Neonatal and adult male Sprague-Dawley (SD) rats (80 g and 250 g, respectively) were obtained from the Medical Institute Animal Center of Zhejiang University (Hangzhou, Zhejiang, China). The experiments were approved by the Animal Care and Use Committee of the Zhejiang Province Medical Institute and were in compliance with the “Guide for the Care and Use of Laboratory Animals” (
12), published by the National Institutes of Health (USA).
Cell culture
MSCs were obtained from the femora and tibiae of male SD rats, using a modified method as previously described (
7,
8). The bones were dissected free, and the proximal and distal ends were removed to reveal the marrow cavity, which was aspirated with 10 mL of Dulbecco’s modified Eagle’s medium (DMEM) via a 21 G needle. The aspirant was layered over Percoll solution (1.073 g/mL; GE Healthcare Bio-Sciences AB, Sweden) and centrifuged at 900
g for 25 min at room temperature. The mononuclear cells were recovered at the interface, resuspended in growth medium (DMEM, 10% fetal bovine serum, 100 U/mL penicillin G and 100 U/mL streptomycin; Gibco, USA), and then plated on 50 cm
2 flasks and left for 24 h. The flasks were then washed with phosphate-buffered saline to leave an adherent layer of cells containing MSCs. The cultures were maintained at 37°C in a 5% CO
2 incubator; the medium was changed every three to four days. With three to five passages, MSCs negative for CD45 and positive for CD44 and CD90 were used for the experiments. For in vitro experiments, MSCs were seeded in the 3 μm Transwell insert (Corning, USA). For transplantation, MSCs were labelled with 4,6-diamidino-2-phenylindole (DAPI; Sigma, USA) as previously described (
13,
14) to track engrafted cells. Briefly, MSCs were incubated in growth medium containing DAPI (50 μg/mL) for 3 h. Then, the cells were rinsed six times in phosphate-buffered saline to remove unbounded DAPI, detached with 0.25% (weight/volume) trypsin and suspended in DMEM for cell transplantation.
Primary cardiomyocyte cultures were prepared by the method originally described by Simpson and Savion (
15), with minor modifications. Briefly, the hearts from neonatal male rats were minced and dissociated with 0.125% trypsin (Gibco) and 0.1% collagenase type II (Worthington, USA). After incubation of dispersed cells on a 25 cm
2 flask for 60 min in a normoxic incubator (95% air and 5% CO
2), unattached viable cells were collected and seeded into a 25 cm
2 flask or Transwell lower plates (Corning) and incubated. The cells were then incubated with DMEM supplemented with 20% fetal calf serum plus 0.1 mmol/L 5-bromo-2-deoxyuridine (Sigma) for 72 h to prevent low-level nonmyocardial cell proliferation, and then replaced with DMEM plus 20% calf serum.
AP
For AP of MSCs (AP-MSCs), MSCs were incubated in a modular incubator chamber (Billups-Rothenberg, USA) for 3 h in serum-free DMEM; normal air was replaced by 95% nitrogen and 5% CO2. AP-MSCs for transplantation were incubated in serum-free DMEM containing DAPI (50 μg/mL) in the modular incubator chamber.
Apoptosis of AP-MSCs was evaluated by a chromatin dye (5 μg/mL; Hoechst 33342; Sigma). Cells were fixed for 1 h in 4% paraformaldehyde at room temperature, and then exposed to the dye for 30 min in the dark. All samples were observed by using a fluorescence microscope. Apoptotic cells were characterized by morphological alteration such as condensed nuclei and cell shrinkage.
Hypoxia/reoxygenation
Cardiomyocytes in the lower plates of the Transwells were randomly divided into three groups: the DMEM (control) group, the MSCs group and the AP-MSCs group. For coculture experiments, MSCs or AP-MSCs along with cardiomyocytes were cultured in two individual chambers separated by a semipermeable membrane (3 μm hole). This system allowed for the sharing of the culture medium between two chambers but prevented cell contact. Each was treated with hypoxia/reoxygenation (H/R). To mimic hypoxia, the cardiomyocytes were incubated at 37°C in the anoxic chamber for 24 h; normal air was replaced by 95% nitrogen/5% CO2. Then, cardiomyocytes were moved into a normoxic incubator (95% air/5% CO2) for 3 h to mimic the reoxygenation process. The control group was incubated in DMEM supplemented with 20% fetal calf serum under standard cell culture conditions (95% air/5% CO 2).
Apoptosis of cardiomyocytes was determined using an Annexin V fluorescein isothiocyanate apoptosis detection kit (BioVision, USA) according to the manufacturer’s instructions. Cells were visualized directly on glass slides under a fluorescence microscope and analyzed by flow cytometry.
MI model and transplantation
Adult male SD rats (250 g) were included in the studies and anesthetized with 4% chloral hydrate (4 mg/kg, administered intraperitoneally), then mechanically ventilated using a small-animal ventilator (Zhejiang University apparatus). An MI was created by permanently ligating the left anterior descending coronary artery with a 7-0 silk suture. Successful coronary occlusion was verified by blanching of the distal myocardium. The sham-operated group was subjected to thoracotomy without coronary ligation. The chest was closed, and rats were weaned from the ventilator and allowed to recover for one week. Then, the MI rats were randomly divided into three groups (16 rats per group): control animals that received a DMEM injection, cell-treated animals that received MSCs (5×106) and cell-treated animals that received AP-MSCs (5×106). The cells suspended in 150 μL DMEM were prepared individually and injected into peri-infarcted regions of anterior left ventricles at three to five locations. The sham group received the same injected volume of DMEM as transplant subjects. The rats were allowed to live for another one or four weeks.
Infarct size measurement
The rats (eight in each group) were euthanized and the hearts were removed one week after transplantation. The excised heart was cut into three transverse sections and embedded in paraffin. Sections (5 μm thick) were cut and mounted on slides; heart sections at the papillary muscle level were selected to determine infarct size. After deparaffinization and dehydration, the sections were stained with hematoxylin and eosin (
13,
16). Images were digitized using a computerized image analysis system (NIH Image; National Institutes of Health, USA). Infarct size was calculated by dividing the sum of the planimetered endocardial and epicardial circumferences of the infarcted area by the sum of the total epicardial and endocardial circumferences of the left ventricle, and expressing the result as a percentage (
13,
16).
Terminal deoxynucleotidyltransferase-mediated 2′-deoxyuridine 5′-triphosphate nick end labelling analysis
Apoptotic cardiomyocytes in the border zone of the ischemic region were evaluated by terminal deoxynucleotidyltransferase-mediated 2′-deoxyuridine 5′-triphosphate nick end labelling (TUNEL) assay with an in situ cell death detection kit (Roche, Germany) according to the manufacturer’s instructions. The percentage of TUNEL-positive cells was assessed in five randomly selected fields in each heart section. A total of 16 sections were analyzed, each from the four groups (ie, sham plus three MI groups).
Western blotting analysis
Protein (40 μg to 150 μg) prepared from the cardiomyocytes or infarcted heart was loaded per lane and electrophoresed in sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene difluoride Immobilon-P membrane (Bio-Rad, USA) using a transblot apparatus (Bio-Rad). The membranes were blocked in 10 mmol/L Tris-HCL (pH 8.0), 150 mmol/L sodium chloride and 0.05% Tris-buffered saline Tween 20 (TBST) with 5% (weight/volume) nonfat milk at room temperature, followed by overnight incubation at 4°C with primary antibodies diluted in TBST (1:1000 for Bcl-2, Bax, cleaved caspase-3 and beta-actin; Cell Signal, USA). After washing with TBST, the membranes were incubated for 1 h with a horseradish peroxidase-conjugated secondary antibody diluted (1:5000) in TBST, and the labelled proteins were detected by using the enhanced chemiluminescence reagents and exposed to the film (Kodak, USA).
Measurement of cardiac function
Cardiac function was assessed by transthoracic echocardiography (Vivid 7; GE Healthcare, USA) in MI rats four weeks after transplantation. Briefly, a two-dimensional short-axis view of the left ventricle was obtained at the level of the papillary muscles, and M-mode tracings were recorded and analyzed to evaluate cardiac function.
Identification of implanted cells
Animals were sacrificed after echocardiography. The hearts were quickly harvested and tissues from the free wall of the left ventricle including the infarct and peri-infarct regions were then embedded in optimal cutting temperature tissue-freezing medium. Frozen sections (6 μm thick) of left ventricular samples were made for identification of implanted cells. Subsets at the papillary muscle level were stained with hematoxylin and eosin for measurement of infarct size, as described above.
Statistical analyses
Data are expressed as mean ± SEM. Statistical significance between groups was assessed by one-way ANOVA followed by the Student-Newman-Keuls test, using SPSS 11.5 (SPSS Inc, USA). P<0.05 was considered to be statistically significant.
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