Inhibition of the Notch1 pathway induces peripartum cardiomyopathy

Abstract Increased expression and activity of cardiac and circulating cathepsin D and soluble fms‐like tyrosine kinase‐1 (sFlt‐1) have been demonstrated to induce and promote peripartum cardiomyopathy (PPCM) via promoting cleavage of 23‐kD prolactin (PRL) to 16‐kD PRL and neutralizing vascular endothelial growth factor (VEGF), respectively. We hypothesized that activation of Hes1 is proposed to suppress cathepsin D via activating Stat3, leading to alleviated development of PPCM. In the present study, we aimed to investigate the role of Notch1/Hes1 pathway in PPCM. Pregnant mice between prenatal 3 days and postpartum 3 weeks were fed with LY‐411575 (a notch inhibitor, 10 mg/kg/d). Ventricular function and pathology were evaluated by echocardiography and histological analysis. Western blotting analysis was used to examine the expression at the protein level. The results found that inhibition of Notch1 significantly promoted postpartum ventricular dilatation, myocardial hypertrophy and myocardial interstitial fibrosis and suppressed myocardial angiogenesis. Western blotting analysis showed that inhibition of Notch1 markedly increased cathepsin D and sFlt‐1, reduced Hes1, phosphorylated Stat3 (p‐Stat3), VEGFA and PDGFB, and promoted cleavage of 23k‐D PRL to 16‐kD PRL. Collectively, inhibition of Notch1/Hes1 pathway induced and promoted PPCM via increasing the expressions of cathepsin D and sFlt‐1. Notch1/Hes1 was a promising target for prevention and therapeutic regimen of PPCM.

tively. We hypothesized that activation of Hes1 is proposed to suppress cathepsin D via activating Stat3, leading to alleviated development of PPCM. In the present study, we aimed to investigate the role of Notch1/Hes1 pathway in PPCM. Pregnant mice between prenatal 3 days and postpartum 3 weeks were fed with LY-411575 (a notch inhibitor, 10 mg/kg/d). Ventricular function and pathology were evaluated by echocardiography and histological analysis. Western blotting analysis was used to examine the expression at the protein level. The results found that inhibition of Notch1 significantly promoted postpartum ventricular dilatation, myocardial hypertrophy and myocardial interstitial fibrosis and suppressed myocardial angiogenesis. Western blotting analysis showed that inhibition of Notch1 markedly increased cathepsin D and sFlt-1, reduced Hes1, phosphorylated Stat3 (p-Stat3), VEGFA and PDGFB, and promoted cleavage of 23k-D PRL to 16-kD PRL. Collectively, inhibition of Notch1/ Hes1 pathway induced and promoted PPCM via increasing the expressions of cathepsin D and sFlt-1. Notch1/Hes1 was a promising target for prevention and therapeutic regimen of PPCM.

K E Y W O R D S
Cathepsin D, Notch1, PPCM, PRL, sFlt1 soluble fms-like tyrosine kinase-1 (sFlt-1) has also been demonstrated to impair cardiac capillary network through inhibiting pro-angiogenic vascular endothelial growth factor (VEGF) and placental growth factor (PIGF) activities. 3 The combination of bromocriptine (PRL inhibitor) and recombinant VEGF is a curative option for PPCM. 1,4 As a canonical target gene of Notch pathway, Hes1 has been found to regulate angiogenesis. 5,6 Our previous work has suggested that Hes1 is able to protect ischaemic myocardium via mediating the phosphorylation of Stat3. 7 Reportedly, Stat3 modulates proliferation, differentiation, survival, oxidative stress and/or metabolism in cardiomyocytes, fibroblasts, endothelial cells, progenitor cells and various inflammatory cells. 8 Indeed, Hilfiker-Kleiner 2 and colleagues have found that activated Stat3 can inhibit cathepsin D, which then suppresses PPCM development. Furthermore, our team has also found that Notch1 can promote VEGF-mediated cardiac angiogenesis in ischaemic regions 9 and inhibit myocardial fibrosis. 10,11 Bioinformatic analysis shows that there are multiple Hes1 binding sites in the promoter region of cathepsin D and sFlt-1, indicating a potential role of Hes1 in PPCM.
We hypothesized that inhibition of Notch1/Hes1 induced and promoted PPCM via increasing cathepsin D and sFlt-1. In the present study, we used LY-411575 (γ-secretase inhibitor) to suppress Notch1 pathway and decrease Hes1 expression 12 to explore the potential role of Notch1/Hes1 in PPCM.

| Animal experiments
Female C57BL/6J mice (6-8 weeks of age) were purchased from Slaccas Co., Ltd. All animal studies were performed at Experimental Animal Center of Nanchang University in accordance with the Guideline of US National Institutes of Health (NIH), and animal-related protocols were approved by the Institutional Committee for Use and Care of Laboratory Animals of Nanchang University. The mice with PPCM during pregnancy and breastfeeding were assigned into the peripartum group, while the nulliparous mice were used as the control group. Mice were administered by gavage with LY-411575 (10 mg/ kg/d, diluted in 0.4% methylcellulose) daily starting 3 days before delivery until 3 weeks after delivery. The blank mice were dosed with 0.4% methylcellulose vehicle. The PPCM phenotype was verified as previously described. 13 At the end of the dosing period, mice were sacrificed by CO 2 asphyxiation, total blood was collected and centrifuged at 1500 g for 10 minutes at room temperature to obtain serum.
Serum samples were stored at −80°C until analysis. The heart tissues were surgically isolated for further analysis.
The heart rate and body temperature were maintained and recorded.

| Measurement of cathepsin D and sFlt1 in serum
Cathepsin D and sFlt1 were detected using commercial ELISA kits for cathepsin D and sFlt1 according to the manufacturer's instructions. All samples were simultaneously detected. Serum concentrations of cathepsin D and sFlt1 were determined using standard curves and expressed as units per litre (mg/L). The linear ranges for cathepsin D and sFlt1 were 0-50 mg/L.

| Histological analyses
For histological analyses, mouse hearts were fixed in situ by retrograde perfusion with PBS (pH 7.4) containing 50 mM KCl and 200 U/ mL heparin for 2 minutes at 80 mm Hg, followed by in situ paraformaldehyde fixation. Sections were embedded in paraffin and stained with H&E and wheat germ agglutinin (WGA, Alexa Fluor 488 conjugate; Thermo Fisher). Masson (HT15-1KT; Sigma-Aldrich) staining was performed to determine collagen deposition following the manufacturer's instruction. Tissue morphometry was performed in a blinded fashion using the Quantimet 500MC digital image analyzer.

| Western blotting analysis
The left ventricle tissues were lysed in cell lysis buffer (Beyotime Institute of Biotechnology) at 4°C. Equal amounts of proteins were subjected to 8%-10% SDS-PAGE and then transferred onto nitrocellulose membranes (Millipore). The blots were blocked in 10% non-fat milk in TBST. Membranes were incubated with primary antibodies at 4°C overnight, followed by incubation with secondary antibodies at room temperature for 1 hour. The immunoreactive bands were visualized using ECL kit (Thermo Scientific) and analysed by ImageQuant LAS4000 (GE).

| Quantitative real-time PCR
Total RNA was extracted with TRIzol reagent (Thermo Fisher).

| Statistical analysis
Data were expressed as mean ± SD and analysed by SPSS 18.0 package (SPSS Inc). The obtained data conform the ANOVA assumptions as evaluated using Shapiro-Wilk normality test and Levene's test for the equality of variances. Comparisons between groups were analysed by two-way ANOVA with Bonferroni's post-test. P < .05 was considered statistically significant.

| Inhibition of Notch1 induces and promotes postpartum ventricular dilatation
To explore the role of Notch1 in PPCM, we randomly formed four groups (n = 6) as follows: nulliparous, peripartum, nulliparous LY-411575 and peripartum LY-411575 . Figure 1 shows that LVEDD Collectively, these data suggested that the Notch1 pathway played a protective role in PPCM, and inhibition of Notch1 could induce and promote PPCM.

| Inhibition of Notch1 promotes ventricular hypertrophy and myocardial interstitial fibrosis
Ventricular hypertrophy and myocardial interstitial fibrosis are remarkable pathological changes in PPCM. To confirm whether Notch1 was involved in these histopathological changes, we compared the HW/BW ( Figure 3A) and HW/TL ( Figure 3B) among different groups and evaluated histological changes of myocardium ( Figure 3C). It demonstrates that there was just interstitial fibrosis ( Figure 3D

| Inhibition of Notch1 suppresses postpartum myocardial angiogenesis
Myocardial angiogenic imbalance is essential for PPCM. Here, we evaluated myocardial capillary density and detected angiogenic factors among different groups.

F I G U R E 3
Inhibition of Notch1 promotes myocardial hypertrophy and interstitial fibrosis in the left ventricle. A, The ration of heart weight/bodyweight, HW/BW. B, The ratio of heart weight/tibia length, HW/TL. C, Haematoxylin-eosin was used to evaluate the ventricular wall thickness and cavity. D, Masson's trichrome staining was used to evaluate the fibrosis. E, Wheat germ agglutinin staining was used to analyse the cardiomyocyte surface area. F, The relative expression of ANP, BNP, β-MHC and COL1A1 mRNA was evaluated by real-time PCR. All data were presented as mean ± SD (n = 6). *P < .05, **P < .01 versus indicated group. Comparisons between groups were analysed by two-way ANOVA with Bonferroni's post-test

| Inhibition of Notch1 decreases N1ICD, Hes1 and p-Stat3 and increases cathepsin D
The above-mentioned findings showed that Notch1 was involved in PPCM. As a canonical target gene of Notch1, we wondered whether Hes1 mediated Notch1-regulated PPCM.

| D ISCUSS I ON
The aetiology and aetiopathogenesis of PPCM remain elusive.
However, myocardial hypertrophy, interstitial fibrosis and ventricular dilatation are common pathological changes. 4  Our data here showed that inhibition of Notch1 by LY-411575 significantly down-regulated the expressions of N1ICD, Hes1, p-Stat3 and pro-angiogenic factors, such as VEGFA, PDGFB and BFGF, and increased the production of cathepsin D and anti-angiogenic factors, such as sFlt-1 and 16-kD PRL. Consistent with these changes, we found significant myocardial hypertrophy and myocardial interstitial fibrosis as well as reduced myocardial capillary density in LY-411575-treated mice. Overall, these findings strongly indicated that the sequential activation of Notch1/ Hes1/Stat3 might contribute to correcting angiogenic imbalance and alleviating PPCM.

| CON CLUS ION
Our data strongly supported the idea that imbalances in angiogenic signalling contribute to PPCM, and Notch1/Hes1pathway may play a protective role in such disorder via regulating cathepsin D and sFlt-1.

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and analysed during the current study are available from the corresponding author on reasonable request.