Fibrosis, Connexin-43, and Conduction Abnormalities in the Brugada Syndrome

Koonlawee Nademanee, Hariharan Raju, Sofia V de Noronha, Michael Papadakis, Laurence Robinson, Stephen Rothery, Naomasa Makita, Shinya Kowase, Nakorn Boonmee, Vorapot Vitayakritsirikul, Samrerng Ratanarapee, Sanjay Sharma, Allard C van der Wal, Michael Christiansen, Hanno L Tan, Arthur A Wilde, Akihiko Nogami, Mary N Sheppard, Gumpanart Veerakul, Elijah R Behr, Koonlawee Nademanee, Hariharan Raju, Sofia V de Noronha, Michael Papadakis, Laurence Robinson, Stephen Rothery, Naomasa Makita, Shinya Kowase, Nakorn Boonmee, Vorapot Vitayakritsirikul, Samrerng Ratanarapee, Sanjay Sharma, Allard C van der Wal, Michael Christiansen, Hanno L Tan, Arthur A Wilde, Akihiko Nogami, Mary N Sheppard, Gumpanart Veerakul, Elijah R Behr

Abstract

Background: The right ventricular outflow tract (RVOT) is acknowledged to be responsible for arrhythmogenesis in Brugada syndrome (BrS), but the pathophysiology remains controversial.

Objectives: This study assessed the substrate underlying BrS at post-mortem and in vivo, and the role for open thoracotomy ablation.

Methods: Six whole hearts from male post-mortem cases of unexplained sudden death (mean age 23.2 years) with negative specialist cardiac autopsy and familial BrS were used and matched to 6 homograft control hearts by sex and age (within 3 years) by random risk set sampling. Cardiac autopsy sections from cases and control hearts were stained with picrosirius red for collagen. The RVOT was evaluated in detail, including immunofluorescent stain for connexin-43 (Cx43). Collagen and Cx43 were quantified digitally and compared. An in vivo study was undertaken on 6 consecutive BrS patients (mean age 39.8 years, all men) during epicardial RVOT ablation for arrhythmia via thoracotomy. Abnormal late and fractionated potentials indicative of slowed conduction were identified, and biopsies were taken before ablation.

Results: Collagen was increased in BrS autopsy cases compared with control hearts (odds ratio [OR]: 1.42; p = 0.026). Fibrosis was greatest in the RVOT (OR: 1.98; p = 0.003) and the epicardium (OR: 2.00; p = 0.001). The Cx43 signal was reduced in BrS RVOT (OR: 0.59; p = 0.001). Autopsy and in vivo RVOT samples identified epicardial and interstitial fibrosis. This was collocated with abnormal potentials in vivo that, when ablated, abolished the type 1 Brugada electrocardiogram without ventricular arrhythmia over 24.6 ± 9.7 months.

Conclusions: BrS is associated with epicardial surface and interstitial fibrosis and reduced gap junction expression in the RVOT. This collocates to abnormal potentials, and their ablation abolishes the BrS phenotype and life-threatening arrhythmias. BrS is also associated with increased collagen throughout the heart. Abnormal myocardial structure and conduction are therefore responsible for BrS.

Keywords: gap junction; myocardial fibrosis; right ventricular outflow tract; sudden arrhythmic death syndrome; sudden unexpected death.

Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Figures

Central Illustration
Central Illustration
Pathophysiology of Brugada Syndrome: Conduction Delay Due to Fibrosis and Connexin-43 Abnormalities Conduction delay in the right ventricular outflow tract (RVOT) is caused by myocyte electrical uncoupling due to a reduction in connexin-43 at endplates and subtle interstitial and replacement fibrosis. As a result, epicardial electrograms are abnormal, slowed, and fragmented. This provides the substrate for the Brugada type 1 electrocardiographic (ECG) pattern, re-entry, and the generation of polymorphic ventricular tachycardia (VT) and ventricular fibrillation.
Figure 1
Figure 1
Morphometric Analysis of Histological Stained Sections (A) Morphometric analysis of a single tissue section. (A1) Visually defined tissue zones (yellow polygons) defining the epicardium, mid-myocardium, and endocardium. (A2) Collagen (black). (A3) Fat cells (black). (B) Representative serial myocardial strips from the post-mortem control group for collagen correction for Cx43 morphometric analysis. (B1) Myocardial strip of Cx43 expression. (B2) Serial section aligned to B1, stained with PSR. (B3) A threshold drawing generated from the PSR-stained myocardium image (as created by ImageJ). Scale bar = 200 μm; Cx43 = connexin-43; PSR = picrosirius red.
Figure 2
Figure 2
Computed Tomography Scan, Epicardial Electrograms, and Histology of RVOT of In Vivo BrS Patient Computed tomography scan of the heart (center) of in vivo BrS patient V2 showing an anatomical grid over the anterior RVOT. ECG lead II and a distal bipolar (0.4 mV/cm voltage scale at 30- to 300-Hz filter settings) and unipolar (5 mV/cm voltage scale at 0.05- to 300-Hz filter settings) electrogram at labeled sites are given in surrounding panels, with pacing stimuli indicated by red arrowheads. Abnormal fractionated electrograms are on the (A to C) left and normal electrograms on the (D to E) right. (F) Epicardial biopsy and histology (PSR) at the site of the abnormal electrogram shows epicardial fibrosis with focal finger-like projections of collagen into myocardium. ABL d = distal bipolar ablation catheter electrogram; ABL uni = unipolar ablation catheter electrogram; BrS = Brugada syndrome; RVOT = right ventricular outflow tract; other abbreviations as in Figure 1.
Figure 3
Figure 3
Right Precordial ECG Traces From Blood Relatives of Post-Mortem BrS Cases During Ajmaline Provocation ECG traces acquired following cranial displacement of electrode positions V1 and V2 into 2ics. 2ics = second intercostal space; other abbreviations as in Figures 1 and 2.
Figure 4
Figure 4
RVOT Histological Sections Stained for Collagen and Immunoconfocal Images of Cx43 Expression RVOT histological sections stained for collagen (purple-red) with PSR and immunoconfocal images of gap junction protein Cx43 expression (green fluorescence). Sections from (A) post-mortem control, (B) post-mortem BrS cases, and (C) in vivo BrS patients. (A) Post-mortem control. PSR: (A1) normal epicardial collagen thickness, with (A2) linear collagen between myocytes and (A3) around blood vessels, but no evidence of complete circumscription of myocytes by collagen. Cx43: (A4) normal appearance of gap junction signal concentrated to form transverse stripes, with an organized parallel orientation. (A5) Clusters of gap junctions in a typical ring-like formation at the intercalated disc, with large gap junctions circumscribing the periphery of the disc and smaller junctions in the inner region. (B) Post-mortem BrS. PSR: (B1) thickened epicardial collagen layer, with (B2) evidence of interstitial fibrosis, identified by collagen circumscribing myocytes, and (B3) replacement fibrosis, identified by replacement of myocytes by collagen in a region of infiltration by fat. Cx43: (B4) notable dispersion of the signal along the axis of the cell and (B5) sparse junctional plaque with an ill-defined border. (C) In vivo BrS. PSR: (C1) thickened epicardial collagen layer with (C2) evidence of interstitial fibrosis, identified by collagen circumscribing myocytes, and (C3) replacement fibrosis, identified by replacement of myocytes by collagen. Abbreviations as in Figures 1 and 2.
Figure 5
Figure 5
Scatterplot of Collagen and Cx43 Quantification in the Epicardial Myocardium of the Right Ventricular Outflow Tract of BrS and Control Post-Mortem Cases (A) PSR quantification of collagen content and (B) immunofluorescence quantification of Cx43. Orange data points represent distribution means. Blue data points represent individual cases and controls. Abbreviations as in Figures 2 and 3.

References

    1. Priori S.G., Wilde A.A., Horie M. Executive summary: HRS/EHRA/APHRS expert consensus statement on the diagnosis and management of patients with inherited primary arrhythmia syndromes. Europace. 2013;15:1389–1406.
    1. Brugada P., Brugada J. Right bundle branch block, persistent ST segment elevation and sudden cardiac death: a distinct clinical and electrocardiographic syndrome. A multicenter report. J Am Coll Cardiol. 1992;20:1391–1396.
    1. Behr E., Wood D.A., Wright M., for the Sudden Arrhythmic Death Syndrome (SADS) Steering Group Cardiological assessment of first-degree relatives in sudden arrhythmic death syndrome. Lancet. 2003;362:1457–1459.
    1. Raju H., Behr E.R. Unexplained sudden death, focussing on genetics and family phenotyping. Curr Opin Cardiol. 2013;28:19–25.
    1. Hedley P.L., Jørgensen P., Schlamowitz S. The genetic basis of Brugada syndrome: a mutation update. Hum Mutat. 2009;30:1256–1266.
    1. Yan G.X., Antzelevitch C. Cellular basis for the Brugada syndrome and other mechanisms of arrhythmogenesis associated with ST-segment elevation. Circulation. 1999;100:1660–1666.
    1. Corrado D., Nava A., Buja G. Familial cardiomyopathy underlies syndrome of right bundle branch block, ST segment elevation and sudden death. J Am Coll Cardiol. 1996;27:443–448.
    1. Corrado D., Basso C., Buja G. Right bundle branch block, right precordial ST-segment elevation, and sudden death in young people. Circulation. 2001;103:710–717.
    1. Ohkubo K., Watanabe I., Okumura Y. Right ventricular histological substrate and conduction delay in patients with Brugada syndrome. Int Heart J. 2010;51:17–23.
    1. Nademanee K., Veerakul G., Chandanamattha P. Prevention of ventricular fibrillation episodes in Brugada syndrome by catheter ablation over the anterior right ventricular outflow tract epicardium. Circulation. 2011;123:1270–1279.
    1. Morita H., Zipes D.P., Morita S.T. Epicardial ablation eliminates ventricular arrhythmias in an experimental model of Brugada syndrome. Heart Rhythm. 2009;6:665–671.
    1. Papadakis M., Raju H., Behr E.R. Sudden cardiac death with autopsy findings of uncertain significance: potential for erroneous interpretation. Circ Arrhythm Electrophysiol. 2013;6:588–596.
    1. Frustaci A., Priori S.G., Pieroni M. Cardiac histological substrate in patients with clinical phenotype of Brugada syndrome. Circulation. 2005;112:3680–3687.
    1. Coronel R., Casini S., Koopmann T.T. Right ventricular fibrosis and conduction delay in a patient with clinical signs of Brugada syndrome: a combined electrophysiological, genetic, histopathologic, and computational study. Circulation. 2005;112:2769–2777.
    1. Wilde A.A.M., Postema P.G., Di Diego J.M. The pathophysiological mechanism underlying Brugada syndrome: depolarization versus repolarization. J Mol Cell Cardiol. 2010;49:543–553.
    1. Waldo K.L., Lo C.W., Kirby M.L. Connexin 43 expression reflects neural crest patterns during cardiovascular development. Dev Biol. 1999;208:307–323.
    1. Elizari M.V., Levi R., Acunzo R.S. Abnormal expression of cardiac neural crest cells in heart development: a different hypothesis for the etiopathogenesis of Brugada syndrome. Heart Rhythm. 2007;4:359–365.
    1. Raju H., Papadakis M., Govindan M. Low prevalence of risk markers in cases of sudden death due to Brugada syndrome: relevance to risk stratification in Brugada syndrome. J Am Coll Cardiol. 2011;57:2340–2345.
    1. Govindan M., Batchvarov V.N., Raju H. Utility of high and standard right precordial leads during ajmaline testing for the diagnosis of Brugada syndrome. Heart. 2010;96:1904–1908.
    1. Basso C., Burke M., Fornes P., for the Association for European Cardiovascular Pathology Guidelines for autopsy investigation of sudden cardiac death. Pathologica. 2010;102:391–404.
    1. Royer A., van Veen T.A.B., Le Bouter S. Mouse model of SCN5A-linked hereditary Lenègre’s disease: age-related conduction slowing and myocardial fibrosis. Circulation. 2005;111:1738–1746.
    1. Jeevaratnam K., Rewbury R., Zhang Y. Frequency distribution analysis of activation times and regional fibrosis in murine Scn5a+/- hearts: the effects of ageing and sex. Mech Ageing Dev. 2012;133:591–599.
    1. Zhang Y., Guzadhur L., Jeevaratnam K. Arrhythmic substrate, slowed propagation and increased dispersion in conduction direction in the right ventricular outflow tract of murine Scn5a+/- hearts. Acta Physiol (Oxf) 2014;211:559–573.
    1. Hoogendijk M.G., Potse M., Linnenbank A.C. Mechanism of right precordial ST-segment elevation in structural heart disease: excitation failure by current-to-load mismatch. Heart Rhythm. 2010;7:238–248.
    1. Meregalli P.G., Tan H.L., Probst V. Type of SCN5A mutation determines clinical severity and degree of conduction slowing in loss-of-function sodium channelopathies. Heart Rhythm. 2009;6:341–348.
    1. Hao X., Zhang Y., Zhang X. TGF-β1-mediated fibrosis and ion channel remodeling are key mechanisms in producing the sinus node dysfunction associated with SCN5A deficiency and aging. Circ Arrhythm Electrophysiol. 2011;4:397–406.
    1. Lambiase P.D., Ahmed A.K., Ciaccio E.J. High-density substrate mapping in Brugada syndrome: combined role of conduction and repolarization heterogeneities in arrhythmogenesis. Circulation. 2009;120:106–117.
    1. Sacher F., Jesel L., Jais P. Insight into the mechanism of Brugada syndrome: epicardial substrate and modification during ajmaline testing. Heart Rhythm. 2014;11:732–734.
    1. Postema P.G., van Dessel P.F.H.M., de Bakker J.M.T. Slow and discontinuous conduction conspire in Brugada syndrome: a right ventricular mapping and stimulation study. Circ Arrhythm Electrophysiol. 2008;1:379–386.
    1. Zhang J., Sacher F., Hoffmayer K. Cardiac electrophysiological substrate underlying the ECG phenotype and electrogram abnormalities in Brugada syndrome patients. Circulation. 2015;131:1950–1959.
    1. Papavassiliu T., Wolpert C., Flüchter S. Magnetic resonance imaging findings in patients with Brugada syndrome. J Cardiovasc Electrophysiol. 2004;15:1133–1138.
    1. Van Hoorn F., Campian M.E., Spijkerboer A. SCN5A mutations in Brugada syndrome are associated with increased cardiac dimensions and reduced contractility. PloS One. 2012;7:e42037.
    1. Zumhagen S., Spieker T., Rolinck J. Absence of pathognomonic or inflammatory patterns in cardiac biopsies from patients with Brugada syndrome. Circ Arrhythm Electrophysiol. 2009;2:16–23.
    1. Hoogendijk M.G., Opthof T., Postema P.G. The Brugada ECG pattern: a marker of channelopathy, structural heart disease, or neither? Toward a unifying mechanism of the Brugada syndrome. Circ Arrhythm Electrophysiol. 2010;3:283–290.

Source: PubMed

Sponsor e collaboratori

Condizioni mediche

Interventi farmacologici

3
Sottoscrivi