Clinical research model
30 patients with MI and 30 normal volunteers were collected from Zhongshan People’s Hospital.Serum was collected and saved at -80 ˚C. This experiment was performed in accordance with the Guide for the Care and Use of US National Institutes of Health. Experimental protocols were approved by the Zhongshan People’s Hospital. Each cancer patient provided their written informed consent for study participation.
Mice were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. C57BL/6 male mice were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). All aspects of the animal care and experimental protocols were approved by the Zhongshan People’s Hospital Committee on Animal Care.
All mice anaesthetized using 50 mg/kg of pentobarbital sodium and the left anterior descending arteries (LAD) were ligated to induce myocardial infarction. Mice were ventilated by a rodent ventilator (Shanghai Alcott Biotech Co., Shanghai, China), then LAD was ligated by an 8.0 suture followed by the thoracotomy. At 2 weeks of modeling, mice were sacrificed and executed other experiment. Left ventricular internal diameter, left ventricular ejection fraction, left ventricular fractional shortening and left ventricular stroke volume were obtained from Nillar pressure-volume system (MPVS-400).
Vitro experimental design
H9c2 rat cardiomyocytes (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were grown in Dulbecco’s modified Eagle medium (DMEM, Gibco, Grand Island, NY, United States), with 5% fetal bovine serum (FBS, Gibco, Grand Island, NY, United States), in a humidified 5% CO2 incubator at 37°C. HSMECs were performed transfections using Lipofectamine 2000 (Thermo Fisher Scientific). MEF2D (0.4 µg/ml), HDAC5 (0.4 µg/ml), MEF2D (20 nmol/ml) or HDAC5 siRNAs (20 nmol/ml) were transfected in the serum-free and antibioticfree media. After 48 h of transfection, H9c2 cells were stimulated with 5% oxygen (O2) and 5% carbon dioxide (CO2) and 90% N2 for 24 h.
Quantitative polymerase chain reaction (qPCR)
Total RNAs were isolated with RNA isolator total RNA extraction reagent (Takara) and cDNA was synthesized using PrimeScipt RT Master Mix (Takara). qPCR were performed with the ABI Prism 7500 sequence detection system according to the Prime-ScriptTM RT detection kit. Relative levels of the sample mRNA expression were calculated and expressed as 2-DDCt.
Aneurysm tissue or cells samples were lysed with ice-cold RIPA buffer with complete protease and phosphatase inhibitors. The protein concentrations were measured using BCA protein assay kit. Total proteins were separated by SDS–PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with primary antibodies: MEF2D (1:1000, abcam), HDAC5 (1:1000, Cell Signaling Technology, Inc.) and β-Actin (1:5000, Santa Cruz Biotechnology) after blocking with 5% BSA in TBS, followed by incubation with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). The signals were detected with the ECL system and exposed by the ChemiDoc XRS system with Image Labsoftware (Bio-rad).
Enzyme-linked immunosorbent assay (ELISA)
Total protein was diluted using PBS, and measurement was performed according to the instructions of the manufacturer. CK, LDH, SOD, GSH, GSH-PX and MDA levels were measured from cell samples or tissue samples using commercial ELISA kits.
Heart tissue were harvested from each group and immersion fixated in 4% formaldehyde, embedded in paraffin. Heart sections (4 µm) were cut from Aneurysm tissue and stained with HE. Sections were viewed with a fluorescent microscope.
Microarray experiments were performed at the Genminix Informatics (China). Gene expression profiles were analyzed with the Human Exon 1.0 ST GeneChip (Affymetrix).
H9c2 cells were stimulated with 5% oxygen (O2) and 5% carbon dioxide (CO2) and 90% N2 for 24 h. Next, H9c2 cells were fixed with 4% paraformaldehyde for 15 min and incubated with using 0.15% Triton X100 for 15 min at room temperature. H9c2 cells was incubated with MEF2D (1:500, abcam) and HDAC5 (1:500, Cell Signaling Technology, Inc.) at 4˚C overnight after blocking with 5% BSA for 1 h. hASMCs was incubated with goat anti-rabbit IgG-cFL 555 antibody (1:100) for 2 h at room temperature and stained with DAPI for 15 min and washed with PBS for 15 min. The images of hASMCs obtained using a Zeiss Axioplan 2 fluorescent microscope (carl Zeiss AG, Oberkochen, Germany).
The data were entered into GraphPad Prism 5.01 Software and represented as mean values ± SD. Comparisons of data between groups were followed using Student’s t test or one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. P < 0.05 was considered to denote a statistically significant difference