Buccal cell proteomics reveals myocardial ATP1B1 deficiency in brugada syndrome

Introduction Brugada syndrome (BrS) is a major cause of sudden cardiac death (SCD) worldwide. The most commonly mutated gene in BrS is SCN5A, encoding the α-subunit of the cardiac sodium channel; however, this accounts for only 20% of cases, and despite decades of research, most remain genetically and mechanistically unexplained. BrS has traditionally been regarded as a cardiac channelopathy without overt structural abnormalities. Recent studies suggest subtle histological changes in BrS hearts, yet these are insufficient for reliable post-mortem diagnosis. There is therefore an unmet clinical need for cardiac biomarkers of BrS. Buccal cells have been shown to represent a suitable surrogate for cardiac tissue in arrhythmogenic cardiomyopathy (ACM). Aim The aim of this project is to identify novel biomarkers and potential aberrantly affected pathways in BrS. Methods Buccal cell suspensions from 2 BrS patients and 2 age-matched controls underwent subcellular proteome fractionation followed by quantitative mass spectrometry (MS). Both patients presented with a spontaneous type 1 ECG pattern and had ICDs implanted. One patient (no variant identified) manifested with syncope, while the other patient (SCN5A variant identified) manifested with cardiac arrest. Formalin-fixed paraffin-embedded (FFPE) myocardial sections from SCD victims and controls were analysed by immunohistochemistry (IHC) and confocal microscopy to validate candidate biomarkers identified by MS-based pathway enrichment. Results MS revealed dysregulation of the transporter complex in buccal cells from BrS patients versus controls. The most affected protein within the pathway was the sodium/potassium-transporting ATPase subunit beta-1 (ATP1B1), which was selected for validation. Immunostaining was optimized to yield a binary outcome; presence versus near/total absence of ATP1B1 at the intercalated discs (IDs) and/or lateral membranes (LMs). In control myocardium (n=6), ATP1B1 localized to both IDs and LMs. In BrS-related SCD (n=11), ATP1B1 was virtually absent from the IDs in 73% and from LMs in 82% (Figure 1). To assess specificity, myocardial sections from SCD victims with ACM (n=7), a known BrS phenocopy, were analysed. ATP1B1 distribution was indistinguishable from controls in 71% of ACM samples both at the IDs and LMs (Figure 1). Conclusions Our results suggest that MS-based analysis of buccal cell proteome fractions is a powerful strategy to identify novel biomarkers and mechanistic therapeutic targets in heritable cardiac disease. Expression of ATP1B1 is markedly reduced in BrS myocardium but preserved in ACM, suggesting ATP1B1 as a potential biomarker to distinguish BrS from overlapping entities at post-mortem in SCD victims.