Cell culture and treatments
H9C2 cardiomyocytes were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), which were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37℃ and 5% CO2. To detect the cytotoxicity of Metoprolol in H9C2 cardiomyocytes, cells were stimulated with 0.5, 1, 5, 10, 50, 100, and 500 μM for 24 hours. To determine the protective effects of Metoprolol, cells were stimulated with AVP (10 μM) in the presence or absence of Metoprolol (5, 10 μM) for 24 hours.
LDH release assay
Briefly, the treated H9C2 cardiomyocytes were planted on a 96-well plate, followed by collecting the supernatant for LDH release assay. The supernatant was incubated with LDH reagents (Jiangcheng, Nanjing, China) for 1.5 h, followed by detecting the absorbance at 450 nm using a Bio-Rad 680 Microplate Reader.
A CCK-8 assay kit (Jiangcheng, Nanjing, China) was used to detect the cell viability of H9C2 cardiomyocytes treated with different strategies according to the instructions of the manufacturer. In brief, the cells were seeded on 96-well plates followed by adding 10 μL CCK-8 solution in each well. After incubation at 37°C for 3 hours, the absorbance at 450 nm was detected utilizing a PerkinElmer microplate reader (PerkinElmer, Massachusetts, USA).
The concentration of 8OHdG released by the treated H9C2 cardiomyocytes was detected using ELISA assay. Briefly, the supernatants were collected following centrifugation and added to the 96-well plates. After removing the non-specific binding proteins by incubating with 1% BSA for 1 hour, the sample was supplemented with the primary antibodies to be incubated at room temperature for 1 h. Subsequently, the plates were washed with PBS buffer and added with streptavidin-horseradish peroxidase (HRP) conjugated secondary antibodies, followed by being incubated for 20 min at room temperature. Lastly, the microplate spectrophotometer (Thermo, Massachusetts, USA) was used to measure the absorbance at 450 nm.
SA-β-gal staining assay
The treated H9C2 cardiomyocytes were fixed with 2% formaldehyde and 0.2% glutaraldehyde for 10 min at room temperature, followed by being washed using PBS buffer and added with SA-β-gal staining solution (CST, Massachusetts, USA), which was consisted of 40 mM citric acid/sodium phosphate (pH 6.0), 150 mM NaCl, 2 mM mgCl2, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 1 mg/mL of 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside. After incubation at 37℃ for 16 h, the percentage of SA-β-gal-positive cardiomyocytes was counted under a microscope (Olympus, Tokyo, Japan).
After lysing the treated H9C2 cardiomyocytes using RIPA lysis buffer (Beyotime, Shanghai, China), the supernatant was collected after centrifugation. The telomerase activity was detected utilizing the telomerase ELISA kit (Mlbio, Shanghai, China) according to the description reported previously 21.
The iNampt activity in the cardiomyocytes was detected using the NAMPT Activity Assay kit (Abcam, Cambridge, UK) according to the instruction of the manufacturer. In brief, the lysis of the treated H9C2 cardiomyocytes was added with two-step reaction mix 1, followed by being incubated for 60 min at 30 ℃. After adding the two-step reaction mix 2, the samples were incubated for 30 min at 30 ℃. Lastly, the absorbance at 450 nm was detected using the PerkinElmer microplate reader (PerkinElmer, Massachusetts, USA).
Measurement of NAD+/NADPH ratio
The NAD+/NADPH quantification kit (Bio Vision, San Francisco, USA) was used to detect the cellular concentration of NAD+ according to the instruction of the manufacturer. In the commercial kit, the concentration of NAD+ and NADPH was detected based on an enzyme cycling reaction, which is a sensitive method for the measurement of NAD+ and NADPH levels.
The cardiomyocytes were homogenized with the lysis buffer containing Tris-HCl, NaCl, phenylmethylsulfonyl fluoride, leupeptin, and aprotinin, followed by collecting the total proteins after centrifugation for 10 min at 15000 rpm and 4℃. The histone deacetylase colorimetric assay kit (Enzo Life Science, New York, USA) was utilized to detect the Sirt1 activity according to the instruction of the manufacturer.
Immunoprecipitation of acetylated p53
Cardiomyocytes were lysed and protein concentration was evaluated using the bicinchoninic acid (BCA) method. 1 mg total protein was mixed with 5 µg anti-acetylated-lysine antibody (CST, Massachusetts, USA) and incubated at 4 ℃ overnight to form antigen-antibody complex. 50 µL magnetic beads was then added into the complex solution and incubated at 4℃ for 4 h. After centrifuged by 2500 rmp at 4℃, the complex was collected and stored at -80℃ for subsequent western blot analysis using p53 antibody.
Real time PCR assay
The total RNA was isolated from the treated cardiomyocytes using the Trizol reagents (Thermo, Massachusetts, USA), followed by quantifying the total RNA and transcribing the RNA into cDNA utilizing the RT Master Mix kit (Takara, Tokyo, Japan). In the present study, the PCR reaction was conducted utilizing the SYBR Master Mix kit and the StepOne-Plus system (Takara, Tokyo, Japan) according to the following procedure: denaturing at 95 °C for 30 s, annealing at 60 °C for 1 min and extending at 95 °C for 5 s. Finally, the 2−ΔΔCt method was used to determine the relative expression level of target genes with GAPDH used for normalization.
Western blot assay
After extracting the total proteins from the treated cardiomyocytes with the RIPA Lysis Buffer (Beyotime, Shanghai, China), the proteins were quantified with a BCA kit (Beyotime, Shanghai, China), followed by being loaded and separated by the SDS-PAGE. Then, the separated proteins were transferred to the PVDF membranes (Beyotime, Shanghai, China), followed by incubation with 5% BSA for the removal of non-specific binding proteins. Subsequently, the membrane was incubated with the primary antibody against p53 (1:1000, CST, Massachusetts, USA), p21 (1:1000, CST, Massachusetts, USA) and β-actin (1:1000, Massachusetts, Boston, USA) at 4℃ overnight, followed by being incubated with secondary antibody at room temperature for 1.5 hours. Finally, the membrane was incubated with ECL solution (Beyotime, Shanghai, China), followed by being exposed to Tanon 5200-multi (Tanon, Shanghai, China). Image J software was used to quantify the relative expression level of target proteins.
Data are expressed as means ± standard deviation (S.D.). Statistical analyses were performed using analysis of variance (ANOVA). p<0.05 was considered statistically significant.