Abstract
BACKGROUND The right ventricular outflow tract (RVOT) is acknowledged to be responsible for arrhythmogenesis in
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) .
B
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) .
B
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.
METHODS STUDY SETTING AND COHORTS. P o s t -m o r t e m B r S c o h o r t .
From 2005 to 2010, 1,304 unexpected 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 STUDY SETTING AND COHORTS. P o s t -m o r t e m B r S c o h o r t .
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.
METHODS STUDY SETTING AND COHORTS. P o s t -m o r t e m B r S c o h o r t .
Myocardial Abnormalities in Brugada Syndrome 
Myocardial Abnormalities in Brugada Syndrome
Brugada ECG pattern, with other relatives identified following ajmaline provocation ( Figure 3) . No relatives had evidence of structural or functional myocardial disease on cardiac imaging. Figure 4A2 and 4A3) . This collagen distribution pattern is normal in the RV. other abbreviations as in Figure 1 .
Myocardial Abnormalities in Brugada Syndrome
N O V E M B E R 3 , 2 0 1 5 : 1 9 7 6 -8 6
Myocardial Abnormalities in Brugada Syndrome
In the post-mortem BrS group, there was an appearance of increased epicardial surface collagen that was thicker than that in control hearts, indicating epicardial fibrosis ( Figure 4B1 ). There was infiltration of the epicardial surface fibrosis into the underlying epicardial myocardium, with individual myocytes surrounded by collagen, which was considered interstitial myocardial fibrosis ( Figure 4B2 ). There was also evidence of replacement of myocytes by collagen, focal replacement fibrosis, admixed with fat in the epicardial myocardium ( Figure 4B3 ). The in vivo tissue samples taken in the regions of late potentials showed similar epicardial and myocardial fibrosis patterns ( Figure 4C1 to 4C3) . The epicardial fibrosis appeared to be separated from the underlying myocardium by fat in some sections, whereas in others, it infiltrated directly into the underlying myocardium.
MORPHOMETRIC ANALYSIS OF POST-MORTEM
COLLAGEN BY PSR. The BrS cohort had greater collagen content than control hearts, with maximal differences seen in the RVOT epicardium (13.9% vs.
MORPHOMETRIC ANALYSIS OF POST-MORTEM
10.5%; p ¼ 0.024) ( Figure 5A ). Multivariable analysis ( Similarly, a gradient of decreasing collagen content was seen from the epicardial to endocardial zones (OR: 2.00; p ¼ 0.001) in all chambers.
MORPHOMETRIC ANALYSIS OF POST-MORTEM FAT
CELLS. Regression analysis for the proportion of fat content in the myocardium showed no significant difference between BrS and control hearts (p ¼ 0.133).
MORPHOMETRIC ANALYSIS OF POST-MORTEM FAT
POST-MORTEM Cx43 SIGNAL DISTRIBUTION AND QUANTIFICATION. In control myocardial tissue, Cx43 localized to the intercalated disc ( Figure 4A4 and 4A5) .
MORPHOMETRIC ANALYSIS OF POST-MORTEM FAT
BrS cases showed a reduced Cx43 signal and a decreased punctate pattern in the intercalated disc ( Figure 4B4 and 4B5) . Nademanee et al.
MORPHOMETRIC ANALYSIS OF POST-MORTEM FAT
ventricular walls. This was over and above the normal collagen seen in age-and sex-matched control hearts.
MORPHOMETRIC ANALYSIS OF POST-MORTEM FAT
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.
MORPHOMETRIC ANALYSIS OF POST-MORTEM FAT
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.
MORPHOMETRIC ANALYSIS OF POST-MORTEM FAT
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).
MORPHOMETRIC ANALYSIS OF POST-MORTEM FAT
Our study only included symptomatic BrS cases.
MORPHOMETRIC ANALYSIS OF POST-MORTEM FAT
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) . 
CONCLUSIONS


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