Disruption of cardiac cholinergic neurons enhances susceptibility to ventricular arrhythmias

13 Pages·2017·2.16 MB·English

by  Christiane Jungen

ARTICLE
Received31Mar2016 |Accepted 28Nov2016 |Published 27 Jan 2017 DOI:10.1038/ncomms14155 OPEN
Disruption of cardiac cholinergic neurons enhances
susceptibility to ventricular arrhythmias
Christiane Jungen1,2, Katharina Scherschel1,2, Christian Eickholt1, Pawel Kuklik1, Niklas Klatt1,2, Nadja Bork2,3,
Tim Salzbrunn1, Fares Alken1, Stephan Angendohr1, Christiane Klene4, Janos Mester4, Nikolaj Klo¨cker5,
Marieke W. Veldkamp6, Udo Schumacher7, Stephan Willems1,2, Viacheslav O. Nikolaev2,3 & Christian Meyer1,2
Theparasympatheticnervoussystemplaysanimportantroleinthepathophysiologyofatrial
fibrillation. Catheter ablation, a minimally invasive procedure deactivating abnormal firing
cardiac tissue, is increasingly becoming the therapy of choice for atrial fibrillation. This is
inevitably associated with the obliteration of cardiac cholinergic neurons. However, the
impact on ventricular electrophysiology is unclear. Here we show that cardiac cholinergic
neurons modulate ventricular electrophysiology. Mechanical disruption or pharmacological
blockade of parasympathetic innervation shortens ventricular refractory periods, increases
the incidence of ventricular arrhythmia and decreases ventricular cAMP levels in murine
hearts. Immunohistochemistry confirmed ventricular cholinergic innervation, revealing
parasympatheticfibresrunningfromtheatriatotheventriclesparalleltosympatheticfibres.
In humans, catheter ablation of atrial fibrillation, which is accompanied by accidental
parasympathetic and concomitant sympathetic denervation, raises the burden of premature
ventricular complexes. In summary, our results demonstrate an influence of cardiac
cholinergic neurons on the regulation of ventricular function and arrhythmogenesis.
1DepartmentofCardiology-Electrophysiology,cardiacNeuro-andElectrophysiologyResearchGroup(cNEP),UniversityHeartCenter,UniversityHospital
Hamburg-Eppendorf,Martinistrasse52,20246Hamburg,Germany.2DZHK(GermanCenterforCardiovascularResearch),partnersiteHamburg/Kiel/
Lu¨beck,13347Berlin,Germany.3InstituteofExperimentalCardiovascularResearch,UniversityMedicalCenterHamburg-Eppendorf,Martinistrasse52,
20246Hamburg,Germany.4DepartmentofNuclearMedicine,UniversityMedicalCenterHamburg-Eppendorf,Martinistrasse52,20246Hamburg,
Germany.5InstituteofNeuralandSensoryPhysiology,MedicalFaculty,UniversityofDu¨sseldorf,Universita¨tsstrasse1,40225Du¨sseldorf,Germany.
6AcademicMedicalCenter,UniversityofAmsterdam,DepartmentofClinicalandExperimentalCardiology,Meibergdreef9,1105AZAmsterdam,The
Netherlands.7InstituteofAnatomyandExperimentalMorphology,UniversityCancerCenterHamburg,UniversityMedicalCenterHamburg-Eppendorf,
Martinistrasse52,20246Hamburg,Germany.CorrespondenceandrequestsformaterialsshouldbeaddressedtoV.O.N.(email:[email protected])
ortoC.M.(email:[email protected]).
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T
he autonomic nervous system of the heart and its impact PAD reduces dispersion of ventricular wave propagation.
on heart rate and rhythm are known for more than a Next, we determined wave propagation velocity and dispersion
century1. Recent advances demonstrate the importance of in conduction direction by using multi-electrode epicardial
the cardiac neural network in the pathophysiology of atrial activation mapping. Ventricular mapping during fixed pacing
fibrillation (AF). This is the most common sustained human indicatedthatmeanepicardialwavepropagationvelocitydidnot
arrhythmia,witharisingincidenceofupto10%inoctogenarians, differ between PAD (0.77±0.05ms(cid:2)1) and control hearts
and leads to significant morbidity and mortality2,3. Cardiac (0.89±0.05ms(cid:2)1,P¼0.09,unpairedt-test;Fig.1e).Ofinterest,
neurons are inevitably obliterated during catheter ablation4, colour-coded reconstruction of isochrones in combination with
which is the therapy of choice for most patients suffering from arrows representing the direction of conduction showed that
symptomatic AF. This technique is a minimally invasive dispersioninconductiondirectionwassignificantlylowerinPAD
procedure performed to deactivate specific areas of abnormal (0.52±0.07 radian) compared with control hearts (0.91±0.12
firing heart tissue especially in the left atrium around the radian, P¼0.026, Mann–Whitney test; Fig. 1f) indicating that
pulmonary veins. As the atrial neural network contains mostly cardiaccholinergicneuronsimpacttheuniformityofconduction.
cholinergic neurons, an increase in heart rate after catheter
ablation is widely recognized to result from this partial atrial
CholinergicPADincreasesventriculararrhythmiaoccurrence.
denervation (PAD)2. Although PAD during catheter ablation
To investigate whether modulation of the intrinsic autonomic
of AF seems to alter atrial electrophysiology beneficially4,5, its
nervous system of the heart increases arrhythmia susceptibility,
impact on ventricular electrophysiology remains unclear.
standardizedprogrammedstimulationwasperformed(Fig.2a–d).
Text book knowledge states that parasympathetic axonal
Compared with control hearts, we observed an increase in
supply is limited to the atria6, but this paradigm has recently
susceptibility to ventricular arrhythmias after PAD (10% versus
beenchallenged7,8.Thereissomeevidencesupportingthenotion
100%, Po0.0001, Fisher’s exact test; Fig. 2b). In control hearts,
that extracardiac parasympathetic efferents are able to modulate
ventriculararrhythmiasoccurredmorefrequentlyafterganglionic
ventricular function9,10 and electrophysiology11 by involving
blockade (10% versus 100%, P¼0.002, Fisher’s exact test),
centraland peripheral neuralpathways12–14.In addition,cardiac
muscarinergic blockade (10% versus 100%, P¼0.002, Fisher’s
ganglia have been shown to influence ventricular repolarization
exacttest)orcholinergicstimulation(10%versus80%,P¼0.017,
properties15. However, the relevance of cardiac cholinergic
Fisher’s exact test) in line with previous findings16. b-Blockade
neurons for ventricular electrophysiology is not yet understood.
(10% versus 20%, P¼1.0, Fisher’s exact test) did not alter
Here, we present clinical data demonstrating impaired
arrhythmia susceptibility. In PAD hearts, ganglionic blockade
ventricular electrophysiology following catheter ablation in
(100% versus 100%, P¼1.0, Fisher’s exact test), muscarinergic
patients. Experimental data confirm that mechanical disruption
blockade (100% versus 100%, P¼1.0, Fisher’s exact test) or
orpharmacologicalblockadeofcardiacparasympatheticinnerva-
b-blockade (100% versus 60%; P¼0.095, Fisher’s exact test) did
tionshortensventricularrefractoryperiods(VRPs),increasesthe
not influence arrhythmia occurrence. Cholinergic stimulation
incidence of ventricular arrhythmia and decreases ventricular
reversed the increased susceptibility to arrhythmias in PAD
cAMP levels in mouse hearts. Confirmingly, immunohistochem-
hearts (20% versus 100%, P¼0.004, Fisher’s exact test).
icalstainingsshowthatcardiaccholinergicnervefibresrunfrom
Comparedwithcontrols(4.9±1.5),theventriculararrhythmia
the atria to the ventricles in parallel to sympathetic fibres. The
score was higher after PAD (25.4±5.8, P¼0.003, unpaired
results imply a vital role of atrial cholinergic neurons in the
t-test;Fig.2d).Incontrolhearts,ganglionicblockade(36.8±11.4,
modulation of ventricular electrophysiology.
P¼0.002, unpaired t-test) and muscarinergic blockade
(33.4±16.2, P¼0.010, Mann–Whitney test) increased the
Results
arrhythmia score, while it was not altered during b-blockade
PAD decreases ventricular refractory periods. To investigate
(4.8±3.3, P¼0.975, unpaired t-test) or cholinergic stimulation
whether disruption of cardiac neurons influences baseline
(4.0±1.4, P¼0.702, unpaired t-test). In PAD hearts, ganglionic
electrophysiology,wefirstperformedelectrophysiologicalstudies
(38.8±20, P¼0.745, Mann–Whitney test) or muscarinergic
in mouse hearts (Fig. 1a–f). We determined standard electro-
blockade(15.6±4.9,P¼0.228,unpairedt-test)didnotinfluence
physiologicalparametersincludingtheVRP,whichdescribesthe
the arrhythmia score, while b-blockade (5.6±1.7, P¼0.011,
duration of time in which the cardiac cell is unable to initiate
Mann–Whitney test) or cholinergic stimulation (5.0±3.4,
another action potential. VRPs were shorter in PAD hearts
P¼0.036, unpaired t-test) reduced it. This suggests that PAD
(23.4±0.9ms) compared with control hearts (31.6±2.2ms,
or pharmacological modulation of cardiac autonomic tone
P¼0.003, unpaired t-test; Fig. 1d), similar to atrial refractory
impacts arrhythmia susceptibility.
periods (see Supplementary Table 1), demonstrating that dis-
ruption of cardiac neurons distant from the ventricles influences
ventricular baseline electrophysiology. In control hearts, the Cardiac cholinergic neuronsdecrease ventricular cAMP levels.
VRPs were shortened by ganglionic (22.4±1.0ms, P¼0.002, Toanalyseeffectsoflocalatrialnicotinergicstimulationonatrial
Mann–Whitney test) or muscarinergic blockade (24.4±0.7ms, and left ventricular cAMP levels, we used a newly established
P¼0.045, unpaired t-test) but not altered by b-blockade whole-heart Fo¨rster Resonance Energy Transfer (FRET)-based
(34.8±2.4ms, P¼0.391, unpaired t-test) or cholinergic stimu- imaging method (Fig. 3a,b). Topical application of nicotine
lation (35.6±1.2ms, P¼0.248, unpaired t-test). In PAD hearts, (ganglionic stimulant) reduced isoproterenol (ISO)-stimulated
ganglionic(22±0.9ms,P¼0.345,unpairedt-test),muscarinergic atrial ((cid:2)16±3.3%; Fig. 3c left panel) and ventricular
(24.4±0.7ms, P¼0.484, unpaired t-test) or b-blockade ((cid:2)16±3.5%; Fig. 3cright panel) cAMP levels in control hearts.
(25.2±0.5ms, P¼0.265, Mann–Whitney test) did not alter The effect of nicotine on ventricular cAMP levels was almost
VRPs. However, cholinergic stimulation in PAD hearts completely abolished after PAD ((cid:2)3.3±0.8%, P¼0.018,
(37.2±2.6ms versus 23.4±0.9ms, Po0.0001, unpaired t-test) unpaired t-test; Fig. 3c). Ganglionic ((cid:2)3.5±1.7%, P¼0.023,
prolongedrefractoryperiodstosimilarvaluesasincontrolhearts unpaired t-test) or muscarinergic blockade ((cid:2)3.7±3.2%,
suggesting reversibility of PAD induced shortening of refractory P¼0.036, unpaired t-test; Fig. 3c) also blocked the response.
periods. Electrophysiological parameters obtained in vivo and Intheatrium,cAMPlevelswereaswellreducedbylocalnicotine
ex vivo are displayed in Supplementary Tables 1–4. application ((cid:2)16±3.3%). This effect was not altered by PAD
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a b c
LA
6 mm
28 mm
37 mm
CS
RV
9 mm
LV
14 mm
23 mm
30 mm
d
50 * 50 ****
**
ms) ms)
P ( 30 P ( 30 #
R R
V V
10 10
Control Ganglionicblockade uscarinergicblockade β-Blockade Cholinergicstimulation PAD Ganglionicblockade uscarinergicblockade β-Blockade Cholinergicstimulation
M M
e
Control PAD
1.5
3 ms 3 ms
–1) 1.0
s
m
V (
P 0.5
W
0 ms 0 ms
0.0
Control PAD
f
1.5 * Control PAD
3 ms 3 ms
n)
a 1.0
di
a
D (r
C 0.5
D
0.0 0 ms 0 ms
Control PAD
Figure1|Disruptionofatrialcholinergiccontrolmodifiesventricularelectrophysiology.(a)Schematicdrawingofamurineheartinposteriorview.Light
anddarkorangesurfacesdepictareasofatrialfatwithganglionatedplexi,whichhavebeenmechanicallyremovedinpartialatrialdenervation(PAD)
experiments.CS,coronarysinus;LA,leftatrium;LV,leftventricle;RV,rightventricle.(b)ExampleofamurineheartwithintheLangendorffsetup.
Intracardialandepicardialcatheters,aswellastheepicardialmulti-electrodearray,aredepicted.(c)Theepicardialmulti-electrodearrayisdepictedwithan
enlargementoftheschematicelectrodelayout.(d)Theventricularrefractoryperiods(VRPs)arepresentedforcontrolandPADheartswithdifferent
pharmacologicalinterventions.Incomparisonwithcontrol(n¼10),theVRPwasdecreasedbyPAD(denotedby#;n¼10;P¼0.003;unpairedt-test),by
ganglionicblockade(hexamethonium,5(cid:3)10(cid:2)4M;n¼5;P¼0.002;Mann–Whitneytest)andbymuscarinergicblockade(atropine,1(cid:3)10(cid:2)5M;n¼5;
P¼0.045;unpairedt-test).b-Blockade(propranolol,1(cid:3)10(cid:2)6M;n¼5)andcholinergicstimulation(acetylcholine,1(cid:3)10(cid:2)5M;n¼5)didnotinfluence
VRPsincontrols.AfterPADganglionic,muscarinergicorb-blockadedidnotreducetheVRP.However,cholinergicstimulationinPADheartsraisedtheVRP
(n¼5;Po0.0001;unpairedt-test)suggestingreversibilityofPAD-inducedshorteningofVRPs.(e)Wavepropagationvelocity(WPV)wasnotinfluenced
byPADasdepictedinthecolour-codedreconstructionwithisochrones(2ms(cid:2)1distancebetweenisochrones)ofepicardialmulti-electrodeactivation
mapping.(f)Dispersionofconductiondirection(DCD)wassmallerafterPAD(n¼16)comparedwithcontrols(n¼19;P¼0.026;Mann–Whitneytest).
Notethemorehomogeneousarrowalignmentintherightimage.Allthevaluesshownaremean±s.e.m.*Po0.05,**Po0.01,****Po0.0001.
((cid:2)12±3.1%, P¼0.45, unpaired t-test). However, ganglionic changesofcAMPlevelsafternicotinergicstimulationwere2.29in
((cid:2)2.4±1.1%, P¼0.009, unpaired t-test) or muscarinergic the atrium and 0.89 in the ventricle. Similar effects have been
blockade ((cid:2)2.6±2.2%, P¼0.011, Mann–Whitney test) reduced observedafterdirectadenylylcyclaseactivationusingthesoluble
atrial cAMP levels after local nicotinergic stimulation. Absolute forskolin analogue NKH477 (Fig. 3d). Here, nicotine reduced
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NKH477stimulatedcAMPlevelsintheatrium((cid:2)24±5.9%)as (Fig. 4a–l; Supplementary Tables 5 and 6). Choline acetyl-
well as in the ventricle ((cid:2)16±3.6%), which were reduced after transferase (ChAT)-positive ganglia were mainly located around
muscarinergicblockade(atrium: (cid:2)11±3.1%,P¼0.019,pairedt- the pulmonary veins. Both, sympathetic (tyrosine hydroxylase
test; ventricle: (cid:2)4.9±2.1%, P¼0.039, paired t-test). Experi- (TH)-positive) and parasympathetic (ChAT-positive) structures
mentsinisolatedcardiomyocytesconfirmedchangesinmaximal were located in close proximity to each other in the atria and
cAMP levels (after ISO stimulation) due to cholinergic stimula- along the coronary sinus. From there, axons traversed towards
tion ((cid:2)55±8.4%) or muscarinergic blockade ((cid:2)3.8±6.7%, both ventricles via different routes (Fig. 5a–f). Up to the
Supplementary Fig. 1). These results demonstrate that activation ventricularapex,TH-positivefibreswerepredominantandtightly
of atrial cholinergic neurons by nicotine modulates atrial and intertwinedwithChAT-positivefibres(Fig.5f).Westernblotting
ventricular cAMP levels. against ChATand againstthe vesicular acetylcholine transporter
(VAChT)confirmedthepresenceofa70kDaband(knowntobe
Cholinergic nerves traverse from the atria to the ventricle. To the neuronal ChAT isoform17) in the ventricles that was also
investigate the interconnectivity of the atrial and ventricular detectable in brain tissue (Fig. 5h; Supplementary Fig. 2), which
intracardiacautonomicnervoussystem,weexaminedtherelation indicates the presence of a cholinergic system in the ventricles.
between cholinergic ganglia and sympathetic/parasympathetic NativeeGFPfluorescenceofChATBAC-eGFPmicedemonstrated
neural fibres. Immunohistochemical staining of whole mount adelicatenetworkofparasympatheticfibressurroundingcardiac
murine hearts revealed fibres originating from atrial ganglia, myocytes (Fig. 5g). PAD resulted in a 61% removal of ganglia
passingthecoronary sinus towardsventricularmyocardial tissue which was revealed in haematoxylin and eosin-stained sections
(7.7±1.9 versus 19.7±3.2 ganglia, P¼0.1, Mann–Whitney test;
a Fig.6)andwhole-mountChATstainingsofPADhearts(6.2±0.3
Control PAD ganglia). Quantitative real-time polymerase chain reaction
(qRT-PCR) of the atria after PAD indicated a clear reduction of
EG EG ChatmRNA(0.11±0.08-foldtoCdkn1bandfattissue,P¼0.063,
RA I RA I Wilcoxonsigned-ranktest),aswellasThmRNA(0.55±0.2-fold
to Cdkn1b and fat tissue, P¼0.078, Wilcoxon signed-rank test)
Sinus rhythm VT induction
and RBfox3 mRNA (also known as NeuN), which is expressed
EG EG exclusively in neuronal nuclei18 (0.41±0.09-fold to Cdkn1b and
V I V I fat tissue, P¼0.011, paired t-test; Fig. 6d).
R R
100 ms 100 ms
PVC occurrence after AF ablation. To investigate whether
modulationoftheintrinsiccardiacautonomicnervoussystemat
b
*
** ** Figure2|Disruptionofatrialcholinergiccontrolincreasesventricular
** # arrhythmogenesis.(a)Exampleofarrhythmiasusceptibilitytestingusing
100 100
burststimulationintherightventricle(RV;cyclelength10ms,duration5s.)
miae (%) 75 miae (%) 75 without(leftpanel:control)orwith(rightpanel:partialatrialdenervation
Arrhythoccurenc 5205 Arrhythoccurenc 5205 (mePleoAcrDter)of)rgeirnqadumue.nc(ttbiloy)niSnoufPsAcaeDvpethnibetiraliircttyusl.taoRrAvtae,cnrhitgryihcctauraldatriraiua(rmVrh;TyI)Et.hGAm,riiranhstyraitnhccamrrediaaisasecodccauftrererd
0 0
PAD(rightpanel,blackbar,100%,n¼10)comparedwithcontrolhearts
Control Ganglionicblockade uscarinergicblockade β-Blockade Cholinergicstimulation PAD Ganglionicblockade uscarinergicblockade β-Blockade Cholinergicstimulation mh(leeuafstrctpsaa,ringneaelnr,ggwilcihobintleioccb(kahare,dxe1a0m(%aet,rtohnpo¼inni1eu0,m;1,d(cid:3)e5n1(cid:3)0ot(cid:2)1e0d5(cid:2)Mb4y,Mn#¼,;Pn5o¼;P05¼.;0P00¼0.0100)..20I)n0i2nc)cornoetrarsoeld
M M arrhythmiasusceptibility.b-Blockade(propranolol,1(cid:3)10(cid:2)6M;n¼5)did
notsignificantlyinfluencearrhythmiainducibilityincontrols.Cholinergic
c
Re-entry Focal activation stimulation(acetylcholine,1(cid:3)10(cid:2)5M,n¼5;P¼0.017)ledtoanincrease
inarrhythmiasusceptibilityincontrolheartsinlinewithpreviousfindings16.
11.4 ms 3.9 ms InPADhearts,ganglionic(n¼5),muscarinergic(n¼5)orb-blockade
(n¼5)didnotsignificantlyinfluencearrhythmiainducibility.Cholinergic
stimulationreducedVToccurrenceinPADhearts(n¼5;P¼0.004).
Fisher’sexacttestwasusedforallanalysesinb.(c)Examplesofventricular
arrhythmiaswithdifferentunderlyingmechanisms(left:re-entry;right:
0 ms 0 ms
focal).Redcolourdisplaysareasofearliestactivation,purpleoflatest
activation.(d)Thearrhythmiascoreclassifiestheinducedventricular
arrhythmias.AfterPAD,ventriculararrhythmiastendedtobelongerand
d
** * moreseverecomparedwithcontrols(denotedby#;n¼10;P¼0.003;
Arrhythmia score (n) 864200000 ** Arrhythmia score (n) 864200000 # * whtug(hnaenhena¼pilrgaaet5lirsiror;i,ethnPdgywia¼cttanh-(tsg0menl.snii0¼oato)1nt05,siccw;a;ofPM(hfrnei¼ecac¼.hnt0ben5i.-ds0–)BWr0bloeo2yflrch;ekibmtuacn-ntudbeepselydocac(aitbrnkereyais¼nddthe)e5itrg-ig;tnohiePccresr¼rcteb)shal0ocosooc.el0rikrnde1mae1dt;vruhegaMseilcc(uanaaesnrrs¼tirn.nihm–Ie5ynWr)utghcldhaiomcitindtiibontarlnneoosy.oclctIkhtoneaaersdPleattA,eer)trDs,
Control Ganglionicblockade uscarinergicblockade β-Blockade Cholinergicstimulation PAD Ganglionicblockade uscarinergicblockade β-Blockade Cholinergicstimulation oianurdtciochnaootliminngeicrtgthoiacntsePtiiAmmDuplaoacrttiospnhaar(rrnhmy¼athc5om;lPoiag¼iscu0as.l0cme3p6otd;ibuuilnlaitptyiao.inrAeldolfttht-ehteevsatc)laurredesidasuchcoewdnit,are
M M mean±s.e.m.*Po0.05,**Po0.01.
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the atrial level impacts ventricular electrophysiology in humans, heterogeneity increased after catheter ablation. In one of these
we studied 111 consecutive patients (60±1 years, 60% male) patients, a non-sustained ventricular tachycardia (VT) was
undergoing catheter ablation of paroxysmal AF (Supplementary detected during follow-up. In age- and gender-matched controls
Table7).Duringablationatanatomicalsitesofcardiacgangliain (n¼6) without a significant increase in PVCs per hour after AF
the left atrium (Ligament of Marshall region, n¼5; left superior ablation (1.4±1.3PVCh(cid:2)1 versus 2.4±2.0PVCh(cid:2)1, P¼0.5,
ganglionated plexus region, n¼1; Fig. 7a) premature ventricular Wilcoxon signed-rank test), the ventricular repolarization
complexes (PVCs) occurred in six patients with concomitant heterogeneity did not change (QT dispersion: 22±3ms versus
vagal responses in five patients. 24±3ms, P¼0.222, paired t-test).
In20%ofallpatients,atrialarrhythmiasweredetectedduring
follow-up after a blanking period of 3 months, irrespective AFablationreducesparasympatheticandsympatheticactivity.
of PVC incidence. During follow-up, in six patients a PVC The mean heart rate increased from 61±1b.p.m. before to
increase was documented irrespective of AF recurrence (before 69±1b.p.m. (Po0.0001; paired t-test) 48h after AF ablation
catheter ablation: 7±3.3PVCh(cid:2)1; after catheter ablation: in all patients with sinus rhythm, indicating a reduction of
144±70PVCh(cid:2)1, P¼0.031, Wilcoxon signed-rank test; cholinergic control. Heart rate variability (HRV) and HRV
Fig. 7c). Meanwhile, QT dispersion (28±5ms versus 40±6ms, measurement during deep breathing (HRV-DB) indicated a
P¼0.011, paired t-test; Fig. 7d) as a marker of repolarization reduced tonic and phasic parasympathetic activity after catheter
ablation of AF (Supplementary Table 8).
In patients with an increase in PVCs after AF ablation, the
a heart rate increased Z10b.p.m. in four out of six patients
(59±4.4b.p.m. versus 64±2.5b.p.m., P¼0.34, paired t-test)
suggesting parasympathetic denervation, while it did not change
in controls (66±5.4b.p.m. versus 67±3.3b.p.m., P¼0.81,
Wilcoxon signed-rank test).
Cardiac I-123-meta-iodo-benzylguanidine (MIBG) single-
photon emission computed tomography (SPECT) imaging
YFP CFP demonstrated a heart-to-mediastinum ratio of 1.61±0.05 in
patients with new onset of PVCs (n¼4) and 1.56±0.12
b (P¼0.83; Mann–Whitney test) in patients without PVCs after
1.2 ISO Nicotine AF ablation (n¼4). Semiquantitative analysis of sympathetic
application
m.) denervation revealed a summed defect score of 3.0±1.8 with
nor regional defects of sympathetic innervation at the inferior and
YFP ( 1.1 inferolateral left ventricular wall in patients with PVCs after AF
P/ ablation (Fig. 7b). Sympathetic innervation was less impaired in
Ratio CF VAetrniutrmicle p0.a5t±ien0t.s5,wPith¼o0u.t43P;VMCsananft–erWAhFitnaebylattieosnt)(sinudmicmateidngdeafecrtelsactioorne
1.0
0 100 200 300 Time (s) Figure3|AtrialcholinergicmodulationaltersventricularcAMPlevels.
(a)Cyclicadenosinemonophosphate(cAMP)biosensorfluorescencein
c yellow(YFP)andcyan(CFP)channelsduringFRETmeasurementsina
Atrium c Ventricle c Langendorff-perfusedheart.Thered/bluesquaresattheventricle/atrium
Control PAD GanglionicblockadeMuscarinergiblockade Control PAD GanglionicblockadeMuscarinergiblockade idCtnaeFdrpPgici/ecaYttteiFendPgtahrttaeirmtiiaaoelr)-earadpesusporloiilcnfvagemtdipoeecnarAsfouuMfrseniPomicndoeywntnitnia.tehm(b(iisc6)os.R2p(erpop(cid:3)rtreee1s0rseee(cid:2)nnnto6tealdMt(iIvaS)es.Ocen,Axo1pMr(cid:3)merP1ai0mlliez(cid:2)even7edtlMsi)natnhde
alSO) 0 0 leftatriumandventricleweredecreasedbynicotine.(c)Relativechangesin
atri% I cAMPlevels(%ofISOresponse)inatriaandventriclesduringlocalatrial
FRET response to nicotine stimulation ( ––2400 ** * ––2400 * * * aig(nnapaitctnperoglortivclipineaoinentnietoiaic,onp1(npho(cid:3)l(eficn1xna0¼aitcmi(cid:2)oo8en5t)it,nhMraeeofd;tnieunniurc¼cmepoda5,nr5)rtteri(cid:3)aaolarlle1tahi0vtdere(cid:2)ieaapr4altitdscMrteiwaen;ldinetc.rh¼AvIonaM5uttt)ihoPopenlrhe(aamvPtrerAmuilussDamci;cano,nrlitc¼onaogern5girctg)eraitoaclelndsbdlaoadntcurdkiraailndge
PADhearts.Thiseffectwasreducedafterganglionicblockade(n¼5;
d P¼0.009;unpairedt-test)ormuscarinergicblockade(n¼5;P¼0.011;
Atrium c Ventricle c Mann–Whitneytest).Intheventricle,relativechangesincAMPlevels
ergi ergi duringtargetedatrialapplicationofnicotinewasobviousincontrols(n¼8)
ontrol uscarinockade ontrol uscarinockade bduutrinagbogliasnhgeldioninicP(AnD¼h5e;aPrt¼s0(n.0¼235;;uPn¼pa0i.r0e1d8t;-utenspta)iroerdmt-utsecsat)rinaenrdgic
7) C Mbl C Mbl blockade(n¼5;P¼0.036;unpairedt-test),indicatinganinhibitionof
alH47 0 0 parasympatheticactivity.(d)Afterstimulationwiththewater-soluble
atriNK forskolinanalogueNKH477(1(cid:3)10(cid:2)5M;n¼5),relativechangesincAMP
o %
FRET response totine stimulation ( ––2400 * ––2400 * Nlcb(ePolKvon¼ecHtlkrs40oa7(.dl0%7eh1e9saoa;tpfripmptNsaliuiKerwledHaedt4eirne7td-7ttrhheereesceatos)srrptadasomen.nddeAselfhvlo)eetrdnhatutrehrtriseivcnalraegelutdlr(eoiuPuscc¼maeslhd0aao.tnc0wrAdi3anM9lvaae;Prpnpeptalrelmiiirvcceeleaedlats.inotMi-±nnteuotsssh.ftcee)na.mioraciftn.oretiuirngmeicin
nic *Po0.05,**Po0.01.
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a RA b RA c RA
g
CS
LAA LAA
CS CS
LAA
NF TH ChAT
d * * e * * f * *
* * *
* * *
CS CS CS
TH ChAT Merge
g * h i l
*
NF ChAT
j k
CS
TH Hoechst Merge
Figure4|Interconnectivityoftheatrialandventricularintracardiacneuralnetwork.(a–c)Representativewhole-mountstainingsagainstneurofilament
(NF;a),tyrosinehydroxylase(TH;b)andcholineacetyltransferase(ChAT;c).Fibrestraversedfromtheposteriorleftatriumalongthepulmonaryveinsand
thecoronarysinus(CS)totheapexoftheventricle.ArrowheadsmarkthecourseoftwoChAT-positivefibres.CS,coronarysinus;LAA,leftatrial
appendage;RA,rightatrium.(d–f)ConfocalimagingofTH(d)andChAT(e)whole-mountco-stainingrevealedinterwovenparasympatheticand
sympatheticfibresoriginatingfromtheparasympatheticganglia(markedwithasterisks).(g)MagnificationfrombdepictingTH-positivefibresoriginating
fromatrialganglia(markedwithasterisks)withadensenetworkentwiningtheCS.(h–l)Immunohistochemicalstainingofatrialgangliaillustratedthe
predominanceofChAT-positivecells.Scalebars,1mm(a–c),200mm(d–g),25mm(h–l).
a b c f g h Brain Ventricle
cs TH 70
GAPDH 35
NF TH VACht 70
GAPDH 35
d e
70
f ChAT 55
35
NF ChAT Merge Merge eGFP GAPDH 35
(kDa)
Figure5|Sympatheticandparasympatheticventricularinnervation.(a)Neurofilament(NF)stainingdepictingtherichinnervationoftheventricle.
Thesquaremarksthelocationofthefibresshowninb–f.(b–f)ConfocalimagingofventricularNF(b),tyrosinehydroxylase(TH;c)andcholine
acetyltransferase(ChAT;d).ThewhitearrowheadsdisplayaTH-butnotChAT-positivefibre.Thegreatermagnification(f)emphasizesthetight
entanglementoffibres,withthemajoritybeingTH-positiveandasmalleramountofparallelrunningChAT-positivefibres.ArrowsdepictChAT-positive
fibres.(g)Nativeenhancedgreenfluorescentprotein(eGFP)fluorescenceofChATBAC-eGFPmicedemonstratedadelicateinterconnectivitybetween
parasympatheticventricularfibresandcardiacmyocytes.(h)Westernblotanalysisofventriculartissue,usingantibodiesagainstTH,ChATandVAChT
revealedbandsatthesamemolecularweightasinthebrain(positivecontrol),indicatingthepresenceofacholinergicsystemintheventricles.Scalebars
1mm(a),100mm(b–e),10mm(f),25mm(g).
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a b
Control PAD
30
n)
PV glia ( 20
n
F a
g
PV # # PV ber of 10
F m
u
N
0
Control PAD
c d
Rbfox3 Chat Th Nppa
PV one) 3 * 100 ****
CS NA expressi1b and fat tissu 2 7550
e mRo Cdkn 1 25
Relativ(x-fold t 0 Fat Atria Fat Atria Fat Atria 0 Fat Atria
Figure6|QuantificationofcardiacgangliaafterPAD.(a)Haematoxylinandeosin(H&E)-stainedsectionsofcontrol(leftimage)andpartialatrial
denervated(PAD)hearts(rightimage)aredepicted.Cholinergicgangliaaremarkedwitharrows.InPADhearts,agreatamountofepicardialfatandthe
ganglialocatedwithinhavebeenremoved.F,fat;PV,pulmonaryvein;#,posterioratrialwall.Scalebar,300mm.(b)Thetotalnumberofgangliacountedin
theH&E-stainedsectionsisreducedby61%inPADheartscomparedwithcontrols(n¼3;P¼0.100;Mann–Whitneytest).(c)Whole-mountstaining
againstcholineacetyltransferase(ChAT)depictingdisruptedcholinergicnervefibres(arrows)afterPAD.CS,coronarysinus.Scalebar,500mm.(d)Gene
expressionanalysisofatrialmyocardialtissueandepicardialfatviaqRT–PCR(n¼7)revealedsignificantdifferencesinneuronalmRNAlevelsbetweenthe
epicardialfatandcardiacatria.ComparedwithatrialtissueRbfox3mRNA(P¼0.011;pairedt-test),ChatmRNA(P¼0.063;Wilcoxonsigned-ranktest)and
ThmRNA(P¼0.078;Wilcoxonsigned-ranktest)seemedtobeslightlymoreprominentintheepicardialfat.AtrialNppaexpression(representingatrial
natriureticpeptide)confirmscorrectfatpreparationasitisonlyexpressedwithintheatria(n¼7;Po0.0001;pairedt-test).Allthevaluesshownare
mean±s.e.m.*Po0.05,****Po0.0001.
between the modulation of atrial cholinergic neurons, concomi- reduce AF recurrence. Importantly, PVCs and VTs have been
tant sympathetic denervation and the occurrence of ventricular reported following AF catheter ablation in patients without any
arrhythmias. evidence of structural heart disease or inherited ion channel
diseases23–25. Our results support reports having speculated that
these arrhythmias might reflect autonomic influences26.
Discussion Atrial and ventricular neural structures are tightly
The major findings of the present study are as follows: interconnected27,28. In addition, some studies support textbook
(i) morphological and functional evidence support that cardiac knowledge stating that beyond sensory afferents, ventricular
cholinergic neurons regulate ventricular electrophysiology; electrophysiology is primarily controlled by postganglionic
(ii) modulation of the atrial neural network changes left sympathetic efferent axons6,29. Although controversial,
ventricularcAMPlevels;(iii)accidentalpartialatrialdenervation ventricular innervation by parasympathetic fibres7,8 and
during catheter ablation of AF is characterized by both reduced changes in ventricular repolarization following cervical vagal
parasympathetic activity and disturbed sympathetic ventricular nerve stimulation have been described11,14,30. This influence of
control. This can translate into increased susceptibility to extracardiacvagalstimulationonventricularelectrophysiologyis
ventricular arrhythmia after catheter ablation of AF. relayed via different intracardiac ganglia15,30 but it is not fully
It is well known that catheter ablation of AF inevitably understood whether this effect is mediated via central or
modulates the intracardiac neural network as supported by peripheral neural pathways. Here, we present morphological
morphologicalandfunctionalstudies2.Themostobviousclinical and functional evidence demonstrating that cardiac cholinergic
parameter is an increased heart rate, which is often used as a neurons modulate ventricular electrophysiology.
surrogate parameter for withdrawal of sinus nodal or global Stimulation of post-ganglionic neurons decreased ISO- and
cardiac parasympathetic activity following catheter ablation of Forskolin-stimulated cAMP levels in the ventricle, which was
AF19,20. Our indirect measurements of neural cardiac control blocked by PAD, the ganglion nicotinic antagonist hexametho-
including heart rate, HRV at rest and HRV-DB, support a nium, or the muscarinic antagonist atropine, indicating that
decrease in both tonic and phasic parasympathetic activity after cholinergic neurons modulate ventricular function. This is also
catheter ablation of AF in our patient cohort. The former is in supported by PAD-induced shortening of the VRP and immuno-
linewithseveralpreviousreportshavingfoundanimpairmentof histochemical demonstration of intertwined TH- and ChAT-
HRV following AF ablation21. Although still controversial, positivefibresintheventricle.However,oneshouldkeepinmind
observational studies22 and one randomized controlled trial5 that the behaviour of these neurons may be completely different
support that atrial denervation (accidentally or targeted) can from in vivo conditions, as central inputshavebeendisrupted.
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It also needs to be taken into account, that isolated myocytes31,32, it seems unlikely that non-neuronal acetylcholine
cardiomyocytes themselves have been shown to synthesize explains the functional effects observed in our study.
acetylcholine31,32. Although it is not clear whether and how Itiswellknown,thatbesidesparasympatheticalsosympathetic
this non-neuronal acetylcholine acutely modulates ventricular activity can influence ventricular electrophysiology. Our experi-
electrophysiology,itmighthaveinfluenced ourresultsatleastin mental and clinical data suggest that both the reduction of
part. However, since acetylcholine was only detectable after parasympatheticactivityaswellastheconcomitantheterogeneity
the addition of acetylcholine esterase inhibitors in isolated inventricularsympatheticpredominanceareaccountableforthe
a
LSPV
LAA RSPV LSPV
LAA
LIPV LIPV
Left Left
atrium atrium
Ablation site
LoM
CS ablation site CS
II 1,000 ms
LSPV LSPV
RSPV
RSPV
LIPV LAA
LIPV
Left RIPV RIPV Ablation site
atrium Left
atrium
CS GP
CS
ablation site
II 1,000 ms
b
3D geometry 3D geometry 17-Segmental
(anterior) (posterior) Polar plot analysis
Anterior
Anterior
Anterior 0
Basis Basis
0
PVCs SIenpfetarilor LateralApex Apex Lateral Inferior Septal Lateral 00 00 0 0000 00..320.±31.±00±.20.51.0±0.5
0.5±0.3
0% 100% 0% 100% Inferior
Anterior Anterior Anterior
0
No PVCs Basis SeIpntfaelriorLateral Apex Apex Lateral Basis Septal Lateral 00 00 0 00000 0000.03±0.2
Inferior 0.3±0.2
0% 100% 0% 100% Inferior
c d
30 300 * 60 *
Before
ms) ablation
–1h) 20 200 on ( 40
PVC ( 10 100 spersi 20 Aabftleartion
di
T
Q
0 0 0
No PVCs PVCs PVCs
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increaseinarrhythmiasusceptibility.Althoughtheimportanceof sinusnoderecoverytime(maximumreturncyclelengthafter10sfixed-rate
sole decreased parasympathetic activity in structurally healthy pacing),Wenckebachperiodicity(longestcyclelengthwithlossof1:1
hearts is less well defined, it is widely accepted that disturbed atrioventricular-nodalconduction),atrioventricular-nodalrefractoryperiod
sympathetic/parasympatheticinteractionsareproarrhythmic33,34. (longestextrastimuluscyclelengthwithlossofatrioventricular-nodalconduction),
atrialandventricularrefractoryperiods(longestextrastimuluscyclelengthwith
It has been described that muscarinic receptors on adrenergic absentatrialorventricularresponse)weredeterminedbyusingfixed-rateor
nerve terminals attenuate norepinephrine release35, leading to extrastimuluspacing.InexvivoLangendorffstudies,theepicardialatrialfatpads,
parasympathetically mediated inhibitory effects on cardiac inwhichatrialgangliaaremainlylocated,wereeitherremovedbycareful
dissection40(PAD)orleftintact(control).Forelectrophysiologicalstudiesina
sympathetic activity. This indirect parasympathetic ventricular
Langendorffsetup,heartsweresubmergedinice-coldmodifiedKrebs-Henseleit
control by opposing the effects of heightened adrenergic tone solution(mM:NaCl119,NaHCO 25,KCL4.6,KHPO 1.2,MgSO 1.1,CaCl
3 2 4 4 2
might influence ventricular electrophysiology in Langendorff- 2.5,glucose8.3andNa-pyruvate2;pH7.4,95%O/5%CO)anddissected
2 2
perfused hearts. However, b-blockade36 did not significantly carefully.Theaortawascannulated,quicklyattachedtotheperfusionsystem
(HugoSachsElektronik/HarvardAparatus,Germany)andretrogradelyperfusedat
influencearrhythmogenesisinPADheartsinourstudies.PADas
constantpressure(80mmHg;ref.41).Theheartwassurroundedbyafullyclosed
well as ganglionic or muscarinergic blockade led to an increased chamber,ensuringaprecisetemperaturecontrol.A2Foctapolarelectrophysiology
susceptibility to ventricular arrhythmias. This indicates that catheterwith0.5mmelectrodespacing(CIB’ERMouse,NuMedInc.,Hopkinton,
intracardiac neurons continue to fire action potentials in an NY,USA)wasinsertedintotherightatriumandrightventricleforrecordingof
ex vivo setup and are able to communicate with each other36,37. intracardiacelectrogramsviaanECGamplifier(F104,ADInstruments).Asecond
2Foctapolarcatheterlocatedepicardiallyattheleftventriclestimulatedtheheart
In addition, FRET imaging confirmed the influence of atrial
withacyclelengthof100msfortheinitial20minequilibrationperiod.Heartsthat
neurons via a reduction of ventricular cAMP levels during local didnotregainapinkcolourationoraspontaneousheartbeatafterequilibration
application of nicotine, although these experiments were wereexcluded.Perfusionpressure,aorticflowandheartratewerecontinuously
performedunderconditionsofsympatheticstimulationordirect recordedusingadigitaldataacquisitionsystemandcorrespondingsoftware
(Powerlab8/30&Labchart,ADInstruments).Programmedstimulationwas
cAMP activation. This influence on ventricular control was
appliedviathedistalorproximalelectrodesofthecatheterusingadesignated
abolished by muscarinergic or ganglionic blockade, while digitalstimulusgenerator(STG4002,MultiChannelSystems,Reutlingen,
cholinergicstimulationofPADheartspreventedVTinducibility. Germany)attwicetheatrialorventricularpacingthresholdtodeterminestandard
However,althoughsomemajorpatternsaresimilarinmouseand electrophysiologicalparameters42.InlinewiththeLambethConventions43,
programmedextrastimulationtechniques(baselinecyclelengths100msand
man,extrapolatingmurinedatatohumanstudiesmaybedifficult
120ms)withuptothreeextrastimuliandburstpacingwitha2msor10ms
as cardiac neuroanatomy is known to exhibit interspecies stepwisereductionwereusedtodetectarrhythmiasusceptibility44–46.VTwas
variability38. definedasZ4consecutiveprematureventricularcomplexes43.Ventricular
In conclusion, modulation of atrial cholinergic neurons arrhythmiashavebeenclassifiedbyanestablishedscoringsystem47.
InadditiontoPAD(n¼10)andcontrolhearts(n¼10),furtherexperiments
can acutely influence ventricular electrophysiology. Reduced
(eachn¼5)wereperformedwiththefollowingdrugsaddedtotheperfusion
parasympathetic activity and concomitant spatial heterogeneity
solutionbeforestartingtheexperiment:hexamethonium(ganglionicblockade,
of dominant sympathetic activity can translate into increased 5(cid:3)10(cid:2)4M,Sigma-Aldrich,St.Louis,MO,USA;n¼5),atropine(muscarinergic
ventricular arrhythmogenesis in some patients after catheter blockade,1(cid:3)10(cid:2)6M,Sigma-Aldrich;n¼5),propranolol48(b-receptorblockade,
1(cid:3)10(cid:2)6M,mibeGmbH,Sandersdorf-Brehna,Germany;n¼5),and
ablation of AF.
acetylcholine(cholinergicstimulation,1(cid:3)10(cid:2)5M,Sigma-Aldrich;n¼5).
Pharmacologicalinterventionswereperformedinadditionalexperimentsandonly
Methods onedrugwastestedperheart(controlorPAD)tokeepexaminationtimesimilar
Animals.ThestudyprotocolwasapprovedbythelocalauthoritiesoftheStateof andtoguaranteecomparabilityinallperformedexperiments49.
Hamburg,theUniversityofHamburgAnimalCareandUseCommitteesand
conformstotheGuidefortheCareandUseofLaboratoryAnimals,EighthEdition,
NationalAcademyPress,updatedbytheUSNationalResearchCouncilCommittee EpicardialmappingofLangendorff-perfusedhearts. Myocardialwavepropa-
in2011. gationcharacteristicsweredeterminedbyepicardialactivationmappingwithtwo
32-electrodearrays(EcoFlexMEA36,MultiChannelSystems,Reutlingen,Ger-
many;inter-electrodedistance:300mm;1.8(cid:3)1.8mm)positionedatbothventricles
Electrophysiologicalstudies.Weexaminedmalewild-typeC57/BL6mice(10to duringepicardialpacing(cyclelength100ms)andinducedventriculararrhyth-
16weeksofage;StocknumberJacksonLaboratories:000664)electrophysiologically mias.Unipolarelectrogramswererecorded(ME128-FAI-MPA-System,Multi
byasinglecathetertechnique39.Toobtainbaselinecardiacelectrophysiological, ChannelSystems,Reutlingen,Germany)withasamplingrateof25kHz.Datawere
electrocardiographic(ECG)andechocardiographicparameters,invivostudies bandpassfiltered(50Hz),digitizedwith12bitandasignalrangeof20mV.Wave
(n¼10)wereperformed(SupplementaryTables1–4)39.Themicewere propagationvelocityanddispersioninconductiondirectionweredetermined50.
anaesthetizedwith2.4Vol.%isofluraneandpositionedonatemperature-
controlledplatformtomaintainbodycoretemperatureat36(cid:2)C.Surfaceresting
ECGwasobtainedandthenanalysedofflinebyusingPowerlab8/30& FRET-basedcAMPimaginginwholeheartsandcardiomyocytes. Fo¨rster
Labchart(ADInstruments,Dunedin,NewZealand).Forechocardiography,the resonanceenergytransfer(FRET)-basedmeasurementswereperformedinLan-
parasternallong-axisview,shortaxisviewandtwo-dimensional-guidedM-mode gendorff-perfusedheartsharvestedfromCAG-Epac1-campstransgenicmice51.
imageswereobtainedatthelevelofthepapillarymusclesforthemeasurementof Aftermeasurementofbaselineactivity,theheartswereperfusedwithisoproterenol
wallthicknessandchamberdimensions.Electrophysiologicalparameterslikethe (ISO,1(cid:3)10(cid:2)7M)ortheNKH477(1(cid:3)10(cid:2)5M,bothfromSigma-Aldrich)and
Figure7|Atrialneuroablationaffectsventricularelectrophysiology.(a)Examplesofthree-dimensional(3D)guidedablationattheendocardialligament
ofMarshallregion(LoM;upperpanel)andleftsuperiorganglionatedplexi(GP)region(lowerpanel)inducesprematureventricularcomplexes(PVCs)
duringcatheterablationofAF.Reddots(VisiTagpoints)illustratethesitesofradiofrequencyablation.Leftlateral(left)andanterolateral(right)viewsare
shown.NotethePVCsoccurringduringablationdepictedintheECG.Acircularmappingcatheterislocatedwithintheleftsuperiorpulmonaryvein(LSPV;
lowerpanel).Thefluoroscopyimageattherightshowsthepositionoftheablationcatheterandthecircularmappingcatheterpositionedwithintheleft
inferiorpulmonaryvein(LIPV;upperpanel)demonstratingthecloseproximitybetweenthisvein,thecoronarysinusandtheablationsite.CS,coronary
sinus;LA,leftatrium;LAA,leftatrialappendage;rightinferiorpulmonaryvein,RIPV;rightsuperiorpulmonaryvein,RSPV.Scalebar,each1,000ms.
(b)I-123-MIBGSPECTimagingrevealedareducedsympatheticinnervationattheinferiorandinferolateralpartsoftheleftventricleaftercatheterablation
ofAFinpatientswithanincreasedPVCburdenorsymptomaticpalpitationsafterAFablation(n¼4).InthosewithoutanincreaseinPVCsafterAF
ablation(n¼4),regionalleftventricularsympatheticinnervationwaslessimpaired.Therightpaneldisplaysmeansummeddefectscorevalues,whereas
redpartsdepictareasofreduceduptake(meansummeddefectscoreforonesegmentZ0.5).(c)ThemajorityofpatientshadnoincreaseofPVCsper
hour(NoPVCs,n¼105).Asubsetofpatients(PVCs,n¼6;P¼0.031;Wilcoxonsigned-ranktest)reportedpalpitationsorshortnessofbreathafter
catheterablationofAFconcomitantwithanincreaseinPVCsperhour,irrespectiveofAFrecurrence.(d)QTdispersionaftercatheterablationwas
increasedinpatientswithanelevatedPVCburden(n¼6;P¼0.011;pairedt-test).Allthevaluesshownaremean±s.e.m.*Po0.05.
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subsequentlystimulatedwithnicotine(40mlof6.2(cid:3)10(cid:2)6M,Sigma-Aldrich), anddonkeyachickenIgYAlexa647(1:500;ThermoFisherScientific;seealso
whichwaspipettedontheupperpartoftheleftatrium52.Theseexperimentswere SupplementaryTables5-6)in1%BSA/TBS.Incubationwasperformedovernight
performedincontrol(n¼8)orPADhearts(n¼5)andwithperfusionofatropine at4(cid:2)C(primaryantibody)or2hatroomtemperature(secondaryantibodies)
(muscarinergicblockade,1(cid:3)10(cid:2)6M,Sigma-Aldrich;n¼5)orhexamethonium with3(cid:3)5minwashesTBSinbetween.1mgml(cid:2)1bisBenzimideH33342
(ganglionicblockade,5(cid:3)10(cid:2)4M,Sigma-Aldrich;n¼5).Inheartsthatwere trihydrochloride(Hoechst,#B2261,Sigma-Aldrich)wasaddedtothesecondary
prestimulatedwithNKH477,atropine(muscarinergicblockade,1(cid:3)10(cid:2)6M, antibodysolution.TheslidesweremountedinVectashieldHardSetmounting
Sigma-Aldrich)wasaddeddirectlytotheperfusionbufferbeforeperformingan medium(#VEC-H-1400,VectorLaboratories).
additionalnicotinestimulation.ForsinglecellFRETmeasurements,adultmurine Forquantificationofcardiacganglionremoval,controlandPADheartswere
cardiomyocyteswerefreshlyisolatedviaretrogradecardiacperfusionwithan cutin5mmthicksections,haematoxylinandeosinstained,partitionedinfour
enzymesolutioncontaining1.25mgliberaseDH(RocheDiagnosticsDeutschland regionsandthenumberofgangliawascountedmicroscopicallyinastandardized
GmbH,Mannheim,Germany)and300mltrypsin(2.5%,ThermoFisherScientific, waybytwoindependentscientists(K.S.andN.B.)59.
Waltham,MA,USA).Afterdigestion,thecalciumlevelwasadaptedandthecells
wereplatedontoglass-bottomed,laminin(Sigma-Aldrich)coateddishes53.
Microscopy. Confocalimagesoffluorescentwholemountandparaffinsection
TomonitorFRET,weusedself-builtimagingsystemsaroundLeicaM165FC
stainingsweretakenwitheitheraLeicaTCSSP5(LeicaMicrosystemsGmbH)
(LeicaMicrosystemsGmbH,Wetzlar,Germany)stereomicroscope(for
using (cid:3)10NA¼0.3HCPLFluotar, (cid:3)20NA¼0.7,HCPLApoCSImm/Corroil
Langendorffexperiments)orLeicaDMI3000B(LeicaMicrosystemsGmbH)
and (cid:3)40NA¼1.3HCXPLAPOCSobjectivesora2-photonBX61WIupright
invertedfluorescencemicroscope(forsingle-cellrecordings).cAMPsensorwas
microscope(OlympusCooperation,Tokyo,Japan)usingaplan-Neofluar (cid:3)5
excitedwith440nmLED(pE-100,CoolLED,Andover,UK).Emissionlightwas
NA¼0.15objective.Three-dimensionalmulticolourimageswerecollectedover
splitintodonorandacceptorchannelsusingtheDV2DualViewequippedwiththe
thefullrangeofthesignalwithsequentialchannelacquisitionofeachstackfor
565dcxrdichroicmirrorandD480/30andD535/40emissionfilters(Photometrics,
wholemounts.AmaximumprojectionimagewascreatedusingtheLeicaLASAF
Tucson,AZ,USA).ImagesweretakenusingoptiMOScamera(Photometrics,
software.Imagesofchromogenicstainingsandoverviewofwholefluorescent
Tucson,AZ,USA)withMicroManager1.4opensourceimagingsoftwareand
heartswerephotographedusinganOlympusSZX16widezoomstereomicroscope
analysedbyImageJ(NIH,USA).Rawdatawerecorrectedofflineforthe
bleedthroughfactorofthedonorintotheacceptorchannel54. (OlympusCooperation).Threedifferentfocalplaneswerecollectedforchromo-
genicstainingsandstackedusingtheHeliconFocusSoftware(HeliconSoftLtd.).If
necessary,brightnessandcontrastwasadaptedusingGNUImagemanipulation
Histologyandimmunohistochemistry. Whole-mountimmunostainingswere programme(GIMP).Forquantificationofgangliainwhole-mountChAT-stained
performedinintactmurineheartstoexaminetheinterconnectivityofatrial hearts,gangliawerecountedmanuallyontheSZX16microscope.
andventricularneuralstructureswithintheintracardiacneuralnetworkand
cholinergicventricularinnervation55.Neurofilamentstaining(chickenantiNF-H, Immunoblotting. Expressionofsympatheticandparasympatheticmarkerswas
1:3,000;EMDMillipore)wasusedtocharacterizetheintracardiacneuralnetwork
analysedincardiacventriculartissueviaimmunoblotting.Braintissueservedas
traversingalongthepulmonaryveins,theposteriorleftatriumandbothventricles.
positivecontrol.TissueswerelysedinRIPAbuffer(50mMTrisbasepH8.0,
WeusedfluorescentandchromogeniclabellingforTHandChAT(goataChAT,
150mMsodiumchloride,0.5%sodiumdeoxycholate,0.1%sodiumdodecylsulfate
1:50;EMDMillipore;rabbitaTH,1:1,000;EMDMillipore)tocharacterize
(SDS),1%TritonX-100,1mMdithiothreitol,plusproteaseinhibitors(Complete
adrenergicandcholinergicstructures35,55,56.MaleChATBAC-eGFPtransgenicmice
Mini,RocheDiagnosticsDeutschlandGmbH)andphosphataseinhibitors(phos-
werepurchasedfromJacksonLaboratories(BarHarbor,ME,USA;stocknumber
STOP,RocheDiagnosticsDeutschlandGmbH)usingaglass-teflonpotterand
007902;ref.57).ForimagingofnativeeGFPfluorescence,heartswereextracted centrifugedfor30minat15.000g,4(cid:2)C(ref.60).Proteinconcentrationsinthe
andperfusedwithice-coldPBS.
homogenateswerequantifiedusingthePierceBCAProteinAssayKit(Thermo
FisherScientific)accordingtothemanufacturer’sinstructions.Theseparationof
Immunohistochemistry—whole-mountstaining. AfterLangendorffperfusion, 25mgtotalproteinwasperformedunderdenaturingconditionsbysodiumdodecyl
sulfate-polyacrylamidegels(SDS–PAGE)usingprecastgels(4–20%,Bio-Rad,
mouseheartswerefixedinformalin(Sigma-Aldrich)for24hat4(cid:2)Candstoredin
Hercules,CA,USA)andtransferredtonitrocellulosemembranesviawetblotting.
phosphate-bufferedsaline(PBS,BiochromGmbH,Germany)untilthestaining
Themembraneswereblockedin5%skimmilkpowderinTBS.Forgoat-derived
wasperformed.HeartswerebleachedinDent’sbleach(4:1:1MeOH:HO:DMSO,
2 2 antibodies,blockingwasperformedin5%BSA/TBS(receptorgrade,Serva,
MerckKGA,Darmstadt,Germany)for1weekat4(cid:2)Candsubsequentlyrehydrated
toPBSinaseriesofdescendingMeOHinPBS(100,75,50,25%,1heach)58.The Heidelberg,Germany).Themembraneswereincubatedovernightat4(cid:2)Cwith
primaryantibodiesasfollows:goataChAT(1:500,EMDMillipore),rabbitaTH
followingincubationswereperformedin24-well-plateformatwithgentleagitation
(1:1,000;EMDMillipore),goataVAChT(1:500,EMDMillipore);seealso
at4(cid:2)C.Theheartswerepermeabilizedin1%TritonX-100/PBS(PBS-T,Sigma-
SupplementaryTable5.AfterthreewashingstepswithTBSþ0.5%Tween,sec-
Aldrich)for3(cid:3)1hatroomtemperaturebeforeblockingovernightinblocking
ondaryantibodyincubationfollowedforonehouratroomtemperature:POX-
buffer(5%BSA/PBS-T(Biomol,Hamburg,Germany)þ0.2%sodiumazide).
Antibodiesweredilutedasfollows:goataChAT(1:50;EMDMillipore),rabbita conjugatedhorseagoatigG(1:10,000;VectorLaboratories)andPOX-conjugated
TH(1:1,000;EMDMillipore),chickenaneurofilament(1:3,000;EMDMillipore); goatarabbitigG(1:10,000;VectorLaboratories).IncubationwithHRP-conjugated
forfluorescentlabelling:donkeyarabbitIgGAlexa488(1:500;ThermoFisher rabbitaGAPDH14C10(1:2,000;CellSignaling)for1hwasperformedtodetect
Scientific),donkeyagoatIgGAlexa568(all1:500;ThermoFisherScientific)and GAPDHasaloadingcontrol.TheFusionSoloSgeldocumentationsystem(VRW
donkeyachickenIgYAlexa647(all1:500;ThermoFisherScientific);for International,Radnor,PA,USA)wasusedtodetectreactiveproteinbandswith
chromogeniclabelling:biotin-conjugateddonkeyarabbitigG(1:200;EMD enhancedchemiluminescence.
Millipore),biotin-conjugateddonkeyagoatigG(1:200;R&DSystems),
biotin-conjugatedgoatachickenigY(1:200;R&DSystems);seealso RNAisolationandqRT–PCR. RNAwasisolatedusingTrizol(ThermoFisher
SupplementaryTables5and6. Scientific)andfurtherpurifiedusingtheRNeasyMinikit(Qiagen,Hilden,
Specimenswereincubatedinprimaryantibodiesdilutedinblockingbufferfor Germany)accordingtothemanufacturer’sinstructions.TotalRNAwasreverse
1week.Afterwards,theheartswerewashed3(cid:3)15mininPBS-Tbeforesecondary transcribedusingtheHigh-CapacitycDNAReverseTranscriptionKit(Thermo
antibodyincubationinblockingbufferfor4days.Subsequently,thehearts FisherScientific).qRT-PCRwasperformedona7900TaqMansystem(Applied
werewashed3(cid:3)15mininPBS-TandstoredinVectashieldmountingmedium Biosystems,FosterCity,CA,USA)usingTaqMangeneexpressionmastermixand
(#H-1000,VectorLaboratories,Burlingame,CA,USA)forfluorescentstainingor geneexpressionassays(Rbfox3Mm01248771_m1;ChatMm01221880_m1;Th
incubatedinVectastainABCkit(#PK-4000,VectorLaboratories)accordingtothe Mm00447557_m1;ThermoFisherScientific).Nppa(mNPPA_Mm01255748_g1)
manufacturer’sinstructionsfor3hatroomtemperature.Subsequently,thehearts wasusedasacontrolforatrialtissuepreparation,10-40ngofcDNAwereusedas
werepre-incubated1hinSteadyDAB/Plus(#ab103723,Abcamplc,Cambridge, reactiontemplate;experimentswereperformedinduplicates.Cyclingconditions
UK)withoutDAB,beforedevelopingundervisualcontrolinSteadyDAB wereasfollows:50(cid:2)Cfor2min;95(cid:2)Cfor10min(onecycle),95(cid:2)Cfor15sand
accordingtothemanufacturer’sinstructions.Thespecimenswerestoredin 60(cid:2)Cfor1min(40cycles).Relativegeneexpressionwascalculatedusingthe
double-distilledH2O. 2(cid:2)DDCtmethod,normalizingtoCdkn1b(Mm00438167_g1)andfattissue61.
Cdkn1bisnotdifferentiallyexpressedinfatandatrialtissue.
Histology—paraffinsections.Mouseheartswerepreparedandfixedasdescribed
aboveandsubsequentlydehydratedandembeddedinparaffin.Fourmicrometre- Studyprotocol. Allpatientsgavewritteninformedconsent.Patientstudieswere
thicksectionswerecutanddeparaffinizedusingHistoClear(#HS2002,DiaTec, approvedbythelocalEthicsCommittee(No.:WF-01/16)andinaccordancewith
Bamberg,Germany)andethanol.Noantigenretrievalwasperformed.Theslides theDeclarationofHelsinki.Theclinicaltrialregistrationcanbefoundwiththe
werepermeabilizedfor10minin0.2%TritonX-100/Tris-bufferedsaline(TBS), followingURL(https://clinicaltrials.gov)undertheuniqueidentifier
followedby3(cid:3)5minwashesinTBS.Blockingwasperformedwith3%BSA/TBS NCT02699255.Theburdenofventriculararrhythmiaswasinvestigatedinan
for1hatroomtemperature.Antibodiesweredilutedasfollows:goataChAT observationalstudyincluding111consecutivepatientswithparoxysmalAF
(1:50;EMDMillipore),rabbitaTH(1:500;EMDMillipore),chickenaneurofi- undergoingcatheterablation.AllpatientsunderwentcatheterablationofAFusing
lament(1:1,000;EMDMillipore),donkeyarabbitIgGAlexa488(1:500;Thermo radiofrequencyorcryoablationbyanexperiencedelectrophysiologist(41,000left
FisherScientific),donkeyagoatIgGAlexa568(1:500;ThermoFisherScientific) atrialcatheterablationprocedureseach)andwerecontinuouslymonitoredfor48h
10 NATURECOMMUNICATIONS|8:14155|DOI:10.1038/ncomms14155|www.nature.com/naturecommunications