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. 2014 Dec;7(6):1214-22.
doi: 10.1161/CIRCEP.114.001973. Epub 2014 Nov 11.

Loss of microRNA-106b-25 cluster promotes atrial fibrillation by enhancing ryanodine receptor type-2 expression and calcium release

David Y Chiang  1 , Natee Kongchan  1 , David L Beavers  1 , Katherina M Alsina  1 , Niels Voigt  1 , Joel R Neilson  1 , Heinz Jakob  1 , James F Martin  1 , Dobromir Dobrev  1 , Xander H T Wehrens  1 , Na Li  2
Affiliations

Loss of microRNA-106b-25 cluster promotes atrial fibrillation by enhancing ryanodine receptor type-2 expression and calcium release

David Y Chiang et al. Circ Arrhythm Electrophysiol. 2014 Dec.

Abstract

Background: Enhanced sarcoplasmic reticulum Ca(2+)-leak via ryanodine receptor type-2 (RyR2) contributes to the pathogenesis of atrial fibrillation (AF). Recent studies have shown that the level of RyR2 protein is elevated in atria of patients with paroxysmal AF, suggesting that microRNA-mediated post-transcriptional regulation of RyR2 might be an underlying mechanism. Bioinformatic analysis suggests that miR-106b and miR-93, members of the miR-106b-25 cluster, could bind to RyR2-3'-untranslated region and suppress its translation. Thus, we tested the hypothesis that loss of the miR-106b-25 cluster promotes AF via enhanced RyR2-mediated sarcoplasmic reticulum Ca(2+)-leak.

Methods and results: Quantitative real-time polymerase chain reaction showed that the levels of mature miR-106b, miR-93, and miR-25 were lower in atria of patients with paroxysmal AF when compared with patients in sinus rhythm. In vitro assay showed that miR-93 reduced RyR2-3'-untranslated region luciferase activity. Total RyR2 protein in atrial tissue of miR-106b-25(-/-) mice was increased by 42% when compared with wild-type littermates but still maintained a normal subcellular distribution. Ca(2+)-spark frequency and total sarcoplasmic reticulum Ca(2+)-leak were increased in atrial myocytes of miR-106b-25(-/-) mice. Telemetry ECG recordings revealed that miR-106b-25(-/-) mice exhibited more frequent atrial ectopy and were also more susceptible to pacing-induced AF than wild-type littermates. Increased sarcoplasmic reticulum Ca(2+)-release and AF susceptibility in miR-106b-25(-/-) mice were abolished by the RyR2 blocker K201.

Conclusions: These results suggest that miR-106b-25 cluster-mediated post-transcriptional regulation of RyR2 is a potential molecular mechanism involved in paroxysmal AF pathogenesis. As such, the miR-106b-25 cluster could be a novel gene-therapy target in AF associated with enhanced RyR2 expression.

Keywords: atrial fibrillation; microRNA; ryanodine receptor calcium release channel.

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Conflict of interest statement

Conflict of Interest Disclosures: X.H.T.W. is a founding partner of Elex Biotech, a company that develops new drugs targeting intracellular calcium leak.

Figures

Figure 1
Figure 1
Upregulation of RyR2 and downregulation of the miR-106b-25 cluster in pAF patients. A) WB analysis revealed increased RyR2 protein levels in sarcoplasmic reticulum preparation of atrial biopsies from patients with pAF versus control patients in SNR. B) qRT-PCR showing unchanged levels of RYR2 mRNA in atrial samples of pAF patients. C) qRT-PCR showing downregulation of the miR-106b-25 cluster and miR-106a in pAF patients. Numbers in bars indicate number of patients. *P<0.05, **P<0.01 vs. SNR.
Figure 2
Figure 2
miR-93 target RYR2-3′UTR. A) Sequences of mature miR-106b, miR-93, miR-25, and miR-106a. B) Predicted interaction between human (h) Ryr2-3′UTR and miR-93 (i) and mutation of the binding site (ii). C) Predicted interaction between mouse (m) Ryr2-3′UTR and miR-93 (i) and mutation of the binding site (ii). D) Dual luciferase assay of 3′UTR constructs in the presence of scramble, miR-106b, miR-93, miR-25, and miR-106a mimics. n=4 or 8 per group. **P<0.01.
Figure 3
Figure 3
Increased RyR2 protein levels in miR-106b-25−/− atria. A) qRT-PCR showing unchanged levels of Ryr2 mRNA in miR-106b-25−/− atria. B) Representative western blots and dot plot showing upregulated RyR2 protein levels in miR-106b-25−/− atrial lysates. *P<0.05. C) Representative western blots and bar graph showing unchanged expression of other major Ca2+-handling proteins and phosphorylation of RyR2 at serine (S)2814 in miR-106b-25−/− atria. N=3–4 animals per group.
Figure 4
Figure 4
Enhanced RyR2 Ca2+-release activity in miR-106b-25−/− atrial myocytes. A) Representative line-scan images of Ca2+-spark recordings in isolated atrial myocytes. B) Bar graph showing increased CaSF normalized to SR load in miR-106b-25−/− atrial myocytes, normalized by K201. C) Bar graph showing unchanged Ca2+-spark amplitude comparing miR-106b-25−/− and WT atrial myocytes. D) Bar graph showing an increase in the FWHM of Ca2+-sparks in miR-106b-25−/− atrial myocytes, normalized by RyR2 blocker K201. Numbers inside bars indicate number of cells studied from 3 mice per group. *P<0.05, **P<0.01, ***P<0.001.
Figure 5
Figure 5
Enhanced SR Ca2+-leak and arrhythmogenic Ca2+-release in miR-106b-25−/− atrial myocytes. A) Representative SR Ca2+-leak measurements in atrial myocytes. NT=normal Tyrode solution; TTC=tetracaine. B) Bar graph showing increased relative levels of SR Ca2+-leak in miR-106b-25−/− atrial myocytes. Representative tracing (C) and summary bar graph (D) showing increased spontaneous Ca2+-wave frequency in high Ca2+-buffer with isoproterenol in atrial myocytes from miR-106b-25−/− versus WT littermates (n=3–4 mice per group). Red arrow marks a spontaneous Ca2+-wave. Numbers inside bars indicate number of cells studied. *P<0.05, **P<0.01.
Figure 6
Figure 6
miR-106b-25 deficiency leads to increased atrial ectopy and atrial weight. A) Representative ECG telemetry recordings and B) dot plot showing premature atrial contractions (PACs) per hour in miR-106b-25−/− mice. C) Representative whole mount photographs and atrial weight normalized to tibial length (AW/TL; D) showing enlarged atria in miR-106b-25−/− mice. **P<0.01 vs WT.
Figure 7
Figure 7
miR-106b-25 deficiency leads to enhanced AF susceptibility. Representative recordings of surface ECG and intracardiac electrograms in WT (A), miR-106b-25−/− (B), and miR-106b-25−/− mice treated with RyR2 blocker K201 (C). D) Bar graph summarizing the incidence of pacing-induced AF showing an increase in miR-106b-25−/− mice, suppressed by K201. Numbers inside bars indicate numbers of mice studied. *P<0.05.
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