Materials
Sodium hydrogen sulfide (NaHS), 4-PBA (an inhibitor of ERS), GSK2656157 (an inhibitor of PERK), STF083010 (an inhibitor of IRE 1α) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The primary antibodies for anti-Cyclin D1, p21Cip/WAF−1, cleaved caspase-3 and -9, Bcl-2, cytochrome c (Cyt c), GRP 78, CHOP, cleaved caspase-12, ATF 4, ATF 6, XBP-1 and GAPDH were from Proteintech (Wuhan, China). Hoechst 33342 was from Beyotime (Wuhan, China). The anti-PERK, eIF 2α and IRE 1α antibodies were obtained from Cell Signaling Technology (Denver, USA). H2O2 and PBS solution were from Sigma Chemical Co. (St. Louis, MO, USA). Western blotting relative solutions were from Santa Cruz Biotechnology (Dallas, USA).
Culture of cardiomyocytes
Rat cardiomyocytes line H9C2 was purchased from the American Type Culture Collection. H9C2 cells were cultured in growth medium DMEM containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. The experiments were performed when the cells reached 70–80% confluence between passages 6 and 10.
H2O2 treatment of the aged H9C2 cells
The treatment for H2O2 induction was as previously described [18]. Briefly, the DMEM supplemented with 10% FBS was removed, and DMEM supplemented with different concentrations (0, 5, 10, 30, 50, 80, 100 μM) H2O2 was added to the H9C2 cells in the culture cluster for 2 h and subsequently cultured for 3 days. The degree of the aged cells was observed through SA β-Gal Staining, AGEs ELISA Assay and the expression of Cyclin D1 and p21Cip/WAF−1. In the present study, we selected 30 μM H2O2 concentrations for 2 h and subsequently cultured for 3 days.
Hypoxia/reoxygenation (H/R) of the aged H9C2 cells
H/R model of the aged H9C2 cells was established as described previously [3, 4, 12]. A hypoxic condition was produced by D-Hank solution (in mM: 5.37 KCl, 0.44 KH2PO4, 136.89 NaCl, 4.166 NaHCO3, 0.338 Na2HPO4, 5 d-glucose, pH 7.3–7.4 at 37 °C) saturated with 95% N2 and 5% CO2. The pH was regulated to 6.8 with lactate to mimic ischemic solution. The aged cardiomyocytes were put into a hypoxic incubator that was equilibrated with 1% O2/5% CO2/94% N2. After hypoxia, the culture medium was rapidly replaced with fresh DMEM with 10% fetal bovine serum (normoxic culture solution) for initiating reoxygenation.
Experimental protocols of the aged H9C2 cells
The aged H9C2 cells were randomly divided into the following 6 groups. Each group included 8 samples (n = 8) (Fig. 1): (1) Control group (Control); The aged H9C2 cells were cultured for 9 h with 10% FBS-DMEM; (2) Hypoxia/reoxygenation group (H/R): The aged H9C2 cells were exposed to hypoxic culture medium for 3 h and reoxygenated for 6 h by replacing the hypoxic culture medium with fresh DMEM with 10% FBS; (3) H/R + NaHS group: The procedure was similar to that for group 2, except that 100 μM NaHS were added in 6 h reoxygenation; (4) PC group: At the end of 3 h of hypoxia, the aged H9C2 cells were exposed to normoxic culture solution for 5 min, after which cells were placed in hypoxic solution for 5 min. The PC cycle was repeated three times and followed by 6 h of reoxygenation; (5) PC + NaHS group: At the end of 3 h of hypoxia, initiated immediately at the onset of reoxygenation, 100 μM NaHS were given at the onset of reoxygenation for 5 min following with 5 min hypoxia. This protocol was repeated for another two times. The cells were then treated as those of group 3; (6) PC + 4-PBA (or GSK2656157, or STF083010) group: 0.5 mM 4-PBA (or 10 µM GSK2656157 or 50 µM STF083010) were added to the medium 40 min before the end of hypoxia. The cells were then treated as those of group 4.
AGEs ELISA assay
The rat advanced glycation end products (AGEs) assay was performed with AGEs ELISA kit according to the instructions from the manufacturer and was as previously described [4, 19, 20]. The reagents of the test kit were placed at room temperature for 30 min and diluted 1:20 with distilled water. Aliquots of 100 µl of the standards and samples were added to blank micropores and 50 µl enzyme marker solution was added. Microtiter plates were incubated at 37 °C for 60 min and then washed five times and put aside for 10–20 s each time. The A and B substrate solutions (50 µl) were added into the microtiter plates for 15 min dark reactions at 37 °C. The reaction was terminated by the addition of 50 µl stop solution, and the optical density (OD) at 450 nm was determined by an ultra microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). An AGEs standard curve was generated and the AGEs values of the samples were calculated from the standard curve.
SA β-gal staining
Senescence-associated β-gal (SA β-gal) activity was measured with the β-gal staining kit at pH 6.0 according to the instructions from the manufacturer [4, 19, 20]. Briefly, the cells were washed in phosphate buffered saline (PBS), fixed for 10–15 min at room temperature with 1 ml of fixative solution and incubated overnight at 37 °C with the staining solution mix. Cells were observed for development of the blue coloration with a microscope at a magnification of ×400. Aging cardiomyocytes were assessed by counting the number of cells that displayed blue coloration.
Caspase-3 activity assay
Caspase-3 activity was determined using the ApoAlert Caspase Colorimetric Assay kit in accordance with the manufacturer’s protocol [18]. In brief, at least 1 × 106 cells per sample were lysed in 50 µl of lysis buffer, and the protein concentrations in the samples were estimated using the Bio-Rad protein assay. After incubation on ice for 10 min, the samples were centrifuged at 16,000×g for 3 min at 4 °C. Each supernatant was mixed with 50µl 2 × Reaction Buffer/DTT mix and 5 µl of 1 mM Caspase-3 Substrate (DEVD-pNA, 50 µM final concentration), and the samples were then incubated for 1 h at 37 °C in the dark. Developed colour was measured at 405 nm, and caspase-3 activity was calculated in terms of absorbance units per µg protein.
Cell viability assay
Cell viability was measured by Cell Counting Kit-8 (CCK-8). Cells were seeded in 96-well plates at a concentration of 3 × 103 cells/well. After 24 h of each treatment, 10 μl was added to each well of CCK-8 immediately. Subsequently, they were incubated for 2 h at 37 °C. Using a microplate spectrophotometer, the plates were read at 570 nm (A 570) to determine their optical density.
Apoptotic rate of cells by Hoechst 33342 staining
Cells were analyzed for apoptosis after visualization of nuclei morphology with fluorescent DNA-binding dye Hoechst 33342, as described previously [4, 12]. After treatment, cells were rinsed with PBS and incubated with 5 µg/ml Hoechst 33342 for 10 min. Nuclei were visualized at 400× magnification using fluorescent microscopy at an excitation wavelength of 330–380 nm. Apoptotic nuclei of cells were assessed by counting the number of cells that displayed nuclear morphology changes, such as chromatin condensation and fragmentation.
Apoptotic rate of cells by flow cytometry assay
The apoptotic rate was measured by flow cytometry as described previously [4]. Cells were washed three times with ice-cold PBS, and then stained with annexin V-fluorescein isothiocyanate for 15 min at room temperature in 200 μl binding buffer. Next, 300 μl binding buffer was added, and the cells were stained with propidium iodide for 30 min at 4 °C. The fluorescence of the cells was analyzed by flow cytometry. The percentage of apoptotic cells was determined using Mod Fit LT software (Verity Software House Inc., Topsham, ME, USA).
Western blotting analysis
The related protein expressions were measured by Western blot as described previously [2,3,4,5,6]. Brifely, equal amounts of proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted on polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies. The secondary antibody was goat anti-rat immunoglobulin G. The intensities of the protein bands were quantified by a Bio-Rad ChemiDoc™ EQ densitometer and Bio-Rad Quantity One software (Bio-Rad Laboratories). The protein concentration was quantified using the BCA Protein Assay kit (Beyotime, Nantong, China).
Detection of Cyt c release from mitochondrial
Western blot analysis of Cyt c in the cytosolic fraction was performed as described previously [3, 4, 12]. Briefly, cells were harvested, washed twice with ice-cold PBS, and incubated in ice-cold Tris-sucrose buffer (0.35 mM sucrose, 10 mM Tris–HCl at pH 7.5, 1 mM EDTA, 0.5 mM dithiothreitol, 0.1 mM phenylmethylsulphonyl fluoride). After a 40 min incubation, cells were centrifuged at 1000×g for 5 min at 4 °C and the supernatant was further centrifuged at 40,000×g for 30 min at 4 °C. The supernatant was retained as the cytosolic fraction and analyzed by Western blot with a primary rat anti-Cyt c monoclonal antibody and a secondary goat anti-rat immunoglobulin G (Promage). GAPDH expression was used as the control.
Statistical analysis
All data were expressed as the mean ± SE and represented at least three independent experiments. Statistical comparisons were made using student’s t test or one-way ANOVA followed by a post hoc analysis (Tukey test) where applicable. Significance level was set at p < 0.05.