Abstract 14386: RNA Binding Protein HuR Regulates Cardiac Sodium Channel Gene (SCN5A) Expression Through Transcriptional and Post-Transcriptional Pathways
Introduction: In patients, deletions or loss-of-function mutations of SCN5A have been associated with a wide range of arrhythmias. Our previous results have shown that the expression of SCN5A decreases in failing hearts and the reduced expression of SCN5A is associated with the arrhythmia risk in heart failure. mRNA levels are determined by the balance between transcription and mRNA degradation. While transcriptional regulation of SCN5A expression has been studied and is controlled in part by MEF2C, a cardiac transcription factor, little is known about regulation of SCN5A mRNA degradation. Here, we examined how the degradation of SCN5A mRNA is regulated by HuR, an AU-rich element binding protein.
Methods: Human fetal cardiomyocyte cells were infected with lenti-viral particles carrying inducible HuR or MEF2C over expression constructs using TransDux virus transduction reagent. siRNA was transfected into cardiomyocyte using RNAiMAX reagent. Human SCN5A promoter fragments were cloned into pGL3-Basic vector and transfected into cardiomyocyte using SuperFect reagent. Total RNA from cultured cells was isolated using RNAeasy mini plus kit. Real-time quantitative RT-PCR (RT-qPCR) was conducted to detect the target gene mRNAs.
Results: SCN5A mRNA expression increased in a dose-dependent manner by doxycycline-induced overexpression of MEF2C. Knockdown of MEF2C by siRNA reduced SCN5A mRNA level by 22.3% (P=0.001). Luciferase mRNA driven by human SCN5A 2kb promoter increased 3.81 fold by overexpression of Mef2C compared to control (3.81± 0.71 vs 1.00 ± 0.12, P=0.002). Overexpression of HuR in human cardiomyocyte increased MEF2C mRNA by 43.7% (P=0.001) and SCN5A mRNA expression by 46.3% (P=0.004). Knockdown of HuR reduces MEF2C mRNA by 24.48% (P=0.01) and SCN5A mRNA by 27.5% (P=0.000). Actinomycin D inhibition assay shows that HuR protected both SCN5A and MEF2C mRNAs from decay.
Conclusions: RNA binding protein HuR regulates SCN5A expression by stabilizing SCN5A mRNA and also by promoting SCN5A gene transcription through increasing the expression of transcription factor MEF2C. Therefore, the loss of HuR in cardiomyopathy contributes to the proarrhythmic downregulation of SCN5A by at least two mechanisms.
Author Disclosures: A. Zhou: None. N. Jiang: None. G. Shi: None. A. Xie: None. H. Liu: None. M. Liu: None. E. Jeong: None. S. Dudley: None.
- © 2016 by American Heart Association, Inc.