Introduction
Cardiovascular disease is one of the leading causes of morbidity and mortality in the western world [
23]. Following myocardial infarction (MI), damaged myocardium is replaced with scar tissue, which may result in heart failure for which limited therapeutic options are available. One potential important signal transduction pathway involved in regulating cardiac repair and potentially stem cell maintenance and differentiation is the Wnt pathway, which plays an essential role in normal cardiac development [
8]. Wnt signaling has been shown to be a key regulator of stem cell growth, differentiation, and proliferation in both normal homeostasis and diseased state [
10,
34]. Wnt proteins form a family of highly conserved secreted signaling molecules in which the canonical Wnt/β-catenin pathway is mostly studied. Upon binding of Wnt to the seven-transmembrane domain spanning frizzled (Fzd) receptor and the co-receptor lipoprotein receptor-related 5/6 (Lrp5/6) proteins GSK3β is inactivated, thereby preventing the breakdown of β-catenin. After stabilization and accumulation, β-catenin enters the nucleus, where it binds to LEF/TCF transcription factors to activate the transcription of Wnt target genes.
Although extensively studied in cardiac development, the exact role of Wnt signaling after MI still needs to be unraveled. Infarct size reduction was achieved by both stimulation and inhibition of Wnt/β-catenin signaling [
2,
14], and both downregulation [
4] and upregulation [
32] of β-catenin gene levels were observed in cardiac hypertrophy. Moreover, epicardium-derived progenitor cells lacking β-catenin displayed impaired coronary artery formation [
41], while β-catenin depletion in cardiac progenitor cells enhanced differentiation during cardiac remodeling [
42]. Recent data demonstrates that β-catenin is involved in expansion of resident cardiac progenitor cells, but its role in differentiation of these cells remains controversial [
20,
22,
33,
42]. Most studies investigating Wnt signaling during pathological conditions (e.g. myocardial infarction) focused on proteins participating in Wnt signal transduction such as Dishevelled-1 [
9,
38] and β-catenin [
7,
40]. However, this not necessarily means that active Wnt/β-catenin signaling, including transcription of Wnt target genes, is present.
Considering its potency to act as a therapeutic target, fundamental insights on Wnt time-dependency and cell specificity upon injury in the adult injured myocardium are necessary. Therefore, we evaluated the presence of active Wnt signaling in vivo in the heart following MI. Moreover, we investigated in which cell types active Wnt signaling was present during different phases following MI.
After binding of β-catenin to LEF/TCF transcription factors in the nucleus, several Wnt target genes are activated, including the Axin2 gen [
21]. Axin2 is able to downregulate β-catenin and acts as a negative regulator of Wnt signaling [
16]. We used Axin2
+/LacZ reporter mice in which the LacZ gene is under the control of the Axin2 promoter, providing a reliable way to detect Wnt activity by visualizing LacZ-reporter-positive cells [
24].
Materials and methods
Animals
Male and female C57BL/6 Axin2
+/LacZ reporter mice were bred and used at 8–10 weeks of age. For generation of the Axin2-lacZ mouse [
24], the β-galactosidase (NLS-lacZ) gene was introduced in frame to the endogenous Axin2 promoter by homologous recombination, thereby replacing most of exon 2 (MGI Ref ID J:74286) but leaving the Axin2 promoter intact. All experiments were approved by the Animal Experimentation Committee of the Utrecht University and were in accordance with the
Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
Myocardial infarction
The MI was induced by ligation of the left anterior descending (LAD) coronary artery under isoflurane anesthesia, as described previously [
37,
39]. Briefly, mice were anesthetized with isoflurane, orally intubated, and ventilated. A left thoracotomy was performed at the third intercostal space, and muscles and pericardium were dissected. LAD ligation was performed with an 8-0 non-absorbable ethilon suture. After verification that coronary occlusion had occurred by the change of color and kinesis of the apex and anterior-lateral wall, the thorax was closed in layers. After detubation, mice were kept warm until fully recovered. Mice were sacrificed at baseline (0 day, controls) and at 1, 3, 7, 14, and 21 days after MI. The hearts were flushed with phosphate-buffered saline (PBS) and dissected. The left ventricle was cut in two halves through the center of the infarct along the longitudinal axis. One half was snap-frozen in liquid nitrogen and stored at –80°C; the other half was kept in PBS for further processing. Remote area was defined as the non-infarcted part of the interventricular septum.
Cell isolation and flow cytometry analysis
Freshly dissected hearts, containing the infarcted and non-infarcted area, were perfused with PBS and washed, minced into 1–2 mm
2 pieces, digested for 45 min at 37°C with 10 mg/ml collagenase A (Roche), and passed through a 70-μm filter. Remaining cells were plated on a 12-well plate DMEM with 10% fetal bovine serum (FBS) as described previously [
36] or aliquoted after centrifugation for flow cytometric analysis. After centrifugation, cells were resuspended in PBS containing 4% FBS, and aliquots containing 1.0 × 10
6 cells were stained. Single cell suspensions were stained with FDG (Fluoreporter
® LacZ Kit F-1930, Molecular Probes) to detect β-galactosidase activity. Next, isolated cells were labeled with antibodies against Sca-1 (BD Pharmingen 553108, PE conjugated), isotype control (BD Pharmingen, 553930), CD31 (Biolegend 102417, Pe-Cy7 conjugated), isotype control (Biolegend, 400521), ckit (Abcam 46790, APC-Cy5.5 conjugated), isotype control (Abcam 46745), and CD45 (Abcam 51482, PE-Texas Red conjugated). To exclude dead cells from analysis (including cardiomyocytes), 7-AAD (BD 559925) was used. Samples were analyzed by flow cytometry (Beckman Coulter Cytomics FC500 FACS), collecting 15,000–50,000 events per sample.
Histology and immunohistochemistry/immunocytochemistry
Frozen sections of 5–7 μm were cut in a cryostat (Microm HM560, Cryo-Star) and mounted on silane-BSA-coated slides. Histologic analysis of MI was performed by H&E staining. Sections were fixed in acetone, blocked with 10% normal goat serum (NGS), and incubated overnight with primary antibody. After incubation with secondary antibody for 1 h, sections were washed in PBS and mounted in Fluoromount (Southern Biotech). For immunocytochemistry, coverslips with cultured cells were fixed in 4% paraformaldehyde at room temperature and permeabilized with 0.2% Triton X-100 in PBS. Cells were blocked with 2% bovine serum albumin (BSA) for 15 min and incubated overnight at 4°C with primary antibody with 10% NGS. The coverslips were then incubated with secondary antibody in PBS with 10% NGS for 1 h and subsequently mounted. For both IHC and ICC, Hoechst dye was used to visualize nuclei. Primary antibodies for Sca-1 (BD Pharmingen, 57403), CD31 (BD Pharmingen, 550274), CD45 (BD Pharmingen 553774), tropomyosin (Sigma, T9283), β-galactosidase (Abcam ab616), and von Willebrand Factor (Dako, M0616) were used with matching isotypes and a FITC-conjugated antibody against alpha-smooth muscle actin (Sigma, A2547). Secondary antibodies were conjugated with AF555 (Invitrogen A21429), AF488 (Invitrogen A11001), and AF647 (Invitrogen A21247). Slides and coverslips were examined with a Nikon light microscope equipped for epifluorescence or Zeiss LSM 510 Meta confocal microscope for high magnification capturing. All images illustrated are representative of least three independent experiments.
β-Galactosidase (LacZ) staining on cultured cells
Cells were fixed in 0.2% paraformaldehyde for 10 min, followed by a 10-min wash in PBS with 2 mM MgCl2 on ice. Cells were then placed in Xgal staining solution (2 mM MgCl2, 0.1% sodium deoxycholate and 0.02% NP-40, 5 mM K3FE(CN)6, 5 mM K4Fe(CN)6, and 1 mg/ml X-gal (Invitrogen) in PBS) at 37°C for 30 min. Thereafter, cells were washed twice in PBS with 2 mM MgCl2 at room temperature and covered with coverslips and examined by light microscopy.
Statistics
Data (mean ± SEM) were analyzed by Mann–Whitney U test, using a significance level of P < 0.05 (SPSS for Windows, v15.0).
Discussion
Wnt signaling is an important regulatory pathway in biology and behavior of stem cells [
10,
34], and is required for normal cardiac development [
8,
11]. In response to cardiac stress and injury, reactivation of Wnt/β-catenin signaling takes place, probably being part of a fetal gene reprogramming required for tissue repair [
18,
30]. Considering its potency to be used as a therapeutic target, it is of great interest to gain more information on the function of Wnt signaling in the adult heart, especially in the context of cardiac tissue repair. However, during the last few years, contradictory results were reported upon inhibition and stimulation of Wnt with respect to the adaptive response on hypertrophic and ischemic stimuli [
2‐
4,
14,
38]. It is becoming clear that Wnt molecules do not simply drive proliferation or differentiation, but rather regulate and fine-tune these processes in a cell-type and time-dependant manner. There are 19 Wnt proteins, 10 Fzd receptors, and 2 Lrp co-receptors in mammals, representing a complex regulatory family.
In our study, cell-specific Wnt signaling and its localization was visualized in response to cardiac injury using Axin2
+/LacZ reporter mice. Although an indirect measurement of the active Wnt cascade, the not cell-specific role of Axin2 in the Wnt response [
10] makes it a reliable way to detect the canonical Wnt activity. We demonstrate that active Wnt signaling is significantly upregulated in those cell populations that are considered to play a role in cardiac wound healing after MI and in cardiomyocytes. Active Wnt signaling was significantly increased in the border zone and remote area after cardiac injury. Together with an increase in total progenitor, endothelial, and leukocyte cell populations, the number of LacZ+ cells in these populations increased significantly. In addition, a significant increase in Sca+/CD31− progenitor and Sca−/CD31+ endothelial cell number was found within the population that showed active Wnt signaling. Moreover, we observed active Wnt signaling in fibroblasts, vWF-positive endothelial cells, vascular smooth muscle cells (αSMA+), fibroblasts (αSMA+), and cytoplasm of cardiomyocytes.
Whether we can enhance the cardiac repair by modulating Wnt remains the question since active Wnt signaling was present in all the cell populations studied. Different compounds are available already that can interfere with Wnt signaling, but up to now Wnt modulation after myocardial infarction did not result in consistent results. Frizzled or secreted frizzled-related proteins (sFRPs) can compete for Wnt binding, thereby antagonizing Wnt signaling. Transgenic mice overexpressing Frizzled-A, a member of the frizzled family, displayed less apoptosis and a higher capillary density resulting in reduced infarct size as compared to wild-type animals [
2]. Others reported that mesenchymal stem cells (MSCs) overexpressing sFRPs showed better survival after injection into the peri-infarct region. This resulted in increased engraftment and vascularized granulation tissue after MI [
1,
27], probably by antagonizing Wnt3a which resulted in less apoptosis [
43]. On the contrary, direct β-catenin injection in the border zone of rats after coronary artery ligation decreased infarct size and promoted cell survival in both cardiomyocytes and cardiac fibroblasts [
14]. Furthermore, cardioprotection by ischemic preconditioning was shown after GSK3β inhibition [
17], due to induction of neovascularization and inhibition of apoptosis. The opposite effect was demonstrated by sFRP1 overexpression [
3]. Recently, fibrosis was shown to be limited in sFRP1 null mice after myocardial infarction [
19] although regulated via mechanisms independently of Wnt signaling. As indicated, different Wnt-dependent and -independent stimuli seem to interact with GSK3β and sFRPs with respect to cell survival and repair [
15,
28].
In our study, Sca+ progenitor, as well as endothelial cells, and cardiomyocytes showed an increased LacZ expression, and these LacZ+ cells increased in number. This increase in number suggests that increased Wnt signaling might influence survival or proliferation of these cells in response to cardiac injury. Furthermore, ckit+ and CD45+ cell populations showed increased LacZ expression as well. Although this association was observed, our study is purely descriptive and does not allow to conclude any causal relationship between an increased presence of LacZ and the different cell numbers. Interestingly, studies aiming at wound healing after MI reported an upregulation of β-catenin in vascular endothelial cells during neovascularization [
7,
9] and in proliferating and migrating myofibroblasts [
9] after MI. Consistent with these studies, our findings indirectly suggest that Wnt signaling drives expansion of different cell types, including Sca+/CD31− progenitor and Sca−/CD31+ endothelial cells, as well as in ckit+ and CD45+ cell populations, in response to cardiac injury. The upregulation of β-catenin observed in these studies does not necessarily mean that a higher promoter activity is present though. Whether increased Wnt signaling directly influences cardiac wound healing or cell survival remains unanswered.
A second question is whether Wnt signaling modulates differentiation after cardiac injury. Several studies reported that canonical and non-canonical Wnt signaling are required for the differentiation of cardiac progenitor cells [
20], and enhances the cardiomyogenic potential of bone marrow cells [
13]. Different progenitor cell populations, resident in the adult heart, have been shown to differentiate into cardiomyocytes or vascular structures both in vitro and in vivo [
5,
6,
29]. Although stimulation of endogenous regeneration capacity might offer a new treatment modality, activation of the resident cardiac progenitor cells to repair the injured myocardium still remains a matter of debate. From this point, it is at least striking that we observed a significant increase in Sca+/CD31− progenitor cells and Sca−/CD31+ endothelial cells within the LacZ+ population. LacZ+ cells expressing both Sca+ and CD31+ decreased after MI. These shifts within the LacZ+ populations might suggest the involvement of Wnt signaling in the process of differentiation in response to MI. Several other reports have been published supporting a role for Wnt signaling in the expansion and proliferation of cardiac progenitor cells during development [
11,
12,
22]. Canonical and non-canonical Wnt signaling has been shown to stimulate the cell growth and survival in isolated endothelial cells in vitro [
26,
35,
40]. Moreover, β-catenin stabilization via lithium stimulation and administration of different Wnt molecules induced muscle regeneration via differentiation of Sca+/CD45+ cells, which was reduced upon sFRP2/3 injection [
31]. In contrast, cardiac-specific β-catenin depletion was shown to attenuate cardiac remodeling, mainly through enhanced differentiation of Sca+ progenitor cells [
42].
In conclusion, the present study demonstrates a temporal upregulation of the active Wnt signaling after MI which is not restricted to a particular area of the heart. LacZ+ cells were shown to co-express progenitor, endothelial, leukocyte, and fibroblast markers, suggesting a broad role of Wnt in reaction to cardiac injury. Interestingly, different cell populations displayed a distinct LacZ response in time, suggesting a time- and cell-specific activation of Wnt after MI. When considering Wnt reactivation as a therapeutic approach to enhance cardiac regeneration, one should consider this time- and cell-specific expression window. Based on the significant upregulation of the progenitor and endothelial cell populations that show the active Wnt signaling, it is possible that Wnt modulates cardiac regeneration and neovascularization during the healing phase after MI. However, as more populations are involved, this also illustrates its complexity.