Generation of Nkx2.5-YFP and Nkx2.5 enhancer–Cre/Itgb1flox/flox mice
Isolation of embryonic and adult cardiomyocytes
Cardiomyocytes were prepared from E12.5–13.5 mouse embryos by the conventional preplating method (Ieda et al., 2007
). Briefly, hearts were minced and digested with collagenase type II (Worthington) solution. To enrich for cardiomyocytes, the cells were preplated for 2 h to remove nonmyocytes. For mixed population culture, cells were directly plated on culture dish without preplating. By preplating, the percentage of nonmyocytes was reduced from 40% to 10% as reported (Engel et al., 1999
). In either case, cells were cultured in DMEM/M199 medium containing 10% FBS at a density of 104
Adult cardiomyocytes were isolated from 8- to 12-week-old mice by Langendorff perfusion and Thompson’s procedure (O’Connell et al., 2007
). For isolation of adult cardiac fibroblasts, hearts were digested with collagenase/dispase (Roche) solution and plated for 2 h. Attached fibroblasts were cultured for 7 days, treated with mitomycin C, and stored in liquid nitrogen.
Isolation of Nkx-YFP+ cells
To isolate Nkx-YFP+ cells, we began with 20–30, E12.5–13.5 Nkx2.5-YFP embryos. YFP+ ventricles were cut into small pieces and digested with collagenase type II solution. A single-cell suspension was obtained by gentle triturating and passing through a 40-μm cell strainer. Nkx-YFP+ live cells (as defined by the lack of propidium iodine staining) were isolated by FACS Diva flow cytometer and cell sorter (BD Biosciences). Embryonic cardiac fibroblasts (YFP− cells) were plated onto plastic dishes for 2 h, cultured for 7 days, and treated with mitomycin C. FACS-sorted Thy1+CD31− cells were treated with mitomycin C. Fibroblasts were stored in liquid nitrogen for the co-culture experiment.
Culture of Nkx-YFP+ cells
Nkx-YFP+ cells were cultured in DMEM/M199 medium containing 10% FBS at a density of 2×104/cm2 on noncoated, PLL-, fibronectin-, laminin-(Sigma Aldrich), collagen III-(BD Biosciences), periostin, or hapln1-(R&D) coated dishes according to the manufacturer’s protocols. In some experiments, cells were maintained in 0.1% FBS media either with or without viral infection for 24h, and stimulated with HBEGF (50 ng/ml), Ptn (1 μg/ml), or FGF2 (50 ng/ml, R&D). For co-culture experiments, mitomycin-treated cardiac fibroblasts or Thy1+CD31− cells were plated at 104/cm2, and Nkx-YFP+ cells (104/cm2) were added 24 h later and cultured for 3 days. An anti-β1 integrin-blocking antibody (10 μg/ml, BD Biosciences), PD98059 (5 μM), SB203580 (5 μM), or LY294002 (10 μM, Calbiochem) was added after a 1-day incubation in some experiments. Unless otherwise stated, cells were cultured on noncoated plates.
FACS analyses and sorting
For Thy1+ cell expression analyses and sorting, single cells were isolated from ICR mouse ventricles, incubated with APC-conjugated anti-Thy1 antibody (eBioscience), analyzed and sorted by FACS Diva with FlowJo software. For sorting of Thy1+CD31− cells from cardiac fibroblasts, APC-conjugated anti-Thy1 antibody and FITC-conjugated anti-CD31 antibody (eBioscience) were used. For vimentin and DDR2 expression analyses, Thy1+CD31− cells were fixed with BD Cytofix/Cytoperm Kit (BD Bioscience), stained with anti-vimentin and DDR2 antibodies, followed by secondary antibodies conjugated with Alexa 488 and 647, and analyzed on a FACS Calibur (BD Biosciences) with FlowJo software.
H&E and Masson-trichrome staining were performed on paraffin-embedded sections, according to standard practices. The hearts were sectioned longitudinally in 5-μm thickness near the central conduction system to show the 4 chambers. Trabecular and compact layers were determined by their morphology. For each slide stained with H&E, we defined trabecular myocardium as bundles of cardiomyocytes surrounded by endocardium that project across the lumen of the ventricular chamber, and compact myocardium as concentrically organized layers of tightly adherent cardiomyocytes not surrounded by endocardium that constitute the outer ventricular chamber wall. Five parts per section were randomly selected for measuring wall thickness. The proportion of the compact layer to the heart size was the ratio between the length of the compact myocardium and the longest diameter of the ventricle, each measured by Image J software. Data for each mouse were calculated from 10 serial sections, and we observed 4–5 mice in each group. For immunohistochemical studies, hearts or whole embryos were fixed in 4% paraformaldehyde overnight, and then embedded in OCT compound and frozen in liquid nitrogen. Hearts were cut longitudinally in 7-μm sections in the middle to show the four chambers. Sections were stained with primary antibodies against actinin, vimentin, BrdU, β1 integirn (Chemicon), fibronectin (Thermo Scientific), p-ERK, with secondary antibodies conjugated with Alexa 488 or 546, and DAPI. For BrdU labeling, pregnant mice were injected intraperitoneally with BrdU (100 μg/g body weight) 1 h before sacrifice. The numbers of BrdU+ and p-ERK+ cells relative to the total number of total nuclei were counted in randomly selected three fields per section. The datafor each mouse were calculated for 5–10 sections. The numbers of vimentin+ or DDR2+ cells per area were calculated as the ratio between the number of vimentin+ or DDR2+ cells and the myocardial area, measured by Image J software. The data for each mouse were calculated from 10 sections.
Cells were fixed in 4% paraformaldehyde for 15 min at room temperature, blocked, and incubated with primary antibody against α-actinin (Sigma Aldrich), vimentin (Progen), BrdU (Accurate), GFP (Invitrogen), Ki67 (Novocastra), DDR2, Raldh2 (Santa Cruz), Thy-1, CD31 (BD Biosciences), SM-MHC (Biomedical Technologies), p-ERK, p-p38MAPK or p-Akt (Cell Signaling), with secondary antibodies conjugated with Alexa 488 or 546 (Molecular Probes), and DAPI (Invitrogen). TUNEL staining was performed according to the manufacturer’s protocol (Roche). To determine cell number, actinin+ cardiomyocytes were counted in six randomly selected fields in triplicate. YFP+ cell area was measured by image J software. For BrdU labeling, cells were incubated with 10 μM BrdU (Roche) for the last 2 day. To determine p-ERK+ and p-p38MAPK+ cells, the percentage of cells with nuclear p-ERK and p-p38MAPK were counted. In each experiment, the numbers of immunopositve cells were counted in six randomly selected fields, and 500–1000 embryonic cardiomyocytes or fibroblasts were counted in total.
Mitomycin-treated cardiac fibroblasts were transfected with siRNA against mouse Fn1, Col3a1, Fn1/Col3a1 or scramble siRNA (Dharmacon) with Lipofectamine 2000 (Invitrogen), according to the standard protocol. After a 1-day incubation, cells were used for co-culture experiment. qRT-PCR was performed after a 2-day incubation. We confirmed that the transfection efficiency was more than 90% by FACS Calibur with BLOCK-iT Fluorescent Oligo (Invitrogen).
Adenoviruses were generated as described (Ross et al., 1998
cells were infected with adenovirus expressing LacZ, β1A, or TAC-β1 for 24 h prior to the experiments. Viral titer was determined on all stocks using the Adeno-X titer kit (Clontech), and cells were infected at matched multiplicities of infection of 10 to maximize expressed protein, but prevent viral toxicity and detachment of cells. LacZ infection resulted in >90% transfection efficiency.
Mouse genome-wide gene expression analyses were performed using Affymetrix Mouse Gene 1.0 ST Array. YFP+
cells were collected just after sorting, and embryonic, and adult cardiac fibroblasts were collected after plating for 2 h without further incubation. RNA was extracted using Trizol (Invitrogen). Microarray analyses were performed in triplicate from independent biologic samples, according to the standard Affymetrix Genechip protocol. Data were analyzed using the Affymetrix Power Tool (APT, version 1.8.5). Linear models were fitted for each gene on the sample group to derive estimated group effects and their associated significance using the limma package (Smyth, 2004
) in R/Bioconductor. Moderated t-statistics and the associated p-values were calculated. P-values were adjusted for multiple testing by controlling for false-discovery rate using the Benjamini-Hochberg method. Gene annotations were retrieved from Affymetrix (version Nov 12, 2007). Differential gene expression was defined using the statistics/threshold combination. Genes differentially expressed >2 fold and p<0.05 were shown in , Supplementary figure 3
and Supplementary figure 6F
, and genes differentially expressed >1.2 fold or <0.8 fold, and p<0.05 were shown in Supplementary figure 6G
Differences between groups were examined for statistical significance using Student’s t-test or ANOVA. P values of <0.05 were regarded as significant.
Other Experimental Procedures