Cardiac arrhythmia induced by genetic silencing of &lsquo

15 Pages·2014·2.21 MB·English

by  Pietro Mesirca

ARTICLE
Received7May 2014|Accepted 14Jul2014|Published 21 Aug 2014 DOI:10.1038/ncomms5664
Cardiac arrhythmia induced by genetic silencing
of ‘funny’ (f) channels is rescued by GIRK4
inactivation
Pietro Mesirca1,2,3, Jacqueline Alig4, Angelo G. Torrente1,2,3, Jana Christina Mu¨ller5, Laurine Marger1,2,3,
Anne Rollin1,2,3, Claire Marquilly1,2,3, Anne Vincent1,2,3, Stefan Dubel1,2,3, Isabelle Bidaud1,2,3,
Anne Fernandez6, Anika Seniuk5,7, Birgit Engeland4,8,9, Jasmin Singh10, Lucile Miquerol11, Heimo Ehmke5,7,
Thomas Eschenhagen7,10, Joel Nargeot1,2,3, Kevin Wickman12, Dirk Isbrandt4,8,9,* & Matteo E. Mangoni1,2,3,*
The mechanisms underlying cardiac automaticity are still incompletely understood and
controversial. Here we report the complete conditional and time-controlled silencing of the
‘funny’ current (I) by expression of a dominant-negative, non-conductive HCN4-channel
f
subunit (hHCN4-AYA). Heart-specific I silencing caused altered [Ca2þ] release and Ca2þ
f i
handling in the sinoatrial node, impaired pacemaker activity and symptoms reminiscent of
severe human disease of pacemaking. The effects of I silencing critically depended on the
f
activity of the autonomic nervous system. We were able to rescue the failure of impulse
generation and conduction by additional genetic deletion of cardiac muscarinic G-protein-
activated (GIRK4) channels in I-deficient mice without impairing heartbeat regulation. Our
f
studyestablishestheroleoff-channelsincardiacautomaticityandindicatesthatarrhythmia
relatedtoHCNloss-of-functionmaybemanagedbypharmacologicalorgeneticinhibitionof
GIRK4 channels, thus offering a new therapeutic strategy for the treatment of heart rhythm
diseases.
1De´partementdePhysiologie,InstitutdeGe´nomiqueFonctionnelle,LabExICST,F-34094Montpellier,France.2UMR-5203,Centrenationaldelarecherche
scientifique,Universite´sdeMontpellier1&2,F-34094Montpellier,France.3INSERMU661,Universite´sdeMontpellier1&2,F-34094Montpellier,France.
4ExperimentalNeuropediatrics,UniversityMedicalCenterHamburg-Eppendorf,20246Hamburg,Germany.5DepartmentofCellularandIntegrative
Physiology,CenterforExperimentalMedicine,UniversityMedicalCenterHamburg-Eppendorf,20246Hamburg,Germany.6Centrenationaldelarecherche
scientifique,UPR-1142,InstitutdeGe´ne´tiqueHumaine,F-34094Montpellier,France.7CardiovascularResearchCenterHamburg(CVRC)andDZHK
(GermanCenterforCardiovascularResearch),UniversityMedicalCenterHamburg-Eppendorf,partnersiteHamburg/Kiel/Luebeck,Martinistrasse52,
20246Hamburg,Germany.8ExperimentalNeurophysiology,UniversityHospitalCologne,50924Cologne,Germany.9GermanCenterfor
NeurodegenerativeDiseases(DZNE),53175,Bonn,Germany.10DepartmentofExperimentalPharmacologyandToxicology,CenterforExperimental
Medicine,UniversityMedicalCenterHamburg-Eppendorf,20246Hamburg,Germany.11DevelopmentalBiologyInstituteofMarseille,Universite´
Aix-Marseille,CNRSUMR7288,13288Marseille,France.12DepartmentofPharmacology,UniversityofMinnesota,Minneapolis,Minnesota55455,USA.
*Theseauthorsarethejointseniorauthors.CorrespondenceandrequestsformaterialsshouldbeaddressedtoM.E.M.(email:[email protected]).
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H
eartautomaticityisafundamental physiologicalfunction genetic inactivation of G-protein-activated (GIRK4) channels.
inanimals.Pacemakeractivityisgeneratedbyspecialized, In summary, our study provides a new animal model of human
spontaneously active myocytes localized to the sinoatrial pathologies of HR and rhythm, and identifies a new potential
node (SAN). The SAN with its fastest spontaneous beating rate pharmacological target for the treatment of bradycardia.
generates the cardiac impulse and controls the heartbeat. In the
caseof SANfailure,other components oftheconduction system
such as the atrioventricular node (AVN) and the His-Purkinje Results
fibre (PF) network can also generate viable pacemaking1. Generation of conditional hHCN4-AYA-expressing transgenic
Dysfunction in SAN automaticity underlies congenital or mice. To suppress I conductance in a cardiac-specific and time-
f
acquired bradycardia and bradycardia-associated conditions. controlled manner, we generated double-transgenic mutant
Furthermore, it contributes to debilitating symptoms such as (Mut)miceexpressinghHCN4-AYAdominant-negativesubunits
syncope, atrial fibrillation and heart failure, and causes sudden and a tetracycline-sensitive transactivator (tTA24) under the
death2–5. SAN disease and conduction block account for more controlofana-myosinheavychain (aMHC)promoter25.In the
than 450,000 electronic pacemaker implantations each year in absence of doxycycline (DOX), tTA binds to its promoter and
Europe and North America6. Thus, there is a strong interest in drives the expression of an hHCN4 pore mutant construct in
elucidating the mechanisms underlying cardiac pacemaking and whichahaemagglutinin(HA)epitopetagwasaddedtotheamino
impulse conduction at both the fundamental and clinical levels, terminus and theselectivityfilter motifwas mutatedto hHCN4-
and in finding alternative treatment options. G480A/G482A (hHCN4-AYA; Fig. 1a). Two-electrode voltage-
The ability of SAN cells to generate the pacemaker action clamp recordings in Xenopus oocytes after coexpression of
potential is due to the diastolic depolarization phase during hHCN4-AYA cRNA with wild-type mHCN4 or mHCN2
whichthemembranevoltage,fromtheendofanactionpotential, cRNAs showed that mutated hHCN4-AYA protein affected
slowlydepolarizestoreachthefiringthresholdforthesucceeding mHCN2andmHCN4subunit-mediatedcurrentsinadominant-
one. The slope of the diastolic depolarization continuously negative manner (Supplementary Fig. 1a).
modulates the heart rate (HR) and is regulated in opposite Heart-specific expression of hHCN4-AYA during pregnancy
directions by the sympathetic and parasympathetic branches of resultedinembryoniclethality,whichissimilartothatdescribed
the autonomic nervous system. Different classes of membrane inhomozygousHCN4(cid:2)/(cid:2) mice26,becausenodouble-transgenic
ion channels, as well as ryanodine receptors of the sarcoplasmic mutantmicewereborn(þ/T þ/T,Fig.1b.Thelikelihoodofthe
reticulum, are involved in the generation and regulation of other possible genotypes was B30% (n (þ/þ þ/þ)¼33, n
the diastolic depolarization (see ref. 1 for review), but their (þ/þ þ/T)¼24 and n (þ/T þ/þ)¼27). When hHCN4-
specificfunctionalrolesarestillincompletelyunderstood.Among AYA expression was suppressed by administering DOX to the
cardiac ion channels, f- (HCN) channels, underlying the mothers during pregnancy, the animals were born at a normal
hyperpolarization-activated I current, are thought to play a Mendelian ratio (n (þ/þ þ/þ)¼62, n (þ/þ þ/T)¼52, n
f
major role in the generation and autonomic regulation of the (þ/T þ/þ)¼52andn(þ/T þ/T)¼54).DOXwithdrawalat
diastolicdepolarizationintheSANandinthecardiacconduction weaning induced expression of hHCN4-AYA, which was
system7,8,butitsfunctionalroleisstillamatterofcontroversy9.A detectable after 1 week (Fig. 1c and Supplementary Fig. 1b).
family of four homologous HCN-channel subunit isoforms Theexpressionlevelincreasedand resultedinastrongerprotein
(HCN1–4) was identified in mammals10–13. HCN4 is the signal after 2–3 weeks, reaching a maximum level after 4 weeks.
predominant f-channel isoform in the mouse SAN and in the The mutant hHCN4-AYA protein was detected in all cardiac
conduction system14. HCN1 and HCN2 are also present chambers, with the highest expression in the atria (Fig. 1d and
in the conduction system at varying expression levels14,15. Supplementary Fig. 1b). To verify that the aMHC promoter
Recent studies have indicated that human HCN4-channel effectively induced expression of hHCN4-AYA protein in
mutations underlie congenital alterations in SAN pacemaking pacemaker myocytes, we studied anti-HCN4 and anti-HA
in humans, with variable symptoms ranging from mild or immunoreactivities by macro-confocal microscopy in
moderate bradycardia16–18 to chronotropic incompetence19, or preparations of the whole supraventricular stage of the heart
atrioventricular conduction block with ventricular tachycardia20. containing the SAN and AVN of control (Supplementary Fig. 2)
HCN4 knockout mice show a reduction of B70% in I current and mutant mice (Supplementary Fig. 3). Individual cells in the
f
associated with varying degrees of dysfunction in heart intact mutant SAN showed strong membrane-bound anti-HA
automaticity, ranging from recurrent SAN pauses with no immunoreactivity consistent with hHCN4-AYA protein
significant change in HR and autonomic responsiveness21,22 expression (Fig. 1e). SAN–AVN preparations of the mutant
to lethal ventricular bradycardia and heart block23. heart displayed expression of hHCN4-AYA protein in all
These discrepancies could be explained by the different identifiable myocytes of the two heart pacemaker centres
strategies of gene targeting adopted and/or by the presence of (Fig. 2). Inside the SAN region, the density of hCN4-AYA-
the other HCN-channel isoforms that can compensate for the expressing cells was particularly high in the site of origin of the
loss-of-functionofHCN4channelsinboththeSANandcardiac pacemaker activity described in previous studies27,28. Similar to
conduction system. Furthermore, deletion of the HCN4 protein that in SAN, hHCN4-AYA-expressing cells were densely
may lead to alterations in the function of channel-associated distributed throughout the whole AVN region (Fig. 2 and
proteins. SupplementaryFig.3).Noanti-HAimmunoreactivitywasfound
We thus developed a conditional transgenic mouse model in preparations obtained from control mice (Supplementary
expressing non-conducting hHCN4 subunits to suppress I Fig.4).AsignificantpopulationofhHCN4-AYA-expressingcells
f
conductance, independent of the endogenously expressed HCN in the SAN and AVN stained positively against anti-HCN1
subunits,tostudytheimportanceofthiscurrentinthegeneration immunoreactivity (Fig. 2), which was also expressed in
and conduction of the cardiac impulse. We show that complete pacemaker cells of the SAN and AVN14. In addition, we
silencing of I alters intracellular Ca2þ handling in pacemaker performed immunostaining of enzymatically isolated SAN and
f
myocytes and thus causes a complex disorder of cardiac AVN myocytes of control and mutant mice29. All myocytes
rhythm, including SAN failure, atrioventricular block (AVB) isolated from mutant mice showed strong plasma membrane
and ventricular arrhythmia, which could be prevented by anti-HA immunoreactivity (Supplementary Fig. 5).
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40 (33) Water
DOX
(27)
Promoter line 30 (62) (24)
αMHC tTA (A)n e (%) (52) (52) (54)
No Dox (on) Dox (off) otyp 20
n
e
G
10
TRE HA hHCN4-AYA (A)n
Responder line
0 (0)
(+/+ +/+) (+/+ +/T)(+/T +/+) (+/T +/T)
Control (+/T +/+) Mutant (+/T +/T)
Weeks on water
kDa LA RA LV RV LA RA LV RV
0 1 2 3 4 5 160
130 kDa hHCN4-AYA Ab: Anti-HA
Ab: Anti-HA
50
55 kDa CSQ
Ab: Anti-CSQ
Ab: Anti-CSQ
HCN4 HCN4 DNA
HA DNA Visible
50 μm
Figure1|Generationoftransgenicmutantmice.(a).SchematicillustrationoftheTet-offsystem:Thetetracycline-dependenttransactivator(tTA)is
expressedunderthecontroloftheaMHCpromoter.Intheheartofdouble-transgenic(mutant)animals,tTAbindstothetetracycline-responsiveelement
(TRE),resultingintheexpressionofHA-hHCN4-AYA(hHCN4-AYA).AconformationalchangeoftTAafterthebindingofDOXinhibitstransgene
expression.(b)Percentageofgenotypesobtainedafterbreedingofthepromoterline(firstlocus)andresponderline(secondlocus)onwater(blackbars)
orDOX(whitebars).Absoluteanimalnumbersaregiveninparenthesesabovethebars.(c)DetectionoftransgenichHCN4-AYAproteinafter0to5weeks
afterDOXwithdrawalbywesternblotting.TotalheartlysateswereanalysedwithantibodiesagainsttheHA-Tagandcalsequestrin(CSQ).(d)Western
blottinganalysisofproteinisolatedfromtheleftatrium(LA),rightatrium(RA),leftventricle(LV)andrightventricle(RV)ofcontrolandmutant
mice.DifferenttissueslysateswereanalysedwithantibodiesagainsttheHA-TagandCSQ.(e)CellularexpressionofhHCN4-AYAproteinin(n¼4)SAN
preparations(seeSupplementaryFig.2)isolatedfrommutantmice.Expressionwasevaluatedbyusinganti-HCN4(red,top-leftpanel)andanti-HA
(green,bottom-leftpanel)antibodies.Stainingofnucleiisshowninblue(top-rightandbottom-leftpanels).Bottom-rightpanel:bright-fieldimageoftissue.
Scalebar,50mm.
Expression of hHCN4-AYA in mutant mice silenced I con- In Mut/Cx40þ/EGFP mice, PF myocytes were identified by
f
ductance in SAN and conduction system. To evaluate the enhanced green fluorescent protein (EGFP) epifluorescence. In
functional effects of hHCN4-AYA expression on I in the SAN comparison with controls, the averaged cellular capacitance was
f
and in the cardiac conduction system, we recorded I in control increased in mutant myocytes, indicating an increase in cell size
f
and mutant mice deprived of DOX after weaning. To unam- intheconductionsystemofmutantmice(SupplementaryFig.6).
biguously quantify current densities, we defined I as the net In myocytes isolated from control mice, the density of I was
f f
Ba2þ-insensitive and Csþ-sensitive current29,30 (Fig. 3). To highestinSANandlowerinAVNandPFmyocytes,asreported
record I in PF myocytes, we crossed control and mutant mice previously32. Unlike in control myocytes, I was drastically
f f
with Cx40þ/EGFP mice (Mut/Cx40þ/EGFP, see Methods)31. reduced at all membrane voltages tested in SAN, AVN and PF
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HA DNA HCN1 DNA HA HCN1
SAN
CT 0.2 mm
PNE
Figure2|TransgeneexpressionintheSANandAVNregionsofheartnodaltissues.Representativewhole-mountstainingsofn¼4mouseheart
nodaltissueswithclose-upviewsofthemutantSAN(a)andAVN(b)regionsstainedwithanti-HA(green)andanti-HCN1(red)antibodies.Stainingsofnucleiare
showninblue.SeeSupplementaryFig.3forthelocalizationoftheclose-upviewsinwhole-mountrightatrialpreparationscontainingthenodes.Scalebar,200mm.
myocytes isolated from mutant mice. We did not detect I at
V (mV) f
–135 –85 –35 voltagesspanningthediastolicdepolarizationrangeinallmutant
myocytes tested ((cid:2)75/(cid:2)35mV, Fig. 3).
A
p ****
500 *******–*75 –65 –55 –45 –35 –10 A/PF) IdfelasiyleedncainftgerdsleopwoeladrizbaatsiaolnpinaceSmAaNk,erAVaNctivaintyd PanFdmiynodcuycteesd.
**** –1 –20 I (p MratuetsatnhtaSnAcNonmtryoolcmytyeoschyatedssaingndisfihcoawnteldylaoswtreornsgpornedtauncetioounsifinritnhge
1 s **** **************––*23 –30 diastolic depolarization slope, both during the linear and expo-
**** *** nentialphases,aswellasintheupstrokevelocity(Supplementary
Table 1). b-Adrenergic receptor stimulation with submaximal
V (mV)
–135 –85 –35 (2nM) or maximal (100nM) concentrations of isoproterenol
(ISO)increasedthespontaneousbeatingrateinbothcontroland
A *** mutant cells in a concentration-dependent manner. The differ-
500 p ************ –10 PF) eanllcIeSiOnrcaotnecbeenttwraeteinonmsutteasntetdan(dFicgo.n4tar,obl).mAyoncaylytesisswoafstphreesaecnttioant
**** –75 –65 –55 –45–35 pA/ potential properties of mutant SAN myocytes under basal non-
**** –20 I ( stimulated conditions revealed a more negative action potential
1 s * * * * *––12 threshold, prolonged action potential duration and reduced
–3 –30 action potential amplitude. Control and mutant SAN myocytes
showed spontaneous diastolic depolarizations of the membrane
V (mV) potential (delayed afterdepolarizations, DADs)thatdid notelicit
pA –135 –85 –35 an action potential (Fig. 4a,c). Under basal conditions (no ISO),
00 0 thefrequencyofDADswassignificantlyhigherinmutantthanin
5 ** ** ** ** ** control myocytes (Fig. 4c). ISO significantly reduced the fre-
** –10 F) quencyofDADsincontrolbutnotinmutantmyocytes(Fig.4c).
**–75**–6*5*–*5*5**–*45*–*3–51 –20 I (pA/P AImSsOyoacwcyoatesnssm.eqTourheeentchdeai,ansthttoeelnifcfroeilqnduteehrnivgcahyleorbfethDtwaAneDetnhsaiatnrDmecAuoDtradneatdncdienlltschouenntfdroeolr-l
–2 lowing action potential under ISO was significantly longer than
1 s –3 –30
the cycle length between the two consecutive action potentials
Figure3|I incontrolandmutantpacemakermyocytes.Representative preceding the DAD (Fig. 4d), suggesting that DADs delayed the
f
examplesofI recordings(Csþ-sensitivecurrent,(a))averagedcurrent-to- formation of the following pacemaker impulse. Similar results
f
voltage(I/V)curve(b)incontrol(n¼11)andmutant(n¼14)SAN were obtained in mutant AVN and PF myocytes under basal
pacemakersmyocytes;(c,d)I recordingsandI/VcurveinAVNmyocytes conditions or ISO (Fig. 4e–l). DADs were consistently recorded
f
fromcontrol(n¼16)andmutant(n¼10)mice.(e,f)IfrecordingsandI/V alsoinAVNandPFmyocytesanddisplayedpropertiessimilarto
curveinmyocytesfromcontrol(n¼12)andmutant(n¼9)PFmyocytes.In those seen in SAN cells. Apart from longer action potential
(a,b,e)thetoppanelshowsI incontrolmyocytesandthebottompanel durations in mutant AVN cells, no differences were found in
f
showsI inmutantmyocytes.Inb,d,f,blackcirclesindicatecontrolandred action potential parameters between mutant and control AVN
f
squaresindicatemutantmyocytes.Statisticalsignificancewastestedat and PF myocytes at baseline (Supplementary Table 1). The very
eachvoltageusingtheunpairedt-test.*Po0.05,**Po0.01,***Po0.001, low basal pacemaker activity recorded in mutant PF myocytes
****Po0.0001.Errorbarsindicates.e.m.Thevoltage-clampprotocol was also increased by ISO application, but—similar to our find-
usedforalltherecordingsisshownine. ings in SAN and AVN—the difference in spontaneous beating
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a b c d
400 25 2.0
CL DAD *** 20
Rate (b.p.m.) 321000000 *** *** –1DAD min11055 * ** Time (s) 101...550 D **D
L A L A
C D C D
0 0 0.0
0 2 100 0 100 0 100
ISO (nM) ISO (nM) ISO (nM)
e f g h
400 25 2.0
CLDAD Rate (b.p.m.) 321000000 ** * ** –1DAD min1120505 * * Time (s) 011...505 D *D
L A L A
C D C D
0 0 0.0
0 2 100 0 100 0 100
ISO (nM) ISO (nM) ISO (nM)
i j k l
400 25 2.0
CL DAD
V Rate (b.p.m.) 321000000 * * ** –1DAD min112050 * * Time (s) 011...505
m 5 D D
50 0 0 0.0 CL DA CL DA
0 2 100 0 100 0 100
1 s
ISO (nM) ISO (nM) ISO (nM)
Figure4|PacemakeractivityandDADsincontrolandmutantmyocytes.(a)Actionpotentialrecordingsofcontrol(left)ormutant(right)SAN
myocytesinTyrode’ssolution(blackline)andISO(100nM,greyline).(b)AveragedspontaneousbeatingratesofSANmyocytesinTyrode’ssolution(0;
n¼11control,n¼11mutant),ISO2nM(n¼10control,n¼11mutant)or100nM(n¼7control,n¼10mutant)expressedinbeatsmin(cid:2)1(b.p.m.).
(c)FrequencyofDADs(definedasavariationofatleast5mVofthemembranepotential)incontrolandmutantSANmyocytesinTyrode’ssolution(n¼10
control,n¼14mutant)orISO100nM(n¼10control,n¼12mutant).(d)AveragedtimeintervalbetweenaDADandthefollowingactionpotential
(DAD)incomparisonwiththecyclelength(CL)ofthetwoconsecutiveactionpotentialsprecedingtheDADinn¼8mutantSANmyocytes.(e)Action
potentialsofcontrol(left)ormutant(right)AVNmyocytesinTyrode’ssolution(blackline)andafterISO(100nM)perfusion(grayline).(f)Spontaneous
ratesofAVNmyocytesinTyrode’sandISO(0:n¼12control,n¼9mutantcells;2:n¼9control,n¼7mutant;100:n¼6control,n¼7mutant).
(g)FrequencyofDADsincontrolandmutantAVNmyocytesinTyrode’sandISO(0:n¼11control,n¼11mutant;100:n¼11control,n¼13mutant).
(h)SamedataasindforAVNmutantmyocytes(0,n¼9)andinISO(100,n¼6).(i)SpontaneousactionpotentialsrecordedfromPFmyocytesfrom
control(left)ormutant(right)miceinTyrode’ssolution(blackline)andafterperfusionofISO(100nM,greyline).(j)Spontaneousbeatingrates
ofPFmyocytes(0:n¼10control,n¼7mutant;2:n¼9control,n¼6mutant;100:n¼7control,n¼5mutant).(k)FrequencyofDADsincontroland
mutantPFmyocytes(0:n¼10controlandn¼8mutant;100:n¼5control,n¼7mutant).(l)SamedataasindandhforPFmutantmyocytesin
Tyrode’ssolution(n¼6)andISO(n¼3)inmutantPFmyocytes.Statistics:two-wayanalysisofvariancefollowedbySidakmultiplecomparisonstestand
unpairedorpairedt-test.*Po0.05,**Po0.01,***Po0.001.Errorbarsindicates.e.m.Dottedlineindicatesthe0mV.Openbars
representdatafromcontrol,filledbarsfrommutantmice.
rates between control and mutant cells remained (Fig. 4e,f). myocytes (Fig. 5). Quantification of voltage-dependent Ca2þ
Taken together, our data show that I is critical for setting the currents revealed an increased density of I , while the density
f Ca,L
basal spontaneous beating rate in SAN, AVN and PF myocytes, and activation of I were unchanged (Supplementary Fig.7).
Ca,T
butisnotrequiredforthesympatheticregulationofpacemaking Consistent with our current clamp recordings (Fig. 4), the
inthesestructures.Inaddition,I silencingfavouredtheinduction frequency of spontaneous [Ca2þ] transients was reduced in
f i
of DADs in spontaneously beating myocytes. mutantSANcells(Fig. 5a).Under basal conditions,mutantcells
showed a significantly higher frequency of local Ca2þ release
events (LCRs, Fig. 5b) and increased amplitude of [Ca2þ]
i
I silencinginducedaugmentedSRCa2þ loadandanincreased transients(Fig.5c),indicatingaugmentedsarcoplasmicreticulum
f
frequency of local Ca2þ release events in SAN myocytes. The (SR)Ca2þ loadinthesemyocytes.PerfusionwithISO(100nM)
high frequency of DAD occurrence in I -deficient mutant myo- reduced the frequency of LCRs, but increased the frequency and
f
cytes isolated from the three rhythmogenic centres suggested reduced the amplitude of [Ca2þ] transients (Fig. 5d) in mutant
i
altered Ca2þ handling and release in these cells33. We therefore SAN cells. We did not observe differences in the upstroke
recorded in control and mutant SAN cells voltage-dependent velocity, duration or recovery time of spontaneous [Ca2þ]
i
Ca2þ currentsusingvoltage-clampexperimentsandintracellular transients (Supplementary Fig. 8). We then directly tested the
Ca2þ release ([Ca2þ]) by line-scan imaging of Fluo-4-loaded status of the SR Ca2þ load in isolated control and mutant SAN
i
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min) 200 ** m 400 8 ****
Rate2+]transients per ([Cai115500000 *0*** *2* 100 μ–1 per 40 LCRs min 3210000000 *0 2 100 F/F0 (a.u.) 0246 0 *2* 1*00
ISO (nM) ISO (nM) ISO (nM)
***
8 8 8 5,000
*
Cont
F/F0 (a.u.) 246 F/F0 (a.u.) 246 F/F0 (a.u.) 246 (cid:2) (ms) 2,500 F/F081 M
Mut μ
0
0 0 0 0 4
0 100 0 100 5 s
ISO (nM) ISO (nM)
M
μ
0
4
1 s
CL DAD CL DAD 8
0
F
F/
1
25 ** 25 1.0 1.0
–1n 20 –1n 20 * 0.8 0.8 *
mi 15 mi 15 s) 0.6 s) 0.6
avelet 10 avelet 10 Time ( 0.4 Time ( 0.4
W 5 W 5 0.2 D 0.2 D
CL DA CL DA
0 0 0.0 0.0
Figure5|[Ca2þ] handlinginI –deficientSANmyocytes.(a)Averagedfrequencyofspontaneous[Ca2þ] transientsofSANmyocytesinTyrode’s
i f i
solution(n¼20control,n¼29mutantcells),ISO2nM(n¼16control,n¼29mutantcells)andinISO100nM(n¼14control,n¼12mutantcells).
(b)FrequencyofLCRsinTyrode-perfusedSANmyocytes(n¼19control,n¼24mutantcells),inISO2nM(n¼18controlandn¼26mutantcells)andISO
100nM(n¼14control,n¼13mutantcells).(c)Averagedamplitudeofspontaneous[Ca2þ]itransientsofSANmyocytesinTyrode’ssolution(n¼19control,
n¼19mutantcells),inISO2nM(n¼18,n¼28mutantcells)andISO100nM(n¼14control,n¼13mutantcells).PerfusionwithISOreduced
transientamplitudeinmutantbutnotincontrolSANcells(d).Statistics:two-wayanalysisofvariance,followedbySidakmultiplecomparisonstest.
(e)Histogramsofcaffeine-evoked(10mM)Ca2þ release(n¼12control,n¼14mutantcells,e-left)anddecay(n¼9control,n¼11mutantcells,e-middle)
inSANmyocytes.e-Right:Eeamplesoflinescanincontrol(top)andmutant(bottom)cellduringcaffeineapplication.Statistics:unpairedt-test;confocal
imagesofspontaneouslyactivecontrol(f)andmutant(g)SANmyocytesloadedwithFluo-4perfusedwithTyrode’ssolution(left)or100nMISO(right).Red
circleindicates[Ca2þ] wave;(h,i)averagedfrequencyof[Ca2þ] waves(definedasa[Ca2þ] releaselargerthan4mmand/orwithanintrinsiclight
i i i
intensityof410%oftheintensitymeasuredduringthefollowingspontaneous[Ca2þ] transient)recordedincontrolandmutantSANmyocytesunder
i
Tyrode’ssolution(n¼12control,n¼11mutantcells,h)andISO100nMperfusion(n¼12control,n¼11mutantcellsi);(j,k)histogramscomparing
theaveragedintervalofthecyclelength(CL)betweentwoconsecutivespontaneous[Ca2þ] transientsprecedingthewaveandthosebetweenthewaveand
i
thefollowingspontaneous[Ca2þ]itransient(DAD)inTyrode’ssolution(n¼11,j)andinISO100nM(n¼6,k)inmutantSANmyocytes.Statistics:t-test.
*Po0.05,**Po0.01,***Po0.001,****Po0.0001.Errorbarsindicates.e.m.
myocytes under ISO by rapid caffeine (10mM) application. myocytes from the two mouse strains (Fig. 5e). As it is known
Caffeine-evokedCa2þ releasewassignificantlyhigherinmutant that SR Ca2þ overload favours the formation of [Ca2þ] waves
i
thanincontrolcells,butthedecayofCa2þ releasewassimilarin leading to Naþ/Ca2þ exchanger activation-mediated DADs in
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cardiomyocytes34, we quantified the frequency of [Ca2þ] waves hHCN4-AYA protein expression (Fig. 1c). To investigate
i
(Fig. 5f,g). Under basal conditions and ISO, the frequency of the reversibility of the phenotype of mutant mice, we reintro-
[Ca2þ] waves in mutant SAN myocytes (Fig. 5h,i) was ducedDOXtothedietattheendofweek8torepresstransgene
i
comparable to the frequency of DADs (Fig. 4c). Under ISO, the expression. After 4 weeks of DOX reintroduction, AVBs dis-
diastolic interval between a [Ca2þ] wave and the following appeared and the HRs of control and mutant mice were com-
i
spontaneous [Ca2þ] transient was significantly longer than the parable (Supplementary Fig. 9a,b). Furthermore, voltage-clamp
i
cycle length between the two consecutive spontaneous [Ca2þ] recordings of mutant SAN myocytes showed normalized cell
i
transientsprecedingthe[Ca2þ] wave(Fig.5k).Thisobservation capacitance and I density 4–5 weeks after renewed administra-
i f
is consistent with the hypothesis that [Ca2þ] waves induced tion of DOX to the food (Supplementary Fig. 9c,d). These
i
DADs in mutant myocytes. observations demonstrate that the effects of hHCN4-AYA
protein expression on HR are completely apparent after
5 weeks following DOX withdrawal and can be reversed by
I silencing induced bradycardia, AVBs and ventricular suppression of mutant subunit expression, indicating a causal
f
tachycardia inmutant mice. To investigatethe consequences of relationship.
I silencing in rhythmogenic centres in vivo, we performed tele- ECG recordings of mutant mice deprived of DOX after
f
metric electrocardiography (ECG) recordings in freely moving weaning showed that I silencing induced multiple and severe
f
control and mutant mice. As western blot analysis of hHCN4- alterations in HR and rhythm. SAN activity of mutant mice was
AYA protein indicated that the transgene expression reached a characterized by a prolonged SAN rate (PP intervals) and the
plateau after 5 weeks following DOX withdrawal (week 0), we presenceofSANpauses.Ventricular(RR)beatingratesofmutant
continuously analysed the time course of ECG alterations for mice were also considerably lower in comparison with control
8 weeks after induction of transgene expression following DOX animals (Fig. 6d). We recorded strongly prolonged atrioventri-
withdrawal.HRofmutantmiceconstantlydecreasedandreached cular conduction (PR) intervals in mutant mice (Fig. 6d). Both
aplateauphaseinweek6(Fig.6a).Wedidnotrecorddifferences MobitztypeIandIIAVBswereobservedinmutantmice,butthe
in the HR of control mice during the entire 6-week period frequencyoftypeIIMobitzAVBswashigherthanthatoftypeI
(495±45 at week 0 and 508±49beatsmin(cid:2)1 at week 6, n¼3 AVBs (Fig. 6e). During a 24-h recording period, maximum and
P40.05).Atthesametime,weobservedastrongincreaseinthe minimum HRs were lower in mutant than in control mice, but
frequency of AVBs in mutant mice (Fig. 6b). The decrease in the absolute difference between maximum and minimum HRs
HR (Fig. 6a) and increase in the occurrence of conduction did not differ, showing a preserved degree of HR regulation
abnormalities (Fig. 6b,c) approximately paralleled the kinetics of (Fig. 6f). Consistent with the prominent effect of hHCN4-AYA
0 50
40
Δ b.p.m. –1–0500 –1AVB min 321000 *
**
–150 0
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 9
W W W W W W W W W W W W W W W W W W W
Week 0 Week 8
PRPRP R PRPRPR
500 ms
800 300
CTRL Mut P 20 ******** **
QRPPRRRPS 111311(m8830±±±±s8511) n7777 117138(8643m±±±±s5612)0 n7777 <<0000....0000010055001211 –1AVBII min11550 HR (b.p.m.) 246000000 ** Δ b.p.m. 120000
QQTTc 5424±±22 77 5467±±23 77 00..13756648 0MI 2:1MI 3:M1II 2:1MII 3:1 0 Max Min 0
Figure6|TimecourseofHRslowinginmutantmicefollowingDOXwithdrawal.(a)TimecourseoftheHR(inb.p.m.)slowinginmutantmiceexpressed
asthedifferenceinHRbetweenn¼3controlandn¼5mutantmice.DOXwasremovedfromthedietatweek0(W0).ThemaximumHRdifference
wasobservedatweek6(from525±16b.p.m.atweek0to429±6b.p.m.atweek6;ttest,Po0.001).(b)Relationshipbetweentheaverageincreasein
thenumberofAVBandthetimeelapsedafterDOXwithdrawalinn¼5mutantmice(0.2±0.2AVB/60satweek0versus15±6AVB/60satweek6;
ttest,Po0.05).(c)RepresentativeexamplesofECGtracesrecordedfrommutantmicebefore(left)andafter(right)withdrawalofDOX.(d)Meanvalues
ofECGintervalsrecordedfromcontrolandmutantmice.Statistics:t-test.(e)AveragednumberofMobitztypeIandtypeII2:1(twoP-wavesforone
QRScomplex)and3:1(threeP-wavesforoneQRScomplex)AVBsduring30sofECGrecording(n¼9mutantmice).(f-left)Averagedmaximumand
minimumHRsincontrol(openbars)andmutant(filledbars)mice.Statistics:two-wayanalysisofvariancefollowedbySidakmultiplecomparisons
test.(f-right)RangeofHRregulationcalculatedasdifferencesbetweenmaximumandminimumHRsduringcontinuous24-hECGrecordingsofn¼7
controlandn¼9mutantmice(ttest,P40.05).*Po0.05,**Po0.01,***Po0.001.Errorbarsindicates.e.m.
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expression on PF myocyte rhythmicity and DAD occurrence a *** b
(Fig.4),mutantmicealsoshowedprolongedQRScomplexesand 600 *** 600
*
frequent episodes of ventricular tachycardia in comparison with
controlmice(seebelow).Ventricularrepolarization(QTandQTc
intervals) was not affected in mutant mice (Fig. 6d). As chronic m.) 400 m.) 400
bibnrrvaaeddsyytccigaaarrddteiidaa loewfdthetneothgcehoreasngloaelnsogni-ngterlwemfitthvtercnaatnrrisdcgiuaelcnaerhsyiepzxeeprtarrenosdpsihofyun3n5c,taiwnonde HR (b.p. 200 HR (b.p. 200
in mutant mice. In line with telemetric recordings,
ecocardiography showed slower HRs in mutant mice, but
normal left ventricular and atrial mass to body weight ratios 0 0
0 1 3 6 0 1 3 6
(Supplementary Fig. 10). Left ventricular end-diastolic volume,
IVA (mg kg–1) IVA (mg kg–1)
strokevolumeandareashorteningundercontrolandstimulated
conditions did not differ between control and mutant mice c 600 d 600
(Supplementary Fig.10).Recordingsshowedatendencytowards
a reduced cardiac output in mutant mice, but this difference did
not reach statistical significance (Supplementary Fig. 10).
Furthermore, mutant mice did not show alterations in blood m.) 400 m.) 400
p. p.
pressure measured by means of radiotelemetry as a function of b. b.
locomotor activity in the home cage (Supplementary Fig. 11). R ( 200 R ( 200
H H
HR of mutant mice was insensitive to the I inhibitor
f
ivabradine. To investigate whether If silencing in mutant mice 0 0
correspondedtotheeliminationofthepharmacologicalsensitivity 0 1 3 6
of HR to I inhibition, we recorded ECGs of control and mutant IVA (mg kg–1)
f
mice on injection of ivabradine (IVA), a selective I inhibitor36.
f
Administration of IVA (6mgkg(cid:2)1) reduced the HR in control, e ***
250 **
butnotinmutantmice(Fig.7a,b).NoIVAeffectwasdetectedin ##
mutant mice when considering the ventricular (RR interval,
200
FSAigN.7rba)teosroSfAIVNAr-attreesa(tePdPcionntetrrovlalm,Ficige.a7ncd).uRnetmreaatrekdabmlyu,tHanRtsmaincde p.m.) 150
were similar (Fig. 7d), indicating that the negative chronotropic b.
eeffffeecctt ionf iIsVoAlatewdasspcoonmtapnleeoteulyslyabboelaitsihnegd.riLgihktewatirsiea,lIpVrAepahraadtionnos Rate ( 15000 0 mV
frommutantmice(SupplementaryFig.12),butitcausedaslowly 5
developing, concentration-dependent decrease in spontaneous
0 500 ms
beating rates in control atria, which were comparable to those
CTRL Mut
observed in mutant SAN myocytes perfused with ISO
(Supplementary Figs 12 and 13). In agreement with these Figure7|Lossofivabradine-mediatedHRreductioninmutantmice.
results, IVA slowed pacemaker activity of isolated control SAN Dose–responserelationshipofHR(RR)tointraperitonealadministrationof
myocytes by B33% but did not affect the spontaneous beating IVAinn¼13control(a)andn¼7mutant(b)mice(analysisofvariance
rateofmutantmyocytes(Fig.7e).Theseobservationsindicatethat (ANOVA)followedbyTukey’smultiplecomparisonstest).(c)Averaged
no residual, physiologically relevant I current was present in SANrate(PPinterval)inmutantmiceafterIVAadministration.
f
hCN4-AYA-expressing mutant SAN myocytes. (d)ComparisonbetweentheSANrateofcontrol(whitebar)andmutant
(blackbar)miceascomparedwiththeventricularrateofmutantmice
Autonomic nervous system activity modulated the functional (greybar)afterinjectionofthehighestdoseofIVA(6mgkg(cid:2)1,ANOVA
impactofI silencingonHRandimpulseconduction.Wethen followedbyTukey’smultiplecomparisonstest).(e-left)Averaged
f
investigated the impact of I silencing on in vivo HR regulation. spontaneousbeatingratesofSANmyocytes,isolatedfromn¼9control
f
Pharmacological inhibition of the sympathetic branch of the (whitebars)andn¼8mutantmice(blackbars),inTyrode’ssolution
autonomic nervous system by propranolol (5mgkg(cid:2)1) sig- (emptybars)andafterperfusionwithivabradine(1mM,dottedbars).
nificantlydecreasedtheHRsofcontrolmice,buthadnoeffecton Statistics:two-wayANOVAfollowedbySidakmultiplecomparisonstest.
those of mutant mice (Fig. 8a). Propranolol reduced the HR of ##Po0.01,*Po0.05,**Po0.01,***Po0.001.Errorbarsdefinethes.e.m.
control mice to levels that did not differ from those of mutant (e-right)Sampleactionpotentialsrecordedfromcontrol(top)andmutant
mice, either under basal conditions or in the presence of pro- (bottom)SANmyocytesbefore(blackline)andafter(greyline)application
pranolol (Fig. 8a). Likewise, combined injection of propranolol of1mMIVA.
and atropine (0.5mgkg(cid:2)1), an inhibitor of the parasympathetic
input,didnotsignificantlyaffecttheHRofmutantmice(Fig.8b).
Interestingly, pharmacological inhibition of the autonomic input theirHRunderconditionsofmaximalsympatheticinput,thatis,
by combined injection of atropine and propranolol strongly direct pharmacological stimulation of b-adrenergic receptors or
reduced the number of AVBs in mutant mice (Fig. 8c,d). These physicalexercise.InjectionofISOoratropinesimilarlyincreased
observationsindicatethatthephysiologicalimpactofI silencing theHRinbothcontrolandmutantmice(Fig.8e–g).Contraryto
f
onHRdeterminationandimpulseconductionwasreducedinthe theinhibitionoftheautonomicnervoussystem(Fig.8d),ISOdid
absence of autonomic input. not reduce cardiac dysrhythmia due to SAN pauses and AVBs
(Fig.8e).ComparableresultswereobtainedfromISO-stimulated,
I silencingdidnotimpairmaximalb-adrenergicregulationof spontaneously beating right atria preparations (Supplementary
f
HR. We then assessed the capability of mutant mice to increase Fig. 10c).AlthoughthemaximumHRinISO-stimulatedmutant
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800 800 25
** * **
HR (b.p.m.) 246000000 ## HR (b.p.m.) 246000000 ### ## –1AVB min 2115005
0 0 0
Pre Post Pre Post Pre Post
ISO
600 A+P 400 **** 800
m.)400 m.) 300 ** 600 m.)
p. p. 200 400 p.
b. b. b.
HR (200 23::11 5 min s.d.( 100 * 200 HR (
0 0 0
–100 0 100 200
Time (min)
****
800 *******#*## 800 ****#### 800 ****## 250
R (b.p.m.) 460000 #### R (b.p.m.) 460000 #### R (b.p.m.) 460000 ## Δ b.p.m. 211005000
H 200 H 200 H 200 50
0 0 0 0
Pre Post Pre Post Pre Post ISO Atro Stress
Figure8|AutonomicregulationofHRandimpulseconductioninmutantmice.AveragedHRrecordedfromn¼11control(openbars)andn¼11mutant
(filledbars)micebefore(pre)andafter(post)intraperitonealinjectionofpropranolol(a),oratropineandpropranolol(b).Statisticsinaandb:two-way
analysisofvariance(ANOVA)followedbySidakmultiplecomparisonstest.(c)NumberofAVBsmeasuredina30-swindowevery5minfora30-mintotal
recordingperiodbefore(pre)andafter(post)injection.AVBcountingstarted30minafterinjectiontoallowtheeffecttostabilize.Statistics:t-test.
(d)Sampledotplotofbeat-to-beatvariability(b.p.m.)ofHRofmutantmicebeforeandafter(dottedline)injectionofatropineandpropranolol.Notethe
slowventricularbeatsinthepresenceofAVBbeforeinjection.ArrowsindicateAVBs(twoP-wavesforoneQRScomplex,2:1;threeP-wavesfor
oneQRScomplex,3:1).(e)TimecourseofHRandthes.d.ofHRbeforeandafterinjectionofISO.(f–h)HRincontrolandmutantmicebeforeandafter
injectionofatropine(f)orISO(g)inn¼8controlandn¼12mutantmice.(h)HRbeforeandafter5-minswimmingtest,inn¼5control(openbars)and
n¼7mutant(filledbars)mice.(i)DeltaofHRfollowingdifferentb-adrenergicstimulations.Valuesrepresentthemeandifferencebetweenpre-and
post-adrenergicpathwayactivation(pharmacologicalorphysiological).Statistics:two-wayANOVAfollowedbySidakmultiplecomparisonstest.*Po0.05,
**Po0.01,***Po0.001,****Po0.0001,##Po0.01,###Po0.001,####Po0.0001.Errorbarsindicates.e.m.
mice did not reach that of control mice (Fig. 8f), the relative improved SAN function. The PR interval was significantly
extent of ISO-induced increase did not differ between the two shorter in Mut/GIRK4(cid:2)/(cid:2) mice, although it was still longer
strains (Fig. 8h). Likewise, during a swimming test combining than that in control mice (Fig. 9c). The occurrence of AVBs
mental stress with physical activity (Fig. 8g,h), the HR was was drastically reduced in Mut/GIRK4(cid:2)/(cid:2), suggesting that
increased to an extent similar to that in control mice. Taken the activity of GIRK4 channels in the AVN contributed to
together,thesedatashowthatinspiteofcompleteI silencingthe conductiondysfunctioninmutantmice(Fig.9d).LossofGIRK4
f
autonomic nervous system was still capable of regulating HR channels did not normalize QRS intervals, which were similarly
(Fig. 8i). prolongedinMut/GIRK4(cid:2)/(cid:2) andmutantmice(Fig.9e).Inmost
cases, the ventricular tachycardia/tachyarrhythmia observed in
Genetic inactivation of GIRK4 channels ameliorated mutant mice was preceded by dysfunction of atrial rhythm or
SAN failure, atrioventricular dysfunction and ventricular AVBs.Inparticular,SANpausesandAVBsprecededventricular
arrhythmiainmutantmice.AstheimpactofI silencingonHR tachycardia in 470% of cases, suggesting a causal relationship
f
and atrioventricular dysfunction depended on the status of the (Fig. 9j). In line with the absence of SAN pauses and AVBs in
autonomic nervous system, we investigated the possibility of Mut/GIRK4(cid:2)/(cid:2) mice,wedidnotrecordventriculartachycardia
counterbalancing I loss-of-function in mutant mice by reducing or extrasystoles in these animals (Fig. 9k, Table 1). Similar to
the influence of thef parasympathetic nervous system on HR and control and mutant mice, Mut/GIRK4(cid:2)/(cid:2) animals showed a
atrioventricular conduction. We thus crossed mutant mice with preserved degree of HR regulation and a normal recovery
GIRK4 (Kir3.4) knockout mice (GIRK4(cid:2)/(cid:2)) deficient in the of resting HR following direct b-adrenergic stimulation
cardiac G-protein-gated inwardly rectifying Kþ current I 37 (Supplementary Fig. 13). These data indicate that the
KACh
toobtain double-mutantMut/GIRK4(cid:2)/(cid:2) mice.Telemetric24-h inactivation of GIRK4 channels was effective in improving SAN
ECGrecordingsinMut/GIRK4(cid:2)/(cid:2) miceshowedahigherHRin automaticity and atrioventricular conduction, and in preventing
comparisonwithmutantanimals,althoughthemeanRRinterval ventricular arrhythmias in If-deficient mice (Table 1).
measured in double-mutant mice was still significantly longer
than that of controls (Fig. 9a). The SAN rates (PP interval) of Discussion
Mut/GIRK4(cid:2)/(cid:2) and of mutant mice did not differ from each Cardiac pacemaker activity is a complex phenomenon requiring
other (Fig. 9b). However, SAN pauses were absent in Mut/ the interaction of different classes of membrane ion channels1,
GIRK4(cid:2)/(cid:2) mice, indicating that GIRK4 loss-of-function [Ca2þ] cycling38 and probably other mechanisms yet to be
I
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****
250 *** * 250 90 **** * 40 ******** 30
ms 211005000 ms 211005000 * * ms60 –1B min 2300 ms20 **
30 V 10
50 50 A 10
0 0 0 0 0
RR PP PR AVB QRS
1 s 1 s 1 s
50 *
%) 40
s ( 30
ent 20
v
E 10
0
1 s SV +S PAVB IIAVB IBIigEeSmVinisNmormal 1 s
E
Figure9|ImprovementofHRinmutantmicebyGIRK4geneknockout.AveragedRR(a),PP(b),PR(c),numberofAVBs(d)andQRS(e)inn¼7
control(whitebar),n¼7mutant(blackbar)andn¼8Mut/GIRK4(cid:2)/(cid:2)(blackbarwithwhitestripes)mice.Statistics(a–e):analysisofvariance(ANOVA)
followedbyTukey’smultiplecomparisonstest.*Po0.05,**Po0.01,****Po0.0001.Errorbarsdefinethes.e.m.(f)RepresentativeexampleofECG
recordingsfromcontrolmice.(g–i).DifferentexamplesofECGrecordingsshowingHRdysfunctionrecordedfrommutantmice.(g)Second-degreeAVB;
(h)ventricularextrasystoles;(i)ventriculartachycardia.(j)RelativepercentagesofSANorAVNdysfunctionsprecedingaperiodofventriculartachycardia
inn¼5mutantmice(SP,sinuspauses;ESV,ventricularextrasystoles).(k)ECGrecordedfromMut/GIRK4(cid:2)/(cid:2) mice.Smallcirclesindicate
P-waves.
conduction system. We show that chronic functional ablation of
Table1| Summarytableof thefractionof CTRL,Mut or
I conductance induces severe abnormalities of both atrial and
Mut/GIRK4(cid:2)/(cid:2) mice withheart rhythmdisturbances. vfentricular rhythms by altering Ca2þ handling in pacemaker
myocytes. The resulting complex cardiac arrhythmia was
CTRL Mut Mut/GIRK4(cid:2)/(cid:2) P-value prevented by inactivation of GIRK4 channels.
Sinuspauses 0/12 5/13 0/8 0.0408 We used the Tet-off strategy to express mutant hHCN4-AYA
Bradycardia 0/12 13/13 8/8 0.0009 non-conductingsubunitstosuppressf-channelconductanceina
AVBIdegree 0/12 13/13 0/8 0.0009 dominant-negative manner. Using this experimental approach,
AVBIIdegree 0/12 13/13 0/8 0.0009
we achieved I silencing without deletion of HCN-channel
AVBIIIdegree 0/12 13/13 0/8 0.1293 f
subunitsfromtheplasmamembraneandthuswithoutdepletion
ESV 0/12 4/13 0/8 0.0715
of HCN-associated proteins. Embryonic lethality observed when
VT 0/12 5/13 0/8 0.0408
mutant hHCN4-AYA channels are expressed during embryonic
AVB,atrioventricularblock;CTRL,control;ESV,ventricularextrasystole;Mut,mutant;VT, development suggests that I conductance is required for the
f
ventriculartachycardia.
Statistics:w2-test. embryonichearttobeviable(Fig.1),whichiscompatiblewiththe
phenotype of both global and heart-specific HCN4 knockout
mice26. Western blotting experiments demonstrated that the
hHCN4-AYA protein was highly expressed in the atria and
identified. The ‘funny’ current I is thought to play a key role in ventricles of adult mutant mice (Fig.1). Similar to findings from
f
thegenerationandregulationofautomaticityintheadultheart8, immunostainings of native HCN4 channels of SAN and AVN
but its functional role and importance are still incompletely preparations (Fig. 2), or of isolated myocytes, for hHCN4-AYA
understood9,21. Pharmacological39 and genetic studies in protein(SupplementaryFig.5)indicatedthatthemutantchannel
humans16–19 indicate a role for I in the determination of HR protein was targeted to the cell membrane. Consistent with the
f
and rhythm. However, a body of data obtained from mouse immunostaining in mutant pacemaker tissue and the dominant-
modelsinwhichHCN4subunitsweredeletedshowedcontrasting negative effect on HCN4- and HCN2-mediated currents by
results in relation to the importance of these channels in SAN hHCN4-AYA recorded in Xenopus oocytes, I was abolished at
f
pacemakingandimpulseconduction21–23.Furthermore,although voltages spanning the diastolic depolarization range of myocytes
several groups described the phenotype of mice deficient in from the SAN and the conduction system (Fig. 3). I silencing
f
HCN1 (ref. 15), HCN2 (ref. 40) or HCN4 (refs 21,23,41) throughout the conduction system suggested that mutant
subunits, no mouse model has yet been available that shows a hHCN4-AYA channels exerted a dominant-negative effect, also
suppressionofI conductanceofupto495%ofitscontrolvalue. on native HCN1 channels. In agreement with the complete
f
In this study, we show for the first time heart-specific and time- silencingofI,pacemakeractivityofmutantSANmyocytes,intact
f
controlled silencing of I conductance in both the SAN and the atrial preparations and the in vivo HR of hHCN4-AYA mutant
f
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