A total of 23 male C57/BL6 mice (age 8–10 weeks; weight 20–23 g) were used for the purposes of this study. All mice were purchased from Heze Better Biotechnology Co., Ltd. (Shangdong, China) and had free access to normal chow diet and water, temperature and humidity were kept at 22–24°C, 40–60% with a 12 h light/12 h dark cycle. Mice were divided into 4 groups (5–7 mice per cage) with the same average body weight. Out of these, two groups were used for sham (control group) operations with the administration of either saline (n=5) or telmisartan (n=5). Aortic binding (AB) operations were performed on mice in the remaining two groups, together with the administration of either saline (n=6) or telmisartan (n=7). All mice were sacrificed at 4 weeks post-AB operation.

The afterload-induced pressure model was generated by AB under abdominal anesthesia as described previously (23). Briefly, the chest was first opened, and AB of the aortic arch between the brachiocephalic artery and left common carotid artery was performed using a 27-gauge needle as the standard binding level to lead to the aortic arch narrow. Following successful binding, the needle was removed and the chest cavity was closed. The same operation without AB was performed on mice in the sham group.

Wistar rats (age, 1–3 days) were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences (Beijing, China). Hearts were harvested under aseptic conditions on a clean bench. Following 3 washes with sterilized phosphate-buffered saline to remove excess blood, the hearts were minced, washed with Hank's Balanced Salt Solution at 4°C and digested in cardiomyocyte balance suspension liquid containing 0.1% trypsin and 0.025% collagenase (Sigma-Aldrich; Merck KGaA; Darmstadt, Germany) for 15 min. The undigested heart tissue was re-digested 4 or 5 times. Following complete digestion, cardiomyocytes were separated and isolated by centrifugation using the Percoll gradient system (upper, 45.3% percoll; bottom, 58.5% percoll; Sigma-Aldrich; Merck KGaA) as described previously (24). The separated rat cardiomyocytes were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and penicillin (0.2%) (Sigma-Aldrich; Merck KGaA) at a density of 1×10⁵ cells/cm² in a 5% CO₂ incubator at 37°C. At 2 days following isolation, and when cardiomyocyte beating was confirmed, the cells were used for the purposes of downstream experiments. All animal experiments were approved by the Ethics Review Committee of Harbin Medical University (Harbin, China).

Telmisartan (Boehringer Ingelheim Shanghai Pharmaceuticals, Co., Ltd., Shanghai, China) was dissolved in saline prior to in vivo administration at a dose of 50 mg/kg/day. Saline or telmisartan were administered to the mice once-a-day via oral gavage, commencing 5 days prior to AB operations and continuing until they were sacrificed at 4 weeks post-AB operation. For the treatment of neonatal primary rat cardiomyocytes in vitro, telmisartan was first dissolved in dimethyl sulfoxide. Three different concentrations of telmisartan (5, 20 and 50 µM) were used for these experiments. In addition, Ang II (10 µM; Sigma-Aldrich; Merck KGaA) was used to induce cardiomyocyte hypertrophy in vitro. Rat cardiomyocytes were treated with the vehicle (dimethyl sulfoxide), telmisartan and Ang II for 12 h.

Mouse heart samples were fixed in 4% formaldehyde overnight at room temperature and embedded in paraffin. Blocks were cut into 5-µm thick sections and de-waxed in xylene for 10 min and stained with hematoxylin and eosin (H&E) at room temperature. Briefly, the sections were placed in distilled water, stained with alum hematoxylin at room temperature for 10 min, rinsed under running tap water, differentiated with 0.3% acid alcohol at room temperature for ~30 sec, rinsed in running tap water again and rinsed in Scott's tap water substitute (Sigma-Aldrich; Merck KGaA) prior to rinsing in tap water again. The sections were subsequently stained with eosin for 2 min. Following that, sections were observed (Olympus FluoView™ FV1000) to evaluate cardiac morphology and measure cardiomyocyte size. The thickness of the anterior, posterior, lateral and septum heart walls was measured, and the average of these four parameters was calculated as the average wall thickness. Similarly, the internal dimensions (presented as an average of the horizontal and vertical measurements) of left ventricles were measured and an average value was calculated as average internal dimensions. To investigate cardiomyocyte size, ~50 cardiomyocytes stained by H&E were measured and the average value was calculated as the average area of myocytes per sample. All of these measurements were performed using ImageJ software (v1.48; National Institutes of Health, Bethesda, MD, USA). Heart weight was measured by an experimental scale.

TUNEL assay was performed on heart sections (5-µm) with a TdT-FragEL DNA Fragmentation Detection kit to quantify apoptosis. Counterstaining with fluorescence mounting medium containing DAPI (blue; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was performed to visualize normal nuclei. Sections were observed by use of a fluorescence microscope Olympus FluoView™ FV1000. Measurements of the apoptotic nuclei percentage were obtained from the border zone area of sections from each heart and 8 fields were randomly selected from the border zone of each heart and the average TUNEL-positive percentage (%) was calculated.

Cardiomyocytes were collected and cultured on glass bottom coverslips in 4-well dishes that had been previously coated with collagen II, as described above for rat cardiomyocyte primary culture. Following treatment with Ang II or telmisartan, cells were fixed with 10% acetone at 4°C for 20 min. After the glass slides were washed by TBS with 0.5% Tween-20, 3 times, double immunofluorescent staining was performed using a specific antibody against NFATc3 (cat. no. sc-8405, 1:500; Santa Cruz Biotechnoloy, Inc., Dallas, TX, USA) at room temperature for 2 h, followed by the addition of a donkey anti-rabbit IgG (red; Alexa Fluor^® 488; cat. no. ab150077; 1:500; Abcam, Cambridge, UK) for 1 h at room temperature. Finally, following 3 washes with TBS, slides were mounted in fluorescence mounting medium containing DAPI (blue; Thermo Fisher Scientific, Inc.). Cells were observed by use of a fluorescence microscope, Olympus FluoView™ FV1000. °C.

Total RNA was extracted from the mouse hearts using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), and total RNA (2,000 ng) was used, cDNA (50 ng/µl) was synthesized using oligo (dT) primers and the Transcriptor First Strand cDNA Synthesis kit (cat. no. 04896866001; Roche Applied Science, Penzberg, Germany). PCR amplifications were performed using LightCycler^® 480 SYBR-Green I Master (cat. no. 04887352001; Roche Applied Science). Target gene expression levels were normalized to the expression of the gene encoding the 18S ribosomal RNA subunit by the ΔΔCq method, as described (25). The primers used were as follows: Myosin heavy chain α isoform (Myh6), forward, 5′-AGATCATCAAGGCCAAGGCA-3′, and reverse, 5′-CGCTGGGTGGTGAAATCATT-3′; GATA binding protein 4 (Gata4), forward, 5′-AGCTCCGTGTCCCAGACG-3′, and reverse, 5′-TCTGTGGAGACTGGCTGACG-3′; collagen I, forward, 5′-ATGTTCAGCTTTGTGGACCTC-3′, and reverse, 5′-TCCCTCGACTCCTACATCTTC-3′; collagen III, forward, 5′-TGGTTTCTTCTCACCCTTCTTC-3′, and reverse, 5′-TGCATCCCAATTCATCTACGT-3′.

Mouse ventricular cytoplasmic protein was extracted using lysis buffer (50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 25 mM EDTA, 0.25% sodium deoxycholate, and 1 mM dithiothreitol). The nuclear protein precipitate was extracted using radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 1 mM EDTA and 1% NP-40). Total protein was extracted from cultured cells using RIPA lysis buffer containing 2% phenylmethylsulfonyl fluoride, 10% complete protease inhibitor cocktail, 10% PhosSTOP (Sigma-Aldrich; Merck KGaA), 5% NaF and 1% Na₃VO₄. The protein concentration was determined using a Pierce BCA Protein assay kit (cat. no. 23225; Pierce; Thermo Fisher Scientific, Inc.). Proteins (20 µg) were electrophoresed by 10% SDS-PAGE (cat. no. NP0301BOX; Invitrogen; Thermo Fisher Scientific, Inc.) and subsequently electrotransferred to a polyvinylidene difluoride membrane (cat. no. IPVH00010; Merck Millipore). Membranes were blocked in TBS with 0.5% Tween-20 containing 5% skim milk for 60 min at room temperature. Membranes were subsequently incubated with the following primary antibodies overnight at 4°C: NFATc3 (cat. no. sc-8405; 1:1,000), NFATc4 (cat. no. sc-13036; 1:1,000), ANP (cat. no. sc-20158; 1:2,000), BNP (cat. no. sc-67455; 1:2,000), caspase 3 (cat. no. sc-7148; 1:1,000), GAPDH (cat. no. sc-365062; 1:2,000) and lamin (cat. no. sc-20680; 1:2,000). All primary antibodies were purchased from Santa Cruz Biotechnology, Inc. The membrane was incubated at room temperature for 1 h with Peroxidase-affiniPure goat anti-mouse IgG (H+L; cat. no. 115-035-003; 1:2,000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or Peroxidase-affiniPure goat anti-rabbit IgG (H+L; cat. no. 111-035-003; 1:2,000; Jackson ImmunoResearch Laboratories, Inc.). A FluorChem E (Cell Biosciences Pty, Ltd., Heidelberg, VIC, Australia) imaging system was used to visualize the signals. GAPDH was used as a loading control.

Results are expressed as the mean ± standard deviation. Comparisons between 2 groups were analyzed using a Student's t-test. Comparisons among >2 groups were analyzed using one-way analysis of variance and Turkey's multiple comparisons test. Statistical analysis was performed using GraphPad Prism (v6.0; GraphPad Software, Inc., La Jolla, CA, USA) P<0.05 was considered to indicate a statistically significant difference.

In order to determine whether telmisartan inhibits cardiac hypertrophy, an in vivo study using a mouse AB model was performed. At 4 weeks following the AB operation, mice administered with saline exhibited a hypertrophied heart with increased wall thickness, an enlarged left ventricular dimension and increased heart weight (HW)/body weight (BW) ratio, when compared with the saline-treated sham group (Fig. 1A, C and E). By contrast, mice administered with telmisartan exhibited reduced cardiac hypertrophy, with a significantly reduced wall thickness, smaller left ventricular dimensions and a lower HW/BW ratio when compared with the saline-treated AB group (Fig. 1A, C and E). In addition, cardiomyocyte size was significantly lower in the hearts of mice in the telmisartan-treated group when compared to that of the saline-treated AB group (Fig. 1B and D). The mRNA expression levels of cardiac hypertrophy markers, Myh6 and Gata4, as well as the markers of cardiac fibrosis, collagen I and collagen III were increased when compared with the sham telmisartan-treated AB group, but at a significantly lower level when compared with the saline-treated AB group (Fig. 1F and G). These results indicated that telmisartan may inhibit cardiac hypertrophy and fibrosis in a mouse model of cardiac afterload.

NFAT is an important nuclear transcriptional factor involved in cardiac hypertrophy (26). Thus, the authors investigated whether telmisartan inhibited NFAT nuclear translocation. There are five subtypes of NFAT in cardiomyocytes, however, it is thought that NFATc3 and NFATc4 are the most highly expressed in cardiomyocytes, and therefore NFATc3 and NFATc4 may be the two most important subtypes (27). Following the extraction of cardiomyocyte nuclear protein, the expression levels of NFATc3 and NFATc4 were significantly increased in the AB group when compared with the sham group (P<0.01; Fig. 2A and B). However, this expression was significantly reduced in the telmisartan-treated AB group when compared with the untreated AB group (P<0.05) (Fig. 2A and B).

Activated NFAT has been reported to stimulate the expression of ANP and BNP (28,29). Thus, the present study investigated whether ANP/BNP expression may be inhibited by telmisartan treatment. ANP and BNP expression significantly increased in the hearts of mice from the AB group when compared with the sham group (P<0.01; Fig. 2C and D). However, telmisartan administration significantly lowered ANP and BNP levels when compared to the untreated AB group (P<0.01; Fig. 2C and D). The inhibitory effect of telmisartan on ANP expression was marginally more pronounced than its effect on BNP protein levels (Fig. 2C and D).

As ANP and BNP reportedly induce apoptosis (30,31), and cardiac afterload may induce damaging levels of apoptosis in heart tissues (32), the anti-apoptotic effects of telmisartan were investigated in the present study. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stain revealed that the percentage of TUNEL-positive nuclei induced by cardiac afterload were significantly reduced by telmisartan administration when compared with the untreated AB group (P<0.05; Fig. 3). These results indicate that telmisartan may inhibit NFAT nuclear translocation, ANP and BNP expression and cardiomyocyte apoptosis to protect against cardiac afterload-induced cardiac hypertrophy.

The results presented so far provide evidence to suggest that telmisartan may inhibit cardiac hypertrophy, NFATc3 and NFATc4 nuclear translocation, ANP expression and cardiac cell apoptosis in a mouse model of cardiac afterload. In order to investigate whether these biochemical effects are detectable in cardiomyocytes directly, a primary neonatal rat cardiomyocyte culture was established and stimulated with Ang II. As shown in Fig. 4A, telmisartan appeared to inhibit Ang II-induced cardiomyocyte hypertrophy, which was consistent with the results observed in the in vivo mouse AB model (Fig. 1A-E). In order to determine whether telmisartan may inhibit the nuclear translocation of NFAT, ANP expression and cardiomyocyte apoptosis, cardiomyocytes were stimulated with three different concentrations of telmisartan (5, 10 and 50 µm). A high concentration of telmisartan (50 µm) inhibited Ang II-induced NFATc3 nuclear translocation (Fig. 4B). In addition, ANP expression was significantly inhibited by telmisartan in a dose-dependent manner (P<0.01, 50 µM vs. 0 µM telmisartan; Fig. 4C and D). Furthermore, the Ang II-stimulated increase in caspase 3 protein expression levels, which is a known marker for apoptosis (33), was inhibited by telmisartan in a dose-dependent manner (P<0.01, 50 µM vs. 0 µM telmisartan; Fig. 4C and D). These results indicated that telmisartan may inhibit NFAT nuclear translocation, ANP expression, Ang II-induced cardiomyocyte apoptosis and suppress cardiomyocyte hypertrophy in vitro.