Transcriptional mechanisms of sino-atrial node specific channel HCN4 in the heart.

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We aimed to clarify the transcriptional mechanism of hyperpolarization activated, cyclic nucleotide sensitive channel Hcn4, which is specifically expressed in cardiac sinoatrial node (SAN). We identified 16 conserved non coding sequences (CNS1-16) in the genomic locus of Hcn4. CNS13 possessed enhancer activity on the minimal promoter of Hcn4. CNS13 possessed MEF2- and AP1 binding motif. The binding activity was confirmed using chromatin immunoprecipitation and electrophoresis mobility sift assay. Hcn4 expression was inhibited when dominant negative MEF2 was overexpressed in fetal cardiac myocyte. We therefore concluded that Hcn4 was a direct transcriptional target of MEF2. However, transgenic mouse carrying CNS13-lacZ failed to reproduce SAN specific expression pattern, suggested that multiple CNSs are required for SAN specific expression. We next identified 25 transcription factors highly expressed in SAN including Isl1, Shox2, and Tbx3 with microarray. Overexpression of Shox2 in cultured cardiomyocytes increased Hcn4 expression, only when NRSF expression was simultaneously knocked down, stimulating Hcn4 minimal promoter activity.