Activation of endothelial β-catenin signaling induces heart failure

Activation of β-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/β-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of β-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of β-catenin, which activates Wnt/β-catenin signaling in Bmx-positive arterial endothelial cells (Bmx/CA mice) led to progressive cardiac dysfunction and 100% mortality at 40 weeks after tamoxifen treatment. Electron microscopic analysis revealed dilatation of T-tubules and degeneration of mitochondria in cardiomyocytes of Bmx/CA mice, which are similar to the changes observed in mice with decreased neuregulin-ErbB signaling. Endothelial expression of Nrg1 and cardiac ErbB signaling were suppressed in Bmx/CA mice. The cardiac dysfunction of Bmx/CA mice was ameliorated by administration of recombinant neuregulin protein. These results collectively suggest that sustained activation of Wnt/β-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling, and that the Wnt/β-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure.

Scientific RepoRts | 6:25009 | DOI: 10.1038/srep25009 phosphorylation/ubiquitination site responsible for its proteasomal degradation. Knock-in mice with LoxP sequence flanking exon 3 of β -catenin gene (Ctnnb1 lox(ex3)/lox(ex3) mice) generates β -catenin protein lacking its exon 3 (β -catenin (Δ ex3)) after Cre-mediated recombination and is widely used as a mice model for conditional activation of Wnt/β -catenin signaling together with cell type-specific Cre mice 12 . Previous reports used Ctnnb1 lox(ex3)/lox(ex3) mice to investigate the roles of endothelial Wnt/β -catenin signaling in angiogenesis during embryonic development and ischemia. Sustained activation of Wnt/β -catenin signaling in ECs blocks vascular remodeling in early embryonic development 13 whereas activation of Wnt/β -catenin signaling in ECs promotes angiogenesis after myocardial infarction 14 . However, the role of endothelial Wnt/β -catenin signaling in the biological process other than angiogenesis is poorly understood.
We here demonstrate the novel role of endothelial Wnt/β -catenin signaling in the heart. Using a transgenic mouse model with tamoxifen (TAM)-inducible, endothelial-specific expression of β -catenin (Δ ex3), we found that sustained activation of β -catenin signaling in ECs impairs cardiac function leading to severe heart failure. Mechanistically, activation of endothelial β -catenin signaling suppressed the expression of neuregulin (NRG), resulting in reduced activity of ErbB signaling of cardiomyocytes. Administration of recombinant NRG1 ameliorated ErbB signaling and cardiac function of the transgenic mice. These results suggest that sustained activation of β -catenin signaling in ECs causes heart failure in a NRG-ErbB signaling dependent manner.

Inducible activation of Wnt/β-catenin signaling in arterial ECs. A previous report crossed Tie2-Cre
or VE-cadherin-Cre mice with Ctnnb1 lox(ex3)/lox(ex3) mice to induce EC-specific expression of β -catenin (Δ ex3) but these mice were embryonic lethal 13 . We therefore used Bmx-CreER T2 transgenic mice to achieve inducible expression of degradation-resistant β -catenin (Δ ex3) in an arterial EC-specific manner and to investigate the role of β -catenin signaling in ECs of adult mice. Bmx is a member of the Tec tyrosine kinase gene family and is highly expressed in the ECs of arteries and in the endocardium, but not in the venular endothelium 15 . We first crossed Bmx-CreER T2 mice with enhanced green fluorescent protein reporter mice (CAG-CAT-EGFP mice) 16 , and confirmed that all the EGFP-positive cells were also CD31-positive and approximately a quarter of CD31positive cells were EGFP-positive in the heart (Fig. 1a). Immunofluorescence analysis also showed that EGFP was ECs were collected from the heart of Bmx-CreER T2 mice (Ctrl) or from Bmx-CreER T2 crossed with CAG-CAT-EGFP mice (Bmx/EGFP) 1 week after the TAM treatment. (b) Immunofluorescent staining of cardiac tissue for GFP (red), CD31 (green), and TO-PRO-3 (blue). Scale bars: 20 μ m. (c, d, e) Genotyping PCR (c), western blot (d) and quantitative RT-PCR analysis (e) of cardiac ECs isolated from Ctrl (Bmx-CreER T2 with Ctnnb1 +/+ ) mice (Ctrl ECs) and Bmx/CA mice (Bmx/CA ECs). (c) Floxed allelle of β -catenin (= 500 bp) was detected in Bmx/CA ECs but not in Ctrl ECs. (d) β -catenin protein lacking exon3 (= 75 kDa) was detected in Bmx/CA ECs but not in Ctrl ECs. (e) Expression levels of Wnt/β -catenin signaling target genes (Axin2 and Lef1) were higher in Bmx/CA ECs compared with Ctrl ECs. **P < 0.01. expressed in the endothelium of coronary arteries and endocardium, but not in the capillary vessels (Fig. 1b). We then crossed Bmx-CreER T2 mice with Ctnnb1 lox(ex3)/lox(ex3) mice (Bmx/CA mice) to observe the effect of conditional activation of β -catenin signaling in endothelial cells. Floxed Ctnnb1 allele and β -catenin protein lacking exon3 were detected in cardiac ECs of Bmx/CA mice but not of control Bmx-CreER T2 Ctnnb1 +/+ (Ctrl) mice (Fig. 1c,d). β -catenin protein lacking exon3 were not detected in cardiomyocytes of Bmx/CA mice (Supplemental Fig. S1) suggesting that Cre-mediated recombination did not directly affected cardiomyocytes. We also observed the up-regulation of Wnt/β -catenin target genes such as Axin2 and Lef1 in cardiac ECs of Bmx/CA mice (Fig. 1e), indicating that β -catenin (Δ ex3) protein in ECs are functional and activates Wnt/β -catenin signaling in Bmx/CA mice.
Activation of β-catenin signaling in ECs causes heart failure. Since ECs play critical roles in regulation of vascular functions, we first analyzed the blood pressure of Bmx/CA mice. Blood pressure of Bmx/CA mice was comparable to that of Ctrl mice (Fig. 2a). In clear contrast, cardiac function was progressively impaired in Bmx/CA mice compared with Ctrl mice (Fig. 2b,c, Supplementary Table 1), resulting in nearly 100% mortality at 60 weeks of age (50 weeks after TAM treatment) (Fig. 2d). The heart weight/body weight ratio was significantly higher in Bmx/CA mice ( Supplementary Fig. S2a), and expression levels of genes related to heart failure such as Nppa and Nppb as well as fibrosis marker gene Col1a1 were also higher in the heart of Bmx/CA mice compared with Ctrl mice (Fig. 2e, Supplementary Fig. S2b). TUNEL staining revealed that the number of apoptotic cardiomyocytes was not increased in the heart of Bmx/CA mice ( Supplementary Fig. S2c). Capillary density was also comparable between Ctrl and Bmx/CA mice ( Supplementary Fig. S2d) and no signs of tissue hypoxia was observed in the heart of Bmx/CA mice ( Supplementary Fig. S2e). These results suggest that sustained activation of β -catenin signaling in ECs induces fatal heart failure without the involvement of cardiomyocyte death or cardiac ischemia. Gross histological analysis revealed that there was neither inflammatory nor fibrotic changes in the heart of Bmx/CA mice ( Fig. 2f,g), however, electron microscopic analysis revealed characteristic changes such as dilatation of T-tubules and degeneration of mitochondria in the cardiomyocytes of Bmx/CA mice ( Fig. 2h-j). These changes are similar to the changes in hearts of cardiomyocyte-specific ErbB2 receptor 17,18 and ErbB4 receptor knockout mice 19 and of the patients with trastuzumab-induced 20 cardiomyopathy, suggesting that ErbB signaling might be involved in the cardiac phenotype of Bmx/CA mice.

Endothelial Nrg1 expression and cardiac ErbB signaling are suppressed in Bmx/CA mice. NRG1
is produced mainly by ECs 21 and exerts cardioprotective effects through binding to ErbB2/ErbB4 receptors 4-6 . Mice deficient for either NRG1 22 , ErbB2 23 , or ErbB4 24 showed embryonic lethality due to absence of trabeculae formation in the ventricle. Cardiomyocyte-specific deletion of either ErbB2 17,18 or ErbB4 19 resulted in dilated cardiomyopathy-like phenotype, e.g. cardiac dysfunction and increased expression of heart failure marker genes, with abnormal electron microscopic finding such as vacuole formation and dilation of T-tubules. We therefore hypothesized that reduced NRG-ErbB signaling might be involved in the mechanism of heart failure in Bmx/CA mice. Expression levels of Nrg1 were significantly lower in cardiac ECs prepared from Bmx/CA mice compared with Ctrl mice. Expression levels of Nrg1 in non-endothelial cells were lower compared with endothelial cells and were comparable between Ctrl and Bmx/CA mice (Fig. 3a). Recombinant human Wnt3a increased the expression of Axin2 whereas it suppressed the expression of Nrg1 in cultured human coronary arterial endothelial cells (HCAECs) (Fig. 3b), suggesting that activation of Wnt/β -catenin signaling is tightly associated with decreased Nrg1 expression in endothelial cells. Moreover, phosphorylation levels of ErbB2 and ErbB4 proteins were lower in the heart of Bmx/CA mice as compared with Ctrl mice (Fig. 3c). These results indicate that activation of β -catenin signaling in ECs suppressed the activity of cardiac ErbB signaling through down-regulating the expression of Nrg1 in ECs.
Administration of NRG1 protein rescued the cardiac phenotype of Bmx/CA mice. We next examined whether decreased Nrg1 expression in endothelial cells and reduced neuregulin/ErbB signaling are involved in the mechanism of cardiac dysfunction in Bmx/CA mice. Administration of recombinant NRG1 recovered the phosphorylation of cardiac ErbB2 and ErbB4 receptors (Fig. 4a). Cardiac function of Bmx/CA mice was also improved after administration of NRG1 (Fig. 4b, Supplementary Table 2.), which was accompanied by significant improvement of electron microscopic findings, i.e., decreased number of dilated T-tubules and degenerated mitochondria in cardiomyocytes (Fig. 4c,d). These results suggest that decreased expression of Nrg1 and activity of ErbB signaling were responsible for cardiac dysfunction of Bmx/CA mice.

Discussion
Wnt/β -catenin signaling plays an important role in cardiac hypertrophy and ischemic injury [25][26][27] , however, little is known about the role of Wnt/β -catenin signaling in heart failure. In this study, we showed that sustained activation of β -catenin signaling in ECs induces heart failure in adult mice. Activation of β -catenin signaling in ECs decreased the expression levels of endothelial Nrg1 and suppressed the activity of cardiac ErbB signaling. Administration of NRG protein rescued the cardiac dysfunction that is caused by activation of β -catenin signaling. These results collectively suggest that activation of Wnt/β -catenin signaling in cardiac ECs suppresses cardiac NRG-ErbB signaling, resulting in development of heart failure.
As Wnt/β -catenin signaling in ECs regulates angiogenesis during embryogenesis 13 , we first postulated that sustained activation of endothelial Wnt/β -catenin signaling impairs the angiogenesis in the heart, thereby inducing hypoxia. However, there were no significant differences in vascular density ( Supplementary Fig. S2d), and in myocardial hypoxia between Bmx/CA and Ctrl mice ( Supplementary Fig. S2e), indicating that angiogenesis or tissue hypoxia were less likely to be the cause of cardiac dysfunction in Bmx/CA mice. Angiotensin II or phenylephrine treatment has been reported to decrease the expression of NRGs in ECs in vitro 28 , however, it is not known how endothelial NRG production is regulated in vivo. In the present study, we showed that activation of β -catenin signaling suppressed NRG1 production from ECs both in vitro and in vivo. Whether activation of β -catenin signaling suppress the expression of Nrg1 gene through Tcf-dependent canonical Wnt signaling or through interaction with other signaling cascade such as Foxo signaling 29 , Notch signaling 30 , or Hippo signaling 31 require further investigations. We observed T-tubule dilatation and mitochondria degeneration that appear like "vacuoles" through electron microscopic analysis of Bmx/CA mice. Vacuole-like structure formation in the cardiomyocytes is also observed in the heart of ErbB knockout mice 32 , doxorubicin-induced heart failure model mice 33,34 and in the patients with trastuzumab (Herceptin)-induced cardiomyopathy 20 , suggesting that activation of β -catenin or Wnt/β -catenin signaling in endothelial cells might be involved in the molecular mechanism of those diseases. However, as Ctnnb1 lox(ex3)/lox(ex3) mice might increase the cytosolic β -catenin and induce/suppress the expression of Wnt target genes to the supraphysiological level, whether there are relevant human cardiac diseases that are related with high level of β -catenin or Wnt/β -catenin signaling in endothelial cells remains to be elucidated in the future study.
Dysfunction of the cardiomyocytes plays a central role in the pathophysiology of heart failure. There are many "heart failure model mice" with cardiomyocyte specific gene modification 35 . Recent reports, however, highlight the role of non-cardiomyocytes in the heart, e.g. endothelial cells, fibroblasts and blood cells, in maintaining ventricular homeostasis and proper cardiac function 1,36 . In the present study, heart failure was observed in the mice with endothelial cell specific gene modification, suggesting that dysfunction of endothelial cells may play a primary role in the pathogenesis of heart failure (Fig. 5). Approaches targeting the signaling cascades in non-cardiomyocytes, e.g. Wnt/β -catenin signaling in endothelial cells, might become a novel therapeutic target for heart failure.
loxP-mediated recombination in Bmx-CreER T2 mice, TAM dissolved in corn oil was injected intraperitoneally for 5 consecutive days (1 mg/50 μ L/day). Blood pressure was measured in conscious mice by the tail-cuff system using with BP98A (Softron) according to manufacturer's protocol. Left ventricular size and function of conscious mice were assessed by echocardiography using with the Vevo770 system (VisualSonics) with a 40MHz probe. Human recombinant NRG1-β 1 was dissolved in phosphate buffer saline (PBS) containing 0.1% bovine serum albumin (BSA) and administrated intraperitoneally for 25 consecutive days (2.5 μ g/day). Pimonidazole hydrochloride was dissolved in normal saline and administrated intraperitoneally 30 minutes before sampling (0.6 mg/ kg). All animals were maintained in a virus-free facility on a 12-h light/12-h dark cycle and fed a standard diet.   heart tissue was minced and lysed in buffer containing 20 mM Tris-HCl (pH: 7.4), 150 mM NaCl, 1% NP-40, 3 mM EDTA, and protease/phosphatase inhibitors. Protein concentration was measured by BCA method. Densitometric analysis of the image was performed using ImageJ.
Histological analysis. For morphological analysis, heart tissues were fixed with neutralized formaldehyde and embedded in paraffin. For fluorescent immunostaining, 5 μ m-thick fresh frozen sections were stained and the nuclei were counterstained with TO-PRO-3. TUNEL staining was performed with TUNEL Apoptosis Detection Kit (Millipore: 17-141). Images were acquired by LSM700 confocal microscope (Zeiss) or FSX100 (Olympus). For electron microscope analysis, heart tissues were fixed with 2.5% glutaraldehyde solution (Wako: 072-02262). Septum and posterior free wall were dissected to 1-2 mm cube and ultrathin section was made using Reichert Ultracut S (Leica). Images were acquired with H-7650 (Hitachi).
Statistical analysis. All data are presented as mean ± s.e.m. Statistical analysis was performed using Excel2013 (Microsoft, USA) with the add-in software Statcel3 (OMS, Japan). All variables were tested for distribution normality using Chi square test. When the data do not fit normal distribution in a group or normal variance between groups, non-parametric tests are used. In case of analyzing two groups, statistical difference was determined by the unpaired two-sided Student's t-test (parametric) or the unpaired two-sided Mann-Whitney U-test (non-parametric). In case of analyzing multiple groups, the statistical difference was determined by Steel-Dwass test. Cumulative survival data was evaluated by Kaplan-Meier non-parametric regression analysis and the log-rank test. Significant differences were defined as P < 0.05.