Animal model of DCM and experimental design
All animal experiments were approved by the Animal Care and Utilization Committee of Fudan University (201802021S). We obtained the male BALB/c mice aged 6 weeks from Fudan University Experimental Animal Center. The animal model of DCM was established in BALB/c mice by immunization with porcine cardiac myosin (Sigma). Cardiac myosin was emulsified with an equal volume of complete Freund’s adjuvant (Sigma) to the concentration of 2 mg/ml. The solution was subcutaneously injected into the groin of BALB/c mice at days 0 and day 7. The total dose of porcine cardiac myosin for DCM induction was 0.2 mg per mouse. The mice in control group were injected with complete Freund’s adjuvant alone. As previously reported , we confirmed myosin-induced DCM model by histomorphological study and echocardiographic assessments in the present study. Eight normal mice and twenty-four DCM mice were divided into the following four experimental groups as follows: control group (normal + PBS), DCM group (DCM +PBS), rapamycin group (DCM + rapamycin) and 3-MA group (DCM +3-MA). Eight weeks after immunization, rapamycin was then administered at a dose of 2 mg/kg/d for 2 weeks. The mice in 3-MA group received 3-MA at a dose of 15 mg/kg/d, while the mice in control group were injected with PBS alone. After intraperitoneal injection of sodium pentobarbital (75 mg/kg body weight), all mice were sacrificed by cervical dislocation while anesthetized.
M-mode transthoracic echocardiography was performed using a 30-MHz imaging transducer to evaluate the cardiac function. The mice were anesthetized with 2% isoflurane and their chests were epilated. M-mode images were obtained at the level of papillary muscles in the long-axis view. The left ventricular ejection fraction (LVEF), fractional shortening (FS), left ventricular end-diastolic dimension (LVEDD), and left ventricular end-diastolic volume (LVEDV) were measured, which were acquired by the technician who was blinded to the present experimental groups.
After sacrifice, myocardial tissues were fixed in 4% formaldehyde, embedded in paraffin and cut into 5μm thick slices. Specimens were stained with picrosirius red, and microscopic images were evaluated. The collagen volume fraction (CVF) was determined by quantitative morphometry of specimens with IMS Cell Image Analysis System (Shen Teng, Shanghai, China). Images were viewed under confocal scanning microscope (Leica, TCS-SP2, Germany). For quantitation of cardiac fibrosis areas, 5 random fields of view per mouse were evaluated for CVF analysis across the left ventricular section (Each group, n=8). Consequently, there were 40 quantitative data for statistical analysis in each group.
Transmission electron microscopy
Transmission electron microscopy (TEM) for morphological evaluation was performed at Electron Microscopy Core Laboratory, Shanghai medical college, Fudan University (Philips CM120, Nethelands), according to standard operating procedures. As previously reported for morphological TEM , cardiac tissues were fixed in 2.5% glutaraldehyde in phosphate buffer overnight at 4°C. After sample preparation, 90-100nm thick sections were mounted onto a 200 mesh copper grid and examined under a Philips CM120 electron microscope. Cardiomyocyte nucleus, mitochondria, myocardial fibers, and autophagosomes were evaluated under TEM respectively.
After being harvested, the left ventricular myocardium specimens were stored at - 80°C. Proteins were extracted from the myocardial tissues homogenized in RIPA Lysis (Beyotime) and Extraction Buffer with a protease inhibitor cocktail, and proteins were quantified using the bicinchoninic acid method according to the manufacturer’s instructions. Samples of 25μg protein were loaded into 8% SDS-PAGE gels for electrophoresis then transferred to PVDF membranes over night at 30V. Antibodies specific for LC3 II (dilution 1:1000; Cell Signaling), p-mTOR (dilution 1:1000; Cell Signaling), and p-4EBP1 (dilution 1:1000; Cell Signaling) were incubated at 4˚C overnight, and GAPDH (dilution 1:5000; Santa Cruz) was used as a loading control to normalize gel loading and protein expression. HRP-conjugated secondary antibodies plus ECL were incubated at 37˚C for 1 hour for protein visualization. The densitometric values of immunoreactive bands were measured using Image J (NIH, USA).
The data are presented as mean ± standard deviation. Values of P less than 0.05 were considered statistically significant. Normal distribution was confirmed in all experimental groups, and differences in means between two groups were analyzed by unpaired Student’s t test when the data were normally distributed. Multiple group comparison was performed by one-way ANOVA followed by Newman-Keuls multiple comparison test. GraphPad Prism version 6.0 software (GraphPad Software Inc., USA) was used for data analysis.