Notum Protects Against Myocardial Infarction-Induced Heart Dysfunction By Alleviating Cardiac Fibrosis


 Cardiac fibrosis is a pathological reparative process that occurs subsequent to myocardial injury. It is associated with cardiac systolic and diastolic dysfunction and reduced cardiac compliance that eventually leads to heart failure. Delaying or inhibiting the progression of pathological myocardial fibrosis is of great significance for the treatment of many cardiovascular diseases. The Wnt signaling pathway is closely related to the occurrence of organ fibrosis, and Notum is a highly conserved secreted feedback inhibitor of Wnt signaling. It has been shown that Notum acts as a regulator in many organs, such as the aging intestinal epithelium, adult ventricular-subventricular zone neurogenesis, and mouse tooth root development. However, the role and mechanism of Notum on cardiac fibrosis are not well-understood. In this study, we found that Notum significantly increased survival rate and improved cardiac function following myocardial infarction in mice. More importantly, Notum inhibited the Wnt/β-catenin signaling pathway and senescence of cardiac fibroblasts, thereby decreasing the activation of cardiac fibroblasts, reducing the excessive deposition of extracellular matrix, and ultimately inhibiting the occurrence of cardiac fibrosis. Taken together, our findings demonstrated the anti-fibrotic effects of Notum on maladaptive cardiac fibrosis, and suggest that it may be a new strategy for the treatment of cardiac fibrosis.

occurrence and development of brosis [12][13][14]. However, the pathogenesis of cardiac brosis still needs in-depth study, and there are currently limited means to effectively reduce or reverse this process.
Wnt pathway plays an important role in the development and homeostasis of many organs, including liver and heart [15,16]. Numerous studies have shown that the Wnt pathway participates in the process of organ brosis, such as cardiac brosis [17][18][19][20], renal brosis [21], etc. The results from Yang et al. found that CGX1321 administration blocked the secretion of Wnt proteins, inhibited both canonical and noncanonical Wnt signaling pathways, then reduced myocardial infarct size and brosis, and nally improved cardiac function in MI mice [16]. Qian et al. found that downregulation of S100A4 alleviates cardiac brosis via Wnt/β -catenin pathway in mice [22]. In addition, Jeong and colleagues found that Cdon de ciency causes hyperactive Wnt signaling leading to aberrant intercellular coupling and cardiac brosis [23].
Notum, an endogenous feedback inhibitor of the Wnt signaling pathway, is a carboxylesterase with the speci c activity of deacylating Wnts by removing an essential palmitoleic moiety [24]. Pentinmikko et al. con rmed that senescent Paneth cells can produce Notum and inhibit the regeneration of aging intestinal epithelial cells. In addition, inhibiting Notum in mice can enhance the regeneration ability of aging stem cells and promote the regeneration of aging tissue [25]. However, whether Notum can regulate cardiac brosis, and through what kind of mechanism, has not been con rmed.
In this study, we determined the cardioprotective effect of Notum in mice with myocardial infarction and preliminarily clari ed the mechanisms by which Notum exerted an anti-brotic effect in the myocardium.
These results support a new strategy for the prevention and treatment of myocardial brosis.

Animal model and treatment
The procedures for the use of animals in this work were in accordance with the regulations of the Primary cardiac broblasts were isolated from 2 to 3-day-old Kunming mice. The hearts of the mice were taken out and placed in a petri dish with ice-cold DMEM (Biological Industries, Israel).Then the heart tissues were cut into 1mm cubed pieces with sterilized scissors and digested by trypsin (0.5mg/ml) completely. The supernatant were centrifuged at 1300rpm for 7min after ltration. Furthermore, the cells were re-suspended in DMEM containing 10% fetal bovine serum (FBS, Biological Industries) and seeded into culture plates. After 1.5h, the non-adherent cells were removed. The cardiac broblasts (adherent cells) were cultured at 37℃ with 5% CO 2 .

Western blot
Protein of heart tissues and cardiac broblasts were extracted with RIPA lysis buffer (Beyotime, Jiangsu, China). Protein samples (80 or 50µg) were separated by SDS-PAGE and then transferred to polyvinylidene uoride membranes (Pall Life Sciences, Ann Arbor, MI, USA). After 2 hours of blocking in 5% skim milk, the membranes were incubated with primary antibodies at 4°C overnight. The next day, these membranes were washed with PBST 3 times and incubated with the secondary antibody for 1h. The Odyssey Infrared Imaging System was used to scan the membranes. The Western blot results were analyzed by Image Studio Ver 5.2 software.

qRT-PCR
Total RNA was extracted from heart tissues or cardiac broblasts using a Trizol standard protocol. The concentration and purity of RNA was detected by Nano-Drop 8000 Spectrophotometer (Thermo, USA).
According to the manufacturer's instructions, RNA was reverse transcripted into cDNA with High Capacity cDNA Reverse Transcription Kit (Transgene, AT341-02) and the relative mRNA levels were detected by qRT-PCR with SYBR Green I (Roche, 4913914001). The Ct values were calculated to analyze mRNAs relative levels, and these data were normalized to GAPDH.

Histology analysis
Hearts were xed in 4% paraformaldehyde for 3 days before dehydrating, then they were embedded in para n and cut into 6µm slices. According to the manufacturer's instructions, the slices were stained with H&E and Masson's trichrome to evaluate the degree of brosis. The brotic areas were calculated with Image-Pro plus 6.0 software.

Immunohistochemistry staining
Dewaxing and rehydrating cardiac sections with dimethylbenzene and gradient ethanol, then these sections were treated with 3% H 2 O 2 for 10 min before repairing antigen with Sodium Citrate Buffer in microwave. Sections were blocked with 50% goat serum at 37 °C for 1h and incubated with primary antibodies against FN1 purchased from Proteintech, α-SMA and β-catenin from Abcam and p16 from Wanlei. HRP conjugated secondary antibodies (ZsBio, Beijing, China) were used to combine with the primary antibodies. Then the sections were stained with DAB (ZsBio) and the nucleus was stained with hematoxylin (Solarbio, China).

Immuno uorescence staining
After treatment of TGF-β1 and notum, cardiac broblasts were washed with PBS for three times and xed in 4% paraformaldehyde for 30 min at temperature. Then the cells were penetrated with 0.4% Triton X-100 for 1h at room temperature, and blocked with 50% normal goat serum at 37 °C for 1 h .The cells were washed for three times and incubated with antibody against α-SMA (1:200, Abcam) at 4 °C overnight. The next day, after three times' washing, cells were incubated with FITC-conjugated goat anti-mouse antibody in the dark for 1 h. The nucleuses were stained with DAPI (Roche Molecular Biochemicals). Immuno uorescence was observed with microscope (Olympus, IX73, Japan).

Proliferation assay
Primary cardiac broblasts were cultured and treated in 24-well plates, Cell-Light EdU DNA Cell Proliferiation Kit (RiboBio, Guangzhou, China) was used to detect cell proliferation. All experimental procedures were based on manufacturer's instructions. The images were observed with a uorescence microscope (Olympus, IX73, Japan).

Wound-healing Scratch assay
Scratch assay was performed to evaluate the migration of cardiac broblasts. Cells were seeded in 6-well plates, wounds were created by scratching the cell monolayer with 10-μl pipette tips. Then cells were washed with PBS for two times and treated as indicated for 24h. Images were captured at 0h, 12h and 24h with the Nikon TS100 microscope (Nikon, Japan).And the relative cell migration was analyzed by Image J.

Statistical analysis
Data are presented as mean ± SEM. One way analysis of variance (ANOVA) followed by Bonferroni or Dunnett's post-hoc test was used for multiple group comparisons. P< 0.05 was considered statistically signi cant. Graph Pad Prism 8.0 was used for statistical analyses.

Notum increases survival and improves cardiac function in MI mice
To investigate the effects of Notum on the heart, C57BL/6 mice underwent ligation of the left coronary artery surgery for 7 days to induce myocardial infarction (MI), and Notum was injected into the heart at the same time ( Figure 1A). As shown in Figure 1B, the survival rate post-MI was observed up to 7 days post-surgery, which was signi cantly higher in the MI+Notum group (23/25; 92%) than that in the MI group (18/25; 72%). Furthermore, cardiac function was tested by echocardiography on 7 day post-MI ( Figure 1C), and the data showed that. cardiac systolic dysfunction was signi cantly noted in MI mice compared to that in Sham group, as evidenced by decreased LVEF Sham 52.26±5.25% vs. MI 40.38±4.51% and LVFS (Sham 26.11±3.33% vs. MI 19.43±2.21%). However, in MI+Notum group, the LVEF (57.35±11.81%) and LVFS (30.05±7.49%) were signi cantly higher in comparison with mice in the MI group ( Figure 1D and 1E). Further analysis of the data reveals that treated with Notum was also associated with signi cant decreases in LV systolic (MI 43.35±17.43µl vs. MI+Notum 26.54±12.57µl) and diastolic volume (MI 79.10±22.08µl vs. MI+Notum 59.30±14.70µl) relative to MI group ( Figure 1F and 1G). These results suggested that treated with Notum could improve cardiac function and attenuate cardiac remodeling in MI mice model.

Notum prevents MI-induced cardiac brosis in mice
To explore the potential role of Notum on cardiac brosis post-MI, at the 7 day post-operation, the mice hearts were obtained and processed for histological analysis. According to H&E and Masson staining, compared with the sham group, the myocardial tissue around the infarct region in MI group was arranged in disorder with a large number of in ammatory cells necrosis and in ltration, but this situation was signi cantly improved in Notum+MI group (Figure 2A). Consistently, the Masson staining exhibited less brosis areas in the Notum-treated mice when compared to the MI group ( Figure 2B).
The transformation of broblasts into myo broblasts is an important feature of brosis, α-Smooth muscle actin (α-SMA) and Fibronectin 1 (FN1) are the widely used markers of CF to cardiac myo broblasts (CMF) differentiation [27,28]. In our study, the western blot results of cardiac tissue in the infarct border zone showed that the expression of α-SMA and FN1 were signi cantly up-regulated in MI mice compared with that in sham operation mice, and which could be inhibited after treatment with Notum ( Figure 2C). Furthermore, immunohistochemistry assay showed that treated with Notum eliminated the MI-induced up-regulation of α-SMA and FN1, suggesting that Notum diminished the differentiation of myo broblasts ( Figure 2D and 2E). Meanwhile, compared with the MI group, the high mRNA levels of FN1, Col 1α1 and Col 3α1 were strikingly reversed in Notum+MI group ( Figure. 2F).

Notum attenuates cardiac broblasts activation induced by TGF-β1
TGF-β1 is a classic promotes brogenic cytokine, which can cause the transformation of CF to MFs and lead to the deposition of ECM [29]. In vitro, to determine the role of Notum on cardiac broblast, primary cultures of cardiac broblasts were treated with TGF-β1 and TGF-β1+Notum for 24 hours. As shown in Figure 3A, the addition of Notum could effectively inhibit the high expression of FN1 and the production of collagen 1 induced by TGF-β1. The results of real-time polymerase chain reaction (RT-PCR) also showed that the addition of Notum could inhibit the up-regulation of FN1, Col 1α1, Col 3α1 and α-SMA at mRNA level induced by TGF-β1 ( Figure 3B). Moreover, Notum alleviated the TGF-β1-induced proliferation of CFs ( Figure 3C). We used scratch assay to detect the effect of Notum on the migration ability of broblasts. As illustrated in Figure3D, the addition of Notum can signi cantly inhibit the cell migration induced by TGF-β1. Furthermore, the expression of α-SMA in cardiac broblasts treated with TGF-β1 was signi cantly increased, and the well-organized α-SMA laments could be clearly seen in immuno uorescence results, indicating the transformation of myo broblasts. However, Notum could inhibit the TGF-β1-induced MF transformation and the overexpression of α-SMA ( Figure 3E).

Notum inhibits Wnt/β-catenin signaling activation in CFs
To determine the role of the Notum in the process of cardiac brosis, we detected whether Notum can inhibit the activation of Wnt/β-catenin signal pathway in cardiac broblasts in vivo and in vitro. As illustrated in Figure 4A, the results of immunohistochemistry staining showed that the expression of βcatenin was signi cant increase in the infarcted border zone of MI mice, whereas that was obvious inhibited after Notum injection. Consistent with the immunohistochemistry results, the qRT-PCR and Western blot results also showed that the mRNA and protein level of β-catenin were strikingly decreased after injection of Notum in MI mice ( Figure 4B&C). Meanwhile, as shown in Figure 4D, treated with Notum abolished TGF-β1-induced elected β-catenin expression, and also the glycogen synthase kinase 3β (GSK3β), an important component of β-catenin destruction complex [30]. Moreover, Notum reversed the upregulation of mRNA levels of β-catenin induced by TGF-β1 in CFs ( Figure. 4E).

The effect of Notum on cardiac broblasts senescence
Aging is not a disease but a physiological and pathological process involving attenuated cellular function and weakened stress resistance [31]. The pathological senescence of myocardial broblasts after myocardial infarction is one of the key factors inducing brosis. In order to further explore the mechanisms of Notum regulating cardiac brosis, primary cultured cardiac broblasts were treated with H 2 O 2 for 2 hours. Western blot results showed that the increased expression of p53 revealed the activation of aging pathway. Meanwhile, the upregulation of p53 was signi cantly reversed when Notum were pretreatment for 22 hours ( Figure 5A). In addition, the RT-PCR results showed that, compared with the H 2 O 2 group, treated with Notum decreased the expression of the aging markers p21 and p16 at mRNA level ( Figure 5B and 5C). Furthermore, as illustrated in Figure 5D, treatment with Notum prevented p16 upregulation in the infarcted border zone of MI mice.

Discussion
In this study, we evaluated the important role of Notum, and its mechanisms of action, in governing cardiac brosis. Our results revealed that, after intervention with Notum, the cardiac function and survival of mice was signi cantly improved within seven days post-myocardial infarction. Furthermore, we showed that Notum inhibited the senescence of cardiac broblasts and their pathological transformation into cardiac myo broblasts, reduced the production of collagen, and blocked activation of the Wnt/βcatenin pathway. We preliminarily demonstrated the therapeutic e cacy of Notum in broblasts, and in vivo in a mouse model with cardiac brosis induced by myocardial infarction. These experimental ndings led us to hypothesize that Notum could be considered a new therapeutic strategy for the prevention and treatment of cardiac brosis ( Figure 6).
Cardiac brosis is a pathological condition that occurs after injury and during aging and is present in almost all types of heart disease. It is caused by the excessive net accumulation of ECM, resulting in cardiac interstitial dilatation, decreased compliance, arrhythmias, and heart failure [8]. Currently, there are no effective anti brotic therapies that speci cally target the cardiac broblast [32]. In recent years, numerous studies have shown that the mechanism of cardiac brosis involves cardiomyocytes emitting stress signals that trigger in ammatory cell in ltration and cardiac broblast activation during cardiac pathological states, such as pressure overload, metabolic dysfunction, or myocardial infarction. The activation of cardiac broblasts leads to their differentiation into myo broblasts and subsequent increase in proliferation, as well as matrix deposition, expression of a rich array of cytokines and growth factors, and formation of brous scar tissues. Thus, activated cardiac myo broblasts are the main effector cells of cardiac brosis [33,34], and modulating the signals of early broblast activation and late remodeling have obvious therapeutic implications for cardiac brosis [35].
Wnt signaling is involved in the process of embryonic development and plays an important role in the dynamic balance of adult tissues. Wnt proteins are secreted ligands that transmit signals across the plasma membrane by interacting with Frizzled receptors and low density lipoprotein receptor-related protein co-receptors 5/6. When Wnt proteins bind to their receptors, a series of intracellular signaling events are induced, including the scaffold protein Axin and glycogen synthase kinase-3β that nally stabilize β-catenin. β-Catenin then translocates to the nucleus where it binds to T-cytokines/lymphokines and induces the transcription of Wnt target genes [36,37]. Duan's team demonstrated that the Wnt signaling pathway plays an important role in the pathogenesis of cardiac brosis [19]. Moreover, Huang et al. found that the Wnt/β-catenin axis promoted the activation and proliferation of broblasts during cardiac brosis [38,39].
Notum, which belongs to the α/β-hydrolase superfamily, is a Wnt ligand deacylase that can remove the functional palmitoleic acid moiety and act as a secreted feedback antagonist of Wnt [19]. Our results showed that the expression of β-catenin was signi cantly increased in a mouse model of myocardial infarction, and cardiac broblast brosis induced by TGF-β1 stimulation. Importantly, intervention with Notum could reverse overexpression of the β-catenin protein. These results suggest that Notum inhibits the Wnt/β-catenin signaling pathway in cardiac broblasts, which may be one of the mechanisms of its anti-brotic effect.
The mouse model of myocardial infarction is a classical method to induce pathological cardiac brosis [40]. Under pathologic conditions, cardiac myo broblasts secrete extracellular matrix proteins (mainly collagen I) that promote progressive cardiac brosis [41], as well as the ECM-related bronectin 1 and a-smooth muscle actin, a marker of the transformation of broblasts into myo broblasts; these are important indicators of brosis [42,43]. Our results showed that, in vivo, intramyocardial injection of Notum downregulated the expression of a-smooth muscle actin and bronectin 1 in the infarct marginal zone, inhibited the pathological generation of myo broblasts, and reduced the production of collagen I, thereby mitigating cardiac brosis caused by myocardial infarction.
Substantial evidence indicates that TGF-β is a key regulatory cytokine causing cardiac brosis [44]. TGF-β1 can initiate the transformation of broblasts to myo broblasts and regulate the expression of collagen and other ECM proteins, ultimately promoting myocardial brosis [45]. In vitro, pharmacological intervention with Notum reversed the differentiation of broblasts to myo broblasts induced by TGF-β1, inhibited the proliferation and migration of broblasts, and post-transcriptionally regulated the expression of collagens 1α1 and 3α1. Both the in vitro and in vivo results showed that Notum could regulate the pathological activation of broblasts after injury and inhibit the secretion of ECM. In addition to inhibiting the Wnt/β-catenin signal pathway, Notum may also be involved in the regulation of other brosis-related mechanisms.
Cellular senescence is a stable cell-cycle arrest that can be caused by many physiological and pathological conditions, including oncogene-induced and replicative senescence and can promote tissue remodeling during development and after injury [46,47]. Senescent cells have their own unique markers, including senescence-related β-galactosidase activity and up-regulation of proteins, such as p16, p19, p21, and p53. Early studies suggested that aging was a tumor suppressor mechanism. Rayess et al.
demonstrated that P16 was a tumor suppressor gene [48]. Furthermore, Zhu's team found that myocardial infarction could induce cardiac broblast senescence, inhibit the production of reparative myo broblasts, interfere with heart repair, and even lead to heart rupture, all of which are related to activation of the p53 signaling pathway [25]. In our study, Notum inhibited the increased p53 expression in cardiac broblasts induced by H 2 O 2 stimulation, and regulated the expression of P21 and P16 at the mRNA level. This indicated that Notum modi ed the senescence pathway of cardiac broblasts. In addition, the results of an immunohistochemical experiment showed that the expression of P16 was inhibited by Notum in the infarct border zone of myocardial infarction mice. This result further reveals the mechanism by which Notum plays a protective role in cardiac brosis. Because Notum is an extracellular enzyme, its chemical structure and characteristics have been fully recognized. Speci cally, Notum has a well-de ned and large active site pocket. Thus, pharmacological interventions utilizing Notum are diverse, and this agent has the potential to be an ideal therapeutic agent.
In conclusion, our present work con rmed the protective effect of Notum on cardiac function in mice with myocardial infarction. In addition, we demonstrated that Notum regulated the Wnt/β-catenin pathway and inhibited senescence of cardiac broblasts to alleviate their abnormal activation and phenotypic differentiation to myo broblasts thus inhibiting the occurrence of brosis. These ndings provide support for a new strategy for the treatment of cardiac brosis.

Availability of Data and Materials
All data that support the ndings of this study are available from the corresponding author upon reasonable request.

Author Contributions
Tianyu   Notum inhibits Wnt/β-catenin signaling activation in cardiac broblasts. (A) Immunohistochemistry analysis showed decreased protein expression of β-catenin in MI+Notum-treated mice compared MI mice.

Figure 6
Schematic diagram for the proposed mechanism involving in the anti-brotic effect of Notum in heart.