Developmental, cardiac-specific PDGFR-β–knockout mice.
Mice expressing Cre recombinase driven by the Nkx2.5 promoter (C57BL/6 background) were crossed with Pdgfrbfl/fl mice (129 background) to create Nkx2.5-Cre:Pdgfrbfl/fl (PdgfrbNkx-Cre) mice or littermate control, non–Cre-expressing Pdgfrbfl/fl mice (mixed C57BL/6 × 129 background). To study the effects of the Nkx2.5-Cre transgene on basal cardiac function in an identical genetic background, we crossed mice expressing Cre recombinase driven by the Nkx2.5 promoter (C57BL/6 background) with wild-type 129 mice to generate Nkx2.5-Cre mice in a mixed C57BL/6 × 129 background. Nkx2.5-Cre mice were provided by R.J. Schwartz (Institute of Biosciences and Technology, Houston, Texas, USA). Pdgfrbfl/fl mice were provided by P. Soriano (Mount Sinai School of Medicine, New York, New York, USA).
Inducible, cardiac-specific Pdgfrb-knockout mice.
To create inducible, cardiac-specific PDGFR-β–knockout mice, we crossed heterozygous α-MHC-MerCreMer mice (C57BL/6 background) with Pdgfrbfl/fl mice (129 background), resulting in Pdgfrbfl/fl mice and α-MHC-MerCreMer:Pdgfrbfl/fl (PdgfrbMerCre) mice in a mixed C57BL/6 × 129 background. To study any possible contribution of the α-MHC-MerCreMer transgene, we used heterozygous α-MHC-MerCreMer mice in a similar, mixed C57BL/6 × 129 background. All groups were treated with 4-hydroxytamoxifen beginning at age 6–8 weeks. 4-Hydroxytamoxifen was administered intraperitoneally at a dosage of 20 mg/kg/d for a total of 5 days. Mice were given 7–10 days of rest after the last dosage of 4-hydroxytamoxifen prior to functional characterization or to exposure to pressure overload–induced stress induced by TAC.
The University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee approved all animal protocols used in this study. Eight- to 12-week-old male mice were used for functional experiments. TAC surgeries were performed based on previously described protocols (45
). The sham procedure was similar, except that the aorta was not ligated.
Ultrasound measurement of aortic, carotid, and coronary flows.
Carotid velocities were measured 24 hours, 7 days, and 14 days after TAC using a 20-Mhz ultrasound probe connected to a signal processing unit (Indus Instruments) using our previously published protocol (46
). CFR was measured as previously described (33
). Briefly, the ultrasound probe tip was placed at the left side of the chest of the anesthetized animal and stabilized using a micromanipulator (model MM3-3, World Precision Instruments). Peak coronary velocities were identified, and the probe was fixed in position. Baseline coronary flow was measured at 1.0% isoflurane. As a coronary vasodilator stimulus, the inhaled isoflurane concentration was increased to 2.5%. The coronary vasodilatory properties of isoflurane in general are well described (47
), and we have previously validated its use in the measurement of CFR in conjunction with a noninvasive ultrasound technique (33
). Ratios of peak velocities at low- (1%) and high-dose (2.5%) isoflurane were reported as CFR.
In vivo myocardial perfusion studies.
For myocardial perfusion studies, short- and long-axis echocardiographic images were obtained using the Vevo 770 machine (Visual Sonics). Baseline short-axis images were obtained, followed by injection of a 50-μl bolus containing 1.2 × 107 DEPO Micromarker microbubbles (Visual Sonics) via tail vein. After 12–15 minutes of contrast agent bolus injection, short-axis images were again captured without moving the probe. Global perfusion was only assessable in the anterior and lateral portions of the left ventricle, due to shadowing cast on the septum and posterior aspect of the heart, rendering this data uninterpretable. Quantification of overall perfusion was performed by integrating contrast values obtained from 9 separate measurements from each of at least 3 mice in each group.
Cardiac function in experimental mice was measured by magnetic resonance (MR) scanning using a retrospectively gated imaging method (49
). MR scanning experiments were performed using a Bruker 7T MR scanner (BioSpin MRI). Mice were anesthetized using 1%–3% isoflurane in oxygen, and 0.5%–1.5% isoflurane was delivered to maintain anesthesia. During MR scanning, heart rates and respiratory rates were 350–500 beats/minute and 25–40/minute, respectively. Each MR scan included a scout; single-slice sagittal and coronal long-axis cine images of the heart; and a series of axial short-axis cine acquisitions. Sagittal and coronal images were used to establish the cardiac axis and to identify the apex for prescribing short-axis cine slices. Short-axis cine images were used to quantify LV end-diastolic and -systolic volumes. LV volumes, anterior and posterior wall thickness, and LV internal diameter at different phases of the cardiac cycle were measured using ParaVision software (Bruker BioSpin). The total cross-sectional area in the 1-mm-thick short-axis slices (typically 6–8 slices) from apex to base was integrated to calculate ventricular volumes and ejection fractions.
We used a previously published method adopted to arrest hearts of inducible mutant mice or controls in diastole and fix them in 10% formalin (50
). Five-micrometer sections of formalin-fixed, paraffin-embedded tissue were used for TUNEL assay, H&E staining, Masson’s trichrome staining, and immunohistochemistry.
Myocyte cross-sectional area.
Horizontal short-axis cut hearts were fixed in 10% buffered formalin solution for 24 hours. Paraffin-embedded hearts were sectioned at 5-mM thickness. All the sections were stained according to previously standard methods. Briefly, sections were deparaffinized, rehydrated, and then incubated for 1 hour with TRITC-labeled lectin (100 μg/ml; Sigma-Aldrich). Multiple ×400 magnified pictures were captured using a fluorescence microscope (Olympus BX41) from epicardial and endocardial areas of the left ventricle. Myocytes (40–60/heart) that showed round capillaries and clear membrane staining were included in the analysis. The Scion Image (Scion Corp.) program was used to calculate cross-sectional area.
Measurement of microvascular density.
We used an antibody directed against CD31 (BD Biosciences) to stain cardiac microvessels. Detection, staining, and counterstaining with hematoxylin was performed using standard methods. For quantification of microvessels, at least 3 random fields (×400) from each of 2 independent sections for each heart assessed were analyzed. To normalize microvessel density to the number of cardiomyocytes present, the same images used to quantify microvessel number were used to count cardiomyocyte nuclei, which were identified based upon their morphologic appearance made visible by counterstaining.
TUNEL staining was performed on formalin-fixed, paraffin-embedded tissue using the In Situ Cell Death Detection Kit (Roche Applied Science). Sections were counterstained with DAPI, and apoptotic cells per 105
cardiomyocytes were quantified using previously described methods (51
Tissue hypoxia measurement.
To analyze tissue hypoxia 7 and 14 days after TAC, we treated all mice intraperitoneally with 60 mg/kg pimonidazole hydrochloride (Hypoxyprobe, Natural Pharmacia International) 3 hours prior to sacrifice. Immunodetection of Hypoxyprobe binding, indicative of tissue hypoxia, was performed using a previously described method (52
). For quantification of cardiac hypoxic area, at least 6 high-magnification (×400) randomly chosen fields from the left ventricle were selected for quantification of immunopositive cells, and the average number of positive staining cells per field was used as an index of tissue hypoxia. Quantification of hypoxic area in hearts of inducible mutant or control mice after TAC was performed by an investigator blinded to the sample identification, using NIH ImageJ software (http://rsbweb.nih.gov/ij/) incorporating standard, grid-based quantitative morphometric techniques (53
). For determination of statistical significance between groups at each day, we fit a mixed-effects linear model, with genotype as the fixed effect and animal as the random effect. We used an F-test to determine the significance of differences between genotypes.
Primary antibodies from Cell Signaling Technology were directed at the following antigens: Akt, phospho-Akt (Ser473), ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), PDGFR-β, JNK, phospho-JNK (Thr183/Tyr185), p38, and phospho-p38 (Thr180/Tyr182). Primary antibodies obtained from Santa Cruz Biotechnology Inc. were directed at the following antigens: VEGF-A, GAPDH, GATA4, and phospho–PDGFR-β (Tyr 1021). Anti–HIF-1α antibody was obtained from Novus Biologicals. Protein bands were quantified via densitometry using Fluorchem 8900 imaging software (Alpha Innotech).
RNA isolation for quantitative RT-PCR and gene expression microarrays.
Total RNA was isolated from heart tissues using TRIzol (Invitrogen) reagent using the manufacturer’s recommended protocol. RNA integrity was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies), with samples run against an RNA ladder of specified molecular weights (Eukaryote Total RNA Nano Series II, Agilent Technologies).
Gene expression microarrays.
Mouse genome 430 2.0 array gene chips were purchased from Affymetrix and probed with RNA according to the manufacturer’s recommended protocol using a double in vitro transcription method, and data were processed for analysis as previously described (54
Genotyping by PCR.
Genomic DNA was extracted from mouse tails with the RedExtract-N-Amp Tissue PCR Kit (Sigma-Aldrich). Primers were as follows: Nkx2.5-Cre forward, 5′-GGCGTTTTCTGAGCATACCT-3′, and Nkx2.5-Cre reverse, 5′-CTACACCAGAGACGGAAATCCA-3′; internal control forward, 5′-CTAGGCCACAATTGAAAGATCT-3′, and internal control reverse, 5′-GTAGGTGGAAATTCTAGCATCATCC-3′; MerCreMer forward, 5′-ATACCGGAGATCATGCAAGC-3′, and MerCreMer reverse, 5′-AGGTGGACCTGATCATGGGAG-3′; internal control forward, 5′-CTAGGCCACAATTGAAAGATCT-3′, and internal control reverse, 5′-GTAGGTGGAAATTCTAGCATCATCC-3′. To check for the Pdgfrb floxed/deleted allele, the forward primer was 5′-GGAAAAGCAGGTTTGTGC-3′, the floxed reverse primer was 5′-TACCAGGAAGGCTTGGGAAG-3′, and the deleted allele reverse primer was 5′-CCAGTTAGTCCACCTATGTTG-3′.
Neonatal murine cardiomyocyte isolation, adenoviral infection, and hypoxia.
Neonatal mouse cardiomyocytes were isolated using a commercially available kit from Cellutron (55
). Briefly, hearts from 2- to 3-day-old wild-type or Pdgfrbfl/fl
mice were removed and incubated with digestion medium and then centrifuged to collect the dissociated cells from the medium. Cell cultures were at least 95% pure of cardiomyocytes as determined by flow cytometry after immunostaining of cardiac myosin, a marker of cardiomyocytes. Cells were infected with 25 MOI of adenoviral CMV-GFP or adenoviral CMV-CreGFP (gifts of M.D. Schneider, Imperial College, London, United Kingdom) in growth medium for 48 hours, and then cells were incubated at 37°C with serum-free DMEM F12 (Invitrogen) for 16–24 hours. After 16 hours exposure of cells to hypoxia (1% O2
, 5% CO2
, at 37°C) or normoxia (21% O2
, 5% CO2
, at 37°C), condition medium was collected and stored at –80°C. Cells were collected in TRIzol
reagent for RNA isolation.
Endothelial cell proliferation and tube formation assays.
HUVECs were cultured in growth medium (5,000 cells/well of 96-well plate). After 3 hours of incubation, growth medium was replaced with condition medium and then incubated for 24 hours. Cells were counted using a Coulter Z series device. The in vitro angiogenesis assay kit was purchased from Millipore, and the assay method was adapted from previous reports (56
). Briefly, HUVECs were cultured using endothelial growth medium for 12 hours. HUVECs (5 × 103
/well of a 96-well plate) were resuspended in 150 μl condition medium (from 16-hour hypoxia or normoxia) supplemented with 2% heat-inactivated FBS. Resuspended cells were seeded onto the surface of the ECM-coated wells. After 4–8 hours incubation, 3–5 random view fields (×40 magnified) were pictured for each well. Tube length was calculated using ImageJ software.
In all figures and within the text, data are presented as mean ± SEM. We tested the data for normality using the Kolmogorov-Smirnov test. To assess statistical significance between groups, we used the unpaired 2-tailed Student’s t test or, when appropriate, we used the nonparametric, 2-sided Wilcoxon rank-sum test. A P value less than 0.05 was considered statistically significant. Changes between multiple groups were calculated using ANOVA, and Tukey’s test was then used to test for significant changes between individual groups.
For identification of genes of interest between mutant and control mice over time after TAC using gene expression microarrays, we fit an ANOVA model that accounts for differences due to genotype differences between the 3 time points (day 0, 3, or 7), as well as potential interactions between genotype and day. We used a false discovery rate of 1% to identify genes of interest using this method. To better understand the behavior of the genes found to be differentially expressed by the model (with interaction between treatment and day), we used clustering to group these genes into sets that exhibited similar expression patterns. Gene annotation enrichment analysis of clustered genes was performed using the NIH DAVID annotation tool (http://david.abcc.ncifcrf.gov).