Hes1 antibody was purchased from Abcam (Cambridge, UK), and VDAC1 and β-actin antibodies were obtained from Santa Cruz (Dallas, Texas, USA). AD-Hes1-shRNA and AD-NTAP/Hes1 were provided by Hanbio Biotechnology (Shanghai, China), and the NTAP tag was a tandem combination of streptavidin binding peptide (SBP) tag and calmodulin binding peptide (CBP) tag, which loaded onto the pNTAP vector through the InterPlay Adenoviral TAP system. ROS assay kits and mitochondrial staining kits (5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine, JC-1) were purchased from Invitrogen. CPK and LDH assay kits were supplied by Jiancheng Bioengineering Institute (Nanjing, China).
Cell culture and model establishment
H9c2 cardiomyocytes, obtained from the Cell Bank of Shanghai Institutes for Biological Sciences, were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone) containing 4.5 g/L glucose and supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 100 U/ml penicillin and 100 mg/ml streptomycin (New Cell Molecular Biotech, Suzhou, China) in a humidified incubator (37 ℃, 95% air, 5% CO2).
The experimental design was as follows: (1) Control group: H9c2 cardiomyocytes routinely cultured as described previously. (2) A/R group: The A/R model was established as previously described 28. Briefly, H9c2 cells underwent anoxia using a simulated anoxia buffer (6.0 mM NaHCO3, 98.5 mM NaCl, 0.9 mM NaH2PO4, 10.0 mM KCl, 1.8 mM CaCl2, 1.2 mM MgSO4, 20 mM HEPES, and 40.0 mM Sodium lactate, pH = 6.8) in a tri‐gas incubator with 95% N2 and 5% CO2 for 3 h at 37 °C.Then, anoxia buffer was changed to reoxygenation buffer (20 mM NaHCO3, 129.5 mM NaCl, 0.9 mM NaH2PO4, 5.0 mM KCl, 1.8 mM CaCl2, 1.2 mM MgSO4, 20 mM HEPES, 5.5 mM Glucose, pH = 7.4) incubated with an 95% O2 and 5% CO2 for 2 h at 37 °C. (3) IPC group: The IPC model was established following the published protocols with a minor modification 12, 29, 30. Briefly, under sterile conditions, H9c2 cardiomyocytes were preincubated for 15 min in the simulated anoxia buffer in a hypoxic incubator (95% N2 - 5% CO2) as described above and then incubated in simulated reoxygenation buffer in a high oxygen incubator (95% O2-5% CO2) for another 15 min. This process was repeated 3 times to induce anoxic preconditioning and then followed by the A/R treatment as previously described in the A/R group. (4) IPost group: According to our previously published protocols with a minor modification 12, 31-33. The IPost model was established as follows: At the end of 3 h of hypoxia, subsequently, H9c2 cardiomyocytes were exposed to 3 cycles of 15 min of reoxygenation and then 15 min of hypoxia, and at last, followed by 2 h of reoxygenation. (5) A/R+AD-NTAP/Hes1 group: H9c2 cardiomyocytes were treated as the cells in the A/R group then subsequently transfected with AD-NTAP/Hes1. (6) IPC+AD-Hes1-shRNA group: H9c2 cardiomyocytes were transfected with AD-Hes1-shRNA for 48 h then subjected to the same treatment as cells in the IPC group. (7) IPost+AD-Hes1-shRNA group: H9c2 cardiomyocytes were transfected with AD-Hes1-shRNA for 48 h then treated the same way as cells in the IPost group.
CPK, LDH release, and Cell viability assay
CPK and LDH release and the viability of cells in each experimental group were measured using an LDH assay kit (Jiancheng Bioengineering Institute, Nanjing, China), CPK assay kit (Jiancheng Bioengineering Institute, Nanjing, China), and a commercial CellTiter 96® Aqueous One solution cell proliferation assay (Promega), respectively, in accordance, in each case, with the manufacturer's instructions.
Extraction and purification of Hes1 ligand-protein by TAP
Total proteins were extracted from AD-NTAP/Hes1 infected H9c2 cardiomyocytes after 48-72 h, in accordance with the InterPlay TAP purification kit ( Agilent Technologies,USA) instructions. The total number of infected H9c2 cardiomyocytes was approximately 3×108, while the quantity of reagents for this experimental step was calculated based on 107 cells.
The peptides were separated using an LC-20AD nanoliter liquid chromatograph in accordance with the manufacturer's instructions. The peptides were separated by liquid phase separation into a Q-EXACTIVE mass spectrometer (primary mass spectrometry resolution: 70,000, secondary resolution: 17,500). Fifteen parent ions with charges of 2+~5+ and peak intensities greater than 20000 were selected for secondary analysis. The peptides were fragmented in HCD mode using a collision energy of 27, after which the fragments were detected in Orbi software. The following parameters were used: Dynamic exclusion time: chromatographic half-peak width duration; ion source voltage: 1.6 kV. Automatic gain control was achieved using Orbi, by setting the mass-to-charge ratio range of the scan (primary: 350-2000; secondary: 100-1800).
Standard bioinformatics analysis
The original mass spectrometry files were converted into mass spectrometry peak files and then searched against sequences in protein databases, in this case the ipi_rat database (39925 sequences), using protein identification software Mascot 2.3.02. The search results were filtered and quality controlled in order to identify the proteins. The results of the identified proteins were analyzed by functional annotation, including gene ontology (GO) and Clusters of Orthologous Groups (COG) databases.
Proteins were collected from H9c2 cardiomyocytes, lysed in pre-cooled RIPA buffer then incubated with an anti-VDAC1 or anti-GST-tag antibody ( as negative control) overnight at 4 °C. The proteins were then incubated for 4-5 h at 4 °C with protein A/G agarose suspension (Merck Millipore, Germany) which was pre-washed with cold PBS. The mixture was then centrifuged for 5 min at 4,000 g at 4 ℃, from which the supernatant was collected for subsequent analysis and the pellet washed 3 times in pre-cooled PBS followed by recentrifugation for 5 min at 4,000 g at 4 ℃ to obtain the immunoprecipitation complex. The supernatant and immunoprecipitation complex were boiled in 1× SDS-PAGE sample buffer and then prepared for Western blot analysis.
Western Blotting Assay
Proteins were isolated from H9c2 cardiomyocytes in accordance using a method described previously 34. Protein concentration was measured using a Bradford assay (Beyotime). Protein samples (40 μg) were separated using 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride membranes. Following blocking with 5% skimmed milk for 2 h at 37 ℃, the membranes were then incubated with primary antibodies overnight against Hes1 (Abcam, Cambridge, MA, USA), VDAC1 (Abcam), and β-actin (Cell Signaling Technology). The molecular weight of exogenous NTAP/Hes1 and endogenous Hes1 is about 50 kDa and 35 kDa, respectively.The membranes were then washed with 1×TBST 3 times then incubated with a secondary antibody (Cell Signaling Technology) for 2 h at room temperature. Finally, protein bands were visualized using enhanced chemiluminescence (GE Healthcare, USA) after incubation with an enhanced chemiluminescence substrate and quantified using a Quantity One System image analyzer (Bio-Rad Laboratories, Hercules, CA, US).
ROS production assay
The production of ROS was measured using a ROS assay kit (Invitrogen, USA) in accordance with the manufacturer's instructions, as we have described in our previous study 34. Briefly, treated H9c2 cardiomyocytes were mixed with DMEM supplemented with 10 μM DCFH-DA (Invitrogen, USA) and then incubated for 20 min at 37 °C in the dark. Fluorescence was monitored at excitation and emission wavelengths (ex/em) of 485 and 528 nm by flow cytometry (Becton Dickinson, USA).
Mitochondrial Membrane Potential (ΔΨm) assay
A mitochondrial staining kit (JC-1) (Invitrogen, USA) was used to measure ΔΨm concentration, following the manufacturer's instructions, as we have described in a previous study 34. JC-1 was added to a final concentration of 200 μM in H9c2 cardiomyocytes and then incubated in the dark for 20 min at 37 °C from which fluorescence was measured using flow cytometry (Becton Dickinson, USA) at ex/em of 530/580 nm (red) and then at 480/530 nm (green), respectively. MMP was calculated from the ratio of red to green fluorescence.
Flow cytometric analysis of apoptosis
An Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, San Jose, CA, USA) was utilized to detect the apoptosis of H9c2 cardiomyocytes. Briefly, the H9c2 cardiomyocytes cells were trypsinized, resuspended in 100 μL 1x binding buffer, and then incubated with 5 μL annexin V-FITC and 5 μL PI in the dark for 15 min. Apoptosis was identified in cells using a FACScan flow cytometer using CellQuestTM software (BD Biosciences, San Jose, CA, USA) by virtue of their green but not red fluorescence.
All values were presented as mean ± SEM. One-way ANOVA was used to compare the biochemical indices of different groups. SPSS 18.0 was used for statistical analysis. A p-value less than 0.05 was considered statistically significant.