After myocardial infarction (MI), the heart is difficult to repair because of great loss of cardiomyoctyes and lack of cardiac regeneration. Novel drug candidates that aim at reducing pathological remodeling and stimulating cardiac regeneration are highly desirable. In the present study, we identified if and how a novel porcupine inhibitor CGX1321 influenced MI and cardiac regeneration. Permanent ligation of left anterior descending (LAD) coronary artery was performed in mice to induce MI injury. Cardiac function was measured by echocardiography, infarct size was examined by TTC staining. Fibrosis was evaluated with Masson’s trichrome staining and vimentin staining. As a result, CGX1321 administration blocked the secretion of Wnt proteins, and inhibited both canonical and non-canonical Wnt signaling pathways. CGX1321 improved cardiac function, reduced myocardial infarct size, and fibrosis of post-MI hearts. CGX1321 significantly increased newly formed cardiomyocytes in infarct border zone of post-MI hearts, evidenced by the increased EdU+ cardiomyocytes. Meanwhile, CGX1321 increased Ki67+ and phosphohistone H3 (PH3+) cardiomyocytes in culture, indicating enhanced cardiomyocyte proliferation. The mRNA microarray showed that CGX1321 up-regulated cell cycle regulating genes such as Ccnb1 and Ccne1. CGX1321 did not alter YAP protein phosphorylation and nuclear translocation in cardiomyocytes. In conclusion, porcupine inhibitor CGX1321 reduces MI injury by limiting fibrosis and promoting regeneration. It promotes cardiomyocyte proliferation by stimulating cell cycle regulating genes with a Hippo/YAP-independent pathway.

Introduction

Heart failure following myocardial infarction (MI) remains a major medical problem and a leading cause of deaths in the world [1]. The mainstay therapies for MI are to rapidly re-establish sufficient reperfusion. Survivors of MI usually die from post-MI heart failure due to segmental loss of cardiomyocytes and reduced cardiac function. It is now recognized that human and mammalian cardiomyocytes are able to regenerate but with a very limited efficacy [2,3]. Novel drug candidates that aim at reducing pathological remodeling and stimulating cardiac regeneration are highly desirable.

The Wnt signaling pathways are a network of proteins that passes signals which have been implicated in a wide spectrum of important biological phenomena. Wnt signalings include the canonical Wnt/β-catenin pathway, the non-canonical planar cell polarity pathway, and the non-canonical Wnt/Ca2+ pathway [4]. Wnt pathways play crucial roles in cardiac development and especially in the formation of the myocardium [5]. The canonical Wnt/β-catenin signaling controls ventricular myocyte proliferation during development and perinatal period. Differential activation of the Wnt/β-catenin pathway accounts for the observed differences in the proliferation rates of the compact compared with the trabecular myocardium during normal cardiac development [6]. Wnt/β-catenin signaling induces cardiac specification during early developmental stages but inhibits it later [5]. Wnt signaling also promotes the expansion of the second heart field progenitor cells that ultimately give rise to the majority of cardiomyocytes [7].

In normal adult mammalian heart, Wnt signaling is quiescent, but is activated in response to cardiac injury [8]. Wnt-1 is robustly induced 2 days post injury and sustained in entire post-MI hearts, whereas Wnt-4 and Wnt-7a are only up-regulated transiently at later stages [9]. These Wnt ligands can stimulate both canonical and non-canonical Wnt pathways. Moreover, the beneficial effects of Wnt inhibitory secreted Frizzled-related protein 1 (sFRP-1) are mediated via the Wnt pathway during MI [10]. These findings indicate that both canonical and non-canonical Wnt pathways play a role in MI injury. However, Wnt-5 is the exclusive protein in cardiomyocyte [11], and whether Wnt signaling plays a role in the proliferation in adult cardiomyocytes (ACMs) is unclear.

Recent breakthrough in the research of the Wnt pathway has revealed new points of intervention that may lead to novel drug targets for small or large molecular weight compounds. Amongst them, porcupine, an acyltransferase that enables secretion and activity of all Wnt proteins [12], is highly druggable. Indeed, an inhibitor of porcupine, LGK974 has entered human clinical trial for treating several tumors and has been proven safe in human subjects [12]. Porcupine inhibitors have been recently found to exert beneficial effect against MI [11,13]. However, if and how porcupine inhibitors promote cardiac regeneration is not fully clear. The source of the new cardiomyocytes in adult mammals has been debating. We [14] and others [1517], using fate-mapping technologies, demonstrate that the newly formed cardiomyocytes in adult mammals are predominantly derived from pre-existing cardiomyocytes. Thus it is important to explore if porcupine inhibitors can promote cardiomyocyte proliferation.

In the present study, we used a novel porcupine inhibitor CGX1321 developed by our contributing author (Curegenix Co. Ltd.). CGX1321 has entered phase I human clinical trial (NCT02675946) for treating several types of solid tumors and has been proven safe in human subjects. Mouse MI model was induced by permanent ligation of left anterior descending (LAD) coronary artery. Our data showed that CGX1321 reduced infarct size and promoted cardiac function in post-MI mice. The therapeutic effect was associated with enhanced cardiomyocyte proliferation mediated cardiac regeneration. CGX1321 blocked both canonical and non-canonical Wnt pathways, and stimulated the expression of cell cycle regulating genes with a Hippo/YAP-independent pathway.

Materials and methods

Wnt inhibitor

CGX1321 is a novel inhibitor of the Wnt pathway discovered by Guangzhou Curegenix Co. Ltd. Mechanistically, it is a potent and specific inhibitor of porcupine that is required for acylation and secretion of Wnt proteins. It was derived through an extensive medicinal chemistry campaign from a lead compound series (published as an issued patent US9556144) (http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=1&f=G&l=50&co1=AND&d=PTXT&s1=9556144&OS=9556144&RS=9556144). CGX1321 has completed all preclinical studies and entered human clinical trials for the treatment of cancer (identifier: NCT02675946, https://clinicaltrials.gov/ct2/show/NCT02675946?term=CGX1321&rank=1).

Experimental animals

Adult male C57BL/6J mice (3 months old) were used for the animal study. The experimental protocol was approved by the Institutional Animal Care and Use Committee at the Third Military Medical University. All surgeries were performed under anesthesia with 2% isoflurane inhalation, and all efforts were made to minimize animal suffering. All experiments were carried out in accordance with the recommendation in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

MI model

Permanent ligation of LAD coronary artery was performed on mice as described previously [18]. Briefly, anesthesia was maintained with 2% isoflurane inhalation. A permanent ligation was performed around the LAD coronary artery 2–3 mm from its origin with a 7-0 silk suture. Sham-operated animals were subjected to the same surgical procedures except that the suture was passed under the LAD but not tied. These mice were intraperitoneally injected with CGX1321 or vehicle for different time periods. In our previous work, a daily dosage range of 0.03–2.5 mg/kg CGX1321 per body weights were tested in mice to inhibitory efficacy and safety (issued patent: US9556144). Administration with 2.5 mg/kg/day CGX1321 could significantly inhibit the target genes of Wnt signaling, and did not show significant toxicities in vital organs. Therefore, intraperitoneal dosing of 2.5 mg/kg/day was used in the present study.

Echocardiographic evaluation

Echocardiographic recordings (GE vivid E9) were performed to determine cardiac structure and function in mice under sedation with 2% isoflurane. The heart was imaged using the 2D and M-mode in the parasternal long-axis and short-axis views. LVIDd is diastolic left ventricle (LV) internal diameter and LVIDs is systolic LV internal diameter. LV fractional shortening (LVFS) was calculated by FS (%) = (LVIDd − LVIDs)/LVIDd × 100, and LV ejection fraction (LVEF) was calculated by the cubic method: LVEF (%) = ((LVIDd)3 − (LVIDs)3) (LVIDd)3 × 100. The functional status of the heart was also assessed by left ventricular diastolic posterior wall thickness (LVDPW). Data from three consecutive cardiac cycles were analyzed using software resident on the ultrasonography by an experienced researcher, who was blinded to treatment.

Measurement of myocardial infarct size

The infarct size was defined by phosphate-buffered 1% TTC staining as we have described earlier [18]. In brief, the heart was snap-frozen and cut into six 1-mm-thick transverse slices from the apex to the base, parallel to the atrioventricular groove. Each slice was stained in a 1% solution of phosphate-buffered TTC at 37°C for 20 min and then fixed in 4% paraformaldehyde. The infarct area (pale) and normal area (brick red) on both sides were delineated and calculated using the ImageJ software. The ratio of the area of the infarct band to the total area of the LV was calculated and presented as a percentage. The values obtained were averaged and assay was performed in a blinded fashion.

Apoptosis detection

To detect cardiomyocyte apoptosis, the terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assay using an In Situ Cell Death Detection Kit (POD; Roche Applied Bio Sciences, Switzerland) was carried out according to the manufacturer’s protocols, as we previously described [19]. The sections were also counterstained with DAPI, and the cardiomyocyte cytoplasm was identified by sarcomeric tropomyosin antibody. The slices were observed with a Nikon fluorescence microscope (Nikon, Tokyo, Japan). The numbers of apoptotic nuclei were counted in ten non-adjacent images of peri-infarct areas, and a total of five sections per animal were analyzed. All the manual counts were performed in a blinded fashion.

Determination of interstitial fibrosis

Interstitial fibrosis was assessed by Masson’s trichrome staining [11] and fibroblast marker (vimentin) [20] for the detection of connective tissue accordingly. Briefly, hearts from each group were excised, washed in PBS, fixed in 4% formaldehyde at room temperature, embedded in paraffin after conventional processing (alcohol dehydration), and further processed for histology. For Masson’s trichrome staining, 3-µm-thick sections were stained with Masson’s trichromic solution according to standard procedures, and analyzed for extent of fibrosis. For immunostaining of vimentin, the sections were incubated with 10% normal goat serum for 60 min and then treated with antibody against vimentin and tropomyosin overnight at 4°C. The sections were washed with PBS twice and incubated with secondary antibody (1:500; Beyotime) for 1 h in the dark at 37°C. Nuclei were labeled with DAPI (Sigma). Images of stained cells were obtained by using an Olympus confocal laser scanning microscope (FluoView 1000, Japan) and calculated using the ImageJ software.

EdU incorporation analysis

The EdU incorporation in mice and the analysis of isolated ACM by enzyme digestion were performed, according to a modified protocol as previously described [21]. EdU was administered intraperitoneally (500 μg per animal) every 2 days for a period of 10 days. The hearts were harvested 28 days post MI. EdU detection in isolated cardiomyocytes and in tissues were performed using a commercially available kit (Invitrogen), according to the manufacturer’s instructions.

Isolation of neonatal rat ventricular myocytes and cardiomyocyte proliferation detection

Neonatal rat ventricular myocytes (NRVMs) were prepared from less than 24 h old neonatal rats according to previously published procedures [22]. Briefly, NRVMs were pretreated with fresh FBS-free DMEM for 12 h, and then were treated with CGX1321 (0.1 μmol/l) for 48 h. Each treatment was performed in triplicate wells. For the detection of proliferation markers Ki67 and phosphohistone H3 (PH3), NRVMs were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for another 20 min at room temperature. The cells were incubated with 10% normal goat serum for 30 min. NRVMs were then treated with antibody against Ki67 or PH3 overnight at 4°C and incubated with secondary antibody (1:500; Beyotime) for 1 h in the dark at 37°C. Nuclei were labeled with DAPI (Sigma).

Wnt signaling detection

For the detection of inhibition of Wnt secretion by CGX1321, NRVMs were pretreated with fresh FBS-free DMEM for 12 h and then treated with CGX1321 (0.1 μmol/l) for 48 h, the medium and lysate proteins were isolated. SDS/PAGE was conducted with antibodies against Wnt5a/b and GAPDH. For the analysis of inhibition of canonical or non-canonical Wnt signaling by CGX1321, NRVMs were pretreated with fresh FBS-free DMEM for 12 h. Then cardiomyocytes were randomly divided into four groups: control; CGX1321 (0.1 μmol/l); hypoxia; hypoxia + CGX1321 (0.1 μmol/l). CGX1321 was added into medium for 48 h, the cells were then treated with hypoxia for 6 h. The total and nucleus proteins were isolated and prepared with 6× sample loading buffer. SDS/PAGE was conducted with antibodies against β-catenin, NFATc3, Histone-H3, GAPDH. A LiCor Odyssey Fc instrument was used to detect chemiluminescent signal.

Detection of YAP activity and localization

YAP activity and localization were detected with a similar method of previous studies [23,24]. Briefly, NRVMs pretreated with or without CGX1321 (0.1 μmol/l for 48 h) were induced with hypoxia for 6 h. Then cells were harvested and SDS/PAGE was conducted with antibodies against YAP1, p-S127-YAP1, GAPDH. A LiCor Odyssey Fc instrument was used to detect chemiluminescent signal. For the detection of YAP localization, the heart tissue sections were harvested at 4 weeks post MI and incubated with antibody against YAP1 and tropomyosin overnight at 4°C. The sections were washed in PBS and stained with secondary antibody (1:500; Beyotime) for 1 h in the dark at 37°C. Nuclei were incubated with DAPI (Sigma). The percentage of tropomyosin-positive cardiomyocytes with nuclear expression of YAP was counted.

mRNA expression arrays

mRNA expression was analyzed using GeneChip 3′IVT express arrays (Affymetrix). Fifty nanograms of total RNA for each sample was prepared and analyzed on expression arrays following manufacturer’s instructions. Validation of mRNA expression levels was performed using TaqMan Gene Expression Assays (Life Technologies). Relative quantitation was performed according to manufacturer’s recommendations on ABI 7900HT. Data analysis were performed using the ΔΔCT method, as part of the SDS 2.3 software package.

Statistical analysis

All the experimental data are presented as the mean ± S.E.M. Data for each group are mean values of at least three different experiments. Statistical analyses were performed by one-way ANOVA followed by Holm–Sidak’s post hoc multiple comparison test (for comparison of more than two groups) or Student’s ttest (for comparison of two groups). Kaplan–Meier survival curves for different groups of mice were constructed and compared (with the log rank test) using StatView software. P<0.05 was considered statistically significant.

Results

CGX1321 inhibited the secretion of Wnt-5a/b in cardiomyocytes

It is known that most of the Wnt excretion is difficult to be detected. Based on the findings in a recent study [11], only Wnt-5a/b expression is detectable in conditioned medium of cardiomyocytes using quantitative PCR and Western blot analysis. In the present study, Western blot analysis showed that Wnt-5a/b protein in the medium of NRVMs treated by CGX1321 was significantly decreased, suggesting that CGX1321 could inhibit the secretion of Wnt-5a/b protein (Supplementary Figure S1). In addition, Wnt-5a/b protein in the lysate of NRVMs was also significantly reduced by CGX1321 (Supplementary Figure S1). This result indicated the effective inhibition of CGX1321 on Wnt-5a/b production and secretion in cardiomyocytes.

CGX1321 promoted the cardiac function in post-MI hearts

To determine whether CGX1321 could exert a therapeutic effect against MI injury, we evaluated the effect of CGX1321 on animal survival and cardiac function in post-MI mice hearts. As shown in Figure 1A, daily injections of CGX1321 for 4 weeks in mice did not affect the survival rate in the sham-operated mice. The survival rate of post-MI mice was 66.7% (24 out of 36 mice) at 4 weeks post-MI. Administration of CGX1321 significantly increased it to 82.9% (29 out of 35 mice). Echocardiographic analysis was used to examine the cardiac function and ventricular chamber dilation in post-MI hearts. Representative echocardiographic images are shown in Figure 1B. MI significantly decreased LVEF and LVFS. LVEF and LVFS were greater in post-MI hearts at 4 weeks treated with CGX1321 than in post-MI hearts treated with vehicle (Figure 1C). LVDPW of post-MI hearts at 4 weeks was significantly thinner than that of sham hearts, indicating LV dilation induced by MI injury. CGX1321 significantly limited the decrease in LVDPW induced by MI (Figure 1C). These results showed that CGX1321 reduced the post-MI deterioration of cardiac function and ventricular structure.

CGX1321 improved cardiac function in mice subjected to MI injury

Figure 1
CGX1321 improved cardiac function in mice subjected to MI injury

(A) CGX1321 improved the survival of mice subjected to MI injury. CGX1321 or vehicle was administrated daily for 4 weeks post-MI. n=7 for sham; n=10 for sham + CGX1321; n=36 for MI; n=35 for MI + CGX1321. *P<0.05 compared with sham; #P<0.05 compared with MI. (B) Representative images of M-mode echocardiographic analysis. (C) Heart rates were controlled to be similar in different groups (C1). The quantitations of LVEF (C2), LVFS (C3), LVDPW (C4) are presented. n=10. *P<0.05 compared with sham; #P<0.05 compared with MI.

Figure 1
CGX1321 improved cardiac function in mice subjected to MI injury

(A) CGX1321 improved the survival of mice subjected to MI injury. CGX1321 or vehicle was administrated daily for 4 weeks post-MI. n=7 for sham; n=10 for sham + CGX1321; n=36 for MI; n=35 for MI + CGX1321. *P<0.05 compared with sham; #P<0.05 compared with MI. (B) Representative images of M-mode echocardiographic analysis. (C) Heart rates were controlled to be similar in different groups (C1). The quantitations of LVEF (C2), LVFS (C3), LVDPW (C4) are presented. n=10. *P<0.05 compared with sham; #P<0.05 compared with MI.

CGX1321 reduced infarct size and fibrosis in post-MI hearts

Meanwhile, the TTC staining was performed to evaluate the infarct size of post-MI hearts. Myocardial infarct size was 53.6 ± 5.7% at 4 weeks in post-MI mice. But it was significantly smaller (34.7 ± 5.0%) in mice treated with CGX1321 than those with vehicle at 4 weeks post-MI (Figure 2A). TUNEL assay was used to measure apoptotic cell death in the infarct border zone. As shown in Figure 2B, TUNEL+ cardiomyocyte nucleus number per 106 nuclei was significantly increased in infarcted hearts at 2 days post-MI. However, CGX1321 treatment did not alter the percentage of TUNEL+ apoptotic cardiomyocytes. Masson’s trichrome staining was performed at 4 weeks post-MI to evaluate the fibrosis in heart tissue. MI induced significant fibrosis in the infarct border zone and infarct area. There were increased islands of viable cardiac muscle at 4 weeks after CGX1321 treatment. The percentages of fibrotic area in the infarct border zone (Figure 2C) and infarct area (Figure 2D) were both significantly reduced in CGX1321-treated hearts. Similar results were shown by using immunostaining analysis of a fibrosis marker vimentin (Supplementary Figure S2).

CGX1321 reduced infarct size and fibrosis of mice subjected to MI injury but did not alter cardiomyocyte apoptosis

Figure 2
CGX1321 reduced infarct size and fibrosis of mice subjected to MI injury but did not alter cardiomyocyte apoptosis

(A) The infarct size was detected with TTC analysis at 4 weeks post-MI. The representative images (A1) and quantitation (A2) of heart infarct size were shown. n=6. *P<0.05 compared with sham; #P<0.05 compared with MI. (B) Representative images TUNEL staining (B1) and quantitation of TUNEL+ cells (B2) and TUNEL+ cardiomyocytes (B3) in infarct border zone at 2 days post-MI. Scale bar =20 µm. n=7. *P<0.05 compared with sham; #P<0.05 compared with MI. (C) Representative images (C1) and quantitation (C2) of fibrotic area in infarct border zone by Masson’s trichrome staining at 4 weeks post-MI. Scale bar =20 µm. n=7. *P<0.05 compared with sham; #P<0.05 compared with MI. (D) Representative images (D1) and quantitation (D2) of fibrotic area in infarct area by Masson’s trichrome staining at 4 weeks post-MI. Scale bar =40 µm. n=7. *P<0.05 compared with sham; #P<0.05 compared with MI.

Figure 2
CGX1321 reduced infarct size and fibrosis of mice subjected to MI injury but did not alter cardiomyocyte apoptosis

(A) The infarct size was detected with TTC analysis at 4 weeks post-MI. The representative images (A1) and quantitation (A2) of heart infarct size were shown. n=6. *P<0.05 compared with sham; #P<0.05 compared with MI. (B) Representative images TUNEL staining (B1) and quantitation of TUNEL+ cells (B2) and TUNEL+ cardiomyocytes (B3) in infarct border zone at 2 days post-MI. Scale bar =20 µm. n=7. *P<0.05 compared with sham; #P<0.05 compared with MI. (C) Representative images (C1) and quantitation (C2) of fibrotic area in infarct border zone by Masson’s trichrome staining at 4 weeks post-MI. Scale bar =20 µm. n=7. *P<0.05 compared with sham; #P<0.05 compared with MI. (D) Representative images (D1) and quantitation (D2) of fibrotic area in infarct area by Masson’s trichrome staining at 4 weeks post-MI. Scale bar =40 µm. n=7. *P<0.05 compared with sham; #P<0.05 compared with MI.

CGX1321 increased cardiac regeneration in post-MI hearts

Next we asked if CGX1321 promoted the cardiac regeneration in the post-MI hearts. EdU incorporation analysis of myocytes was performed in heart tissue with or without CGX1321 administration. EdU was injected every 2 days after 3 days post-MI, and the heart tissue was observed at 4 weeks post-MI. EdU+ cardiomyocytes were predominantly found in the infarct border zone of post-MI heart, similar to the previous studies [21]. Compared with non-treatment in infarcted heart, CGX1321 significantly increased the percentage of EdU+ cardiomyocytes in infarct border zone (Figure 3A). We further performed EdU incorporation analysis of myocytes isolated from hearts with or without CGX1321 administration 4 weeks post-MI. This approach not only unequivocally identifies the co-localization of EdU and nuclei within a cardiomyocyte, but also allows the analyses of EdU+ ACMs with different number of nuclei. A total number of 2243200 ACMs from four hearts treated with vehicle and 1782400 ACMs from four hearts treated with CGX1321 were screened. As shown in Figure 3B, significantly more EdU+ ACMs were found in CGX1321-treated hearts. Mononuclear EdU+ ACMs were also found significantly more often in CGX1321-treated hearts. These data showed that CGX1321 induced three-fold more EdU+ ACMs, indicating increased newly formed cardiomyocytes. The increased cardiac regeneration might, at least partially, contribute to the improved cardiac structure and function.

CGX1321 increased newly formed cardiomyocytes in post-MI heart

Figure 3
CGX1321 increased newly formed cardiomyocytes in post-MI heart

(A) Representative images (A1) and quantitation (A2) of EdU+ cardiomyocytes at 4 weeks in post-MI heart tissue. Scale bar =20 μm. n=4. *P<0.05 compared with sham; #P<0.05 compared with MI. (B) Representative images (B1) and quantitation (B2,B3) of EdU+ cardiomyocytes isolated from hearts 4 weeks after MI. Both mononuclear and binuclear EdU+ cardiomyocytes were shown. Scale bar = 20 μm. n=4. *P<0.05 compared with sham; #P<0.05 compared with MI.

Figure 3
CGX1321 increased newly formed cardiomyocytes in post-MI heart

(A) Representative images (A1) and quantitation (A2) of EdU+ cardiomyocytes at 4 weeks in post-MI heart tissue. Scale bar =20 μm. n=4. *P<0.05 compared with sham; #P<0.05 compared with MI. (B) Representative images (B1) and quantitation (B2,B3) of EdU+ cardiomyocytes isolated from hearts 4 weeks after MI. Both mononuclear and binuclear EdU+ cardiomyocytes were shown. Scale bar = 20 μm. n=4. *P<0.05 compared with sham; #P<0.05 compared with MI.

CGX1321 increased the proliferation of cardiomyocytes in vitro

The source of cardiac regeneration post ischemic injury remains unclear so far. It could be the differentiation of cardiac progenitor cells or/and proliferation of pre-existing cardiomyocytes [25]. Recent studies have indicated that the proliferation of pre-existing cardiomyocytes predominates the newly formed cardiomyocytes [1417]. We further identified if CGX1321 promoted cardiomyocyte proliferation in vitro. NRVMs were cultured with or without CGX1321. Proliferation marker Ki67 and PH3 were stained. Cells co-expressing both cardiomyocyte marker tropomyosin and Ki67/PH3 were counted as proliferating cardiomyocytes. As shown in Figure 4, CGX1321 treatment significantly increased the percentage of Ki67+ cardiomyocytes (CGX1321: 13.3 ± 0.8% compared with Control: 5.3 ± 0.6%, P<0.05), and increased the percentage of PH3+ cardiomyocytes (CGX1321: 0.9 ± 0.1% compared with Control: 0.4 ± 0.1%, P<0.05). These results suggested that CGX1321 was able to induce cardiomyocyte proliferation, contributing to its promotion of cardiac regeneration.

CGX1321 increased cardiomyocyte proliferation

Figure 4
CGX1321 increased cardiomyocyte proliferation

The proliferation of neonatal mouse cardiomyocytes was detected with Ki67 and PH3 staining. Tropomyosin staining was used to identify cardiomyocytes. Representative images (A1 and B1) and quantitations (A2 and B2) of Ki67+ or PH3+ cardiomyocytes were shown. Scale bar =20 μm. n=3. *P<0.05 compared with control.

Figure 4
CGX1321 increased cardiomyocyte proliferation

The proliferation of neonatal mouse cardiomyocytes was detected with Ki67 and PH3 staining. Tropomyosin staining was used to identify cardiomyocytes. Representative images (A1 and B1) and quantitations (A2 and B2) of Ki67+ or PH3+ cardiomyocytes were shown. Scale bar =20 μm. n=3. *P<0.05 compared with control.

CGX1321 inhibited both canonical and non-canonical Wnt signaling in cardiomyocytes

A previous report has suggested that inhibition of β-catenin-mediated transcription is linked with cardiomyocyte proliferation in zebrafish [26]. We performed experiments to investigate the changes in canonical and non-canonical proteins by CGX1321. The activation of canonical Wnt pathway is dependent on β-catenin nuclear translocation, and the activation of non-canonical Wnt pathway is dependent on NFAT nuclear translocation [27]. As shown in Figure 5A,B, hypoxia increased nuclear translocation of β-catenin in NRVMs, which was significantly inhibited by CGX1321, indicating that CGX1321 inhibited β-catenin-mediated transcription. Besides, CGX1321 also inhibited the nuclear translocation of no-canonical molecule NFAT induced by hypoxia in NRVMs. These results suggested that CGX1321 exerted an inhibitory effect on both canonical and no-canonical Wnt pathways.

CGX1321 inhibited both canonical and non-canonical Wnt signaling in cardiomyocytes

Figure 5
CGX1321 inhibited both canonical and non-canonical Wnt signaling in cardiomyocytes

(A) Representative images (A1) and quantitation (A2,A3) of nucleus and total β-catenin protein expression in NRVMs, analyzed by Western blotting. GAPDH and histone H3 were used as controls. n=3. *P<0.05 compared with control; #P<0.05 compared with hypoxia. (B) Representative images (B1) and quantitation (B2,B3) of nucleus and total NFATc3 protein expression in NRVMs, analyzed by Western blotting. GAPDH and histone H3 were used as controls. n=3. *P<0.05 compared with control; #P<0.05 compared with hypoxia.

Figure 5
CGX1321 inhibited both canonical and non-canonical Wnt signaling in cardiomyocytes

(A) Representative images (A1) and quantitation (A2,A3) of nucleus and total β-catenin protein expression in NRVMs, analyzed by Western blotting. GAPDH and histone H3 were used as controls. n=3. *P<0.05 compared with control; #P<0.05 compared with hypoxia. (B) Representative images (B1) and quantitation (B2,B3) of nucleus and total NFATc3 protein expression in NRVMs, analyzed by Western blotting. GAPDH and histone H3 were used as controls. n=3. *P<0.05 compared with control; #P<0.05 compared with hypoxia.

CGX1321 did not alter YAP activity and localization in vitro and in vivo

It has been reported that Hippo/YAP pathway restrains cardiomyocyte proliferation through interaction with β-catenin [28]. We next determined whether YAP activity and localization were involved in CGX1321-induced cardiomyocyte proliferation. The pYAP (S127) and total YAP were detected with Western blot analysis in NRVMs subjected to hypoxia. As a result, CGX1321 did not change both protein expressions (Supplementary Figure S3A). Besides, the YAP nuclear translocation in cardiomyocytes was detected in post-MI heart with immunostaining. CGX1321 did not change the percentage of cardiomyocytes with nuclear expression of YAP in the infarct border zone (Supplementary Figure S3B). These results indicated that the CGX1321 induced cardiomyocyte proliferation might not be dependent on Hippo/YAP signaling pathway.

The elevation of cell cycle regulating genes mediates CGX1321-induced ACM proliferation

To explore the underlying mechanism response for CGX1321 induced ACM proliferation, mRNA microarray was performed. As shown in Figure 6, three post-MI hearts with vehicle and three post-MI hearts with CGX1321 treatment were used for the array. GO analysis showed that the genes regulating the function of mitotic nuclear division, cell division were significantly altered. Amongst those genes, the regulatory genes for the attachment of spindle microtubules to kinetochore changed most significantly. Meanwhile, pathways analysis also showed the signaling pathways regulating cell cycle were one of the most significant change. Further, the mRNA expression of cell cycle regulating genes Ckdn2a, Cdk1, Bub1, Ccna2, Ccnb1, Ccne1, Cdc20, Mcm2, Mcm5, and PLK1 in post-MI hearts treated with CGX1321 were significantly increased by over two-fold compared with control post-MI hearts.

CGX1321 elevated the expressions of cell cycle regulating genes in post-MI heart tissue

Figure 6
CGX1321 elevated the expressions of cell cycle regulating genes in post-MI heart tissue

Heart tissues from three post-MI hearts with vehicle and three post-MI hearts with CGX1321 treatment were harvested for mRNA microarray. (A) The heat-map image of hierarchical clustering based on the most variable genes amongst post-MI hearts with or without CGX1321 administration. The scale extends from 0.25- to 4-fold over mean (−2 to +2 in log2 scale) as indicated on the bottom. (B,C) GO analysis of the mRNA microarray. (D) Pathway analysis of the mRNA microarray. (E) Profiling of cell cycle regulatory gene expression in post-MI hearts with or without CGX1321 administration. The genes up-regulated at least two-fold in the post-MI hearts with CGX1321 compared with those without CGX1321 by microarray analyses are listed with their fold enrichment. The ratios (fold-changes) of gene expressions in the two groups are also shown.

Figure 6
CGX1321 elevated the expressions of cell cycle regulating genes in post-MI heart tissue

Heart tissues from three post-MI hearts with vehicle and three post-MI hearts with CGX1321 treatment were harvested for mRNA microarray. (A) The heat-map image of hierarchical clustering based on the most variable genes amongst post-MI hearts with or without CGX1321 administration. The scale extends from 0.25- to 4-fold over mean (−2 to +2 in log2 scale) as indicated on the bottom. (B,C) GO analysis of the mRNA microarray. (D) Pathway analysis of the mRNA microarray. (E) Profiling of cell cycle regulatory gene expression in post-MI hearts with or without CGX1321 administration. The genes up-regulated at least two-fold in the post-MI hearts with CGX1321 compared with those without CGX1321 by microarray analyses are listed with their fold enrichment. The ratios (fold-changes) of gene expressions in the two groups are also shown.

Discussion

Although modern therapies with rapid establishment of sufficient reperfusion have significantly improved clinical outcomes for MI, the high mortality of MI patients from post-MI heart failure due to segmental loss of ventricular myocardium remains a major problem [29]. In adult patients, the damaged myocardium is eventually replaced mainly by non-contractile fibrous tissue (scar). Albeit some new myocytes can be seen in the border zones around the infarct core, the rate of myocyte formation is too low to result in significant myocardial repair after heart injury [25]. In the present study, we applied a novel porcupine inhibitor CGX1321, which has entered phase I clinical trial, to the treatment of MI in a mice model. CGX1321 exerted a therapeutic effect against MI, proved by increased cardiac function and reduced infarct size. CGX1321 did not limit the apoptosis of the cardiomyocytes in post-MI heart, but increased the proliferation of cardiomyocytes and promoted the cardiac regeneration. A bunch of genes promoting cell cycle re-entry of cardiomyocytes were stimulated, which might be associated with increased new cardiomyocytes.

In clear contrast with reparative healing or scar formation in the adult, regeneration of new functional cardiomyocytes is the major healing mechanism in the fetus in response to MI [30]. The differential response between the fetus and the adult to MI spurs significant interest in researchers and clinicians in cardiovascular medicine in endogenous cardiac regeneration of damaged heart [31,32]. Wnt signaling pathway is vital for heart development but plays a complex role. Activation of Wnt/β-catenin signaling has been shown to be crucial for mesoderm cells to be specified into the cardiac lineage, whereas later inhibition of the Wnt/β-catenin pathway is critical for defining size or maturation status of the heart field [5,6]. In the adult heart, Wnt pathway is thought to be silent but it is activated during cardiac injury including MI [8]. Both activation and inhibition of Wnt signaling play a complex and necessary role in the repair of cardiac tissue post MI [33]. Both positive and negative roles for the Wnt pathway are observed depending on the model used, and manipulation of Wnt pathways leads to mixed results in post-MI animals [11,13,3336]. Our study showed that CGX1321 blocked the secretion of Wnt proteins, and inhibited both canonical and non-canonical Wnt signaling pathways. CGX exerted a therapeutic effect against MI, agreeing with previous studies that inhibiting WNT pathways benefits cardiac repair. On the other hand, some of the discrepant observations by some studies [3436] may be explained by ligand and cell-type-dependent effects (on endothelial cells, smooth muscle cells, and neonatal/ACMs) of Wnt signaling on healing. Besides, it has been reported that mutation of Wnt5a and Wnt11 results in increased angiogenesis and these ligands elicit RMC responses via a non-canonical Wnt pathway [37], implying that Wnt inhibition could promote angiogenesis. Therefore the therapeutic effect of CGX1321 on MI could be synergetic effects including angiogenesis.

Wnt signaling has been considered to be a drug target for the treatment of heart failure following MI. Potential points of intervention on the Wnt signaling pathway are: (i) outside the cell (cell membrane, preventing the formation of the Wnt/frizzled/LRP complex); (ii) in the cell cytoplasm (affecting the destruction complex); or (iii) within the nucleus (modulating the effect of β-catenin on the gene transcription) [33]. Unfortunately, due to the difficulty in developing potent and specific compounds targetting the previously known points of intervention, few drugs have been successfully developed. Nonetheless, tool compounds of Wnt signaling inhibitor have shown proof-of-concept. For example, Pyrvinium, a non-selective inhibitor of Wnt signaling improves cardiac remodeling after MI [38]. Small-molecule compounds are identified to control proliferation or differentiation of early beating cardiomyocytes through modulation of the Wnt/β-catenin signaling pathway [39]. Another compound, UM206 that antagonizes the effect of Wnt-3a and Wnt-5a, reduces infarct expansion and preserves cardiac function after MI [40].

Porcupine, an acyltransferase that enables secretion and activity of all Wnt proteins, is highly druggable. An inhibitor of porcupine, LGK974 has entered human clinical trial for treating several tumors and has been proven safe in human subjects [12]. Wnt-974 has been found to exert a therapeutic effect on MI by decreasing aberrant remodeling and fibrosis [11]. Another porcupine inhibitor GNF-6231 can also treat MI and has been reported to promote proliferation of cardiac progenitors and other interstitial cells [13]. Wnt inhibitor ICG-001 enhances cardiac regeneration indicated as increased BrdU positive cardiomyocytes [41], but the source of the newly formed cardiomyocytes is still unclear. By using fate-mapping technologies, accumulating evidence by us [14] and others [1517] demonstrate that the major source of the newly formed cardiomyocytes is pre-existing cardiomyocytes. This raises the possibility that therapeutic alteration of the signaling environment of the injured heart might activate compensatory proliferation of cardiomyocytes. Thus it is meaningful to explore if porcupine inhibitors could promote cardiomyocyte proliferation. Our study not only confirmed the therapeutic function of porcupine inhibitor against MI, but also showed that CGX1321 was able to enhance cardiomyocyte proliferation and promote cardiac regeneration. These findings were consistent with a previous report that inhibition of Wnt/β-catenin pathway promoted cardiomyocyte proliferation in zebrafish [26].

Hippo/YAP signaling pathway plays an important role in myocyte proliferation [23,42]. Hippo signaling can suppress the β-catenin/YAP interaction in differentiating cardiomyocytes, thus leading to decreased proliferation of cardiomyocytes [28]. However, these experiments were mostly performed in embryonic stage of heart. Our data showed that CGX1321 did not alter YAP activity and localization, indicating that its pro-proliferation effect was not dependent on Hippo/YAP signaling pathway. Thus how Wnt signaling regulates cardiomyocyte proliferation is largely unknown. CGX1321 exerted an inhibitory effect on both canonical and non-canonical Wnt pathways. CGX1321 inhibited β-catenin-mediated transcription, which was indicated to be associated with cardiomyocyte proliferation in zebrafish [26]. Cell cycle regulating genes Ckdn2a, Cdk1, Bub1, Ccna2, Ccnb1, Ccne1, Cdc20, Mcm2, Mcm5, and PLK1 in post-MI hearts treated with CGX1321 were significantly increased by over two-fold compared with control post-MI hearts. It has been reported that pro-proliferative genes Ccna2 and Cdc20 are Wnt/β-catenin target genes [28,43]. Thus, CGX1321 increased cardiomyocyte proliferation might be associated with β-catenin-mediated suppression of Ccna2 and Cdc20.

In conclusion, a novel Wnt signaling inhibitor CGX1321 exerted a therapeutic effect on MI injury by limiting fibrosis and promoting cardiac regeneration. Stimulation of post-MI cardiomyocyte proliferation may at least partially account for the CGX1321-induced cardiac regeneration, which may be associated with the regulation of cell cycle related genes.

Clinical perspectives

  • For MI, the current treatments do not address the underlying cardiac tissue remodeling as a result of cell death, tissue fibrosis, and lack of cardiomyocyte regeneration. Novel drug targets and drug candidates that aim at the underlining pathologic changes in remodeling are highly desirable. In the present study, we identified the therapeutic effect of a novel Wnt signaling inhibitor (CGX1321) against MI and its role in cardiac regeneration.

  • Our study demonstrated that CGX1321 administration improved cardiac function and reduced myocardial infarct size of post-MI heart mainly through enhancing cardiomyocyte proliferation.

  • Our study suggested that inhibition of Wnt signaling reduced MI injury by limiting fibrosis and promoting cardiomyocyte proliferation mediated regeneration. Wnt pathway inhibitors might be explored further as drug candidates for treating heart failure following MI.

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This study was supported by grants from the National Natural Science Foundation of China [31730043; 81400256], and the Program of Innovative Research Team by National Natural Science Foundation [81721001].

Author contribution

D.Y., W.F. designed the study, performed experiments and analyzed the data. L.L., X.X., Q.L., R.Y., H.C. bred the mice and performed experiments. S.A., X.C. provided valuable comments and reagents, analyzed the data and edited the manuscript. C.Z., W.W. (China) conceived and supervised the study, analyzed the data and wrote the manuscript.

Abbreviations

     
  • ACM

    adult cardiomyocyte

  •  
  • FS

    fractional shortening

  •  
  • LAD

    left anterior descending

  •  
  • LV

    left ventricle

  •  
  • LVDPW

    left ventricular diastolic posterior wall

  •  
  • LVIDs

    systolic LV internal diameter

  •  
  • LVIDd

    diastolic LV internal diameter

  •  
  • LVEF

    LV ejection fraction

  •  
  • LVFS

    LV fractional shortening

  •  
  • MI

    myocardial infarction

  •  
  • NRVM

    neonatal rat ventricular myocyte

  •  
  • PH3

    phosphohistone H3

  •  
  • TUNEL

    terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling

  •  
  • TTC

    Triphenyl-tetrazolium chloride

  •  
  • YAP

    Yes Associated Protein

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Author notes

*

These authors contributed equally to this work.

Supplementary data