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

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.

B rugada syndrome (BrS) is an inherited arrhythmia syndrome diagnosed by the presence of the type 1 Brugada electrocardiogram (ECG) (1). It was initially described in survivors of cardiac arrest without structural disease (2), and it is partly responsible for sudden arrhythmic death syndrome (SADS) (1,3,4). Potential causal variants in the cardiac sodium channel gene SCN5A are identified in 20% of cases (5). It was initially proposed that the basis for BrS was an abnormal transmural repolarization in the right ventricular outflow tract (RVOT) due to heterogeneous loss of the cardiomyocyte action potential dome in the epicardium (6). However, electrophysiological, imaging, and histopathological studies have identified subtle structural abnormalities in patients with BrS (7)(8)(9). Myocardial fibrosis has been suggested by abnormal, low-voltage, fractionated electrograms localized to the RVOT at the epicardium (9,10). Ablation at these sites has eliminated the type 1 Brugada ECG pattern and successfully reduced arrhythmic events (10), as was seen in a previous experimental model (11).
A study of sudden cardiac death (SCD) cases associated the type 1 ECG with arrhythmogenic right ventricular cardiomyopathy (8). Furthermore, SCD cases with a familial diagnosis of BrS showed structural abnormalities that were insufficient to fulfill the diagnostic criteria for cardiomyopathy or myocarditis (12). Other myocardial anomalies have been reported in selected cases (13,14). Therefore, there is significant debate about the underlying substrate in BrS (15).
To resolve this controversy, we tested the hypothesis that BrS is associated with fibrosis in the RVOT and altered expression of the gap junction protein connexin-43 (Cx43), which may be critical for correct cellular migration and maintenance of RVOT zonation (16,17). We expected this to manifest as abnormal late and fractionated potentials at the RVOT epicardium. pected SCD cases were referred for specialist cardiac autopsy. We studied 6 male cases (B1 to B6; mean age 23.2 years) ( Table 1), which fulfilled the following criteria for SADS (1): 1) age 1 to 64 years; 2) unexpected sudden death; 3) whole heart available; 4) heart morphologically normal at coronial/medical examiner and specialist cardiac autopsies; 5) no antemortem cardiac conditions; and 6) negative toxicological analysis. In addition, 1 or more first-degree blood relatives had to be diagnosed with BrS (Online Methods) following familial evaluation (1,18,19   Epicardial, mid-myocardial, and endocardial zones and fat cells were defined by consensus ( Figure 1A).

METHODS
Regions of collagen and fat were defined by color threshold, with proportions calculated by cardiac wall and tissue zone relative to tissue area.  Nademanee et al.    Figure 4A2 and 4A3). This collagen distribution pattern is normal in the RV.   In control myocardial tissue, Cx43 localized to the intercalated disc ( Figure 4A4 and 4A5).
BrS cases showed a reduced Cx43 signal and a decreased punctate pattern in the intercalated disc ( Figure 4B4 and 4B5). Ablation at these sites eliminated the type 1 ECG pattern with successful suppression of VT/VF recurrence, giving support to the hypothesis that conduction delay is responsible for the BrS phenotype.  ventricular walls. This was over and above the normal collagen seen in age-and sex-matched control hearts.
In addition, the in vivo cases all had normal cardiac imaging, including computed tomography/magnetic resonance imaging, as well as macroscopically normal hearts on direct visualization during thoracotomy.
These cases, therefore, represent minimally structurally perturbed candidates for the diagnosis of BrS, yet they showed distinctive patterns of fibrosis. This reveals the limitations of current imaging technology for detecting subtle changes in the myocardium that can still give rise to physiologically detectable changes.
We have identified previously that one-third of unexplained SCDs with idiopathic fibrosis and/or hypertrophy had familial diagnoses of BrS (12). LV and RV free-wall, age-related fibrosis has also been seen in mouse models of BrS (21,22). In addition, we    Retrospective investigation by molecular autopsy was not possible in our cases, although the absence of a mutation would not exclude BrS due to the low molecular genetic yield (5).
Our study only included symptomatic BrS cases.
Thus, our observations may reflect a biased population of high-risk subjects. However, myocardial fibrosis has also been identified in low-risk living patients on magnetic resonance imaging (31,32) and histopathology (33).