miR-208b Reduces the Expression of Kcnj5 in a Cardiomyocyte Cell Line

Abstract

MicroRNAs (miRs) contribute to different aspects of cardiovascular pathology, among them cardiac hypertrophy and atrial fibrillation. Cardiac miR expression was analyzed in a mouse model with structural and electrical remodeling. Next-generation sequencing revealed that miR-208b-3p was ~25-fold upregulated. Therefore, the aim of our study was to evaluate the impact of miR-208b on cardiac protein expression. First, an undirected approach comparing whole RNA sequencing data to miR-walk 2.0 miR-208b 3'-UTR targets revealed 58 potential targets of miR-208b being regulated. We were able to show that miR-208b mimics bind to the 3' untranslated region (UTR) of voltage-gated calcium channel subunit alpha1 C and Kcnj5, two predicted targets of miR-208b. Additionally, we demonstrated that miR-208b mimics reduce GIRK1/4 channel-dependent thallium ion flux in HL-1 cells. In a second undirected approach we performed mass spectrometry to identify the potential targets of miR-208b. We identified 40 potential targets by comparison to miR-walk 2.0 3'-UTR, 5'-UTR and CDS targets. Among those targets, Rock2 and Ran were upregulated in Western blots of HL-1 cells by miR-208b mimics. In summary, miR-208b targets the mRNAs of proteins involved in the generation of cardiac excitation and propagation, as well as of proteins involved in RNA translocation (Ran) and cardiac hypertrophic response (Rock2).

Keywords: Kcnj5; Ran; Rock2; cardiomyocytes; heart; miR-208b.

Figure 1

Expression of miR-208b in whole hearts from mice with severe heart hypertrophy. (a) Real-time qRT-PCR and (b) digital droplet PCR of male and female (N = 19–20 animals/group, * p ≤ 0.05 vs. WT).

Figure 2

Differentially expressed target genes of miR-208b in the hearts of mice with severe heart hypertrophy. (a) Comparison of predicted miR-208b target genes (3′-UTR, miR-walk 2.0) with downregulated genes identified by next-generation whole RNA sequencing (whole-heart WT versus KO, N = 6 animals/group) that revealed 58 common genes. (b) Real-time qRT-PCR for selected genes in additional whole-heart samples of mice with severe heart hypertrophy (N = 15–19 animals/group, * p ≤ 0.05 vs. WT). (c) Real-time qRT-PCR for selected miR-208b target genes in HL-1 cells transfected either with miR-208b mimics or mimic control (N = 3–7 samples/group, * p ≤ 0.05 vs. mimic control).

Figure 3

(a) HEK293 cells were transfected with either an empty vector or a luciferase vector containing a miR-208b-binding site. Co-transfection with mimic control had no significant effect on firefly luciferase activity, while co-transfection with the mir-208b mimic reduced firefly luciferase activity significantly (N = 3–12 experiments/group, n = 12–36 wells/group, * p ≤ 0.05 vs. mimic control). (b) To evaluate if the miRNA binds to the 3′-UTR dual luciferase constructs containing the 3′-UTR from the L-type Ca2+ channel subunits (Cacna1c, Cacnb2) or the GIRK4 potassium channel subunit (Kcnj5), the seed sequence or an empty vector were transfected in HEK293 cells either with or without mimic control or miR-208b mimic. miR-208b mimic reduced the luciferase activity of the Cacna1c-II and the Kcnj5 construct (N = 3–8 experiments/group, n = 9–24 wells/group, * p ≤ 0.05 vs. mimic control). (c) To analyze the effect of miR-208b on the L-type Ca²⁺ channel, we performed patch clamp analysis in HL-1 cells transfected with mimic control or miR-208b mimics. miR-208b had no effect on current density in HL-1 cells (N = 7 experiments, n = 55–60 cells/group). Representative current tracings for control and mimic are given. (d) HL-1 cells were transfected either with mimic control or miR-208b mimics, and GIRK4-dependent ion flux was measured through the fluorescence changes of a thallium-sensitive dye. In HL-1 cells transfected with miR-208b mimics, the area under the curve and thereby, the ion flux over time were significantly reduced compared to control cells. (N = 5 experiments, n = 15 wells, * p ≤ 0.05 vs. mimic control).