Yveline Henchoz, Saviana Di Giovanni, Bruno Bard et Sophie Martel pour les moments de bonne humeur partagés au cours de mes séjours annuels et visites inopinées.
Un grand merci à tous mes collègues côtoyés au Département de Pharmacologie et Toxicologie de même qu’au Département de Recherche Clinique. Je tiens à témoigner ma profonde reconnaissance aux membres du « groupe HA », d’avant et après le déménagement, avec qui j’ai partagé l’essentiel de mes journées de thèse, mais soirées aussi. Un merci particulier aux ‘‘patcheurs’’ – à mon parrain le Docteur Jean‐Sébastien Rougier pour l’enseignement de son art, à ma moitié de tous les voyages, le Docteur Séverine Petitprez, et à Cédric Laedermann – avec lesquels de nombreuses discussions, d’ordre scientifique ou pas, ont agréablement comblé le
‘‘biochimistes’’ pour le partage de leurs connaissances et leur bonne humeur, que j’adresse spécialement au Docteur Bruno Gavillet et à notre Smooth du laboratoire, alias Anne‐Flore Zmoos, pour sa gentillesse à toute épreuve et son professionnalisme. Merci à Maria Essers pour son dévouement et son implication à simplifier notre installation dans le nouveau laboratoire d’outre‐Sarine. Merci à tous mes collègues qui ont rendu ce séjour parmi eux si agréable.
J’aimerais exprimer également mes remerciements aux ‘‘touristes HA’’ que la science ou l’amitié amenait plus souvent à se retrouver. J’adresse un merci particulier aux Docteurs Christine Gonzales, Armelle Takeda et Stéphany Gardier, à SarahPedretti, Luca Cariolato et Liliane Abuin pour leur contribution au bon déroulement de cette thèse, par leurs conseils avisés et leur expérience, scientifiques mais avant tout humains.
Je remercie TOUS ceux qui m’ont accompagné durant ces années de thèse sur les différents sites, sans oublier celles qui ont toujours su m’aider efficacement et agréablement dans les différentes démarches administratives, c’est‐à‐dire Sylvia Passaquay‐Rion, Isabelle Rivier‐Flühmann, Chantal Demont, Sabrina Kittel et Verena Frazao.
Merci aux membres du jury de thèse, le Docteur Antoine Daïna, le Docteur Esther Schenker, le Docteur Stephan Kellenberger et le Docteur Olivier Michielin, pour leur lecture attentive, leur regard critique et leurs précieuses recommandations.
Ma profonde gratitude va naturellement à ma famille et à mes amis – en particulier à mes parents et à ma sœur Daniela – pour leur présence, leurs encouragements et leur soutien irremplaçable au cours de ces années (qui ont dû leur paraître plus longues qu’à moi). Et finalement, j’adresse des remerciements particuliers à Christophe, pour la sérénité qu’il a su m’apporter dans les moments plus difficiles, pour son amour et sa présence à mes côtés pour écarter les doutes, soigner les blessures et partager les joies.
Merci !
Tableofcontent
Avantpropos ...iv
Résumédelathèse ... v
Abstractofthethesis ...vii
Listofabbreviations ...ix
–PartI– I. Background ... 3
I.A. Theheart ... 3
I.A.1. Heartandcirculatorysystem ...3
I.A.2. Themyocardium...5
I.A.3. Electricalpropertiesoftheheart...7
I.A.3.a. Electricalconductionsystemoftheheart ...7
I.A.3.b. CardiacactionpotentialandECG ...8
I.B. Cardiacchannelopathies... 14
I.B.1. LQTS...15
I.B.1.a. CongenitalLQTS...15
I.B.1.b. AcquiredLQTS...20
I.B.1.c. DruginducedLQTS...21
I.B.2. SQTS ...23
I.C. ThehERGpotassiumchannel ... 24
I.C.1. DiscoveryofhERGandtheEAGchannelfamily ...24
I.C.2. Structureandfunction...25
I.C.2.a. PredictedtopologyofhERG...25
I.C.2.b. GatingofhERG ...30
I.C.3. RegulationofhERGchannel...36
I.C.3.a. RegulationofcellsurfacedensityofhERGchannels(N) ...37
I.C.3.b. Regulationoffunction(g,Po)...40
I.D. Aimsofthethesis ... 50
II. hERGchannelregulationbytheubiquitinligaseNedd42... 53
II.A. Introduction... 53
II.A.1. Ubiquitinandubiquitylationforms ...53
II.A.2. Deubiquitylation ...56
II.A.3. Ubiquitylation ...57
II.A.4. RegulationofionchannelsbytheNedd4familyligases ...59
II.B. Publication1(underreviewattheJournalofBiologicalChemistry) Nedd42dependentubiquitylationandregulationofthecardiacpotassium channelhERG1... 64
II.C. Complementarydiscussion ... 87
–PartIII– III. CharacterizationofcongenitalLQTStype2mutations... 95
III.A. Introduction... 95
III.A.1. GenetictransmissionofLQTS...95
III.A.2. LocationandcodingtypeofLQT2mutations...97
III.A.3. Riskstratificationandtreatment ...98
III.A.4. MechanismsinvolvedinthelossoffunctionofhERG ...99
III.A.4.a. Defectivesynthesis(class1) ...100
III.A.4.b. Defectivetrafficking(class2) ...101
III.A.4.c. Defectivegating(class3) ...103
III.A.4.d. Defectiveionpermeation(class4)...104
III.B. Publication2(submittedtoAnnalsofNoninvasiveElectrocardiology) Patient with syncope and LQTS carrying a mutation in the PAS domain of thehERG1channel:acasereportandmutationcharacterization ... 105
III.C. Publication3(HeartRhythmJournal) Takotsubo cardiomyopathy and congenital LQTS in a patient with a novel duplicationinthePerArntSim(PAS)domainofhERG1 ... 118
III.D. Complementarydiscussion ... 142
–PartIV–
IV. DruginducedLQTSandstereoselectiveblockofhERG ... 149
IV.A. Introduction... 149
IV.A.1. DrugblockofhERGchannel...149
IV.A.1.a. MoleculardeterminantsofhERGchannelblock ...150
IV.A.1.b. CharacteristicsofhERGchannelblock ...152
IV.A.2. MolecularModeling ...154
IV.A.3. Chiraldrugsandstereoselectivity ...159
IV.A.3.a. Stereoselectivityandionchannels...160
IV.A.3.b. StereoselectivityandQTintervalprolongation ...161
IV.A.4. Pharmacogenetics ...164
IV.B. Publication4(ClinicalPharmacologyandTherapeutics) Stereoselective block of hERG channel by (S)methadone and QTinterval prolongationinCYP2B6slowmetabolizers ... 168
IV.C. Publication5(inpreparation) Electrophysiological and molecular modeling investigation of the hERG channel’sstereoselectiveblockbybupivacaineenantiomers ... 180
IV.D. Complementarydiscussion ... 229
–PartV– V. Conclusionandperspectives ... 241
Appendices... 247
AppendixI:CompoundsknowntoblockthehERGchannel... 247
AppendixII:ChiralcompoundsknowntoblockthehERGchannel ... 249
AppendixIII:Overviewofpreclinical“QTmodels”andparametersevaluated... 250
AppendixIV:Maleandfemaleequality:stillfarfromgoal(J.Physiol.) ... 252
AppendixIV:StereoselectiveblockofhERGchannel bybupivacainescrutinizedat molecularlevel(CHIMIA)... 245
References……… 261
Avantpropos
La présente thèse a été effectuée conjointement au sein de la Section des Sciences Pharmaceutiques (Université de Genève, Université de Lausanne) et du groupe Canaux Ioniques (Université de Lausanne, Université de Berne), sous la direction du Professeur PierreAlainCarrupt duProfesseur Hugues Abriel. Le travail de thèse présenté dans ce manuscrit se divise en plusieurs parties distinctes qui disposent d’une introduction théorique spécifique et des articles scientifiques, publiésouenpréparation,réalisésdanslesdifférentsdomainesderechercheayant poursujetprincipallecanalpotassiquehERG.
Résumédelathèse
Le canal hERG ouhuman ether àgogo related gene est responsable de la composante rapide du courant potassique rectifiant retardé (IKr) qui détermine en partie la durée du potentiel d'action dans une cellule cardiaque. Il est désormais établiquececanalpotassiquedépendantduvoltageestforméparl'assemblagede quatresousunitésDcodéesparlegèneKCNH2.
En réduisant les courants de repolarisation, de nombreuses mutations de type“perte de fonction” dans le gèneKCNH2 peuvent allonger l'intervalle QT sur l'électrocardiogramme;cesmaladiesgénétiquessontappeléessyndromeduQTlong (SQTL) de type congénital. Un intervalle QT prolongé est un facteur de risque d'arythmies potentiellement mortelles désignées sous le nom de Torsades de Pointes. Une autre forme de SQTL liée au canal hERG est source de préoccupation dans le domaine pharmaceutique. En effet, de nombreux médicaments ont la capacité indésirable de bloquer le canal hERG; il en résulte une forme de SQTL induite par le médicament. Puisque les structures et classes thérapeutiques des médicamentsimpliquéssontvariées,lasusceptibilitédetoutnouveau médicament enverslecanalhERGdoitêtreévaluée.Cecipeutconduireàuneperteéconomique considérablesilamolécules’avèrebloquerlecanalpotassiqueaprèsdesannéesde développement.IlexisteégalementuneformeacquisedeSQTLquines'expliquepas par des mutations dans les gènes liés au SQTL ni par un blocage du canal hERG. Le SQTL acquis est également reconnu pour augmenter le risque d'arythmies ventriculairesdangereuses,bienquelesmécanismessousjacentssoientmoinsbien connus.
DepuisladécouverteducanalhERGdanslemilieudesannées90etdesonrôle central dans le SQTL, de nombreux travaux ont été entrepris afin d’élucider ses mystères. Le présent travail de thèse a cherché à mettre en lumière différents aspectsdecefascinantcanalpotassique.
La première étude présentée dans ce manuscrit traite de la régulation du canal hERG. Dans un système de surexpression, il a été montré que le canal potassique hERGinteragitavecl'ubiquitineligaseNedd42viaunmotifparticulier(lemotifPY)
protéinehERGparl’enzyme.Cettemodificationdéclenchelarégulationnégativedu canal présent à la membrane plasmatique et la diminution du courant mesuré.
L’améliorationdesconnaissancessurlaphysiologienormaleducanalhERGestun premierpasverslacompréhensiondemodificationsphysiopathologiquesquisous tendraientleSQTLacquis.
Le deuxième aspect traité et présenté dans ce manuscrit concerne la forme congénitaleduSQTL.DeuxmutationsdanslegèneKCNH2,nondécritesauparavant, ont été caractérisées fonctionnellement et présentées dans des rapports de cas distincts.Lesdeuxsujetssontdesfemmes,portanttoutesdeuxunemutationdansla partie aminoterminale de la protéine hERG, plus précisément dans une région hautementstructurée,ledomainePAS.Lesmutationsontétéreproduitesdansdes systèmes de surexpression à l'état hétérozygote et ont montré une réduction d’environ50%et75%ducourant.Cettediminutionestvraisemblablementdueàun défautdansleroutagedescanauxmutantsnouvellementsynthétisés.Danslesdeux cas,lapertedefonctionducanalhERGpeutexpliquerlephénotypeSQTLdupatient.
La dernière section de ce mémoire aborde, par deux travaux différents, la question de la stéréosélectivité dans le blocage du canal hERG. La composante stéréosélective a été négligée en ce qui concerne l’allongement de l'intervalle QT induitpardesmédicament.Néanmoins,danslapremièreétude,l’énantiomère(S) delaméthadoneadémontréêtreuninhibiteurpluspuissantquelaforme(R),cette dernière étant l'énantiomère actif pour les cibles pharmacologiques. Une reévaluationdelaprescriptiondumédicamentsoussaformeracémiqueenfaveur de la seule (R)méthadone est également proposée. En effet, près de 6% de la population de type caucasienne et africaine serait à risque plus élevé de prolongation de l'intervalle QT dû à une métabolisation lente de la méthadone touchant préférentiellement la forme (S). La deuxième étude a tiré parti de la techniquedemutagenèseetdesimulationsd’ancragepourévaluerlesfondements moléculairesquirégissentleblocstéréosélectifducanalhERGparlabupivacaïne.En outre, l'ancrage d'énantiomères dans un modèle d'homologie s’est révélé être un moyenélégantdetesterlastratégiedéveloppée.
Abstractofthethesis
The potassium hERG channel orhuman ether àgogo related gene channel is responsiblefortherapidcomponentofthedelayedrectifierpotassiumcurrent(IKr), whichisanimportantdeterminantoftheactionpotentialdurationintheheart.Itis now established that this functional voltagegated potassium channel is formed by assemblyoffourDsubunitsencodedbytheKCNH2gene.
Byreducingrepolarizingcurrents,manylossoffunctionmutationsinKCNH2gene can prolong the QT interval on the ECG; such genetic disorders are referred to as congenitalLongQTSyndrome(LQTS).LengtheningoftheQTintervalisawellknown risk factor for potentially lethal arrhythmias namedTorsades de Pointes. Another hERGrelatedformofLQTShasbeenidentifiedandcausesimportantconcerninthe pharmaceutical area. Indeed, a variety of medications have the undesired abilityof blocking the hERG channel and resulting in adruginduced form of LQTS. Since structures and therapeutic classes are diverse,hERG liability has to be assessed for all new drugs, sometimes leading to considerable economic loss if, after years of development,thedrugrevealstoblockthepotassiumchannel.Anacquiredformof LQTS,whichisnotexplainedbyvariantsinLQTSgenesorduetohERGblockerdrugs, is also recognized as increasing the risks of malignant arrhythmias, although its underlyingmechanismsarelesswellunderstood.
SincediscoveryofthehERGchannelinthemid1990’sanditscentralroleinLQTS, a lot of work has been undertaken in the attempt of unveiling its mysteries. The present thesis work focused on bringing some light on different aspects of this fascinatinghERGchannel.
The first study presented in this manuscript deals with regulation of the hERG channel. In an overexpressing system, the potassium channel has been shown to interact with the ubiquitinligase Nedd42via a particular motif at its carboxy terminus, the PY motif. This proteinprotein interaction promotes ubiquitylation of the hERG protein that leads to downregulation of the channel at the plasma membrane accompanied by decrease of current. Understanding normal physiology
modificationsunderlyingtheacquiredLQTS.
The second aspect of the hERG channel investigated and presented in the manuscript regards the congential form of LQTS. Two yetundescribed KCNH2 mutations were functionally characterized and presented in separate case reports.
Bothprobandsarefemalescarryingmutationsintheaminoterminusofthechannel, morepreciselyinthehighlystructuredPASdomain.Themutationswerereproduced at the heterozygous state in overexpression systems and yielded about 50 to 75%
decreaseofcurrent,resultingfromadefectintraffickingofthesynthesizedmutant channel. In both cases, the reduced function of hERG may explain the LQTS phenotypeofthepatient.
ThelastsectionofthismanuscriptdiscussesthestereoselectivityofhERGblockby drugs in two differentworks. The stereoselective property of channel blockade has beenneglectedasregardsdruginducedprolongationoftheQTinterval.However,in the first study, (S)methadone demonstrated to be a more potent blocker than the (R)form, this latter being the active enantiomer for the intended pharmacological targets. Moreover, reevaluation of the racemic prescription in favor of the single (R)methadone is suggested, since in addition about 6% of Caucasian and African population may be at higher risks for QT interval prolongation due to a slow metabolism affecting preferentially the (S)form. The second study took advantage of mutagenesis analysis and docking simulations to assess the molecular determinants that govern the stereoselective block of hERG by bupivacaine.
Moreover, docking of enantiomers in a homology model proved to be an elegant waytochallengethecomputationalstrategydeveloped.
Listofabbreviations
ALLN:NAcytylLLeucylLLeucylLNorleucinal(proteasomeinhibitor) AP:Actionpotential
APD:Actionpotentialduration AVnode:Atrioventricularnode
C2domain:Calcium/lipidbindingdomain cAMP:cyclicAdenosineMonophosphate Cavchannels:Voltagegatedcalciumchannels CHOcells:ChineseHamsterOvarycells
cNBD:cyclicNucleotideBindingDomain(inhERGCterminus) CPVT:CatecholaminergicPolymorphicVentricularTachycardia DAAM:DextroDAcetylmethadol
DAD:DelayedAfterDepolarization DAG:Diacyglycerol
DM:DiabetesMellitus
DUB:Deubiquitylatingenzyme E1:Ubiquitinactivatingenzyme
E2:Ubiquitinconjugatingenzymeorubiquitincarrierenzyme E3:Ubiquitinproteinligase
EAD:EarlyAfterDepolarization ECG:Electrocardiogram EM:ExtensiveMetabolizer ENaC:EpithelialsodiumChannel ER:EndoplasmicReticulum ERP:EffectiveRefractoryPeriode
HECTdomain:HomologoustoE6associatedproteinCterminusdomain HEK293:HumanEmbryonicKidney293cells
hERG:humanEtheràgogoRelatedGene IC50:HalfmaximalInhibitoryConcentration ICD:ImplantableCardioverterDefribrillator IP3:1,4,5InositolTriphosphate
JLNS:JervellLangeNielsenSyndome [K+]e/i:external/internalK+concentration KOmouse:KnockOutmouse
Kvchannels:Voltagegatedpotassiumchannels LAAM:LevoDAcetylmethadol
LQT2:LongQTSyndrometype2 LQTS:LongQTSyndrome
Listofabbreviations(continued)
MD:MolecularDynamics
MiRP1:MinkRelatedPeptide1(KCNE2) MMT:MethadoneMaintenanceTreatment Navchannels:Voltagegatedsodiumchannels
Nedd4:NeuralprecursorcellexpressedDevelopmentallyDownregulatedgene4 NMD:NonsenseMediatedmRNADecay
PASdomain:PerArntSimdomain(inhERGNterminus) PD:Pharmacodynamics
PK:Pharmacokinetics
PKA:ProteinKinaseA(cAMPdependentProteinKinase) PKC:ProteinKinaseC
ROS:ReactiveOxygenSpecies RT:RoomTemperature RWS:RomanoWardSyndrome SAnode:Sinoatrialnode SM:SlowMetabolizer SQTS:ShortQTSyndrome TdP:TorsadesdePointes
TTCM:TakotsuboCardiomyopathy UBD:Ubiquitinbindingdomain WT:WildType
WW:Doubletryptophan(W)residues
Currentandrespectiveionchannel(official*andalternativenomenclatures)
INa Nav1.5 SCN5A
Ito Kv4.3 KCND3
IKur Kv1.5 KCNA5orHK2
ICa,L Cav1.2 CACN2(TypeL)
IKr Kv11.1 hERG1(hERG)
IKs Kv7.1(+MinK) KvLQT1orKCNQ1(+KCNE1orIsK)
IK1 Kir2.1Kir2.3 IRK1IRK3
IK,Ach Kir3.1Kir3.4 GIRK1GIRK4
IK,ATP Kir6.2 KATP(+SUR2)
*officialnameaccordingtotheIUPHARdatabase.
PartI
Background
I. Background
I.A. Theheart
The heart has always been a mysterious organ for the human being. It has long been,andisevennowadays,consideredastheemotionalorgan.Indeed,weallhave experienced that intense emotions influence the heart beating rate. The Greek Roman physician Galen (2nd century AD) was one of the first to describe the heart anatomyandtomakethepostulatethatarteriescarry“vitalblood”andnotairasit was previously thought (Furley & Wilkie 1984). However, Galen failed to recognize the heart's true role as a circulatory pump. It was William Harvey, physician of the 17th century, that finally related the anatomy of the heart to its bloodpumping function (Bylebyl 1979). Although great scientists, over two thousand years time, have made significant breakthroughs in our understanding of the heart and circulation system, a lot of unknown remains for modern time scientists to investigate.
The current knowledge of cardiac anatomy and function, which is a necessary prerequisiteforthereadingofthisthesis,issummarizedinthefollowingsections.
I.A.1. Heartandcirculatorysystem
Theheartissituatedobliquelyinthemiddle,slightlytotheleft,mediastinum(the centralcompartmentofthethoraciccavity),betweenthelungs,behindthesternum and above the diaphragm. To protect the heart and anchor it to the diaphragm, a doublewalledsac,namedpericardium,surroundstheorgan.However,majorvessels connect the heart to the entire body, which can be separated into those arriving (vena cava and pulmonary veins) and those leaving the heart (aorta and the pulmonary trunk or arteries,Figure 1). The heart is a hollow organ constituted of four chambers: two atria and two ventricles. A septum separates the left and the right side of the heart into two distinct pumps. In each side, specialized valves are presentatthejunctionsofatriumandventricle(tricuspidandmitralvalves),aswell as ventricle and artery (pulmonary and aortic valves, for left and right side,
respectively,Figure 1). The role of this double pump is to ensure the correct circulationofoxygenatedbloodandnutrimentstothedifferentorgansofthebody, including the heart itself. Oxygenated blood from the lungs, coming through the pulmonaryveinsintotheleftatrium,ispumpedtotheleftventricle.Bycontracting, theleftventricledrivestheoxygenrichbloodintotheaortaandbackflowofblood is prevented by the presence of the mitral valve and papillary muscles (Figure 1).
Simultaneously, the right side of heart contracts. Deoxygenated blood from the organs arrives in the right atrium through both superior and inferior vena cava.
Contractionoftheatriumexpelsthebloodintotherightventricle.Thentheventricle pumps out the blood to the lungs via the pulmonary trunk to terminate the cycle.
Here again, valves and papillary muscles ensure the correct route of blood. As mentionedabove,thehearttoohastobeprovidedwithoxygenrichblood;another circulatory system, the coronary circulation, allows for efficient irrigation of the pumpingorgan(Marieb1998).
Figure1:Detailedstructureoftheinnerheartandbloodcirculation.
Red arrows standing for oxygenated blood and blue for nonoxygenated blood.
Modifiedfrom(Internetsource1).
I.A.2. Themyocardium
As mentioned before, the heart’s function is essentially to contract in order to ensurebloodcirculation.Contractioncanonlybeachievedbyamusculartissue,the cardiacmuscle,whichisaninvoluntarystriatedmuscletissuefoundonlywithinthis organ. The name myocardium, literally themuscle of the heart(myos kardia in Greek), is internally covered by the endocardium and externally by the epicardium (Figure 2). These layers constitute the wall of the heart that isolates the heart chambers.
Theepicardium is also known as the visceral pericardium and is involved in the pericardiumdoublesacstructurethatanchorstheheartandprotectsitfromfriction duringbeating(Figure2).Thislayerenclosesthecoronarybloodvesselsandnerves whichsupplytheheart.
Theendocardium is the innermost layer of tissue that lines the chambers of the heartandthevalves(Figure2).Itisanendotheliumbasedonathinlooseconnective tissue,anditconnectswiththeendotheliumofthevessels.Bycreatingaflatcoating, itdecreasesfrictionofbloodagainstthecardiacwalls.
The myocardium is the middle layer of the wall and the most important constituentoftheheart(Figure2).Itmightfurtherbedividedintosubendocardial, midmyocardialandsubepicardialregions.Thismyocardiumismadeofdiversetypes ofcells,butprimarilycontractilecellsprovidedwithactinandmyosinmyofilaments:
cardiomyocytesandMcells(inthemidmyocardium).Thecardiacmusclefibersare short, thick and often branched cells, containing one or at most two nuclei. Long fingerlikeextensionsoftheplasmamembrane,thetransverse(T)tubules,penetrate deeplyintothecell.Ttubulesarebroaderandlessabundant(fewornoTtubulesin atrial cells) than in skeletal fibers, but play similarly a critical role in excitation contractioncoupling.Thecardiacmyocyteisthemostphysicallyenergeticcellinthe body, contracting constantly, without tiring, 3 billion times or more in an averagehuman lifespan (Severs 2000). Intracellular spaces are filled with
endomysium,alooseconnectivetissue.Thecardiomyocytesareassociatedwiththe fibrous skeleton in circular or spiral bundles that will produce a torsion movement that helps to efficiently squeeze the blood out from the heart (Marieb 1998). In contrastwithskeletalmusclecellsthatarestructurallyandfunctionallyindependent, cardiomyocytes are connected through an undulating membrane structure separating adjacent cells, theintercalateddiscs. These specialized areas of the membrane contain different junctional complexes (desmosomes, adherens junctions, gap junctions) that allow force transmission during muscle contraction andenable the myocardium to function as a syncytium, with mechanical and electricalcouplingofallfibers(Gutsteinetal.2003).Theysupporttherapidspread of the electric impulse and the synchronized contraction of the myocardium.
Coordinationofcontractionisachievedbyaspecializedconductingsystemthatwill bediscussedhereafter.
Figure2:Sectiondepictingpericardiumandlayersoftheheartwall.
Thepericardiumiscomposedbythefibrouspericardium,theparietalpericardium and the parietal cavity. The three layers of the heart wall are, from outside to inside, the epicardium or visceral pericardium, the myocardium and the endocardium.Figuremodifiedfrom(Internetsource2).
I.A.3. Electricalpropertiesoftheheart
I.A.3.a. Electricalconductionsystemoftheheart
Althoughtheheartrhythmislargelycontrolledbytheautonomicnervoussystem, a heart devoid of all nervous connections keeps beating in a regular rhythm. The independent and coordinated activity of the heart is due to the already mentioned couplingofthecardiomyocytesandtotheelectricalconductionsystemoftheheart, also known asnodal system(Marieb 1998). The role of these specialized cells is to produce impulses and propagate them through the heart to obtain a synchronized contractionofthewholemuscle,bycoordinatingitsbeatingactivitywiththatofits3 billion neighbors (Severs 2000). Cells of the conducting system – derived from myocytesbutwithrelativelylowpercentageofmyofilaments(Opthof1988)–jointo formdistinctlydefinedtissues:thenodesandconductingpathways(Figure3).These cellshaveincreasedpermeabilitytosodium.Theslowentryofsodiumproducesthe pacemakerpotential, which will gradually change polarity of the membrane until a threshold and thus automatically generates an excitation wave (Solc 2007; Marieb
Figure3:Excitatoryelementsandconductionsystemoftheheart(green). Arrowsindicatethedirectionoftheimpulse.Modifiedfrom(Internetsource3).
1998). It is noteworthy to mention that each portion of the conduction system exhibits the property of automaticity, with its own frequency. If a rhythmogenic centerfails,thenext(slower)mightpacetheheart.
Propagation of the excitation follows the electrical conducting pathways (Figure3),withthestartingpointfoundinaclusterofcellslocatedintherightatria and known as the sinoatrial (SA) node. Because it spontaneously depolarizes
~75times per minute, the sinus rhythm determines the heart rate and is thus consideredasthe“pacemaker”oftheheart.Theexcitationwave,oractionpotential, rapidly spreads through the gap junctions to the atria and propagates to the other important excitatory element, theatrioventricular (AV)node. At the AV node, just above the tricuspid valve, the influx is delayed by 100ms to allow termination of atrial contraction before the ventricles start their own (Marieb 1998). It is noteworthythattheAVnodeistheonlyelectricalconnectionbetweentheatriaand the ventricles (Meijler & Janse 1988). From this node, the excitation spreads along theHis bundle and cross the ventricular septum through the right and left bundle branches. Then,Purkinjefibers finally carry the contraction impulse from both bundle branches to the myocardium of the ventricles (Figure3). Purkinje fibers are larger thancardiac muscle fibers, thus the impulse conduction along these fibers is extremely rapid. The impulse spreads without delay throughout the ventricular myocardium, followed almost immediately by contraction of the ventricles (Marieb 1998).
I.A.3.b. CardiacactionpotentialandECG
Actionpotentialandionchannels
Acardiomyocyte,asanycellofthebody,issurroundedbyaphospholipidbilayer:
the plasma membrane. Electric impulses propagate easily in an electrolyte solution bymeansofionmovements,butnotinlipidicmedium,whichisanelectricinsulator.
Ionictransferthroughgapjunctionsisacurrentthatrapidlydecreasesasafunction ofdistanceandcouldnotensureproperexcitationandthereforecontractionofthe whole heart. Propagation of the impulse coming from the conducting system is
relayed from cell to cell in the myocardium, because cardiomyocytes are excitable cells. Their plasma membrane encompasses specialized poreforming proteins, namedvoltagegatedionchannelsbecausetheirpermeabilitiestoionsaresensitive tothevoltage(orpotential)acrossthemembrane.Itisthesequentialactionofion channels, involving influx and efflux of multiple ions, that produces theaction potential(AP)ofthecell.Thedifferenceinionconcentrationbetweentheinsideand the outside of the cardiomyocyte creates electrical gradients and generates the membranepotential.Therestingpotentialofmyocardialcellsisaround85mV(i.e.
the inside of the cell being negative), but under a propagating impulse from an adjacent cell the potential across the membrane becomes less negative; this phenomenon is known asdepolarization. Depolarization is sensed by the voltage gated sodium channels (Nav1.5) that trigger a rapid sodium (Na+) influx into the cardiomyocyte(Figure).Theinwardsodiumcurrent(INa)istypicallyresponsiblefor theAPphase0.Nav1.5channelswillthenrapidlyenterintoanonconductingstate (inactivatedstate).Thereafterfollowsphase1withpartialrepolarizationduetothe effluxofpotassium(K+)ionsthroughoutwardK+channelsmediatingIto,fortransient outward current(Figure). In the meantime, calcium (Ca2+) channels, mainly Ltype, are activated. Importantly, the change in intracellular calcium resulting from these currents(ICa,L)isessentialforactivatingthecontractionmachineryofcardiomyocytes through a process called excitationcontraction coupling. The cardiac AP is characterized by a plateau (phase 2). The plateau phase reflects the balance between inward currents (mostly ICa,L) and outward currents, mainlyvia delayed rectifier K+ channels (KCNQ1 associated with KCNE1, and hERG) as presented in Figure . Other currents, the sodium/calcium exchanger current (INa,Ca) and the sodium/potassiumpumpcurrent(INa,K)alsoplayminorrolesduringphase2.Thenet repolarization of the membrane during the followingphase 3 is due to increased currents of the delayed rectifier K+ channels (responsible for IKr and IKs) along with inactivationoftheCa2+channels(Figure).Finallytheinwardpotassiumcurrent,IK1, endstherepolarizationphaseandsetstherestingmembranepotential(phase4).
Importantly, after an action potential initiates, the cardiac cell is unable to start anotherAPuntilmidphase3,mainlyduetotheinactivationprocessofdepolarizing
channels that need time to recover. This period of time is referred to as the refractory period, divided into a firsteffective refractory period (ERP, during which even strong stimuli are blocked) and relative refractory period (might allow depolarizationbyastrongstimulus).ERPactsasaprotectivemechanism,keepsthe heartrateinchecktopreventarrhythmiasduetoearlyafterdepolarizations(EADs) or delayed after depolarizations (DADs), and coordinates muscle contraction. In addition, it is a crucial condition to conduct the electrical signal in one unique direction.
DespitegeneralsimilarityinthemechanismsofAPgeneration,actionpotentials exhibit distinct shapes in atrial and ventricular myocardium (Figure ). Major differences are that the plateau phase occurs at more negative potentials in atrial cellsandoveralldurationoftheAPisshorterwhencomparedwithventricularcells.
Note, for instance, that IKur is only present in atria (Ravens & Cerbai 2008). These Figure4:Inward,depolarizingandoutward,repolarizingcurrentsthatunderlie atrialandventricularactionpotential.
Phase0,rapiddepolarization;phase1,rapidearlyrepolarizationphase;phase2,
plateau phase;phase 3, late repolarization phase;phase 4, resting membrane potential.Modifiedfrom(Ravens&Cerbai2008).
K
differences are due to the heterogeneous distribution of ion channels and other proteinsthatconstitutecardiacioncurrents(Rodenetal.2002).
CardiacECG
Electriccurrentsareeasilytransmittedinthebodyliquids, andelectricpotential differences generated in the heart can be measured by an electrocardiographic deviceatthesurfaceofthebody.TheDutchphysiologistWillemEinthovenisknown as the creator of theelectrocardiograph, which was at the time (1902) known as
Figure 4 : Electrocardiographic leads and electrodes location, and ECG signal withcharacteristicfeatures.
(A)Einthoven’s triangle with bipolar (blue arrows: I, II, III) and unipolar (orange arrows:aVR,aVL,aVF)limbleads.Adaptedfrom(Internetsource4).
(B)Precordialchestleads (V1V6) usedtorecord theheart'selectricalactivityin thehorizontalplane(orangesquare).Adaptedfrom(Internetsource4).
(C)Thestandardandaugmentedleadsreflectthelimbelectrodes(leftarm,right arm,leftfoot)usedtorecordtheheart'selectricalaxisinthefrontalplane.Image from(Internetsource5).
(D)
A B
C D
stringgalvanometer,andistodayoneofthemostfrequentlyusedcardiacdiagnostic devices (RiveraRuizet al. 2008). Einthoven was awarded a Nobel Prize in 1924 for his contributions to the field of electrocardiography, and his name remained in the triangle’s name (Figure 4A) that explains the relationship between thestandard leads.
In modern electrocardiographs, 12 leads are typically used to record the signal, whichiscalledelectrocardiogramorECG.Aleadcanbeseenasthevoltagebetween two electrodes (positive and negative). The principle behind the way the electrical impulse is recorded in an ECG is simple, although interpretation of the results by untrained people is not always the case. When the overall electrical current of the heart goes towards a particular lead, it registers a positive deflection. Conversely, whencurrentgoesawayfromthelead,itproducesanegativedeflection,andfinally, currentperpendiculartothevectoroftheleadisrecordedasanisoelectricline.By placingoneelectrodeoneacharm(right,R,andleft,L)andonthelowerlimb(left foot,F),thelimbleadscanbeobtained(Figure4C).Theyarefurtherseparatedinto bipolarlimbleads(I,II,III)andaugmentedunipolarlimbleads(aVR,aVL,aVF);their vectors are presented respectively in blue and in orange arrows inFigure 4A. Whereaslimbleadsallowtoviewtheelectricalactivityoftheheartfromthefrontal plane (see Einthoven’s triangle), electrodes placed directly on the surface of the chest(overdifferentregionsoftheheart)recordprojectionsofthetimedependent changesinvoltagesoftheheartonthehorizontalplane.Sixelectrodescanbeused andplacedaspresentedinFigure4BtoprovideunipolarprecordialleadsnamedV1 toV6,whichfollowthesamerulesofinterpretationasforthelimbleads.
Consequently, a typical ECG signal that reflects complete cardiac contraction presents five deflections (Figure 4D). The first deflection is the P wave, which representsthewaveofdepolarizationthatspreadsfromtheSAnodethroughoutthe atria (nearly simultaneous depolarization of both atria). Deflections Q, R and S are considered altogether and referred to as the QRS complex, which reflects the ventricular depolarization. Atrial repolarization occurs during ventricular depolarization.However,becausethesignalofatrialrepolarizationisrelativelysmall inamplitude(i.e.lowvoltage),itismaskedbythelargeQRScomplexsignal.Finally,
theTwaveistheresultoftheventricularrepolarization.SometimesasmallUwave may be seen following the T wave. The origin of this wave is still not clear and controversial,althoughthreehypothesisarefrequentlyquoted:i)therepolarization of the Purkinje fibers, ii) the prolonged repolarization of the M cells in the mid myocardium or iii)afterpotentials, possibly caused by mechanical forces in the ventricularwall(RitsemavanEcketal.2005).Uwavesappearoften,butnotalways, in normal ECGs. Nevertheless, inverted or prominent U waves indicate underlying pathology or conditions affecting repolarization (Girishet al. 2005; Chikamoriet al.
1996;Antzelevitchetal.1995;Kirchhofetal.2009;PerezRieraetal.2008).
Assessmentof the morphology and duration of the different waves serves as an important diagnosis tool. Similarly, different portions of the ECG recording have been defined (Figure4D) and their duration provides useful information about the heart’sactivity(Marieb1998):
PR interval: time from the onset of the P wave to the beginning of the QRS complex. It reflects the period between the onset of atrial depolarization and the onsetofventriculardepolarization.
PR segment: isoelectric tracing that follows the P wave and ends with the beginningoftheQwave.ItrepresentsthedelayoftheelectricalimpulseattheAV node.
QT interval: time between deflection of the Q wave and end of the T wave. It corresponds to both ventricular depolarization and repolarization, and its value roughly estimates the duration of an average ventricular AP. Importantly, at high heartrates,ventricularactionpotentialsshorteninduration,whichdecreasestheQT interval. In order to assess the QT interval independently of heart rate, the QT intervalisexpressedasacorrectedQTinterval(QTc),usinge.g.theBazett'sformula thatdividestheQTintervalbythesquarerootoftheRRinterval.
STsegment:isoelectrictracingbetweenendoftheQRSandTwavedeflection.
ItsdurationroughlycorrespondstotheplateauphaseoftheventricularAP.
RR interval: time between consecutive R waves of the ECG recording. It represents the duration of a ventricular cardiac cycle and is an indicator of ventricularrate.
I.B. Cardiacchannelopathies
Asmentionedearlier,everyelectricalactivityoftheheart,henceeveryheartbeat, is dependent on the finely orchestered activity of diverse ion channels. Disorders involving ion channels – orchannelopathies – form a key group of heart diseases (Marban 2002), and resting or underexercise ECGs are pivotal for their diagnosis (Schimpfetal.2009).In1957,JervellandLangeNielsonreportedonfourboysofa familywhosufferedcongenitaldeafnessinassociationwithprolongationoftheQT interval and syncope (Jervell & LangeNielsen 1957). Both parents were asymptomatic, had a normal ECG and no hearing problems. The JervellLange Nielsen syndrome (JLNS) was rapidly defined as an autosomal recessive disorder.
Soon after, Romano and Ward described independent reports of prolonged QT intervalassociatedwithsuddencardiacdeath(SCD),thoughinabsenceofdeafness (Romanoetal.1963;Ward1964).ThisRomanoWardsyndrome(RWS)isnowknown tobeinherited(autosomaldominant),morecommonthanJLNS,andtheseverityof thediseasevariesconsiderably(Perrinetal.2008;MedeirosDomingoetal.2007).
EventhoughthegeneticbasisoftheprolongationoftheQTintervalwasevident,it was only in 199596 that the connection was achieved with the three main genes causingthesocalledcongenitalLongQTSyndrome(LQTS)(Wangetal.1995;Wang et al. 1996; Curranet al. 1995). The genesKVLQT1,KCNH2 andSCN5A encode respectively the ion channels KCNQ1, hERG and Nav1.5, and are now known to account for at least twothirds of LQTS (Ackerman 2004). As often, the function of proteins is unraveled by their dysfunction (Jentschet al. 2004), and since the mid
Gene Ionchannel Lossoffunction Gainoffunction
KVLQT1 KCNQ1
x LongQTsyndrome(LQTS)
x Familialatrialfibrillation x ShortQTsyndrome(SQTS)
KCNH2 hERG1 x LongQTsyndrome(LQTS)
x ShortQTsyndrome(SQTS)
SCN5A Nav1.5
x Brugadasyndrome(BrS1) x Idiopathicventricularfibrillation x Progressivecardiacconduction
disease x
x LongQTsyndrome(LQTS)
Table1:Additionalheritablearrhythmicsyndromeslinkedwiththethreeprincipal ionchannelsaccountingforLQTS.Adaptedfrom(Ackerman2004).
1990’s, the burst in translational research –e.g. highthroughput DNA sequencing, genetic linkage analyses, gene cloning, electrophysiological characterization, mathematical and molecular modelings – broadened the knowledge on cardiac channelopathies. The list of potentially heritable primary electrical diseases is not anymorerestrictedtoLQTS,andlossorgainoffunctionmutationsofasamegene can lead to different symptomatic abnormalities of the cardiac rhythm (Table 1).
Moreover, channelopathies are nowadays not restricted to the plasma membrane ion channels (Dsubunits) but also to accessory (E)subunits or interacting proteins (Ackerman 2004). Intracellular ion channels can also be affected, such as the ryanodine receptorcalcium release channel (RYR2 gene) present at the cardiac muscle sarcoplasmic reticulum, which is important for the mediation of the excitationcontractioncoupling.MutationsoftheRyR2proteincanleadtoincreased intracellular Ca2+ levels, increasing thereby dispersion of repolarization through the ventricularwall(transmuraldispersion)responsibleforarrhythmiasobservedinthe Catecholaminergic Polymorphic Ventricular Tachycardia or CPVT. Individuals with CPVTcharacteristicallypresentwithexerciseinducedsyncopeand/orsuddencardiac death. Unfortunately, patients show a normal resting ECG, which makes the diagnosisofdifficult(Schimpfetal.2009).
I.B.1. LQTS
I.B.1.a. CongenitalLQTS
ThelongQTsyndromeisthefirstcardiacchannelopathygeneticallydescribedandis maybethemoststudied,withabnormalprolongationoftheQTintervalasitsmain hallmark. The incidence of congenital LQTS is estimated at 1/30005000 individuals (MedeirosDomingoet al. 2007). As explained earlier, prolongation of the interval ranging from the beginning of the QRS complex and end of the T wave represents the total time for ventricular depolarization and repolarization. If ventricular depolarization is affected and/or repolarizing currents are decreased (Figure 5A), ECG recordings will present longer QT interval values. Ventricular repolarization of the female heart is characterized by a longer heart ratecorrected QT interval. For thisreason,definitionofaprolongedQTcintervalisgenderspecific.Forwomen,QTc
intervalsareconsiderednormalwhen450ms,borderlinewhenpresentingwith451 to 470ms, and prolonged if >470 ms; for men, the cutoff is 20ms lower,i.e.QTc intervals are normal when 430ms, borderline between 431 and 450ms, and prolongedwhendurationis>450ms(CommitteeforProprietaryMedicinalProducts 1997).AbnormallylongQTintervalspresentahigherriskofgeneratingamalignant type of ventricular tachycardia commonly called Torsades de Pointes or TdP (Dessertenne 1966). This was symbolically termed “twistings of the points” (about the isoelectric axis, seeFigure 5B) because it reminded the original author of the torsades de pointes movement in ballet. Despite the nice image it refers to, TdP causessyncopethatcaneithersolvespontaneously,leadtoseizuresor,intheworst case, lead to sudden cardiac death (Ackerman 2004). Mechanisms underlying arrhythmogenesis in LQTS are complex. However, it is widely accepted that prolongation of repolarization may lead to activation of an inward depolarization
Figure 5: Ventricular action potentials and related ECG signals. Prolonged QT intervalisariskfactorforTorsadesdePointes.
(A)Prolongation of ventricular action potential duration (red) is reflected in prolongationofQTintervalontheECGrecording(redtrace).
(B)ECG recording presenting onset of TdP in a patient with long QT syndrome.
A
B
current (ICa,L and INa) that generates EADs (due to the relative shortening of ERP), which in turn promotes triggered activity at the end of the repolarization. When accompanied with marked dispersion of repolarization, this may induce reentry phenomena and provoke TdP, sustained by further reentry or spiral wave activity (Cammetal.2004).
Formanyyears,itwastakenforgrantedthateachpatientaffectedbyLQTShada prolonged QT interval (Prioriet al. 1999). However, some patients do not present clinical symptoms and up to 36% of patients with a genetic subtype of LQTS (affectingKVLQT1) present with normal QTc intervals (Prioriet al. 2003). The congenital long QT syndrome (cLQTS) is far from being a homogeneous disease, notablybecauseofthevariablepenetranceofthedisease,i.e.patientswhohavethe mutation and manifest the phenotype (Priori et al. 1999), and the genetic heterogeneity. Indeed, several hundreds of mutations distributed in 12 genes have been described in this condition:KVLQT1, KCNH2, SCN5A, KCNE1, KCNE2, ANKB, KCNJ2, CACNA1, CAV3, SCN4B,and morerecently AKAP9 (Chenet al. 2007) and SNTA1 (Uedaet al. 2008). The genes involved incLQTS are conventionally named LQT112asdetailedinTable2.Itisnotsurprisingtoseethatthemaingenesrelated to congenital LQTS are encoding ion channels and associated subunits. Genetic
Table 2: First ten described genes involved in the long QT syndrome. Adapted from(MedeirosDomingoetal.2007).
TwoadditionalformsofLQTS(withfrequencies<1%)shouldbeaddedtothistable:
LQT11 AKAP9 gene (locus 7q21q22) encoding the AKAP9/Yotiao protein, and LQT12SNTA1gene(locus20q11.2)encodingtheD1syntrophinprotein.
testing in LQTS can detect an underlying mutation (genetic diagnosis) in ~70% of patients(Schimpfetal.2009).However,negativegeneticscreeningcannotruleout thedisease.
Interestingly, for the most common and betterknown forms of RomanoWard syndrome (LQT13,Table 2), specific characteristics can be mentioned at the ECG level,aswellaspotentialtriggersforTdPandresponsetotreatment(Schimpfetal.
2009;MedeirosDomingoetal.2007;Khan2002a;Schwartzetal.1995).
Patients with LQT1 often present with broadbase T wave of very prolonged duration.Physicalactivitywasidentifiedasthetriggerin>70%ofarrhythmicevents in patients with an LQT1. More precisely, swimming is a typical predisposing factor forcardiacevents.Asregardslongtermtreatment,Eblockers(e.g.atenolol),which reduce the adrenergic stimulation, are particularly effective in patients with IKs channelmutations.
IndividualsdiagnosedwithLQT2commonlypresentabiphasic(withnotching)and lowamplitude T wave on the ECG recordings. They characteristically have syncope or sudden death following sudden auditory stimuli (~50%) or with strong emotion, andlessfrequentlyduringsleep(~20%)orexercise(~30%).WomenwithLQT2inthe postpartum period are a particularly susceptible subset of patients. As regards preventive treatment,Eblocker medication seems also effective to reduce TdP events, though less than for LQT1. Oral potassium supplementation was shown to correcttheabnormalitiesofrepolarizationdurationandshortentheQTcintervalin thecLQTStype2(Etheridgeetal.2003).
Incontrast,theECGsinLQT3patientsusuallyshowadelayed,pointedTwaveand allow clear observation of the ST segment prolongation. Patients with LQT3 have a higherincidenceofsuddendeathduringrest(sleep)orbradycardia.Thesepatients usually have fewer symptoms than those with LQT1 or LQT2, but the events are characteristicallymorelethal.TheunderstandingofthemolecularbasisoftheLQT3 allowed new therapeutic treatments. Sodium channel blockers of the anaesthetics class such asmexiletine andflecainide – drugs that significantly shorten the QTc interval – were reported to be beneficial to this subtype ofcLQTS (Schwartzet al.
1995;Benhorinetal.2000).BecausetheyaremorepronetoSCDatlowheartrates, LQT3patientsmaybemorelikelytobenefitfromimplantationofpacemakerspacing atrelativelyhighrates.
Regarding the longterm preventive therapy, which aims at shortening the QTc interval and preventingTorsades de Pointes, many other drugs have been used sporadically, and among them Ca2+ channel blockers (verapamil), K+ channel activators forcLQTS subtype 1 and 2 (nicorandil,pinacidil,chromakalim) and other Na+ channel blockers for LQT3 (pentisomide,lidocaine,phenytoin), but their use needs further investigation (Khan & Gowda 2004). Implantable cardioverter defribrillators (ICDs) are an invasive solution for highrisk LQTS patients with documentedmalignantarrhythmias,recurrentsyncope,oranabortedcardiacarrest.
However,anICDdoesnotpreventTorsadesdePointes:itprecludessuddencardiac deathwhenTdPareprolongedordegenerateintoventricularfibrillation(veryrapid, uncoordinated and ineffective contractions). Thus, combination of firstline treatmentEblockers and ICD should be continued to prevent occurrence of TdP.
Moreover,unnecessaryshocksfromthedevicemayproduceemotionaldistressand cause adrenergic stimulation, sufficient to precipitate TdP. This is another reason that motivatesEblocker cotreatment. For patients who experienced frequent shocks from their ICD, or for whom firstline therapies failed (high doses of Eblockers, pacemakers and ICDs), the left cervicothoracic sympathectomy is a highly effective method for adrenergic therapy (Khan 2002a). It consists in the removal of part of the stellate ganglia and T2T4 left thoracic ganglia of the sympathetic chain. Beneficial effects were confirmed for LQT1, but are likely to be smallerforLQT2andLQT3patients(MedeirosDomingoetal.2007).
Moreover, it is important to recommend exercise restriction, for both symptomaticandasymptomaticpatients,asitmighttriggerTdP.Moreimportantly, aggravating factors, such as modified health conditions or consumption of several drugs that prolong the QT interval, have to be strictly controlled if not avoided. By themselves, theses factors are known to cause theacquired (aLQTS) or, when involvingdrugs,thedruginduced(diLQTS)formoflongQTsyndrome.
I.B.1.b. AcquiredLQTS
Studies on channelopathies have yielded important insights into the pathophysiology of some far more common diseases (Marban 2002), such as congestive heart failurethatafflicts hundreds of millions of people worldwide. We now know that heart failure represents a commonacquired form of the long QT syndrome.Myocytesfromfailingheartsshowprolongationofactionpotentials,and theirrepolarizationinvivoisabnormallylabile(Marban2002).Othercardiovascular diseases, such as coronary artery disease, left ventricular hypertrophy or cardiomyopathy, produce remodeling of cardiac ion channel expression resulting in electricalimbalanceofAPgeneratingcurrents,thusleadingtoaLQTS(Swynghedauw
&Chevalier1995;Tsujietal.2002).
Electrolyte disturbances have been linked with the acquired LQTS. Low extracellular potassium is known to prolong the AP duration (APD)– an effect not predictable from simple electrochemical considerations (Roden 2006). Apart from hypokalaemia, other electrolytic imbalances have also been reported, such as hypomagnesaemiaorhypocalcaemia(Khan2002b).
Many other conditions – connected directly or not with the cardiovascular system(Khan2002b)–havebeenrelatedtotheacquiredformofthesyndromeand increased risks for arrhythmic events, although in the absence of mutations in the knownLQTSgenesordrugsprolongingtheQTinterval.Thesehealthconditionsare
Heartdisease
Coronaryarterydisease Heartfailure
Ventriculartachyarrhythmias Dilatedcardiomyopathy LeftVentricularhypertrophy Hypertension
Bradycardia(SAnodaldysfunction,AVblock) Myocarditis
Metabolicabnormalities Hypokalaemia
Hypocalcaemia Hypomagnesaemia Liverdisease
Cirrhosis Hepaticfailure
Renaldisease Endocrinedisorder
Hypothyroidism Hyperparathyroidism Pheochromocytoma Hyperaldosteronism Intracranialpathology
Subarachnoidhemorrhage Headinjury
Encephalitis Diabetesmellitus
Anorexianervosa/starvation Bulimia
Obesity
Liquidproteindiet
HumanImmunodeficiencyVirus(HIV)infection Table3:SelectedcausesofacquiredLQTS,modifiedfrom(Cammetal.2004).
namely hypothyroidism, stroke or CNS lesions, malnutrition, human immuno deficiencyvirusinfections,hypothyroidismormyocardialischemia(Table3).
I.B.1.c. DruginducedLQTS
From what has been explained, drugs that cause an electrolyte imbalance, e.g.potassiumwasting diuretics, could be considered as a factor for bothdrug induced andacquired LQTS. In fact, drugs prolonging the LQTS have long been included as an additional risk factor for the acquired form. Moreover, drug interaction with any ion channel that contributes to cardiac repolarization could in theory produce LQTS. However, clinically relevant prolongation of QT interval is invariablytheresultofdrugblockadeofauniquetarget:thehERGchannel(Perrinet al. 2008). Nowadays, druginduced LQTS is regarded as a separate form of this arryhthmogenicdisorderalmostrestrictedtoblockersofthehERGchannel.
The first described case of diLQTS is probably attributable to quinidine (antiarrhythmic agent class Ia, intended to act on Na+ channels) in the 1920’s,i.e.
much before description of the disease and the understanding of the molecular bases involved. Indeed, syncope on initiation of quinidine therapy was recognized shortly after the introduction of the drug (reviewed in Roden 2006). Later in the development of antiarrhythmic therapy, molecules have been designed to specificallyblockK+currents(classIII):dofetilideandibutilide(forstructure,referto Appendix I). Unfortunately, such drugs predictably evoke prolongation of the QTc interval,whichissufficienttocausepotentiallylethalventriculararrhythmiasin57%
of recipients (Marban 2002). Theses drugs, along with other molecules of the methaneulfonanilidegroup,werereportedtoblockthehERGchannel(Spectoretal.
1996a;Yangetal.1997).Infact,manystructurallyunrelateddrugs(seeAppendicesI andII),designedtoactasnoncardiactargets,unintentionallyblocktherepolarizing currentIKr.Inthepast15years,themostcommoncauseofwithdrawalorrestriction of use of drugs already marketed has been the prolongation of the QT interval associated withpolymorphic ventricular tachycardia or TdP. The rare incidence of thesearrhythmiasexplainswhytheywerenotreadilyobservedduringclinicaltrials orpostmarketingsurveillance.Manydrugshavethereforebeenremovedfromthe
marketafteryearsofuseorhadtheiravailabilityseverelyrestrictedbecauseofthis rareformofpotentiallyfataltoxicity.Thesecompounds(seeAppendicesIandIIfor structures) can be antimuscarinic drugs like terodiline (withdrawal in 1991) or antianginal agents likemibefradil (1998); drugs used to treat allergy symptoms like the antihistaminic terfenadine and astemizole (withdrawal in 1998 and 1999, respectively); antibiotics like grepafloxacin (1999); gastrointestinal prokinetic cisapride(2000)andanalgesiclevoDacetylmethadol(2003)wereconcerned;some neuroleptic drugs are no more commercialized, likesertindole (since 1998) or thioridazine (2005). This is not an exhaustive list of hERG channel blockers. Many drugs known to prolong the ventricular repolarization are still on the market (see www.QTdrugs.org), because their benefits are considered higher than the cardiac risk. As regards newly developed drugs, regulatory authorities demand systematic assessment of the TdP risk (for preclinical tests, see Appendix III) to avoid other disastrousconsequencesforbothpharmaceuticalcompaniesandpublichealth.
It is noteworthy that prolongation of the QT interval is not always linked with occurrence of Torsades de Pointes. As mentioned previously, patients with prolonged QTc interval can live without symptoms; only 12% of asymptomatic patientswilldeveloparrhythmiasandmayexperienceSCD(MedeirosDomingoetal.
2007).Adecadeago,Rodenproposedtheprincipleofrepolarizationreserve,i.e.that a normal heart includes multiple, redundant mechanisms to accomplish normal repolarization. Hence, lesion of one of these mechanisms may not be sufficient to elicit an absolute LQTS phenotype (Roden 1998). For example, a subject with subclinicalcongenitalLQTScanbecomemanifestonlyonexposuretoanIKrblocker.
Similarly, a female patient presenting with anorexia or under strict diet – factors reducing the repolarization reserve – might experience TdP underfluconazole (an antifungal treatment that blocks the hERG channel). Interestingly, the concept of repolarizationreservehastheadvantageofdrawingconnectionsbetweenallforms of LQTS – congenital, acquired and druginduced – and allowing a better understandingoftherisksforasymptomaticcLQTSdiagnosedpatients.
I.B.2. SQTS
In 2000, the link between a persistently shortened QT interval and arrhythmia was first described in a sporadic case suffering from SCD and paroxysmal atrial fibrillation(Gussaketal.2000).Sincethen,thearrhythmicpotentialofshortenedQT interval was confirmed, andShort QT Syndrome (SQTS) is now considered a new cardiac channelopathy. The risk of arrhythmic events is high in patients with SQTS and cardiac arrest is the most frequent clinical presentation. Atrial fibrillation was also documented in about 30% of patients (Schimpf et al. 2009).
Electrocardiographic hallmarks of SQTS are constantly short QTc intervals, short or even absent ST segment, and often tall, narrow and symmetrical T waves in chest leads (Schimpfet al. 2009). In contrast to prolonged QTc values, there is no clear consensusforthelowerlimitoftheQTcinterval,andthereforenothresholdvalueis available to differentiate between pathological and healthy individuals (Schimpfet al.2009).
RegardingthegeneticbaseoftheSQTS,thediseaserevealstobealsogenetically heterogeneous with, until now, five genes affected. Underlying gainoffunction mutationsinrepolarizingK+channelsareresponsibleforSQT1(KCNH2gene),SQT2 (KVLQT1gene)andSQT3(KCNJ28gene,forIK1current);lossoffunctionmutationsin D1subunit (CACNA1c gene) and Esubunits (CACNB2b gene) of cardiac Ltype calcium channels, give rise to SQT4 and SQT5, respectively (Patel & Antzelevitch 2008). Note that, likewise tocLQTS, KCNQ1 and hERG channelencoding genes are oneofthemolecularbasesofthearryhthmogenicdisorder.
J
Inthefirsttwochapters,theessentialsregardingcardiacfunctionandionchannel disorders were presented. Description of the cardiac activity and AP fundamentals highlighted that the outward IKr current, mediated by hERG channels, is one of the most important determinants for termination of the plateau phase. Either gainof function or lossoffunction mutations in the KCNH2 gene (encoding hERG) demonstrated to be responsible for congenital cardiac disorders (LQTS, SQTS) that can lead to potentially lethal ventricular tachyarrhythmias. Moreover, the drug
inducedformofLQTSseemstobealmostuniquelylinkedtotheblockofthehERG channel. The following chapter will get to the heart of the matter and introduce in more details the main protagonist of this thesis work. Some important advances in the knowledge of this puzzling, though fascinating, potassium channel will be summarized.
I.C. ThehERGpotassiumchannel
I.C.1. DiscoveryofhERGandtheEAGchannelfamily
DiscoveryofthehERGchannelstartedwiththeidentificationofthemutatedgene (theetheràgogo locus EAG) responsible for legshaking behavior ofDrosophila melanogasterduringetheranesthesia.Thestrikinghyperexcitabilityofeagmutants demonstrated the importance of these channels in maintaining normal neuronal excitability in the fly. Using homology screening, related cDNA sequences were obtained from different species, namely human and mice (Warmke & Ganetzky 1994). They were later divided into three different subfamilies of theEAG gene family:i)eag,ii)eaglikeK+channel(elk)andiii)eagrelatedgene(erg)(Trudeauet al. 1995). The hERG channel, or the humanetheràgogo related gene channel, belongstothelastsubfamily.
In the human, theeagrelated gene orerg subfamily comprises three members, namedhERG1,hERG2andhERG3thatareencodedbythreedifferentgenes(KCNH2, KCNH6andKCNH7,respectively).Accordingtoanothernomenclaturesystem,these proteinscanbereferredtoasKv11.1,Kv11.2andKv11.3(forKvfamily11,members1, 2 and 3, respectively). The strong conservation of sequences fromDrosophila to mammals suggests preservation of neuronal functions as well. Indeed in humans, hERG channels are expressed in neurons (Emmiet al. 2000), but also in a range of other tissues including neuroendocrine glands (Rosatiet al. 2000), various cancer cellslines(amongothersgastric,breast,lung,colon,lymphocytes)(Chenetal.2005;
Bianchiet al. 1998) and smooth muscle cell (Shoebet al. 2003). However, hERG1 channels have been better characterized in the heart, where their expression is higherinventriclesthanatria(Pondetal.2000).
