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Mechanism of right precordial ST-segment elevation in structural heart disease: Excitation failure by current-to-load mismatch

Published:October 12, 2009DOI:https://doi.org/10.1016/j.hrthm.2009.10.007

      Background

      The Brugada sign has been associated with mutations in SCN5A and with right ventricular structural abnormalities. Their role in the Brugada sign and the associated ventricular arrhythmias is unknown.

      Objective

      The purpose of this study was to delineate the role of structural abnormalities and sodium channel dysfunction in the Brugada sign.

      Methods

      Activation and repolarization characteristics of the explanted heart of a patient with a loss-of-function mutation in SCN5A (G752R) and dilated cardiomyopathy were determined after induction of right-sided ST-segment elevation by ajmaline. In addition, right ventricular structural discontinuities and sodium channel dysfunction were simulated in a computer model encompassing the heart and thorax.

      Results

      In the explanted heart, disappearance of local activation in unipolar electrograms at the basal right ventricular epicardium was followed by monophasic ST-segment elevation. The local origin of this phenomenon was confirmed by coaxial electrograms. Neither early repolarization nor late activation correlated with ST-segment elevation. At sites of local ST-segment elevation, the subepicardium was interspersed with adipose tissue and contained more fibrous tissue than either the left ventricle or control hearts. In computer simulations entailing right ventricular structural discontinuities, reduction of sodium channel conductance or size of the gaps between introduced barriers resulted in subepicardial excitation failure or delayed activation by current-to-load mismatch and in the Brugada sign on the ECG.

      Conclusion

      Right ventricular excitation failure and activation delay by current-to-load mismatch in the subepicardium can cause the Brugada sign. Therefore, current-to-load mismatch may underlie the ventricular arrhythmias in patients with the Brugada sign.

      Keywords

      Abbreviations:

      APD90 (action potential duration at 90% of repolarization), ARVC (arrhythmogenic right ventricular cardiomyopathy), DSC2 (desmocollin-2), DSG2 (desmoglein-2), DSP (desmoplakin), dV/dtmax (maximal action potential upstroke velocity), GNa (sodium channel conductivity), INa (sodium current), JUP (junction-plakoglobin), LV (left ventricle), PKP2 (plakophilin-2), RV (right ventricle)

      Introduction

      The Brugada sign of right precordial ST-segment elevation followed by a negative T-wave is associated with ventricular tachyarrhythmias and sudden cardiac death.
      • Antzelevitch C.
      • Brugada P.
      • Borggrefe M.
      • et al.
      Brugada syndrome: report of the second consensus conference: endorsed by the Heart Rhythm Society and the European Heart Rhythm Association.
      In the Brugada syndrome, the sign occurs in the absence of gross structural abnormalities.
      • Antzelevitch C.
      • Brugada P.
      • Borggrefe M.
      • et al.
      Brugada syndrome: report of the second consensus conference: endorsed by the Heart Rhythm Society and the European Heart Rhythm Association.
      However, the Brugada sign is not limited to structurally normal hearts. Right ventricular (RV) structural abnormalities have been demonstrated in a significant portion of patients with the Brugada sign.
      • 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.
      • Corrado D.
      • Basso C.
      • Buja G.
      • Nava A.
      • Rossi L.
      • Thiene G.
      Right bundle branch block, right precordial ST-segment elevation, and sudden death in young people.
      • Frustaci A.
      • Priori S.G.
      • Pieroni M.
      • et al.
      Cardiac histological substrate in patients with clinical phenotype of Brugada syndrome.
      • Zumhagen S.
      • Spieker T.
      • Rolinck J.
      • et al.
      Absence of pathognomonic or inflammatory patterns in cardiac biopsies from patients with Brugada syndrome.
      In addition, sodium channel blockers can provoke the Brugada sign in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC)
      • Peters S.
      • Trümmel M.
      • Denecke S.
      • Koehler B.
      Results of ajmaline testing in patients with arrhythmogenic right ventricular dysplasia-cardiomyopathy.
      and Chagas disease,
      • Chiale P.A.
      • Przybylski J.
      • Laino R.A.
      • et al.
      Electrocardiographic changes evoked by ajmaline in chronic Chagas' disease without manifest myocarditis.
      conditions characterized by severe structural derangements. The role of structural derangements in the mechanism of the Brugada sign and the associated arrhythmias is unknown.
      To date, two mechanisms of ST-segment elevation have been proposed for the Brugada sign: (1) early repolarization
      • Yan G.X.
      • Antzelevitch C.
      Cellular basis for the Brugada syndrome and other mechanisms of arrhythmogenesis associated with ST-segment elevation.
      and (2) late activation
      • 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.
      in the RV wall. Sodium channel function is important in both mechanisms because sodium channel blockers can provoke the Brugada sign,
      • Miyazaki T.
      • Mitamura H.
      • Miyoshi S.
      • Soejima K.
      • Ogawa S.
      • Aizawa Y.
      Autonomic and antiarrhythmic drug modulation of ST segment elevation in patients with Brugada syndrome.
      and loss-of-function mutations in the gene encoding the cardiac sodium channel (SCN5A) can be identified in approximately 20% of patients with Brugada syndrome.
      • Antzelevitch C.
      • Brugada P.
      • Borggrefe M.
      • et al.
      Brugada syndrome: report of the second consensus conference: endorsed by the Heart Rhythm Society and the European Heart Rhythm Association.
      Neither hypothesis has been confirmed in patients.
      • Meregalli P.G.
      • Wilde A.A.M.
      • Tan H.L.
      Pathophysiological mechanisms of Brugada syndrome: depolarization disorder, repolarization disorder, or more?.
      Structural abnormalities cause geometric variation in myocardial organization. Sites where structural abnormalities lead to sudden expansion of myocardium are susceptible to conduction block by current-to-load mismatch, especially when the available sodium current (INa) is reduced.
      • Sasyniuk B.I.
      • Mendez C.
      A mechanism for reentry in canine ventricular tissue.
      • Rohr S.
      • Salzberg B.M.
      Characterization of impulse propagation at the microscopic level across geometrically defined expansions of excitable tissue: multiple site optical recording of transmembrane voltage (MSORTV) in patterned growth heart cell cultures.
      • Mendez C.
      • Mueller W.J.
      • Merideth J.
      • Moe G.K.
      Interaction of transmembrane potentials in canine Purkinje fibers and at Purkinje fiber-muscle junctions.
      • Fast V.G.
      • Kléber A.G.
      Cardiac tissue geometry as a determinant of unidirectional conduction block: assessment of microscopic excitation spread by optical mapping in patterned cell cultures and in a computer model.
      If excitation fails at these sites, the potential gradient between the unexcited myocardium and the myocardium proximal to the site of conduction block will cause ST-segment elevation. Therefore, we hypothesized that RV excitation failure by current-to-load mismatch can cause the Brugada sign in patients with structural abnormalities, especially when INa is reduced by loss-of-function mutations in SCN5A or sodium channel blockade. To test this hypothesis, we determined the activation and repolarization characteristics of the explanted heart of a patient with a loss-of-function mutation in SCN5A and structural discontinuities in the setting of dilated cardiomyopathy, before and after induction of ST-segment elevation by sodium channel blockade. Furthermore, we simulated the effect of INa reduction and structural discontinuities on ECG in a computer model encompassing the heart and thorax.

      Methods

      The study was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from the patient's guardians. Cardiac transplantation was performed in May 2007 at the Erasmus Medical Center, Rotterdam, The Netherlands. The explanted heart was submerged in ice-cold modified Tyrode's solution
      • 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.
      and transported (within 1 hour) to the Laboratory of Experimental Cardiology (Academic Medical Center, Amsterdam, The Netherlands).

       Genetic study

      Genomic DNA was extracted from lymphocytes using standard protocols. The entire coding regions of SCN5A and LMNA were screened for mutations by denaturing high-performance liquid chromatography followed by sequencing of amplicons displaying an aberrant elution profile. Screening of genes associated with ARVC [plakophilin-2 (PKP2), junction-plakoglobin (JUP), desmoglein-2 (DSG2), desmocollin-2 (DSC2), desmoplakin (DSP)] was performed by direct sequencing of the entire coding region.

       Experimental setup

      The right and left coronary arteries were cannulated, and the heart was connected to a perfusion setup. The heart was perfused with a mixture of washed erythrocytes (800 mL) and modified Tyrode's solution.
      • 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.
      The potassium concentration was 4.0 mmol/L. Coronary flow was set to 300 mL/min. The heart was suspended in a cylindrical container (diameter 20 cm, height 14 cm) filled with perfusion fluid. Myocardial temperature was 37.0°–37.5°C throughout the experiment. The pH of the oxygenated perfusate was 7.34.

       Electrophysiologic studies

       Mapping experiment

      Seven electrode strips (14 electrodes per strip in two rows, interelectrode distance 1.5 cm, distance between strips ≈2 cm) were equally distributed over and attached to the ventricular epicardium. An inflatable balloon (64 electrodes, interelectrode distance ≈1.5 cm) was inserted through the mitral orifice into the left ventricle (LV), and an inflatable balloon (32 electrodes, interelectrode distance ≈1.5 cm) was inserted through the tricuspid orifice into the RV. Three electrodes at the side of the container were used to record a pseudo-ECG. One electrode was positioned at the bottom below the apex of the heart, one faced the lateral LV, and one faced the lateral RV. A reference electrode was placed at the bottom of the container. Electrode positions were documented with digital photography.
      The heart was stimulated from the basal RV septum at twice diastolic threshold (steady-state pacing at cycle length 2,000–1,500–1,000–800 ms). The stimulation protocol was repeated after addition of ajmaline 2.4 μmol/L (Solvay Pharmaceuticals GmbH, Hannover, Germany), a rate-dependent blocker of the cardiac sodium channel.
      • Heistracher P.
      Mechanism of action of antifibrillatory drugs.

       Data acquisition and analysis

      Simultaneous recordings (sampling rate 2 kHz) were made from all electrodes during selected episodes. Signals were analyzed using a custom-made analysis program
      • Potse M.
      • Linnenbank A.C.
      • Grimbergen C.A.
      Software design for analysis of multichannel intracardial and body surface electrocardiograms.
      based on MATLAB R2006b (The MathWorks, Inc., Natick, MA, USA). The instant of maximal negative dV/dt in the QRS complex and the instant of maximal positive dV/dt in the T wave of the unipolar electrogram were used as local activation and repolarization time, repectively.
      • Coronel R.
      • de Bakker J.M.T.
      • Wilms-Schopman F.J.G.
      • et al.
      Monophasic action potentials and activation recovery intervals as measures of ventricular action potential duration: experimental evidence to resolve some controversies.
      The local contribution to unipolar electrograms was determined by calculation of coaxial electrogram by subtracting the mean of values of neighboring electrodes from a central electrogram at each sample.

       Cellular electrophysiology

      After the mapping procedure, LV myocytes were isolated as previously described.
      • Den Ruijter H.M.
      • Berecki G.
      • Verkerk A.O.
      • et al.
      Acute administration of fish oil inhibits triggered activity in isolated myocytes from rabbits and patients with heart failure.
      INa and action potentials were recorded using an Axopatch 200B amplifier (Molecular Devices Corp., Sunnyvale, CA, USA). Voltage control, data acquisition, and analysis were accomplished using custom software. Signals for INa were low-pass filtered with a cutoff frequency of 5 kHz and digitized at 20 kHz. Action potentials were filtered and digitized at 10 and 40 kHz, respectively. Cell capacitance and series resistance were compensated for by at least 80%. Potentials were compensated for the calculated liquid junction potential.
      INa was recorded at room temperature (20°C) in the ruptured whole-cell configuration of the patch-clamp technique. The bath solution contained the following (in mmol/L): 7.0 NaCl, 133 CsCl, 1.8 CaCl2, 1.2 MgCl2, 11.0 glucose, 5.0 HEPES, and 0.005 nifedipine; pH 7.4 (CsOH). Patch pipettes (1.5–2.0 MΩ) were filled with the following (in mmol/L): 3.0 NaCl, 133 CsCl, 2.0 MgCl2, 2.0 Na2ATP, 2.0 TEACl, 10 EGTA, and 5.0 HEPES; pH 7.3 (CsOH). INa amplitude and (in)activation properties were measured using a double-pulse protocol (see Figure 2A). Voltage dependence of (in)activation was determined by fitting a Boltzmann function to the individual curves. Current density was calculated by dividing whole-cell current amplitude by the cell capacitance.
      Action potentials were recorded at 36°C ± 0.2°C with the amphotericin B perforated patch-clamp technique. Action potentials were elicited at 1 Hz by 3-ms, 1.5× threshold current pulses through the patch pipette. The bath solution contained the following (in mmol/L): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 5.5 glucose, and 5 HEPES; pH 7.4 (NaOH). The pipette solution contained the following (in mmol/L): 125 K-gluc, 20 KCl, 5 NaCl, 0.22 amphotericin-B, and 10 HEPES; pH 7.2 (KOH). We analyzed resting membrane potential, action potential duration at 90% repolarization (APD90), and maximal action potential upstroke velocity (dV/dtmax), a measure of INa availability. Values from 10 consecutive action potentials were averaged.

       Histology

      After the electrophysiologic studies, the remainder of the heart was fixed in 4% buffered formalin. Transmural tissue samples from the RV outflow tract, basal RV free wall, and basal LV free wall were routinely processed and embedded in paraffin. Sections 7-μm thick were stained with picrosirius red F3A for visualization of collagens in fibrous tissue. The content of adipose and fibrous tissue in the subepicardial rim of myocardium (outer 1 mm) was quantified by computerized morphometry on 10× objective fields of each section (mean 11.4 fields per section). Red (fibrous tissue) and white (adipose tissue) areas were expressed as a percentage of the total area using Image-Pro 6.2 (Media Cybernetics, Inc., Bethesda, MD, USA). The epicardial fat and large areas of perivascular adipose tissue were excluded from measurements. Transmural sections of six structurally normal hearts sampled from the same sites and obtained at autopsy from patients without a history of cardiac disease served as control samples.

       Computer simulations

      Propagating action potentials were simulated with a whole-heart reaction-diffusion model containing 90 million nodes, each represented by a membrane model of the human ventricular myocyte.
      • Potse M.
      • Dubé B.
      • Richer J.
      • Vinet A.
      • Gulrajani R.M.
      A Comparison of monodomain and bidomain reaction-diffusion models for action potential propagation in the human heart.
      Transmural fiber rotation was represented in the model. Membrane ionic currents were computed with a human membrane model that included the differential characteristics of subendocardial, midmyocardial, and subepicardial myocytes.
      • ten Tusscher K.H.W.J.
      • Noble D.
      • Noble P.J.
      • Panfilov A.V.
      A model for human ventricular tissue.
      The ECG was computed using a bidomain model of the human heart and torso, including lungs and intracavitary blood volumes. Structural discontinuities were simulated by the introduction of barriers (thickness 0.4 mm) in the outer 50% of the RV wall. In these barriers, no intercellular coupling was present, but the interstitium was unaffected. The barriers contained gaps of 0.2 mm width in which intercellular coupling was present. Intercellular coupling in the gaps was reduced in steps from 100% to 8% of normal to simulate smaller gaps. Sodium channel conductivity (GNa) was reduced in steps from 100% to 20% of normal in the entire heart. Single cardiac cycles were simulated in 12 hours on 128 processors of an SGI Altix 4700 supercomputer.

       Statistical analysis

      Values are given as mean ± SD. Unpaired, two-tailed, Student's t-tests were used for statistical comparison of normally distributed data. P <.05 was considered significant.

      Results

       Patient data

      The female patient (age 13 years) presented for medical attention after experiencing syncope during exercise in 2004. She had a low exercise tolerance and reported two episodes of syncope over the last year, one of which occurred at rest. Her ECG showed atrial flutter with a flutter rate of 258/min and ventricular rate of 43/min, left axis with QRS duration of 150 ms, poor R-wave progression in the precordial leads, and negative T waves in leads I, aVL and V2–V6 (Figure 1). Echocardiography revealed LV fractional shortening was reduced (21%) and LV end-diastolic diameter was increased (53 mm, >95th percentile). No RV abnormalities were reported. A dual-chamber pacemaker was implanted. Two weeks after discharge, the patient was readmitted due to fatigue, loss of ventricular capture of the pacemaker, and pericardial effusion. Pericardiocentesis was performed, and the ventricular lead was repositioned. Over the next year, the patient developed episodes of exercise intolerance during paroxysms of atrial flutter. A trial radiofrequency ablation to terminate the atrial flutter was performed. However, the symptoms progressed, and cardiac transplantation was conducted 2.5 years after initial presentation for end-stage heart failure in dilated cardiomyopathy. No sodium channel provocation test was performed, no spontaneous Brugada sign was observed, and the patient was not diagnosed with the Brugada syndrome. The patient did not receive antiarrhythmic drugs prior to transplantation.
      Figure thumbnail gr1
      Figure 1ECG recorded at initial presentation in 2004.

       Family history and genetic data

      The patient had a heterozygous mutation in SCN5A (c.2254G>A; numbering according to NM_198056.2) with substitution of glycine by arginine at position 752 (p.Gly752Arg) located in the second transmembrane segment of channel domain II. This mutation previously has been shown to reduce cardiac sodium channel function and was associated with the Brugada syndrome in a French family.
      • Potet F.
      • Mabo P.
      • Le Coq G.
      • et al.
      Novel Brugada SCN5A mutation leading to ST segment elevation in the inferior or the right precordial leads.
      No mutation was found in LMNA or in the ARVC–associated genes PKP2, JUP, DSG2, DSC2, and DSP. The patient's mother and grandmother also carried the G752R mutation in SCN5A. The family history revealed that a great-uncle and a great-aunt (brother and sister of the grandfather on the mother's side of the family) had died suddenly at the age of 35 years and ∼45 years, respectively. The mother's ECG did not show any abnormalities, but the Brugada sign was induced by flecainide.

       Cellular electrophysiologic studies

      Figure 2A shows typical examples of INa in an isolated ventricular myocyte. V1/2 of activation and inactivation was −40.0 ± 4.4 mV and −85.2 ± 8.3 mV, respectively (n = 6, Figure 2B). Figure 2C shows representative action potential upstrokes. The dV/dtmax is compared to that of single LV myocytes from a patient with a heterozygous mutation (G1935S) in SCN5A with unaltered sodium channel characteristics except for enhanced slow inactivation as previously reported.
      • 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.
      The average dV/dtmax in G752R ventricular myocytes was 165 ± 101 V/s versus 337 ± 74 V/s of those in G1935S ventricular myocytes (n = 3 and n = 7, respectively, P <.05). Resting membrane potential was −79.0 ± 6.3 mV, and APD90 was 502 ± 124 ms.
      Figure thumbnail gr2
      Figure 2Sodium channel characteristics and action potential upstrokes of isolated left ventricular myocytes. Typical whole cell-current recordings (A) and corresponding current–voltage relations (mean ± SEM) (B) show a normal voltage dependency of the sodium current. C: Typical action potential upstrokes and dV/dt show significantly reduced upstroke velocities compared to myocytes of a previously reported control patient. Insets illustrate the voltage and stimulus protocols.

       Isolated heart

      At baseline, ST segments of the pseudo-ECG were isoelectric during stimulation at any cycle length. At a cycle length of 800 ms, QRS and QT duration were 150 and 710 ms, respectively. After ajmaline, the ST segment in pseudo-aVR was elevated by 0.3 mV at a cycle length of 800 ms, whereas QRS and QT duration increased to 180 and 750 ms, respectively (Figure 3).
      Figure thumbnail gr3
      Figure 3Pseudo-ECG of the isolated heart before (black) and after (red) sodium channel blockade (stimulation from right ventricular septum at basic cycle length = 800 ms). Note ST-segment elevation in pseudo-AVR after sodium channel blockade. Bars = 200 ms, 1 mV.

       Activation and repolarization mapping

      Figure 4 shows the activation (panel A) and repolarization (panel B) times projected on pseudo-aVR and a three-dimensional reconstruction of the activation and repolarization pattern after ajmaline. Reference times were earliest activation or repolarization, respectively. ST-segment elevation was present after the moment of latest activation and before the moment of earliest repolarization. Latest recorded activation coincided with the J point, and earliest repolarization occurred at the start of the T wave, 250 ms after the J point. A three-dimensional reconstruction shows the spread of activation from the RV septum over both ventricles. Latest activation was recorded at the RV free wall and LV free wall, and crowding of isochrones was most pronounced at the endocardium of the RV. Earliest repolarization occurred at the basal endocardium of the RV and LV. Of note, epicardial repolarization was later than endocardial repolarization at the RV (mean 128 ms and 84 ms, respectively).
      Figure thumbnail gr4
      Figure 4Activation and repolarization after sodium channel blockade (right ventricular septal stimulation at cycle length of 800 ms). Activation (A) and repolarization (B) times are depicted on the corresponding complex in pseudo-aVR and on a three-dimensional reconstruction of the heart. Anterior view of the heart is depicted on the left; posterior view is depicted on the right. The epicardium is viewed as transparent. No sign of activation or repolarization was found throughout the ST segment. Lines are 20-ms isochrones.

       ST-segment elevation in unipolar electrograms

      ST-segment elevation after ajmaline in pseudo-aVR coincided with ST-segment elevation in unipolar electrograms (Figure 5A, site indicated by asterisk in Figure 5B). A voltage map of epicardial unipolar electrograms 100 ms after the J point in pseudo-aVR illustrates that ST-segment elevation in unipolar electrograms was limited to the basal epicardial RV (Figure 5B, time indicated by arrow in Figure 5A).
      Figure thumbnail gr5
      Figure 5Regional ST-segment elevation in unipolar electrograms after sodium channel blockade. A: Pseudo-aVR (top row), unipolar electrogram (middle row), and calculated coaxial electrogram (bottom row) from the epicardial basal right ventricle (site indicated by asterisk in panel B) during stimulation from the right ventricular septum at increasing frequencies (cycle length indicated at top). ST-segment elevation in pseudo-aVR increased with higher stimulation frequencies and coincided with disappearance of the main activation signal followed by monophasic ST-segment elevation in the unipolar electrogram. The local origin of ST-segment elevation was confirmed using coaxial electrograms. B: Three-dimensional reconstruction of ST-segment amplitude on unipolar epicardial electrograms 100 ms after the J point in pseudo-aVR (moment indicated by arrows in panel A) at cycle length of 800 ms. ST-segment elevation was limited to the basal epicardial right ventricle. Right ventricular view is shown on the left; left ventricular view is shown on the right. Color scale is given in millivolts. LV = left ventricle; RV = right ventricle.
      Calculated coaxial electrograms confirmed that the local ST-segment elevation was not the result of far-field effects, contrary to the initial deflection in the unipolar electrogram. ST-segment elevation increased with stimulation frequency. Note that the local activation signal in the unipolar and coaxial electrogram virtually disappeared when ST-segment elevation was observed (Figure 5A).

       Pathology of the heart

      Macroscopically, the heart showed hypertrophy and dilation of both ventricles. Pronounced endocardial fibroelastosis was seen, particularly in the LV. A thick layer of epicardial fat covered the RV. The coronary arteries, including coronary ostia, and all heart valves were normal. Microscopically, the myocardium showed cytonuclear features of hypertrophy and multizonal distinct cytoplasmatic vacuolization of myocytes. The subepicardial myocardium of the RV outflow tract and basal RV free wall, but not of the basal LV free wall, was locally interspersed with adipose tissue. The fatty infiltration focally reached the subendocardium but was not transmural at any site (Figures 6A,6B, and 6C). Both ventricles showed focal increase of lymphocytes, including sparse clusters, indicating borderline myocarditis. Morphometric analysis showed that the subepicardium of the RV outflow tract, RV free wall, and LV free wall of the patient (red) contained more fibrous tissue than did any of the controls. The adipose tissue content of the RV outflow tract and RV free wall was greater than of the LV free wall in both the patient and the controls (Figures 6D and 6E).
      Figure thumbnail gr6
      Figure 6Subepicardial histologic sections and quantification of subepicardial fibrous and adipose tissue. A–C: Subepicardial sections from the right ventricular outflow tract (RVOT; A), basal right ventricular free wall (RVFW; B), and basal left ventricular free wall (LVFW, C). Low-magnification images show fatty infiltration (white) in the subepicardial myocardium of the RVOT and RVFW and interstitial-type fibrosis (red) at all locations. Picrosirius red staining; bar = 500 μm. Note that the epicardial rim of collagen seen in panel C was excluded from measurements. D, E: Morphometric analysis of fibrous (D) and adipose (E) tissue content as percent of total measured myocardial area in the heart of the patient (red dots) and in the hearts of controls (black dots). Analysis reveals that the heart of the patient contained more fibrous tissue at any location than did any of the control hearts, and that the adipose tissue content of the RVOT and RVFW was greater than in the LVFW. However, no difference in adipose tissue content was found between the patient and the control hearts. Bars in panel D indicate mean ± SD of controls.

       Computer simulations

      The ECG derived from the simulated heart without structural abnormalities had QRS duration of 80 ms and isoelectric ST segments (Figure 7A, black ECG). Reduction of GNa in the normal heart to 30% of normal caused global activation delay, and QRS duration increased to 130 ms but did not lead to ST-segment elevation (Figure 7A, red ECG). The introduction of structural discontinuities with 30% of normal coupling in the gaps at normal GNa resulted in QRS prolongation to 100 ms and negative T waves in the right precordial leads but not in ST-segment elevation (Figure 7B, black ECG). The negative T wave was caused by activation delay of the RV subepicardium (Figure 7C). Reduction of GNa after introduction of these structural discontinuities caused excitation failure at the anterior RV subepicardium (Figure 7D, black sites). The current received from and given to surrounding elements and corresponding action potentials at five neighboring sites in and immediately distal to the gaps are shown in the left and right graph, respectively. Subepicardial excitation failure occurred when insufficient depolarizing current was received distal of the introduced structural discontinuities to reach threshold potential. The potential gradient between the excited and unexcited myocardium caused ST-segment elevation in the right precordial leads. Activation at other RV subepicardial sites was delayed, leading to a negative T wave in the right precordial leads (Figure 7B, red ECG).
      Figure thumbnail gr7
      Figure 7Simulated subepicardial discontinuities and sodium channel dysfunction. A, B: ECGs of the heart without (A) and with structural discontinuities in the right ventricular subepicardium (B) at baseline (black) and after (red) reduction of sodium channel conductivity (GNa) to 30% of normal.Bars = 200 ms, 1 mV. C, D: Basal short-axis view of the heart with structural discontinuities at the right ventricular subepicardium before (C) and after (D) reduction of GNa to 30% of normal. Colors indicate activation time; sites that failed to excite throughout the cardiac cycle are depicted in black. The current received from and given to surrounding elements and the corresponding action potentials at five neighboring sites are depicted in the left and right graphs, respectively. The locations of these elements were 0.2 mm (black) and 0.4 mm in (brown) and 0.2 mm (red), 0.4 mm (orange), and 1.0 mm (yellow) behind the gaps in the barriers. After reduction of GNa, insufficient current was received by many elements behind the introduced structural discontinuities to reach threshold potential. This resulted in excitation failure and activation delay of the right ventricular subepicardium and in ST-segment elevation followed by a negative T wave in the right precordial leads. LV = left ventricle; RV = right ventricle.
      In the presence of structural discontinuities with 30% of normal coupling in the gaps, the percentage of elements that were not excited throughout the cardiac cycle depended on GNa and correlated well with cumulative ST-segment elevation in leads V1 and V2 of the third and fourth intercostal spaces. Both increased markedly when GNa was reduced below 50% of normal (Figure 8A). Likewise, a decrease of the size of the gaps in the barriers at normal sodium conductance resulted in a reduction in excited elements. The amplitude of ST-segment elevation by excitation failure after reduction of the size of the gaps was limited by the increased resistance between the excited and unexcited myocardium (Figure 8B).
      Figure thumbnail gr8
      Figure 8Simulated ECGs in structurally abnormal heart during reduction of sodium conductance (A) or of simulated size of the gaps in introduced barriers by reduction of coupling in the gaps (B) and corresponding graphs relating excited elements with ST-segment elevation in the right precordial leads. Reduction of sodium channel conductance (GNa) below 50% of normal resulted in a rapid reduction in excited elements and ST-segment elevation on the ECG. A reduction in the size of the gaps in the barriers at normal sodium conductance also resulted in a reduction in excited elements and ST-segment elevation on the ECG. However, the increased resistance between the excited and unexcited elements limited the ST-segment amplitude.

      Discussion

      This study shows for the first time the activation and repolarization characteristics of the complete heart of a carrier of a loss-of-function mutation in SCN5A after provocation of right-sided ST-segment elevation by sodium channel blockade. ST-segment elevation coincided with the local disappearance of the initial activation and the appearance of monophasic ST-segment elevation in unipolar electrograms at the basal epicardial RV. At these sites, fibrosis and fatty infiltration caused discontinuities in the subepicardium. In a computer model encompassing the heart and thorax, structural discontinuities were simulated by the introduction of nonconducting barriers containing gaps in the RV subepicardium. Successful conduction through these gaps depended on their simulated size and was modulated by the available cardiac sodium current. Excitation failure and activation delay of the RV subepicardium resulted in ST-segment elevation and a negative T wave in the right precordial leads of the ECG, respectively. Therefore, current-to-load mismatch may underlie the Brugada sign in patients with RV structural discontinuities.
      In the isolated heart, we found no support for either of the preexisting hypotheses of ST-segment elevation in the Brugada sign.
      • 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.
      • Yan G.X.
      • Antzelevitch C.
      Cellular basis for the Brugada syndrome and other mechanisms of arrhythmogenesis associated with ST-segment elevation.
      Latest activation coincided with the end of the QRS complex and earliest repolarization with the start of the T wave on pseudo-ECG, leaving 250 ms of ST-segment elevation that could not be explained by either late activation or early repolarization. Other causes of ST-segment elevation that were not assessed directly in this heart are regional differences in resting membrane potential or in plateau potential amplitude.
      • Holland R.P.
      • Brooks H.
      Precordial and epicardial surface potentials during myocardial ischemia in the pig A theoretical and experimental analysis of the TQ and ST segments.
      A regional difference in resting membrane potential generates TQ-segment depression that cannot be distinguished from true ST-segment elevation by AC electrograms. However, the resting membrane potentials in isolated myocytes were similar to those previously found in dilated and ischemic cardiomyopathy,
      • Den Ruijter H.M.
      • Berecki G.
      • Verkerk A.O.
      • et al.
      Acute administration of fish oil inhibits triggered activity in isolated myocytes from rabbits and patients with heart failure.
      and INa reduction has no direct effect on the resting membrane potential. Likewise, regional differences in plateau potential amplitude likely did not play a role as local ST-segment elevation was accompanied by the virtual disappearance of the QRS complex in unipolar electrograms.
      The computer model is based on a membrane model of human ventricular myocytes that incorporates transmural differences in the density of the transient outward current.
      • Potse M.
      • Dubé B.
      • Richer J.
      • Vinet A.
      • Gulrajani R.M.
      A Comparison of monodomain and bidomain reaction-diffusion models for action potential propagation in the human heart.
      Reduction of INa alone did not cause either early repolarization or a reduced plateau potential amplitude at the RV subepicardium and did not result in ST-segment elevation. However, reduction of INa did result in the Brugada sign after the introduction of discontinuous barriers in the RV subepicardium. The cause was the combination of excitation failure (ST-segment elevation) and activation delay (negative T-wave) of the subepicardium by current-to-load mismatch. The plateau potential amplitude was reduced proximal to the site of excitation failure. However, this was caused by electrotonic interaction with the unexcited myocardium.
      Whether current-to-load mismatch can also cause the Brugada sign in patients with the Brugada syndrome is unclear because its diagnosis requires exclusion of structural heart disease.
      • Antzelevitch C.
      • Brugada P.
      • Borggrefe M.
      • et al.
      Brugada syndrome: report of the second consensus conference: endorsed by the Heart Rhythm Society and the European Heart Rhythm Association.
      Several histologic and imaging studies recently demonstrated that a variety of myocardial changes, not recognized during standard clinical investigation, are present in many patients with 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.
      • Frustaci A.
      • Priori S.G.
      • Pieroni M.
      • et al.
      Cardiac histological substrate in patients with clinical phenotype of Brugada syndrome.
      • Zumhagen S.
      • Spieker T.
      • Rolinck J.
      • et al.
      Absence of pathognomonic or inflammatory patterns in cardiac biopsies from patients with Brugada syndrome.
      • Papavassiliu T.
      • Wolpert C.
      • Flüchter S.
      • et al.
      Magnetic resonance imaging findings in patients with Brugada syndrome.
      In addition, late potentials on signal-averaged ECGs, usually associated with structural heart disease, are a common finding in the Brugada syndrome.
      • Ikeda T.
      • Sakurada H.
      • Sakabe K.
      • et al.
      Assessment of noninvasive markers in identifying patients at risk in the Brugada syndrome: insight into risk stratification.
      Therefore, lack of sensitivity of standard imaging modalities may render structural abnormalities at the RV subepicardium undetected. Thus, excitation failure in discontinuous myocardium can be the mechanism of the Brugada sign in the Brugada syndrome as well.
      The heart of our patient showed the gross pathology of an end-stage dilated cardiomyopathy. Histologically, we found structural abnormalities that have been described as occurring in biopsy samples from patients with Brugada syndrome and proven SCN5A mutations (fibrosis, cardiomyopathic cellular changes);
      • 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.
      • Frustaci A.
      • Priori S.G.
      • Pieroni M.
      • et al.
      Cardiac histological substrate in patients with clinical phenotype of Brugada syndrome.
      • Zumhagen S.
      • Spieker T.
      • Rolinck J.
      • et al.
      Absence of pathognomonic or inflammatory patterns in cardiac biopsies from patients with Brugada syndrome.
      however, these pathologic changes also have been reported in patients with Brugada syndrome but no such a mutation, particularly lymphocytic myocarditis.
      • Frustaci A.
      • Priori S.G.
      • Pieroni M.
      • et al.
      Cardiac histological substrate in patients with clinical phenotype of Brugada syndrome.
      Therefore, whether the current-to-load mismatch in the RV subepicardium relates to structural pathology resulting from the SCN5A mutation, other coincidental inflammatory pathology, or both remains unsettled.
      An argument against the involvement of excitation failure by current-to-load mismatch in the Brugada syndrome is the observation that ST-segment amplitude and risk of ventricular arrhythmias appear largest during slow heart rhythms (at night or at rest).
      • Mizumaki K.
      • Fujiki A.
      • Tsuneda T.
      • et al.
      Vagal activity modulates spontaneous augmentation of ST elevation in the daily life of patients with Brugada syndrome.
      • Matsuo K.
      • Kurita T.
      • Inagaki M.
      • et al.
      The circadian pattern of the development of ventricular fibrillation in patients with Brugada syndrome.
      In our study, ST-segment elevation increased with stimulation frequency. This can be explained in part by the use of ajmaline, which has a rate-dependent capacity to block sodium channels.
      • Heistracher P.
      Mechanism of action of antifibrillatory drugs.
      Another explanation is the absence of autonomic innervation in the isolated heart. When the safety factor for conduction is reduced, the depolarizing current of the L-type calcium channel can be crucial for conduction success.
      • Shaw R.M.
      • Rudy Y.
      Ionic mechanisms of propagation in cardiac tissue: roles of the sodium and L-type calcium currents during reduced excitability and decreased gap junction coupling.
      • Rohr S.
      • Kucera J.P.
      Involvement of the calcium inward current in cardiac impulse propagation: induction of unidirectional conduction block by nifedipine and reversal by Bay K 8644.
      The L-type calcium current decreases during increased vagal and decreased sympathetic activity. Therefore, the autonomic nervous system may modulate the success of conduction in current-to-load mismatch and ST-segment elevation by excitation failure. In addition, the decrease of ST-segment elevation in the Brugada syndrome by beta-adrenergic stimulation and the increase by acetylcholine can be explained by the effect of the L-type calcium current on conduction.
      • Miyazaki T.
      • Mitamura H.
      • Miyoshi S.
      • Soejima K.
      • Ogawa S.
      • Aizawa Y.
      Autonomic and antiarrhythmic drug modulation of ST segment elevation in patients with Brugada syndrome.
      The rapid variability of the Brugada sign over time likely is not related to structural changes. Besides modulation by pharmacologic agents and the autonomic nervous system, other factors may influence the Brugada sign by excitation failure. Fish oil reduces the cardiac sodium current after acute administration and reduces the L-type calcium current both after acute administration and in feeding experiments.
      • Den Ruijter H.M.
      • Berecki G.
      • Opthof T.
      • Verkerk A.O.
      • Zock P.L.
      • Coronel R.
      Pro- and antiarrhythmic properties of a diet rich in fish oil.
      Therefore, intake of fish oil may reduce the success of conduction in current-to-load mismatch and may augment the Brugada sign by this mechanism. Furthermore, the load at sites of sudden myocardial expansion depends on the intercellular resistance with the surrounding myocardium and is modulated by electrical coupling via connexin43. The half-life of connexin43 is short (∼1.3 hours).
      • Beardslee M.A.
      • Laing J.G.
      • Beyer E.C.
      • Saffitz J.E.
      Rapid turnover of connexin43 in the adult rat heart.
      Changes in the turnover of connexin43 can have a rapid effect on excitation failure by current-to-load mismatch and on the Brugada sign by this mechanism.
      Excitation failure by current-to-load mismatch provides a unifying hypothesis for the ST-segment elevation and predisposition to ventricular arrhythmias seen in patients with the Brugada sign. Previous studies have demonstrated that unexcited tissue shortens the action potential duration and effective refractory period of myocardium proximal to the site of block by electrotonic interaction.
      • Sasyniuk B.I.
      • Mendez C.
      A mechanism for reentry in canine ventricular tissue.
      • Rohr S.
      • Salzberg B.M.
      Characterization of impulse propagation at the microscopic level across geometrically defined expansions of excitable tissue: multiple site optical recording of transmembrane voltage (MSORTV) in patterned growth heart cell cultures.
      • Mendez C.
      • Mueller W.J.
      • Merideth J.
      • Moe G.K.
      Interaction of transmembrane potentials in canine Purkinje fibers and at Purkinje fiber-muscle junctions.
      This combination of unidirectional block and a local decrease in refractoriness can initiate reentry even without the need for a premature beat.
      • Sasyniuk B.I.
      • Mendez C.
      A mechanism for reentry in canine ventricular tissue.
      It also explains why the duration of ST-segment elevation in unipolar electrograms observed in this study was shorter than the activation-recovery interval, which is a measure of action potential duration, before sodium channel blockade.

       Study limitations

      No sodium channel provocation protocol was performed prior to cardiac explantation, which hinders extrapolation of the pseudo-ECG to the patient's ECG. However, the concentration of ajmaline used in our study was similar to those used clinically to provoke the Brugada sign, and our simulations confirm that subepicardial excitation failure and activation delay at the RV shows as the Brugada sign in the right precordial leads. Therefore, the mechanisms described in our study likely would have occurred in the patient as well.

      Conclusion

      Structural discontinuities at the RV subepicardium can cause excitation failure and activation delay by current-to-load mismatch, especially when the available cardiac sodium current is reduced. Excitation failure and activation delay at the RV subepicardium show as the Brugada sign on ECG. Therefore, current-to-load mismatch may underlie ventricular arrhythmias in patients with the Brugada sign and RV structural discontinuities.

      Acknowledgments

      We thank Wim ter Smitte, Charly Belterman, M.E. Campian, Antonius Baartscheer, and Carlo Marcelis for invaluable assistance.

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