artcles arrhythmogenic mechanisms of autoimmune...

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4 The Official Journal of Korean Heart Rhythm Society

Introduction

Myocarditis and subsequent dilated cardiomy-

opathy(DCM)aremajorcausesofheartfailurein

Received: March 20, 2014Revision Received: June 16, 2014Accepted: August 2, 2014Correspondence: Boyoung Joung, MD, PhD, Cardiology Division, Department of Internal Medicine, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul, Republic of Korea 120-752Tel: 82-2-2228-8460, Fax: 82-2-393-2041E-mail: [email protected]

연세대학교 의과대학 내과학교실 정 보 영

Hyelim Park1,2; Hyewon Park1,2; Jisu Lim1, Sujung Oh1; Sungha Park, MD, PhD1; Hui-Nam Pak, MD, PhD1; Moon-Hyoung Lee, MD, PhD1; Boyoung Joung, MD, PhD1,2

Division of Cardiology, Yonsei University College of Medicine1, Brain Korea 21 PLUS Project for Medical Science, Yonsei University2, Seoul, Korea

Arrhythmogenic Mechanisms of Autoimmune Myocarditis Associated with Inflammation and Ca2+/Calmodulin-Dependent Protein Kinase II Activation

ABSTRACT

Rationale: Fatal arrhythmia is a frequent cause of death in patients with acute myocarditis.

Objective: To investigate the arrhythmogenic mechanisms of myocarditis.

Methods and Results: Autoimmune myocarditis was induced by injection of porcine cardiac myo-

sin (2 mg) into the footpads of adult Sprague-Dawley rats on days 1 and 8 (Myo; n=15). Uninjected

rats were used as controls (control; n=15). In an additional group of rats, corticosteroid (6 mg) was

injected into the gluteus muscle prior to injection of porcine cardiac myosin on days 1 and 8 (MyoS;

n=15). In the Myo group, 5 out of 15 rats (33%) died suddenly at 17 ± 2 days after induction of

myocarditis, and 5 of the 10 surviving rats (56%) exhibited arrhythmia. No control rats died or exhib-

ited arrhythmia (p=0.02). Compared with the control group, action potential duration (APD; 98 ± 7

vs. 152 ± 52 ms, p=0.03), APD dispersion (6 ± 2 vs. 24 ± 4 ms, p<0.001), the pacing cycle length

for discordant alternans (90 ± 9 vs. 198 ± 22 ms, p<0.001), and the frequency of ventricular tachy-

cardia (p=0.003) were increased in the Myo group. These arrhythmogenic effects were attenuated

in the MyoS group, however. Expression of high-mobility group box protein 1 (HMGB1), interleukin

6 (IL-6), and tumor necrosis factor-α (TNF-α) was increased in Myo rats compared with controls,

and this was attenuated in MyoS rats. An increase in phosphorylated Ca2+/calmodulin-dependent

protein kinase II (CaMKII), ryanodine receptor type 2 and phospholamban activity were also ob-

served in the Myo group relative to controls, though this was similarly attenuated in MyoS animals.

Conclusions: The arrhythmogenic mechanisms of myocarditis involve increased APD, APD dis-

persion and discordant alternans. These arrhythmogenic effects may be related to the increase in

inflammatory markers and CaMKII activation, as indicated by the attenuating effects of an anti-

inflammatory steroid.

Key Words: ■ myocarditis ■ arrhythmia ■ inflammation ■ Ca2+/calmodulin-dependent protein kinase II

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youngpatients.1Myocarditisischaracterizedbyin-

filtrationofinflammatorycellsintothemyocardium,

withconsequentlossofmyocytesanddevelopmentof

fibrosisandnecrosis.2Inasignificantfractionofpa-

tients,thelossofcardiomyocytesleadstoventricular

remodeling,permanentventricularwalldysfunction,

DCM,heartfailure,and/orarrhythmias.Myocarditis

isinducedbyavarietyofmeans,includinggenetic

susceptibility, toxins, viruses, bacteria, and para-

sites.3-4Itisassociatedwitharrhythmiasandsudden

death,andthetreatmentofventriculararrhythmias

iscriticalforimprovementinpatientprognosis.5

Experimental autoimmunemyocarditis (EAM) in

theratisauniqueandusefulmodelforunderstand-

inggiantcellmyocarditisandsubsequentDCM.6EAM

ratsarehighlysusceptibletoventriculararrhythmia

andprolongedactionpotentialduration(APD).Re-

ducedexpressionofIto-relatedmolecules,including

Kv4.2,Kv1.5, frequeninandKChIP2 isconsidered

toplayakeyroleinventricularremodeling,andto

causethecharacteristicclinicalfindingsofEAM.7-8

Cardiacinflammation–ahallmarkofmyocarditis

–isknowntoincreaseoxidativestress.Niwanoet

al.9demonstratedpreviously that theanti-oxidant

N-acetylcysteinesuppressesventricularremodeling

inEAMrats,suggestingthatoxidativestressplaysa

roleinremodelingandthedevelopmentofmyocardi-

tisitselfduringtheacutephaseofmyocarditis.Itwas

recentlyreportedthatoxidativestresscanactivate

Ca2+/calmodulin-dependentproteinkinaseII(CaM-

KII),10prolongAPDandinduceafterdepolarizationin

cardiomyocytes.11 Therefore, we hypothesized that

EAMcaninducearrhythmiaviaCaMKIIactivation

causedbyinflammationandoxidativestress.Totest

thishypothesis,weevaluatedarrhythmogenicevents

and survival in an EAM model. Arrhythmogenic

mechanismsunderlyingEAMwerefurtherinvesti-

gatedinLangendorff-perfusedisolatedhearts,and

thelevelofinflammationandCaMKIIactivationin

myocarditisweresubsequentlyassessed.Finally,we

confirmedthatarrhythmogeniceventsandCaMKII

activation were suppressed following pretreatment

withananti-inflammatorycorticosteroidagent.

Materialsandmethods

This study protocolwas approved by the Insti-

tutionalAnimalCareandUseCommitteeofYonsei

University College ofMedicine and Cardiovascular

ResearchInstitute,andconformstotheguidelinesof

theAmericanHeartAssociation.

Inductionofexperimentalautoimmunemyo-

carditis

Purified cardiac myosin (M0531, Sigma Aldrich,

Schnelldorf,Germany)12wasemulsifiedinanequal

volume of complete Freund’s adjuvant (BD bio-

sciences,Heidelberg,Germany) supplementedwith

mycobacteriumtuberculosisH37Ra(Difco,Detroit,

USA)ataconcentrationof10mg/mL.Six-week-

oldmaleLewisratswereimmunizedbysubcutaneous

injectionof2mgpurifiedcardiacmyosinineachrear

footpadondays1and8(Myogroup;n=15).Control

ratsreceivedinjectionsof0.5mLcompleteFreund’s

adjuvantinthesamemanner(Controlgroup;n=15).

Inaseparategroupof15rats,6mgsteroidwasad-

ministered simultaneously with cardiacmyosin on

days1and8(MyoSgroup;n=15).AmbulatoryHolter

monitoringwasperformedusingatelemetricsystem

(TelemetryResearch,Auckland,NewZealand).

Histologyandinflammatorycytokineassay

Following measurement of hemodynamic param-

eters,heartswereimmediatelyexcisedandweighed;

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heartweighttobodyweightratioswerecalculated.

Heartswerestainedwithhematoxylinandeosin,and

Masson’strichromestain.Immunostainingwasper-

formedusingatumornecrosisfactor-α(TNF-α)an-

tibodytoevaluatethedegreeofinflammation.

Bloodwasobtainedfromtheabdominalaortaof

eachratonday21.Anenzyme-linkedimmunosorb-

entassay(ELISA)wasperformedtodetermineserum

levelsofhigh-mobilitygroupboxprotein1(HMGB1),

interleukin6(IL-6),andTNF-α.Serumproteinlev-

elswerequantifiedwithkitsforHMGB1(IBLInter-

national,Hamburg,Germany),IL-6(R&DSystem,

Minneapolis,MN,USA)andTNF-α(R&DSystem,

Minneapolis,MN,USA),accordingtomanufacturer’s

instructions.

Opticalmapping

Onthe21stdayaftertheinitialimmunization,rats

(250–300g)wereanesthetizedbyintraperitonealin-

jectionofketamine(80mg/kg)andxylazine(4mg/

kg).Chestswereopenedviamediansternotomyand

theheartswererapidlyexcisedandimmersedincold

Tyrode’ssolution(compositioninmmol/L:125NaCl,

4.5KCl,0.25MgCl2,24NaHCO3,1.8NaH2PO4,1.8

CaCl2,and5.5glucose).Theascendingaortawasim-

mediatelycannulatedandperfusedwithTyrode’sso-

lutionprewarmedto37°Candequilibratedwith95%

O2and5%CO2tomaintainapHof7.4.Coronary

perfusionpressurewasmaintainedbetween80and

95mmHg.Twowidelyspacedbipolarelectrodeswere

usedforcontinuouspseudo-ECGmonitoring.

Foropticalrecording,cardiaccontractilitywasin-

hibitedwith10–17µmol/Lblebbistatin.13Heartswere

stainedwithdi-4-ANEPPS(Invitrogen,California,

USA)andexcitedwithquasi-monochromatic light

(520±30nm)fromtwogreenLEDlamps.Emitted

lightwascollectedbyanimage-intensifiedcharge-

coupleddevicecamera(DalsaInc.,Waterloo,Canada)

witha610-nmlongpassfilter.Dataweregathered

at3.75-mssamplingintervals,acquiringsimultane-

ouslyfrom125×125pixels,each0.08mm×0.08

mm.Themappedareaincludedpartsoftherightand

leftventricularfreewalls.

Opticalrecordingswereperformedduringsteady-

state and programmed stimulation. Programmed

stimulation was induced with bipolar electrodes

atthelateralsideoftheleftventricle(LV).Pacing

wasinitiatedatacyclelength(CL)of300ms,us-

ingstimulioftwicethepacingthreshold,andwas

subsequentlyreducedindecrementsof10msuntil

2:1capturewasachieved.APDat90%repolarization

(APD90)wasmeasuredatthebaseandapexofthe

LV.APDdispersionwasdefinedasthedifferencebe-

tweenmaximumandminimumAPD.Aftertheinitial

electrophysiological study,we attempted to induce

ventriculartachycardia(VT)orventricularfibrillation

(VF)usingastandardpacingtechnique(burstpac-

ingatCLsdownto70ms).Allsustained(＞30s)and

non-sustainedVTorVFepisodesweredocumented.

OpticalmappingandVTinductionstudieswereper-

formedin6ratsfromeachgroup.

ImmunoblotanalysisofCa2+handlingpro-

teins

ImmunoblottingforCaMKII,ryanodinereceptor

type2(RyR2),phospholamban(PLB)andthephos-

phorylatedformofeachproteinwasperformedusing

the following monoclonal antibodies: anti-CaMKII

andanti-p-CaMKII(1:1,000;SantaCruzBiotech-

nology);anti-RyR2(1:1000;AbcamReagents);anti-

p-RyR2(1:1000;Badrilla);andanti-p-PLB(1:1000;

SantaCruzBiotechnology).Targetedantigenswere

visualizedbylabelingwithcorrespondingHRP-con-

jugatedsecondaryIgG(1:5,000;SantaCruzBiotech-

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VOL.15 NO.2

nology) followed by enhanced chemiluminescence

assay(ECLPlus,Amersham,Piscataway,NJ).Blots

werescanned,andbandintensitywasquantifiedus-

ingtheImageJsoftware.

Statisticalanalysis

Dataareexpressedasmean±SEM.TheStudent’s

t-testwithBonferronicorrectionwasusedtocom-

parethemeansoftwonumericvalues.ThePearson's

chi-squaretestwasusedtocomparetwocategorical

variables.Pairedt-testswereusedtocomparethe

meansofmaximumslopeofrestitutioncurvesbe-

tweenbaselineandmyocarditis.pvalues＜0.05were

consideredstatisticallysignificant.

Results

ArrhythmiaandsurvivalofEAM

For the control, Myo and MyoS groups, heart

weightswere1.2±0.1g,1.7±0.1g,and1.3±0.1

g,respectively(Figure1A).Heartweightwassignifi-

Figure 1. Histological findings, arrhythmia and survival associated with EAM. (A) Four-chamber sections of hearts from the 3 groups. (B) Histological analysis at 21 days after immunization. Increased inflammation, fibrosis and TNF-α expression were particularly notable in the Myo group. (C) Kaplan-Meier Survival curves, showing lower survival rates in the Myo group compared with the control group (p=0.03). (D) Ambulatory Holter monitoring in Myo rats: a. AV block (asterisks indicate blocked P-waves), b. PVC and VT (asterisks indicate PVCs; arrow indicates initiation of VT).

A C

B

D

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cantlygreaterintheMyogroupthaninthecontrol

(p=0.002)andMyoS(p=0.03)groups.Comparedwith

controls,theratioofheartweighttobodyweightwas

significantlyincreasedintheMyogroup(5.2±0.5

vs.7.1±0.4,p＜0.001),butnotintheMyoSgroup

(5.8± 0.3, p=0.31). Figure 1B shows histological

findings21daysaftertheinitialimmunizationwith

cardiacmyosin.Whilenoinflammatorychangeswere

observedinthecontrolrats,therewasinfiltrationof

inflammatorycellsinMyorats,includinggiantcells

mainlyintherightventricleandepicardiallayerof

LV.SevereinterstitialfibrosisandincreasedTNF-α

expressionwerealsoobservedinMyorats.Infiltration

ofinflammatorycells,fibrosisandincreasedTNF-α

expressionwereattenuatedintheMyoSgroup.Fig-

ure1CshowstheKaplan-Meiersurvivalcurvesfor

thethreegroups.While5outof15rats(33%)inthe

Myogroupdiedsuddenlyat17±2daysafterinduc-

tionofmyocarditis,noratsandonerat(6%)diedin

thecontrolandMyoSgroups,respectively.TheMyo

grouphadalowercumulativesurvivalratethanthe

controlgroup(p=0.02).Figure1Dshowsthevarious

arrhythmiasrecordedbyambulatoryHoltermonitor-

ingintheMyogroup.VT,sinuspauseandatriov-

entricularblockwereobservedin4ratsintheMyo

group,3inthecontrolgroup,and3intheMyoS

Figure 2. Increased APD and APD dispersion in EAM. (A) Typical action potential traces for control (black), Myo (red) and MyoS (green) rats at pacing CLs of 300 ms and 200 ms. Asterisks indicate action potential recording sites. (B) Comparison of APD90 between control (white), Myo (red) and MyoS (green) rats from the LV base. (C) Activation (upper panels) and APD maps (lower panels). The dotted line marks the interventricular septum. (D) Optical action potentials showing early afterdepolarization (EAD) in Myo rats during sinus rhythm.

A

C

B

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group.Whilearrhythmiaswerenotobservedincon-

trolrats,theywereobservedin5ofthe9surviving

rats(56%)intheMyogroup,andinnoneofthe15

ratsintheMyoSgroup(p=0.03).

InflammatorymarkersinEAM

SerumlevelsofHMGB1,IL-6andTNF-αarepre-

sented in Supplementary Table 1. Compared with

controls,serumlevelsofHMGB1,IL-6andTNF-α

intheMyogroupwereincreased1.1-fold(p＜0.001),

2.1-fold(p＜0.001),and4.0-fold(p＜0.001),respec-

tively.Conversely,nosignificantchangeinexpres-

sionlevelswasobservedintheMyoSgroup.

IncreasedAPDandAPDdispersioninEAM

Opticalmappingwasperformedin6ratsfromeach

group.Figure2Ashowstheactionpotentialtraces

obtainedfromthebaseoftheLVduringpacingCLs

of300msand200msinLangendorff-perfusedrat

hearts.MeanAPDintheMyogroupwasrelatively

prolongedcomparedtothecontrolandMyoSgroups.

AcomparisonofmeanAPD90measuredatLVinall

Figure 3. Aggravated spatially discordant alternans in EAM. A, Spatially discordant alternans at pacing CLs of 80 ms, 150 ms and 110 ms in control (A), Myo (B) and MyoS (C) groups, respectively. Upper panel shows an action potential trace from sites 1 (black) and 2 (gray). Middle panels show activation maps; lower panels, APD maps. The dotted line marks the interventricular septum.

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threegroupsispresentedinFigure2B.MeanAPD90

atapacingCLof300mswassignificantlylonger

intheMyothaninthecontrolgroup(152±52ms

vs.98±7ms,p=0.03).APD90wasnotprolongedin

theMyoSgroup(89±7ms,p=1.0),however.Mean

APD90wasalsolongerinMyoratsthancontroland

MyoSratsatpacingCLsof200and160ms.Figure

2Cshowscorrespondingactivationandrepolarization

maps.Theconduction timeofbothventricleswas

prolongedintheMyogroupcomparedwithcontrol

andMyoSgroups.APDdispersionwassimilarlyin-

creasedintheMyogroup(23.6±3.6ms)compared

to controls (5.8±2.0ms,p＜0.001), though this

increasewasattenuatedintheMyoSgroup(10.5±

1.7ms,p＜0.001).Figure2Dshowstheopticalaction

potentialsofearlyafterdepolarization(EAD)inMyo

ratsduringsinusrhythm.

APDalternansandventriculararrhythmiain

EAM

APDalternanswereevaluatedinall3groups(Fig-

ure3).Discordantalternanswereobservedduringa

pacingCLof80msincontrolrats(Figure3A),150ms

inMyorats(Figure3B)and110msinMyoSrats(Fig-

ure3C).Comparedwithcontrols,thepacingCLre-

quiredtoinducediscordantalternansinMyoratswas

increasedfrom80±9msto178±22ms(p＜0.001);

Figure 4. APD restitution curves measured at LV base (A) and apex (B). Myo rats exhibited steeper APD restitution slopes than control rats (p<0.001). This increase was not observed in the MyoS group (p=1.0).

A B

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VOL.15 NO.2

nosignificantdifferencewasobservedbetweencon-

trolandMyoSrats(114±5ms,p=0.06).Thesefind-

ingssuggestthatspatiallydiscordantalternanswere

moreeasilyinducedintheMyogroup,thaninother

groups.Conductionblockwasalsomorefrequent-

lyobservedatlongerpacingCLsintheMyogroup

(107±10ms)thaninthecontrolgroup(73±6ms,

p＜0.001),whichwasnotsignificantlydifferentfrom

theMyoSgroup(84±10ms,p=0.24).

Ventriculararrhythmiaswereevaluatedin6rats

fromeachgroup.For thecontrol,MyoandMyoS

groups,triggeredactivitywasobservedin1(17%),5

(83%)and2(33%)rats,respectively.Ofthe6ratsin

eachgroup,ventriculararrhythmiaswereinducedin

0,6(100%)and1(27%),respectively.TheMyogroup

exhibitedtriggeredactivity(p=0.02)andventricular

arrhythmia(p=0.03)morefrequentlythanthecon-

trolgroup.Conversely,theMyoSgroupshowedno

significantdifferenceintriggeredactivity(p=0.55)or

ventriculararrhythmia(p=0.57)comparedwithcon-

trols.

IncreasedAPDrestitutionslopeanddomi-

nantfrequencyinEAM

Figure4showsatypicalexampleofanAPDresti-

Figure 5. Increased dominant frequency (DF) of VF in EAM. DF map of control (A), Myo (B) and MyoS (C) groups. Upper panels show action potential traces; middle panels, DF maps; and lower panels, DF distributions.

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tutioncurvemeasuredattheLVbaseandapex.For

thecontrol,MyoandMyoSgroups,themaximum

APDrestitutionslopeswere0.23±0.09,0.70±0.10

and0.28±0.04attheLVbase,respectively.Slopes

attheLVapexwere0.26±0.07,1.19±0.11and

0.24±0.09,respectively.TheAPDrestitutionslopes

intheMyogroupweresteeperthancontrolsatboth

theLVbase(p＜0.001)andapex(p＜0.001).Asig-

nificantincreaserelativetocontrolwasnotobserved

intheMyoSgroup,however(p=1.00).

Thedominantfrequency(DF)mapsofVFepisodes

wereevaluatedinthe3groups.DFwas6Hzinthe

controlgroup(Figure5A),8HzintheMyogroup

(Figure5B)and5HzintheMyoSgroup(Figure5C).

Comparedwiththecontrolgroup,meanDFwassig-

nificantlyhigherintheMyogroup(8.1±1.0vs.6.3

±0.3Hz,p=0.006)butnotintheMyoSgroup(5.8

±0.5Hz,p=0.82).

Increased p-CaMKII, ryanodine and p-

phospholambaninEAM

Figure 6 shows a Western blot of Ca2+ han-

dlingproteins.Comparedwithcontrols,p-CaMKII,

RyR2,p-RyR2andp-PLBwereincreased2.5-fold

(p＜0.001), 2.9-fold (p＜0.001), 5.1-fold (p＜0.001)

Figure 6. Western blot of Ca2+ handling proteins. (A) Western blot analysis. (B) Quantification of band intensity showing increased expres-sion of p-CaMKII, RyR2, p-RyR2 and p-PLB in EAM. These increases are not evident in the MyoS group. Asterisks denote p<0.05.

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and 2.3-fold (p＜0.001), respectively, in the Myo

group.Thissignificantincreasewasnotobservedin

theMyoSgroup.CaMKIIexpressionwasnotdifferent

betweenthe3groups.

Discussion

Thesalientfindingsofthisstudywerefour-fold.

Firstly,EAMratswere found to exhibit fatal ar-

rhythmiaanddecreasedsurvivalrelativetocontrols.

Secondly,theEAMmodelwascharacterizedbypro-

longedAPDwith increasedAPD dispersion, easily

induciblespatiallydiscordantalternans,steeperAPD

restitutionslopesandincreasedventriculararrhyth-

mia.Thirdly,increasedactivityofcalciumhandling

proteins,includingp-CaMKII,p-RyRandp-PLB,is

inducedintheEAMmodel.Finally,EAM-relatedar-

rhythmiaandtheactivationofcalciumhandlingpro-

teinswereattenuatedbypretreatmentwithananti-

inflammatorysteroid.OurresultsindicatethatEAM

caninducearrhythmiaviaCaMKIIactivationunder

conditionsofinflammationandoxidativestress.

Ventriculararrhythmiasanddecreasedsur-

vivalinEAMrats

Ventricular arrhythmia is a significant cause of

death,alongwithheartfailure,inacutemyocarditis.

Themyosin-inducedEAMratisoneoftheanimal

modelsusedtostudytheeventsthatoccurinhuman

giantcellmyocarditis.6,14-17TheEAMmodelcomprises

anacuteinflammatoryphaseevoked2weeksafter

myosininjection,andasubsequentrecoveryphase

initiatedaroundthe25thdayafterinjection,followed

byadilated cardiomyopathy-likephaseassociated

withchronicheartfailure.Inthisstudy,9outof15

EAMrats(60%)exhibitedcardiacevents,including

suddendeathandarrhythmia.Theseresultssuggest

thatthelowsurvivalrateassociatedwiththeEAM

modelislikelyrelatedtothedevelopmentofarrhyth-

mia.Interestingly,inadditiontoVT,sinusdysfunc-

tionandatrioventricularblockwerealsocommonly

observedintheEAMmodel.

IncreasedrepolarizationgradientandCaM-

KIIactivationinEAM

APDwasprolongedinmyosin-inducedEAM.7This

prolongationmaybeexplainedbyaninitialreduc-

tion inIto-relatedcurrents,followingdownregula-

tionofKv4.2,Kv1.5,frequeninandKChIP2.7-8The

roleofCa2+handlingproteinsinEAMhasnotyet

beenestablished,however.Ourresultsindicatethat

theactivationofCa2+handlingproteinsmayplayan

importantroleinEAM-inducedarrhythmia.Weob-

servedincreasedexpressionofCaMKIIandphospho-

rylatedCaMKII following inductionofmyocarditis,

suggestingthatCaMKIIactivationmayfacilitateAPD

prolongation, representinganovel arrhythmogenic

mechanismofEAM.

EAM-associatedinflammationincreasesoxidative

stress.Inadditiontoactivationbyelevatedintrac-

ellularCa2+ levels (following β-adrenergic receptor

stimulation18)CaMKII activity is alsoknown to be

enhancedunderpro-oxidantconditions.10,19-20Oxi-

dationofpairedregulatorydomainmethionineresi-

duessustainsCaMKIIactivityintheabsenceofCa2+/

CaM.10H2O2-inducedafterdepolarizationsdependon

bothimpairedINainactivation,toreducerepolarization

reserve,andenhancedICa,L,toreverserepolarization,

bothofwhicharefacilitatedbyCaMKIIactivation.11

Consistent with elevated p-CaMKII, p-RyR2 and

p-PLBlevelswerealsoincreasedinEAM.TheRyR,

orcalciumreleasechannel,onthesarcoplasmicre-

ticulumisthemajorsourceofcalciumrequiredfor

excitation-contraction coupling in cardiac muscle.

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Hyperphosphorylation of RyR2 results in defective

functionduetoincreasedsensitivitytoCa2+-induced

activation.21

Untilnow,ithasnotbeenclearwhetherAPDpro-

longationinmyocardialcellsishomogeneousorhet-

erogeneousinnature,becausepreviousstudieshave

onlyevaluatedasinglesiteintheheartusingamac-

roscopicelectrophysiologicaltechnique.Inthisstudy,

usingopticalmapping,weobservedheterogeneous

APDprolongationandincreasedAPDdispersionin

EAM.AlthoughAPDprolongationisthemainmech-

anismoflongQTsyndrome,enhanceddispersionof

repolarizationiscriticalfortheinductionoffatalar-

rhythmia.22Transmuralandapicobasaldispersionof

repolarizationwasshowntoberesponsibleforthe

initiationofreentrantactivationinlongQTsyndrome

patients.Itwasreportedthatnumerousmammalian

species,includinghumans,exhibitapex–basedif-

ferencesincardiacrepolarization.23Increasedspatial

dispersionofrepolarizationacrosstheanteriorepi-

cardialsurfacewasalsodemonstratedtorepresent

theelectricalbasis for spontaneousmalignantar-

rhythmiasinlongQTtype2rabbits.24

Mechanisms of ventricular arrhythmia in

EAM

Spatiallydiscordantalternanswereeasilyinduced

inEAM.Thesecauseanincreaseinthespatialdis-

persionofrepolarization,andarethoughttoresultin

T-wavealternans.25T-wavealternansareprecur-

sorsofcardiacelectricalinstability,andconsequently

suddencardiacdeath.26Spatiallydiscordantalternans

canbeexplainedbytheincreasedsteepnessofAPD

restitutionslopesinEAM.Asteepslopeofelectri-

calrestitutionfacilitatesthebreakupofsinglespiral

wavesintomultiplespiralwaves,aprocessthatmay

accountforthetransitionfromVTtoVF.27-28Aslope

of electrical restitution≥1 is especially associated

withVF.29Furthermore,theincreasedprevalenceof

discordantalternansmayalsohavebeentheresult

offibrosisandreducedgapjunctionconductancein

EAM;thesehavepreviouslybeenshowntolowerthe

thresholdforspatiallydiscordantalternans.30-31Fi-

nally,thedevelopmentofdiscordantalternansmay

alsoberelatedtoalteredexpressionandactivityof

Ca2+handlingproteins.TheneteffectsofEAMre-

modelingpromoteCa2+alternansviaphosphorylation

ofRyRsandCaMKIIsignalingtoincreasetheirCa2+

sensitivity(increasingbothgainandleak).32-33

Attenuation of EAM-related arrhythmia by

anti-inflammatorytherapy

In thismodel, the overexpression of inflamma-

torycytokines,suchasHMGB-1,TNF-αandIL-6

can inducemyocardial damageandpossibly cause

ventricularremodeling.6,34Becausetheseinflamma-

torycytokinesarestronginducersofnitricoxideand

reactiveoxygenspecies,theymaypromotecardiac

injuryandelectricalremodelingthroughprecipita-

tion of hyper-oxidative conditions.Niwano et al.9

previouslyreportedthattheanti-oxidantN-acetyl-

cysteinesuppressedventricularremodelinginEAM

rats.Concordantly,wehavedemonstratedthatanti-

inflammatorysteroidtherapysuppressesinfiltration

of inflammatory cells and EAM-induced electrical

remodeling.Moreover,steroidpretreatmentwasas-

sociatedwithimprovedsurvival.Thisindicatesthe

importanceofinflammationandoxidativestressin

remodelingandtheprogressionofmyocarditis.

Studylimitations

Inthisstudy,weinducedmyocarditisbyinjection

of cardiac myosin. Therefore, the arrhythmogenic

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VOL.15 NO.2

mechanismsdelineatedmaynotberepresentativeof

thoseunderlyingmyocarditiscausedbyviralinfection

orotheretiologies.Nonetheless,autoimmunizationto

myosinmayrepresentafinalcommonpathwayof

myocarditis.Moreover, inflammationandoxidative

stressarefrequentlyobservedindiseasedhearts.

Secondly, electrophysiological tests and optical

mappingwereperformedonthe14thdayafterini-

tialimmunization.Toidentifyindividualdifferences

afterinductionofmyocarditis,itwouldbeusefulto

performopticalmappingatdifferenttimepointsaf-

ter immunization. Because an electrophysiological

testcouldnotbeperformedinratsthatsuccumbed

tosuddendeath,wecouldnotprovidedirectevidence

thatAPDdispersionanddiscordantalternanswere

relatedtomortality.

Finally,ventricularfibrillationwasrecordedduring

suddendeathinratswithmyocarditis.However,be-

causeHoltermonitoringwasperformedinonly5rats

thatdiedsuddenly,wecannotruleoutthepossibility

thattheAVblockwasresponsibleforsuddendeath.

Conclusion

EAM-inducedarrhythmia isassociatedwith in-

creasedrepolarizationdispersionandspatiallydis-

cordantalternans.Theseelectricalchangesmaybe

relatedtoalteredCa2+handlingproteinactivity,in-

cludingphosphorylationofCaMKIIandRyR2.These

arrhythmogeniceffectsareassociatedwithincreased

inflammationandoxidativestress,andinthisstudy

wereattenuatedbyanti-inflammatorytherapy.

Sourcesoffunding

Thisstudywassupportedinpartbyresearchgrants

fromYonseiUniversityCollegeofMedicine(8-2011-

0250,7-2011-0758,7-2011-0702,7-2011-0015),a

grantfromtheKoreanHeartRhythmSociety(2011-

3)andtheBasicScienceResearchProgram(2012-

0007604, 2012-045367) through theNationalRe-

searchFoundationofKorea,fundedbytheMinistry

ofEducation,ScienceandTechnology.

Disclosures

None.

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