Transcriptional Control of Pacemaker Channel Genes <i>HCN2</i> and <i>HCN4</i> by Sp1 and Implications in Re-expression of These Genes in Hypertrophied Myocytes

Abstract

Cardiac hypertrophy is characterized by electrical remolding with increased risk of arrhythmogenesis. Enhanced abnormal automaticity of ventricular cells may contribute to hypertrophic arrhythmias. The pacemaker current I_f, carried by the hyperpolarization-activated channels encoded mainly by the HCN2 and HCN4 genes in the heart, plays an important role in rhythmogenesis. Their expressions reportedly increase in hypertrophic and failing hearts, contributing to arrhythmogenesis under these conditions. However, how their expressions are controlled remained unclear. We performed a study to characterize the regulatory elements and transcriptional control of HCN2 and HCN4 genes. We identified the transcription start sites by 5’RACE and core promoter regions of these genes using luciferase reporter assay, and revealed the ubiquitous Sp1 protein as a common transactivator of HCN2 and HCN4 genes. We further unraveled robust increases in HCN2/HCN4 transcripts and protein levels, using real-time RT-PCR and Western blot analyses, in a rat model of left ventricular hypertrophy and in angiotensin II-induced neonatal ventricular hypertrophy. The upregulation of HCN2 and HCN4 transcription was accompanied by pronounced elevations of Sp1 and silencing of Sp1 by siRNA prevented overexpression of HCN2/HCN4 in hypertrophic cardiomyocytes. Our data indicate that Sp1 drives HCN2/HCN4 transcription and determines the functional level of HCN2/HCN4 mRNAs, and upregulation of Sp1 underlie the abnormal re-expression of HCN2/HCN4 genes in hypertrophied myocytes. This study also provides the first evidence for the role of Sp1 in the reactivation of ‘fetal’ cardiac genes, HCN2 and HCN4, in ventricular myocytes, and thereby in the pathological electrical remodeling in hypertrophied myocytes.

MicroRNA-26 governs profibrillatory inward-rectifier potassium current changes in atrial fibrillation

“…Fragments corresponding to the promoter regions spanning the putative NFAT-binding sites upstream of the TSSs of Ctdspl (for miR-26a-1), Ctdsp2 (for miR-26a-2) and Ctdsp1 (for miR-26b) were synthesized by Invitrogen. The fragments were subcloned into luciferase-containing pGL3-promoter vector (Promega) for study of the role of NFAT in regulating transcription of the miR-26 family members and their host genes, according to procedures described elsewhere (44,45). The sequences of the promoter fragments used were as follows (boldface letters indicate the NFAT-binding sites): Ctdspl (for miR-26a-1), 5′-ATTATTCAGTCTATCTTGAATGTGCT-GTAAGGACTGGGATAAAGATATATTTTTTCCTCATGGATAGG-TACTTGTCCCAACAATTTTTGA; Ctdsp2 (for miR-26a-2), 5′-TGTTTC-CAAATTGCCTCTTACCAACCATGCAGTCAGAGAGGCCAGAG-GAAAGGGGATACAGCAGGTAGGGAACCAAGTGAGAGTCAGTGG; Ctdsp1 (for miR-26b), 5′-AAGACCCATTTTACAGATGAGGTAGTGC-TATCTCCAAGTCCTCAACGAGGAAACCGAGAAGCCTCTAGTCCC-GGGTCTTCAGAAAACGCA.…”

“…The binding sites for various transcription factors in the promoter regions of the host genes of miR-26a-1, miR-26a-2, and miR-26b from different species (human, canine, rat, and mouse) were analyzed with MatInspector V2.2 (Genomatix) (44). Analyses were made to the 5′ flanking regions 5 kb upstream of the transcriptional start sites of the host genes (Supplemental Table 4).…”

“…Single-stranded phosphorothioate dODNs were synthesized by Integrated DNA Technologies Inc. (Supplemental Figure 11). The ODN was dried and dissolved in sterilized Tris-EDTA buffer (10 mM Tris + 1 mM EDTA) (44,47). The supernatant was purified using Micro Bio-spin30 columns (Bio-Rad) and quantified by spectrophotometry.…”

“…For luciferase assay involving miRNA function, H9c2 cells were transfected with the pMIR-REPORT luciferase miRNA expression reporter vector carrying the 3′ UTR of KCNJ2 or promoterluciferase fusion plasmid pGL3, as previously described in detail (1,44,45).…”

An Approach to Identify Nerve Injury-Evoked Changes that Contribute to the Development or Protect Against the Development of Sustained Neuropathic Pain

“…As expected when comparisons were done only between one of the SNI groups (Sural-SNI or Tibial-SNI) and Sham it resulted in a large number of genes that displayed changes in their expression in that SNI group; however, when comparisons were done by selecting the genes that their expression changed in one of the SNI groups but not in the Sham and in the other SNI group; the number of genes that changed was highly reduced. Some genes of interest that changed only in the dorsal root ganglia from Tibial-SNI (presumably genes that could be involved in limiting the level of sustained mechanical allodynia) include a reduction in Hcn2 which encodes a hyperpolarization activated cyclic nucleotide-gated K channel 2 that has been shown to contribute to spontaneous rhythmic activity in both heart and brain (Dibbens et al, 2010;Lin et al, 2009); an increase in Map3k10 a kinase that functions preferentially on the JNK signaling pathway, and that has been reported to be involved in nerve growth factor (NGF) induced neuronal apoptosis (NGF is increased following nerve injury) (see ref in (Castillo et al, 2011)). A decrease in Rbm9, a gene that encodes an RNA binding protein that is believe to be a key regulator of alternative exon splicing in the nervous system.…”