Atorvastatin protects cardiomyocytes against OGD/R‑induced apoptosis by inhibiting miR‑199a‑5p.

The present study aimed to evaluate the protective effects of atorvastatin against myocardial ischemia/reperfusion (I/R) injury in cardiomyocytes and its underlying mechanisms. The direct cytotoxic effects of oxygen‑glucose deprivation/reperfusion (OGD/R) on cardiomyocytes with and without atorvastatin pretreatment were evaluated. The effects of atorvastatin on the expression of glycogen synthase kinase‑3β (GSK‑3β) and microRNA (miR)‑199a‑5p were determined using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analyses. In addition, the expression levels of GSK‑3β in cells with miR‑199a‑5p upregulation and downregulation were detected using RT‑qPCR, western blot and immunohistochemical analyses. Pretreatment with atorvastatin significantly improved the recovery of cell viability from OGD/R (P<0.05). In addition, atorvastatin pretreatment significantly increased the expression of GSK‑3β at the mRNA and protein levels, and the expression of miR‑199a‑5p at the mRNA level (all P<0.05). The upregulation and downregulation of miR‑199a‑5p respectively decreased and increased the expression of GSK‑3β at the mRNA and protein levels. These results suggested that atorvastatin provided cardioprotective effects against I/R injury via increasing the expression of GSK‑3β through the inhibition of miR‑199a‑5p.

Introduction

Percutaneous coronary intervention is a critical component of the intervention for acute myocardial infarction in patients (1). However, myocardial tissue suffers from myocardial ischemia/reperfusion (I/R) injury during revascularization, which results in poor clinical outcomes (2). The apoptosis of cardiomyocytes is known to be an important mechanism of I/R injury (3–5). Accumulating evidence has shown that the apoptosis of cardiomyocytes is suppressed by glycogen synthase kinase-3β (GSK-3β) (6–8). Therefore, manipulation of GSK-3β may be a promising strategy for myocardial protection in I/R injury. The GSK-3 family of serine/threonine kinases is important in various pathological processes of the heart, including pressure overload and ischemic injury. It is comprised of two isoforms (α and β), which are encoded by distinct genes and are ubiquitously expressed. Several studies have shown that GSK-3β, but not GSK-3α is cardioprotective (9,10).

MicroRNAs regulate gene expression at the posttranscriptional level by degrading or inhibiting the translation of target messenger RNA (mRNA) (11,12). Various studies have shown that the increased expression of microRNA (miR)-199a-5p is responsible for pathophysiological alterations contributing to and promoting the development of heart diseases, including atrial fibrillation and heart failure (13,14). Therefore, therapeutic interventions targeting miR-199a-5p may be promising strategies for the treatment of I/R injury. Statins have been found to prevent heart failure and acute coronary syndrome, and reduce cardiovascular risk (15–17). Several studies have demonstrated the cardioprotective effects of statins against myocardial I/R injury in animal experiments and clinical studies (18,19). However, the major molecular mechanisms underlying the cardioprotective effect of statins remain to be fully elucidated. The present study hypothesized that cardioprotection by statins may be associated with inhibition of apoptosis in myocardial I/R injury through the miR-199a-5p/GSK-3β pathway.

The present study examined the effects of atorvastatin on H9c2 cells and neonatal rat cardiac ventricular cardiomyocytes by detecting changes in the mRNA and protein expression levels of GSK-3β. In addition, the effect of atorvastatin on the expression levels of miR-199a-5p and GSK-3β were examined. The mechanism underlying the effects of atorvastatin on myocardial I/R injury was further analyzed, to provide a theoretical basis and novel intervention targets for the clinical prevention and treatment of myocardial I/R injury.

Materials and methods

Drugs and reagents

Atorvastatin and lithium chloride (LiCl) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Antibodies targeting GSK-3β, Caspase-9 and B-cell lymphoma 2 (Bcl-2) were provided by BD Biosciences (Franklin Lakes, NJ, USA). Antibody targeting Cytochrome c (Cyt c) was from Abcam (Cambridge, MA, USA). Antibodies targeting α-actin, β-actin and GAPDH were provided by Sigma-Aldrich; Merck KGaA. LY294002 was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). The syn-rno-miR-199a-5p miScript miR mimic, anti-rno-miR-199a-5p miScript miR inhibitor, AllStars negative control small interfering (si)RNA, AllStars Mm/Rn cell death control siRNA, and HiPerFect transfection reagent were purchased from Qiagen, Inc. (Valencia, CA, USA) 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was purchased from Promega Corporation (Madison, WI, USA).

Cell culture and treatment

A total of forty neonatal rat cardiac ventricular cardiomyocytes were prepared from 1–3-day-old neonatal SD rats weighing 8–12 g (20 male, 20 female), bred at 25°C and a humidity of 60% on a 12/12-h dark/light cycle with free access to food and water, and cultured as previously described (20,21). The study was approved by the ethics committee of Wujin People's Hospital (Changzhou, China). The cells were cultured in a humidified incubator (37°C with 5% CO2). Brdu (0.1 mM) was added to the Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich; Merck KGaA), containing 2 mM L-glutamine, 100,000 IU per L penicillin, 100 mg L21 streptomycin, 5% fetal bovine serum and 200 nM tetradecanoyl phorbol acetate (Sigma-Aldrich; Merck KGaA) for the first 2 days to inhibit fibroblast growth. The neonatal rat myocytes were cultured to 70% confluence and then serum starved in basal medium (DMEM) for 24 h prior to drug treatment. The H9c2 rat myocardial cells (cat. no. CRL 1446; American Type Culture Collection) were cultured in DMEM supplemented with 10% FBS (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), penicillin G (100 U·ml−1), streptomycin (100 µg·ml−1) and glutamine (2 mM). The neonatal rat cardiac ventricular cardiomyocytes and H9c2 cells were treated with oxygen-glucose deprivation/reperfusion (OGD/R) with or without the pretreatment, as described previously with minor modification (22). The pretreatment comprised incubation with 1 µM atorvastatin for 3 h or 10 µM GSK-3β inhibitor (LiCl) for 1 h at a density of 1×109 cells/ml. The cells were then rinsed twice, incubated at a density of 2×105 cells/well in a 6-well plate containing glucose-free DMEM and subsequently placed in an anaerobic chamber containing a mixture of 95% N2 and 5% CO2 at 37°C for 6 h. Following OGD, glucose was added to normal levels (final concentration, 4.5 mg·ml−1), and cells were incubated under normal growth conditions (95% air and 5% CO2) for an additional 18 h as OGD/R, unless otherwise specified.

Identification of neonatal rat cardiac ventricular cardiomyocytes

Following culture for different time periods, the samples were rinsed with PBS, fixed with 4% paraformaldehyde for 20 min, and then permeabilized with glycine/triton solution (PBS/0.1% Triton/10 mM glycine). The samples were incubated with mouse monoclonal anti-α-actin (1:300 dilution; cat. no. A7811; Sigma-Aldrich; Merck KGaA) at 4°C overnight. Following three PBS rinses, the samples were incubated with anti-mouse IgG-FITC (1:300 dilution; cat. no. F2012; Sigma-Aldrich; Merck KGaA) for 60 min at 37°C. The nuclei were counterstained with 100 nM DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) for 30 min, followed by rinses with PBS. Fluorescent images were captured with a spectral confocal microscope imaging system (Leica TCS SP2; Leica Microsystems, Inc., Buffalo Grove, IL, USA).

Cell viability assays

Cell viability was assessed using CellTiter vt96 Aqueous One Cell Proliferation assays (Promega Corporation). The cells were seeded at 5×103 per well in 96-well plates overnight. Following treatment, the cells were incubated with 20 µl/100 µl MTS for 1 h at 37°C. Cell viability was determined by measuring the absorbance at 490 nm using a microplate reader (M200PRO; Tecan Austria GmbH, Grödig, Austria).

Measurement of lactate dehydrogenase (LDH) activity

Supernatant was obtained from the culture medium following centrifugation for 1 min (15,550 × g) at 37°C. The activity of LDH in the supernatant was detected using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing China).

Analysis of the mRNA expression of GSK-3β

Total RNA was extracted with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The cDNA was synthesized from 0.5 µg of total RNA using a reverse transcription system (Promega Corporation) according to the manufacturer's protocol. Subsequently, the cDNA was subjected to PCR using GoTaq qPCR master mix (Promega Corporation). Reactions were performed on a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Included in the 25 µl reaction system was 300 nmol/l primers. Thermal cycling conditions consisted of 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. The primers used for RT-qPCR were as follows: GSK-3β, forward 5′-TCCATTCCTTTGGGATCTGCC-3′ and reverse 5′-ATCAGCTCTGGTGCCCTGTAGTAC-3′; β-actin, forward 5′-CAGGGTGTGATGGTGGG-3′ and reverse 5′-GGAAGAGGATGCGGCAG-3′. The fold change in expression of each gene was calculated using the 2−ΔΔCq method (23).

Analysis of the expression of miR-199a-5p

Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RT-qPCR was performed using the All-in-One miRNA qRT-PCR detection kit (GeneCopoeia, Inc., Rockville, MD, USA). The reactions were performed on a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers used for RT-qPCR were as follows: the specific primer for rno-miR-199a-5p was 5′-CCCAGTGTTCAGACTACCTGTTC-3′ and 5′-GACTATCATATGCTTACCGT-3′ for U6 (GeneCopoeia, Inc.). The thermal cycling conditions were the same as described above. The fold change in the expression of each gene was calculated using the 2−ΔΔCq method.

Transfection of H9c2

The H9c2 cells at a density of 2×105 cells/well were seeded in 6-well plates for 12 h. Prior to transfection, the medium was replaced with DMEM without serum and antibiotics. The AllStars Mm/Rn Cell Death Control siRNA (Qiagen, Inc.) at 10, 20 and 50 nM were added and incubated for 24, 48, and 72 h to determine the optimal conditions. Transfection efficiency was observed and evaluated under a light microscope (Olympus Corporation, Tokyo, Japan). Transfection with 20 nM was selected due to the highest efficiency, which was >70%. HiPerFect transfection reagent (Qiagen, Inc.) was used as a transfection reagent. Syn-rno-miR-199a-5p miScript miR mimic and anti-rno-miR-199a-5p miScript miR inhibitor were used (20 nM) to respectively overexpress and inhibit the expression of miR-199a-5p. AllStars negative control siRNA was used as a negative control.

Western blot analysis

Total cell lysates were obtained by incubation of cells in radioimmunoprecipitation assay lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA and 0.1% SDS) supplemented with 50 µg/ml aprotinin and leupeptin. Protein concentration was determined using the Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein extracts (30 µg) were separated by 10% SDS-PAGE, and then transferred onto PVDF membranes. The membrane was blocked with 5% milk at 37°C for 1 h. The membranes were blotted with specific antibodies against GSK-3β (1:2,000), Caspase-9 (1:2,000), B-cell lymphoma 2 (BCL-2; 1:2,000), cytochrome c (1:5,000), GAPDH (1:1,000) and β-actin (1:1,000) at 4°C overnight, and then incubated with the horseradish peroxidase-conjugated secondary antibody (1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-2020) at 37°C for 1 h. The membranes were then processed for chemiluminescent detection according to the manufacturer's protocol (Santa Cruz Biotechnology, Inc.). All bands were evaluated using densitometry with Quantity One V4.6.2 software (Bio-Rad Laboratories, Inc.). Bands of interest were normalized against GAPDH and β-actin. Data are presented as relative density ratios.

Immunofluorescence assay

The cells were fixed with 4% paraformaldehyde for 20 min, and then permeated with glycine/triton solution (PBS/0.1% Triton/10 mM glycine). The samples were incubated with mouse monoclonal anti-GSK-3β (1:100 dilution; BD Biosciences) at 4°C overnight. Following rinses, the samples were then incubated with anti-mouse IgG-FITC (1:300 dilution; BD Biosciences) for 40 min at 37°C. The nuclei were counterstained with 100 nM DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) for 30 min followed by rinses with PBS. Fluorescent images were captured using a spectral confocal microscope imaging system (Leica TCS SP2).

Statistical analysis

IBM SPSS 17.0 software (IBM SPSS, Armonk, NY, USA) was used to perform statistical analyses. The data are expressed as the mean ± standard deviation. Differences between groups were analyzed using one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference. The 2−ΔΔCq method was used for the RT-qPCR analysis.

Results

Atorvastatin protects H9c2 myocardial cells from OGD/R by increasing cell viability, and decreasing the release of LDH and expression of Caspase-9, Bcl-2 and Cyto C

To evaluate whether atorvastatin protects H9c2 myocardial cells from OGD/R, the present study examined the direct cytotoxic effect of OGD/R on H9c2 cells with and without atorvastatin pretreatment. The data shown in Fig. 1A indicates that atorvastatin protected against OGD/R. The cell viability was significantly decreased following OGD/R, compared with that in the control group (P<0.05). As shown in Fig. 1, pretreatment with atorvastatin increased cell viability and reduced the OGD/R-induced LDH release and expression of cell damage-associated proteins, including Caspase-9, Bcl-2 and Cyto C.

Figure 1.

Effects of atorvastatin on H9c2 myocardial cell viability, LDH release, and expression of Caspase-9, Bcl-2 and Cyto C. (A) Graph showing cell viability in the three groups. (B) Graph showing LDH release in the three groups. (C) Protein levels of Caspase-9, Bcl-2, and Cyto C in the three groups. (D) IOD values of the Caspase-9, Bcl-2, and Cyto C proteins in the three groups. *P<0.05, compared with the control group; #P<0.05, compared with the OGD/R group. LDH, lactate dehydrogenase; Bcl-2, B-cell lymphoma 2; Cyto C, cytochrome c; OGD/R, oxygen-glucose deprivation/reperfusion; Ator, atorvastatin; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; IOD, integrated optical density.

Atorvastatin increases the expression of GSK-3β and miR-199a-5p in H9c2 myocardial cells

The present study examined the effect of OGD/R on the expression of GSK-3β and miR-199a-5p in H9c2 cells with and without atorvastatin pretreatment. As shown in Fig. 2A-C, the mRNA and protein expression levels of GSK-3β were significantly decreased following OGD/R, compared with those in the control group and normoxia group (P<0.05). Pretreatment with atorvastatin increased the mRNA and protein expression levels of GSK-3β. As shown in Fig. 2D, the mRNA level of miR-199a-5p was significantly increased following OGD/R (P<0.05), whereas pretreatment with atorvastatin significantly reversed this effect (P<0.05).

Figure 2.

Effects of atorvastatin on the expression of GSK-3β and miR-199a-5p in H9c2 myocardial cells. (A) Graph showing mRNA levels of GSK-3β in the three groups. (B) Protein levels of GSK-3β in the three groups. (C) IOD values of GSK-3β proteins in the three groups. (D) Graph showing mRNA levels of miR-199a-5p in the three groups *P<0.05, compared with the control group; #P<0.05, compared with the OGD/R group. GSK-3β, glycogen synthase kinase-3G; OGD/R, oxygen-glucose deprivation/reperfusion; Ator, atorvastatin; miR, microRNA; IOD, integrated optical density.

Atorvastatin exerts its protective effects against OGD/R through increasing the expression of GSK-3β

To analyze the exact mechanism of atorvastatin protecting H9c2 cardiomyoblast cells from OGD/R, the present study examined the effect of GSK-3β inhibitor (LiCl) on H9c2 cells with and without atorvastatin pretreatment. As shown in Fig. 3A, the cell viability was significantly decreased in the LiCl+atorvastatin+OGD/R group, compared with that in the control group (P<0.05). As shown in Fig. 3B-D, pretreatment with LiCl significantly increased the atorvastatin-inhibited release of LDH and expression of cell damage-associated proteins Caspase-9, Bcl-2 and Cyto C, compared with the control group (P<0.05).

Figure 3.

Protective effects of atorvastatin on H9c2 myocardial cells through GSK-3ou (A) Graph showing cell viability in the five groups. (B) Graph showing LDH release in the five groups. (C) Protein levels of Caspase-9, Bcl-2, and Cyto C proteins in the five groups. (D) IOD values of Caspase-9, Bcl-2, and Cyto C proteins in the five groups. *P<0.05, compared with the control group; #P<0.05, compared with the OGD/R group. LDH, lactate dehydrogenase; Bcl-2, B-cell lymphoma 2; Cyto C, cytochrome c; OGD/R, oxygen-glucose deprivation/reperfusion; Ator, atorvastatin; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; IOD, integrated optical density.

Upregulation and downregulation of miR-199a-5p decreases and increases the expression of GSK-3β, respectively

To evaluate the effect of miR-199a-5p on the expression of GSK-3β, the present study examined the effects of miR-199a-5p mimic and inhibitor transfection on the expression of GSK-3β. As shown in Fig. 4A-C, miR-199a-5p mimic transfection significantly decreased the expression of GSK-3β at the mRNA and protein levels, whereas transfection with the miR-199a-5p inhibitor increased their expression (P<0.01). The mRNA levels of miR-199a-5p in the mimic and inhibitor groups are shown in Fig. 4D.

Figure 4.

Effects of miR-199a-5p on the expression of GSK-3β. (A) Graph showing mRNA levels of GSK-3β in the four groups. (B) Protein levels of GSK-3β in the three groups. (C) IOD values of GSK-3β proteins in the three groups. (D) Graph showing mRNA levels of miR-199a-5p following transfection.**P<0.01, compared with the control group. GSK-3β, glycogen synthase kinase-3β; OGD/R, oxygen-glucose deprivation/reperfusion; Ator, atorvastatin; miR, microRNA; IOD, integrated optical density.

Downregulation of miR-199a-5p protects H9c2 myocardial cells from OGD/R by increasing cell viability and decreasing the release of LDH and expression levels of Caspase-9, Bcl-2 and Cyto C

To evaluate whether the miR-199a-5p inhibitor protected the H9c2 cardiomyoblast cells from OGD/R, the present study examined the direct cytotoxic effect of OGD/R on H9c2 cells with and without miR-199a-5p inhibitor pretreatment. As shown in Fig. 5, pretreatment with miR-199a-5p inhibitor increased cell viability and reduced the OGD/R-induced release of LDH and expression of cell damage-associated proteins and mRNA levels of Caspase-9, Bcl-2 and Cyto C.

Figure 5.

Effects of miR-199a-5p inhibitor on H9c2 myocardial cell viability, LDH release, and expression of Caspase-9, Bcl-2 and Cyto C. (A) Graph showing cell viability in the four groups. (B) Graph showing LDH release in the four groups. (C) Protein levels of Caspase-9, Bcl-2, Cyto C and GSK-3β, in the four groups. (D) Immunohistochemical images showing the expression of GSK-3β in the four groups. *P<0.05, compared with the control group; #P<0.05, compared with the OGD/R group. (E) Protein levels of Caspase-9, Bcl-2, Cyto C and GSK-3β in the four groups. LDH, lactate dehydrogenase; Bcl-2, B-cell lymphoma 2; Cyto C, cytochrome c; OGD/R, oxygen-glucose deprivation/reperfusion; GSK-3β, glycogen synthase kinase-3β; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; IOD, integrated optical density.

Atorvastatin protects neonatal rat cardiac ventricular cardiomyocytes from OGD/R by increasing cell viability and decreasing LDH release and the expression of Caspase-9, Bcl-2 and Cyto C via increasing GSK-3β

To evaluate whether atorvastatin protected neonatal rat cardiac ventricular cardiomyocytes from OGD/R as was shown for H9c2 myocardial cells, the present study first verified neonatal rat cardiac ventricular cardiomyocytes (Fig. 6A). The direct cytotoxic effects of OGD/R on neonatal rat cardiac ventricular cardiomyocytes with and without atorvastatin pretreatment were then examined. The data, as shown in Fig. 6B indicated that atorvastatin protected against OGD/R. The cell viability was significantly decreased following OGD/R, compared with that in the control group (P<0.05). As shown in Fig. 6B and C, pretreatment with atorvastatin increased cell viability and reduced the expression of OGD/R-induced cell damage-associated proteins Caspase-9, Bcl-2 and Cyto C. As was observed in the H9c2 myocardial cells, the protective effects of atorvastatin acted through the miR-199a-5p/GSK-3β pathway (Fig. 6D-G) The expression levels of Caspase-9 and Bcl-2 in the OGD/R group were significantly upregulated compared with those in the control group, as illustrated in Fig. 6H.

Figure 6.

Effects of atorvastatin on H9c2 myocardial cell viability, LDH release, and expression of Caspase-9, Bcl-2 and Cyto C. (A) Image of cardiac ventricular cardiomyocytes (magnification, ×200). (B) Graph showing cell viability among the three groups. (C) Protein levels of Caspase-9, Bcl-2, Cyto C and GSK-3β, among the three groups. (D) Expression levels of GSK-3β mRNA among three groups. (E) Graph showing mRNA levels of miR-199a-5p following transfection. (F) Graph showing LDH release in the five groups. (G) Protein levels of Caspase-9, Bcl-2, Cyto C and GSK-3β. (H) Quantitative levels of Caspase 9, Bcl-2, Cyto C and GSK-3β, in the five groups. *P<0.05, compared with the control group; #P<0.05, compared with the OGD/R group. LDH, lactate dehydrogenase; Bcl-2, B-cell lymphoma 2; Cyto C, cytochrome c; OGD/R, oxygen-glucose deprivation/reperfusion; GSK-3β, glycogen synthase kinase-3β; Ator, atorvastatin; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; IOD, integrated optical density.

Discussion

Apoptosis is a vital pathophysiological mechanism associated with myocardial I/R injury. The prevention and treatment of myocardial apoptosis is an important field of investigation in myocardial infarction. The present study found that atorvastatin protected cardiomyocytes from apoptosis caused by OGD/R. The results showed that the cardiomyocytes exhibited a high level of apoptosis following OGD/R, which was significantly attenuated by atorvastatin pretreatment, suggesting that the attenuation of apoptosis may be a mechanism, which enables atorvastatin to protect against myocardial I/R injury.

The results of the present study also suggested that GSK-3β may be involved in the cardioprotective mechanism of atorvastatin on myocardial I/R injury. GSK-3β, a serine/threonine kinase, is involved in various cell activities through the phosphorylation of the substrate protein (24). GSK-3β has received increasing attention due to its involvement in certain serious diseases, including neurological disease, cancer and myocardial I/R injury. In the cardiovascular system, GSK-3β is involved in glucose metabolism, cardiomyocyte hypertrophy (25) and cell death (26). The data obtained in the present study showed that the mRNA and protein levels of GSK-3β were decreased by OGD/R, whereas atorvastatin pretreatment reversed these effects. This finding is consistent with those of previous reports (24–26) demonstrating the downregulation of GSK-3β in response to myocardial I/R injury.

MicroRNAs are small (15–22 nucleotide) non-coding RNAs, which regulate gene expression and are important in numerous cardiovascular diseases (27). They function as endogenous intracellular regulators of mRNA translation. Although the significance of miR-199a-5p in myocardial I/R injury has not been reported previously, it is recognized as a hypoxia-sensitive microRNA (28). Therefore, the present study hypothesized that the anti-apoptotic mechanism of atorvastatin may be responsible for the downregulation of miR-199a-5p. The results of the present study showed that GSK-3β was decreased in the miR-199a-5p mimic transfection group, whereas it was increased in the miR-199a-5p inhibitor transfection group. Therefore, the downregulation of miR-199a-5p appeared to contribute to the cardioprotective effect of atorvastatin against myocardial I/R injury.

To the best of our knowledge, the present study is the first showing that atorvastatin pretreatment altered GSK-3β via miR-199a-5p following OGD/R, which indicates the possibility that atorvastatin protects the myocardium from I/R injury through modulating miR-199a-5p. Wang et al (29) showed that the inhibition of miR-199a-5p reduced the replication of hepatitis C virus via regulating the pro-survival pathway. Sun et al (30) showed that the inhibition of miR-199a-5p reduced cell proliferation in autosomal dominant polycystic kidney disease through targeting cyclin-dependent kinase inhibitor 1C. These previous studies suggest that miR-199a-5p may be a novel target for the treatment of various diseases. The present study, for the first time, showed that the mRNA level of miR-199a-5p was significantly increased following OGD/R (P<0.05), whereas pretreatment with atorvastatin significantly reserved this effect (P<0.05). In addition, it was shown that the downregulation and upregulation in the expression levels of miR-199a-5p by transfection respectively increased and decreased GSK-3β at the mRNA and protein levels. This suggested that miR-199a-5p mRNA exerted its effects via GSK-3β.

In conclusion, the results of the present study suggested a potential mechanism underlying the beneficial effect of atorvastatin in attenuating OGD/R-induced apoptosis in cardiomyocytes by upregulating specific survival proteins, including GSK-3β, via the downregulation of miR-199a-5p. For the first time, to the best of our knowledge, these data provide novel insight into the mechanisms underlying the effectiveness of atorvastatin in reducing apoptosis following myocardial I/R injury. Statins are a standard component of the therapeutic regimen in treating hypercholesterolemia and have been found to prevent cardiovascular diseases, although the exact mechanism remained unclear. The results presented in the present study demonstrated that atorvastatin increased the cardioprotective effects of GSK-3β by inhibiting miR-199a-5p.