The Sprague-Dawley (SD) rats were obtained from the Animal Center of Academy of Military Medical Sciences, Beijing, China. A total of 120 healthy adult SD rats (age, 6–8 weeks; 200±20 g; both males and females) and 15 healthy neonatal rats (aged 24–48 h; 6.3±1.4 g; both males and females) were maintained under controlled conditions (20–25°C; relative humidity 40–60%; 12 h light/dark cycle with food and water ad libitum). Animals were handled as per the guidelines of the Institutional Animal Care and Use Committee of Capital Medical University. All experiments were performed following the National Institutes of Health guide for the care and use of laboratory animals and the guidelines of the International Association for the Study of Pain. The rats were anesthetized with isoflurane (adult SD rats with 1.87%; neonatal rats with 1.6%) and euthanized by cervical dislocation.
Isolation and culture of cardiomyocytes
Primary cultures of neonatal rat cardiac ventricular myocytes were performed as described previously[20, 21]. Briefly, the hearts from 1-2 day(s) old SD rats were removed after hypothermia anesthesia by immersion in ice water and placed in ice-cold 1× phosphate-buffered saline solution. After repeated rinsing, the atria were cut off, and the ventricles were minced with scissors. The minced tissue and ventricular cells were dispersed by digestion with collagenase type IV (0.45 mg/ml), 0.1% trypsin, and 15 μg/ml DNase I. Cardiomyocytes were cultured in the cardiac myocyte culture medium containing Dulbecco's modified Eagle's medium supplemented with 10% serum, 4 μg/ml transferrin, 0.7 ng/ml sodium selenite, 2 g/L bovine serum albumin (fraction V), 3 mmol/L pyruvic acid, 15 mmol/L HEPES, 100 μmol/L ascorbic acid, 100 μg/ml ampicillin, 5 μg/ml linoleic acid, 1% penicillin and 1% streptomycin, and 100 μmol/L 5-bromo-2′deoxyuridine. The cells were seeded into six-well plates. The myocytes were passaged and used for in vitro studies.
Adenoviral vectors and cardiomyocyte gene transfection
The adenovirus vector carrying rat miR-486 gene was generated from Adeno-XTM Expression Systems (Clontech) according to the manufacturer's protocols. The replication-defective recombinant adenovirus was amplified in HEK293 cells and purified by cesium chloride gradient ultracentrifugation. The final plaque-forming units (pfu) were estimated by titration in HEK293 cells using agarose overlay. The cardiomyocytes were infected with the recombinant adenovirus at MOI (multiplicity of infection) of 150. The infection efficiency was determined based on the percentage of GFP+ cells in transduced cardiomyocytes.
For miR-486 inhibitor transfection, cardiac myocytes were cultured and transfected with siRNA by nucleofection. The transduced cardiac myocytes were transferred to the completed medium, cultured for 24 h, and then subjected to proliferation and apoptosis assays.
RNA Extraction and TaqMan miRNA assay
Total RNA was extracted from tissues/cells using the Trizol Reagent and cDNA synthesis was performed with the [email protected] MicroRNA Reverse Transcription Kit (Thermo, USA). miR-486 and U6 (internal control) were amplified according to the TaqMan MicroRNA assay protocol (Applied Biosystems, USA). The assays were carried out in triplicates and the relative quantitative evaluation of the target gene levels was performed by comparing △Ct.
The rat AMI model and treatment
A schematic of our experimental approach is shown in Fig. 1. Experimental AMI was induced as described previously. Briefly, to establish the rat AMI model, adult SD rats were anesthetized and surgery was performed to ligate the left anterior descending coronary artery. The rats (number 120, weight 200±20 g) were divided into four groups, namely the sham group, AMI group, adenovirus group (Ad-ctr), and Ad-miR-486 group. In the last two groups, the AMI rats were treated with control virus and Ad-miR-486 respectively. The adenovirus was injected into the border zone surrounding the infarcts using a 27G needle.
Measurement of left ventricles ejection fraction (LVEF) and left ventricular fractional shortening (LVFS)
Four weeks after establishing AMI in rats, echocardiographic examinations were performed after intraperitoneal injection with Chloral hydrate (10%, 3ml/100g) and using the Vevo770 ultrasound system (Visualsonics Inc., Toronto, Canada). The electrode of the Vevo770 ultrasound system was attached to the animal using adhesive plaster and the ECG was monitored. The hairs on the rat chest were removed with depilatory creams. Left ventricular end-diastolic diameters (LVEDD) and left ventricular end-systolic diameters (LVESD) were measured using the ultrasound coupler (Fig. 2). Based on the measurements, LVEF and LVFS were calculated. At least three successive records were used to calculate the average value.
Infarct size, collagen deposition, and vessel density
The rats were sacrificed at 4 weeks after AMI. After hemodynamic studies, the animals were sacrificed to remove hearts. These were perfused with PBS for rinsing and frozen quickly. Myocardial infarct size was determined by pathological staining, a gold standard in AMI studies. The heart ventricles were sliced transversely into 2 mm thick slices and incubated in 1% triphenyltetrazolium chloride (TTC) at 37°C for 15 min to identify the noninfarcted and infarcted areas. TTC staining displayed a red color. TTC unstained area (white color) defined the infarcted area and the size was expressed as a percentage of the ischemic area.
Microvessel density was evaluated based on CD-31 expression and calculated. The microvessel endothelial cell membranes were highlighted by staining using the anti-CD-31 antibodies. Any cluster of endothelial cells that was markedly separated from the adjacent microvessel was considered a vessel. Then, five random nonoverlapping 400x high-power fields around the MI area were selected and on average, 5 such records were counted on each slide.
Measurement of collagen volume fraction (CVF) by Masson's trichrome staining
Rats were sacrificed at 4 weeks after AMI. Hearts were excised and soaked immediately in saline to remove the excess blood from the ventricles. These were fixed in 4% triformol. Paraffin-embedded samples were sectioned at 5 μm to perform the Masson's trichrome staining. Digital images of the stained sections were captured and CVF was analyzed using the Image Pro-Plus 6.0 software. CVF is expressed as a percentage of the collagen area in the whole field and calculated as the average of 5 random AMI fields in each slide.
Measurement of myocardium apoptotic index (AI)
Rats were sacrificed at 4 weeks after AMI. The tissue sections from the heart were stained with TUNEL. Five consecutive nonoverlapping 400x high-power fields were selected and the average counts of total and apoptotic cells were calculated. The AI was determined using the formula: the number of apoptotic cells/total cells × 100%.
Cell proliferation and apoptosis assays
The proliferation of cardiac myocytes was determined with CCK-8 assay. Cell apoptosis was determined by AnnexinV-APC staining. Briefly, the cardiac myocytes were cultured in a completed culture medium at 37°C for 48 h. The cells were treated with trypsin, washed with cold PBS, and resuspended in Annexin–V binding buffer. Then, the cells were stained with Annexin V-APC to detect apoptotic cells by flow cytometry.
All data are presented as mean ± S.E. p-value < 0.05 was considered statistically significant.