The experimental protocol was approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (No. IACUC-20180223-69). One- to three-day-old neonatal Sprague-Dawley (SD) rats were purchased from the Experimental Animal Center of Xinjiang Medical University and handled in accordance with the recommendations in the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health.
Briefly, rabbit anti-Bax polyclonal antibody (#2772) and rabbit anti-p65 (#8242), anti-p-p65 (#3033), anti-IκBα (#4812), anti-GFP (#2956), anti-Beclin-1 (#3495), and anti-LC3 Ⅱ/Ⅰ (#12741) monoclonal antibodies were all obtained from Cell Signalling Technology (Danvers, MA, USA). Rabbit anti-Bcl-2 (ab196495), anti-Histone H3 (ab1791) and anti-β-actin (ab8227) polyclonal antibodies and horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (ab205718) were obtained from Abcam (Cambridge, UK). RIPA buffer and Halt™ Protease and Phosphatase Inhibitor Cocktail were obtained from Thermo Fisher Scientiﬁc (Waltham, MA, USA). Enhanced chemiluminescence (ECL) reagent and JC-1 were obtained from Millipore (Bedford, MA, USA). Trypsin, PDTC and bromodeoxyuridine (BrdU) were obtained from Sigma (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS), and penicillin-streptomycin solution were obtained from Gibco (Grand Island, NY, USA). Collagenase II was obtained from Worthington (Minnesota, USA). H2O2 was obtained from Sangon (Shanghai, China).
Recombinant AAV-9 vectors generated by a recombinant baculovirus (rBac)-based system in SF9 cells as previously described were purchased from Virovek (Hayward, CA, USA) . The recombinant AAV9 vectors were packaged as double-stranded DNA and contained the enhanced green fluorescent protein (eGFP) gene (dsAAV9-GFP) or the IκBαS32A, S36A gene (dsAAV9-IκBα) driven by the human cytomegalovirus (CMV) promoter.
Isolation and culture of rat cardiomyocytes
The protocol for the isolation and purification of neonatal rat ventricular cardiomyocytes (NRVMs) was reported in our previous study . Briefly, the hearts of 1- to 3-day-old neonatal SD rats were dissected and digested with 0.1% trypsin and 0.08% collagenase II. Following differential adhesion twice for 50 min each time, nonadherent cells were resuspended and cultivated in high-glucose DMEM containing 10% FBS, 1% penicillin-streptomycin, and 0.1 mM BrdU for 48 h. The medium was replaced every 48 h.
AAV9 transfection of cardiomyocytes
After 48 h of culture, NRVMs were transfected with dsAAV9-GFP or dsAAV9-IκBα as previously described . Brieﬂy, cells were first transfected with dsAAV9 (multiplicity of infection, MOI = 5×106 vg/cell) in serum-free medium, and then DMEM at an equal volume containing 20% FBS, 2% penicillin-streptomycin and 0.2 mМ BrdU was added to every dish 3 h later. Images showing GFP were captured using a ﬂuorescence inverted microscope (Leica DMI4000B, Weztlar, Germany), and the green ﬂuorescence intensities were analysed using ImageJ software (National Institutes of Health, NY, USA).
Experimental design and cell grouping
The experiment was designed to explore whether AAV9-delivered IκBαS32A, S36A gene transfection could protect cardiomyocytes against H2O2-induced apoptosis and autophagy via inhibition of NF-κB activation. Cardiomyocytes were starved with serum-free DMEM for 12 h to ensure cell synchronization before H2O2 stimulation. The experimental cardiomyocytes were randomly divided into 5 groups as follows: (1) the control group, which contained primary cardiomyocytes cultivated under normal conditions; (2) the H2O2 control group (H2O2): the model control group, which contained primary cardiomyocytes subjected to 100 μM H2O2 alone ; (3) the GFP control group (GFP): the vector control group, which contained primary cardiomyocytes transfected with dsAAV9-GFP virus for 5 days before being subjected to 100 μM H2O2; (4) the IκBα treatment group (IκBα): the treatment group, which contained primary cardiomyocytes transfected with dsAAV9-IκBα virus for 5 days before being subjected to 100 μM H2O2; and (5) the PDTC treatment group (PDTC): the positive control group, which contained primary cardiomyocytes pretreated with 100 μM PDTC for 60 min before being subjected to 100 μM H2O2.
Measurement of cardiomyocyte viability and cytotoxicity
The Cell Counting Kit-8 (CCK-8; Dojindo, Japan) assay was used to assess cell viability. In brief, 2×104 cells were seeded into each well of a 96-well plate and transfected with GFP or IκBα for five days. After the cells were exposed to H2O2, 10 μL of CCK-8 stock solution was added to each well, followed by incubation at 37 °C for 2 h. The absorbance at 450 nm was measured with a GO microplate spectrophotometer (Thermo Fisher Scientific). The extent of cell death was determined by quantifying lactate dehydrogenase (LDH) released into the culture supernatant with an LDH kit (Jiancheng Bioengineering Institute, Nanjing, China). Intracellular malondialdehyde (MDA), an indicator of oxidative injury, was also measured using an MDA assay kit (Jiancheng Bioengineering Institute).
Flow cytometry analysis
Cell apoptosis was measured using PE Annexin V Kit I (BD Biosciences, NJ, USA). Briefly, cells were collected and resuspended in 1× binding buffer. Then, the solution (1×105 cells) was supplemented with 5 μL of PE Annexin V and 7-AAD and incubated in the dark for 15 min at room temperature. Apoptotic cells were identiﬁed by ﬂow cytometry (Beckman Coulter, CA, USA). All the experiments were performed in triplicate.
Western blotting analysis
Nuclear and cytoplasmic proteins were extracted following the instructions of a Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientiﬁc, USA). Total proteins were extracted with RIPA buffer containing Halt™ Protease and Phosphatase Inhibitor Cocktail. Phosphorylated p65 in the total lysate and the nuclear p65 to cytosolic p65 ratio were both detected to identify activation of the NF-κB signalling pathway . Equal amounts of protein were loaded and separated on precast SDS-PAGE gels (Invitrogen, Grand Island, NY, USA) and transferred to Millipore PVDF membranes. After blocking with 5% skim milk, the membranes were blotted overnight with specific primary antibodies against p65 (1:1000), p-p65 (1:500), IκBα (1:1000), Bax (1:1000), GFP (1:1000), Beclin-1 (1:1000), LC3 Ⅱ/Ⅰ (1:1000), Bcl-2 (1:1000), Histone H3 (1:1000), and β-actin (1:1000) at 4 °C, followed by incubation with anti-rabbit HRP secondary antibody (1:5000) at room temperature for 2 h. ECL solution was added to the membranes to visualize signals. β-actin and Histone H3 were regarded as loading controls. Images were captured and analysed by Image Lab 4.0 software (Bio-Rad Laboratories, Hercules, CA, USA).
Immunofluorescence was employed to identify H2O2-induced nuclear translocation of the NF-κB p65 subunit in cardiomyocytes. Briefly, 2×105 cells were seeded into confocal dishes. After H2O2 treatment, cardiomyocytes were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.25% Triton X-100 for 10 min. After blocking with 1% BSA for 1 h, cells were probed overnight with anti-p65 antibody (1:200) at 4 °C and incubated with Alexa Fluor 594-labelled secondary antibody (Invitrogen, 1:200, labelled with red fluorescence) for 2 h at room temperature, followed by 10 min of DAPI staining of nuclei (labelled with blue fluorescence). Signals were detected using a confocal spectral microscope (Leica SP8, Germany).
Measurement of the mitochondrial membrane potential
JC-1 is an ideal fluorescent probe used to detect the mitochondrial membrane potential (ΔΨm) in cardiomyocytes. Briefly, a 10 nmol/L JC-1 working solution was prepared prior to use, and cardiomyocytes were stained at 37 ℃ in the dark for 15 min. Cells doubly stained with JC-1 were visible by either green or red ﬂuorescence. Fluorescence images and intensities were obtained using a fluorescence microscope and ImageJ software. Generally, ΔΨm is represented by the red to green ﬂuorescence ratio, which decreases in proportion with the severity of cell injury.
All statistical analyses were performed with SPSS 22.0 (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± SEM. Multiple comparisons were carried out using one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. A value of P < 0.05 indicated statistical significance.