A common variant alters SCN5A–miR-24 interaction and associates with heart failure mortality
Xiaoming Zhang, Jin-Young Yoon, Michael Morley, Jared M. McLendon, Kranti A. Mapuskar, Rebecca Gutmann, Haider Mehdi, Heather L. Bloom, Samuel C. Dudley, Patrick T. Ellinor, Alaa A. Shalaby, Raul Weiss, W.H. Wilson Tang, Christine S. Moravec, Madhurmeet Singh, Anne L. Taylor, Clyde W. Yancy, Arthur M. Feldman, Dennis M. McNamara, Kaikobad Irani, Douglas R. Spitz, Patrick Breheny, Kenneth B. Margulies, Barry London, Ryan L. Boudreau
Xiaoming Zhang, Jin-Young Yoon, Michael Morley, Jared M. McLendon, Kranti A. Mapuskar, Rebecca Gutmann, Haider Mehdi, Heather L. Bloom, Samuel C. Dudley, Patrick T. Ellinor, Alaa A. Shalaby, Raul Weiss, W.H. Wilson Tang, Christine S. Moravec, Madhurmeet Singh, Anne L. Taylor, Clyde W. Yancy, Arthur M. Feldman, Dennis M. McNamara, Kaikobad Irani, Douglas R. Spitz, Patrick Breheny, Kenneth B. Margulies, Barry London, Ryan L. Boudreau
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Research Article Cardiology Genetics

A common variant alters SCN5A–miR-24 interaction and associates with heart failure mortality

Abstract

SCN5A encodes the voltage-gated Na+ channel NaV1.5 that is responsible for depolarization of the cardiac action potential and rapid intercellular conduction. Mutations disrupting the SCN5A coding sequence cause inherited arrhythmias and cardiomyopathy, and single-nucleotide polymorphisms (SNPs) linked to SCN5A splicing, localization, and function associate with heart failure–related sudden cardiac death. However, the clinical relevance of SNPs that modulate SCN5A expression levels remains understudied. We recently generated a transcriptome-wide map of microRNA (miR) binding sites in human heart, evaluated their overlap with common SNPs, and identified a synonymous SNP (rs1805126) adjacent to a miR-24 site within the SCN5A coding sequence. This SNP was previously shown to reproducibly associate with cardiac electrophysiological parameters, but was not considered to be causal. Here, we show that miR-24 potently suppresses SCN5A expression and that rs1805126 modulates this regulation. We found that the rs1805126 minor allele associates with decreased cardiac SCN5A expression and that heart failure subjects homozygous for the minor allele have decreased ejection fraction and increased mortality, but not increased ventricular tachyarrhythmias. In mice, we identified a potential basis for this in discovering that decreased Scn5a expression leads to accumulation of myocardial reactive oxygen species. Together, these data reiterate the importance of considering the mechanistic significance of synonymous SNPs as they relate to miRs and disease, and highlight a surprising link between SCN5A expression and nonarrhythmic death in heart failure.

Authors

Xiaoming Zhang, Jin-Young Yoon, Michael Morley, Jared M. McLendon, Kranti A. Mapuskar, Rebecca Gutmann, Haider Mehdi, Heather L. Bloom, Samuel C. Dudley, Patrick T. Ellinor, Alaa A. Shalaby, Raul Weiss, W.H. Wilson Tang, Christine S. Moravec, Madhurmeet Singh, Anne L. Taylor, Clyde W. Yancy, Arthur M. Feldman, Dennis M. McNamara, Kaikobad Irani, Douglas R. Spitz, Patrick Breheny, Kenneth B. Margulies, Barry London, Ryan L. Boudreau

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Figure 2

Effect of miR-24 overexpression on SCN5A function and expression in neonatal rat cardiomyocytes.

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Effect of miR-24 overexpression on SCN5A function and expression in neon...
NRCMs were cotransfected with synthetic pre-miRs (25 nM) and plasmids coexpressing miRs/siRNAs and EGFP. Na+ current in EGFP-positive cells was measured 2–4 days later using patch clamp. Shown are representative traces of cardiac Na+ currents and pulse protocol (A) and the current-voltage relationship of Na+ current (B) in NRCMs transfected with miR-Neg1 (miR-N, negative control), miR-24, or siSCN5A (positive control). (C) Sodium current density at –10 mV is also plotted. These patch-clamp data support the hypothesis that miR-24 potently suppresses sodium current density in NRCMs, to a similar degree as Scn5a-targeted siRNA. Data are represented as the mean ± SEM. Sample numbers combined from 3 separate experiments are denoted in the figure, and P values were calculated using 1-way ANOVA with Dunnett’s post hoc test (versus miR-Neg1). (D) NRCMs were transfected with 25 nM synthetic pre-miR mimics and endogenous NaV1.5 protein levels were measured by Western blot 48 hours later; a representative Western blot image is shown. (E) Combined Western blot (WB) densitometry analysis from 2 separate experiments (n = 6 total biological replicates per treatment; mean ± SEM) is plotted. Pre–miR-29 and -Neg1 serve as negative controls, and β-actin or GAPDH levels were used for normalization. *P value derived using 1-way ANOVA with Dunnett’s post hoc test (versus miR-Neg1).