Primary antibodies against forkhead box protein O1 (FOXO1; #9454; 1:1,000 dilution) and phospho (p)-FOXO1 (#9461; 1:1,000 dilution) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Primary antibodies against von Willebrand factor (VWF;sc-27649; 1:50 dilution), Akt (sc-5298: 1:200), p-Akt (sc-135650: 1:200) and superoxide dismutase 2 (SOD2) (sc-30080:1:200 dilution) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The antibody against vimentin (ab8978) was obtained from Abcam (Cambridge, MA, USA). GAPDH (#MB001; 1:1,000) was purchased from Bioworld Technology (St. Louis Park, MN, USA). Recombinant Mouse Klotho (#1819-KL-050) was purchased from R&D Systems (Minneapolis, MN, USA). Annexin V-fluorescein isothiocyanate (FITC) Apoptosis kit and Lactate Dehydrogenase (LDH) Activity assay kit were purchased from BioVision (Mountain View, CA, USA). A Cell Counting Kit-8 (CCK8) assay kit was purchased from Dojindo (Kunamoto, Japan). 2,7-Dichlorodihydrofluorescein diacetate (DCF) was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). The bicinchoninic acid protein assay kit was purchased from Pierce Biotechnology, Inc. (Rockford, IL, USA).

In the present study, cultured neonatal Sprague-Dawley rat cardiomyocytes were divided into five groups: The control group without any treatment; the H/R group treated with 4-h hypoxia followed by 3-h reoxygenation; and the H/R+Klotho groups separately incubated with 0.1, 0.2 or 0.4 μg/ml Klotho protein for 16 h prior to treatment with 4-h hypoxia and then 3-h reoxygenation. This study was approved by the ethics committee of The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology (Hubei, China).

Primary cultures of neonatal rat cardiomyocytes were prepared as previously described (14,15). Briefly, cardiomyocytes from one- to two-day-old Sprague-Dawley rats (Tongji Medical College, Huazhong University of Science and Technology) were isolated in phosphate-buffered saline (PBS) solution containing 0.03% trypsin and 0.04% collagenase type II (Invitrogen Life Technologies, Carlsbad, CA, USA). Following removal of fibroblasts by the differential attachment technique, neonatal cardiomyocytes were seeded onto six-well culture plates coated with gelatin at a density of 1×10⁶ cells/well in plating medium consisting of Dulbecco’s modified Eagle’s medium: Nutrient mixture F12 (DMEM/F12; Invitrogen Life Technologies) supplemented with 20% fetal calf serum (FCS), 5-bromo-2′-deoxyuridine (0.1 mM, inhibits fibroblast proliferation) and penicillin/streptomycin (Invitrogen Life Technologies). Cardiomyocyte ischemic injury was induced by hypoxia in a serum- and glucose-free medium, followed by reoxygenation. Hypoxia was achieved by placing the cells in a hypoxia chamber filled with 5% CO₂ and 95% N₂ at 37°C for four hours. Following hypoxia exposure, the cells were reoxygenated with 5% CO₂ and 95% O₂ for three hours in DMEM/F12 containing 20% FCS and normal glucose.

Immunofluorescent staining with antibodies was performed in cultured cardiomyocytes as previously described (16). Briefly, cardiomyocytes were washed three times with pre-cooling PBS, fixed with 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). The immunofluorescent staining was performed by incubating cardiomyocytes with VWF, vimentin, α-smooth muscle actin (α-SMA; ab7817; 1:50 dilution; Abcam) and FOXO1 primary antibodies overnight at 4°C. Following incubation with secondary antibodies for 60 min at room temperature, cardiomyocytes were incubated with DAPI for 10 min and mounted with aqueous mounting medium (Sigma-Aldrich). Finally, the stained cells were visualized under a fluorescence microscope (Olympus 1X71; Olympus Corporation, Tokyo, Japan).

Cell viability was assessed by CCK8 assay (Dojindo), performed according to the manufacturer’s instructions. The cardiomyocyte cell suspension was inoculated into each 96-well plate at a density of 1×10⁴ cells/ml. Subsequently, 10 μl CCK8 solution was added into each well to cardiomyocytes treated with or without reoxygenation. The absorbance at 450nm was measured using a Syngery HT plate reader (BioTek Instruments, Inc., Winooski, VT, USA) to evaluate cell numbers and estimate cell viability.

Cardiomyocyte injury due to H/R stress was assessed by measuring LDH release in the supernatants collected at the end of hypoxia or H/R. LDH was measured using the LDH Activity assay kit (BioVision) according to the manufacturer’s instructions.

Apoptosis induced by H/R in cardiomyocytes was detected with Annexin V-FITC/Propidium Iodide (PI) staining on a flow cytometer (EPICS XL/XL-MCL; Beckman Coulter, Brea, CA, USA). Cells were harvested for flow cytometry following exposure to hypoxia, H/R or control conditions. Briefly, cells were washed three times with cold PBS and resuspended in binding buffer (10 mM HEPES, 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl_2, pH 7.4) at a concentration of 1×10⁵ cells/ml. Subsequently, 100 μl solution was transferred to a 5-ml culture tube and labeled with 5 μl Annexin V and 5 μl PI. The solution was gently vortexed and incubated for 15 min at 25°C in the dark, following which, 400 μl binding buffer was added to each tube. Analysis by flow cytometry was conducted within one hour. Excitation wavelength was 488 nm and emission wavelength was 530 nm.

DCF was used to measure intracellular ROS. It is a non-fluorescent, cell permeable compound that is oxidized to a highly fluorescent carboxy-dichlorofluorescein measured at wavelengths of 488 nm excitation/520 nm emission and expressed in arbitrary units (AU) using a Synergy HT microplate reader (Bio-Tek Instruments, Inc.).

Protein extracted from cardiomyocytes which were lysed in radioimmunoprecipitation assay lysis buffer was used for SDS-PAGE. The proteins were subsequently transferred to nitrocellulose membranes and blocked with 5% nonfat dry milk in Tris-buffered saline with Tween 20 (TBST; Cell Signaling Technology, Inc.) for 60 min at room temperature. Membranes were probed with various primary antibodies overnight. The following day, the membranes were washed with 1X TBST, incubated for 60 min with horseradish peroxidase-labeled mouse anti-rabbit antibody and anti-avidin antibodies (Cell Signaling Technology, Inc.) in TBST fluid. Following three washes of the membrane, images were captured on film, which was placed in 10 ml LumiGLO^® solution (Cell Signaling Technology, Inc.) for one minute. Following development, the images were placed into an automatic image analyzer (Bio-Rad Laboratories, Hercules, CA, USA) to determine the function of the proteins as well as the reference grayscale values. A monoclonal GAPDH antibody was used separately as a loading control.

Values are expressed as the mean ± standard error of the mean. Comparisons between four groups were performed using one-way analysis of variance using SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference between values.

Following 72 h of incubation, the majority of cardiomyocytes gathered into a mass and became cross-linked with each other. Identification of cardiomyocytes was performed by immunofluorescent staining using anti-VWF for detection of endothelial cells, anti-vimentin for fibroblasts and anti-α-SMA for cardiomyocytes (17–19). Immunofluorescence staining indicated that these cells were positive for α-SMA which is a marker of cardiomyocytes, but negative for VWF and vimentin, which are markers of endothelial cells and fibroblasts, respectively (Fig. 1). These features indicated that the cultured cells were cardiomyocytes.

The CCK8 assay was used to investigate the effect of Klotho on cell viability of cardiomyocytes following H/R. Cell viability was 92.10±2.80% in the control group, and only 68.60±4.30% in the H/R group. Compared to the H/R group, cell viability was markedly increased in the H/R+Klotho groups to 83.40±1.30, 88.60±0.40 and 88.30±2.30%. However, there was no significant difference between the control group and H/R+Klotho groups (Table I). Collectively, these data indicated that exogenous Klotho protein expression enhanced cell viability of H/R-exposed cardiomyocytes.

LDH release, a marker of cardiomyocyte injury following H/R, was markedly reduced in the H/R+Klotho groups (121.50±14.37, 120.70±13.94 and 118.50±13.28 IU/l, respectively) compared with that of cardiomyocytes in the H/R group (240.30±17.78 IU/l). However, there was no difference between LDH release in the control group and that of the H/R+Klotho groups (Table I). Collectively, these data indicated that exogenous Klotho protein expression reduced cardiomyocyte injury resulting from H/R.

As displayed in Table I, the apoptotic rate of cardiomyocytes was significantly decreased in the H/R groups treated with Klotho protein, compared with that of the H/R group. This result indicated that exogenous Klotho protein may suppress cell apoptosis in cardiomyocytes upon H/R.

As indicated in Table I, H/R treatment resulted in a significant increase in DCF fluorescence from 220.10±16.68 to 797.10±22.24 A.U. Pretreatment with Klotho at 0.1, 0.2 or 0.4 μg/ml attenuated the DCF fluorescence to 595.80±16.84, 510.90±20.26 and 455.40±15.32 A.U., respectively (P<0.05). This result suggested that Klotho pretreatment significantly attenuated intracellular oxidant generation induced by H/R in a dose-dependent manner.

To further study the effect and mechanism of Klotho protein protection of cardiomyocytes against oxidative stress and apoptosis, immunofluorescence and western blot analysis were used in order to observe the changes of FOXO1 and p-FOXO1 protein expression under H/R conditions. Immunofluorescence staining and western blot analysis indicated that in the Klotho protein treatment groups, FOXO1 was diverted from the cytoplasm to the nucleus (Figs. 2 and 3).

Western blot analysis results confirmed that there was no significant difference in total FOXO1 and Akt protein expression between the H/R group and the other groups. However, the phosphorylation levels of FOXO1 and Akt protein decreased significantly in the H/R+Klotho groups compared to those in the H/R group in a dose-dependent manner (Fig. 4A and B). In addition, these results indicated that different concentrations of Klotho protein were capable of effectively increasing SOD2 expression (Fig. 4C and D). Collectively, these data indicated that exogenous Klotho protein inhibited phosphorylation of FOXO1 and Akt resulting from H/R.