M3 subtype of muscarinic acetylcholine receptor promotes cardioprotection via the suppression of miR-376b-5p

Abstract

The M(3) subtype of muscarinic acetylcholine receptors (M(3)-mAChR) plays a protective role in myocardial ischemia and microRNAs (miRNAs) participate in many cardiac pathophysiological processes, including ischemia-induced cardiac injury. However, the role of miRNAs in M(3)-mAChR mediated cardioprotection remains unexplored. The present study was designed to identify miRNAs that are involved in cardioprotective effects of M(3)-mAChR against myocardial ischemia and elucidate the underlying mechanisms. We established rat model of myocardial ischemia and performed miRNA microarray analysis to identify miRNAs involved in the cardioprotection of M(3)-mAChR. In H9c2 cells, the viability, intracellular free Ca(2+) concentration ([Ca(2+)]i), intracellular reactive oxygen species (ROS), miR-376b-5p expression level, brain derived neurophic factor (BDNF) and nuclear factor kappa-B (NF-κB) levels were measured. Our results demonstrated that M(3)-mAChR protected myocardial ischemia injury. Microarray analysis and qRT-PCR revealed that miR-376b-5p was significantly up-regulated in ischemic heart tissue and the M(3)-mAChRs agonist choline reversed its up-regulation. In vitro, miR-376b-5p promoted H(2)O(2)-induced H9c2 cell injuries measured by cells viability, [Ca(2+)]i and ROS. Western blot and luciferase assay identified BDNF as a direct target of miR-376b-5p. M(3)-mAChR activated NF-κB and thereby inhibited miR-376b-5p expression. Our data show that a novel M(3)-mAChR/NF-κB/miR-376b-5p/BDNF axis plays an important role in modulating cardioprotection. MiR-376b-5p promotes myocardial ischemia injury possibly by inhibiting BDNF expression and M(3)-mAChR provides cardioprotection at least partially mediated by the downregulation of miR-376b-5p through NF-κB. These findings provide new insight into the potential mechanism by which M(3)-mAChR provides cardioprotection against myocardial ischemia injury.

Figure 1. MiRNA is involved in M₃-mAChR mediated cardioprotection against myocardial ischemia in rat.

Choline (10 mg/kg, i.v.) was administrated 10 min before occlusion. 4DAMP (0.12 µg/kg) was administered 5 min before choline. (A) Representative infarct photographs. (B) Infarct size. Values were expressed as mean ± SEM; n = 8; *P<0.05 vs. Control; ^# P<0.05 vs. Choline. (C) Choline reversed ischemia-induced miR-376b-5p up-regulation. (D) Choline reversed ischemia-induced miR-539 up-regulation. Values were expressed as mean ± SEM; n = 3; *P<0.05 vs. Control, ^# P<0.05 vs. Ischemia.

Figure 2. MiR-376b-5p promotes H₂O₂-induced H9c2 cells injury.

H9c2 cells were pre-treated with H₂O₂ (50 µM) for 12 h and then transfected with miR-376b-5p negative control (miR-NC), miR-376b-5p, miR-539, and 2′-O-methyl-modified antisense oligoribonucleotides of miR-376b-5p (AMO-376b-5p, miR-376b-5p inhibitor) for 24 h. (A) MTT assay. MiR-376b-5p, not miR-539, reduced cell viability in H₂O₂-treated H9c2 cells. n = 19 values from 5 independent experiments. (B) Intracellular Ca²⁺ concentration ([Ca²⁺]i) fluorescence intensity. MiR-376b-5p significantly increased [Ca²⁺]i and this effect was reversed by AMO-376b-5p (AMO). n = 29 cells from 5 independent experiments. (C) Reactive oxygen species (ROS) fluorescence intensity. MiR-376b-5p significantly increased relative fluoresence intensity of ROS. n = 12 cells from 3 independent experiments. Values were expressed as mean ± SEM; *P<0.05 vs. Control, ^# P<0.05 vs. H₂O₂+miR-NC, ^@ P<0.05 vs. H₂O₂+miR-376b-5p. FI: fluorescence intensity.

Figure 3. M₃-mAChR inhibits the up-regulation of miR-376b-5p stimulated by H₂O₂ in H9c2 cells.

H9c2 cells were co-incubated with H₂O₂ (50 µM), choline (1 mM) and 4DAMP (5 nM) for 12 h. MiR-376b-5p expression was determined by qRT-PCR. U6 served as an internal control for the normalization. Values were expressed as mean ± SEM; n = 4; **P<0.01 vs. Control, ^## P<0.01 vs. H₂O₂+choline, ^@ P<0.05 vs. H₂O₂+choline+4DAMP.

Figure 4. BDNF is a target gene of miR-376b-5p in cardiac myocytes.

(A) MiR-376b-5p binding site in 3′-untranslated region (3′-UTR) of BDNF gene. (B) Inhibitory effects of miR-376b-5p on BDNF expression at the protein level. BDNF level was determined by Western blot after H9c2 cells were transfected with miR-376b-5p negative control (miR-NC), miR-376b-5p and 2′-O-methyl-modified antisense oligoribonucleotides of miR-376b-5p (AMO-376b-5p, miR-376b-5p inhibitor) for 24 h. GAPDH served as loading control. Top, representative Western blots; bottom, quantitation as mean ± SEM. Note: n = 3; *P<0.05 vs. miR-NC, ^# P<0.05 vs. miR-376b-5p. (C) Luciferase assay for direct inhibitory effects of miR-376b-5p on BDNF expression. The reporter construct was transfected into HEK 293T cells with miR-NC, miR-376b-5p, AMO-376b-5p or AMO-376b-5p negative control (AMO-NC). Luciferase activity was normalized by Firefly luciferase signal in HEK 293T cells. Following transfection for 24 h, luciferase activities were measured on a luminometer. Values were expressed as mean ± SEM; n = 3; *P<0.05 vs. miR-NC, ^# P<0.05 vs. AMO-NC.

Figure 5. NF-κB mediates the inhibitory effect of M₃-mAChR on miR-376b-5p expression.

GAPDH as an internal control normalized NF-κB band for western blot. U6 as an internal control normalized miR-376b-5p for quantitative real-time RT-PCR (qRT-PCR). (A) Chromosomal organization of the miRNA-376b gene locus on rat chromosome 6q32 indicating three NF-κB binding sites in the pre-miR-376b gene promoter. (B) Choline activated NF-κB in H9c2 cells. Top, representative Western blots of total NF-κB; Middle, representative Western blots of nuclear NF-κB; bottom, quantitation of nuclear NF-κB as mean ±SEM Note: n = 4; *P<0.05 versus Control. (C) LPS activated NF-κB in H9c2 cells. Top, representative Western blots of total NF-κB; Middle, representative Western blots of nuclear NF-κB; bottom, quantitation of nuclear NF-κB as mean ±SEM. Note: n = 6; *P<0.05 versus Control. (D) LPS inhibited miR-376b-5p expression in H9c2 cells. MiR-376b-5p expression was determined by qRT-PCR. Values were expressed as mean ± SEM; n = 5; *P<0.05 vs. Control.