2.1 Reagents and antibodies
Doxorubicin and cobalt chloride were purchased from MedChemExpress (New Jersey, America) and Sigma, respectively. Primary antibodies for the following proteins were purchased from cell signaling technology (Danvers, MA): β-actin (1:5000), total-PARP (1:1000), total caspase-3 (1:1000), total ERK1/2 (1:1000), phosphorylated ERK1/2 (1:1000), total PI3K (1:1000), phosphorylated PI3K (1:1000) and total AKT (1:1000), phosphorylated AKT (1:1000). Besides, anti-rabbit IgG and HRP-linked antibody were purchased from Biosharp Life Science (Beijing, China). Mitochondrial membrane potential assay kits with JC-1 and a LDH cytotoxicity assay kit, relative oxygen species assay kit, Cell Counting Kit-8, Trypan blue staining cell viability assay kit and one-step TdT-mediated dUTP nick end-labeling apoptosis assay kit were obtained from Beyotime Biotechnology (Shanghai, China). ML221 was purchased from MedChemExpress (New Jersey, United States).
2.2 Peptide synthesis and administration
Apelin-13 CMPLHSRVPFP peptide and scramble MPCLSHPVFPR peptide were synthesized with purity>95% by Shanghai Science Peptide Biological Technology Co. LTD. (Shanghai, China). The peptide powder was dissolved in sterile water to generate a 10 µM stock solution and was diluted to the experimental concentration.
2.3 Cell culture
Rat primary cardiomyocytes were extracted from rat one-half day after birth. The blood, fat and connective tissues were separated from heart tissues and digested with trypsin at 37°C at 60 rpm for 15 min. The solution was removed and repeated the digestion three times. The cell-containing digestive fluids were placed in a centrifuge tube and centrifuged together after passing through 180 mesh. Cells were suspended in 10 ml DMEM containing 10% horse serum (HS, GIBCO, USA) and incubated in a 10 cm2 dish for 1.5h. The cell suspension was removed, and the cells (5~6×105 cells per dish) were inoculated into a new plate as previously described.
Male C57BL/6J mice (6-10 weeks of age, 20~22 g) were obtained from the Model Animal Research Center of Nanjing University (Nanjing, Jiangsu, China) and all procedures were followed in accordance with the ethical committee of Nanjing Medical University. All animals were raised at 20~25°C and in 50~70% relative humidity. These mice were randomly divided into four groups (scramble peptide treated group, apelin-13 treated group, DOX and scramble peptide co-treatment group and DOX and apelin-13 co-treatment group) and 6 mice per group. The mice were treated with 5 mg/kg/week DOX i.p for five consecutive weeks and 1.5µmol/kg apelin-13 were administrated daily i.p. Electrocardiograms were obtained 6 weeks later and the mice were sacrificed. The mice were treated according to the experimental requirements. All animal experiments complied with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publications No. 85-23, revised 1996).
2.5 Cell death rates and CCK-8 assay
Trypan blue staining was used to calculate the mortality of rat primary cardiomyocytes. The cell mortality rate was obtained by counting the stained ones. The cells were collected at different lengths of times (0, 6, 12, 18, 24 and 36h) and stained by a trypan blue staining cell viability assay kit to determine the cell death rate. At different concentrations of DOX (0.1, 0.5, 1.0, 2.0 and 5.0µM) and CoCl2 (200, 400, 600, 800 and 1000µM) were measured according to the manufacturer’s instructions. CCK-8 assay can be used to evaluate cell proliferation and viability. The absorbance of different groups was measured at 450nm and the cell survival rates were calculated according to the manufacturer’s instructions.
2.6 Lactate dehydrogenase (LDH) level detection
Levels of lactate dehydrogenase (LDH) released were detected in serum using an LDH release assay kit according to the manufacturer’s protocol.
2.7 JC-1 assay
The mitochondrial membrane potential was measured by a mitochondrial membrane potential assay kit with JC-1 according to the manufacturer’s instructions. The cells were cultured in serum-free DMEM containing (1×) JC-1 staining working fluid at 37°C for 20 min. Then, washed twice with JC-1 buffer and the cells were photographed by a fluorescence microscope (BX61; Olympus Corporation, Tokyo, Japan) after added 2ml DMEM. The JC-1 density was assessed by ImageJ software and calculated upon normalization to the control.
2.8 TUNEL staining assay
The rate of cell apoptosis can be detected by TUNEL assay. Cells were seeded (1×105 cells per well) in 6-well dishes. Then the cells were washed once with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde. Apoptotic cells were visualized with stained cells according to the manufacturer’s protocol. TUNEL fluorescence intensity/DAPI fluorescence density was used to calculating the percentage of positive cells and the density was evaluated using ImageJ software 1.26 (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).
Male C57BL/6J mice were anesthetized with isoflurane (3% in 2L/min), then they were placed on a heated animal operating table to maintain at 36.5~37.5℃，which was monitored with a YSI telethermometer. Unhaired and detected ejection fraction (EF), fractional shortening (FS) and left ventricular end-systolic diameter (LVEDs) by echocardiography (vevo3100, FUJIFILM VisualSonics) respectively. All animal experiments complied with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publications No. 85-23, revised 1996).
2.10 Enzyme linked immunosorbent (ELISA) assay
Serum CKMB, cTnI, cTnT and apelin-13 level were determined by commercially available ELISA kit (Mlbio, Shanghai, China) according to the manufacturer’s instructions.
2.11 Western blot analysis
Proteins were isolated from cells using lysis buﬀer (containing Radio-Immunoprecipitation Assay (RIPA) and 1% Phenylmethylsulfonyl fluoride (PMSF)). Protein quantification was performed using a BCA protein detection kit (23229; Thermo Fisher Scientific). Protein samples of the same mass were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA), blocked with 5% skim milk for 1h and then incubated with specific primary antibodies. A FluorChem M system was used to quantify the positive bands representing proteins involved in the orchestrated immune responses (ProteinSimple, San Jose, CA, USA).
2.12 Statistical analysis
All results are expressed as the mean±SD. Student's t-tests were used within two groups to determine significant differences. Comparisons between multiple groups were performed by one-way analysis of variance (ANOVA), and p<0.05 (*), p<0.01 (**) and p<0.001(***) were considered significant. All experiments were repeated at least three times unless otherwise specified. All data were analyzed by GraphPad Prism software.