EPHB2 knockdown mitigated MI-induced myocardial injury by inhibiting MAPK signaling

blot to verify the expression of EPHB2 in MI of to the


Abstract Background
Myocardial infarction (MI) is a common disease in the cardiovascular eld. The incidence of ventricular remodeling dysplasia and heart failure increases signi cantly after MI. The objective of this study was to investigate whether EPHB2 could regulate myocardial injury after MI and explore its regulatory pathways.

Methods
RT-qPCR and Western blot were used to verify the expression of EPHB2 in MI mice, and then shRNA knockdown of EPHB2 was used to con rm the relationship between EPHB2 expression and disease progression. The levels of in ammation, apoptosis and brosis were detected by tissue staining. Related factors were detected by RT-qPCR, Western blot or ELISA. Further, the signaling pathway through which EPHB affected MI processes was detected and preliminarily con rmed by Western blot.

Results
EPHB2 was signi cantly overexpressed in heart tissue of MI mice. Knockdown of EPHB2 gene signi cantly downregulated immune factors and apoptotic factors, and alleviated mi-induced cardiac tissue damage and functional decline in mice. The MAPK pathway was found to be a downstream pathway in which EPHB2 acted. Knockdown EPHB2 down-regulates phosphorylation of MAPK pathwayrelated proteins.

Conclusions
In mouse models, knockdown EPHB2 alleviated MI-induced cardiac function decline, in ammation and apoptosis of myocardial tissue, and myocardial brosis. This process may be achieved through the MAPK pathway.

Background
Myocardial infarction (MI) is a common cardiovascular disease with high morbidity and mortality. In recent years, with the popularization and application of vascularization therapy (such as percutaneous coronary intervention, coronary artery bypass grafting, etc.) and other treatment methods, the survival rate of acute myocardial infarction has been greatly improved. However, it also signi cantly increases the risk of ventricular remodeling, which signi cantly increases the incidence of heart failure [1,2]. Among the factors involved in myocardial remodeling, myocardial brosis is the dominant factor. The imbalance of extracellular matrix synthesis and degradation is the main cause of myocardial brosis [3,4]. In some cases, myocardial brosis is abnormally prolonged, resulting in abnormal heart function and hardening of the ventricular wall, increasing the likelihood of heart failure [4,5]. Therefore, it is very necessary to control the degree of myocardial brosis in a certain range.
The pathological basis of myocardial remodeling and impaired cardiac function after MI includes oxidative stress, in ammatory response, cytokine production, neuroendocrine changes, changes in hemodynamic load, etc. [6]. In ammatory response in ischemic and reparative brosis plays an important role, the injury of myocardial cells release a lot of necrosis associated molecular patterns induced myocardial broblasts into proin ammatory phenotype, reactive oxygen species, IL-1 beta, and also induced myocardial broblasts secrete a large number of proin ammatory cytokines and chemokines, collect a large number of white blood cells, macrophages to myocardial locally. Subsequently, the in ammatory response was rapidly suppressed and the cell proliferation phase was transferred [7][8][9].
Oxidative stress occurs during ischemic injury, and the degree of oxidation exceeds the elimination of oxides, resulting in the imbalance between the oxidative system and the antioxidant system. Oxidative stress activates extracellular matrix metalloproteinase (MMPs) in myocardial broblasts by directly acting on cytokine and growth factor signal transduction. MMPs not only play a role in the degradation of cardiac matrix components, but also regulate the synthesis of collagen. The increased activity of MMPs increases the degree of myocardial brosis [10]. In addition, reactive oxygen species can also cause myocardial cell necrosis and apoptosis by activating a variety of signaling pathways, and can cause vascular endothelial dysfunction by inactivating carbon monoxide, further accelerating the development of myocardial brosis.
With the development of gene microarray and sequencing technology, comparing the transcriptome difference between pathological tissue and physiological tissue is bene cial to nd the differentially expressed genes related to myocardial infarction. Immune-related pathways, cell cycle-related pathways, and extracellular matrix remodeling-related pathways were signi cantly increased after MI in mice. Several genes have also been identi ed to be associated with MI, such as Nppa, Serpina3n, and Anxa1, which are signi cantly altered after MI [11]. Recent studies have shown that 5-HTT De ciency Affects Healing after Myocardial Infarction [12]. CTRP1 exacerbates cardiac dysfunction after myocardial infarction by regulating TLR4 in macrophages [13]. In the early stages of MI, TNFR2 agonist treatment improved left ventricular function. Regulation of TNFR1, on the other hand, has adverse effects [14].
Another research in monkeys, pigs, and rats showed that blocking the death checkpoint protein TRAIL improves cardiac function after MI [15].
Based on the above research background, we expected to screen the targets of tissue injury and brosis related proteins after myocardial infarction by bioinformatics methods and verify them in mouse MI model. Studies included detection of changes in the expression of related proteins at the protein level, detection of cardiac tissue apoptosis at the cellular level, assessment of pathological changes and brosis at the tissue level, and detection of blood ow at the overall level, prediction of the downstream pathway. Through the above experiments, we expect to nd the key proteins of tissue damage and brosis after MI, so as to provide theoretical support for treating heart failure after MI with these proteins as targets.

## Bioinformatics analysis
The expression pro le datasets of patients with MI were searched from the publicly available Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo). In the GEO dataset (GSE141512), a transcriptome pro ling in peripheral blood mononuclear cells of 6 MI patients and 6 healthy individuals was performed and the expression of EPHB2 was calculated.
CTD and String databases were used to screen out the signaling pathways through which EPHB2 affected MI. The pathways associated with myocardial infarction were screened from the CTD database. The pathways affected by EPHB2 are retrieved from the String database. The intersection of the two was taken to obtain the signaling pathway that mediated EPHB2's effect on MI, which was prepared for further veri cation.
## Mice feeding and model establishment C57BL/6J mice were purchased from Charles River Co., Ltd, China. Mice were allowed unlimited access to food and water throughout the study. The circadian was twelve hours in the light (7:00 am~ 7:00 pm) and the rest of twelve hours in the dark. The ambient temperature was 22±2°C. Relative humidity was 55±5%.
To knockdown EPHB2 speci cally, AAV9-shEPHB2 was injected into mice via the jugular vein. The detailed procedure is as follows. Mice were anaesthetized with 1% iso urane in oxygen, while viral solution (3×10 11 vector genomes (vg)/mouse) was slowly injected via the jugular vein. MI model was established 4 weeks after administration.
To establish MI mouse model, left anterior descending (LAD) coronary occlusion was performed. As described above [16], The mice were anaesthetized by intraperitoneal injection of 1% phenobarbital sodium (30 mg/kg). After thoracotomy at the fourth intercostal space, the left thoracic cavity was exposed. The LAD coronary artery was ligated with a 6-0 suture at the lower edge of the left atrial appendage. The success of establishing the MI model was con rmed if there was an immediate color change on the heart surface after LAD ligation. Sham animals underwent the same surgical procedures, except the LAD was not occluded. Follow-up tests were conducted 4 weeks after the model was established.

## RT-qPCR
Total RNA was extracted using Trizol reagent. then reverse transcribed into DNA using a RT-PCR Kit (Thermo #K1622), according to the manufacturer's instructions. Real-time PCR was performed with SYBR Green PCR kit (Thermo F-415XL) on a Real-Time PCR System (ABI-7500, USA) with GAPDH gene being used as internal control [17]. The 2 -ΔΔCt method was used to analyze the data [18]. The primers for qPCR were purchased from Sangon Biotech Company (Shanghai, China) .primers were designed through Primer-BLAST tool [19]. Each sample was tested in triplicate. ## Immunohistochemistry EPHB2 expression was detected followed standard immunohistochemical protocol as reported elsewhere [20]. Brie y, para n-embedded slides (5 µM) were baked at 60°C, then dewaxed by xylene and rehydrated by fractionated ethanol. Antigen was extracted, and incubated with 3% H 2 O 2 for 10 min. Then the slides were sealed with bovine serum for 1 h, and then anti EPHB2 antibody (cat. no. ab252935, Abcam, USA; 1:100 dilutions in 1% bovine serum albumin) were added and incubated at room temperature for 3 hours. The slides were covered with the secondary antibody and placed in a humid chamber for 1 hour, and then DAB was added to reveal the staining intensity. Sections were photographed using an Olympus IX81 microscope (Olympus, Inc, Japan).

## Echocardiographic examination
Cardiac physiological analysis of mice was performed as previously described [21,22]. In brief, mice were anesthetized with i.p. injection of 1% phenobarbital sodium (30 mg/kg). A sector scanner (Sonos 1500; Hewlett-Packard) equipped with a 12-MHz transducer was used to record two-dimensionally guided Mmode tracings to assess left ventricle wall thickness, left ventricle dimensions, and fractional shortening. Electrocardiogram recordings were acquired on anesthetized mice with a multichannel ampli er and were converted to digital signals for analysis (PowerLab system; ADInstruments).

## Hemodynamic measurements
Cardiac hemodynamic measurements were performed as described [23]. Mice were anesthetized with i.p. injection of 1% phenobarbital sodium (30 mg/kg), and the right common carotid artery was isolated and cannulated with a 1.4-F micromanometer (Millar Instruments). Maximal values of the instantaneous rst derivative of LV pressure (+dp/dt max, as a measure of cardiac contractility) and minimum values of the instantaneous rst derivative of LV pressure (-dp/dt min, as a measure of cardiac relaxation) were recorded.
## The target pathway of Embelin was predicted by network pharmacology analysis Images were captured by an Olympus IX81 microscope (Olympus, Inc, Japan) in bright-eld mode.

## Statistical analysis
The GraphPad Prism software (version 8.0) was used to perform Statistical analysis and mapping. Oneway analysis of variance followed by a Bonferroni post-hoc test was used to analyze differences between groups. Data were considered signi cantly different when p < 0.05. Image analysis was conducted using ImageJ software (1.53c).

Results
Page 8/20 ##EPBH2 was signi cantly up-regulated after MI To detect genes that might be involved in MI processes, we used the GEO database for screening. The dataset GSE141512 is used. Compared with healthy patients, EPBH2 transcription level was signi cantly increased in the tissues of MI patients, as shown in Figure1A. This result was replicated in the mouse MI model. As shown in Figure1B, qPCR results showed that compared with sham group, the transcription level of cardiac EPHB2 gene in MI group was signi cantly up-regulated. At the protein expression level, the expression of EPHB2 protein in mice in MI group was signi cantly enhanced, as shown in Figure 1C.
The immunohistochemical results were consistent with the results of qPCR and western blot. As shown in Figure1D, the expression of EPHB2 was enhanced in the heart tissue of MI model mice.
##The downregulation of EPBH2 signi cantly alleviated the cardiac function damage after MI To further examine the role of EPBH2 in the MI process, we injected AVV9-shEPHB2 jugular with the expectation of downregulating EPHB2 expression. As shown in Figure2A-B, compared with MI+ AAV 9-shNC group, the transcription level and protein expression level of cardiac EPHB2 in MI+AAV9-shEPHB2 group were signi cantly down-regulated. Heart function was then measured. As shown in Figure2C-E, echocardiography showed that the EF (left ventricular ejection fraction) and FS (left ventricular shortening fraction), were signi cantly decreased in mice after MI, and left ventricular systolic function was reduced and cardiac function was impaired. The hemodynamics results (dp/dt max and dp/dt min) showed the same trend (Figure2F-G). In addition, serum LDH and CK levels in MI model mice were signi cantly increased, as shown in Figure2H-I, which also indicated that heart function was impaired in MI model mice [24,25]. In comparison with the MI+AVV9-shNC group, the heart function of the MI+AVV9-shEPHB2 group was signi cantly improved after the expression of EPHB2 was down-regulated by shEPHB2 ( Figure 2C-I).
## EPBH2 knockdown signi cantly alleviated the in ammation and apoptosis of heart tissue after MI We then examined MI-induced in ammation and apoptosis in mouse heart tissue. As shown in Figure 3A, HE staining results showed that the in ltration level of in ammatory cells in the myocardial tissue of mice increased and edema appeared after the establishment of MI model. The in ammation level was reduced after down-regulation of EPHB2. ELISA results showed that the expression levels of in ammatory factors such as TNF-α, IL-6 and IL-1β in serum of mice were signi cantly up-regulated after the establishment of MI model, while the levels of in ammatory factors were also decreased after the down-regulation of EPHB2 ( Figure 3B-D).
Similarly, after the establishment of MI model, a large number of myocardial cells in mice were apoptotic, and western blot results also showed that the expression of apoptotic factors was enhanced. The expression of apoptotic factors in the MI+AAV-shNC group was down-regulated compared with that in the MI+AAV-shNC group (Figure4A-E).

## Knockdown of EPBH2 signi cantly alleviated MIinduced myocardial brosis
Cardiac brosis is the main cause of heart failure after MI [4]. Masson Trichrome Staining was used to detect the level of myocardial brosis in mice. As shown in Figure 5A, the level of myocardial brosis in mice was signi cantly increased after the establishment of MI model, and decreased after the downregulation of EPHB2 level. The change trend of transcription level of related factors detected by qPCR was consistent with the results of tissue staining ( Figure 5B) ## EPBH2 may act through the MAPK pathway These results indicated that MI induced the up-regulation of EPHB2 expression in the myocarum of mice. Down-regulation of EPHB2 reversed MI-induced cardiac function decline, myocardial tissue in ammation and apoptosis, and myocardial brosis. The signaling pathway EPHB2 is involved in is unclear. We used the CTD database to retrieve MI-related pathways and interacted with the EPHB2-related pathways retrieved from the String database, and ve signaling pathways were detected ( Figure 6A). Western blot analysis showed that the phosphorylation levels of MAPK signaling pathway related proteins such as ERK, JNK, p38 were signi cantly up-regulated after MI, while the phosphorylation levels were partially restored after down-regulation of EPHB2 ( Figure 6B-E).

Discussion
This study con rms that EPHB2 is involved in cardiac function decline after MI. The expression of EPHB2 was up-regulated by MI. Down-regulation of EPHB2 alleviated MI-induced cardiac function decline, myocardial tissue in ammation and apoptosis, and myocardial brosis. EPHB2 was predicted to act through the MAKP pathway by bioinformatics analysis and was validated by Western blot analysis. In conclusion, EPHB2 promotes cardiac function decline after MI through MAKP pathway.
Eph receptors are cell surface molecules that have a wide range of biological functions and affect a variety of cellular behaviors [26,27]. There are 10 EphA receptors, and 6 EphB receptors [28]. Previous studies have found that Eph receptors are involved in ischemia-reperfusion injury. In both in-vivo and invitro mouse model of renal ischemia-reperfusion injury, EphA2 was up-regulated through an Src kinasedependent pathway [29]. The expression levels of EphB4 and EphA2 were up-regulated in hypoxic skin, suggesting that Eph receptor was involved in revascularization after hypoxic injury [30]. EPHB2 is an important member of the Eph receptor family and has previously been shown to be expressed mainly in endothelial and tumor cells [31,32]. More recently, EPHB2 has been found to also be expressed on a number of immune cells, including T cells, monocytes, and macrophages [33,34]. Furthermore, EphB2 speci cally binds to ephrin B1/B2 and activates forward signaling that promotes T cell migration and monocyte activation [34,35]. EphrinB2/EPHB2 promoted synaptic germination and synaptic strengthening in the colonic plexus via the ERK-MAPK pathway in a PI-IBS rat study [36].
MAPK pathway is involved in the regulation of various biological cell processes and becomes active due to various stress responses. It plays an important role in the proliferation and differentiation of stem cells and the regulation of related differentiation genes. Evidence for the involvement of the MAPK pathway in cardiovascular disease has been presented in patients with Noonan syndrome. As a genetic disease, it is mainly caused by multiple gene mutations in the MAPK pathway [37]. In conclusion, silencing EPHB2 mitigated MI-induced cardiac function decline, myocardial tissue in ammation and apoptosis, and myocardial brosis in mouse models. This process may be achieved through the MAPK pathway.

Declarations
Ethics approval and consent to participate The experimental protocol of our study was performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by Jinan Hospital.

Consent for publication
None.
Availability of data and material The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. showed that the transcription level of EPHB2 in MI mice was signi cantly up-regulated compared with that in Sham group; (C) Western blot results showed that the expression level of EPHB2 in MI mice was signi cantly up-regulated compared with that in SHAM group; (D) Immunohistochemistry showed that EPHB2 expression was up-regulated in heart tissue of MI mice. **P<0.01 compared with sham group.

Figure 2
The downregulation of EPBH2 signi cantly alleviated the cardiac function damage after MI. (A) shEPHB2 signi cantly down-regulated the transcription level of EPHB2 in MI mice; (B) shEPHB2 signi cantly downregulated the expression level of EPHB2 protein in MI mice; (C) Ultrasonic cardiogram of mice in each group; (D) Ejection fraction decreased signi cantly in MI group and partially recovered after EPHB2 knockdown; (E) The shortening fraction was signi cantly decreased in the MI group and partially recovered after knockdown of EPHB2; (F) The maximal dp/dt was signi cantly decreased in the MI group and partially recovered after knockdown of EPHB2; (G) The minimal dp/dt was signi cantly decreased in the MI group and partially recovered after knockdown of EPHB2; (H) Serum LDH in MI group increased and partially recovered after inhibiting EPHB2; (I) Serum CK in MI group increased and partially recovered  EPBH2 knockdown signi cantly alleviated the in ammation of heart tissue after MI. (A) HE staining showed that MI induced myocardial tissue in ammation, which was relieved after knockdown of EPHB2; (B) TNF-α was up-regulated in MI mice, and was relieved after knockdown of EPHB2; (C) IL-6 was upregulated in MI mice, and was relieved after knockdown of EPHB2; (D) IL-1β was up-regulated in MI mice, and was relieved after knockdown of EPHB2. **P<0.01 compared with Sham group, ##P<0.01 compared with MI+AAV9-shNC group.