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Wnt-C59 Attenuates Pressure Overload-Induced Cardiac Hypertrophy via Interruption of Wnt Pathway.

Background Cardiac hypertrophy usually results in heart failure and is an important cause of mortality worldwide. Wnt/β-catenin signaling pathway hyper-activation is involved in the pathogenesis and progression of cardiac hypertrophy. Wnt-C59 is a small molecular compound, which strongly and specifically targets at Porcupine to pharmacologically inhibit Wnt palmitoylation, secretion, and other biological activities. However, the role of Wnt-C59 in cardiac hypertrophy remains unknown. Material/Methods We performed transverse aortic constriction (TAC) in adult male mice to induce pressure overload and establish an in vivo model of cardiac hypertrophy. Angiotensin II (Ang-II) was utilized to culture cardiomyocyte to establish a model of in vitro cardiomyocyte hypertrophy. Daily administration of Porcupine inhibitor Wnt-C59 was performed for 4 weeks after TAC surgery. Results Wnt-C59 significantly improved cardiac function and enhanced survival of mice subjected to TAC surgery. Histologically, Wnt-C59 attenuated TAC-induced increase in heart mass, cross-section area of cardiomyocyte, cardiac fibrosis, cardiomyocyte apoptosis, and expression of the hypertrophic biomarkers β-MHC, ANP, and BNP. TAC-induced oxidative stress was also ameliorated by Wnt-C59. Wnt-C59 attenuated Ang-II-induced in vitro cardiomyocyte hypertrophy, as indicated by decreased cell size and lower expression of ANP, BNP, and β-MHC. Moreover, Wnt/β-catenin activation was blocked by Wnt-C59 in cardiac hypertrophy, as indicated by decreased protein expression of Wnt3a and β-catenin and the Wnt target genes cyclin D1 and c-Myc. Conclusions Collectively, Porcupine inhibitor Wnt-C59 attenuates pressure overload-induced cardiac hypertrophic via interruption of the Wnt/β-catenin signaling pathway, and it might be a promising drug for patients with cardiac hypertrophy.


Background
Cardiac hypertrophy is an important characteristic of many cardiovascular diseases; it usually results in heart failure and is a major cause of mortality worldwide [1]. Cardiac hypertrophy results in myocardial adverse remodeling, which includes myocardial fibrosis, cardiomyocyte death, and cardiac dysfunction [2][3][4]. Although remarkable efforts have been made by researchers to unveil the molecular pathophysiology and develop effective methods to prevent or reverse cardiac hypertrophy, to date there has no satisfactory clinical therapy. Therefore, new and safe drugs are urgently needed to improve treatment of patients with cardiac hypertrophy.
The underlying molecular mechanisms of pathologic cardiac hypertrophy are unclear. In the pathogenesis and progression of cardiac hypertrophy, extracellular stresses are translated, resulting in activation of numerous cellular signaling pathways including the PPARg-dependent pathway, insulinlike growth factor-1 (IGF-1)/phosphoinositide-3-kinase (PI3K) pathway, and Wnt/b-catenin signaling pathway, which play important roles in the pathogenesis and progression of maladaptive cardiac hypertrophy [5][6][7]. Among these pathways, the Wnt/b-catenin signaling pathway has recently become an important research focus. Substantial evidence shows that hyper-activation of the Wnt/b-catenin pathway is involved in the pathogenesis of cancers such as colon cancer, and also plays a central role in cardiac hypertrophy [8,9]. It is usually quiescent in the adult heart under normal conditions, but is activated by pathological heart stress such as pressure overload. A growing number of studies showed that hyper-activation of the signaling pathway is correlated with hypertrophic response, and targeting this pathway might be promising strategy to treat cardiac hypertrophy. For example, it is reported that acetyl salicylic acid ameliorates cardiac hypertrophy via inactivation of the Wnt signaling pathway [10,11]. However, there are still no safe and effective drugs that can attenuate cardiac hypertrophy via inhibiting the Wnt/b-catenin signaling pathway.
Many molecules targeting the Wnt signaling pathway have been developed, such as ICG001, XAV939, and Wnt-C59 [12][13][14]. Wnt-C59 strongly and specifically targets Porcupine, which is essential for Wnt palmitoylation, secretion, and other biological activities, and has been demonstrated to be a promising molecular target to attenuate cardiac hypertrophy [15]. Previous studies have demonstrated that Wnt-C59 can effectively ameliorate progression of many tumors via downregulating the Wnt/b-catenin signaling pathway [16]. Wnt-C59 exhibits no apparent toxicity in the gut or other organs when administered at a dose with therapeutically efficacy [17]. These findings indicate that Wnt-C59 is safe and effective and may have translational potential in clinical practice. However, whether Wnt-C59 also inhibits cardiac hypertrophy via targeting the Wnt/b-catenin signaling pathway has not been previously determined.
In the present study, we explored the therapeutic effect of Wnt-C59 on cardiac hypertrophy using both in vivo and in vitro methods. We showed that Wnt-C59 markedly attenuated cardiac dysfunction and enhanced survival of mice subjected to TAC surgery, and hypertrophic response and oxidative stress were also attenuated. Wnt-C59 ameliorated Ang-II-induced cardiomyocyte hypertrophy induced in vitro. We also found that Wnt/b-catenin signaling pathway activation in cardiac hypertrophy was partially reversed by Wnt-C59, indicating that its anti-hypertrophic effects might be mediated by inactivation of the Wnt/b-catenin signaling pathway.

Establishment of cardiac hypertrophy
Adult C57BL/6J male mice were obtained from the Laboratory Animal Center of Chongqing Medical University. Animals experiments were performed in compliance with institutional regulations and were approved by the Experimental Animals Ethics Committee of Chongqing Medical University. TAC was performed to establish pressure overload-induced cardiac hypertrophy, as previously reported [18]. Mice were divided into 4 groups: sham, sham+Wnt-C59, TAC, and TAC+Wnt-C59. Briefly, mice were anesthetized with isoflurane (2%) and a sternotomy was performed to visualize the aortic arch. Then, 7-0 polypropylene suture was used to ligate the aortic arch with a 27G needle as a space holder, which was subsequently removed. The same surgical procedures except for the ligation of the aorta were performed to animals in the sham group. Wnt-c59 was dissolved in saline and administered daily by oral gavage (5 mg/kg/day) for experimental groups for 4 weeks.

Echocardiography analysis
Cardiac function was measured by transthoracic echocardiography using GE vivid 8-dimension ultrasound 4 weeks after TAC surgery. Animals were first anesthetized using isoflurane (2%) and then parasternal short-axis views at the mid-papillary muscle were achieved with M-mode and the results of 3 consecutive heart beats were averaged. Left ventricular fractional shortening (LVFS) and ejection fraction (LVEF) were automatically calculated by the apparatus: FS%=[(LVEDd -LVESd)/LVEDd]×100, EF%=[(LVEDV-LVESV)/LVEDV]×100%. LVESV is the left ventricular end-systolic volume and LVEDV is the left ventricular enddiastolic volume. All measurements were performed by a technician who was unaware of the experimental groups.

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Morphology and histological analysis At 4 weeks after TAC surgery, mice were anesthetized by isoflurane overdose and hearts were harvested. The hearts were weighed and then fixed with 4% polyformaldehyde, followed by embedding in paraffin and sectioning at 4 μm. FITC-conjugated wheat germ agglutinin (WGA) staining was performed for sections to quantitatively analyze the cardiomyocyte CSA. Approximately 100 cardiomyocytes in each section were measured and 5 sections of each heart were counted, with at least 5 different mouse samples assessed in each group. The images were analyzed using Image J software. Cardiac fibrosis was analyzed through Masson trichrome staining following the manufacturer's protocols and quantified using Image J software. Cardiomyocyte apoptosis was detected using the Cell Death Detection kit based on terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) following the manufacturer's protocols, and the percentage of TUNELpositive cardiomyocyte was calculated.

Examination of oxidative stress
Oxidative stress in heart tissue was assessed by detecting ROS generation, glutathione peroxidase (GSH-Px) activities, and malondialdehyde (MDA) and superoxide dismutase (SOD) levels following the instructions of the detection kits (Beyotime Biotechnology, China).

Isolation and culture of neonatal rat cardiomyocytes
We used 1-to 2-day-old neonatal rats to prepare neonatal rat ventricular myocytes (NRVMs) as previously described [19]. NRVMs were cultured with DMEM containing 10% FBS (fresh fetal bovine serum) for 24 h. Then, cardiomyocytes were randomly allocated into a control group, a Wnt-C59 (0.01 μmol/L) group, an angiotensin II (Ang-II, 0.1 μmol/L) group, and an Ang-II+Wnt-C59 group. Cells were treated with Ang-II and Wnt-C59 for 48 h and then fixed with 4% polyformaldehyde followed by immunostaining with cardiac troponin T (cTnT) antibody to identify and measure the cardiomyocyte cell surface area calculated using Image J software. All experiments were performed at least 3 times and about 100 cells were analyzed in each group.

Quantitative real-time PCR
Total RNA was prepared with RNAiso plus (TAKARA) from the heart tissue and NRVM following the manufacturer's protocols, and reverse transcription polymerase chain reaction (RT-PCR) was carried out with TOYOBO RT-PCR kit. SYBR Green quantitative PCR was performed with Applied Biosystems 7500 Fast Real-Time PCR System. Relative quantification of RNA expression normalized against GAPDH was analyzed using double delta Cq method. Each of the PCRs was repeatedly performed at least 3 times.

Western blot
Total protein of heart tissues and cultured NRVMs was extracted and protein concentration was measured with a Bradford protein assay kit (Bio-Rad, Hercules, CA, USA). We separated 50 ug of samples through SDS-PAGE (8% polyacrylamide gel) and then transferred them to polyvinylidene difluoride (PVDF) membranes. The membranes were subjected to antigen blockage for 1 h at room temperature using 5% non-fat milk and incubated with diluted primary antibodies overnight at 4°C. Then, the membranes were washed with TBS (Tris-buffered saline) 3 times and incubated with IR Dye 800-conjugated secondary antibodies (Invitrogen) at room temperature for 2 h. After washing with TBS, the Odyssey Infrared Imaging System was utilized to visualize the protein-binding in membranes, and intensities of the bands were analyzed using Quantity One software. GAPDH was used as an internal control.

Statistical analysis
All data are expressed as means±SD and were analyzed with SPSS 18.0 statistical analysis software. The 2-tailed t test was used to compare means between 2 groups. One-way ANOVA followed by Holm-Sidak's post hoc multiple comparison test was utilized to perform comparison of means among more than 2 groups. Kaplan-Meier survival curves of mice were plotted and compared with the log rank test. Statistical significance was defined as P<0.05.

Results
Wnt-C59 improved cardiac function and enhanced survival of mice subjected to TAC.
Abundant evidence has recently shown a potential maladaptive role of Wnt/b-catenin signaling pathway activation in cardiac hypertrophy. Therefore, many researchers have made attempts to treat cardiac hypertrophy by inhibiting this signaling pathway. Wnt-C59 is a small molecule that can inhibit PORCN enzymatic activity, powerfully blocking activation of the Wnt/b-catenin signaling pathway. To explore whether Wnt-C59 attenuates cardiac dysfunction, adult mice were subjected to TAC surgery to establish a model of cardiac hypertrophy, after which Wnt-C59 was administered orally at dosages of 1 mg/kg·d, 2 mg/kg·d, 5 mg/kg·d, or 10 mg/kg·d for 28 days. Based on a previous study [16], the dosage of 5 mg/kg·d was selected as the optimized dosage because of improvement of cardiac function in our study and safety for use in mice.

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Our results revealed that TAC surgery led to significant decreases in EF and FS ( Figure 1A, 1B), indicating successful establishment of the model of cardiac hypertrophy. Administration of Wnt-C59 did not change EF and FS of mice in the sham group, indicating that Wnt-C59 had no beneficial or toxic effect on cardiac function in physiological conditions. However, Wnt-C59 significantly attenuated cardiac dysfunction of mice subjected to TAC surgery, as demonstrated by higher EF and FS in the TAC + Wnt-C59 group compared to the TAC group ( Figure 1A, 1B). In addition, the data showed that TAC surgery led to significant elevation in left ventricular posterior wall thickness at end-diastole (LVPWd) and posterior wall thickness at end-systole (LVPWs), and these changes were ameliorated by Wnt-C59 ( Figure 1C, 1D). We also analyzed the cumulative survival rate of post-TAC mice for 28 days. The survival rate of animals in the TAC group dropped to 57.54% at 28 days after TAC surgery ( Figure 1E). Notably, the survival rate of mice in the TAC+Wnt-C59 group (72.8%) was significantly higher than with TAC surgery, indicating Wnt-C59 can effectively prevent TAC-induced mortality. These data suggested that Wnt-C59 exerted a beneficial effect on cardiac function and survival in pressure overload-induced cardiac hypertrophy.

Wnt-C59 attenuated hypertrophic response of mice subjected to TAC
Given that Wnt-C59 showed a beneficial effect on cardiac function and survival in TAC-induced cardiac hypertrophy, we next performed histological analysis to investigate whether hypertrophic response was attenuated, which could account for the improved cardiac function. TAC led to marked increase in heart size ( Figure 2A) and heart weight (HW)/body weight (BW), HW/tibial length (TL), and lung weight (LW)/BW ( Figure 2B), which was consistent with cardiac dysfunction of the TAC group ( Figure 1A, 1B), further confirming the successful establishment of cardiac hypertrophy. Wnt-C59 significantly limited the increase in heart size and HW/BW, HW/TL, and LW/BW, indicating the hypertrophic response was attenuated and cardiac dysfunction was ameliorated (Figure 2A, 2B). Further, immunostaining of wheat germ agglutinin (WGA) showed that the CSA was significantly larger in the TAC group compared to the sham group, and this change was blocked by Wnt-C59 ( Figure 2C). Results of Masson trichrome staining and TUNEL staining revealed that Wnt-C59 also attenuated  cardiac fibrosis and cardiomyocyte apoptosis in mice with cardiac hypertrophy ( Figure 2D, 2E), indicating a protective effect of Wnt-C59 on cardiac remodeling. In addition, TAC surgery significantly induced the elevation of mRNA expressions of ANP, BNP, and b-MHC, and these changes were also partially blocked by Wnt-C59 ( Figure 2F), suggesting that hypertrophic response was induced by TAC surgery but was attenuated by Wnt-C59. These data indicated that Wnt-C59 can exert a potential inhibitive effect on pressure overload-induced hypertrophic response in vivo.

Wnt-C59 ameliorated oxidative stress in mice with cardiac hypertrophy
Oxidative stress is an important factor for cardiac injury in multiple cardiac diseases, including pathological cardiac hypertrophy, and therefore is regarded as a target for treatment of cardiac hypertrophy [20,21]. Therefore, we next examined whether Wnt-C59 could ameliorate oxidative stress in cardiac hypertrophy. As shown in Figure 3A, TAC induced a significant increase in ROS level in heart tissue compared to the sham group, and this pathological progress was ameliorated by Wnt-C59. We also found GSH-Px activity was enhanced in the TAC group, and Wnt-C59 treatment partially blocked this change ( Figure 3B). Notably, TAC also enhanced lipid peroxidation, as indicated by the increased MDA level and decreased SOD activity compared with the sham group. Wnt-C59 treatment markedly reversed changes in MDA level and SOD activity ( Figure 3C, 3D). These data indicated that Wnt-C59 can ameliorate oxidative stress in cardiac hypertrophy.

Wnt-C59 attenuated cardiomyocyte hypertrophy in vitro
To further investigate the anti-hypertrophic effect of Wnt-C59, in vitro experiments were performed. As shown in Figure 4A, treatment with Ang-II (0.1 μmol/L) for 48 h resulted in significant hypertrophy of cultured NRVMs, and this result was further confirmed by quantification of cell surface area ( Figure 4B). Wnt-C59 did not show any effect on cell surface area of normal NRVMs, but it significantly attenuated the Ang-II-induced hypertrophic response, as indicated by the limited increase in cell size ( Figure 4A, 4B). We also assessed mRNA and protein expressions of ANP, BNP, and b-MHC. Consistent with in vivo data, Ang-II-induced elevation of mRNA and protein levels of ANP, BNP and b-MHC, and Wnt-C59 significantly blocked these changes ( Figure 4C, 4D). These results suggest that Wnt-C59 can attenuate cardiomyocyte hypertrophy in vitro, supporting its anti-hypertrophic effects in vivo.

Wnt-C59 blocked Wnt/b-catenin signaling pathway activation in mice with cardiac hypertrophy
A large body of evidence has demonstrated that hyper-activation of the Wnt/b-catenin signaling pathway is involve in the hypertrophic response induced by pressure overload. As a small molecule targeting PORCN enzymatic activity, which is essential for Wnt signaling, Wnt-C59 has a powerful inhibitive effect on activation of the Wnt/b-catenin signaling pathway, thus exerting beneficial effects in many diseases, such as preventing tumor growth. To explore whether Wnt-C59 blocks Wnt/b-catenin signaling pathway activation and thus accounts for its protective effect in cardiac hypertrophy, we examined expression of the Wnt signaling molecules Wnt3a and b-catenin and the Wnt target genes cyclin D1 and c-Myc.

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As shown in Figure 5A and 5B1, 5B2, administration of Wnt-C59 inhibited expression of Wnt3a and b-catenin, which were upregulated in the TAC group, indicating that Wnt/b-catenin signaling pathway activation was reduced by Wnt-C59. In addition, cyclin D1 and c-Myc were expressed at higher levels in the TAC group compared to the sham group, but these changes were attenuated by Wnt-C59, further confirming the blockage of Wnt-C5 in Wnt/b-catenin signaling pathway activation ( Figure 5A, 5B3, 5B4). Collectively, these data suggest that Wnt-C59 blocks Wnt/b-catenin signaling pathway activation in cardiac hypertrophy, and this may account for its anti-hypertrophic effect.

Discussion
In the present research, we identified Wnt-C59 as a cardiac-protective molecule in cardiac hypertrophy mice. Wnt-C59 significantly improved function and enhanced survival of mice that underwent TAC surgery. The TAC-induced increase in heart size, CSA, cardiac fibrosis, cardiomyocyte apoptosis, and oxidative stress, as well as increased levels of hypertrophic biomarkers, were blocked by Wnt-C59. Consistent with in vivo results, Wnt-C59 attenuated Ang-II-induced cardiomyocyte hypertrophy in vitro. In addition, Wnt-C59 ameliorated TAC-induced elevation in protein expression of Wnt3a and b-catenin, as well as the Wnt target genes cyclin D1 and c-Myc.
Cardiac hypertrophy can be divided into physiologic hypertrophy and pathologic hypertrophy. Physiologic hypertrophy is usually an adaptive response in the heart, in which hypertrophy is associated with increased contractility and improved function. However, sustained stress leads to pathologic hypertrophy, accompanied with detrimental response, including cardiac fibrosis, myocardial loss, arrhythmia, and cardiac dysfunction. In this phase, embryonic genes, including ANP, BNP, and b-MHC, are reactivated. In clinical practice, pathologic cardiac hypertrophy is typically induced by hypertension, myocardial infarction, and other diseases [22][23][24][25]. Prolonged increase in afterload is the major cause of pathologic cardiac hypertrophy. TAC has been widely used to establish pressure overload-induced cardiac hypertrophy in many studies. In our study, we performed TAC surgery in mice and results showed that TAC induced significant cardiac dysfunction and morbidity in the 4-week study period, which was similar to results of previously published studies [26,27]. In addition, TAC induced a significant increase in heart mass and cross-section area, which were comparable to previously published results [28], indicating TAC-induced

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cardiac hypertrophy was successfully established. Furthermore, hypertrophy biomarker genes ANP, BNP, and b-MHC were all upregulated by TAC, confirming that a hypertrophic response was induced. Therefore, these data make our results obtained from TAC-induced cardiac hypertrophy reliable.
Wnt signaling is involved in many aspects of cell processes, including motility and proliferation, and participates in controlling homeostatic self-renewal in various tissues. Abnormal activation of Wnt signaling is involved in many pathologies, including cancer and other disease states. For example, growing evidence has demonstrated that dysregulation of Wnt/b-catenin signaling is involved in the pathogenesis and progression of gastrointestinal cancers such as colon cancer and liver cancer [29][30][31][32].
Mutations in AXIN and APC can lead to hyper-activation of the Wnt/b-catenin signaling pathway, thus leading to gastrointestinal cancers. Besides cancers, the Wnt/b-catenin signaling pathway is also involved in cardiovascular diseases. Previous studies showed that various cell populations in the heart can mediate interactions after cardiac injury through the Wnt/b-catenin signaling pathway [33]. In addition, Wnt/b-catenin signaling activation can promote retina vascularization [34]. Wnt/b-catenin signaling is a growing focus of researchers due to its important effect on cardiac hypertrophy. In the present study we also observed hyper-activation of the signaling pathway in cardiac hypertrophy induced by TAC surgery, which was consistent with previous studies [9,35]. Interruption of the Wnt/b-catenin signaling pathway has been proved effectively ameliorate cardiac hypertrophy and heart failure [10,36,37].
Attempts have been made to develop small-molecule inhibitors to inactivate Wnt signaling due to its important roles in cancers and cardiovascular diseases. Porcupine is a membrane-bound O-acyltransferase, which is required for Wnt palmitoylation to enable Wnt secretion and produce biological activity. It was demonstrated that the Wnt/b-catenin signaling pathway can be effectively arrested by Porcupine inhibitors.
Wnt-C59 specifically targets Porcupine to inhibit activities of the Wnt/b-catenin signaling pathway and showed therapeutic effect in cancers, including nasopharyngeal carcinoma and breast cancer [17,38]. Therefore, we speculated that Wnt-C59 might exert anti-hypertrophic effects in the heart though blockage of Wnt/b-catenin signaling pathway activation. We found that Wnt-C59 can ameliorate cardiac hypertrophic, and administration of Wnt-C59 can significantly inhibit the Wnt/b-catenin signaling pathway, which is hyper-activated in cardiac hypertrophy. Previous studies demonstrated that Wnt signaling is quiescent in the basal state in the heart but is activated by cardiac hypertrophic stimuli [9,39]. This suggests that Wnt signaling may be difficult to further inhibit in the basal state in the heart, but in the activated state Wnt-C59 can block Wnt signaling activity to some extent, which might be the mechanism underlying the anti-hypertrophic effect of Wnt-C59. Previous studies showed that Wnt-C59 exerts no toxicity at a therapeutically effective dose in the gut or other tissues. Therefore, Wnt-C59 might be a promising molecule which is of translational potential in patients with cardiac hypertrophy. However, more experiments are needed to determine the safety and optimal dose in patients with hypertrophy.

Conclusions
Our results revealed that Porcupine inhibitor Wnt-C59 effectively attenuates pressure overload-induced cardiac hypertrophy both in vivo and in vitro. Additionally, the beneficial effects of Wnt-C59 appear to be mediated via blockage of Wnt/b-catenin signaling pathway activation. As Wnt-C59 exerts no toxicity at a therapeutically effective dose in the gut or other tissues, it may be a promising drug for patients with cardiac hypertrophy.

Conflicts of interest
None.