Increased atrial arrhythmia susceptibility induced by intense endurance exercise in mice requires

14 Pages·2015·2.13 MB·English

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Received4Sep2014|Accepted 2Dec 2014 |Published 19Jan 2015 DOI:10.1038/ncomms7018
Increased atrial arrhythmia susceptibility induced
by intense endurance exercise in mice requires
TNFa
Roozbeh Aschar-Sobbi1,2,*, Farzad Izaddoustdar1,2,*, Adam S. Korogyi1,2, Qiongling Wang3, Gerrie P. Farman4,
FengHua Yang1,2, Wallace Yang1,2, David Dorian1, Jeremy A. Simpson5, Jari M. Tuomi6,w, Douglas L. Jones6,
Kumaraswamy Nanthakumar2,7, Brian Cox1,8, Xander H.T. Wehrens3, Paul Dorian2,9 & Peter H. Backx1,2,7,10
Atrial fibrillation (AF) is the most common supraventricular arrhythmia that, for unknown
reasons,islinkedtointenseenduranceexercise.Ourstudiesrevealthat6weeksofswimming
or treadmill exercise improves heart pump function and reduces heart-rates. Exercise also
increasesvulnerabilitytoAFinassociationwithinflammation,fibrosis,increasedvagaltone,
slowed conduction velocity, prolonged cardiomyocyte action potentials and RyR2
phosphorylation(CamKII-dependentS2814)intheatria,withoutcorrespondingalterationsin
the ventricles. Microarray results suggest the involvement of the inflammatory cytokine,
TNFa,inexercised-inducedatrialremodelling.Accordingly,exerciseinducesTNFa-dependent
activationofbothNFkBandp38MAPK,whileTNFainhibition(withetanercept), TNFagene
ablation, or p38 inhibition, prevents atrial structural remodelling and AF vulnerability in
response to exercise, without affecting the beneficial physiological changes. Our results
identify TNFaas a key factor in the pathology of intense exercise-induced AF.
1DepartmentofPhysiology,UniversityofToronto,1King’sCollegeCir.,Toronto,Ontario,CanadaM5S1A8.2DepartmentofMedicine,UniversityofToronto,1
King’sCollegeCir.,Toronto,Ontario,CanadaM5S1A8.3CardiovascularResearchInstitute,DepartmentofMolecularPhysiologyandBiophysics,Department
ofMedicine,BaylorCollegeofMedicine,OneBaylorPlaza,Houston,Texas77030,USA.4DepartmentofPhysiologyandBiophysics,BostonUniversity,700
AlbanySt,Boston,Massachusetts02118-2526,USA.5DepartmentofHumanHealthandNutritionalSciences,UniversityofGuelph,50StoneRoadEast,
Guelph,Ontario,CanadaN1G2W1.6DepartmentofPhysiologyandPharmacology,WesternUniversity,MedicalScienceBuilding,London,Ontario,Canada
N6A5C1.7DivisionofCardiology, PeterMunkCardiacCentre,UniversityHealthNetwork,200ElizabethSt,Toronto,Ontario,CanadaM5G2C4.8Programin
DevelopmentalandStemCellBiology,HospitalforSickChildrenResearchInstitute,555UniversityAve,Toronto,Ontario,CanadaM5G1X9.9Divisionof
Cardiology,StMichael’sHospital,2300YongeSt,Toronto,Ontario,CanadaM4P1E4.10Heart&StrokeRichardLewarCentreofExcellence,Universityof
Toronto,1King’sCollegeCir.,Toronto,Ontario,CanadaM5S1A8.*Theseauthorscontributedequallytothiswork.wPresentAddress:NorthernOntario
MedicalSchool,Sudbury,Ontario,Canada.CorrespondenceandrequestsformaterialsshouldbeaddressedtoP.H.B.(email:[email protected]).
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A
trialfibrillation(AF)isthemostcommonarrhythmiathat infiltration and fibrosis. This exercise-induced atrial remodelling
is associated with ageing as well as hypertension, is prevented by treatment with the TNFa inhibitor (etanercept)
structural heart disease, hyperthyroidism and several and is not seen in mice lacking TNFa gene.
other cardiovascular conditions1. Although exercise is known to
provide enormous health benefits particularly by improving Results
disease outcomes in cardiovascular patients2, it is now clear that Physiological cardiac remodelling in exercised mice. Although
high intensity endurance sports increase AF susceptibility in the theprimarypurposeofourstudieswastoassessthelinkbetween
absenceofunderlyingcardiovasculardisease3–6.Thebasisforthe endurance exercise and cardiac arrhythmias, we began by char-
associationbetweenintenseexerciseandAFispoorlyunderstood. acterizing our exercised mice. Six-week-old male mice were
AF is generally associated with atrial remodelling often selected for swimming and treadmill running exercise at levels
characterized by increased parasympathetic nerve activity thatexceeded70%oftheVO maxfortheentiredurationoftheir
2
(PNA)7, atrial enlargement8, fibrosis9 and inflammation10. A training protocols (Supplementary Fig. 1A), which is similar to
hallmark of endurance athletes is heart rate slowing due to levels targeted by elite athlete training programmes20. All the
elevated parasympathetic tone11 and atrial hypertrophy12 both mice were trained for 6 weeks (unless otherwise stated), and all
risk factors for AF. Intense physical activity also elevates the exercised mice were used in subsequent analysis (See
inflammatory factors such as CRP13 and TNFa14, which can be Methods). As expected from previous studies examining
activated by atrial stretch following elevated atrial pressures endurance athletes11, heart rates estimated using telemetry ECG
(B35mmHg) as occurs during intense exercise15. Indeed, measurementsdeclined(swimP¼0.009,treadmillP¼0.03,one-
although expression of TNFa was originally thought to way ANOVA) progressively after initiating exercise (Fig. 1 &
originate primarily from macrophages16, other cells such as SupplementaryFig.1C).Theheart-ratedifferences(Po0.01,two-
cardiac myocytes synthesize TNFa in response to myocardial way ANOVA) resulting from exercise were eliminated by
stretch17,18 and elevated cardiac TNFa induces atrial fibrosis, blocking the autonomic nervous system (via combined atropine
myocardial hypertrophy and AF19. and propranolol treatment, Fig. 1c). In addition, there were no
In this study we show that intense endurance exercise differences (P¼0.7, Student’s t-test) in beating rates of isolated
(involvingswimmingortreadmillrunningfor6weeks)increases atria (which lack autonomic nerve inputs) between exercise and
susceptibility to AF in mice and is associated with macrophage sedentary groups (Fig. 1d). These results support the conclusion
Baseline
6 weeks swim exercise
700 Swim
Light Dark m.) 550 n=6
b.p.m.) 600 e (b.p. 525
eart rate ( 500 e heart rat 500 # # #
H g
a
er
400 v 475
A
0 12 Tim24e (h) 36 48 Baseline Week 2 Week 4 Week 6
Sedentary Sedentary
Swim exercised Swim exercised
## NS
600 400
NS
eart rate (b.p.m.) 240000 ating rate (b.p.m.) 123000000
H e
n=12 n=12 n=12 n=12 B n=11 n=13
0 0
Baseline Autonomic
blockade
Figure1|Exercise-inducedheartratereductionsismediatedbytheautonomicnervoussystem.(a)Three-hourmeansofHRacquiredovera48-h
periodinmiceimplantedwithtelemeteryECG,atbaselineandafter6weeksofswimmingexercise,illustratepreservedcircadianoscillationsofHRand
reductionsinHRatalltimepointsoverthe48-hperiodasaresultofexercise.Barindicatesday-nightcycles.(b)Averagedheartrates(over48h)
measuredusingtelemetricECGsinmiceexercisedbyswimmingshowsignificantreductionsinHRafter4weeksofswimmingexercise.(c)Heartrates
derivedfromsurfaceECGsinanesthetizedmicebeforeandafterautonomicblockadeusingatropine&propranolol.Administrationofatropineand
propranololeliminatedheartratedifferencesbetween6-weekexercisedandsedentarymice.(d)Tofurtherassesstheroleofautonomicnerveinputon
exercise-inducedHRreductions,atriawereisolatedandcompletelydenervated.Fieldrecordingsofspontaneouslybeatingatriawereusedtocalculate
beatingrate.Thenvaluesinbarsindicatenumberofmiceused.Beatingratesofdenervatedatriaexvivodidnotdifferbetweensedentaryand6-week
exercisedmice.Datapresentedasmean±s.e.m.,#Po0.05whencomparedwithbaselineusingrepeatedmeasureone-wayANOVAwithDunnett’s
multiplecomparisontest.##Po0.05whencomparedwithbaselineusingrepeatedmeasuretwo-wayANOVAwithSidak’smultiplecomparisontest.
NS,notsignificant.
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and increased APD heterogeneity24 both of which can occur
Table1|Physicalparametersofmiceexercisedfor6weeks.
with activation of muscarinic Kþ currents (I ) following
K,Ach
parasympathetic nerve activity (PNA)25. Consistent with a role
Sedentary Swim
forelevatedPNA,exercisedmiceshowedlargerHRresponsesto
Bodyparametersn 13 14
atropine than sedentary controls (Fig. 3a), and the heart rate
BW(g) 41.5±0.7 36.3±1.0* variability component, which is associated with PNA11, was
Tibialength(mm) 19.3±0.2 19.1±0.2
Atriaweight(mg) 17.7±0.6 19.2±0.4 elevated in exercised mice (Fig. 3b, Supplementary Fig. 9).
Ventricleweight(mg) 156.2±4.8 145.7±2.2 Furthermore, blockade of the PNA with atropine caused the
Atria/Tibialength(mgmm(cid:2)1) 0.90±0.03 0.99±0.02* effective refractory period (ERP) to be longer in exercised mice
Ventricle/Tibialength(m mm(cid:2)1) 8.09±0.26 7.67±0.11 compared with sedentary (Fig. 3c), despite the lack of ERP
Atria/bodyweight(mgg(cid:2)1) 0.43±0.02 0.54±0.2* differences in the absence of drug (Supplementary Table 4).
Ventricle/bodyweight(mgg(cid:2)1) 3.76±0.08 4.06±0.12* However,althoughatropineprolongedERPs,itcouldnotentirely
Atria/ventricleweight(mgg(cid:2)1) 114.4±5.1 132.2±3.6* prevent AF in exercised mice (Fig. 3d). These effects of PNA
Echocardiographyn 32 32
blockadeonERPandAFinexercisedmiceareconsistentwiththe
longer action potential durations (APDs) in isolated exercised
LVDd(mm) 4.21±0.03 4.43±0.03*
atria and cardiomyocytes (Fig. 4a,b), which were associated with
LVDs(mm) 2.67±0.04 2.92±0.04*
SV(ml) 53.2±0.7 56.0±1.0 increased L-type Ca2þ currents (Fig. 4c,d) without changes in
EF(%) 67.1±0.8 63.0±0.9* voltage-gated Kþ currents or IK,Ach (peak amplitude, desensiti-
FS(%) 37.0±0.6 34.1±0.7* zation or deactivation) measured in atrial cardiomyocytes from
CO(mlmin(cid:2)1) 26.8±0.6 24.9±0.8 exercisedmice(SupplementaryFig.10).AsalteredRyR2function
Heartrate(b.p.m.) 508±10 448±12* has been linked to AF in humans26,27 and mice28, pathological
LVPWth(mm) 0.79±0.01 0.76±0.01* cardiac remodelling29, and elevated oxidative stress30 with
exercise31, RyR2 phosphorylation and oxidation levels were
InvasiveHemodynamicsn 7 8
measured. We found increased (Po0.05, One-way ANOVA)
MAP(mmHg) 80.5±2.1 80.4±3.6
RyR2 phosphorylation at S2814 in the atria from mice after 6
BW,bodyweight;CO,cardiacoutput;EF,ejectionfraction;FS,fractionalshortening;LVDd,left weeks of swimming or after only four sessions of swimming
ventriculardiastolicdiameter;LVDs,leftventricularsystolicdiameter;LVPWth,leftventricular (Fig. 4e). Interestingly, the level of phosphorylation at S2814 in
posteriorwallthickness;MAP,meanarterialpressure.Thenvalueshownpresentnumberof
miceused.Datapresentedasmean±s.e.m. theventricleswassignificantlydecreasedinmiceafter6weeksof
*Po0.05,comparedwithsedentaryusingStudent’st-test. swimming.Incontrast,nodifferencesinRyR2oxidation,orRyR2
phosphorylation at position S2808 (PKA-dependent) or CaMKII
that the exercise-induced reductions in heart rate arise primarily phosphorylation at T287 were detected between the groups
fromdifferences inautonomic nerve activity,as typically seen in (SupplementaryFig.11).Inaddition,nochangeintheexpression
athletes11. Ventricles of exercised mice also underwent level of RyR2 or CaMKII was seen between the groups
physiological remodelling, as in athletes21, characterized by left (Supplementary Fig. 11). Elevated RyR2 phosphorylation at
ventricular chamber enlargement (P¼0.04, Student’s t-test) and 2814 correlated with trends towards increased Ca2þ spark
enhanced ventricular contractility at rest (P¼0.002, Student’s activity (P¼0.06, Fig. 4f, Student’s t-test) as well as Ca2þ
t-test)aswellasfollowingintraperitonealinjectionof1.5mgg(cid:2)1 transient amplitudes (P¼0.06, Supplementary Fig. 11, Student’s
dobutamine (P¼0.006, Student’s t-test), as detailed in Table 1, t-test)withnochangeinCa2þ transientdecayratesinswimming
Supplementary Tables 1–3 and Supplementary Fig. 2. exercised atrial myocytes as compared with sedentary mice.
Increasedvulnerabilitytoatrialarrhythmiasinexercisedmice.
As documented in Fig. 2 (also Supplementary Figs 4–6), pro- Structural remodelling in atria of exercised mice.The inability
grammed electrical stimulations (Supplementary Fig. 4) revealed ofPNAblockadetoentirelypreventAFvulnerabilityinexercised
that sustained atrial, but not ventricular, arrhythmias (i.e., bouts mice combined with the increased AF incidence in isolated
of rapid electrical activity lasting 410s) were induced more denervated exercised atria motivated usto examine otherfactors
frequently in mice undergoing swimming (16/43, Po0.01, w2- previouslylinkedtoAFincludingatrialenlargement8,interstitial
test) and treadmill (14/29, Po0.01, w2-test) exercise compared collagen deposition (fibrosis)32 and inflammation10,33. Exercised
with sedentary controls (1/41). These results establish clear dif- mice increased (P¼0.01, Student’s t-test) interstitial collagen
ferential effects of exercise on atria versus ventricles. No spon- deposition (Fig. 5a,b) in atria without differences (P¼0.4,
taneous atrial arrhythmias were observed, however, in telemetry Student’s t-test) in ventricles. As tissue fibrosis is invariably
ECGrecordingsofswimortreadmillexercisedmiceduringthe6- associated with inflammation in AF10,33, cardiac sections were
week training period. To characterize the AF events, electrical examined with toluidine blue (Mast cells), Ly6b.2 antibodies
activity of isolated atria was determined (using Di-4-ANEPPS) (primarily neutrophils and recently activated inflammatory
following programmed field stimulation. As summarized in monocytes34) and Mac-3 antibodies (macrophages and
Fig.2e,f(andSupplementaryFig.5C),AFalsooccurred(Po0.03, monocytes). Although no elevations in mast cells or Ly6b.2-
w2-test) in isolated atria from swimming (5/13) and running (7/ positive cells were observed acutely (2 days) or after 6 weeks of
16) mice versus sedentary mice (1/15). Analysis of AF events in exercisetraining,Mac-3-positivemacrophage/monocytenumbers
isolatedatria(SupplementaryFigs7and8)revealeddynamicand were increased (P¼0.005, Student’s t-test) after 6 weeks of
complex ECG electrical activity characterized by transient peri- exercise (Fig. 5c,d). Increased macrophage/monocyte numbers,
odic rapid focal activity and re-entry circuits accompanied by without neutrophil infiltration, support the conclusion that
variable conduction block, as described in humans and other inflammation in exercised atria does not result from
animal models1,22. inflammatory cell infiltration as might occur with myocyte
damage. No differences (P¼0.67, Student’s t-test) in staining
Autonomicandelectricalremodellinginatriaofexercisedmice. patterns(Ly6b.2andMac-3staining)wereobservedinventricles
Candidate mechanisms for increased AF vulnerability with between exercise (16.26±2 cells mm(cid:2)2) and sedentary mice
exercise include abbreviated action potential durations (APD)23 (14.1±4 cells mm(cid:2)2). We also observed increased (P¼0.03,
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Sedentary
Swim
Treadmill
0.2 s 0.2 s
No AF
No VT
Sustained AF
Sustained VT
*
*
100 100
n=41 n=43 n=29 n=19 n=14 n=19
80 80
60 60
% %
40 40
20 20
0 0
Sedentary Swim Treadmill Sedentary Swim Treadmill
0.4 s No AF
Sustained AF
Sedentary
*
*
100
Swim
n=15 n=13 n=16
80
SR
60
%
40
AF
20
0
0 6 12 18 24 ms Sedentary Swim Treadmill
Figure2|Increasedvulnerabilitytoatrialbutnotventriculararrhythmiasinexercisedanimals.Vulnerabilitytoatrialorventriculararrhythmiaswas
assessedinvivousinganintracardiaccatheterplacedintherightventricle.Representativeintracardiacbipolarelectrogramsshowinginductionofatrial
arrhythmias(a)aswellassurfaceelectrogramrecordingsintheleadIIconfigurationshowinginductionofventriculartachyarrhythmias(VT;b)from
sedentary,swimmingandtreadmillexercisedmice.Thepercentageofanimalswithsustained(410s)atrial(c)butnotVT(d)isincreasedsignificantlyby
exercise.(e)AtypicalrepresentativeAFeventinitiatedinisolated(denervated)atriabyrapidstimulationrecordedusingfieldrecording(top)andoptical
mappingimagesofatrialoadedwithavoltagesensitivedyeduringsinusrhythm(SR)andduringAF(bottom).Notethedisorganizedwavepropagation
duringanAFepisode.Redarrowsindicatethewave-frontpathway.(f)PercentageofisolatedatriafromeachgroupshowingsustainedAFepisodes
(410s).Thenvalueforeachgroupisindicatedonbargraphsandpresentnumberofmice.w2-test*Po0.05.
n¼3,Student’st-test)fibroblastnumbersinatriafrommiceafter increased (P¼0.01, Student’s t-test) atrial capacitance in the
6 week of swimming (887±135 cells mm(cid:2)2) compared with swimming (74.4±2.4pF, n¼46) compared with sedentary
sedentary controls (409±55 cells mm(cid:2)2), without evidence of groups (66.2±2.3pF, n¼48).
myofibroblasts positive for a-smooth muscle actin staining. In These structural changes in atria from exercised mice were
addition to fibrosis and inflammation, we observed atrial associated with slowed (P¼0.02, Student’s t-test) conduction
hypertrophy (P¼0.02, Student’s t-test), measured as atrial-to- velocities and prolonged (P¼0.003, Student’s t-test) P-wave
ventricularweightsoratria-to-bodyweightsratios,inmiceafter6 durations (Fig. 5e–h) without changes in either connexin-40 or
weeks of swimming (Table 1). There was no change in absolute connexin-43 expression in swimming mice compared with
left ventricle weightsor left ventricle weights normalized to tibia sedentary mice (Supplementary Fig. 12). Interestingly, the
length (Table 1), but we did observe a slight increase in left organizational index of ECG recordings during AF episodes in
ventricle weights normalized to body weights (Table 1) as exercised atria were greater, consistent with models of structural
previously shown in exercised mice35. Consistent with atrial remodelling in AF22 (Supplementary Fig. 7). Importantly, if we
hypertrophy,whole-cellpatch-clampmeasurementsalsorevealed examinedmice6weeksaftertheterminationofthe6-weekswim
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Sedentary
Swim exercised Sedentary
e 25
pin * 50 Swim exercised
with atro 1250 2er (ms)3400
nge HR 10 HF pow1200
a 5
h
% C 0 n=8 n=11
300 400 500 600
Heart rate (b.p.m)
#
# **
40 40
% change AERPwith atropine 123000 AF duration (s) 123000
0 0
Sedentary Swim Treadmill (–) Atropine (+) Atropine
n=10 exercised exercised n=15 n=15
n=5 n=10
Figure3|EffectsofatropineonAFsusceptibilityinexercisedmice.(a)Administrationof2mgkg(cid:2)1atropinehadalargereffectonheartrate(HR)in6-
weekswimmingexercisedcomparedwithsedentarymice.(b)Consistentwithincreasedparasympatheticnerveactivity(PNA),6-weekswimming
exercisedmicehadincreasedhighfrequency(HF)powercomparedwithsedentarymice.TherelationbetweenheartrateandHFpowerisshown.
R2¼0.63sedentaryand0.49forexercised,Pearson’sPo0.01inbothgroups.(c)Thechangeinatrialeffectiverefractoryperiod(AERP)afteratropinewas
greaterinexercisedmicecomparedwithsedentary.(d)AtropinereducedthedurationofAFinexercisedmice,suggestingaroleofPNAinAFsusceptibility
inexercisedmice.Treadmillexercisedmiceareshownbycirclesandswimmingexercisedmicebysquares.Thenvaluesindicatenumberofmiceused.
Datapresentedasmean±s.e.m.*Po0.05comparedwithsedentaryusingunpairedStudent’st-test.**Po0.05comparedwithsedentaryusingpaired
Student’st-test.#Po0.05usingone-wayANOVA.
training, the atrial AF vulnerability and fibrosis persisted TNFa etanercept (Enbrel) and mice lacking the TNFa gene
(P¼0.02, Student’s t-test), while HR changes were reversed (TNFa(cid:2)/(cid:2)) were studied. Interference with TNFa abolished
afterdetraining(to492±7beats/minute,P¼0.02comparedwith the exercise-induced increases in NFkB-driven luciferase activity
6 weeks swim exercised, Student’s t-test), establishing that the (Fig. 6a), the increased susceptibility to AF (Fig. 7a,c, Supple-
exercise-induced structural remodelling is not readily reversible mentary Fig. 5D), atrial fibrosis (Fig. 7b,d) and increases in
(Supplementary Fig. 13). Mac-3-positive cells (Fig. 8). Interestingly, when etanercept
treatment was started 3 weeks post swimming exercise, neither
atrialfibrosisnorAFsusceptibilitywasprevented(Supplementary
RoleofTNFainexercise-inducedatrialstructuralremodelling. Fig.13C,D).InterferencewithTNFa(TNFa(cid:2)/(cid:2))alsoprevented
To gain insight into the possible genetic and biochemical the increased atria-ventricular weight ratios (Supplementary
mechanismsunderlyingexercise-inducedAF,weperformedgene Table 6) without affecting either the mild ventricular chamber
set enrichment analysis (GSEA) of microarray gene expression dilation or HR reductions induced by exercise (Supplementary
values that revealed enrichment of several signalling pathways Table 7). Moreover, APD prolongation (Po0.01, Student’s t-test)
(p38 MAPK and NFkB pathways) associated with inflammation wasalsoseenwithexerciseinTNFa(cid:2)/(cid:2) atrialmyocytes,withno
in exercised atria (Supplementary Fig. 14A,B, Supplementary change in the amplitude or resting membrane potentials
Table 5). Although these enriched pathways are regulated by (Supplementary Fig. 10E–H).
severalfactors36,37,theyareneverthelessdownstreamof,orcross- Previous studies have shown that increased fibrosis is linked
talkwith,TNFa36,38.Theseobservations,combinedwithprevious to TNFa and p38 activation40, and we found that p38
studies implicating TNFa in the promotion of AF19,39, suggest phosphorylation was acutely elevated by exercise (Fig. 6d) in
that TNFa plays a role in AF pathogenesis with exercise. In addition to the observed chronic differential expression of p38
support of this possibility, acute bouts of exercise activated pathway related genes (Supplementary Table 5). Consistent with
(P¼0.03, Student’s t-test) NFkB, the canonical downstream a role for p38 activation in exercise-induced remodelling,
transcription factor of TNFa, in atria, but not in ventricles treatment of mice with the p38 inhibitor SB203580 during the
(P¼0.2, Fig. 6a,b, Student’s t-test). Acute bouts of exercise also 6-week exercise period completely prevented AF (P¼0.021,
increasedthephosphorylationlevelsofotherdownstreamfactors Fig.7a,c,w2-test)aswellasatrialfibrosis(Po0.01,Fig.7b,d,one-
of TNFa signalling such as IkB (P¼0.01, Student’s t-test) and way ANOVA). In addition, the acute exercise-induced elevation
p38 MAPK (P¼0.03, Student’s t-test) compared with sedentary inp38 phosphorylationwassignificantlyreduced inTNFa(cid:2)/(cid:2)
controls (Fig. 6c,d). mice (P¼0.5, Supplementary Fig. 14C, Student’s t-test)
To more directly assess the involvement of TNFa in exercise- supporting the conclusion that exercise induces atrial-selective
mediated atrial remodelling, the pharmacological inhibitor of remodelling via TNFa-dependent p38 activation.
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Sedentary
40 Sedentary **
0 mV Swim exercised
s) 30 Swim exercised
m
20 mV on ( 20
ati **
20 ms Dur 10 **
0 n=21 n=23 n=21 n=23 n=21 n=23
APD30 APD50 APD90
+ 50 mV Sedentary
– 40mV –1F 1 Swim exercised
p
– 75 mV A
p
2 –50 –25 25 50mV
100 ms –1
*
–2
*
* –3
*
Sedentary Swim exercised * –4 pA pF–1
Sedentary
Sed 6w 4x Sed 6w 4x
ps2814-RyR2 565 kDa
Swim
RyR2 565 kDa
#
2 1.5 # NS Sedentary
yR # 6 weeks swim NS
Ratio of14-RyR2 to R 01..50 4 session swim μ–1–2s 100 m 112050 SSewdimen etaxreyrcised
ps28 0.0 7 6 4 7 6 4 arks 5
p
Atria Ventricle S 0
Figure4|IncreasedactionpotentialdurationsandI intheatriaofexercisedmice.(a)Representativeaveragedactionpotentialsrecordedusingsharp
Ca
microelectrodeinisolatedexvivoatriafromsedentaryandswimmingexercisedmice.(b)Sixweeksofexerciseincreasedtheaveragetimeto30,50and
90%repolarization(APD30,APD50andAPD90)ofleftatrialmyocytes.N¼21/6miceforsedentary,n¼23/6miceforexercise.(c)RepresentativeICa
recordingsinleftatrialmyocytesusingwhole-cellpatch-clamp.(d)I I-VcurvesinleftatrialmyocytesshowingincreaseinI in6-weekexercisedmice;
Ca Ca
n¼14myocytes/3miceforexercisedandn¼12/3miceforsedentary.(e)IncreasedRyR2phosphorylationatS2814inatriaof6week(6w)andfour
session(4x)swimexercisedmiceascomparedwithsedentary(Sed)mice.PleasenotethereductioninRyR2phosphorylationintheventriclesof6-week
swimexercisedmice.Thenvaluesindicatedonbargraphandrepresentnumberofmice.(f)Ca2þ sparkactivityintheatriaofswimandsedentary
exercisedmicewithrepresentativesparkactivityshownintoppanel;n¼35/3miceforsedentary,n¼42/3miceforexercise.Scalebar,200ms.Data
presentedasmean±s.e.m.*Po0.05comparedwithsedentaryatthesamemembranepotentialusingStudent’st-test.**Po0.01comparedwithsedentary
usingStudent’st-test.#Po0.05usingone-wayANOVA.
Discussion miceundergoingtread-millrunning(thatis,reducedheartrates,
Our results support the conclusion that increased PNA and mild ventricular hypertrophy, increased vagal tone, increased
structural atrial remodelling, characterized by inflammation, incidenceofAFbothinvivoandexvivo,andincreasedfibrosisin
hypertrophy and fibrosis, increase AF vulnerability in intense atria but not ventricles). Although we did not fully explore the
exercised mice. Our studies show that the mechanosensitive role of TNFa in the response of mice to tread-mill running, the
inflammatorycytokineTNFaiscentraltothepathophysiologyof similar responses in the swimming and tread-mill running
AF induced by intense exercise but does not appear to interfere micesuggestedthatinterference withTNFawillalsobeeffective
withthebeneficialphysiologicalventricularremodellingaswellas in treadmill exercised mice that will require further studies to
the electrical remodelling seen with exercise. Involvement of validate.
TNFa in exercise-induced atrial remodelling is supported by the InvolvementofTNFa-dependentpathwaysreadilyexplainsthe
enrichment of the p38 and NFkB pathways36–38 in our structural atrial remodelling with exercise since TNFa is an
microarray analysis, by increased IkB and p38 phosphorylation integral component of inflammatory responses, by recruiting
in atrial lysates, and by elevated NFkB activity in atria, but not circulating monocytes42 and regulating resident tissue
ventricles, following acute bouts of exercise. Although altered macrophages43. In addition, TNFa is a central regulator of
TNFa-dependentsignallingcanoccurwithorwithoutchangesin cardiac hypertrophy19 and fibrosis that involves altered p38
TNFa levels, or TNFa shedding41, we were unable to determine activity in fibroblasts17,44. In this regard, we found that
whether atrial TNFa levels change with exercise because the pharmacological blockade of p38, as with TNFa inhibition, also
specificity of commercially available antibodies could not be preventedAFandstructuralremodellingwithoutinterferingwith
verified by us (using TNFa(cid:2)/(cid:2) heart lysates). the physiological changes in heart rate and the ventricular
Although our studies focused primarily on swimming-based chambers induced by exercise. The underlying mechanisms for
exerciseinmice,wealsoobservedsimilarresponsestoexercisein the activationof these pathways and the differential pathological
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NATURECOMMUNICATIONS|DOI:10.1038/ncomms7018
Sedentary Swim exercised
Sedentary
Swim exercised
20 **
15
A
L
10
n
e 5
g
colla 0 n=9 n=11
e LAA
su 20
s
Ti
% 15
NS
10
V
L
5
n=6 n=11
0
LV
–2m)150 **
m
s 100
ell
c
+ ( 50
3
ac- n=4 n=5
M 0
0 ms 12 ms
*
0.7
0.6
–1m s) 00..45
CV ( 00..23
0.1
n=5 n=4
0.0
**
s) 20
m
n ( 15
o
urati 10
d
ve 5
a
w n=10 n=10
P- 0
20 ms
Figure5|Increasedinterstitialfibrosisandinflammationinatriaofswimmingexercisedmice.(a)Representativeimagesofpicrosiriusred-stained
heartsshowingincreasedmyocardialcollageninatriabutnotventriclesofexercisedmice(red).(b)Summaryof%tissuecollagenderivedfrompicrosirius
redstainedsections.(c)RepresentativeMac-3-stainedatrialsectionsshowingincreasedmacrophagecountswithexercise(shownbyredarrows).
(d)Normalizedmacrophagecounts.(e)Isochronalactivationmapsofpacedleftatrialappendagesshowingtimetoactivationoftheleftatrialappendage
fromsiteofstimulation.(f)Averagedconductionvelocities(CV)calculatedfromactivationmapsoftheleftatrialappendage.(g)Representativesurface
ECGrecordingsshowingprolongedP-wavedurationsinexercisedmice(dashedline).Inall,55%ofexercisedcomparedwith10%ofsedentarymice
showed þ/(cid:2) p-wavemorphology.(h)AverageP-wavedurations.Thenvaluesindicatedonbarsrepresentnumberofmiceused.Datapresented
asmean±s.e.m.*Po0.05whenexercisediscomparedwithsedentaryusingStudent’st-test.**Po0.05whenexercisediscomparedwithsedentary
usingWelch’st-test.Scalebarsindicate100mm.NS,notsignificant.
remodelling on atria versus ventricles with exercise are unclear. exercising humans. A role for stretch in atrial remodelling
However, stretch has been shown to activate TNFa in resulting from intense exercise in our mice is supported by our
cardiac17,18, skeletal45, fibroblasts46 and smooth muscle47 cells, findings that atria have a far greater compliance than ventricles
and we noted that exercise in mice leads to sharp elevations in (Supplementary Fig. 15) as well as by previous studies showing
diastolic atrial pressures exceeding 30mmHg (Supplementary. that atrial fibroblasts undergo transformation to proliferating,
Fig. 15), as seen in humans15. Although the pericardium collagen-secreting myofibroblasts in response to stretch more
constrains heart volumes, the atrium expansion is (B2-fold) easily than ventricular fibroblasts50. However, exercise induces a
greater than ventricular expansion under conditions when the multitudeofmetabolic,biochemicalandphysiologicalchangesin
pericardiumbecomesengaged48,andthismayunderlietherisein multiple cells types that could lead to stretch-independent
serumTNFa14aswellasserumatrialnatriureticpeptide49seenin activation of TNFa and its downstream signalling molecules
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Sedentary
4 session swim exercised
Sedentary
TNFα–/– 4 session swim exercised
TNFα+/– 4 session swim exercised 4 session swim exercised
NS
3 NS 2
#
e e NS
s s
a a
er 2 er
e lucifctivity e lucifctivity 1
ativa 1 ativa
el el
R R
n=7 n=5 n=4 n=4 n=3 n=5
0 0
Atria Ventricle
SedentaryExercised Sedentary Exercised
37 kDa Phospo IκB 37 kDa Phospo p38
37 kDa Total IκB 37 kDa Total p38
Sedentary Sedentary
4 session swim exercised 30 min swim exercised
2.0 * 4 *
Ratio o f phosphoκκIB to total IB 011...505 n=6 n=8 Ratio of phosphop38 to total p38 123 n=4 n=4
0.0 0
Figure6|IncreasedNFjBbindingactivityandp38phosphorylationwithswimmingexercise.(a)NFkB-drivenluciferaseactivitywasmeasuredinatria
andventriclesafterfoursessionsofswimmingexerciseinwildtype,TNFa þ/(cid:2) andTNFa (cid:2)/(cid:2) mice(seemethods).Valueswerenormalizedtoprotein
concentration.(b)NochangeinNFkBluciferaseactivitywasobservedintheventricleofexercisedmice.(c)IkBphosphorylationincreased2hpost
swimmingexercise.Micewereexercisedforfoursessions,andatriawereisolated2hafterfinalexercisesession.Summarybargraphsshowing
phosphorylatedIkBnormalizedtototalendogenousIkB.(d)P38phosphorylationincreasedfollowing30minofswimmingexercise.Micewere
exercisedfor30minandtheatriawererapidlyisolatedandprobedforp38phosphorylationusingwesternblots.Summarydataofphosphorylated
p38normalizedtototalp38areshown.Thenvalueshownonbargraphsandpresentnumberofmiceused.Datapresentedasmean±s.e.m.
*Po0.05usingstudent’st-test.#Po0.05usingone-wayANOVA.NS,notsignificant.
like p38 (ref. 51). Clearly, additional studies will be required to findings are intriguing because acute bouts of exercise elevated
address the underlying mechanisms for TNFa/p38-mediated p38 phosphorylation, and chronic p38 inhibition prevented the
atrial remodelling with exercise. In this regard, we are currently adversestructuralremodellingintheatriaassociatedwith6weeks
examining the effects of tissue-specific TNFa deletion including of exercise.
immunecelldeletionsonexercised-inducedatrialremodellingin ItshouldbementionedthatinhibitionofthePNAreduced,but
our mice. did not eliminate AF susceptibility in exercised mice. PNA
Our findings predict that interference with, or reductions in, involvement in exercise-induced AF is anticipated based on its
TNFa-dependent signalling pathways could prove useful in link to AF in endurance athletes56, which presumably occurs via
minimizing the harmful consequences of intense exercise on effects on atrial ERP (via I ). Consistent with this possible
K,Ach
atria, without interfering with either the performance of mechanism, our exercised mice had signs of elevated PNA (that
enduranceathletesorthemanyotherwisehighlybeneficialeffects is,elevatedheartratevariabilityandlowerheartrates),similarto
of exercise. Nevertheless, since previous clinical trials with that seen in human athletes11. Indeed, following PNA blockade,
etanercept in advanced heart failure patients were terminated atrial ERPs prolonged more in exercised mice compared with
due to a lack of efficacy52, despite showing promise in animal controls and these differences correlated with longer APDs in
studies53, etanercept may have limited efficacy in preventing cardiomyocytesfromexercisedmicethatwerenotassociatedwith
detrimental atrial remodelling with exercise, even though alterations in Kþ currents but were linked to enhanced L-type
etanercept is equally effective in animals and human in other Ca2þ currents (I ), as was also reported previously in
Ca,L
diseases, such as rheumatoid arthritis54. It is worth noting that, ventricular myocytes due to changes in PI3Kinase signalling
although treating mice with human-based etanercept is expected with exercise57.
to evoke an immune response, intraperitoneal injection of OurstudiesalsouncoveredmodestincreasesinRyR2phosphor-
etanercept has been used often in animal models to treat ylation at position S2814 (but not S2808) following swimming
TNFa-dependent inflammatory conditions53,54. The modulation exercise that was accompanied by trends towards increased
of pathways downstream of TNFa such as p38 might also spontaneous Ca2þ spark activity in atria. Increased RyR2
represent viable targets for future interventions. In this regard, phosphorylation and Ca2þ leak as a result of CaMKII activation
p38inhibitorshavebeenusedinheartfailuremodelsandshown has previously been reported in human AF patients26,27 as well as
toreducecardiacapoptosisandfibrosisintheventricles55.These mouse AF models28. In this regard, CaMKII can be activated
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Swim
exercised
Swim
+Etn
Swim
TNF(cid:2)–/–
Swim
+SB203580
0.2 s
Swim +Etn TNF(cid:2)–/– SB203580
No AF
Sustained AF
* **
* **
* 20 **
100
80 n=34 n=8 n=10 n=10 agen 15
oll
% 60 ue c 10
40 ss
Ti 5
20 %
n=11 n=6 n=7 n=6
0 0
Swim +Etn TNF(cid:2)–/– SB203580 Swim +Etn TNF(cid:2)–/– SB203580
Figure7|TNFaandp38inhibitionpreventsarrhythmogenicatrialremodelling.(a)Representativeatrialarrhythmiainductioninuntreated,etanercept
(Etn)treated,TNFaknockout((cid:2)/(cid:2))andp38inhibitorSB203580treatedmiceswimmingexercisedfor6weeks.(b)Representativepicrosirius
red-stainedatrialsectionsshowingpreventionoffibrosis(red)inexercisedetanercepttreated,exercisedTNFa(cid:2)/(cid:2) andexercisedSB203580treated
micecomparedwithuntreatedexercisedmice.(c)PercentageofmiceshowingsustainedAFepisodes.SustainedepisodesofAFwerenotinducedin
etanercepttreated,TNFa(cid:2)/(cid:2) orSB203580-treatedmicewithexercise.(d)Summarydatashowingpercentfibrosisinexercisedetanercepttreated,
exercisedTNFa(cid:2)/(cid:2) andSB203580treatedmice.Thenvalueshownonbargraphsandpresentnumberofmiceused.Datapresentedasmean±s.e.m.
w2-test*Po0.05whenexerciseduntreatediscomparedwithexercisedþEtn,exercisedTNFa(cid:2)/(cid:2) orexercisedSB203580treatedmice.**Po0.05
whenexerciseduntreatediscomparedwithexercisedþEtn,exercisedTNFa(cid:2)/(cid:2),orexercisedSB203580treatedmiceusingone-wayANOVAwith
Dunnett’smultiplecomparisontest.Scalebar,100mm.
by a number of inflammatory factors, including TNFa58, as As seen in our exercised mice, human athletes also show
well as a number of inter-related mechanisms including elevated reductionsinheartratemediatedbychangesinautonomicnerve
Ca2þ and oxidativestress59.Interestingly,wedid notobserveany activity that reverse after long-term deconditioning61, while
changesinCaMKIIphosphorylationatT287orexpressionlevelsof chamber dilatation persists. In contrast, it remains unknown
CaMKII with exercise, although recent studies have questioned whethercessationofexercisecaneliminatetheatrialremodelling
the specificity of CaMKII phospho-specific antibodies60. Future and AF vulnerability. In this regard, we found that reversal of
studies will clearly be required to determine the roles and links atrial fibrosis and enhanced vulnerability to atrial arrhythmias
betweenRyR2,CaMKIIandTNFainatrialremodellinginducedby persisted after 6 weeks after cessation of swimming exercise (for
exercise. 6 weeks), despite the reversal of HR slowing as in human. If
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**
150
**
–2m)
m100
s
ell
c
+ ( 50
3
c-
a
M n=5 n=5 n=6
0
Swim +Etn TNFα–/–
Swim +Etn TNF(cid:2)–/–
Figure8|ReducedinflammationwithTNFinhibition.(a)RepresentativeMac-3-stainedatrialsectionsformacrophages/monocytes(shownbyred
arrows)showingpreventionofmacrophage/monocytecountincreasesinexercisedetanercepttreatedandexercisedTNFa(cid:2)/(cid:2) micecomparedwith
untreatedexercisedmicesummarizedin(b).Thenvalueshownonbargraphsandpresentnumberofmiceused.Datapresentedasmean±s.e.m.
**Po0.05whenexerciseduntreatediscomparedwithexercisedþEtnorexercisedTNFa(cid:2)/(cid:2) usingone-wayANOVAwithDunnett’smultiplecomparison
test.Scalebar,100mm.
these observations are applicable to humans, they highlight the humans (that is, compare Supplementary Fig. 15A with Reeves
criticalimportanceofidentifyingandinterferingwithpro-fibrotic et al.15).
pathwaysactivatedbyexercise.Theseresultsfurthersuggestthat Itshouldbementionedthatourexerciseprogrammeswerenot
exercise as well as other factors inciting atrial structural voluntary. Nevertheless, stress was minimized in our exercised
remodelling (such as hypertension, valve disease, heart failure) miceinseveralways.Miceswamagainstawatercurrentthereby
may accumulate with age, which readily explains the strong avoiding the drowning stress associated with tail-weights,
linkbetweenageandAF62includinginenduranceathletes63.On bubbling or mild detergents to break the surface tension of
the other hand, it is clearly conceivable that ageing could water.Inaddition,withthetreadmillexercise,theuseofelectrical
influencetheresponseoftheatriatostressesinducedbyexercise shocks was limited to the pre-training sessions and not used
and other cardiovascular conditions. Nevertheless, consistent during the remainder of the exercise protocol. We believe that
with the potentially cumulative and irreversible nature of atrial stress is not a major factor in the atrial remodelling in the mice
structuralremodelling,wefailedtopreventtheincreasedfibrosis forseveralreasons.First,theHRinouranimalsdeclineasearlyas
intheatriaofexercisedmicewhentreatmentwithetanerceptwas 1 week after the start of the exercise protocol, whereas mice
started(3weeksafterinitiatingswimmingexercise).Interestingly, subjected to psychosocial stress show chronic HR elevations65.
many studies have established that ageing and cardiovascular Second, though fecal corticosterone levels tended to be higher
diseasearethemselvesaccompaniedbyinflammation,withTNFa in exercised versus sedentary mice, the difference was not
being involved in both conditions64. Future studies that are significant (Supplementary Fig. 1B). The interpretation of these
designed to identify individuals at risk for exercise-induced AF corticosterone measurements is complicated because acute and
shoulddevelopmethodologiesforthelongitudinalassessmentof chronic66 intense exercise in humans independently causes
atrial remodelling in athletes. It is also imperative for future elevated cortisol levels. Additional studies are needed to
studiestoidentifythetrainingandexerciseconditions(intensity, understand the link between stress, exercise and AF induction.
duration, frequency) that most strongly predispose to
pathological atrial remodelling; we are currently engaged in
Methods
studies designed to address precisely these questions in our
Experimentalanimalsandexerciseprotocols. Allmicewerehousedatthe
mouse models.
DivisionofComparativeMedicineanimalfacilityinaccordancewiththestandards
Despite the similar AF and fibrosis between our results and oftheCanadianCouncilonAnimalCare.Ethicalapprovalforallanimal
exercised rats7, the extrapolation of rodent results to human experimentswasgrantedbytheUniversityofToronto.Inpreliminarystudieswe
athletes clearly requires caution. Besides having much smaller foundthatCD1strainofmice(CharlesRiverLaboratories)instinctivelyswim
againstacurrentinourswimmingapparatus,abehaviourthatwasfoundtobe
hearts, mice also have 10-fold greater HRs than humans,
absentinotherstrains.Instudiesusingmicewithwhole-bodyTNFadisruption
thus potentially making mice less susceptible to arrhythmias. (c57b,Taconicmodel#1921)orexpressinganNFkB-Lucreporter(c57b,Jackson
Nevertheless, the ERPs in mice are also proportionally shorter, Laboratoriesstock#006100),asingleback-crosswithCD1micewassufficientto
which probably explains the many previous mouse studies manifestinstinctiveswimmingbehaviour.Miceswaminwatercontainers(30cm
diameter)withasteadywatercurrent(325lmin(cid:2)1)generatedbyacentralwater
that reproduce human cardiac arrhythmias. Another notable
pumpat34(cid:2)C.Swimmingtrainingbeganwith30-minswimmingsessions
difference between humans and mice, of particular relevance to (twicedaily,separatedby4h)thatwereincreasedto90minbyadding10min
our studies, is the relative differences in heart rate increases perday.Trainingthencontinuedfor6weeks.Forrunningexercise,micetrained
during exercise; in humans, heart rate can be increased by 3–4 onatreadmillapparatus(HarvardApparatus)at35cms(cid:2)1witha30(cid:2)incline.
Runningtrainingbeganwith30-minsessions(oncedaily)thatwereincreasedby
times above baseline heart rates while in mice the heart rates
10minperdayto120min.Inthefirst3–4sessions,electricstimulation(0.2mA)
increasebyonlyB50%.Consequently,miceareexpectedtorely wasusedtopromoterunningduringtraining;thereafter,micewereencouragedto
muchmoreonstrokevolumeincreasesinordertoelevatecardiac runusingasoftbrushpressedagainsttheirposteriorregion,toreducethepotential
outputduringexercisecomparedwithhumans.Ifatrialstretchis stressofcontinuedelectricshocking.Malemicewererandomlyassignedto
exercisedorsedentarygroupsat6weeksofage.Nomicewereexcludedfrom
indeed a major factor in atrial remodelling, then exercise is
thestudybasedoninabilitytotrain.Controlanimalswereplacedinwater
predicted to create a far stronger stimulus for atrial remodelling
containerswithoutwatercurrentfor5min,orplacedonthetreadmillfor5min
in mice compared with humans. While these species differences at5cms(cid:2)1.
in response to exercise could explain the appearance of atrial Micetreatedwithetanercept(Enbrel)wereinjectedsubcutaneouslydailyat
hypertrophy and inflammation after only 6 weeks of intense 2.5mgkg(cid:2)1fortheentire6-weekexercisetraining.MicetreatedwithSB203580
(AbMoleBioscience)wereinjectedi.p.at4mgkg(cid:2)1.Allexperimentalassessments
exercise,itisalsoimportanttonotethattheatrialfillingpressures
wereconducted24hafterthefinalexercisesession.Theidentityofthegroups
changes during exercise are remarkably similar in mice and (exercisedversussedentary)wasconcealedtotheinvestigators.Samplesizeswere
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