Simulation of post-MI cardiomyocyte microenvironment
Two types of samples were used to mimic the MI microenvironment: 1. the supernatants of necrotic HL-1 cells (Supernatant-NH), 2. the supernatants of infarcted myocardium (Supernatant-IM) at 8 h post-MI. The generation of necrotic HL-1 cells was performed according to the protocol described by Maekawa et al. [12]. Briefly, HL-1 (a cardiac muscle cell line; SXBIO, Shanghai, China) cells were washed three times with serum-free medium and then processed by five cycles of freezing in liquid nitrogen followed by thawing at 37 °C. Supernatants from infarcted mouse myocardium were obtained from MI-model mice after ligation of the left anterior descending (LAD) artery for 8 h. In brief, the hearts of the mice were removed under sterile conditions, and sections of infarcted myocardium were excised and washed three times in phosphate-buffered saline (PBS) to remove blood cells. Following this, the sections were cut into small pieces that were used to generate single-cell suspensions via the gentleMACS™ system (MACS® Cell Separation, Miltenyi Biotec B.V.& Co., Bergisch Gladbach, Germany). Finally, the supernatants were obtained by centrifugation. Then, 100-µL samples (cell membrane particles were removed by centrifugation at 1500 g for 30 min) were collected from the supernatants of the HL-1 cells, and infarcted myocardial cells were added to 106 bone marrow-derived dendritic cells (BMDCs) for 24 h. All experiments were performed under sterile conditions. All procedures performed in studies involving animals were in accordance with the ethical standards of Laboratory Animal Management and experimental ethics committee of Union Hospital, Fujian Medical University. All methods were performed in accordance with relevant guidelines and regulations.
Cell culture and treatments
The BMDCs obtained from approximately 6-week-old C57BL/6 mice were cultured in RPMI 1640 Media supplemented with 10 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) and 1 ng/mL IL-4 at 37 ∘C in 5% humidified CO2 for 4 h. The medium containing nonadherent cells was replaced with fresh medium every 2 d. On culture day 7, the cells were treated with Supernatant-NH and Supernatant-IM alone, or in combination with 10 𝜇M atorvastatin (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. PBS was used as a control.
Induction of MI models and injection of DCs
Ligation of the left main descending coronary artery (LCA) was performed as described previously [9]. In brief, mice were anesthetized with 2% isoflurane, and their hearts were manually exposed through small chest incisions. The LCA was permanently ligated with a 7–0 silk suture 2-3 mm from the origin of the left atrium. The ligations were validated as successful by characteristic ECG changes. For the DCs transfer experiment, 1×106 DCs treated with Supernatant-IM (mature DCs, mDCs), 1×106 DCs treated with atorvastatin and Supernatant-IM (tolerogenic DCs, tDCs), and PBS were infused intravenously 1 d before and 1 week after MI.
Flow cytometric measurement
BMDCs were washed and resuspended in ice-cold PBS containing 5% fetal bovine serum (FBS) to prevent nonspecific binding, and further incubated with anti-CD80, anti-CD86, and anti-CD40 (BD Pharmingen, San Diego, CA, USA) for 30 min at 4 ∘C. After extensive washing, the stained cells were analyzed using a FACScan™ flow cytometer (BD Biosciences, San Jose, CA, USA) and CellQuest™ software (BD Biosciences, San Jose, CA, USA).
Intracellular levels of reactive oxygen species (ROS) were measured with DCFH-DA molecular probes (Molecular Probes-Invitrogen, Carlsbad, CA, USA). Cells were incubated with 10 𝜇M DCFH-DA for 30 min at 37 ∘C, then washed and resuspended in PBS at 1 × 106 cells/mL. The DCs were analyzed using flow cytometry. The fluorescence was determined at 503/529 nm and expressed as a percentage of the control.
Enzyme-linked immunosorbent assay
The supernatant of the cultured BMDCs was harvested and stored at −70 ∘C. The cytokine concentrations of TNF-𝛼, IL-1, IL-6, IL-12P40, and IL-8 were analyzed using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions. The superoxide dismutase (SOD) activity and malondialdehyde (MDA) contents were measured at 450 and 532 nm by SOD and MDA ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA), respectively.
Western blotting
Protein samples were fractionated with 12% SDS-PAGE (Invitrogen, Carlsbad, CA, USA) and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes with blotted protein were blocked, then probed with the following antibodies: toll-like receptor 4 (TLR-4), nuclear factor-κB (NF-κB), phospho-NF-κB, IκB, phospho-IκB, interleukin-1 receptor-associated kinase 4 (IRAK4), and phospho-IRAK4 at 4 ∘C overnight (Cell Signal Technology, Inc., Danvers, MA, USA). The membranes were washed and incubated at 4 °C for 2 h with diluted (1:5000) secondary HRP-conjugated antibodies. Immunoreactive proteins were identified using Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Franklin, MA, USA). Densitometric analysis of the western blotting was performed using Image J software.
Echocardiography
In vivo cardiac function was determined by echocardiography using the Vevo® 2100 Imaging Platform (VisualSonics, Inc., Toronto, Canada) as described previously [9]. Briefly, the mice were anesthetized with 2% isoflurane and oxygen, and two-dimensional echocardiographic views of the left ventricular long axis through the anterior and posterior LV walls were obtained at the level of the papillary muscle tips below the mitral valve. The left ventricular ejection fraction (LVEF) and fractional shortening (FS) were calculated as previously reported [6]. The echocardiograms were evaluated in a blinded manner.
Measurement of myocardial infarct size and myocardial fibrosis
After freezing at −20 °C for 12 hours, the heart ventricles were sectioned transversely into ~2-mm thick sections. The sections were subsequently incubated in 1% triphenyltetrazolium chloride (TTC) for 15 min at 37 °C to identify the non-infarcted and infarcted areas. Once identified, the areas were fixed in 10% buffered formalin. The infarcted area was displayed as the TTC-unstained area (white). The extent of fibrosis was measured using Masson’s trichrome stain on day 28 after MI. Image-Pro Plus software (v6.0; Media Cybernetics, Rockville, MD, USA) was used to determine the infarct size and extent of fibrosis.
Immunohistochemistry analysis
Immunohistochemical studies were performed by immunoperoxidase staining methods using paraffin-embedded tissue sections (6 mm thick). After inhibiting endogenous peroxidase activity, the sections were incubated with primary anti-myeloperoxidase (MPO; Abbiotec™, Midlothian, UK), anti-Mac3 (BD Biosciences, San Jose, CA, USA), and anti-CD3 (eBioscience, Inc., San Diego, CA, USA) at 4 °C overnight, followed by respective secondary HRP-conjugated antibodies for 1 h at room temperature. The positive cells were visualized with DAB, and nuclei were counterstained with hematoxylin. The numbers of Mac3+ macrophages, MPO+ neutrophils, and CD3+ T lymphocytes were assessed by counting the total cell numbers in the infarcted and border areas in twenty randomly chosen fields in each section.
Cardiomyocyte apoptosis
Cardiomyocyte apoptosis was assessed in the heart sections by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) staining. TUNEL staining was performed using the In situ Cell Death Detection Kit (Roche Applied Science, Upper Bavaria, Germany) according to the manufacturer’s protocol. The apoptosis index was determined by counting TUNEL-positive nuclei in ten random fields per section and expressed as a percentage of the total nuclei.
Statistical analyses
The data are presented as the means ± SD, with 𝑃 < 0.05 considered to be statistically significant. A one-way ANOVA, followed by the Student-Newman-Keuls (SNK) test, was employed for the statistical analysis of our results. All statistical analyses were performed with SPSS 21 statistical software.