M3 Subtype of Muscarinic Acetylcholine Receptor Promotes Cardioprotection via the Suppression of miR-376b-5p

The M3 subtype of muscarinic acetylcholine receptors (M3-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 M3-mAChR mediated cardioprotection remains unexplored. The present study was designed to identify miRNAs that are involved in cardioprotective effects of M3-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 M3-mAChR. In H9c2 cells, the viability, intracellular free Ca2+ concentration ([Ca2+]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 M3-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 M3-mAChRs agonist choline reversed its up-regulation. In vitro, miR-376b-5p promoted H2O2-induced H9c2 cell injuries measured by cells viability, [Ca2+]i and ROS. Western blot and luciferase assay identified BDNF as a direct target of miR-376b-5p. M3-mAChR activated NF-κB and thereby inhibited miR-376b-5p expression. Our data show that a novel M3-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 M3-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 M3-mAChR provides cardioprotection against myocardial ischemia injury.

MicroRNAs (miRNAs) emerge as important regulator of gene expression and have been implicated in numerous cardiovascular pathological processes, including cardiac fibrosis, cardiac hypertrophy, heart failure and cardiac arrhythmia [11][12][13][14]. Specially, several miRNAs including miRNA-320 [15], miRNA-21 [16] and miRNA-494 [17] are involved in myocardial ischemia. However, the potential role of miRNAs in cardioprotection of M 3 -mAChR remains to be elucidated. Therefore, the present study aimed to explore the contribution of miRNAs to cardioprotective effects mediated by M 3 -mAChR against myocardial ischemia. Our results revealed for the first time that miR-376b-5p increased myocardial ischemia injury and was involved in M 3 -mAChR's cardioprotection.

Ethics Statement
All experimental protocols were pre-approved by the Experimental Animal Ethic Committee of Harbin Medical University, China (Animal Experimental Ethical Inspection Protocol No. 2009104). Use of animals was confirmed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Establishment of rat myocardial ischemia model
Male Wistar rats (weight 280-300 g) were randomly divided into four groups: control, ischemia, choline (agonist of M 3 -mAChR), and choline+4DAMP (antagonist of M 3 -mAChR) groups. All treatments were administered via the lingual vein with doses of choline and 4DAMP as described previously [7]. Choline (10 mg/kg, i.v.) was administered 10 min before the occlusion. For choline+4DAMP group, 4DAMP (0.12 mg/kg) was administered 5 min before choline. Rat model of myocardial ischemia was established as previously described [8]. Briefly, the rats were anesthetized intraperitoneal injection with sodium pentobarbital (40 mg/kg) and the respiration of rats was controlled by the volume-controlled rodent ventilator. The ventilator setting and the level of anesthesia were adjusted to maintain the animal in an anaesthetize status without spontaneous breathing efforts. A left thoracotomy was performed at the 3rd-4th rib and a segment of saline-soaked 5-0 sutures was looped around the left anterior descending (LAD) coronary artery, near its origin from the left coronary artery. Successful occlusion was observed by electrocardiographic ST-segment elevation. The chest was closed before the rats were weaned from the ventilator and extubated. Rats were put into cage to breed with normal water and feeds after waking up. Mortality rate of the rats were calculated and compared after 24 h myocardial infarction. After 24 h ischemia, rats were reanesthetized and the hearts were removed for the measurement of myocardial infarct size and microRNA microarray analysis. Control group without drugs was handled in the same manner except that the coronary artery was not ligated.

Measurement of myocardial infarct size
Ventricular tissues were dissected from the animals after 24 h ischemia and kept overnight at 24u. Frozen ventricles were sliced into 2 mm thick sections, and then incubated in 1% triphenyltetrazolium chloride (TTC) at 37uC in 0.2 M Tris buffer (pH 7.4) for 30 min. The non-ischemia area was stained as red and infarct area grey white. Size of the infarcted area was estimated by weight as a percentage of the left ventricle [7].

MicroRNA microarray analysis
After 24 h ischemia, surrounding tissues of infarcted area from ventricular were departed for microRNA microarray analysis by using hybridization to mParafloHmicrofluidics microarrays (LC Sciences) in the version of the Sanger miRBase database Release 10.1 (Release 12, http://microrna.sanger.ac.uk/). Each detection probe consisted of a chemically modified nucleotide-coding segment complementary to the target miRNA or other target and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. Each miRNA probe was represented 5 times on a single microarray, and the control probes were spiked into the RNA samples before the labeling. The detection probes were prepared by in situ synthesis using PGR (photogenerated) chemistry to allow highly sensitive and specific detection of miRNAs. The mParafloH technology enabled on-chip synthesis, ensuring high probe quality and tight process control.

Cell culture
H9c2 myoblast cell line (the rat embryonic ventricular myocardial cell line) were cultured and maintained as monolayer in high glucose DMEM, supplemented with 10% heat inactivated FBS, 100 U/ml penicillin and 100 mg/ml streptomycin, at 37uC in a humidified incubator with 5% CO 2 . H9c2 cells were plated at a density of 5000 cells/cm 2 and allowed to proliferate in growth medium. Medium was changed every 3 days.

MTT assay
H9c2 cells were seeded into 96-well plates at the density of 2610 4 / well. After 24 h, MiR-539, miR-NC, miR-376b-5p and AMO-376b-5p were transfected and incubated for 24 h, followed by treatment with H 2 O 2 (50 mM) for 12 h. Then 10 ml MTT reagents (0.5 mg/ml) was added and incubated for 4 h. After dissolving the formazine granulars with 150 ml dimethyl sulphoxide (DMSO), the absorbance at 570 nm was measured by using a microplate reader. The cell viability = (A control group 2A experimental group )/A control group 6100%.

Measurement of intracellular free Ca 2+
H9c2 cells were seeded into 6-well plates. The cells were treated with H 2 O 2 (50 mM) for 12 h before transfection with miRNAs. The [Ca 2+ ]i levels were measured 24 h after transfection. Pluronic F-127 was used as a dispersing agent to facilitate the loading of cells with a final concentration of 0.012% Fluo-3/AM in the loading medium. Changes in the fluorescence intensity of Fluo-3/ AM-loaded cells were detected by laser scanning confocal microscopy (FV-300; Olympus, Tokyo, Japan) at 488 nm excitation and 530 nm emission wavelengths. The acquisition rate was one frame at 10 s intervals and [Ca 2+ ] i was monitored for 300 s.

Measurement of intracellular ROS
Intracellular reactive oxygen species (ROS) were stained using DCFH-DA. H9c2 cells were seeded into 6-well plates. The cells were treated with H 2 O 2 (50 mM) for 12 h before transfection with miRNAs. The intracellular ROS levels were measured 24 h after transfection. The cells were washed with serum-free DMEM and incubated with 10 mmol/L DCFH-DA in the loading medium in 5% CO 2 /95% air at 37uC for 20 min. After DCFH-DA was removed, the cells were washed twice and the DCF fluorescence intensity was observed and photographed by laser scanning confocal microscopy (FV-300; Olympus, Tokyo, Japan) at 488 nm excitation and 525 nm emission wavelengths. The acquisition rate was one frame at 10 s intervals and ROS was monitored for 300 s.

Western blot analysis
Whole cell extract and nuclear extract were extracted from H9c2 cells. The protein content was determined with Bio-Rad Protein Assay Kit using bovine serum albumin as the standard. Protein sample (50 mg) was fractionated by 7.5-10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked in 5% milk PBS-Tween20 (PBST) for 3 h and incubated overnight at 4uC with the following primary antibodies: BDNF, NF-kB or GAPDH as internal controls, followed by incubation with Alexa FluorH 800 goat anti-mouse IgG or antirabbit IgG (1:10000) for 1 h. The images were captured on the Odyssey Infrared Imaging System (LI-COR Bioscience, Lincoln, NE, USA) and band intensity was quantified using Odyssey v1.2 software.

Statistical analysis
Average data were presented as mean 6 standard error and were analyzed by analysis of variance (ANOVA) followed by Bonferroni's post-hoc test. P,0.05 was considered statistically significant.

MicroRNAs are involved in M 3 -mAChR mediated cardioprotection against myocardial ischemia in rat
We established rat model of myocardial ischemia and examined mortality rate and myocardial infarct size. Choline significantly reduced the infarct size, which was reversed by 4DAMP ( Figure 1A and B). As expected, choline group showed an obvious decrease in mortality rate (8.33%, n = 12, 1 died) compared to control group subjected to the same procedure (28.6%, n = 14, 4 died), and 4DAMP increased mortality rate (14.3%, n = 14, 2 died) compared to choline group. Taken together, these results confirmed that M 3 -mAChR provides cardioprotection against myocardial ischemia.
To determine whether miRNAs are involved in M 3 -mAChR mediated cardioprotection, we performed miRNA microarray analysis and identified 12 differentially upregulated miRNAs upon myocardial ischemia, among which 10 miRNAs were differentially downregulated by M 3 -mAChR agonist choline (Table 1). While miR-199a, miR-376b-5p, miR-539 and miR-106 were all significantly upregulated (more than 3 times compared to the control), only miR-376b-5p and miR-539 were downregulated to the level similar to the control group after treatment with choline.
To confirm the expression changes of miR-376b-5p and miR-539 in myocardial ischemia, next we performed qRT-PCR analysis and found that miR-376b-5p and miR-539 were significantly upregulated upon myocardial ischemia and downregulated by choline (P,0.05, Figure 1C and D). These data demonstrate that miR-376b-5p and miR-539 are significantly upregulated in ischemic heart of the rats and M 3 -mAChR agonist choline could reverse their upregulation. Therefore, miR-376b-5p and miR-539 appear to be involved in M 3 -mAChR mediated cardioprotection against myocardial ischemia.

MiR-376b-5p promotes H 2 O 2 induced H9c2 cells injury
To determine the involvement of miR-376b-5p and miR-539 in myocardial ischemia, we used H9c2 cells as an in vitro model. MTT assay showed that miR-376b-5p significantly enhanced H 2 O 2 -induced inhibition of H9c2 cells viability (P,0.05), which was reversed by AMO-376b-5p. However, miR-539 had no significant effect on H 2 O 2 -induced inhibition of H9c2 cells viability (Figure 2A). These results suggest that miR-539 is unlikely involved in myocardial ischemia and we focused on miR-376b-5p in the following experiments.

M 3 -mAChR inhibits H 2 O 2 induced upregulation of miR-376b-5p in H9c2 cells
To examine further miR-376b-5p expression in ischemic myocardial cells and the effects of M 3 -mAChR on miR-376b-5p expression in vitro, H9c2 cells were co-incubated with H 2 O 2 (50 mM), choline (1 mM) or 4DAMP (5 nM) for 12 h. The results showed that H 2 O 2 significantly upregulated the miR-376b-5p expression (P,0.01) and this effect was antagonized by M 3 -mAChR agonist choline ( Figure 3). Furthermore, M 3 -mAChR antagonist 4DAMP reversed the effect of choline on miR-376b-5p. These results are consistent with the results of gene array analysis verified by qRT-PCR ( Figure 1C) and suggest that M 3 -mAChR inhibits the up-regulation of miR-376b-5p stimulated by H 2 O 2 in H9c2 cells.

BDNF is a target gene of miR-376b-5p in cardiac myocytes
To further explore the role of miR-376b-5p in myocardial ischemia, we predicted the targets of miR-376b-5p by targetScan Release 5.1 online (www.targetscan.org) and focused on BDNF ( Figure 4A). Next we determined the expression of BDNF at the protein level in H9c2 cells and found that miR-376b-5p significantly reduced the expression of BDNF (P,0.05), and this reduction was reversed by AMO-376b-5p ( Figure 4B). These results suggest that BDNF is a target gene of miR-376b-5p.
To confirm that miR-376b-5p could directly bind to BDNF gene and inhibit its expression, a reporter construct with the insertion of a fragment of the 39-UTR of BDNF mRNA containing the predicted miR-376b-5p binding site was transfected into HEK 293T cells. Luciferase assay showed that miR-376b-5p significantly reduced the luciferase activity (P,0.05) while AMO-376b-5p significantly increased the luciferase activity (P,0.05) ( Figure 4C). Taken together, these data prove that miR-376b-5p directly inhibits BDNF expression.
To verify that M 3 -mAChR could activate NF-kB, we determined nuclear NF-kB protein level in H9c2 cells. Western blot analysis showed that nuclear NF-kB level was significantly increased (P,0.05, Figure 5B) after treatment with choline with no significant variance of total NF-kB level, suggesting that M 3 -mAChR could activate NF-kB.

Discussion
In the present study, for the first time, we found that miR-376b-5p inhibited the expression of BDNF and miR-376b-5p promoted H 2 O 2 -induced H9c2 cells injury, indicating that miR-376b-5p promoted myocardial ischemia injury possibly by inhibiting the expression of BDNF. Moreover, we found that M 3 -mAChR inhibited miR-376b-5p expression by activating NF-kB. These findings provide new insight into the potential mechanism of cardioprotection of M 3 -mAChR against myocardial ischemia.
First, we observed that M 3 -mAChR decreased infarct size and mortality rate in rat models, consistent with previous studies showing that M 3 -mAChR has the protective effect against myocardial ischemia [6][7][8][9][10]. Next, to explore the underlying mechanisms responsible for our observation, we focused on miRNAs, given their emerging role in the pathogenesis of the cardiovascular system including myocardial ischemia [11][12][13][14][15][16][17]. Based on microarray analysis and further validation by qRT-PCR, we postulated that the upregulation of miR-376b-5p and miR-539 is involved in myocardial ischemia and M 3 -mAChR may protect against myocardial ischemia via the downregulation of miR-376b-5p and miR-539 expression.
H9c2 cells were treated with H 2 O 2 for 12 h to simulate myocardial ischemia injury, which was employed as in vitro model  to test our hypothesis. In our experiment, the miRNA transfection efficiency was measured by real-time PCR. MiR-376b-5p level was upregulated more than 3 times after transfection ( Figure S1), demonstrating the efficient transfection. Notably, miR-376b-5p but not miR-539 was implicated in reduced H9c2 cells viability induced by H 2 O 2 . Therefore, miR-376b-5p was chosen for further characterization.