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Multisite conduction block in the epicardial substrate of Brugada syndrome

Open AccessPublished:October 31, 2021DOI:https://doi.org/10.1016/j.hrthm.2021.10.030

      Background

      The Brugada pattern manifests as a spontaneous variability of the electrocardiographic marker, suggesting a variability of the underlying electrical substrate.

      Objective

      The purpose of this study was to investigate the response of the epicardial substrate of Brugada syndrome (BrS) to programmed ventricular stimulation and to Na blocker infusion.

      Methods

      We investigated 6 patients (all male; mean age 54 ± 14 years) with BrS and recurrent ventricular fibrillation. Five had no type 1 BrS electrocardiogram pattern at admission. They underwent combined epicardial-endocardial mapping using multielectrode catheters. Changes in epicardial electrograms were evaluated during single endocardial extrastimulation and after low-dose ajmaline infusion (0.5 mg/kg in 5 minutes).

      Results

      All patients had a region in the anterior epicardial right ventricle with prolonged multicomponent electrograms. Single extrastimulation prolonged late epicardial components by 59 ± 31 ms and in 4 patients abolished epicardial components at some sites, without reactivation by surrounding activated sites. These localized blocks occurred at an initial coupling interval of 335 ± 58 ms and then expanded to other sites, being observed in up to 40% of epicardial sites. Ajmaline infusion prolonged electrogram duration in all and produced localized blocks in 62% of sites in the same patients as during extrastimulation. Epicardial conduction recovery after ajmaline occurred intermittently and at discontinuous sites and produced beat-to-beat changes in local repolarization, resulting in an area of marked electrical disparity. These changes were consistent with models based on microstructural alterations under critical propagation conditions.

      Conclusion

      In BrS, localized functional conduction blocks occur at multiple epicardial sites and with variable patterns, without being reactivated from the surrounding sites.

      Graphical abstract

      Keywords

      Introduction

      Brugada syndrome (BrS) is an arrhythmogenic disorder diagnosed by an electrocardiographic (ECG) pattern associated with an increased risk of ventricular fibrillation (VF). The diagnostic ECG is defined by J-point elevation and >2-mm coved ST-segment elevation in the right precordial leads.
      • Brugada P.
      • Brugada J.
      Right bundle branch block, persistent ST segment elevation and sudden cardiac death: a distinct clinical and electrocardiographic syndrome.
      • Wilde A.A.M.
      • Antzelevitch C.
      • Borggrefe M.
      • et al.
      Proposed diagnostic criteria for the Brugada syndrome: consensus report.
      • Antzelevitch C.
      • Yan G.X.
      • Ackerman M.J.
      • et al.
      J-wave syndromes expert consensus conference report: emerging concepts and gaps in knowledge.
      • Nademanee K.
      • Raju H.
      • de Noronha S.V.
      • et al.
      Fibrosis, connexin-43, and conduction abnormalities in the Brugada syndrome.
      • Yan G.X.
      • Antzelevitch C.
      Cellular basis for the Brugada syndrome and other mechanisms of arrhythmogenesis associated with ST-segment elevation.
      • Corrado D.
      • Baso C.
      • Buja G.
      • Nava A.
      • Rossi L.
      • Thiene G.
      Right bundle branch block, right precordial ST-segment elevation, and sudden death in young people.
      • Coronel R.
      • Casini S.
      • Koopmann T.T.
      • et al.
      Right ventricular fibrosis and conduction delay in a patient with clinical signs of Brugada syndrome: a combined electrophysiological, genetic, histopathologic, and computational study.
      • Bezzina C.R.
      • Barc J.
      • Mizusawa Y.
      • et al.
      Common variants at SCN5A-SCN10A and HEY2 are associated with Brugada syndrome, a rare disease with high risk of sudden cardiac death.
      • Behr E.R.
      • Ben-haim Y.
      • Ackerman M.J.
      • Krahn A.D.
      • Wilde A.A.M.
      Brugada syndrome and reduced right ventricular outflow tract conduction reserve: a final common pathway.
      • Kasanuki H.
      • Ohnishi S.
      • Ohtuka M.
      • et al.
      Idiopathic ventricular fibrillation induced with vagal activity in patients without obvious heart disease.
      • Nagase S.
      • Kusano F.K.
      • Morita H.
      • et al.
      Longer repolarization in the epicardium at the right ventricular outflow tract causes type 1 electrocardiogram in patients with Brugada syndrome.
      This ECG pattern has a peculiarity, common to “J-wave syndromes,” to exhibit spontaneous variability. In clinics, the sensitivity of ECG diagnosis can be increased by the use of high electrode positioning or a Na-channel blocker, which amplifies the electrical marker. There is evidence that an abnormal electrophysiological substrate is predominantly located in the epicardial right ventricle (RV) particularly near the RV outflow tract (RVOT), with regionalized ablation resulting in the elimination of the BrS phenotype and prevention of arrhythmia episodes.
      • Viskin S.
      • Wilde A.M.
      • Tan H.L.
      • Antzelevitch C.
      • Shimizu W.
      Empiric quinidine therapy for asymptomatic Brugada syndrome: time for a prospective registry.
      • Haïssaguerre M.
      • Extramiana F.
      • Hocini M.
      • et al.
      Mapping and ablation of ventricular fibrillation associated with long-QT and Brugada syndromes.
      • Nademanee K.
      • Veerakul G.
      • Chandanamattha P.
      • et al.
      Prevention of ventricular fibrillation episodes in Brugada syndrome by catheter ablation over the anterior right ventricular outflow tract epicardium.
      • Zhang P.
      • Tung R.
      • Zhang Z.
      • et al.
      Characterization of the epicardial substrate for catheter ablation of Brugada syndrome.
      • Pappone C.
      • Brugada J.
      • Vicedomini G.
      • et al.
      Electrical substrate elimination in 135 consecutive patients with Brugada syndrome.
      • Chung F.P.
      • Raharjo S.B.
      • Lin Y.-J.
      • et al.
      A novel method to enhance phenotype, epicardial functional substrates, and ventricular tachyarrhythmias in Brugada syndrome.
      We investigated the electrophysiological substrate with multielectrode mapping by using programmed ventricular stimulation and Na blocker infusion in patients with BrS.

      Methods

      Patient group

      We analyzed 12 patients who presented for evaluation of the previously documented BrS pattern and VF between June 2015 and August 2019. Six patients were excluded as they had a marked type 1 BrS pattern precluding ajmaline infusion (n = 4) or a prior epicardial ablation procedure (n = 2). In the remaining 6 patients, the 12-lead ECG at admission (using conventional lead positioning) showed apparently normal QRST or a discrete nondiagnostic BrS pattern in 5 and a type 1 BrS with right bundle branch block left axis pattern in 1 (Figure 1). A family history of sudden cardiac death was reported in 2 patients, one of them with an SCN5A mutation (Table 1). All 6 patients had a spontaneous type 1 BrS ECG pattern documented previously at the time of arrhythmia episodes. The clinical protocol was approved by the institutional review board, and all patients gave written informed consent. The study complies with the Helsinki Declaration.
      Figure thumbnail gr1
      Figure 1Twelve-lead electrocardiogram of patients at admission. Right precordial leads show normal range patterns in patients 1 and 4; nondiagnostic Brugada syndrome (BrS) patterns in patients 2, 3, and 5; and type 1 BrS pattern in patient 6.
      Table 1Characteristics of patients
      Patient no.SexAge at first symptom (y)Age (y)Family historySCN5A mutationTotal no. of VF episodesECG at admissionQRS duration (ms)
      1Male5152NoNo1No BrS type 1102
      2Male2832NoNo5No BrS type 1112
      3Male5968YesYes4No BrS type 1142
      4Male5663NoNo2No type 1 BrS113
      5Male4445NoNo4No BrS type 192
      6Male5864YesNo4Type 1 BrS

      RBBB left axis
      112
      Six males49 ± 1254 ± 14Two with a family history of SCDOne with an SCN5A mutation3.3 ± 1.5No type 1 BrS in 5/6112 ± 17
      BrS = Brugada syndrome; ECG = electrocardiogram; RBBB = right bundle branch block; SCD = sudden cardiac death; VF = ventricular fibrillation.

      Electrophysiology study

      Antiarrhythmic drugs (quinidine in 2 patients) were interrupted for at least 5 half-lives before the electrophysiology study. Two or 3 catheters were introduced percutaneously through the femoral veins: 1 catheter for programmed stimulation and 1 or 2 multielectrode catheters (10 or 20 electrodes) to record electrograms at the endocardial RV apex. An additional catheter was introduced in the pericardial space via a subxyphosternal approach. Multielectrode 20-pole catheters (Lasso with 25 mm diameter or PentaRay, Biosense Webster, Irvine, CA) were used for epicardial mapping; they had electrode pairs (bipoles) spaced by 2 mm to minimize far-field effects, with bipole pairs separated by 5 mm.
      • Sacher F.
      • Jesel L.
      • Jais P.
      • Haissaguerre M.
      Insight into the mechanism of Brugada syndrome: epicardial substrate and modification during ajmaline testing.
      ,
      • Takigawa M.
      • Relan J.
      • Martin R.
      • et al.
      Detailed analysis of the relation between bipolar electrode spacing and far- and near-field electrogram.
      Surface ECG leads and bipolar intracardiac electrograms bandpass filtered at 30–250 Hz were recorded and stored on a recorder system (LabSystem PRO, Boston Scientific, Marlborough, MA). This system had 40 channels that did not allow recordings of all bipolar and unipolar electrograms in 3 multielectrode catheters. Recordings were performed in the bipolar mode in all and in the unipolar mode (filters 0.05–250 Hz) in 2 patients. Electroanatomic mapping of the RV endocardium and epicardium was performed during sinus rhythm to identify sites of abnormal electrogram characteristics and delineate the underlying ventricular substrate.
      • Nademanee K.
      • Veerakul G.
      • Chandanamattha P.
      • et al.
      Prevention of ventricular fibrillation episodes in Brugada syndrome by catheter ablation over the anterior right ventricular outflow tract epicardium.
      • Zhang P.
      • Tung R.
      • Zhang Z.
      • et al.
      Characterization of the epicardial substrate for catheter ablation of Brugada syndrome.
      • Pappone C.
      • Brugada J.
      • Vicedomini G.
      • et al.
      Electrical substrate elimination in 135 consecutive patients with Brugada syndrome.
      Measurements were performed on 3-dimensional ventricular reconstructions (CARTO System, Biosense Webster) using default filter settings: 2–240 Hz and 30–250 Hz in the unipolar mode and bipolar mode, respectively.
      • Langfield P.
      • Feng Y.
      • Bear L.
      • et al.
      A novel method to correct repolarization time estimation from unipolar electrograms distorted by standard filtering.
      Signal voltage was measured automatically by the CARTO software. Electrogram duration during sinus rhythm was measured from onset to offset of the local bipolar electrogram. Abnormal electrograms were defined by previously described criteria for bipolar potentials: low amplitude <1 mV (epicardial) or <1.5 mv (endocardial) and/or long duration ≥70 ms with multiple (≥3) components during sinus rhythm.
      • Nademanee K.
      • Veerakul G.
      • Chandanamattha P.
      • et al.
      Prevention of ventricular fibrillation episodes in Brugada syndrome by catheter ablation over the anterior right ventricular outflow tract epicardium.
      • Zhang P.
      • Tung R.
      • Zhang Z.
      • et al.
      Characterization of the epicardial substrate for catheter ablation of Brugada syndrome.
      • Pappone C.
      • Brugada J.
      • Vicedomini G.
      • et al.
      Electrical substrate elimination in 135 consecutive patients with Brugada syndrome.
      • Chung F.P.
      • Raharjo S.B.
      • Lin Y.-J.
      • et al.
      A novel method to enhance phenotype, epicardial functional substrates, and ventricular tachyarrhythmias in Brugada syndrome.
      Programmed ventricular stimulation was performed from the RV apex in 2 patients and RVOT in 4 patients by using a basic cycle length of 600 ms, followed by 1 extrastimulus (Table 1). A pacing stimulus of 10 mA was routinely used. The extrastimulus was delivered at an initial coupling interval (CI) of 450 ms and decremented by 10-ms steps until refractoriness. During programmed stimulation, the epicardial multielectrode catheter was positioned in a stable area of abnormal substrate to record local electrograms from 10 bipoles (ie, 10 anatomical sites); another catheter was positioned in the endocardial region close to the RV apex. As shown previously, electrograms in the RV epicardial substrate displayed multicomponent electrograms with distinct early components having slower dV/dt indicating remote (far-field) activity and later components indicating local epicardial activity, respectively.
      • Nademanee K.
      • Veerakul G.
      • Chandanamattha P.
      • et al.
      Prevention of ventricular fibrillation episodes in Brugada syndrome by catheter ablation over the anterior right ventricular outflow tract epicardium.
      • Zhang P.
      • Tung R.
      • Zhang Z.
      • et al.
      Characterization of the epicardial substrate for catheter ablation of Brugada syndrome.
      • Pappone C.
      • Brugada J.
      • Vicedomini G.
      • et al.
      Electrical substrate elimination in 135 consecutive patients with Brugada syndrome.
      • Chung F.P.
      • Raharjo S.B.
      • Lin Y.-J.
      • et al.
      A novel method to enhance phenotype, epicardial functional substrates, and ventricular tachyarrhythmias in Brugada syndrome.
      • Sacher F.
      • Jesel L.
      • Jais P.
      • Haissaguerre M.
      Insight into the mechanism of Brugada syndrome: epicardial substrate and modification during ajmaline testing.
      We measured the differences in conduction times in the 10 recording sites during extrastimulation relative to basic cycle length from the pacing stimulus to the offset of the late component. A late component, which disappeared (absent or voltage amplitude < 0.1 mV) at a given CI and did not then reappear at all shorter CIs, was defined as localized block at this specific site. The loss of only a part of the late component (a deflection) was not considered as localized block.
      Ajmaline (0.5 mg/kg body weight) was administered for 5 minutes, and its effect on epicardial electrograms was evaluated during sinus rhythm without performing programmed stimulation. We measured the total duration of epicardial electrograms (from onset to offset) and the occurrence of localized block at the 10 recording sites. Unipolar epicardial electrograms were examined to evaluate specifically the correlation between the coved-type repolarization morphology relative to localized changes in conduction after ajmaline. Coved-type morphology was defined as a fully positive unipolar complex.
      • Vigmond E.
      • Efimov I.R.
      • Rentschler S.
      • Coronel R.
      • Boukens B.J.
      Fractionated electrograms with ST-segment elevation recorded from the human RVOT.
      • Hoogendijk M.G.
      • Potse M.
      • Linnenbank A.C.
      • et al.
      Mechanism of right precordial ST-segment elevation in structural heart disease: excitation failure by current-to-load mismatch.
      • Ten Sande J.N.
      • Coronel R.
      • Conrath C.E.
      • et al.
      ST-segment elevation and fractionated electrograms in Brugada syndrome patients arise from the same structurally abnormal subepicardial RVOT area but have a different mechanism.

      Statistical methods

      Categorical variables are reported as number (percentage). Continuous variables are reported as mean ± SD. The Wilcoxon matched pairs test was used for paired comparisons of continuous variables. Statistical analysis was performed using GraphPad Prism version 5 (GraphPad Software Inc., San Diego, CA), and a P value of <.05 was considered significant.

      Results

      Patient characteristics

      Patient characteristics are listed in Table 1. All were male, and the age at the time of the study was 49 ± 12 years.

      Electrophysiology study and programmed stimulation

      The RV endocardial mapping showed no abnormalities except in 1 patient. In contrast, epicardial mapping showed abnormal multicomponent electrograms in the anterior RV in all patients and in the inferior RV in 1 patient. A spontaneous variability of late components was observed in 1 patient (Online Supplemental Figure 1A).
      The delivery of premature stimuli progressively increased the delay from stimulus to the epicardial component. This increase was significantly higher over the epicardial substrate than in the endocardial RV, with a maximum (longest of the 10 recording sites) prolongation of 59 ± 31 ms in the epicardium compared with 28 ± 12 ms in the endocardium (P = .031). Such duration (59 ± 31 ms) was not compatible with phase 2 reentry associated with repolarization gradients shown in experimental studies, as the latter occur with longer delays, of the order of 175 ms.
      • Szel T.
      • Antzelevitch C.
      Abnormal repolarization as the basis for late potentials and fractionated electrograms recorded from epicardium in experimental models of Brugada syndrome.
      ,
      • Di Diego J.M.
      • Patocskai B.
      • Barajas-Martinez H.
      • et al.
      Acacetin suppresses the electrocardiographic and arrhythmic manifestations of the J wave syndromes.
      In 4 patients, an abolition of epicardial components was observed at distinct sites (Figures 2 and 3). In these 4 patients, the localized blocks occurred initially at a CI of 335 ± 58 ms and, with further CI shortening, extended to up to 40% of all recording sites (24 of 60) (Table 2). Strikingly, the sites showing a disappearance of epicardial components were not reactivated from contiguous and still conducting sites.
      Figure thumbnail gr2
      Figure 2Effect of single endocardial extrastimulation on epicardial activation in patient 1. The 3-dimensional image (left panel) represents the catheter positions, with the epicardium made transparent. From top to bottom (right panels) are shown electrocardiographic leads II and V1, endocardial electrograms from the right ventricular (RV) outflow tract (RVOT) from a PentaRay catheter, and epicardial electrograms over the abnormal RVOT substrate from a Lasso catheter. Epicardial electrograms during S2 extrastimulation (drive cycle 600 ms delivered from the endocardial RVOT) at decremental coupling intervals (CIs) are shown. Far-field early components are indicated by asterisks. At a CI of 300 ms, a separation of the late electrogram component from the bulk of depolarization occurs at bipoles 9-10 and 17-18 (arrow) and the late component disappeared at bipole 19-20 (block [B]) by comparison to a CI of 400 ms (arrow). Extrastimulation at 250 ms shows loss of the epicardial electrogram (B) at bipole 17-18. Extrastimulation at the shortest CI of 220 ms shows no electrical activity arriving at the epicardial surface (B) at 4 bipoles out of the 10 recording sites. Small potentials are still visible at bipoles 5-6 and 9-10 with an amplitude of >0.1 mV. In this patient, induction of ventricular fibrillation was provoked by inadvertent ventricular premature complexes occurring during programmed stimulation (full tracing shown in ); localized blocks are observed in 6 of the 10 recording sites with a clear distinction of early far-field components (asterisks). The panel on the right shows a schematic representation of epicardial conduction blocks with respect to the Lasso catheter.
      Figure thumbnail gr3
      Figure 3Decremental endocardial extrastimulation in 2 patients. A: Patient 6: Electrocardiographic leads, epicardial electrograms (arrows—during sinus rhythm) from a PentaRay catheter in the right ventricular outflow tract (RVOT) substrate, and endocardial electrograms from the right ventricle (RV). Epicardial electrograms during S2 extrastimulation (drive cycle 600 ms delivered from the endocardial RV apex) at 440 ms show a block (B) of the late electrogram component at bipoles 17-18 and 19-20. Extrastimulation at 360 ms shows loss of epicardial electrograms at 3 additional bipoles (B). Extrastimulation at 320 ms shows that no electrical activity arrives at the epicardial surface at all 10 recording sites (arrows). B: Patient 4 shows similar findings. At 280 ms, loss of epicardial electrograms is observed at 8 bipoles; similar blocks are shown at 260 ms using a smaller gain recording.
      Table 2Conduction changes during single extrastimulation and ajmaline infusion
      Programmed stimulationAjmaline infusion
      Patient no.Site of endocardial stimulationMaximal EGM prolongation in the epicardial substrate (ms)Maximal EGM prolongation in the endocardial RV (ms)Coupling interval associated with first component abolition (ms)Shortest coupling interval of S2 (ms)No. of sites with the abolition of the epicardial EGMEGM duration in sinus rhythm on the epicardial substrateMaximal prolongation of EGM duration in the epicardial substrateNo. of sites with epicardial EGM abolition
      1RVOT36243502204106958
      2RVOT1512220098240 (partial EGM abolition)
      3RV apex781625023021421810
      4RVOT56382802608178569
      5RV apex6436250098570 (partial EGM abolition)
      6RVOT10442360320101625410
      Total59 ± 3128 ± 12335 ± 58250 ± 3824 out 60 (40%)131 ± 3451 ± 2837 out 60 (62%)
      EGM = electrogram; RV = right ventricular; RVOT = right ventricular outflow tract.
      VF was induced inadvertently in 1 patient after spontaneous or mechanical RVOT beats interspersed during programmed stimulation. Localized blocks observed during prior extrastimulation were also observed during VF initial beats, resulting in slower activities on the epicardial side than on the endocardial side of the RVOT (Figures 2 and 4). There was a marked asynchrony of endocardial, far-field, and local epicardial electrograms, without a discernible pattern (2:1, 3:1, etc) of conduction between them, and their average cycle lengths were 171, 164, and 276 ms, respectively. The activation sequences between endocardial and epicardial electrograms were different for each of the 15 initial VF beats, indicating continuously changing patterns of transmural activations.
      Figure thumbnail gr4
      Figure 4Ventricular fibrillation (VF) initiation in the same patient illustrated in . VF occurred inadvertently after the occurrence of spontaneous or mechanical-induced premature beats (red asterisks). Note the first appearance of the premature beat at the right ventricular (RV) endocardium and the second one at the RV outflow tract (RVOT) epicardium (red stars). Induction of VF shows the absence of epicardial electrograms (block [B]) at the same bipoles than during extrastimulation () and the clear distinction of far-field components. During VF, local epicardial electrograms are much slower than far-field components while endocardial electrograms were associated 1:1 with VF beats. The VF cycle lengths (VFCLs) show activation rate in epicardial (276 ms) and endocardial (171 ms) sides of the RVOT. A marked asynchrony of endocardial, far-field epicardial, and local epicardial electrograms is observed, and none of the initial cycles show a similar sequence of electrograms (eg, see the last 4 cycles delimited by blue dashed vertical lines).

      Ajmaline infusion

      Low-dose ajmaline infusion prolonged QRS complexes in all patients and converted the ECGs of 5 patients into a type 1 pattern (the remaining patient having a baseline type 1 ECG pattern). A prolongation of epicardial electrograms of 51 ± 28 ms was observed in the initial 4 minutes of infusion. Then, an abolition of late epicardial components indicating epicardial block was observed in the same 4 patients as during extrastimulation. Overall, 62% of recording sites (37 of 60) (Table 2) displayed localized block. In the remaining 2 patients, only parts (deflections) of the late component were abolished.
      In patients with epicardial component abolition, conduction recovered intermittently in 2 patients after discontinuing ajmaline. Conduction recovery was differentiated from focal firing activity as the electrograms were tightly coupled with endocardial electrograms as measured manually for all events (Figure 5). These changes were not apparent on the 12-lead ECG. Conduction recovery was apparently random, occurring in 1 or several sites, for a single or several beats, with each sequence being different. Electrograms appeared in full integrity or in fragments with variable arrangement of microcomponents. Thus, continuous recordings during sinus rhythm showed an extreme variety in the patterns of epicardial activations, indicating severe impairment of endo-epicardial propagation.
      Figure thumbnail gr5
      Figure 5Intermittent recovery of epicardial activation 3 minutes after the end of ajmaline infusion (patient 3). A: Electrograms before ajmaline infusion. B: Conduction recovery after ajmaline either as small isolated components (dashed arrows) or in several contiguous sites (circle). These electrograms are not dissociated activities due to ectopic firing as they are consistently coupled at the same interval with the initial far-field electrogram component (circles in panel C). RV = right ventricle.
      Unipolar recordings showed simultaneously varying ST-segment morphologies. A coved-type ST segment was observed in sites with localized block. The occurrence of conduction recovery was associated with the disappearance of the coved-type ST segment by conversion into a diphasic pattern with negative T waves. All these beat-to-beat changes in local conduction and repolarization resulted in a variety of simultaneous activation-repolarization potentials in a small area (Figure 6).
      Figure thumbnail gr6
      Figure 6Relationship between conduction recovery and unipolar ST-segment morphology after ajmaline infusion (patient 1). A and B: Bi- and unipolar recordings from 2 opposed parts of the mapping catheter in the epicardial substrate. Conduction recovery is present intermittently (asterisk) and associated with changed unipolar ST-segment morphology and negative T waves (arrows). C: Considerable dispersion of epicardial conduction recovery and its effect on unipolar ST-segment morphologies.

      Discussion

      The present study describes the electrophysiological changes occurring in the epicardial substrate for BrS during extrastimulation and ajmaline infusion. It shows that localized block occurs at multiple discontinuous sites from a single premature stimulus or during sodium blocker infusion.

      Variability of the arrhythmogenic substrate in BrS

      The mechanism of VF and of ECG pattern variability in patients with BrS remains controversial. There is evidence indicating the presence of structural substrate in the RV epicardium of humans on the basis of the presence of conduction slowing, interstitial fibrosis, and reduced connexin 43/gap junction expression.
      • Nademanee K.
      • Raju H.
      • de Noronha S.V.
      • et al.
      Fibrosis, connexin-43, and conduction abnormalities in the Brugada syndrome.
      ,
      • Coronel R.
      • Casini S.
      • Koopmann T.T.
      • et al.
      Right ventricular fibrosis and conduction delay in a patient with clinical signs of Brugada syndrome: a combined electrophysiological, genetic, histopathologic, and computational study.
      ,
      • Behr E.R.
      • Ben-haim Y.
      • Ackerman M.J.
      • Krahn A.D.
      • Wilde A.A.M.
      Brugada syndrome and reduced right ventricular outflow tract conduction reserve: a final common pathway.
      ,
      • Ten Sande J.N.
      • Coronel R.
      • Conrath C.E.
      • et al.
      ST-segment elevation and fractionated electrograms in Brugada syndrome patients arise from the same structurally abnormal subepicardial RVOT area but have a different mechanism.
      Animal models using pharmacological agents have demonstrated that abnormal repolarization gradient can also recapitulate the ST-segment elevation without underlying structural alteration.
      • Antzelevitch C.
      • Yan G.X.
      • Ackerman M.J.
      • et al.
      J-wave syndromes expert consensus conference report: emerging concepts and gaps in knowledge.
      ,
      • Yan G.X.
      • Antzelevitch C.
      Cellular basis for the Brugada syndrome and other mechanisms of arrhythmogenesis associated with ST-segment elevation.
      In the present clinical study, we analyzed the epicardial substrate electrograms in response to endocardial stimulation and then ajmaline infusion using 20-pole catheters.
      • Sacher F.
      • Jesel L.
      • Jais P.
      • Haissaguerre M.
      Insight into the mechanism of Brugada syndrome: epicardial substrate and modification during ajmaline testing.
      Such substrate mapping has been performed previously in a number of BrS studies, but the electrogram response was characterized regionally or globally on whole heart patterns rather than locally; ajmaline infusion was notably used for the delineation of substrate as an ablation target.
      • Coronel R.
      • Casini S.
      • Koopmann T.T.
      • et al.
      Right ventricular fibrosis and conduction delay in a patient with clinical signs of Brugada syndrome: a combined electrophysiological, genetic, histopathologic, and computational study.
      ,
      • Nademanee K.
      • Veerakul G.
      • Chandanamattha P.
      • et al.
      Prevention of ventricular fibrillation episodes in Brugada syndrome by catheter ablation over the anterior right ventricular outflow tract epicardium.
      • Zhang P.
      • Tung R.
      • Zhang Z.
      • et al.
      Characterization of the epicardial substrate for catheter ablation of Brugada syndrome.
      • Pappone C.
      • Brugada J.
      • Vicedomini G.
      • et al.
      Electrical substrate elimination in 135 consecutive patients with Brugada syndrome.
      • Chung F.P.
      • Raharjo S.B.
      • Lin Y.-J.
      • et al.
      A novel method to enhance phenotype, epicardial functional substrates, and ventricular tachyarrhythmias in Brugada syndrome.
      • Sacher F.
      • Jesel L.
      • Jais P.
      • Haissaguerre M.
      Insight into the mechanism of Brugada syndrome: epicardial substrate and modification during ajmaline testing.
      ,
      • Hoogendijk M.G.
      • Potse M.
      • Linnenbank A.C.
      • et al.
      Mechanism of right precordial ST-segment elevation in structural heart disease: excitation failure by current-to-load mismatch.
      ,
      • Ten Sande J.N.
      • Coronel R.
      • Conrath C.E.
      • et al.
      ST-segment elevation and fractionated electrograms in Brugada syndrome patients arise from the same structurally abnormal subepicardial RVOT area but have a different mechanism.
      ,
      • Postema P.G.
      • van Dessel F.H.M.
      • de Bakker J.M.T.
      • et al.
      Slow and discontinuous conduction conspire in Brugada syndrome: a right ventricular mapping and stimulation study.
      • Lambiase P.D.
      • Ahmed K.
      • Ciaccio E.J.
      • et al.
      High-density substrate mapping in Brugada syndrome: combined role of conduction and repolarization heterogeneities in arrhythmogenesis.
      • Leong K.M.W.
      • Ng F.S.
      • Yao C.
      • et al.
      ST-elevation magnitude correlates with right ventricular outflow tract conduction delay in type I Brugada ECG.
      We observed that extrastimulation caused a disappearance of epicardial electrograms at multiple localized sites, instead of a gradual prolongation, which strikingly indicated the absence of reactivation from surrounding contiguous activated sites. This phenomenon began at a few sites at relatively long CIs and then extended to other sites, ultimately resulting in loss of epicardial activation at 40% of all sites. This was not observed in endocardial RV regions where electrograms prolonged at short CIs but remained present. Similarly, low-dose ajmaline infusion during sinus rhythm resulted in a high incidence of inactivated sites concordant with those observed during extrastimulation. This indicated that epicardial activation was similarly affected by the Na blocker or premature stimulation. The absence of reactivation from the surrounding sites appears to be an uncommon phenomenon in human arrhythmogenic substrates. Isolated late potentials expressing conducting channels are well known in post–myocardial infarction substrate associated with ventricular tachycardia. It has been reported that 13% of low-voltage deflections present in the infarct area were blocked during single extrastimulation.
      • de Riva M.
      • Naruse Y.
      • Ebert M.
      • et al.
      Targeting the hidden substrate unmasked by right ventricular extrastimulation improves ventricular tachycardia ablation outcome after myocardial infarction.
      This incidence is significantly lower than the one reported here in BrS, and the sharper deflections recorded from infarct regions also appear different.
      The regional heterogeneity of conduction in BrS has been demonstrated by many prior authors describing dominant epicardial location and manifestation of prolonged fragmented electrograms.
      • Nademanee K.
      • Raju H.
      • de Noronha S.V.
      • et al.
      Fibrosis, connexin-43, and conduction abnormalities in the Brugada syndrome.
      ,
      • Coronel R.
      • Casini S.
      • Koopmann T.T.
      • et al.
      Right ventricular fibrosis and conduction delay in a patient with clinical signs of Brugada syndrome: a combined electrophysiological, genetic, histopathologic, and computational study.
      ,
      • Behr E.R.
      • Ben-haim Y.
      • Ackerman M.J.
      • Krahn A.D.
      • Wilde A.A.M.
      Brugada syndrome and reduced right ventricular outflow tract conduction reserve: a final common pathway.
      ,
      • Nagase S.
      • Kusano F.K.
      • Morita H.
      • et al.
      Longer repolarization in the epicardium at the right ventricular outflow tract causes type 1 electrocardiogram in patients with Brugada syndrome.
      ,
      • Nademanee K.
      • Veerakul G.
      • Chandanamattha P.
      • et al.
      Prevention of ventricular fibrillation episodes in Brugada syndrome by catheter ablation over the anterior right ventricular outflow tract epicardium.
      • Zhang P.
      • Tung R.
      • Zhang Z.
      • et al.
      Characterization of the epicardial substrate for catheter ablation of Brugada syndrome.
      • Pappone C.
      • Brugada J.
      • Vicedomini G.
      • et al.
      Electrical substrate elimination in 135 consecutive patients with Brugada syndrome.
      • Chung F.P.
      • Raharjo S.B.
      • Lin Y.-J.
      • et al.
      A novel method to enhance phenotype, epicardial functional substrates, and ventricular tachyarrhythmias in Brugada syndrome.
      • Sacher F.
      • Jesel L.
      • Jais P.
      • Haissaguerre M.
      Insight into the mechanism of Brugada syndrome: epicardial substrate and modification during ajmaline testing.
      ,
      • Hoogendijk M.G.
      • Potse M.
      • Linnenbank A.C.
      • et al.
      Mechanism of right precordial ST-segment elevation in structural heart disease: excitation failure by current-to-load mismatch.
      ,
      • Ten Sande J.N.
      • Coronel R.
      • Conrath C.E.
      • et al.
      ST-segment elevation and fractionated electrograms in Brugada syndrome patients arise from the same structurally abnormal subepicardial RVOT area but have a different mechanism.
      ,
      • Postema P.G.
      • van Dessel F.H.M.
      • de Bakker J.M.T.
      • et al.
      Slow and discontinuous conduction conspire in Brugada syndrome: a right ventricular mapping and stimulation study.
      • Lambiase P.D.
      • Ahmed K.
      • Ciaccio E.J.
      • et al.
      High-density substrate mapping in Brugada syndrome: combined role of conduction and repolarization heterogeneities in arrhythmogenesis.
      • Leong K.M.W.
      • Ng F.S.
      • Yao C.
      • et al.
      ST-elevation magnitude correlates with right ventricular outflow tract conduction delay in type I Brugada ECG.
      The unique developmental origin of the RVOT is a potential explanation for the increased heterogeneity and vulnerability to arrhythmias of the RV.
      • Boukens B.J.
      • Christoffels V.M.
      • Coronel R.
      • Moorman A.F.
      Developmental basis for electrophysiological heterogeneity in the ventricular and outflow tract myocardium as a substrate for life-threatening ventricular arrhythmias.
      The epicardium may also be involved as a particularly reactive cell region with a major role for remodeling the underlying muscle.
      • von Gise A.
      • Pu W.T.
      Endocardial and epicardial epithelial to mesenchymal transitions in heart development and disease.
      A resistive barrier has also been reported in the subepicardium as well as a helical fiber orientation in right and left ventricles, which may affect transmural propagation patterns.
      • Nishitani S.
      • Torii N.
      • Imai H.
      • et al.
      Development of helical myofiber tracts in the human fetal heart: analysis of myocardial fiber formation in the left ventricle from the late human embryonic period using diffusion tensor magnetic resonance imaging.
      ,
      • Yan G.X.
      • Shimizu W.
      • Antzelevitch C.
      Characteristics and distribution of M cells in arterially perfused canine left ventricular wedge preparations.
      Our study highlights a severe conduction discontinuity in the epicardium that juxtaposes activated and inactivated sites, the latter representing 40%–62% of sites. Such a loss of electrical connection, as opposed to conduction slowing, is not reported in mapping using animal models of conduction defects without fibrosis, as in SCN5A- or annexin A7–deficient mice, in which epicardial conduction is shown to be prolonged.
      • Martin C.A.
      • Guzadhur L.
      • Grace A.A.
      • Lei M.
      • Huang C.L.H.
      Mapping of reentrant spontaneous polymorphic ventricular tachycardia in a Scn5a+/− mouse model.
      • Schrickel J.W.
      • Brixius K.
      • Herr C.
      • et al.
      Enhanced heterogeneity of myocardial conduction and severe cardiac electrical instability in annexin A7-deficient mice.
      However, conduction block is described in modeling studies that showed that microstructural discontinuities, such as distinct fibrosis densities, can create delayed activation, conduction block, or wavefront “disintegration” within conditions of the cell network near the “percolation threshold.” Percolation indicates the movements through a fine-grained structure with narrow passages to propagation; the “threshold” indicates critical conditions where abrupt conduction changes may occur after minimal variations.
      • Vigmond E.
      • Pashaei E.
      • Amraoui S.
      • Cochet H.
      • Haissaguerre M.
      Percolation as a mechanism to explain atrial fractionated electrograms and reentry in a fibrosis model based on imaging data.
      • Alonso S.
      • Bar M.
      Reentry near the percolation threshold in a heterogeneous discrete model for cardiac tissue.
      • Kudryashova N.
      • Nizamieva A.
      • Tsvelaya V.
      • Panfilov A.V.
      • Konstantin I.
      • Agladze K.I.
      Self-organization of conducting pathways explains electrical wave propagation in cardiac tissues with high fraction of non-conducting cells.
      These microstructural alterations cannot be discerned by clinical imaging and are exquisitely sensitive to functional conditions influencing propagation such as activation rate or direction and ionic or cell-coupling characteristics. Such sensitivity is consistent with the multiple factors, extrinsic or intrinsic, that influence the day-to-day ECG expression of BrS.
      • Behr E.R.
      • Ben-haim Y.
      • Ackerman M.J.
      • Krahn A.D.
      • Wilde A.A.M.
      Brugada syndrome and reduced right ventricular outflow tract conduction reserve: a final common pathway.
      ,
      • Hoogendijk M.G.
      • Potse M.
      • Linnenbank A.C.
      • et al.
      Mechanism of right precordial ST-segment elevation in structural heart disease: excitation failure by current-to-load mismatch.
      ,
      • Ten Sande J.N.
      • Coronel R.
      • Conrath C.E.
      • et al.
      ST-segment elevation and fractionated electrograms in Brugada syndrome patients arise from the same structurally abnormal subepicardial RVOT area but have a different mechanism.
      A reduced Na channel function is likely an essential factor of functional block in addition to microstructural basis. A recent study in mice has shown that normal microstructural heterogeneities present in the RV (intramural clefts) are sufficient to underlie conduction block when the Na current is acutely reduced by tetrodotoxin.
      • Kelly A.
      • Salerno S.
      • Connolly A.
      • et al.
      Normal interventricular differences in tissue architecture underlie right ventricular susceptibility to conduction abnormalities in a mouse model of Brugada syndrome.

      Repolarization link with conduction

      In addition to demonstrating specific conduction properties, our study shows examples of the influence of instantaneous conduction changes on the unipolar ST-segment morphology. The aftermath of ajmaline infusion produced a recovery of epicardial activation occurring with abrupt temporal and spatial variations, resulting in a mosaic of electrical activities in a small area. The ability to observe beat-to-beat changes was applied to analysis of the coved-type pattern, a hallmark of BrS. Localized coved-type repolarization was dependent on localized block, supporting a primary role of conduction in shaping coved-type repolarization in BrS and specifically the source-sink mismatch mechanism demonstrated by the group from Amsterdam
      • Coronel R.
      • Casini S.
      • Koopmann T.T.
      • et al.
      Right ventricular fibrosis and conduction delay in a patient with clinical signs of Brugada syndrome: a combined electrophysiological, genetic, histopathologic, and computational study.
      ,
      • Hoogendijk M.G.
      • Potse M.
      • Linnenbank A.C.
      • et al.
      Mechanism of right precordial ST-segment elevation in structural heart disease: excitation failure by current-to-load mismatch.
      ,
      • Ten Sande J.N.
      • Coronel R.
      • Conrath C.E.
      • et al.
      ST-segment elevation and fractionated electrograms in Brugada syndrome patients arise from the same structurally abnormal subepicardial RVOT area but have a different mechanism.
      ,
      • Postema P.G.
      • van Dessel F.H.M.
      • de Bakker J.M.T.
      • et al.
      Slow and discontinuous conduction conspire in Brugada syndrome: a right ventricular mapping and stimulation study.
      and others.
      • Behr E.R.
      • Ben-haim Y.
      • Ackerman M.J.
      • Krahn A.D.
      • Wilde A.A.M.
      Brugada syndrome and reduced right ventricular outflow tract conduction reserve: a final common pathway.
      ,
      • Lambiase P.D.
      • Ahmed K.
      • Ciaccio E.J.
      • et al.
      High-density substrate mapping in Brugada syndrome: combined role of conduction and repolarization heterogeneities in arrhythmogenesis.
      ,
      • Leong K.M.W.
      • Ng F.S.
      • Yao C.
      • et al.
      ST-elevation magnitude correlates with right ventricular outflow tract conduction delay in type I Brugada ECG.
      ,
      • Kataoka N.
      • Nagase S.
      • Kamakura T.
      • Noda T.
      • Aiba T.
      • Kusano K.
      Local activation delay exacerbates local J-ST elevation in the epicardium: electrophysiological substrate in Brugada syndrome.
      It should be noted that percolation models also report micro-source–sink mismatch within the microfibrotic region.
      • Vigmond E.
      • Pashaei E.
      • Amraoui S.
      • Cochet H.
      • Haissaguerre M.
      Percolation as a mechanism to explain atrial fractionated electrograms and reentry in a fibrosis model based on imaging data.
      A primary conduction mechanism however does not exclude a contributory role of repolarization in BrS arrhythmogenesis, in view of marked repolarization heterogeneity, observed here and in prior reports on overt structural heart diseases
      • Engelman Z.J.
      • Trew M.L.
      • Smaill B.H.
      Structural heterogeneity alone is a sufficient substrate for dynamic instability and altered restitution.
      and animal models of BrS.
      • Antzelevitch C.
      • Yan G.X.
      • Ackerman M.J.
      • et al.
      J-wave syndromes expert consensus conference report: emerging concepts and gaps in knowledge.
      ,
      • Yan G.X.
      • Antzelevitch C.
      Cellular basis for the Brugada syndrome and other mechanisms of arrhythmogenesis associated with ST-segment elevation.

      Clinical implications

      Our results expand on prior studies by showing the multiplicity of epicardial sites with functional block in resting conditions and the decomposition of electrograms into smaller elements. The variability of BrS substrate in relation to multisite conduction block improves our understanding of this pathology and its varying ECG expression. The methods to challenge conduction departed little from clinical conditions by using single extrastimuli (mimicking a premature beat) or low-dose ajmaline. The effect of beat-to-beat changes on local conduction was less perceptible on the surface ECG than the effect on repolarization. Our results highlight the multiplicity of transmural pathways with inconsistent conduction and variable sensitivity, likely to be extendable to the full substrate scale. This offers multiple scenarios for epicardial activation and repolarization dependent on the mosaic of activated, delayed, or inactivated sites, which explains the variability of the ECG pattern.
      Further investigations of the epicardial substrate can be similarly applied using other protocols. They can test for the presence of local blocks as an indication of “concealed” BrS; they can evaluate various clinical, autonomic, or pharmacological factors to characterize the individual substrate sensitivity and the arrhythmogenic risk. The present study also points out the practical importance of using multielectrode and unipolar recordings
      • Coronel R.
      • Casini S.
      • Koopmann T.T.
      • et al.
      Right ventricular fibrosis and conduction delay in a patient with clinical signs of Brugada syndrome: a combined electrophysiological, genetic, histopathologic, and computational study.
      ,
      • Nagase S.
      • Kusano F.K.
      • Morita H.
      • et al.
      Longer repolarization in the epicardium at the right ventricular outflow tract causes type 1 electrocardiogram in patients with Brugada syndrome.
      ,
      • Ten Sande J.N.
      • Coronel R.
      • Conrath C.E.
      • et al.
      ST-segment elevation and fractionated electrograms in Brugada syndrome patients arise from the same structurally abnormal subepicardial RVOT area but have a different mechanism.
      ,
      • Kataoka N.
      • Nagase S.
      • Kamakura T.
      • Noda T.
      • Aiba T.
      • Kusano K.
      Local activation delay exacerbates local J-ST elevation in the epicardium: electrophysiological substrate in Brugada syndrome.
      to distinguish activation-repolarization components in complex local electrograms (Online Supplemental Figure 1B).
      Finally, the presence of multiple sites of blocks open many options for unidirectional propagation and reentries and may constitute a substrate more favorable for VF than for ventricular tachyarrhythmia, like in BrS. Such a peculiar substrate may also be present in other heart diseases, potentially in individuals more prone to VF. In our study a single example illustrated the potential relationship between conduction heterogeneity and VF initiation. Localized epicardial blocks were present during VF initial beats, and a remarkable asynchrony of activations was observed across the RVOT associated with continuously changing patterns. The epicardial side showed slower activity, suggesting that deeper myocardial or transmural activities played a significant role in VF arrhythmogenesis, rather than the sole epicardium. Further studies are needed to specify the mechanism between conduction impairment and arrhythmogenicity and its application for risk quantification.

      Study limitations

      The number of patients was small, and more studies are clearly needed to generalize the conclusions. Our study included patients with an absent or discrete BrS pattern, in whom ajmaline infusion could be safely administered. A significant number of events were however recorded to substantiate the conclusions, with the convergence of data providing important points for further studies.
      The wavefront propagation to the epicardium could not be better characterized since a limited field of substrate was studied, as we were principally concerned with measuring conduction variations under stable mapping. Similarly, technical limitations prevented us from quantifying repolarization data as have been reported previously in detail.
      • Ten Sande J.N.
      • Coronel R.
      • Conrath C.E.
      • et al.
      ST-segment elevation and fractionated electrograms in Brugada syndrome patients arise from the same structurally abnormal subepicardial RVOT area but have a different mechanism.

      Conclusion

      We report the electrophysiological changes occurring in the epicardial substrate of BrS during extrastimulation and ajmaline infusion. We show that multiple sites exhibit variable patterns of localized conduction block without being reactivated from the surrounding sites.

      Appendix. Supplementary data

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