P579Novel insights into the regulatory mechanisms of scn5a expression

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Abstract

Brugada Syndrome (BrS) is a life-threating arrhythmogenic disease associated with a high risk of Sudden Cardiac Death. Genetic alterations in SCN5A, which encodes the alpha subunit of the cardiac voltage-gated sodium channel (Nav1.5), are the most common cause of BrS. Nav1.5 channel is responsible for the rapid influx of sodium ions that initiate the propagation of the action potential in cardiomyocytes. However, mutations in SCN5A gene only explain 20-25% of the cases with BrS. Our working hypothesis is that a deregulation of SCN5A expression could cause BrS and explain, at least in part, some cases of BrS with no mutation identified. Nevertheless, little is known regarding the regulation of SCN5A expression. Therefore, it is the goal of this study to gain insight into the molecular mechanisms that regulate SCN5A expression at the transcriptional level, and how alterations of these mechanisms contribute to cardiac arrhythmias.

The human SCN5A promoter contains putative binding sites for the zinc finger transcription factor GATA-4. We performed GATA-4 overexpression and knockdown studies in cardiac H9c2 cells, and analyzed the effect on the SCN5A promoter by luciferase reporter experiments and real-time PCR. Our data showed that GATA-4 is a novel transcriptional activator of the SCN5A promoter. We also analyzed the effect of other GATA family members, GATA-5 and -6. Whereas GATA-6 did not have any effect on the SCN5A promoter, GATA-5 increased SCN5A transcriptional activity although to a less extent than GATA-4. Moreover, we observed that GATA-4 significantly synergizes with GATA-5 on SCN5A transcriptional activation. Chromatin Immunoprecipitation (ChIP) experiments from human cardiac tissue showed that GATA-4 and GATA-5 bind to the SCN5A proximal promoter region, confirming a critical role of these factors in regulating SCN5A expression in vivo. Our current experiments are also focused on studying the interaction between GATA-4 and GATA-5 transcription factors.

We performed co-transfection experiments of GATA-4 with p300 acetyltransferase and observed a further increase on SCN5A promoter activity. These results suggest that GATA-4 transcriptional activity on the SCN5A promoter is modulated by acetylation. We are now investigating which histones acetyltransferases (HDACs) could participate in the activity of GATA-4 on the SCN5A promoter.

Our findings provide novel insights on the regulation of Nav1.5 expression, which will ultimately contribute to further understanding unexplored causes of BrS and other type of cardiac arrhythmias.

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