Thirty male SHRs and 15 Wistar-Kyoto (WKY) rats (age, 8 weeks) were obtained from the Animal Center of Fuwai Hospital (Beijing, China). The rats had free access to water and a regular diet, and were housed at 22±1°C under a standard 12-h light/dark cycle to acclimate for one week prior to the experiments. All animals in the study were cared for, and the experiments were performed according to, the established Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996). The study was approved by the Institutional Review Board and the Animal Care and Use Committee of the General Hospital of Lanzhou Military Area Command (Lanzhou, China).

The systolic pressure (mmHg) of the rat tail artery was measured using a rat sphygmomanometer (RBP-1B-type, China Japan Friendship Institute of Clinical Medicine, Beijing, China). All measurements for the rat tail artery systolic pressure were performed by the same investigator. The blood pressure of each animal was measured prior to the initiation of the experiment, and once a week until the end of the experiment.

Simvastatin (Wuhan C-bons Group, Wuhan, China) has been widely administered clinically to prevent cardiovascular and cerebrovascular diseases (11). Each animal received simvastatin (10 mg/kg) once a day over a 10-week study period. Control animals received the same volume of saline during the same schedule.

Following weighing, the animals were anesthetized with an intraperitoneal injection of 3% pentobarbital sodium (40 mg/kg). The rat was sacrificed by exsanguination whilst still under anaesthesia. The heart was removed immediately following euthanasia and rinsed with 0.9% saline (4°C). Subsequently, the vessels, cardiac atrium and right ventricular were quickly removed. After blotting the saline with filter paper, the left ventricular and interventricular septum were weighed, from which the LVM/BM ratio was calculated. Finally, the tissue samples were cut into sections at 6-µm thickness with an interval of 300 µm between each section and stored in liquid nitrogen until required.

Cardiomyocytes were cultured and identified according to a previously reported method with slight modifications (12). Briefly, the ventricles of the Sprague Dawley rats (age, 1–3 days; either gender; Research Center of Experimental Animals in the Fourth Military Medical University, Xi'an, China) were harvested aseptically and cut into 1-mm³ pieces. The tissue samples were digested with 0.1% type I collagenase (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 5–10 min with the assistance of magnetic stirring. The digestion procedures were repeated 5–7 times. Following centrifugation at 550 × g for 5 min at 37°C, the cell pellets were collected and suspended in Dulbecco's modified Eagle's medium (DMEM; Gibco Life Technologies, Grand Island, NY, USA) containing 15% neonatal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China). The cells were seeded into culture flasks and allowed to stand for 2 h at 37°C in 5% CO₂ (CO₂ incubator; NuAire, Hong Kong, China). After 2 h, the difference in the adhesion ability of the cells enabled the isolation of the cardiomyocytes. The isolated cardiomyocytes were seeded into culture flasks or plates at a density of 4×10⁵ cells/ml. After 48 h, 0.1 mmol/l bromodeoxyuridine (Sigma-Aldrich) was added to inhibit the growth of the cells other than the cardiomyocytes. The medium was changed every 2 days. The cells were identified as cardiomyocytes by spontaneous beating after 24 h and positive staining for α-smooth muscle actin. The staining procedure is shown as previously described (13). The purity of the cells reached 95%, which was satisfactory for the further experiments.

Stably cultured cells were divided into three groups. In the control group, the cells were cultured with serum free DMEM, while in the serum group, the cells were cultured with DMEM containing 15% neonatal bovine serum. In the simvastatin intervention group, the cells were cultured with DMEM containing 15% neonatal bovine serum, in addition to various concentrations of simvastatin (10⁻⁵, 10⁻⁶, 10⁻⁷ and 10⁻⁸ mol/l). After 24 h, cell cultivation was terminated for further evaluation of the indices.

Cardiomyocytes were seeded at a density of 4×10⁵ cells/ml into six-well plates, with a cover slip placed previously. The cells were subsequently cultured with medium containing a variety of intervention factors. After 24 h of culture, the cultivation was terminated and the cells on the cover slip were fixed with 95% alcohol for 15 min in preparation for hematoxylin and eosin staining. A Leica-Q500 image analysis system (Leica Camera AG, Wetzlar, Germany) was used to determine the cardiomyocyte surface area. Five observation fields were selected randomly on each cover slip, and 5–8 cells within each observation field were selected for the determination of the mean cardiomyocyte surface area according to the image analysis system. The average of the measurements was calculated.

Cardiomyocytes were seeded into 24-well plates at a density of 4×10⁵ cells/ml, with 1 ml in each well. Next, 3H-leucine, with a final concentration of 3.7×10⁴ Bq/ml (China Institute of Atomic Energy, Beijing, China) that had been prewarmed to 37°C, was added to each well together with the intervention factors. Following termination of the cultivation, the cells were washed twice with phosphate-buffered saline (PBS) and 10% trichloroacetic acid for 5 min, which had both been precooled to 4°C. Subsequently, 0.5 ml NaOH-1% SDS (0.3 mol/l) was added, and the mixture was allowed to stand for 30 min at room temperature. The cell lysates were harvested, transferred to glass-fiber filter paper, and subjected to drying at 42°C. The radioactivity (cpm/cell) of the cells was evaluated using a LS6500 liquid scintillation counter (Beckman Coulter, Brea, CA, USA).

After 48 h cultivation, the cells were washed with cold PBS, and lysed with 100 µl 1X SDS lysis buffer, which had been prewarmed to 85°C. The lysates were collected in a microtube, heated for 10 min, and then centrifuged at 12,000 × g for 10 min at room temperature. Following collection of the supernatant, the protein concentration was determined according to the Bradford method (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Subsequently, 20 µg protein from each sample was boiled at 100°C for 5 min, after which the proteins were separated through 5–8% SDS-PAGE. The protein band was transferred to a nitrocellulose membrane, and the membranes were blocked with 3% bovine serum albumin solution overnight at 4°C. The detection of the different proteins was conducted by overnight incubation of the membrane at 4°C with the required dilution of a specific primary antibody (rabbit anti-mouse PKB monoclonal antibody; cat. no. SC-7686; dilution, 1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; rabbit anti-mouse PTEN monoclonal antibody; dilution, 1:500; Sigma-Aldrich). The membrane was further incubated with a horseradish peroxidase-coupled goat anti-rabbit IgG (cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) for 1 h. After thoroughly washing with 0.05% Tween 20, the protein bands were visualized using 3,3′-diaminobenzidine (Sigma-Aldrich). LabWorks 3.0 UVP software (UVP, Upland, CA, USA) was used to analyze the optical density, which represented the relative protein concentration.

Results are expressed as the mean ± standard deviation, and differences among the groups were analyzed using univariate analysis of variance and the least significant difference test. SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA) was used for all the statistical analyses, and P<0.05 was considered to indicate a statistically significant difference.

The LVM/BW ratio has been used to indicate cardiomyocyte hypertrophy in certain in vivo experiments. In the control (WKY) group, the LVM/BW ratio was 3.04±0.12 mg/g, while in SHRs, the ratio was determined to be 4.10±0.13 mg/g. Treatment with simvastatin was shown to reduce the extent of cardiomyocyte hypertrophy in the SHRs, with simvastatin treatment decreasing the LVM/BW ratio to 3.73±0.08 mg/g (Fig. 1).

In order to identify the pathway affected by simvastatin with regard to cardiomyocyte hypertrophy, the protein expression levels of PKB and PTEN were investigated in the study. In the SHR group, the PKB protein expression levels were significantly greater when compared with that in the WKY group (P<0.01), while the level in the simvastatin + SHR group was significantly lower when compared with the SHR group (P<0.01). By contrast, the protein expression level of PTEN in the SHR group was significantly lower when compared with that in the WKY group (P<0.01), while expression levels were significantly increased in the simvastatin treatment group, as compared with that of the SHR group (P<0.01; Fig. 2A–C).

Following treatment with 15% neonatal bovine serum for 24 h, the cardiomyocyte surface area was 1,611.16±160.75 µm², which was significantly higher when compared with that of the serum-free control group (538.04±118.60 µm²; P<0.01). Following treatment with different concentrations of simvastatin together with 15% fetal bovine serum, the cardiomyocyte surface area was determined to be significantly lower in the 10⁻⁵ and 10⁻⁶ mol/l simvastatin intervention groups (799.84±167.70 and 1,076.88±199.28 µm², respectively), as compared with the serum group (P<0.01). However, in the 10⁻⁷ and 10⁻⁸ mol/l simvastatin intervention groups, the cardiomyocyte surface areas (1,529.32±212.83 and 1,606.84±220.81 µm², respectively) were not significantly different when compared with that of the serum group (P>0.05; Fig. 3A).

The 3H-leucine incorporation rate in the 15% neonatal bovine serum group (2,360±106 cpm/well) was significantly greater when compared with that in the serum-free group (1,305±92 cpm/well; P<0.01). The incorporation rates were 1,707±101 and 1,962±125 cpm/well in the 10⁻⁵ and 10⁻⁶ mol/l simvastatin intervention groups, respectively, and these values were significantly lower when compared with the serum group (P<0.01). In the 10⁻⁷ and 10⁻⁸ mol/l simvastatin intervention groups, the incorporation rates were 2,280±105 and 2,311±80 cpm/well, respectively; however, these values were not significantly different when compared with that of the serum group (P>0.05; Fig. 3B).

PKB protein expression levels were significantly higher in the serum group when compared with the control group (P<0.01). Furthermore, the PKB protein expression levels were significantly lower in the 10⁻⁵ and 10⁻⁶ mol/l simvastatin intervention groups when compared with the serum group (P<0.01). However, there were no statistically significant differences in the levels when comparing the 10⁻⁷ and 10⁻⁸ simvastatin intervention groups with the serum group (P>0.05). By contrast, simvastatin was found to increase the PTEN protein expression levels in a concentration-dependent manner. The protein expression levels of PTEN in the 10⁻⁵, 10⁻⁶ and 10⁻⁷ mol/l simvastatin + serum groups were significantly higher when compared with the serum group (all P<0.05); however, the expression levels were all lower when compared with the control group (all P<0.01; Fig. 4A–C).

Inhibition of the biological effects of simvastatin on the cardiomyocytes was observed following treatment with PTEN antisense oligodeoxynucleotides. The protein expression levels of PTEN in the PTEN antisense oligodeoxynucleotides group were significantly lower compared with those in the simvastatin group (P<0.01); however, there were no statistically significant differences when comparing the sense or mismatch groups with the simvastatin group (P>0.05; Fig. 5A and B). Accordingly, the cardiomyocyte surface area (Fig. 5C) and the 3H-leucine incorporation rate (Fig. 5D) in the 10⁻⁶ mol/l simvastatin + 5×10⁻⁶ mol/l PTEN antisense oligodeoxynucleotides group were significantly greater compared with the values observed in the simvastatin single treatment group (P<0.01). However, the values were lower compared with those of the serum group (P<0.01). By contrast, there were no statistically significant differences in the protein expression levels when comparing the PTEN sense and mismatch oligodeoxynucleotides group with the simvastatin group (P>0.05).