Animals

Fifteen male SHRs (Shanghai laboratory animal centre), aged 16 weeks and with an average body weight of 39 ± 21 g, were randomized into two groups: an SHR control group ( n = 7), treated with vehicle; and an SHR enalapril group ( n = 8), treated with enalapril (15 mg/kg/day). Treatment lasted for 4 weeks. During the treatment period, body weight and tail blood pressure were measured once per week and the enalapril dose was adjusted based upon body weight. Ten WKYs served as the normotensive control group and were also treated with vehicle. At the end of treatment, the rats were killed and the left ventricles were dissected carefully and frozen and stored in liquid nitrogen for RT-PCR and Western blot protein staining.

The study was approved by the Animal Care and Use Committee of Suzhou University and our study followed the principles in the Declaration of Helsinki.

Isolation of total ribonucleic acid and reverse transcription-polymerase chain reaction

Total RNA was isolated according to the Trizol reagent manual and reverse transcription was performed using Moloney murine leukaemia virus reverse transcriptase in a mixture containing deoxyribonucleotides, random hexamers and ribonuclease inhibitor in reverse transcriptase buffer. Elongation factor 1α (EF1α) was used as the internal standard. The PCR assay was performed using the primers for ACE, ACE2 and EF1α described by Sakima et al. ( Table 1 ). The PCR amplification conditions (30 cycles) were as follows: denaturing, 94 °C for 90 seconds; annealing, 56 °C for 45 seconds; elongation, 72 °C for 45 seconds. Primers for EF1α were added to all reactions at the cycle number indicated in Table 1 . Finally, an elongation was performed at 72 °C for 10 minutes. Amplification products were separated on a 1% agarose gel dyed by Loading Dye I. The bands were visualized and the optical density was quantified by the Bio-Rad computerized densitometry system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The target messenger ribonucleic acid (mRNA) concentration was expressed as the ratio of target gene to the internal standard EF1α gene.

Western blot

The membrane protein was isolated using cell lysis buffer containing a proteinase inhibitor mixture. Protein concentration was determined using the Bradford method. Sample proteins (10 μg/lane) and a prestained protein weight marker were loaded and size fractionated by sodium dodecyl sulphate polyacrylamide gels. The proteins were then transferred from the gel onto nitrocellulose membranes and blocked with 1% non-fat skimmed milk in phosphate-buffered solution (PBS) for 45 minutes at room temperature under gentle agitation on a shaker. The membranes were then incubated overnight at 4 °C with the primary antibody (1:200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) in PBS containing 0.5% non-fat skimmed milk buffer. On the following day, the membranes were thoroughly washed three times (10 minutes each) using PBS containing 0.1% Tween. Positive bands were developed using the Western Blotting Analysis System (Tiandz, Beijing, China) in which horseradish peroxidase-conjugated secondary antibody was diluted at 1/10,000 and incubated for 1 hour at room temperature. The membranes were then visualized by chemiluminescence and photographed using an x-ray. The protein bands on the x-ray films were quantified by densitometry scanning and corrected for β-actin. The target protein was expressed as the ratio of target protein to β-actin.

Statistical analysis

Data were analysed using SPSS 11.0 software. Each value was expressed as mean ± standard error of the mean. Significant differences were obtained when P < 0.05. Differences between two groups were analysed using the t test. One-way analysis of variance was used to analyse the differences between multiple groups.