Informatic and Functional Approaches to Identifying a Regulatory Region for the Cardiac Sodium Channel

Abstract

Rationale: Although multiple lines of evidence suggest that variable expression of the cardiac sodium channel gene SCN5A plays a role in susceptibility to arrhythmia, little is known about its transcriptional regulation. Objective: We used in silico and in vitro experiments to identify possible noncoding sequences important for transcriptional regulation of SCN5A . The results were extended to mice in which a putative regulatory region was deleted. Methods and Results: We identified 92 noncoding regions highly conserved (>70%) between human and mouse SCN5A orthologs. Three conserved noncoding sequences (CNS) showed significant (>5-fold) activity in luciferase assays. Further in vitro studies indicated one, CNS28 in intron 1, as a potential regulatory region. Using recombinase-mediated cassette exchange (RMCE), we generated mice in which a 435–base pair region encompassing CNS28 was removed. Animals homozygous for the deletion showed significant increases in SCN5A transcripts, Na V 1.5 protein abundance, and sodium current measured in isolated ventricular myocytes. ECGs revealed a significantly shorter QRS (10.7±0.2 ms in controls versus 9.7±0.2 ms in knockouts), indicating more rapid ventricular conduction. In vitro analysis of CNS28 identified a short 3′ segment within this region required for regulatory activity and including an E-box motif. Deletion of this segment reduced reporter activity to 3.6%±0.3% of baseline in CHO cells and 16%±3% in myocytes (both P <0.05), and mutation of individual sites in the E-box restored activity to 62%±4% and 57%±2% of baseline in CHO cells and myocytes, respectively (both P <0.05). Conclusions: These findings establish that regulation of cardiac sodium channel expression modulates channel function in vivo, and identify a noncoding region underlying this regulation.