c-kitH2B-tdTomato/+, c-kitnlacZ-H2B-GFP/+ and c-kitMerCreMer/+ knock-in mouse models were generated by inserting LoxP-4XPloyA-LoxP-H2B-tdTomato-FRT-Neo-FRT, LoxP-nlacZ-4XPloyA-LoxP-H2B-GFP-FRT-Neo-FRT and MerCreMer-FRT-Neo-FRT cassettes, respectively, into the start codon of the c-kit locus (with disruption of endogenous ATG) through homologous recombination in 129/SvJ ES cells. In the targeting constructs, the insertion cassettes are flanked by 3.7 kb 5′ and 3.8 kb 3′ homologous arms (Supplementary Figs 1,7,9). The targeting vectors were linearized and electroporated individually in mouse ES cells. ES cells were screened by long-range PCR (Roche, Cat. 04829069001) with two pairs of primers (P1+P2 and P3+P4, Supplementary Figs 1,7,9). The sequences of the PCR fragments from the positive ES cells were further verified by DNA sequencing. The male chimeric mice carrying the targeted cassette in their germ line were crossed with Black Swiss females to generate F1 heterozygous mice. The Neo cassette flanked by two FRT sites was removed by crossing F1 mice with Flippase deleter mice36. c-kitLoxP-4XPloyA-LoxP-H2B-tdTomato/+ (c-kitSTOP-H2B-tdTomato/+) mice were crossed with Protamine-Cre37 to remove the 4XPloyA stop cassette and to obtain c-kitH2B-tdTomato/+. The P1–4 sequences are: P1, 5′-GGGTCTTCCTATATCTCCCTAGCT-3′; P2 (c-kitSTOP-H2B-tdTomato/+), 5′-CCAAATAAGCTTGGATCCGGAACC-3′; P2 (c-kitnlacZ-H2B-GFP/+), 5′-ATTCGCGTCTGGCCTTCCTGTAGC-3′; P2 (c-kitMerCreMer/+), 5′-CTCTTCTTCTTGGGCATGGTCTGC-3′; P3, 5′-TACCTGCCCATTCGACCACCAAGC-3′; and P4, 5′-ACCTCACACAGAACCTCCAGCAAT-3′.
Nkx2.5H2B-GFP/+, cTnTMerCreMer/+ and ROSA26RtdTomato/+ (R26RtdTomato/+) mouse lines were previously described28,35,38. For cTnTnlacZ-H2B-GFP/+ mouse line, a LoxP-nlacZ-4XPloyA-LoxP-H2B-GFP cassette was targeted to the cTnT start codon (manuscript was submitted). The cTnTH2B-GFP/+ mouse was obtained by crossing cTnTLoxP-nlacZ-4XPloyA-LoxP-H2B-GFP/+ (cTnTnlacZ-H2B-GFP/+) mice with Protamine-Cre mice37. Nkx2.5H2B-GFP/+ and cTnTH2B-GFP/+ mice were crossed with c-kitH2B-tdTomato/+ to obtain c-kitH2B-tdTomato/+;Nkx2.5H2B-GFP/+ and c-kitH2B-tdTomato/+;cTnTH2B-GFP/+ compound heterozygous mice. The compound heterozygous mice had normal heart development.
Tamoxifen (Sigma, Cat. T5648) was intraperitoneally injected into mice (0.12 mg g−1 body weight). Genomic DNA was prepared from yolk sacs or tails for genotyping. Mouse husbandry was conducted in accordance with an approved protocol by Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee (IACUC) and was in compliance with institutional and governmental regulations (PHS Animal Welfare Assurance A3111-01).
For whole-mount staining, the tissues were fixed in 4% paraformaldehyde for 30 min on ice. After the tissues were quickly washed twice in PBS, they were stained in X-gal solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, and 1 mg ml−1 X-gal) overnight at room temperature. For section staining, the heart tissues were fixed in 4% paraformaldehyde for 30 min, washed with PBS, soaked in 30% sucrose overnight and then embedded in optimal cutting temperature compound (Tissue-Tek). Coronal sections of hearts were prepared using a cryostat. The sections were re-fixed in 4% paraformaldehyde for 5–7 min followed by staining with X-gal solution at 37 °C overnight. At least three mice from each stage were examined.
Whole-mount RNA in situ hybridization of mouse embryos was performed using Wilkinson’s protocol39.
Mouse tissues were fixed in 4% paraformaldehyde for 30 min, washed with PBS, soaked in 30% sucrose overnight and then embedded in optimal cutting temperature. Cryosections of heart (coronal) were cut to 8 μm thickness. The primary antibodies used in this study were rat anti-PECAM (CD31; 1:100, BD Biosciences, Cat. 553371), goat anti-c-kit (CD117; 1:20 to 1:40 for postnatal hearts and 1:40 to 1:100 for embryonic hearts, R&D systems, AF1356) and mouse anti-α-SMA (1:100, Sigma, Cat. A5228). Alexa Fluor 488- or 594-conjugated secondary antibodies (1:500; Invitrogen) were applied to detect the corresponding primary antibodies. A TSA kit (Perkin Elmer, Cat. NEL741001KT) was applied to amplify fluorescent signals resulting from c-kit antibody staining. Horseradish peroxidase–conjugated anti-goat IgG (1:500; Abcam, Cat. ab97110) was used as a secondary antibody when TSA was applied to enhance immunostaining.
Mouse ventricular endothelial cells were obtained by enzymatic dissociation of the heart following standard perfusion procedures40 with modifications. Briefly, adult mice (4 months old) were injected with heparin 20 min before heart excision and anaesthetized by isoflurane inhalation. Hearts were quickly removed from the chest and perfused with Ca2+-free solution containing collagenase type II (Worthington, Lakewood, NJ, USA). Ventricles were cut into small pieces and gently minced with a Pasteur pipette. Dissociated cells were transferred to a 50 ml Falcon tube and kept in Tyrode’s solution at room temperature for 5–10 min. Ventricular cardiomyocytes settled on the bottom of the tube. Most non-cardiomyocyte cells were then collected without disturbing the cardiomyocyte layer for flow cytometric analysis.
The cells were washed in PBS with 0.5% bovine serum albumin (BSA). The cell suspension was adjusted to a concentration of 1 × 106 cells ml−1, and single cells were incubated in blocking buffer (PBS with Fc blocking IgG and 1% BSA) for 30 min at room temperature. PECAM/CD31-APC–conjugated antibody (BD, Cat. 561814) was added to the blocking buffer (5 μl per 106 cells). The cells were incubated with gentle shaking for 30 min at room temperature in the dark. Red blood cell lysis buffer was added, and then the samples were incubated at room temperature for 10 min to eliminate red blood cells. The cells were subsequently washed twice and then resuspended in PBS with 0.5% BSA for flow cytometry (Beckman Coulter MoFlo Cytomation).
Myocardial infarction was induced by LAD coronary artery ligation in mice of both genders with body weights ranging from 25 to 34 g (2–6 months old)41. Briefly, mice were anaesthetised intraperitoneally with ketamine (0.065 mg g−1 body weight), acepromazine (0.001 mg g−1 body weight) and xylazine (0.013 mg g−1 body weight). After thoracotomy, LAD ligation was performed with a 7-0 silk suture 3–4 mm from the tip of the left auricle. The successful performance of LAD ligation was verified by visual inspection of the colour of the apex. The chest was closed with a 6-0 silk suture, and the skin was closed with 4-0 silk sutures. All mice were housed under identical conditions and were given water and food ad libitum.
Specific genotype mice were applied to count the number of c-kit+ (c-kitH2B-tdTomato/+), Nkx2.5+ (Nkx2.5H2B-GFP/+) and cTnT+ (cTnTH2B-GFP/+) cells in the hearts. Cryosections (10 μm, coronal) were cut through the heart. For embryonic stages, every fifth section was collected. For hearts older than P30, 1 in every 20 sections was collected. Cells from five representative sections were counted both manually and automatically using ImageJ software with images acquired on a fluorescence microscope. By comparing the numbers acquired by manual counting and ImageJ automatic counting, the thresholds of particle size and intensity were set in ImageJ. Cells from the remaining sections were counted by ImageJ with the same threshold. The total cells were calculated by adding the cell numbers for all sections. The number of cardiomyocytes in the adult heart was divided by 2 considering that 85–90% of these cells are binucleated in mice42. As a result, 1.5-1.7 × 105 Nkx2.5+ or cTnT+ cells were calculated in E12.5–13.5 mouse hearts, 1.05 × 106 myocardial cells were calculated in the adult mouse hearts, and 2.1–4.2 × 106 c-kit+ were calculated in the adult mouse hearts (P30–P90).
