MicroRNA-1 accelerates the shortening of atrial effective refractory period by regulating KCNE1 and KCNB2 expression: an atrial tachypacing rabbit model

Abstract

Background: The potential mechanisms of microRNA-1 (miR-1) in the electrical remodeling of atrial fibrillation remain unclear. The purpose of this study was to evaluate the effects of miR-1 on the atrial effective refractory period (AERP) in a right atrial tachypacing model and to elucidate the potential mechanisms.

Methods and results: QRT-PCR and western blot were used to detect the expression of the miR-1, KCNE1, and KCNB2 genes after 1-week of right atrial tachypacing in New Zealand white rabbits. The AERP was measured using a programmable multichannel stimulator, and atrial fibrillation was induced by burst stimulation in vivo. The slowly activating delayed rectifier potassium current (IKs) and AERP in atrial cells were measured by whole cell patch clamp in vitro. Right atrial tachypacing upregulated miR-1 expression and downregulated KCNE1 and KCNB2 in this study, while the AERP was decreased and the atrial IKs increased. The downregulation of KCNE1 and KCNB2 levels was greater when miR-1 was further upregulated through in vivo lentiviral infection. Electrophysiological tests indicated a shorter AERP, a great increase in the IKs and a higher atrial fibrillation inducibility. In addition, similar results were found when the levels of KCNE1 and KCNB2 were downregulated by small interfering RNA while keeping miR-1 level unaltered. Conversely, knockdown of miR-1 by anti-miR-1 inhibitor oligonucleotides alleviated the downregulation of KCNE1 and KCNB2, the shortening of AERP, and the increase in the IKs. KCNE1 and KCNB2 as the target genes for miR-1 were confirmed by luciferase activity assay.

Conclusions: These results indicate that miR-1 accelerates right atrial tachypacing-induced AERP shortening by targeting potassium channel genes, which further suggests that miR-1 plays an important role in the electrical remodeling of atrial fibrillation and exhibits significant clinical relevance as a potential therapeutic target for atrial fibrillation.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. miR-1 expression.

QRT-PCR quantification of miR-1 in each group (A). Immunofluorescence analysis of atrial samples from the rabbit model of right A-TP obtained 1-week after LVs infection with miR-1-LVs (B). * P < 0.05 vs. Ctl, † P < 0.05 vs. Pacing, §P < 0.05 vs. P+miR-1, one-way ANOVA and Tukey’s post-hoc test; n = 6 independent RNA samples for each group.

Figure 2. Evaluation of KCNE1 and KCNB2 expression.

Western blot profiles (A) and qRT-PCR (B) of KCNE1 and KCNB2 proteins levels (33 kDa and 132 kDa, respectively) and mRNAs levels, isolated from rabbits RA. * P < 0.05 vs. Ctl, † P < 0.05 vs Pacing, §P < 0.05 vs. P+miR-1. One-way ANOVA and Tukey’s post-hoc test; n = 6 independent RNA samples for each group.

Figure 3. AERP evaluations.

AERP of RA obtained from animals as described in the Materials and Methods section (A). The AERP values (y axis: ms) were obtained at 3 time points: before pacing (“a”), before infection control/recombinant LVs (“b”), and 1-week after infection (“c”). * P < 0.05 vs. “a”; † P < 0.05 vs. “b”, one-way ANOVA and Tukey’s post-hoc test; n = 6 independent rabbits for each group. AERP of atrial cells were measured by whole cell patch clamp invitro (B).* P < 0.05 vs. Ctl, † P < 0.05 vs. Pacing, §P < 0.05 vs. P+miR-1, one-way ANOVA and Tukey’s post-hoc test; n = 6 independent RNA samples for each group.

Figure 4. AF inducibility evaluation.

The AF inducibility in each group (A). The intracardiac ECGs (B) were obtained from the following sources: (upper ECG) sinus rhythm from the no-pacing rabbits; and (middle ECG) pacing rhythm from the rabbit with pacing; and (lower ECG) AF rhythm from the rabbit with induced AF.

Figure 5. IKs evaluations.

The IKs in atrial cells was measured using whole cell patch clamp invitro. * P < 0.05 Pacing vs. Ctl, † P < 0.05 P+miR-1 vs. Pacing, § P < 0.05 P+siR-KCNE1 vs. Pacing, one-way ANOVA and Tukey’s post-hoc test; n = 6 independent RNA samples for each group.

Figure 6. Sequence alignment between miR-1 and putative binding sites in the 3'UTRs of KCNE1 and KCNB2.

Figure 7. Luciferase assay monitoring of the KCNE1 / KCNB2 - miR-1 interaction.

HEK293 cells were co-transfected with miR-1- or AMO-1-plasmids and KCNE1 or KCNB2- plasmids and the mutation studies by replacing the matching site in miR-1 and the 3’UTRs of KCNE1/KCNB2 were performed as described in the Materials and Methods section. Luciferase activity, measured in the samples indicated on the “X” axis, was normalized to the activity exhibited in the control cells. * P < 0.05 vs. Ctl, † P < 0.05 vs. miR-1, one-way ANOVA and the Tukey’s post-hoc test.