H9c2 cardiomyocytes were obtained from Chi Scientific, Inc., and cultured in complete high-glucose DMEM [cat. no. 06-1055-57-1ACS; Biological Industries (BI)] with 10% FBS (cat. no. 04-001-1ACS; BI) and 1% penicillin/streptomycin (cat. no. 03-031-1B; BI). The cells were incubated with 5% CO₂ at 37°C.

The cells were divided into four groups: i) The 0 µM Ang II group (control); ii) the 0.01 µM Ang II group; iii) the 0.1 µM Ang II group; and iv) the 1 µM Ang II group. After treatment for 72 h, the cells were collected to detect the protein expression levels of two factors associated with cardiomyocyte hypertrophy, such as atrial natriuretic peptide (ANP) and α-actinin, by western blot assay. The optimal concentration to induce cell hypertrophy in subsequent experiments was selected as 0.1 µM because it induced the highest expression of ANP and α-actinin compared with the other concentrations.

H9c2 cells were divided into five groups: i) The control group; ii) the 0.1 µM Ang II group; iii) the 0.1 µM Ang II+1 µM SKF-96365 group; iv) the 0.1 µM Ang II+5 µM SKF-96365 group; and v) the 0.1 µM Ang II+10 µM SKF-96365 group. The cells were pretreated with SKF-96365 for 30 min and subsequently treated with Ang II for an additional 72 h. SKF-96365 was purchased from Selleck Chemicals (cat. no. S7999).

Cells were digested into a cell suspension by 0.25% trypsin for 30 sec at 37°C, and the degree of digestion was controlled to avoid cell shrinkage. A total of five fields were randomly selected for each group, and 20 cells were randomly selected from each field for imaging. The cell surface area (µm²) was measured using ImageJ 2× software (Rawak Software, Inc.), and the mean value was calculated.

The protein synthesis rate of cells in all groups was determined by a [³H] leucine incorporation assay (8). The cultured cells were treated with a [³H] leucine isotope marker (GE Healthcare Life Sciences) 12 h before the test. The medium was removed, and the cells were rinsed twice with PBS. The cells were digested with 0.25% trypsin and repeatedly agitated for 4 min to detach the cells. The cell suspension was added to a glass fiber filter membrane to remove the liquid, dried and fixed with 5% trichloroacetic acid for 30 min at 4°C, and the free isotope markers were washed away. The membrane was dried and placed in a 5 ml flask with scintillation solution. The radioactive intensity was detected by a liquid scintillation counter (MicroBeta; PerkinElmer, Inc.). The data are expressed as counts/min.

Cells were cultured in special confocal dishes and washed with PBS 2–3 times. Then, cells were treated with 100 µl of a 5 µM Fluo-4/AM calcium fluorescent probe solution (Beyotime Institute of Biotechnology), and were incubated at 37°C for 30 min. After incubation, the cells were washed with PBS 2–3 times, and 1 ml PBS was added to the cells. The Fluo-4/AM positive cells were detected by flow cytometry (Sysmex Partec GmbH) at an excitation wavelength of 488 nm and an emission wavelength of 512–520 nm. The fluorescence intensity of the cells was also quantitatively analyzed using FlowMax version 2.8 flow cytometry software (Sysmex Partec GmbH).

The expression of ANP, nuclear factor of activated T-cells (NFAT), α-actinin, β-myosin heavy chain (MHC), TRPC3 and TRPC6 at the mRNA level was determined by RT-qPCR assay. Total RNA was obtained by TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The RNA concentration was measured by a Nanodrop 2000 (Thermo Fisher Scientific, Inc.). Then, cDNA was synthesized from the total RNA with PrimeScript RT Mix Kit (Takara Bio, Inc.) at 37°C for 15 min and then 85°C for 5 sec. The cDNA was amplified with SYBR Green PCR Mix (Thermo Fisher Scientific, Inc.) and an ABI 7300 system (Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for the qPCR: Initial denaturation, 95°C for 10 min; followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. The expression levels were normalized to that of GAPDH. The primers for qPCR are as follows: TRPC3-forward (F), 5′-TGTGGTCTGAGTGCAAGGAG-3′ and TRPC3-reverse (R), 5′-ACCTCTGGTGGGAGTGTGAC-3′; TRPC6-F, 5′-TTTGCTGAAGGCAAGAGGTT-3′ and TRPC6-R, 5′-TTGTTTCTGGCTGCATTCTG-3′; ANP-F, 5′-ATACAGTGCGGTGTCCAACA-3′ and ANP-R, 5′-CGAGAGCACCTCCATCTCTC-3′; brain natriuretic peptide (BNP)-F, 5′-GGAAATGGCTCAGAGACAGC-3′ and BNP-R, 5′-CGATCCGGTCTATCTTCTGC-3′; β-MHC-F, 5′-CCTCGCAATATCAAGGGAAA-3′ and β-MHC-R, 5′-TACAGGTGCATCAGCTCCAG-3′; GAPDH-F, 5′-CTCATGACCACAGTCCATGC-3′ and GAPDH-R, 5′-TTCAGCTCTGGGATGACCTT-3′.

Protein was extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified using a Bicinchoninic Acid Assay kit (Beyotime Institute of Biotechnology). A total of 40 µg of protein was used for 10% SDS-PAGE and transferred to PVDF membranes. Subsequently, the membranes were blocked with 5% skim milk at 4°C overnight. The membranes were incubated with the following primary antibodies overnight at 4°C: anti-ANP (1:1,000; cat. no. A14755; ABclonal Biotech Co., Ltd.), anti-NFAT2 (1:1,000; cat. no. A1539; ABclonal Biotech Co., Ltd.), anti-α-actinin (1:1,000; cat. no. sc-17829; Santa Cruz Biotechnology, Inc.), anti-β-MHC (1:1,000; cat. no. A7564; ABclonal Biotech Co., Ltd.), anti-TRPC3 (1:1,000; cat. no. A7742; ABclonal Biotech Co., Ltd.), anti-TRPC6 (1:1,000; cat. no. bs-2393R; BIOSS) and anti-GAPDH (1:2,000; cat. no. AC033; ABclonal Biotech Co., Ltd.). Subsequently, the membranes were incubated with a horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin (Ig)G secondary antibody (1:5,000; cat. no. bs-0295G-HRP; BIOSS) or HRP-conjugated goat anti-mouse IgG secondary antibody (1:5,000; cat. no. AS003; ABclonal Biotech Co., Ltd.) for 2 h at room temperature. The membranes were detected with Immobilon Western HRP substrate (EMD Millipore). The bands were semi-quantified by ImageJ 2× software (Rawak Software, Inc.).

A total of 1×10⁵ cells were seeded in 6-well plates. After treatment, the cells were fixed in 4% paraformaldehyde (PFA) for 30 min at room temperature, permeabilized with 0.2% Triton X-100 for 5 min at room temperature and then blocked with 5% BSA for 30 min at room temperature. Then, the cells were incubated with the α-actinin antibody (1:100) in PBS, a FITC-conjugated goat anti-mouse IgG antibody (1:50; cat. no. AS001; ABclonal Biotech Co., Ltd.) for 30 min at room temperature, and DAPI (Beyotime Institute of Biotechnology) for 5 min at room temperature. The cells from the different groups were observed by fluorescence microscopy (magnification, ×200; Nikon Corporation).

Data are presented as the mean ± SD. Statistical analysis was determined by one-way ANOVA, followed by Tukey's multiple comparisons test with GraphPad Prism software (version 5.0a; GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

The effect of Ang II on the cardiomyocyte hypertrophy markers ANP and α-actinin in H9c2 cells was detected by western blot assay. As shown in Fig. 1A, Ang II (0.01 and 0.1 µM) significantly promoted the protein expression levels of ANP and α-actinin. The semi-quantitative results of the western blot assay identified that the expression levels of ANP and α-actinin were the highest after treatment with 0.1 µM Ang II (Fig. 1B and C). According to the results of the western blot analysis, the expression levels of TRPC3 and TRPC6 were higher in the Ang II-treated groups compared with the control group, and their expression levels were higher in the 0.1 µM Ang II-treated group than in the 0.01 µM Ang II-treated group. However, there was no difference between the 0.1 µM Ang II-treated group and the 1 µM Ang II-treated group (Fig. 1A, D and E). The present results indicated that TRPC channels may be associated with the process of Ang II-induced cardiomyocyte hypertrophy in H9c2 cells. Moreover, 0.1 µM Ang II for 72 h was the optimal concentration for the induction of cardiomyocyte hypertrophy markers ANP and α-actinin in H9c2 cells.

To determine the inhibitory role of the TRPC channel inhibitor SKF-96365 on cardiomyocyte hypertrophy in H9c2 cells treated with Ang-II, the expression levels of two genes encoding for TRPC channels, TRPC3 and TRPC6, and the cardiomyocyte hypertrophy markers ANP, BNP, and β-MHC by qPCR and western blotting. As shown in Fig. 2A, the present qPCR results showed that SKF-96365 significantly suppressed the increased expression of TRPC3, TRPC6, ANP, BNP and β-MHC induced by Ang II. The western blot results were consistent with the qPCR results (Fig. 2B and C). These results suggested that SKF-96365 suppressed the expression levels of cardiomyocyte hypertrophy markers in Ang II-treated H9c2 cells.

To demonstrate the potential inhibitory role of SKF-96365 in cardiomyocyte hypertrophy induced by Ang II, the protein level of α-actinin was examined by immunofluorescence. In addition, the H9c2 cell surface area and the [³H] leucine incorporation rate were investigated in all groups. As shown in Fig. 3, H9c2 cardiomyocytes were attached and exhibited a triangular or irregular shape, and the α-actinin protein was localized in the cytoplasm. Ang II (0.1 µM) markedly increased the size of the cardiomyocytes and the level of α-actinin. However, SKF-96365 markedly decreased the size of the cardiomyocytes and the level of α-actinin in a dose-dependent manner (Figs. 3 and 4). These results suggested that the TRPC channel inhibitor SKF-96365 may suppress Ang II-induced cardiomyocyte hypertrophy. As shown in Fig. 4A and B, Ang II (0.1 µM) increased the surface area of H9c2 cells, and SKF-96365 significantly attenuated the increased surface area of H9c2 cells induced by Ang II. Ang II (0.1 µM) also induced an increase in the [³H] leucine incorporation rate compared with the control group, but SKF-96365 inhibited the increased [³H] leucine incorporation rate induced by Ang II in a dose-dependent manner (Fig. 4C). The present results suggested that blocking TRPC channels inhibited cardiomyocyte hypertrophy induced by Ang II.

The effect of SKF-96365 on the Ang II-induced Ca²⁺ increase was determined using the Ca²⁺ indicator Fluo-4/AM and flow cytometry. The Fluo-4 percentage is an indicator of the intracellular Ca²⁺ concentration. Ang-II (0.1 µM) significantly increased the intracellular Ca²⁺ concentration compared with the control group, but SKF-96365 inhibited the increased Ca²⁺ concentration induced by Ang-II in a dose-dependent manner (Fig. 5A). In addition, the Ca²⁺ levels were quantified by flow cytometry (Fig. 5B).