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Mechanisms Underlying EpicardialRadiofrequency Ablation to SuppressArrhythmogenesis in ExperimentalModels of Brugada Syndrome

Bence Patocskai, MD,a,b Namsik Yoon, MD,b,c Charles Antzelevitch, PHDb,d,e

ABSTRACT

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OBJECTIVES This study sought to test the hypothesis that elimination of sites of abnormal repolarization,

via epicardial radio frequency ablation (RFA), suppresses the electrocardiographic and arrhythmic manifestations of

Brugada syndrome (BrS).

BACKGROUND BrS is associated with ventricular tachycardia and ventricular fibrillation leading to sudden cardiac

death. Nademanee et al. reported that RFA of right ventricular outflow tract epicardium significantly reduced the

electrocardiographic and arrhythmic manifestations of BrS. These authors concluded that low-voltage fractionated

electrogram activity and late potentials are caused by conduction delay within the right ventricular outflow tract and that

the ameliorative effect of RFA is caused by elimination of this substrate. Szel et al. recently demonstrated that the

abnormal electrogram activity is associated with repolarization defects rather than depolarization or conduction defects.

METHODS Action potentials (AP), electrograms, and pseudoelectrocardiogram were simultaneously recorded from

coronary-perfused canine right ventricular wedge preparations. Two pharmacological models were used to mimic

BrS genotype: combination of INa blocker ajmaline (2 to 10 mM) and IK-ATP agonist pinacidil (1 to 5 mM); or combination of

Ito agonist NS5806 (4 to 10 mM) and ICa blocker verapamil (0.5 to 2 mM). After stable induction of abnormal

electrograms and arrhythmic activity, the preparation was mapped and epicardial RFA was applied.

RESULTS Fractionated low-voltage electrical activitywas observed in right ventricular epicardiumbut not endocardium as a

consequence of heterogeneities in the appearance of the second upstroke of the epicardial AP. Discrete late potentials

developed as a result of delay of the second upstroke of the AP and of concealed phase 2 re-entry. Epicardial RFA of these

abnormalities normalized Brugada pattern and abolished arrhythmic activity, regardless of the pharmacological model used.

CONCLUSIONS Our results suggest that epicardial RFA exerts its ameliorative effect in the setting of BrS by destroying

the cells with the most prominent AP notch, thus eliminating sites of abnormal repolarization and the substrate for ven-

tricular tachycardia and ventricular fibrillation. (J Am Coll Cardiol EP 2017;3:353–63) © 2017 by the American College of

Cardiology Foundation.

B rugada syndrome (BrS) is an inherited cardiacarrhythmia syndrome associated withincreased vulnerability for ventricular tachy-

cardia (VT) and fibrillation (VF) leading to sudden

m the aDepartment of Pharmacology and Pharmacotherapy, University of Sz

dical Research Laboratory, Utica, New York; cDepartment of Cardiology, C

blic of Korea; dLankenau Institute for Medical Research, Wynnewood, Pen

wood, Pennsylvania. Dr. Antzelevitch is supported by National Institutes o

rris Fund. All other authors have reported that they have no relationships

authors attest they are in compliance with human studies committees and

d Food and Drug Administration guidelines, including patient consent whe

nical Electrophysiology author instructions page.

nuscript received June 30, 2016; revised manuscript received September

arrhythmic death, without the manifestation of struc-tural heart disease. BrS is characterized by distinctiveJ-wave or ST-segment elevation in the right precor-dial electrocardiogram (ECG) leads. Three types of

eged Faculty ofMedicine, Szeged, Hungary; bMasonic

honnam National University Hospital, Gwangju, Re-

nsylvania; and the eLankenau Heart Institute, Wyn-

f Health grant HL47678 and the Wistar and Martha

relevant to the contents of this paper to disclose.

animal welfare regulations of the authors’ institutions

re appropriate. For more information, visit the JACC:

26, 2016, accepted October 20, 2016.

ABBR EV I A T I ON S

AND ACRONYMS

AP = transmembrane action

potential

BrS = Brugada syndrome

ECG = electrocardiogram

EDR = epicardial dispersion of

repolarization

EG = electrogram recorded

using bipolar electrodes

Endo = endocardial

Epi = epicardial

P2R = phase 2 re-entry

RFA = radiofrequency ablation

RV = right ventricular

RVOT = right ventricular

outflow tract

TDR = transmural dispersion of

repolarization

VF = ventricular fibrillation

VT = ventricular tachycardia

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ECG morphology are generally recognized,but type I (coved type) is the only one diag-nostic for the syndrome. The electrographicmanifestations are often dynamic or con-cealed and can be unmasked using potent so-dium channel blockers. The most effectiveand most commonly used agents are ajma-line, procainamide, and pilsicainide. Thepathophysiology of the disease has longbeen a subject of debate.

The 2 principal hypotheses are the repo-larization hypothesis and the depolarizationhypothesis. In the repolarization hypothesis,an outward shift in the balance of current inthe right ventricular outflow tract (RVOT)epicardium, the region displaying a highesttransient outward current (Ito) density andaction potential (AP) notch, leads to repolar-ization defects resulting in heterogeneousaccentuation of the AP notch and loss of thedome. The resulting dispersion of repolari-zation is responsible for the development of

phase 2 re-entry (P2R) and polymorphic VT. Thetransmural voltage-gradient created by the manifes-tation of a prominent AP notch in epicardium but notendocardium is responsible for inscription of theJ-wave, which when broad and tall is often referred toas ST-segment elevation. In the depolarizationhypothesis, conduction delay within the RVOT givesrise to the Brugada ECG and to the development ofre-entrant arrhythmias.

SEE PAGE 364

The most compelling evidence in support of thedepolarization hypothesis derives from the recentelegant work of Nademanee et al. (1). They recordedfractionated electrogram (EG) activity and late poten-tials from the anterior RVOT epicardium in patientswith BrS and showed that radiofrequency ablation(RFA) of these epicardial (Epi) sites produced anameliorative effect by reducing the manifesta-tion of the BrS ECG and by suppressing the induction ofVT/VF. Because fragmented EG activity is traditionallyattributed to conduction abnormalities, the authorsconcluded that the underlying electrophysiologicalmechanism in patients with BrS is delayed depolari-zation over the anterior aspect of the RVOT epicardium(1). Similar results and conclusions were arrived at byBrugada et al. (2), who also demonstrated the ability offlecainide to identify the substrate for ablation.

Szel and Antzelevitch (3) recently tested this hy-pothesis and provided evidence in support of analternative pathophysiologic basis for the EG abnor-malities described by Nademanee et al. (1), showing

that fractionated EG activity and late potentials canarise as a consequence of repolarization defects.The present study was designed to test the hypothe-sis that RFA suppresses arrhythmogenesis in thesetting of BrS by abolishing the substrate responsiblefor these repolarization abnormalities.

METHODS

ARTERIALLY PERFUSED RIGHT VENTRICULAR

WEDGE PREPARATION. All experiments were per-formed using arterially perfused canine right ventric-ular (RV) wedge preparations. Methodological detailsare as previously described (4). Transmembrane APswere simultaneously recorded from 2 Epi and 1 endo-cardial (Endo) or midmyocardial sites together withbipolar EG and a pseudo-ECG positioned along thetransmural axis. Bipolar EGs were obtained using aquadripolar catheter (Livewire 7-F with 4-mm tipand 2-5-2-mm spacing, St. Jude Medical, St. Paul,Minnesota) and Teflon-insulated silver electrodes.

BrS MODELS. BrS was pharmacologically mimickedby targeting ion-channel currents affected by muta-tions associated with BrS (5). Two distinct modelswere created using a combination of agents thatinhibit inward (depolarizing) currents and that in-crease outward (repolarizing) currents. The firstmodel was designed to mimic a gain of function of thetransient outward potassium current (Ito) using the Itoagonist NS5806 (4 to 10 mM) and a loss of function ofcalcium channel current (ICa) using the ICa blockerverapamil (0.5 to 2 mM). The second model wasdesigned to mimic a gain of function of the adenosinetriphosphate–sensitive potassium current (IK-ATP) us-ing the IK-ATP agonist pinacidil (1 to 5 mM) and a loss offunction of fast sodium channel current (INa) usingthe class IA INa blocker ajmaline (2 to 10 mM). Theconcentration of these compounds was increaseduntil the development of fractionated EG activity,late potentials, closely coupled premature ventricularcomplexes, and polymorphic tachycardia (VT) and/orfibrillation (VF), either spontaneously or followingprogrammed electrical stimulation.

ARRHYTHMOGENIC SUBSTRATES AND ABLATION

PROCESS. After stable induction of arrhythmo-genesis, the preparations were mapped using thequadripolar catheter to identify the arrhythmic sub-strates. Abnormal EGs were defined as potentials withprolonged duration ($80 ms), fractionated potentials(consisting of 2 ormore distinct components separatedby $20 ms isoelectric segment), or late potentials(discrete high-frequency potentials appearing afterthe end of the QRS complex). RFA was performed

TABLE 1 Electrocardiogram and AP Parameters in Epicardial Ablation Experiments

ControlProvocative

AgentsEpicardialAblation

NS5806 þ verapamil*

J-wave arear 3.07 � 1.04 42.68 � 5.77† 5.11 � 1.06‡

Tpeak-Tend, ms 30.53 � 5.27 115.27 � 7.05† 45.18 � 2.43‡

AP notch arear 4.74 � 2.10 60.05 � 5.24† 18.19 � 2.88‡

EDR, ms 3.6 � 1.24 150.25 � 18.74† 36.38 � 17.18‡

TDR, ms 11.38 � 3.76 107.4 � 14.91† 23.4 � 12.82‡

Pinacidil þ ajmaline§

J-wave arear 2.90 � 0.48 26.75 � 1.83† 5.61 � 3.28‡

Tpeak-Tend, ms 28.65 � 4.61 95.38 � 9.21† 31.55 � 11.99‡

AP notch arear 9.38 � 2.25 54.90 � 14.93† 4.56 � 0.90‡

EDR, ms 8.1 � 5.64 106.2 � 20.49† 12.4 � 9.5‡

TDR, ms 12.33 � 3.44 65.55 � 18.17† 4.43 � 2.49‡

AllkJ-wave arear 3.0 � 0.63 36.31 � 4.28† 5.31 � 1.35‡

Tpeak-Tend, ms 29.78 � 3.48 107.31 � 6.2† 39.73 � 5.11‡

AP notch arear 6.6 � 1.65 57.99 � 6.29† 12.74 � 2.79‡

EDR, ms 5.4 � 2.3 132.63 � 14.99† 26.79 � 11.21‡

TDR, ms 11.76 � 2.51 90.66 � 12.84† 15.81 � 8.07‡

Values are mean � SEM. *n ¼ 6. †p < 0.05 vs. control. ‡p < 0.05 vs. pre-ablation. §n ¼ 4. kn ¼ 10.

AP ¼ action potential; EDR ¼ epicardial dispersion of repolarization; TDR ¼ transmural dispersion of repo-larization; Tpeak-Tend ¼ T wave peak-to-end duration.

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using the previously described catheter and BiosenseWebster–Stockert 70-F radiofrequency generator sys-tem (Biosense Webster, Diamond Bar, California; andStockert GmbH, Freiburg, Germany) at 30 W/65�Cmaximal parameters, avoiding coronary arteries. Thevasculature was further protected by increased coro-nary perfusion rate at the time of ablation. Thevascular resistance was continuously monitored byperfusion pressure. At the time of ablation, the prep-aration was turned sideways so that the epicardiumwas on top, and was covered with a thin (1 to 2 mm)layer of Tyrode solution. Deeper immersion of theablation catheter results in distribution of RFA energyinto the bath solution instead of the tissue surface.Effective convection was controlled by monitoringRFA parameters, using EP-Win software (BiosenseWebster). The endpoint of ablation process was thecomplete elimination of abnormal EGs.

Immediately following ablation, we washed outthe provocative agents to prevent desensitization orhomogeneous loss of the AP dome throughoutepicardium and subepicardium, thus abolishing Epidispersion of repolarization (EDR) responsible for thevulnerable window that permitted the developmentof arrhythmogenesis.

After an average of 1 h of recovery, the provocativeagents were reintroduced to the perfusate in the sameconcentration, as before ablation. Recordings wereobtained for an additional 2 h. As a control, we per-formed the same ablation protocol on the endocardiuminstead of epicardium. In previous studies, we haveperformed time controls demonstrating the viability ofthese preparations over a 4- to 5-h period (4,6–8).

The methods for measurement of electrophysio-logical and electrocardiographic parameters areprovided in the Online Appendix. Online Figure 1schematically illustrates the parameters measured.

STATISTICAL ANALYSIS. Parameters of noninducibleand inducible experiments were compared using Stu-dent t test (2-tailed p values are shown at eachparameter). For multiple comparisons, 1-way repeatedmeasures analysis of variance was used followed byall-pairwise comparisons using the Bonferronimethod. Data are shown as mean � SEM throughoutthe study.

RESULTS

INDUCTION OF THE ELECTROCARDIOGRAPHIC AND

ARRHYTHMIC MANIFESTATIONS OF BrS. Pinac id i l D

a jmal ine BrS model . After the addition of pinacidil(1 to 5 mM) þ ajmaline (2 to 10 mM) to the coronaryperfusate, we observed a significant increase inmaximal J-wave area, AP notch area, EDR, TDR, and

interval between the peak and the end of the T-wave(Figures 1A, 2B, 2C, 6, and 7, Table 1).

Abnormal EGs, P2R activity, and VT/VF developedin 6 of 11, 6 of 11, and 4 of 11 preparations, respectively(Figures 1A, 2B, and 2C, Online Table 1). EGs displayingfractionated low-voltage activity and discrete latepotentials developed exclusively in the presence ofaccentuated spike-and-dome morphology and con-cealed P2Rs, respectively (Figures 1A and 2B). Prepa-rations that failed to develop the Brugada pattern andarrhythmic activity (“noninducible”), either sponta-neously or in response to programmed electricalstimulation, displayed significantly lower J-wave andAP notch area at baseline and after the addition of theprovocative agents, than those in which the provoc-ative agents were successful in inducing the ECG andarrhythmic manifestations of BrS (“inducible”)(Figure 1B). At baseline, inducible versus non-inducible values were 4.5 � 1.1 versus 1.1 � 0.2(p ¼ 0.0238) for J-wave area, and 9.4 � 1.5 versus 2.4� 0.3 (p ¼ 0.002) for AP notch area (Figures 1C and 1D).

Interestingly, in preparations displaying smallbasal AP notch (Figure 1B), ajmaline decreased theJ-wave and AP notch area, presumably because of themultiple effects on other ion currents, including Itoand widening of the QRS engulfing the J-wave(Figures 1 and 3B) (9). These observations are consis-tent with clinical studies reporting improvement inthe ECG manifestation of early repolarization pattern

FIGURE 1 Effect of a Combination of Pinacidil and Ajmaline to Induce Arrhythmogenesis and Late Potentials Depends Exclusively on the

Magnitude of the Epicardial Action Potential Notch and Consequential J-Wave at Baseline

(A and B) Each column shows action potentials simultaneously recorded from an endocardial (Endo) and 2 epicardial (Epi1 and Epi2) sites

together with a bipolar epicardial electrogram (Bipol. Epi EG) and an electrocardiogram (ECG) recorded across the bath. (A) Preparation

exhibiting a large spike and dome action potential morphology at baseline (control). The provocative agents induce a Brugada syndrome ECG

and concealed phase 2 re-entry giving rise to distinct late potentials (Bipol. Epi EG). (B) Preparation exhibiting a relatively small spike and dome

action potential morphology at baseline. The provocative agents do not induce a Brugada syndrome ECG, but diminish the J-wave. (C and D)

Comparison of epicardial action potential notch arear and J-wave arear of preparations vulnerable (inducible) and nonvulnerable (noninducible)

to the induction of Brugada syndrome pattern and arrhythmias. n ¼ 6 for inducible and n ¼ 5 for noninducible preparations. (C) Parameters at

baseline. Inducible preparations showed an average 3.9-fold higher action potential notch and 4.3-fold higher J-wave area at baseline,

compared with the noninducible ones (inducible vs. noninducible, p ¼ 0.002 and p ¼ 0.024 for notch arear and J-wave arear, respectively). (D)

After the addition of provocative agents, inducible preparations showed a pronounced increase (p ¼ 0.004 vs. baseline), whereas noninducible

preparations showed a significant decrease (p ¼ 0.017 vs. baseline) in both J-wave and action potential notch area. The provocative agents

produced an average 60.5-fold higher notch area and 88.7-fold higher J-wave area in inducible compared with noninducible preparations

(inducible vs. noninducible; p < 0.001 and p ¼ 0.004 for notch area and J-wave arear, respectively).

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FIGURE 2 Radiofrequency Ablation of Epi Suppresses the Electrocardiographic and Arrhythmic Manifestations of Brugada Syndrome in

Coronary-Perfused Canine Right Ventricular Wedge Model Generated Using a Combination of Pinacidil þ Ajmaline

Traces are as described in Figure 1. (A) Epi displays pronounced action potential notch at baseline. (B) Addition of pinacidil (2 mM) and ajmaline

(3 mM) to the coronary perfusate induces the typical Brugada syndrome phenotype. The bipolar epicardial electrogram (Bip. Epi EG) shows

fractionated electrogram activity and a late potential due to the development of concealed phase 2 re-entry. (C) Successful conduction of

phase 2 re-entrant beat gives rise to ventricular tachycardia. (D) Recorded 40 min after Epi ablation and withdrawal of the provocative agents.

Action potential recordings were obtained from midmyocardial (Mid) and subepicardial (Subepi) layers due to ablation of the epicardial cells. (E)

Recorded 10 min after reintroduction of the provocative agents to the perfusate (in the same concentration as before). After Epi ablation,

Brugada syndrome phenotype and arrhythmias were no longer inducible. Abbreviations as in Figure 1.

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following ajmaline infusion (Online Figure 2) (10,11).To confirm these findings, we compared the effect ofhigh-dose ajmaline (10 mM) in the absence and pres-ence of the Ito agonist NS5806, in the same prepara-tion. The results supported our conclusion that theeffect of ajmaline is dependent on the magnitude ofAP notch before introduction of ajmaline. This alsoprovides an explanation for the RVOT predominanceof the disease, because this region of the heart dis-plays the most prominent AP notch (12). As illustratedin Figure 3, ajmaline (10 mM), when applied alone,reduced the size of the J-wave and AP notch(Figure 3B). Ajmaline, in this setting, produced littleeffect on the AP dome, ST-segment, or Epi EG despiteprolongation of QRS duration and slowing of trans-mural conduction. The effect was reversible on

washout. When the Epi AP notch was accentuated bypre-treatment with the Ito agonist NS 5806 (7 mM)(Figure 3D), addition of ajmaline (10 mM) led tomarked accentuation of Epi AP notch, leading to thedevelopment of abnormal EG activity, type I ST-segment elevation, and concealed P2R. Theappearance of abnormal EG activity was secondaryto the inhomogeneous accentuation of the AP notchand loss of the AP dome (Figures 3E and 3F). Thisassociation is further supported by the observationthat at the maximal effect of high-dose ajmaline,loss of the Epi AP dome throughout the preparationresulted in loss of the fractionated EG activity,despite the further prolongation of QRS durationand further slowing of transmural conduction(Figure 3G).

FIGURE 3 The Opposite Effects of Ajmaline to Mask or Accentuate the J-Wave (Jw) Depend on the Basal Level of Ito-Mediated Action Potential Notch

Traces are as described in Figure 1. The bipolar electrograms (Bip.-EG-Epi) were recorded from the epicardium using 3 different low cut filter settings (10 Hz, 30 Hz,

and 100 Hz) and 250 Hz “high cut” filter. When action potential notch was small (A), 10 mM ajmaline produced a decrease in J-wave and action potential notch

area (B). The effect was reversible on wash-out (C). However, when the action potential notch was amplified using the Ito agonist NS5806 (D), the same concentration of

ajmaline caused a marked accentuation of the J-wave appearing as an ST-segment elevation (E to G). Fragmented electrogram activity developed progressively as the

repolarization defects became more pronounced and heterogeneous (D to F). Pronounced action potential notch (without re-entry) produced delayed potentials in a

lower frequency range (D), whereas phase 2 re-entry depicted as “high-frequency” spike (E and F). After 15 min of ajmaline, loss of the action potential dome

occurred throughout the preparation, which led to disappearance of the late potentials (G). Subendo/Mid ¼ action potential from the subendocardium/midmyocardium;

other abbreviations as in Figure 1.

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Inducibility of the BrS phenotype was independentof conduction-dependent parameters. There wasno difference in QRS duration and transmural con-duction time between inducible and noninduciblepreparations (inducible vs. noninducible at baseline,p ¼ 0.242 and p ¼ 0.822 for QRS and conduction time,respectively).NS5806 D verapami l BrS model . The addition ofNS5806 (4 to 10 mM) þ verapamil (0.5 to 2 mM) to thecoronary perfusate significantly increased maximalJ-wave area (3.1 � 1.0 ms vs. 42.7 � 5.8 ms), AP notcharea (4.7 � 2.1 ms vs. 60.1 � 5.2 ms), EDR (3.6 � 1.2ms vs. 150.3 � 18.7 ms), TDR (11.4 � 3.8 ms vs. 107.4 �14.9 ms), and interval between the peak and the endof the T wave (30.53 � 5.27 ms vs. 115.27 � 7.05 ms)when compared with control (Figures 6 and 7, Table 1).

Fractionated EG and/or late potentials, P2R activity,and VT/VF developed in 9 of 9 experiments (Figure 4,Online Table 1).

Fractionated electrical activity was observed inRV epicardium but not endocardium as a conse-quence of heterogeneities in the appearance ofthe second upstroke of the Epi APs (Figures 4B and4C). Discrete late potentials developed as a result ofmajor delays in the appearance of the second APupstroke and a result of concealed P2R (Figures 4Band 4C).

Both pharmacological models of BrS exhibitedabnormal EGs diffusely dispersed throughoutmuch of the epicardium, as observed by Nademaneeet al. (1) in the RVOT of hearts of human victimsof BrS. Late potentials associated with delayed

FIGURE 4 Radiofrequency Ablation of Epi Suppresses the Electrocardiographic and Arrhythmic Manifestations of Brugada Syndrome in Coronary-Perfused

Canine Right Ventricle Wedge Model Generated Using a Combination of NS5806 þ Verapamil

Traces are as described in Figure 1. (A) Control. (B to D) The addition of NS5806 7 mM and verapamil 2 mM to the perfusate induced pronounced J-waves and phase 2

re-entry depicting as abnormal electrogram activity when concealed, and giving rise to ventricular fibrillation when it succeeds in propagating out. (E and F) Recovery

period of the preparation after epicardial ablation. Note the normalization of ST-segment elevation after 70 min. Action potential recordings were obtained from

midmyocardial (Mid) and subepicardial layers due to inactivation of the epicardium. (G) With superficially ablated epicardium, the readministration of the provocative

agents (in the same concentration as before) did not produce pronounced J-waves or arrhythmic activity. (H) Photograph of wedge preparation after epicardial

ablation to a depth of 1 to 2 mm, taken at the end of the experiment. Abbreviations as in Figure 1.

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second upstroke of the RV Epi AP and with concealedP2R appeared with a delay of up to 209 ms after theQRS, averaging 103.4 � 32.4 ms (n ¼ 926), very similarto the delays reported by Nademanee et al. (1).

EFFECT OF RFA. Epi ablation prevented the devel-opment of VT/VF in 10 of 10 and P2R in 9 of 10preparations, irrespective of the pharmacologicalmodel used (Figures 2 to 4, Online Table 1), whereasEndo ablation failed to suppress arrhythmogenesisin all preparations tested (0 of 5) (Figure 5F, OnlineTable 1). Epi ablation significantly reduced J-wavearear (36.31 � 4.28 vs. 5.31 � 1.35; p < 0.001), APnotch arear (57.99 � 6.29 vs. 12.74 � 2.79; p < 0.001),EDR (132.63 � 14.99 ms vs. 26.79 � 11.21 ms;p < 0.001), TDR (90.66 � 12.84 ms vs. 15.81 � 8.07ms; p < 0.001), and interval between the peak andthe end of the T-wave (107.31 � 6.2 ms vs. 39.73 �5.11 ms; p < 0.001) compared with pre-ablation, inall preparations tested (Figures 2E, 4G, 6, and 7). Incontrast, Endo ablation did not alter theseparameters.

As expected, Epi ablation caused temporary ST-segment elevation, whereas Endo ablation resulted intemporary ST-segment depression, as a result of thedevelopment of injury currents. Both dissipated overa period of an h or more (but did not always normalizecompletely).

DISCUSSION

The seminal study of Nademanee et al. (1) showing anameliorative effect of Epi ablation of regions of frac-tionated EG activity in the RVOT of patients with BrSwas interpreted to suggest that ablation of regions ofdelayed conduction is responsible for suppression ofarrhythmogenesis. The present study provides a testof an alternative hypothesis. In the 2 different modelsof BrS used in this study, the fractionated bipolar EGactivity and late potentials were not caused by majorconduction delays, but rather by repolarization defectscreated by an outward shift in the balance of currentactive during the early phases of the AP. This outward

FIGURE 5 Radiofrequency Ablation of Endo Fails to Suppress Brugada Syndrome Phenotype

Traces are as described in Figure 1. (A) Control. (B) Recorded 25 min after addition of 8 mMNS5806 and 2 mM verapamil to the coronary perfusate.

Homogeneous delay of the second upstroke of the epicardial action potentials gives rise to a late potential on the bipolar electrogram (EG). (C)

Recorded 50 min after addition of provocative agents. Concealed phase 2 re-entry gives rise to a high-frequency late potential in the bipolar

electrogram. (D) Recorded 10 s later. Successful propagation of phase 2 re-entrant extrasystole initiates polymorphic ventricular tachycardia. (E)

Recorded 80min after Endo ablation andwithdrawal of the provocative agents. (F)Recorded 25min after reintroduction of the provocative agents.

Endo ablation failed to exert any beneficial effect: the reintroduction of provocative agents induced pronounced Brugada syndromephenotypewith

sustained polymorphic tachycardia. Subendo ¼ subendocardium; other abbreviations as in Figure 1.

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shift of current can be achieved either with mutationsor agents that increase outward current or those thatdecrease inward current, or a combination of the 2.

We studied 2 experimental models of BrS using acombination of outward current channel agonists(pinacidil or NS5806) and inward current channelblockers (ajmaline or verapamil), thus mimicking thegenetic defects and ion channel heterogeneitiesknown to be associated with BrS (13).

Although late potentials and fractionated EG ac-tivity are traditionally ascribed to slow or delayedconduction, our results provide further evidence insupport of the hypothesis that in the setting ofBrS, abnormal EG activity can be a consequence ofrepolarization defects, consistent with the recentreport of Szel and Antzelevitch (3). In addition toour previous findings pointing to concealed P2R asthe cause of high-frequency late potentials (3), wereport that delay of the second upstroke of EpiAPs can also manifest as discrete late potentials(“spikes”) (Figures 2B, 2C, 3D, and 5B, Online Figures 3and 4). The magnitude and delay of these

electrophysiological abnormalities depends criticallyon the characteristics of the second upstroke andnotch of the Epi AP (Online Figure 3).

In both models, the extent of repolarization abnor-malities induced by the provocative agents wasdirectly proportional to the degree of phase 1 repolar-ization and AP notch observed under baseline condi-tions (Figure 1). It is noteworthy that homogeneous“loss of the second upstroke” of APs throughout theepicardiumand subepicardium led to disappearance ofthese late potentials, as anticipated (Figure 3G).

The morphology and frequency range of these po-tentials are similar to those described by Nakagawaet al. (14) in early repolarization syndrome–relatedidiopathic VF (Online Figure 4). These observationsmay explain the lower frequency range of latepotentials recorded in signal-averaged electrocardio-gram of patients with BrS when compared withlate potentials associated with arrhythmogenic RVdysplasia, a disease associated with significant con-duction delay secondary to structural defects causedby fibrofatty replacement of cardiomyocytes (15–17).

FIGURE 6 J-Wave and Maximal Action Potential Notch Area Recorded at Each Step of the Epicardial Ablation Experiments

in the 2 Models

Addition of the provocative agents, NS5806 þ verapamil (NS þ Ver; n ¼ 6) or ajmaline þ pinacidil (Ajm þ Pin; n ¼ 4), significantly increased,

whereas epicardial ablation significantly decreased J-wave area (A) and maximal action potential notch area (B). *p < 0.001 vs. control.

†p < 0.001 vs. pre-ablation. ‡p ¼ 0.027 vs. control. §p ¼ 0.017 vs. pre-ablation.

FIGURE 7 Tpeak-Tend Interval and Dispersion of Repolarization Recorded at Each Step of Epicardial Ablation Experiments

in the 2 Models

(A) Addition of the provocative agents, either NS5806 þ verapamil (NS þ Ver; n ¼ 6) or pinacidil þ ajmaline (Pin þ Ajm; n ¼ 4), significantly

increased, whereas radiofrequency ablation of epicardium significantly reduced Tpeak-Tend intervals. (B) Addition of the provocative agents

significantly increased but epicardial ablation significantly reduced both TDR and EDR. Dark colors represent EDR, pale colors represent TDR.

The highest Tpeak-Tend values were associated with the most pronounced delay of the second action potential upstroke giving rise to negative

T waves and appeared just before the start of arrhythmic activity. *p < 0.01 vs. control. †p < 0.01 vs. pre-ablation. ‡p < 0.001 vs. control. §p <

0.001 vs. pre-ablation. kp # 0.015 vs. control. ¶p # 0.028 vs. pre-ablation. EDR ¼ epicardial dispersion of repolarization; TDR ¼ transmural

dispersion of repolarization; Tpeak-Tend ¼ interval between the peak and the end of the T-wave.

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The fractionation of the Epi EG and appearance oflate potentials following the addition of ajmaline isvery similar to that recorded by Sacher et al. (18) inthe epicardium of the RVOT of a BrS patient.Although the authors interpreted this phenomenon asa proof of depolarization abnormality, in our experi-ments, ajmaline exerted these effects via accentua-tion of the AP notch and induction of P2R, and not viaa slowing of conduction (Figures 1 and 3).

In preparations exhibiting a relatively small APnotch, ajmaline failed to produce any sign of BrS, evenat relatively high concentrations (Figures 1B and 3B,Online Figure 2). These observations explain whyajmaline produces ST-segment elevation in the rightprecordial ECG leads of BrS patients, but fails to pro-voke a Brugada pattern in other ECG leads or inhealthy subjects. This observation may also explainthe recent observations of Park et al. (19). These au-thors genetically engineered Yucatan minipigs toheterozygously express a nonsense mutation inSCN5A (E558X) originally identified in a child with BrS.Patch clamp analysis of atrial myocytes isolated fromthe SCN5AE558X/þ pigs showed a loss of function of INa.Conduction abnormalities consisting of prolongationof P-wave, QRS complex, and PR interval wereobserved, but a BrS phenotype was not observed, noteven after the administration of flecainide. These ob-servations are expected because of the lack of Ito andlack of an AP notch in the pig, which is a prerequisitefor the development of the repolarization abnormal-ities associated with BrS. These findings collectivelylend strong support for the repolarization hypothesis.

Zhang et al. (20) recently performed noninvasiveECG imaging on 25 BrS and 6 right bundle branchblock patients. The authors reported the presence ofslow discontinuous conduction and steep dispersionof repolarization in the RVOT of patients with BrS. In6 BrS patients the response to an increase in rate wasexamined. Increasing rate increased fractionation ofthe EG but reduced ST-segment elevation (Brugadaphenotype), indicating that the conduction impair-ment was not the principal cause of the BrS ECG.

If, as suggested by our findings, abnormal EG ac-tivity in the setting of BrS is not caused by major con-duction delay, then why is ablation effective innormalizing the Brugada pattern and preventing thedevelopment of VT? Our results suggest that ablation iseffective because it destroys the cells with the mostprominent AP notch in ventricular epicardium, pre-sumably the cells with the largest AP notch and highestIto density, thus preventing the development ofaccentuated repolarization abnormalities that areresponsible for causing a pronounced Epi and TDR, thesubstrates for the genesis of P2R and VT. Our results

show that pronounced repolarization heterogeneitiescan recapitulate the electrographic manifestations ofthe BrS and the response to Epi RFA.

STUDY LIMITATIONS. It should be emphasized, thatour study is not aimed at proving the exclusivity ofrepolarization hypothesis. It does not deny thepossible contribution of slow or delayed conduction tothe development of arrhythmogenesis in the setting ofBrS, but does provide support for the hypothesis that inexperimental models that mimic the principal geneticfactors and ion channel heterogeneities responsible forBrS, repolarization abnormalities alone are capable ofcausing the ECG and arrhythmic manifestations of thesyndrome.

CONCLUSIONS

There is no doubt that depolarization abnormalitiescan contribute to arrhythmogenesis in BrS and thatseveral factors can modulate both the degree of repo-larization and depolarization abnormalities, includingthe degree of electrotonic coupling, transmural dif-ferences in tissue resistivity, differences in theexpression of connexin-43, and transmural differ-ences in the expression of other currents (7,21–24). Arecent study by Nademanee et al. (25) suggests thatmicroscopic fibrosis plays a role in the pathophysi-ology of BrS. Regardless of whether it is a late-stage by-product or the original primary cause of BrS, this canlead to conduction impairment. To the extent thatpropagation of an AP dome is similar to propagation ofan early afterdepolarization, fibrosis has also beenshown to markedly reduce the number of myocyteswith abnormal repolarization required to overcomethe source-sink effect and thus generate a propagatedimpulse. It is intriguing to speculate that the devel-opment of mild fibrosis in the RVOT of patients withBrSmay increase arrhythmic risk by promoting (25) thepropagation of otherwise silent triggers, as has beendemonstrated in the case of early and delayed after-depolarizations (26,27). It seems reasonable to suggestthat this facilitation can also apply to the propagationof the AP dome giving rise to P2R.

ACKNOWLEDGEMENTS The authors thank BiosenseWebster for making the radiofrequency ablationequipment available for this study; and RobertGoodrow and Dr. José Di Diego for their kind technicalsupport.

ADDRESS FOR CORRESPONDENCE: Dr. CharlesAntzelevitch, Lankenau Institute for Medical Research,100 East Lancaster Avenue, Wynnewood, Pennsylvania19096. E-mail: cantzelevitch@gmail.com.

PERSPECTIVES

COMPETENCY IN MEDICAL KNOWLEDGE: RFA is an

emerging therapy for Brugada syndrome, providing an

adjunct or replacement to implantable cardioverter-

defibrillator. Identification of substrates amenable to RFA

may be helpful in identification of individuals who can

benefit from this procedure.

TRANSLATIONAL OUTLOOK 1: Understanding the

basis for low-voltage fractionated electrogram activity

and high-frequency late potentials in the right ventricular

outflow tract may be helpful in targeting RFA and

adjunctive pharmacological therapy.

TRANSLATIONAL OUTLOOK 2: Demonstration that

low-voltage fractionated electrogram activity and late

potentials can be caused by defects in repolarization

rather than depolarization or conduction, represents a

paradigm shift in the understanding of such activity with

wide implications for both diagnosis and treatment of

cardiac disease.

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KEY WORDS ECG, electrophysiology,J-wave syndrome, sudden cardiac death,ventricular arrhythmias

APPENDIX For supplemental Methods sec-tion, as well as figures and a table, please seethe online version of this article.