Zfhx3 Transcription Factor Represses the Expression of SCN5A Gene and Decreases Sodium Current Density (INa).

Marcos Rubio-Alarcón,Juan Tamargo,Teresa Crespo-García,Eva Delpón,Anabel Cámara-Checa,Jorge Cebrián,José Luis Merino,Jorge Toquero,María Dago,Paloma Nieto-Marín,Ricardo Caballero,María Marín,Rafael Salguero-Bodes

doi:10.3390/ijms222313031

Abstract

The ZFHX3 and SCN5A genes encode the zinc finger homeobox 3 (Zfhx3) transcription factor (TF) and the human cardiac Na+ channel (Nav1.5), respectively. The effects of Zfhx3 on the expression of the Nav1.5 channel, and in cardiac excitability, are currently unknown. Additionally, we identified three Zfhx3 variants in probands diagnosed with familial atrial fibrillation (p.M1260T) and Brugada Syndrome (p.V949I and p.Q2564R). Here, we analyzed the effects of native (WT) and mutated Zfhx3 on Na+ current (INa) recorded in HL-1 cardiomyocytes. ZFHX3 mRNA can be detected in human atrial and ventricular samples. In HL-1 cardiomyocytes, transfection of Zfhx3 strongly reduced peak INa density, while the silencing of endogenous expression augmented it (from −65.9 ± 8.9 to −104.6 ± 10.8 pA/pF; n ≥ 8, p < 0.05). Zfhx3 significantly reduced the transcriptional activity of human SCN5A, PITX2, TBX5, and NKX25 minimal promoters. Consequently, the mRNA and/or protein expression levels of Nav1.5 and Tbx5 were diminished (n ≥ 6, p < 0.05). Zfhx3 also increased the expression of Nedd4-2 ubiquitin-protein ligase, enhancing Nav1.5 proteasomal degradation. p.V949I, p.M1260T, and p.Q2564R Zfhx3 produced similar effects on INa density and time- and voltage-dependent properties in WT. WT Zfhx3 inhibits INa as a result of a direct repressor effect on the SCN5A promoter, the modulation of Tbx5 increasing on the INa, and the increased expression of Nedd4-2. We propose that this TF participates in the control of cardiac excitability in human adult cardiac tissue.

Highlights

ZFHX3 gene encodes zinc finger homeobox 3 (Zfhx3) or the AT motif binding factor (ATBF1), a transcription factor (TF) with multiple homeodomains and zinc finger motifs
Our results demonstrated that WT Zfhx3 inhibits INa as a consequence of a direct repressor effect on the SCN5A promoter, by the modulation of Tbx5-increasing effects on the INa, and by increasing the expression of Nedd4.2
Genotype-Tissue Expression (GTEx) RNA-seq data of ZFHX3 from human atrial (n = 297) and ventricular (n = 303) samples averaged 2.2 ± 0.05 and 1.4 ± 0.04 transcripts per million (TPM), respectively. These data suggest that Zfhx3 is expressed, even though the mRNA expression level, both in the atria and ventricles, is significantly lower (p < 0.01) than that of TBX5 (59 ± 1.5 and 12.6 ± 0.5 TPM in atria and ventricles, respectively) and NKX25 (115 ± 3.3 and 106 ± 3.5 TPM)

Summary

Introduction

ZFHX3 gene encodes Zfhx or the AT motif binding factor (ATBF1), a transcription factor (TF) with multiple homeodomains and zinc finger motifs. Zfhx is widely expressed in many tissues [1] and participates in the regulation of myogenic [2] and neuronal differentiation. Zfhx was reported to inhibit cell proliferation, negatively regulate c-Myb, and trans-activate the cell cycle and cyclin-dependent kinase inhibitor 1A, functioning as a tumor suppressor in several cancers [3]. It was demonstrated that Zfhx participated in some TF networks in the heart. Huang and coworkers showed that Zfhx positively and reciprocally regulated the expression of PITX2, which encodes the Pitx2c TF [4]. Both, Zfhx and Pitx2c, regulate the expressions of NPPA, TBX5 and NKX25 genes which encode the atrial natriuretic peptide, as well as Tbx and Nkx2.5 TFs, respectively [4]

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Results

Conclusion