Natural product derivative BIO promotes recovery after myocardial infarction via unique modulation of the cardiac microenvironment

The cardiac microenvironment includes cardiomyocytes, fibroblasts and macrophages, which regulate remodeling after myocardial infarction (MI). Targeting this microenvironment is a novel therapeutic approach for MI. We found that the natural compound derivative, BIO ((2′Z,3′E)-6-Bromoindirubin-3′-oxime) modulated the cardiac microenvironment to exert a therapeutic effect on MI. Using a series of co-culture studies, BIO induced proliferation in cardiomyocytes and inhibited proliferation in cardiac fibroblasts. BIO produced multiple anti-fibrotic effects in cardiac fibroblasts. In macrophages, BIO inhibited the expression of pro-inflammatory factors. Significantly, BIO modulated the molecular crosstalk between cardiac fibroblasts and differentiating macrophages to induce polarization to the anti-inflammatory M2 phenotype. In the optically transparent zebrafish-based heart failure model, BIO induced cardiomyocyte proliferation and completely recovered survival rate. BIO is a known glycogen synthase kinase-3β inhibitor, but these effects could not be recapitulated using the classical inhibitor, lithium chloride; indicating novel therapeutic effects of BIO. We identified the mechanism of BIO as differential modulation of p27 protein expression and potent induction of anti-inflammatory interleukin-10. In a rat MI model, BIO reduced fibrosis and improved cardiac performance. Histological analysis revealed modulation of the cardiac microenvironment by BIO, with increased presence of anti-inflammatory M2 macrophages. Our results demonstrate that BIO produces unique effects in the cardiac microenvironment to promote recovery post-MI.


MTT assay for cell proliferation
Cell proliferation was assessed using MTT assay. Briefly, 0.1 volume of MTT (1 mg/mL) was added to cell culture medium and incubated for another 4 hrs. The medium was removed and 100 L of DMSO was added into each well, and the plate was gently rotated to completely dissolve the precipitation. The absorbance was measured at 540 nm.

Cardiomyocyte: cardiac fibroblast co-culture system
Primary cardiomyocytes and cardiac fibroblasts were purified and cultured as described above.
Cells were counted and mixed before seeding at a ratio of 1:1 (by cell number). Cells were seeded on sterile glass cover slips (100 mm diameter) in 12-well plates. The seeding density was 30,000 cells/well in 1 mL (for 2 days treatment) and 20,000 cells/well in 1 mL (for 5 day treatment).

Immunofluorescence staining
After treatment of RAW264.7 cells or cardiac fibroblasts, cells were fixed with 4% paraformaldehyde or ice-cold methanol for 10 minutes and washed with PBS three times. After permeabilization by 0.1% Triton X-100 for 10 minutes and blocking with 5% BSA for 1 hour, primary antibodies were incubated overnight at 4 ºC followed by sequential incubation with secondary antibody conjugated with Alexa-fluor-488 or Alexa-fluor-594 (Molecular Probe, USA, 1:200). After washing with PBS, the slides were mounted with medium containing DAPI (Invitrogen, USA) and observed under fluorescence microscopy.

Monolayer scratch assay
The scratch assay was carried out as previously described. 1 Briefly, cardiac fibroblasts were seeded onto six-well plates and allowed to adhere overnight (achieving >90% confluence). The monolayer was wounded with a 200 µL plastic pipette tip. Cells were washed once, and fresh culture medium was added with or without the compound of interest. The cells were allowed to invade the wound and counted by light microscopy 12 hr later. The rate of migration into the scratch was quantified by measuring the width of the wound at five different sites every 24 hr using light microscopy (CKX41, Olympus) and iSolution Lite 9.1 image capture software.
Densitometric analysis was performed using the Scion Image program (Scion Corporation, USA). The PCR primers used in this study are shown in Table 1.

Quantitative PCR
The transcript level of cytokines was analyzed by quantitative real-time RT-PCR using StepOnePlus Real Time PCR System (Applied Biosystems, UK). Cells were washed twice in PBS and were frozen at −70 ºC until the RNA was isolated. Total RNA was isolated using Trizol according to the manufacturer's protocol and was reverse transcribed to prepare cDNA using AccuPower® RT PreMix Results were expressed as the relative expression level for each gene. The synthesized cDNAs were amplified by quantitative real time PCR (qRT-PCR) or standard PCR.

Western blotting
Whole cell lysates were harvested using lysis buffer (20mM Tris-HCl pH 7.4, 0.1mM EDTA, 150mM NaCl, 1mM phenylmethylsulfonyl fluoride (PMSF) and 1mg/ml leupeptin) on a rotation wheel for 1 h at 4℃. After centrifugation at 10,000g for 10min, the supernatant was prepared as a protein extract.
Equal concentrations of proteins were fractionated by electrophoresis on 8% -12% acrylamide gels and were transferred onto a polyvinylidene fluoride membrane (Merck Millipore, Darmstadt, Germany) membrane, followed by blotting with antibodies followed by secondary staining with horseradish peroxidase-conjugated immunoglobulin G. Protein expression was detected using an Image Reader (LAS-3000 Imaging System, Fuji Photo Film, Tokyo, Japan). The expression level was quantified by ensitometric analysis using Scion Image program.

Isolation and culture of primary bone marrow-derived monocytes
Two young adult (6 weeks) B6 female mice were sacrificed by cervical dislocation and the femur and tibia bones were dissected. The ends of the bones were cut and the marrow was washed out with RPMI 1640 media using a 1 mL insulin syringe (26 gauge needle). Marrow cells were transferred to a 50 mL sterile tube and sieved through a 70 µm mesh to remove debris. 10 mL red blood cell lysis buffer (BioLegend, USA) was, followed by incubation on ice for 5 min. Bone marrow cells were harvested by centrifugation at 1200 rpm for 10 min. The supernatant was removed and cells were washed two, times with 10 mL PBS. Cells were counted and seeded in 6-well culture plates at a density of 10 6 cells/well. Cells were cultured with RPMI 1640 media supplemented with 10% FBS, 1% P/S, 2 mM L-glutamine, 1X non-essential amino acids, 1M HEPES and 50 µM -mercaptoethanol. 2 h later, non-adherent cells were transferred to a 6-well ultra-low attachment surfaced plate (Corning incorporated, USA) and stimulated with 20 ng/mL murine macrophage colony stimulating factor (M-CSF) for 4 days, with one media change on the 3rd day of treatment. The non-adherent cells were harvested and monocytes purified using CD117-coated magnetic beads (Miltenyi Biotec, UK), which remove non-monocytes from the cell population.

Cardiac fibroblast and monocyte co-culture
Rat cardiac fibroblasts were derived as described above. The human THP-1 monocyte cell line was seeded in the lower chamber of a 6-well plate transwell system (0.4 m, Corning) at a density of 2x10 5 cells/well. Cardiac fibroblasts were seeded in the upper chamber at a density of 10 5 cells/well. Cells were cultured in 3 mL media per well consisting of THP-1 media and cardiac fibroblast media at a 2:1 ratio.

Arginase assay
The assay was carried out in accordance with the manufacturer's instructions (BioVision Inc., USA). Adherent macrophages were harvested from the co-culture system by cell scraping. 0.25 ug/mL protein lysate was used for the assay.

Zebrafish model of heart failure
The zebrafish model was based on a previous study. 2  For staining with bromodeoxyuridine, zebrafish were treated with 10 mM BrdU with 1% DMSO in 1X E3 buffer for 1 h at 28.5°C, using a fish incubator. The fish were euthanized and fixed in 4% paraformaldehyde for 2 h. Fish larvae were washed 3 times with PBDT buffer (0.1% Tween-20, 1% DMSO in PBS) and dehydrated in 100% methanol for 1 h at -20°C. The larvae were then rehydrated in a graded methanol series: 75%, 50%, 25% for 20 min each and wash in PBDT for 20 min. Larvae were then digested with proteinase K (10 µg/mL) for 20 min. After three washes with PBDT, larvae were re-fixed in 4% parformaldehyde for 20 min. Larvae were then washed with PBDT and incubated with 12. 2N HCl for 1 h at RT, followed by 3 washes with PBDT and incubation with 10% normal goat serum in PBDT for were washed three times with PBDT and incubated in goat anti-mouse IgG conjugated to Alexa-fluor 594 for 5 h in the dark. After washing with PBDT, stained larvae were mounted and imaged using confocal microscopy (Olympus FluoView TM FV1000).

Rat model of acute MI
The study was reviewed and approved by the Chonnam National University Institutional Animal

Histological staining of fibrosis
Cardiac fibrosis after acute MI was measured by Masson's Trichrome staining, and fibrotic areas were measured by visualizing blue-stained fibrosis deposits by using NIS-Elements Advanced Research program (Nikon, Japan). The percentage of ventricular fibrosis was calculated as the blue-stained area divided by left ventricular area.

Immunohistochemistry of cardiac tissue
For immunohistochemical analysis, slides were treated with 3% hydrogen peroxide in PBS for 10 minutes at room temperature to block endogenous peroxidase activity. After nonspecific binding was blocked with 5% normal goat serum (Sigma-Aldrich, USA), the slides were incubated with primary antibodies against CD68 (Biomedicals, Switzerland, 1:100) or Arg-1 (Abcam, USA, 1:100) for overnight.
Sections were washed with PBS three times, and then incubated for 1 hour with Alexa-fluor 488 or 594 secondary antibodies. After washing, the slides were coverslipped with mounting medium (VectaMount mounting medium, Vector Labs Inc., USA). Immunofluorescence was detected by using a Carl-Zeiss confocal microscope. Images were obtained by using Zeiss LSM version 3.2 SP2 software.

Interleukin-6 enzyme-linked immunosorbent assay
Serum levels of interleukin-6 (IL-6) in rats subjected to MI was measured by ELISA (eBioscience, USA). Whole blood was collected from rats by cardiac puncture immediately after sacrifice (between 3 -5 mL was collected) using e-tubes (Axygen, USA) and kept on ice. Blood was centrifuged at 3500 rpm for 15 min at 4 ºC. The serum was transferred to fresh e-tubes and stored at -80 ºC. 100 L undiluted serum was used for ELISA.

Statistical analysis
All experiments were performed a minimum of three times. The data are presented as means ± standard deviation. Differences were analyzed by Student's t-test or one-way ANOVA, followed by Tukey post-hoc test. P-values less than 0.05 were considered significant. Analysis was performed using GraphPad Prism (version 5.03, GraphPad Software Inc.).
Supplementary Figure 1. Neonatal rat cardiomyocytes were treated with BIO for 2 days, and proliferating cardiomyocytes were detected with immunofluorescence staining with antibody against phosphorylated histone H3.