The Serum of MEF2D Expression in Patients with Myocardial Infarction, May be Clinical Indicators and it Mediated ROS Signaling by HDAC5

Objectives: In this study, we explain that the function of MEF2D gene in myocardial infarction and its possible mechanism. Methods: Patients with MI and normal volunteers were collected. The left anterior descending arteries (LAD) of mice were ligated to induce myocardial infarction. H9c2 cells were stimulated with 5% oxygen (O2) and 5% carbon dioxide (CO2) and 90% N2 for 24 h. Quantitative polymerase chain reaction (qPCR), Microarray experiments, Western blot, Enzyme-linked immunosorbent assay (ELISA) and Immunouorescent staining were used at this experiment. Results: MEF2D mRNA and protein expressions in heart tissue of patients and mice with myocardial infarction were reduced. MEF2D protein prevented Myocardial infarction in mice of myocardial infarction. The inhibition of MEF2D aggravated Myocardial infarction in mice of myocardial infarction. MEF2D gene promoted ROS-induced oxidative stress in vitro model of myocardial infarction. MEF2D interlinkage HDAC5 to reduced oxidative stress in vivo model or vitro model. The regulation of HDAC5 adjusted the function of MEF2D in vitro model. Discussion: These data conrmed that the serum of MEF2D expression in patients with Myocardial infarction was reduced, may be clinical indicators and it inhibited ROS-induced oxidative stress signaling by HDAC5. Thus, our results suggest that targeting MEF2D may provide an approach for the treatment of myocardial infarction.


Introduction
The 2017 China Cardiovascular Disease Report published by the National Cardiovascular Disease Center revealed that the mortality rate of cardiovascular disease was higher than that of any other diseases in both urban and rural areas [1]. Among them, myocardial infarction (MI) is one of the important causes of death [2]. The hospitalization cost of acute MI was as high as 15.34 billion RMB in 2019 [3] .
Existing studies have reported that excessive activation of oxidative stress response plays a critical role in myocardial cell damage after MI [4]. Oxygen free radicals are massively released during the activation of the oxidative stress response [5]. And oxygen free radicals, with strong oxidative capacity, can attack the lipid of myocardial cell membrane structure, cause lipid oxidation and destroy cell membrane structure [6,7].
Histone deacetylase 5 (HDAC5) belongs to the class IIa HDAC family, with deacetylase active domain in the carboxyl terminal as well as protein binding domain, nuclear localization and export sequences in the amino terminal [8,9]. HDAC5 can not only catalyze the deacetylation of histones in the nucleus, but also shuttle between the nucleus and cytoplasm, deacetylate other protein molecules or form complexes with other proteins, thereby regulating physiological processes such as cell division, differentiation, and apoptosis as well as participating in the occurrence of multiple diseases [10,11].
MEF2D is a member of the MEF2 family. Previous studies on MEF2 were mostly limited to the muscle and nervous system [12]. At present, a large number of studies have con rmed that chromosome translocation in leukemia can cause abnormally high expression of MEF2D, which in turn promote the occurrence and development of leukemia [12,13]. Meanwhile, MEF2D is speci cally expressed in liver tissue, which may also play an important role in ROS formation [14]. In this study, we explain that the function of MEF2D gene in myocardial infarction and its possible mechanism.

Animals model
Mice were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. C57BL/6 male mice were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). All aspects of the animal care and experimental protocols were approved by the Zhongshan People's Hospital Committee on Animal Care.
All mice anaesthetized using 50 mg/kg of pentobarbital sodium and the left anterior descending arteries (LAD) were ligated to induce myocardial infarction. Mice were ventilated by a rodent ventilator (Shanghai Alcott Biotech Co., Shanghai, China), then LAD was ligated by an 8.0 suture followed by the thoracotomy. Quantitative polymerase chain reaction (qPCR) Total RNAs were isolated with RNA isolator total RNA extraction reagent (Takara) and cDNA was synthesized using PrimeScipt RT Master Mix (Takara). qPCR were performed with the ABI Prism 7500 sequence detection system according to the Prime-ScriptTM RT detection kit. Relative levels of the sample mRNA expression were calculated and expressed as 2-DDCt.

Western blot
Aneurysm tissue or cells samples were lysed with ice-cold RIPA buffer with complete protease and phosphatase inhibitors. The protein concentrations were measured using BCA protein assay kit. Total proteins were separated by SDS-PAGE and transferred onto polyvinylidene di uoride (PVDF) membranes. The membranes were incubated with primary antibodies: MEF2D (1:1000, abcam), HDAC5 (1:1000, Cell Signaling Technology, Inc.) and β-Actin (1:5000, Santa Cruz Biotechnology) after blocking with 5% BSA in TBS, followed by incubation with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). The signals were detected with the ECL system and exposed by the ChemiDoc XRS system with Image Labsoftware (Bio-rad).

Enzyme-linked immunosorbent assay (ELISA)
Total protein was diluted using PBS, and measurement was performed according to the instructions of the manufacturer. CK, LDH, SOD, GSH, GSH-PX and MDA levels were measured from cell samples or tissue samples using commercial ELISA kits.

Histological examination
Heart tissue were harvested from each group and immersion xated in 4% formaldehyde, embedded in para n. Heart sections (4 µm) were cut from Aneurysm tissue and stained with HE. Sections were viewed with a uorescent microscope.

Microarray experiments
Microarray experiments were performed at the Genminix Informatics (China). Gene expression pro les were analyzed with the Human Exon 1.0 ST GeneChip (Affymetrix).

Statistical analysis
The data were entered into GraphPad Prism 5.01 Software and represented as mean values ± SD.
Comparisons of data between groups were followed using Student's t test or one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. P < 0.05 was considered to denote a statistically signi cant difference

The serum of MEF2D expression in patients with Myocardial infarction and mice model of myocardial infarction
To investigate the gene expression of MEF2D in Myocardial infarction, we rstly detected the gene expression of MEF2D in patients with myocardial infarction and mice model of myocardial infarction. As shown in Fig. 1A, a signi cantly lower serum gene expression of MEF2D compared with normal volunteers groups was detected. Serum gene expression of MEF2D was negative correlation with serum CK levels in patients with myocardial infarction and ROC = 0.9011, as shown in Fig. 1B-1C. In myocardial infarction mice models, MEF2D mRNA and protein expressions in heart tissue of mice with myocardial infarction were reduced ( Fig. 1D-1E).

MEF2D protein prevented Myocardial infarction in mice of myocardial infarction
Next, we analyzed the correlation between MEF2D expression and oxidative stress in myocardial infarction models. MEF2D recombinant protein reduced left ventricular internal diameter, increased left ventricular ejection fraction, raised left ventricular fractional shortening and left ventricular stroke volume, prevented myocardial brosis, weakened CK and LDH activity levels in mice with myocardial infarction ( Fig. 2A-2G). In addition, we found remarkably increased activity of SOD, GSH and GSH-PX levels, inhibition of MDA levels in mice with myocardial infarction by MEF2D recombinant protein (Fig. 2H-2K).
The inhibition of MEF2D aggravated Myocardial infarction in mice of myocardial infarction Next, we found that anti-MEF2D body increased left ventricular internal diameter, decreased left ventricular ejection fraction, abated left ventricular fractional shortening and left ventricular stroke volume, aggravated myocardial brosis and heightened CK and LDH activity levels in mice with myocardial infarction (Fig. 3A-3G). When anti-MEF2D body was injected into mice with myocardial infarction, we found that anti-MEF2D reduced SOD, GSH and GSH-PX activity levels, and increased MDA levels in mice with (Fig. 3H-3K).

MEF2D gene promoted ROS-induced oxidative stress in vitro model of myocardial infarction
To investigate the role of MEF2D in the development of MEF2D in vitro model of myocardial infarction, We then transfected MEF2D mimics or mimics control into vitro model of myocardial infarction. MEF2D mimics increased the expression of MEF2D mRNA, aggravated MDA levels and ROS production levels, and decreased SOD, GSH and GSH-PX activity levels in vitro model of myocardial infarction (Fig. 4A-4F). MEF2D siRNA or negative siRNA into vitro model of myocardial infarction, and we found that MEF2D siRNA reduced MEF2D mRNA expression, abated MDA levels and ROS production levels, and promoted SOD, GSH and GSH-PX activity levels in vitro model of myocardial infarction (Fig. 4G-4L).

MEF2D interlinkage HDAC5 to reduced oxidative stress in vivo model or vitro model
To explore the molecular mechanism by which MEF2D in model of myocardial infarction, we examined a range of oxidative stress signaling pathways. Microarray experiments showed that HDAC5 may be a target spot of MEF2D in model of myocardial infarction (Fig. 5A). Network signal diagram showed that downstream passage of MEF2D interlinkage HDAC5 was calculated (Fig. 5B). MEF2D recombinant protein induced HDAC5 protein expression in mice with HDAC5 ( Fig. 5C-5D). Anti-MEF2D body suppressed HDAC5 protein expression in mice with HDAC5 ( Fig. 5E-5F).
Next, immuno uorescence showed that over-expression of MEF2D induced HDAC5 expression in vitro model of myocardial infarction (Fig. 6A). IP showed that MEF2D interlinkage HDAC5 may be form a complex compound in vitro model (Fig. 6B). Over-expression of MEF2D induced MEF2D and HDAC5 protein expressions in vitro model (Fig. 6C-6E). Down-regulation of MEF2D suppressed MEF2D and HDAC5 protein expressions in vitro model (Fig. 6F-6H).
The regulation of HDAC5 adjusted the function of MEF2D in vitro model To assess whether LMP10 regulates the expression of HDAC5 in vitro model by in vitro model, siMEF2D and HDAC5 mimics were co-transfected into vitro model, and we found that HDAC5 induced HDAC5 protein expression, reduced MDA level and ROS production levels, and increased SOD, GSH and GSH-PX activity levels in vitro model by siMEF2D mimics (Fig. 7A-7F). Si-HDAC5 and MEF2D mimics were cotransfected into vitro model and we found that si-HDAC5 suppressed HDAC5 protein expression, promoted MDA level and ROS production levels, and reduced SOD, GSH and GSH-PX activity levels in vitro model by siMEF2D mimics (Fig. 7G-7L).

Discussion
Malignant ventricular arrhythmia (including ventricular tachycardia, ventricular tachycardia, ventricular brillation and ventricular brillation) is the main cause of death in patients after MI, accounting for over half of the deaths in patients after MI [15]. The excessive regeneration of sympathetic nerves after MI plays an important role in the occurrence of ventricular arrhythmias after MI, which provides the electrical substrate for the occurrence of malignant arrhythmias after MI [16]. Therefore, effective inhibition of the excessive regeneration of sympathetic nerves after MI may be one of the effective ways to prevent and to treat arrhythmia after MI [17]. Here, we found that MEF2D mRNA and protein expressions in heart tissue of mice with myocardial infarction were reduced in patients and mice model. Estrella et al. showed that MEF2D de ciency triggers programmed cell death in neonatal cardiomyocytes [18]. Thus, these results suggest that the down-regulation of MEF2D may play a role in myocardial infarction.
MI leads to abnormal intracellular environment caused by insu cient energy supply [19]. The compensatory increase in cardiac contraction can cause the increase in ROS level caused by NADPH in the cell membrane [20]. More seriously, the increased ROS levels triggers the mitochondria to produce a large amount of ROS ("oxygen triggered-oxygen release" mechanism in mitochondria) [ . Therefore, MEF2D -induced HDAC5 might contribute to the inhibition of ROSinduced oxidative stress in myocardial infarction.
Our data revealed a novel anti-oxidation function for MEF2D in myocardial infarction. We identi ed a regulatory mechanism of MEF2D inhibited ROS-induced oxidative stress, leading to an induction of HDAC5 in myocardial infarction model. Thus, our results suggest that targeting MEF2D may provide an approach for the treatment of myocardial infarction.

Ethics approval and consent to participate
This study was support by the Ethics Committee of Zhongshan People's Hospital.

Consent for publication
Not applicable.
Availability of data and material Not applicable.

Funding
None.

Competing interests
There are no potential con icts of interest to disclose.