DEVELOPMENT OF FUNCTIONAL ASSAYS FOR THE STUDY OF SODIUM, CALCIUM, AND POTASSIUM ION CHANNELS ACTIVITY IN BRUGADA SYNDROME

Brugada Syndrome (BrS) is an autosomal dominant channelopathy that presents a specific ECG pattern characterized by an elevation of the ST-segment in the right precordial leads, and it is related to an increased risk of sudden cardiac death (SCD). The BrS ECG patterns can manifest in three different types: type 1, type 2, and type 3. Type 1 is spontaneous and easily recognized by a normal ECG, while type 2 and type 3 are often ambiguous and they require a diagnostic test for their correct identification. The test consists in the administration of a sodium channel blocking agent which, in the presence of the pathology, unmasks the ECG pattern. This test presents several risks and it has to be executed by highly specialized teams. Thus, the development of a new test based on the measurement of specific biomarkers would therefore be crucial. Nowadays, BrS patients’ treatment requires an implantable cardioverter-defibrillator, followed by an epicardial ablation of the BrS arrhythmogenic substrate through a catheter radiofrequency that eliminates the electrical abnormalities in the RVOT. In about 30% of BrS cases, it is possible to determine a genetic component responsible for the onset of the disease; the most frequent cause, among these cases, is a mutation in the SCN5A gene, encoding the asubunit of the cardiac voltage-dependent sodium channel NaV1.5. More than 300 mutations, distributed over the entire sequence, have been found in the SCN5A gene. Usually, these mutations cause a loss-offunction of the channel even if the mechanism and the extent of the dysfunction may vary, according to the mutation; it has also been shown that mutations that cause a reduction of the sodium flux >90% or a truncated protein, can cause more severe clinical phenotypes. Unfortunately, it is currently not easy to determine the direct genotype-phenotype relationship, thus, being able to experimentally define this link would be very important, as it could provide a new parameter useful in risk stratification. In addition to sodium channel dysfunction, calcium and potassium ion channels alterations have been demonstrated as a cause of the BrS manifestation. Therefore, understanding ion channels dysfunctions and their effects could improve BrS risk stratification or diagnosis, which remain clinical challenges that need to be addressed. Based on these premises, the aim of the study was firstly to develop an in vitro functional voltage-sensing assay to measure NaV1.5 activity, carrying BrS SCN5A mutations to ultimately correlate them to the BrS phenotypes; secondly, we optimized fluorescence functional assays for the evaluation of potassium and calcium channel activity in peripheral blood cells to find potential biomarkers for BrS. For the first part of the thesis, several novel mutations were identified through the genetic screening of the SCN5A gene in BrS patients, and four of them were fully characterized by in silico and in vitro analysis. The bioinformatics prediction classified the four mutations as “probably damaging”, and the functional assay confirmed this result, revealing a lower sodium channel activity as compared to wild-type SCN5A. In the second part, we developed and optimized two fluorescence in vitro functional assay for both potassium and calcium channels on peripheral blood cells to find potential biomarkers in BrS patients. The analysis revealed that the calcium channels activity in BrS PBMCs was higher compared to their controls, and the potassium channels activity was higher in BrS T lymphocytes compared to healthy controls. In conclusion, in this Ph.D. thesis, we developed three functional assays to study sodium, calcium, and potassium channels activity. In future, the results obtained would represent the basis for the identification of BrS biomarker, the development of a new diagnostic test, and for a better understanding of BrS etiology.