Down‐regulation of miR‐200c attenuates AngII‐induced cardiac hypertrophy via targeting the MLCK‐mediated pathway

Abstract Background MicroRNAs (miRNAs) have been shown to commonly contribute to cardiac hypertrophy (CH). The aim of this study was to test the hypothesis that miR‐200c plays an important role in the progression of CH by targeting myosin light chain kinase (MLCK/MYLK). Methods and results Cardiac hypertrophy was induced by aortic banding (AB) in rats. Cellular hypertrophy in neonatal rat cardiomyocytes (NCMs) was induced by AngII treatment. Echocardiography, histology and molecular measurements were used to assess the results of the experiments. The levels of apoptosis and reactive oxygen species (ROS) were also measured. Quantitative real‐time PCR (qRT‐PCR) and Western blotting were used to measure mRNA and protein levels respectively. The present results showed that miR‐200c expression was increased in response to CH both in vivo and in vitro. The down‐regulation of miRNA‐200c by a specific inhibitor markedly ameliorated CH resulting from AngII treatment, and the mRNA levels of atrial natriuretic peptide, brain natriuretic peptide and β‐myosin heavy chain were simultaneously decreased. Notably, minimal apoptosis and ROS accumulation were identified in AngII‐induced hypertrophic cardiomyocytes. Conversely, the up‐regulation of miR‐200c using specific mimics reversed these effects. Mechanistic investigations demonstrated that the MLCK gene is a direct target of miR‐200c; an increase in miR‐200c levels led to a decrease in the expression of MLCK and its downstream effector, p‐MLC2, while miR‐200c inhibition increased the expression of these proteins. Furthermore, inhibiting MLCK impaired the anti‐hypertrophic effects contributions produced by the knockdown of miR‐200c. Conclusion Our studies suggest that miR‐200c may serve as a potential therapeutic target that could delay hypertrophy. We have also uncovered a relationship between miR‐200c and MLCK, identifying MLCK as a direct mediator of miR‐200c.


| INTRODUCTION
Cardiac hypertrophy (CH) can be defined as either pathological or physiological hypertrophy. Physiological CH, which responds to stimuli such as exercise, is considered adaptive and beneficial. Conversely, pathological CH is caused by pathological stimuli such as high blood pressure, neurohumoral overactivation or other myocardial injury and is maladaptive. 1 Pathological CH is usually related to complicated pathological processes including oxidative stress, cell apoptosis, inflammation, metabolic dysfunction, sarcomere disorganization and endoplasmic reticulum stress. 2 Extensive studies have suggested that oxidative stress, inflammation and apoptosis play crucial regulatory roles in CH. [3][4][5] Additionally, the excessive production of reactive oxygen species (ROS) has been found to result in cardiac dysfunction or injury. 6 In addition, the inhibition of inflammation and apoptosis has been shown to improve cardiac function. 7 However, sustained or excessive hypertrophic responses may lead to a transition from compensated hypertrophy to decompensation and, eventually, heart failure (HF), which is associated with sudden death. 8 Myosin light chain kinase (MLCK), also known as the MYLK, is involved in the pathology of several cardiovascular disorders such as HF, 9 myocardial infarction (MI) 10 and CH. 11,12 Cardiac MLCK (cMLCK), encoded by the MYLK3 gene, is a Ca 2+ /calmodulin-activated, serine/threonine-specific protein kinase that phosphorylates cardiac myosin regulatory light chain (cMLC2), which potentiates the rate and the force of contraction in cardiac myocytes. 13,14 Previously, studies have shown that the increased MLC2 phosphorylation by itself does not cause CH and, in actuality, likely inhibits CH by contributing to enhanced contractile performance and efficiency. 15 MicroRNAs (miRNAs, miRs) belong to a class of endogenous small non-coding RNAs (an average size of 22 nucleotides) that negatively regulate the expression of target genes through binding to the 3′ untranslated region within miRNA targets. 16 MicroRNAs are critically involved in heart function and heart dysfunction in a number of physiological and pathophysiological conditions such as MI, cardiac arrhythmia, CH and HF. 17 A recent study reported that MLCK in breast cancer cell lines is regulated by miR-200c, which suppresses epithelial mesenchymal transition during cancer invasion and metastasis. 18 Moreover, miR-200c is abundantly expressed in the heart and, in the diabetic heart, is involved in myocardial injury induced by glucose fluctuations that, result in an increase in the levels of ROS. 19 On the basis of these findings, we suggested a possible regulatory role for miR-200c in MLCK expression and in the underlying mechanisms of CH.   20,21 In brief, all animals were anaesthetized with chloral hydrate (300 mg/kg, ip). Aortic banding was created around the abdominal aorta using a 7-0 silk suture and a 22-gauge needle. The needle was removed, yielding an outer aortic diameter of approximately 0.3 mm. Sham-operated rats underwent the same procedure but without aortic constriction. At 4 weeks after surgery, cardiac function was evaluated by echocardiography, and samples of the heart tissue were obtained.

| Echocardiography
Four weeks after the aortic banding (AB) operation, the rats were anaesthetized with 1.5%-2% isoflurane via inhalation. Transthoracic echocardiography was performed with an echocardiography machine (iE33, Philips) equipped with a 15-MHz transducer in order to evaluate CH in rats. Two-dimensional, guided M-mode tracings were recorded from the parasternal short-axis view at the mid-papillary muscle level. 22 Interventricular septal end-diastolic thickness (IVSd), left ventricular posterior wall end-diastolic thickness (LVPWd) and left ventricular end-diastolic volume (LVEDV) were measured using three parasternal long-axis views. Left ventricular fractional shortening (FS) and ejection fraction (EF) were calculated determined by the system and used as direct indicators of cardiac function. All parameters were collected from at least three heartbeats measurements and averaged.

| Histological analysis
After echocardiography detection, the rats were killed by cervical dislocation according to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health. Then, the rat hearts were perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) for global morphometry. For histological analysis, the heart tissues were fixed in 10% formalin, embedded in paraffin or frozen in liquid nitrogen, sectioned at 5-µm thickness and then stained with haematoxylin and eosin (HE). To evaluate CH, a random collection of 10 cardiomyocytes images which contained at least 20 cells from the cardiomyocyte cross-sectional area (CSA) was calculated using a quantitative digital image analysis system (Image-Pro Plus 6.0).

| Neonatal rat cardiomyocyte culture
Neonatal rat cardiomyocytes (NCMs) were isolated and cultured from the ventricles of 0-to 3-day-old Sprague-Dawley rats as previously described. 22 In detail, all neonatal rats were killed by decapitation, and their hearts were quickly removed, homogenized and placed in a dish with ice-cold PBS. After these procedures, NCMs were digested with 0.08% collagenase type II (Sigma, USA) and 0.125% trypsin (Gibco, USA) at 37°C. Then, the cells were centrifuged, followed by differential preplating to enrich the cardiomyocyte population. Next, the cells were cultured in DMEM/F-12 (Gibco) containing 10% foetal bovine serum (FBS) (Gibco), 0.1 mmol/ L bromodeoxyuridine (Sigma) and 1% penicillin/streptomycin (HyClone, USA) in a humidified incubator at 37°C with 5% CO 2 and 95% air.

| Treatments and transfection
Commercially synthesized miRNA-200c mimics, inhibitor and scrambled control ( Table 1) were purchased from GenePharma (Shanghai, China) and transfected into cardiomyocytes using Lipofectamine TM 2000 (Invitrogen) according to the manufacturer's protocol. 23 Briefly, cells were plated into 6-well plates with serum-free DMEM and starved for 12 hours; then, the miRNA-200c mimics (50 nmol/L) or inhibitor (100 nmol/L) and 5 μL of transfection reagent were separately diluted in 250 μL of OPTI-MEM (Gibco) and incubated for 5 minutes. Next, the Lipofectamine-miRNA was incubated for 20 minutes at room temperature and was added to the serum-free medium.
After 6 hours of transfection, the culture medium was replaced with fresh medium containing 10% FBS. The cells were treated with 1 μmol/L AngII or 10 μmol/L ML-7 (a specific inhibitor of MLCK; Sigma) for 48 hours and then collected for further analysis.

| Immunofluorescent staining for α-actinin and cell surface area assay
The surface area of the cardiomyocytes was determined using immunofluorescent staining for α-actinin. 22 After treatment for 48 hours, the cells were fixed in cold 4% PFA for 20 minutes, permeabilized with 0.5% Triton X-100 in PBS solution for 10 minutes and blocked in 10% goat serum in PBS for 1 hour at room temperature. Then, the cardiomyocytes were incubated with anti-actinin primary antibody (at 1:500; Sigma) at 4°C overnight. The cells were then washed with PBS three times and incubated with Alexa Fluor-555 (Molecular Probes, Eugene, OR) secondary antibody for 1 hour.

| Evaluation of apoptosis
The percentage of cells undergoing early apoptosis and necrosis was measured using a FITC-Annexin V/PI apoptosis kit (Beyotime, Nanjing, China) using flow cytometry according to the manufacturer's instructions. 24 After incubation, the cells were washed twice with cold PBS, harvested and were incubated with 5 µL of FITC-Annexin V and 1 µL of PI working solution (100 µg/mL) for 15 minutes in the dark at room temperature. Cellular fluorescence was measured using a flow cytometer (FACSCalibur, BD Biosciences, CA, USA).

| Detection of intracellular ROS production
Intracellular ROS production was determined using a 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) kit (Beyotime) according to the manufacturer's protocol. 25 The cells were washed twice with PBS, incubated in serum-free medium containing 10 μmol/L DCFH-DA for 30 minutes at 37°C and washed twice with PBS. Then, images of the cells were captured using a fluorescence microscope.

| Luciferase reporter assay
To confirm that MLCK was targeted by miR-200c, the full-length 3′-

| Quantitative real-time PCR
Total RNA was extracted from cardiomyocytes using TRIzol (Invitrogen) as previously described. cDNA was synthesized using the Tran-

| Western blot analysis
Cardiomyocytes were collected and lysed in RIPA lysis buffer (Beyotime

| Statistical analysis
All data are presented as the means ± SD of at least three repeated individual experiments for each group. An unpaired, two-tailed Student's t test or one-way ANOVA, followed by Tukey's post hoc test, was used for the statistical comparisons of two or more than two groups respectively. A P-value <0.05 was considered to denote statistical significance. The data were analysed using SPSS version 22.0 software (SPSS, Chicago, IL).

| miR-200c was up-regulated in the heart tissue of AB rats and in AngII-induced hypertrophic cardiomyocytes
To investigate whether miR-200c is involved in the progression of CH, we established an AB rat model and a model of AngII-induced primary cardiomyocyte hypertrophy. Four weeks after the AB surgery, the AB group exhibited increased heart weight/body weight (HW/BW), lung weight/body weight (LW/BW) and heart weight/tibial length (HW/TL) ratios compared with the sham group ( Figure 1A).
Additionally, transthoracic echocardiography revealed that IVSd, IVSs, LVPWd and LVEDV were markedly increased in the AB group compared with the sham group, and FS% and EF% were significantly decreased in the AB group. Meanwhile, HE staining revealed that the CSA was clearly increased following the AB operation in the AB group compared with the sham group. The gene markers of CH, including ANP, BNP and β-MHC, were measured in the hearts of the AB rats, which confirmed that the AB operation was successful in inducing CH in rats. Moreover, we observed that cMLCK protein expression was decreased in the AB rat heart tissue compared with that in the sham group (

| miR-200c inhibition suppressed CH via the regulation of apoptosis
We examined the apoptotic effects of miR-200c in hypertrophic cardiomyocytes by measuring the percentage of early apoptotic and necrotic cells using a FITC-Annexin V/PI apoptosis assay. As shown in Figure 3A and B, the apoptotic percentage was increased in

| miR-200c directly targeted MLCK
To reveal potential miRNAs with the potential to regulate the expression of MLCK, Targetscan was used to predict possible miR-200c targets based on the reverse complementarity of the rno-miR-

| DISCUSSION
In this study, we predicted miR-200c to be a candidate miRNA to regulate cMLCK during CH. The following observations were made: Apoptosis is a common phenomenon observed in cases of CH.
Pathological myocardial hypertrophy is mainly caused by the activation of AngII and other hypertrophic factors, which, stimulate apoptotic genes to promote cardiomyocyte apoptosis and decrease the myocardial contraction force and, lead to an imbalance in the regulation of negative feedback that results from the proliferation and apoptosis of cardiomyocytes. 36   pathway. Previous studies have revealed that cMLCK is involved in the regulation of ventricular myosin light chain 2 (MLC-2v) phosphorylation, sarcomere organization and cardiomyocyte contraction. 39 The phosphorylation of MLC-2v has been shown to play an important regulatory role in maintaining normal cardiac function, with increasing in MLC-2v phosphorylation likely inhibiting CH and HF by contributing to enhanced contractile performance and efficiency. 15,39 In an earlier study, we found that MLCK and p-MLC2 were involved in CH due to their degradation by AngII. 40 The overexpression of cMLCK in cardiac myocytes promotes sarcomere organization, which is an adaptive response to hypertrophic stimuli during early CH, while the knockdown of cMLCK resulted in sarcomeric disorganization. 41 Moreover, the expression level of cMLCK is reduced in animal models of MI. 10 After 1 week, a reduction in the levels of cMLCK and phosphorylated MLC2v were also demonstrated in the hearts subject to pressure overload induced by thoracic aortic constriction (TAC) surgery. 11 In addition, the knockdown of cMLCK in Mylk3-KO mice was associated with HF, which suggested that cMLCK plays a pivotal role in the transition from compensated to decompensated hypertrophy via sarcomeric disorganization. 42 The present findings suggest a protective role for cMLCK against cardiac stress. Consistent with the above study, we found that the expres-

CONF LICT OF I NTEREST
All authors declare no conflicts of interest.