Experimental Animals and Ethics Statement
The animal protocols in this experiment were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University and conformed to the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Male C57BL/6 mice aged 8–10 weeks (weight: 24–29 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd, (Beijing, China). Mice were housed with four to five mice in a cage fed with common food and water and maintained under 12/12 h light/dark cycles with the ambient humidity at 50–80% and the temperature at 21 ± 2 °C.
Animal MI Model And Study Protocols
The MI model was established by ligating the left anterior descending (LAD) coronary artery as described with minor modifications. Briefly, surgery was performed under anaesthesia with 3% sodium pentobarbital at a dose of 80 mg/kg. A left thoracotomy was performed at the third or fourth intercostal space, open the pericardium, and the LAD was ligated with a 7 − 0 silk suture at 2 mm below the end of left atrial appendage, decreased wall motion and paleness of Anterior wall of left ventricle below the ligation were recognized as a sign of successful ligation. For the sham operation, the left coronary artery was encircled but not ligated, after the surgery, the chest wall was closed, a heating pad was applied to maintain the core body temperature of the mouse at 36 ± 1.0 °C. Lastly, the animals were put back in separate ventilated cages after they were fully recovered.
Male C57BL/6 mice aged 8–10 weeks were randomly divided into four groups:(1) Sham group (only received an injection of the same amount of saline); (2) MI group (only received an injection of the same amount of saline); (3) MI + T3 group (received an injection of T3(Sigma-Aldrich, USA) at the dose of 2 µg/100 g/d); (4) MI + T3 + BMS-754807 group(received an injection of T3 at the dose of 2 µg/100 g/d and BMS-754807 (Sellek Chemicals, USA) at the dose of 2 mg/100 g/d). Drugs or vehicle were administered intraperitoneally daily for 3 days before surgery, each mouse received the same total dose every day by adjusting the dose of saline. The number of animal deaths during the 7 days follow-up were carefully recorded.
Echocardiographic Measurements
Cardiac echocardiography was performed for the experimental mice at 7th day post operation. Mice in each group (n = 6) were anesthetised with 1.5–2% isoflurane by inhalation, and echocardiography was then performed using a MyLab30CV ultrasound (Biosound Esaote Inc. Indianapolis, IN, USA) with a 12.5-MHz linear array ultrasound transducer. By adjusting the probe, a clear two-dimensional image of left ventricular short axis was obtained at the level of left ventricular papillary muscle. The left ventricular ejection fraction(LVEF), fractional shortening (FS), the left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter(LVESd) ,left ventricular posterior wall thickness in diastole (LVPWd) and left ventricular posterior wall thickness in systole (LVPWs) were measured from M-mode recording .The parameters were calculated as mean of the measures obtained in at least three continuous cardiac cycles. All of the operators were blinded to the animal groups.
Serum Inflammatory Cytokines Measurements
Blood was collected from the left ventricle after function measurements, and then kept at room temperature for 1 h, centrifuged at 2000 g for 10 minutes at 4 °C, the buffy coat layer was collected and immediately stored at -80 °C. Serum levels of inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-a) were assayed by enzyme-linked immunosorbent assay(ELISA) kits(ELK Biotechnology, China)based on the manufacturer's instructions.
Serum TH Measurements
Serum levels of total T3 (TT3), free T3 (fT3), total T4 (TT4), free thyroxine (fT4) and thyrotropin (TSH) were assayed by ELISA kits based on the manufacturer's instructions.
Tissue Harvest And Histological Analysis
After function measurements were recorded, mice were euthanized with 3% sodium pentobarbital and the hearts were removed immediately, the excised hearts were placed in 10% KCl solution to induce cardiac arrest during diastole, and then washed and excised the vessels and adipose tissue in ice-cold PBS, the weights of the heart and LV were recorded. Lungs were rapidly excised and weighed.
Masson’s trichrome staining. To obtain cardiac pathological samples, the atriums above the ligation of the heart were removed, the remaining tissues were immersion-fixed with 4% paraformaldehyde and then processed by paraffin embedding. Subsequently, the hearts were cut serially from the base to the apex along the short axis. Each slice was cut at 5-µm thickness. Sections were stained with Masson’s trichrome stain (Solarbio Science and Technology, China) according to the manufacturer’s protocol and then imaged. Morphometric analyses of the infarcted tissue were performed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc. Rockville, MD, USA). Infarct size measurements were derived from the midpapillary level of the infarcted heart. Cardiac fibrosis measurements were obtained at the base, midpapillary, and apical regions of the heart. All measurements were made blindly.
Immunofluorescence Staining
TUNEL staining for myocardial apoptosis. For analyses of the activity of cardiomyocyte apoptosis, the paraffin sections were prepared, dried, dewaxed, hydrated and repaired, and then incubated with the following anti-bodies for confocal microscopy: Mouse Anti-Cardiac Troponin T antibody (Abcam, Britain; 1:250 dilution), Goat Anti-Mouse IgG H&L (HRP) (Abcam, Britain; 1:250 dilution). Subsequently, the sections were washed in PBS, the TdT and dUTP in the TUNEL kit (Roche, Switzerland) were added to cover the tissue according to the instructions in the reagent kits, followed by DAPI (Invitrogen, USA) staining to label the nuclei. Lastly, sections were sealed with anti-fluorescence quenching sealant and placed under Olympus fluorescence microscope (Olympus Corporation, Tokyo, Japan) for image collection. whereas quantitative assessment and analysis were performed by counting the number of positive cells in the infarct border zone using Image-Pro Plus 6.0, the ratio of the number of apoptosis-positive nuclei to total nuclei was used to represent the apoptosis rate.
Immunofluorescence of CD31. Sections obtained after preparation as described above were incubated with primary antibody of CD31(Abcam, Britain; 1:250 dilution) and the Cy3-conjugated secondary antibody (Abcam, Britain; 1:250 dilution) and DAPI (Invitrogen, USA) in sequence. Similarly, images were obtained by Olympus fluorescence microscope, the integrated density and area of CD31 of the infarct border zone were computed using Image-Pro Plus 6.0 software. The average optical densities (AODs) of CD31 were used to evaluate the density of angiogenesis and calculated as the integrated density/area multiplied by 100%.
Protein Extraction And Western Blot Analysis
On the 7th day following surgery, the hearts were harvested and washed in PBS, the infarcted border zone of left ventricular were dissected and stored at − 80 °C. Cardiac tissues were minced, lysed with RIPA buffer(with protease and phosphatase inhibitors; Thermo Scientific, China), cracked on ice for 30 min and grinded with a homogenizer. The resulting homogenates were centrifugated at 13000 g for 5 minutes at 4 °C to collect protein supernatant. Protein concentrations were examined with bicinchoninic acid (BCA) protein assay kit (Rockford, USA). Protein extracts (50 µg) were isolated by 12%SDS-PAGE (Invitrogen, USA) and then transferred to PVDF membrane (Millipore, USA). The membranes were sealed with 5% skimmed milk powder for 1 h at room temperature, and then incubated overnight at 4 °C with the primary antibodies(Cell Signaling Technology, Beverly, MA)specific to Bax (1:2000 dilution),Bcl-2 (1:500 dilution),Caspase-3 (1:1000 dilution),p-PI3K (1:500 dilution),p-AKT (1:1000 dilution). Subsequently, the membrane was washed with TBST for three times and incubated with a peroxidase-conjugated secondary antibody for 1 h. Bands were visualized following activation with ECL (Thermo Scientific, USA), and the grey values of the bands represented the protein expression levels. The specific protein expression levels were normalised to β-actin on the same nitrocellulose membrane. Each group of experiments was repeated 3 times independently, and the average values were taken as the results.
Statistical Analysis
The experimental data were expressed by Mean + SEM and analyzed by SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). Student unpaired t test was used to compare two independent samples, and one-way analysis of variance (ANOVA) was used for comparison among multiple groups. If the overall difference was significant, post hoc Tukey’s test was used to analyze the significant difference between the two groups. Values of P<0.05 were considered statistically significant.