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.