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Acta Cardiologica Sinica logoLink to Acta Cardiologica Sinica
. 2026 Mar;42(2):171–185. doi: 10.6515/ACS.202603_42(2).20250815A

miR-138-5p Promotes HF in Rats by Reducing Histone Methylation in the Myd88 Promoter Region through Inhibition of EZH2

Ye Zhang 1, Weihui Lu 1
PMCID: PMC13017553  PMID: 41907038

Abstract

Objective

We aimed to investigate how microRNA-138-5p (miR-138-5p) promotes heart failure (HF) in rats by inhibiting enhancer of zeste homolog 2 (EZH2) and reducing histone methylation in the myeloid differentiation primary response gene 88 (MyD88) promoter region.

Methods

An HF rat model and isoproterenol (ISO)-induced H9c2 cell injury model were established. Echocardiography was used to assess cardiac function in the rats, flow cytometry was used to detect cardiomyocyte apoptosis, and reverse transcription quantitative polymerase chain reaction or Western blotting was performed to detect the expressions of miR-138-5p, EZH2, and Myd88, as well as Bax, Bcl-2, and Caspase-3. The relationship between miR-138-5p and EZH2 was analyzed by luciferase reporter assay. The methylation level of histone H3 lysine 27 trimethylation (H3K27me3) at the Myd88 promoter region mediated by EZH2 was assessed by chromatin immunoprecipitation assay.

Results

The expression of miR-138-5p was increased in myocardial tissue in the HF rats and ISO-induced H9c2 cells. Inhibition of miR-138-5p enhanced cardiac function in the HF rats. Inhibiting miR-138-5p decreased cardiomyocyte apoptosis, downregulated the expressions of Bax and Caspase-3 genes, and upregulated the expression of Bcl-2. miR-138-5p targeted and bound to the 3′-untranslated region of EZH2 mRNA, and promoted cardiomyocyte apoptosis by inhibiting EZH2 expression. EZH2 increased the H3K27me3 methylation level in the Myd88 promoter region, leading to decreased Myd88 expression. Overexpression of Myd88 and high EZH2 expression promoted cardiomyocyte apoptosis.

Conclusions

miR-138-5p targets and inhibits the expression of the EZH2 gene, reducing H3K27me3 methylation in the Myd88 promoter region, thereby enhancing Myd88 expression, promoting cardiomyocyte apoptosis, and exacerbating HF.

Keywords: Cell apoptosis, Enhancer of zeste homolog 2, Heart failure, Histone H3 lysine 27 trimethylation, MicroRNA-138-5p, Myeloid differentiation primary response gene 88

Abbreviations

CDNA, Complementary DNA

ChIP, Chromatin immunoprecipitation

DNA, Deoxyribonucleic acid

EF, Ejection fraction

EZH2, Enhancer of zeste homolog 2

FITC, Fluorescein isothiocyanate

FS, Fractional shortening

GAPDH, Glyceraldehyde-3-phosphate dehydrogenase

H3K27me3, H3 lysine 27 trimethylation

HF, Heart failure

ISO, Isoproterenol

LDH, Lactate dehydrogenase

LVEDD, Left ventricular end-diastolic diameter

LVESD, Left ventricular end-systolic diameter

miRNA, MicroRNA

miR-30a-5p, MicroRNA-30a-5p

miR-138-5p, MicroRNA-138-5p

MyD88, Myeloid differentiation primary response gene 88

NC, Negative control

oe, Overexpression

PI, Propidium iodide

RNA, Ribonucleic acid

RT-qPCR, Reverse transcription quantitative polymerase chain reaction

SIRT1, Sirtuin 1

3′UTR, 3′-untranslated region

INTRODUCTION

Cardiovascular disease poses a significant socioeconomic burden globally and is a leading contributor to mortality and disability, despite considerable advances in treatment and personalized healthcare. Among the manifestations of cardiovascular disease, heart failure (HF) is a prevalent condition that has reached epidemic levels.1 As a clinical syndrome, HF is marked by the inability of the heart to adequately supply the body’s circulatory needs without raising intracardiac pressures, either at rest or during activity.2 HF is a chronic condition marked by periods of symptomatic stability that can be interspersed with exacerbations, even with ongoing treatment.3 Poorly controlled HF is associated with high morbidity and mortality rates, coupled with escalating costs and strain on healthcare systems, largely due to a high frequency of rehospitalizations and extensive resource utilization.4

HF arises due to a disruption in cardiac homeostasis, which relies on the precise regulation of gene expression. This regulation is orchestrated by various ribonucleic acid (RNA) molecules, including those that encode proteins (known as messenger RNAs) and those that do not encode proteins, referred to as non-coding RNAs.1 As conserved single-stranded non-coding RNAs (21-25 nucleotides), microRNAs play crucial regulatory roles in various biological processes.5 Previous studies have demonstrated the involvement of multiple microRNAs in HF pathogenesis. Suppressing microRNA-30a-5p (miR-30a-5p) has been shown to ameliorate the progression of chronic HF through sirtuin 1 (SIRT1)-modulated nuclear factor-κB/ NOD-like receptor 3 signaling cascade,6 while miR-132 has been shown to promote pathological cardiac remodeling by inducing abnormal cardiomyocyte growth.7 Notably, miR-138-5p reduces SIRT1 enzymatic activity by binding to its 3′-untranslated region (3′UTR), subsequently activating the p53 signaling pathway and inducing cardiomyocyte apoptosis, suggesting its potential as a diagnostic/therapeutic target for HF.8 miR-138-5p functions as an inhibitor of tumor growth and exhibits low expression in various types of cancer, exerting its cancer-suppressing effects by regulating the expressions of different target genes.9,10 Although existing studies have partially elucidated the mechanisms of miR-138-5p in HF, its comprehensive regulatory network remains incompletely understood, which motivated our investigation.

Prior research indicates that circSAMD4A, as a molecular sponge of miR-138-5p, upregulates enhancer of zeste homolog 2 (EZH2) expression during preadipocyte differentiation.11 EZH2 is a catalytic subunit of polycomb repressive complex 2, and it has been shown to mediate transcriptional repression through histone H3 lysine 27 trimethylation (H3K27me3) and to participate in deoxyribonucleic acid (DNA) repair, cell cycle, apoptosis, differentiation, and immune regulation.12 Targeting EZH2 has emerged as a novel strategy for treating cardiovascular diseases.13 EZH2 upregulation has been shown to promote vascular smooth muscle cell phenotypic switching via modulating H3K27me3 and SM22α expression during atherosclerosis,14 while its elevation in ischemic cardiomyopathy has been shown to regulate cardiac gene expression.15 In addition, circ-RCCD has been demonstrated to facilitate cardiomyocyte differentiation by drawing YY1 to the promoter region of myeloid differentiation primary response gene 88 (MyD88).16 MyD88 is widely expressed across various cell types in both the immune and cardiovascular systems, with its functions typically varying depending on the specific cardiovascular disease context and cell type.17

Although previous studies have separately investigated miR-138-5p, EZH2, and MyD88 in cardiovascular contexts, significant limitations persist. Most of these studies have focused on single molecules or pairwise interactions, and lacked systematic integration of the miR-138-5p/EZH2/MyD88 axis. Direct evidence establishing the role of this axis in HF is lacking, particularly regarding whether EZH2-mediated epigenetic regulation constitutes the primary mechanism downstream of miR-138-5p. Moreover, the role of MyD88 in atrial fibrillation is understudied, and its crosstalk with miR-138-5p/EZH2 remains uncharacterized. In this study, we take a novel approach and integrate these molecules into a cohesive signaling axis to address these gaps. We hypothesized that the miR-138-5p/EZH2/MyD88 axis may contribute to cardiomyocyte apoptosis in HF, and aimed to elucidate its mechanistic basis for identifying novel therapeutic targets.

EXPERIMENTAL METHODS

Ethics statement

The Animal Ethics Committee granted approval for all experiments involving animals.

Establishment of the rat HF model

Eight-week-old male Sprague-Dawley rats with a body weight ranging from 290 to 330 g were acquired from Shanghai Southern Model Organisms Center Co., Ltd. After anesthesia with isoflurane, the rats were intubated orally and connected to a ventilator (with a respiratory rate of 80 breaths per minute, an inspiration-to-expiration ratio of 1:1, and a tidal volume of 6.4 mL). The left pectoral muscles were bluntly dissected to expose the intercostal space, and the muscle was torn to expose the heart. The pericardium was gently torn slightly, and a 6-0 suture needle with thread was used to ligate the left anterior descending coronary artery between the left auricle and conus arteriosus. Successful ligation was indicated by ST-segment elevation on the electrocardiogram. Following this, the intercostal and pectoral muscles were sutured, and the ventilator was removed once the rats regained spontaneous respiration. The rats were then placed in cages and received an intraperitoneal injection of penicillin over a 2-week period. Following wound recovery, they were subjected to a 2-week swimming protocol in water maintained at 30 ± 1 °C, swimming for 30 minutes each day to induce the HF rat model.18 The forced swimming took place in a tank (60 cm × 100 cm × 60 cm) filled with warm water to a depth of 30 cm, kept at 30 ± 1 °C. The duration of swimming until exhaustion served as an indicator of forced swimming capacity. Rats were deemed exhausted when they were unable to surface and take a breath within 7 seconds.19 Rats in the negative control (NC) antagomir group and the miR-138-5p antagomir group were injected with NC antagomir and miR-138-5p antagomir respectively via the tail vein at a dose of 10 mg/kg after HF modeling, and the treatment was continued for 14 consecutive days. All antagomirs were acquired from Guangzhou RiboBio Co., Ltd. Gene expression and cardiac function were then assessed.20 To adhere to the 3R principle (Replacement, Reduction, and Refinement), 6 animals per group were used.

Echocardiographic examination of the rats

The experimental rats underwent echocardiographic examinations after continuous injections of antagomirs for 14 days. The rats were continuously lightly anesthetized with isoflurane and placed supine on the examination table with their limbs in close contact with electrode patches. The Vevo 2100 (VisualSonics, Canada) ultrasound imaging system equipped with a 13 MHz probe was used to scan the rat hearts, and left ventricular long-axis and short-axis views of the papillary muscles were sourced from the left parasternal position. From the short-axis view of the papillary muscles, measurements were taken to calculate left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), fractional shortening, and ejection fraction.21

Culture, modeling, and transfection of H9c2 cardiomyocytes

H9c2 cells (acquired from the Shanghai Cell Bank of the Chinese Academy of Sciences) were grown in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin mixed antibiotics. When the H9c2 cells reached 80% confluency, they were treated with isoproterenol (ISO; 50 μM) to induce a HF cell mo-del. Normally cultured H9c2 cells served as the control group. In addition, cells were transfected with an NC inhibitor, miR-138-5p inhibitor, NC mimic + overexpression (oe)-NC, miR-138-5p mimic + oe-NC, miR-138-5p mimic + oe-EZH2, as well as oe-NC, oe-EZH2 + oe-NC, and oe-EZH2 + oe-Myd88 using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. The NC inhibitor, miR-138-5p inhibitor, NC mimic, miR-138-5p mimic, EZH2 oe vector, Myd88 oe vector, and empty vectors were all purchased from Guangzhou RiboBio Co., Ltd. After 24 hours of transfection, the cells were treated with 50 μM ISO for 48 hours and then collected for subsequent experiments.

Lactate dehydrogenase (LDH) secretion detection

Cell culture supernatants from each group were collected 48 hours after treatment. An LDH Cytotoxicity Assay Kit (Beyotime) was used following the manufacturer’s instructions to establish a standard curve. The absorbance values of each group were determined at the corresponding wavelength with a microplate reader, from which the LDH content was calculated.22

Cell apoptosis detection

Cell apoptosis was tested using the AnnexinV-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining method: H9c2 cells from each group were digested with trypsin and gathered in 15 mL centrifuge tubes. After centrifugation, the supernatant was discarded, and the precipitate was washed with phosphate-buffered saline. The cells were resuspended in 500 μL of binding buffer, as per the guidelines provided with the BD Apoptosis Detection Kit (AnnexinV-FITC version I). Five μL of FITC and 5 μL of PI were added under dark conditions and mixed well. After incubation, cell apoptosis was measured with a flow cytometer (BD FACSCalibur).23,24 In the scatter diagram, viable cells were positioned in the bottom left quadrant (FITC-Annexin V negative/PI negative), early apoptotic cells in the bottom right quadrant (FITC-Annexin V positive/PI negative), late apoptotic cells in the top right quadrant (FITC-Annexin V positive/PI positive), and necrotic cells in the top left quadrant (FITC-Annexin V negative/PI positive). The apoptosis rate was defined as the ratio of the combined number of early apoptotic cells (FITC-Annexin V positive/PI negative) and late apoptotic cells (FITC-Annexin V positive/PI positive) to the total cell count.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from tissues and cell samples using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), followed by spectrophotometric assessment of its concentration and purity. For mRNA assay, reverse transcription was performed adopting HiScript III RT SuperMix (Vazyme, Nanjing) for qPCR (+gDNA wiper) to harvest complementary DNA (cDNA). For microRNA (miRNA) measurements, reverse transcription was carried out using an miRNA First Strand cDNA Synthesis (Tailing Reaction) Kit (Vazyme, Nanjing) to harvest cDNA. ChamQ Universal SYBR qPCR Master Mix and miRNA Universal SYBR qPCR Master Mix were used for sample loading, and qPCR was used to determine the expression levels of miR-138-5p, EZH2, Myd88, Bax, Bcl-2, and Caspase-3. The primers were synthesized by Sangon Biotech (Shanghai) (Table 1). The relative expression level of mRNA was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference, and the relative expression of mRNA was calculated with the 2(-ΔΔCt) method. The relative expression level of miR-138-5p was normalized to U6 as an internal reference, with the calculation method being the same as above.25-29

Table 1. Reverse transcription quantitative polymerase chain reaction primer sequences.

Name Sequence
miR-138-5p-F 5′-CGAGCTGGTGTTGTGAATC-3′
miR-138-5p-R 5′-GTGCAGGGTCCGAGGT-3′
EZH2-F 5′-TTCCAGCACAAGTCATCCCG-3′
EZH2-R 5′-GTGCCATCCTGATCCAGAACT-3′
Myd88-F 5′-GTCTCCAGGTGTCCAACAGAAGC-3′
Myd88-R 5′-GTCGCAGATAGTGATGAACCGTAGG-3′
Bax-F 5′-GCCTTTTTGCTACAGGGTTTCAT-3′
Bax-R 5′-TATTGCTGTCCAGTTCATCTCCA-3′
Bcl-2-F 5′-GGGGCTACGAGTGGGATACT-3′
Bcl-2-R 5′-GACGGTAGCGACGAGAGAAG-3′
Caspase-3-F 5′-AGCTGGACTGCGGTATTGAGA-3′
Caspase-3-R 5′-CATGACCCGTCCCTTGAATT-3′
U6-F 5′-CTCGCTTCGGCAGCACA-3′
U6-R 5′-AACGCTTCACGAATTTGCGT-3′
GAPDH-F 5′-CGCTAACATCAAATGGGGTG-3′
GAPDH-R 5′-TTGCTGACAATCTTGAGGGAG-3′
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EZH2, enhancer of zeste homolog 2; miR-138-5p, microRNAs-138-5p; Myd88, myeloid differentiation primary response gene 88.

Western blotting

Total proteins were extracted from the cells, and protein concentrations were determined using the Bicinchoninic Acid Assay method. Protein samples were mixed with loading buffer (Thermo Fisher Scientific Inc.) and denatured by heating, followed by separation via SDS-PAGE. Electrophoretically resolved proteins were then transferred onto PVDF membranes. Immunoblotting was performed using the following primary antibodies: EZH2 (1:1000 dilution, Cell Signaling Technology), MyD88 (1:1000, Cell Signaling Technology), and GAPDH (1:1000, Cell Signaling Technology). Detection was achieved using horseradish peroxidase-conjugated secondary antibodies, with protein bands imaged on an ImageQuant LAS 4000 system (GE Healthcare).

Dual-luciferase reporter assay

A sequence containing the miR-138-5p binding site in the 3′UTR of EZH2 was amplified and cloned into the pGL3-basic luciferase plasmid (TaKaRa, Japan) to construct the wild-type EZH2 (EZH2-WT) recombinant plasmid. The miR-138-5p binding site in EZH2-WT was mutated using a site-directed mutagenesis kit (TaKaRa, Japan) to construct the mutant-type EZH2 recombinant plasmid (the design and construction of the plasmids were performed by TaKaRa). NC mimic and miR-138-5p mimic were co-transfected into H9c2 cells with the EZH2 luciferase reporter plasmid to determine whether miR-138-5p could bind to EZH2. Cells were collected and lysed 48 hours post-transfection, and the luciferase reporter gene assay was performed on a Dual-Luciferase Reporter Gene Analysis System (Promega, USA) with a luciferase detection kit (Biovision, USA). Renilla luciferase served as an internal control, and the activation level of the target reporter gene was compared in line with the ratio of the firefly luciferase measurement value to the Renilla luciferase measurement value.30,31

Chromatin immunoprecipitation (ChIP)

Enrichment of H3K27me3 in the Myd88 gene promoter was assayed using a ChIP kit sourced from Saicheng Biotechnology Co., Ltd. (Guangzhou, China). The procedure involved the following steps: When cell confluence reached 70-80%, formaldehyde was added to crosslink intracellular DNA with proteins. After crosslinking, the cells were randomly fragmented by ultrasound to harvest fragments of appropriate size. The supernatant was gathered by centrifugation and separated into two tubes, which were incubated with an NC antibody, rabbit anti-IgG (1:100, Abcam, UK), and a specific antibody for the target protein, rabbit anti-H3K27me3 (1:100, Abcam, UK). Protein agarose/sepharose was used to isolate endogenous DNA-protein complexes. Following centrifugation, the supernatant was removed, and any nonspecific complexes were washed off. The crosslinks were then broken by incubating at 65 °C overnight. Afterwards, the DNA fragments were purified and isolated through phenol/chloroform extraction. qPCR was used to test enrichment in the Myd88 promoter region.32

Data statistics

All data were presented as mean ± standard deviation. The Student’s t-test was used for statistical analysis between two groups, and one-way analysis of variance with post-hoc tests was used to compare more than two groups (LSD test if the variances were equal, and Tamhane’s T2 test if the variances were unequal). All experiments were conducted in triplicate. Values of p < 0.05 were considered significant. All tests were performed with SPSS version 22.0 for Windows (IBM, Armonk, New York, USA) and GraphPad version 6.0 (GraphPad Software, San Diego, California, USA).33

RESULTS

The expression of miR-138-5p was upregulated in the myocardial tissue of the rats with HF

Previous studies have suggested that miRNAs are pivotal in the emergence and progression of HF.34 To investigate the function of miR-138-5p in HF and the mechanisms behind it, we constructed a rat HF model and myocardial cell injury model, and examined the expression levels of miR-138-5p. First, we established a rat model of HF (HF group), with a sham-operated group (Sham group) serving as the controls. The results of cardiac function analysis showed that, in comparison to the Sham group, the LVEF and LVFS in the HF group were diminished (Figure 1A-B). The model of myocardial cell injury was established by exposing H9c2 cells to ISO (ISO group), with untreated cells (Control group) serving as the controls. The success of the cell model was verified by cell apoptosis rates and LDH release levels. The results revealed elevated cell apoptosis rates and LDH release levels in the ISO group (Figure 1C-D). We then used RT-qPCR to measure the expression levels of miR-138-5p in both the animal and cell models. The findings disclosed that in the animal model, the expression of miR-138-5p in the myocardial tissue in the HF group was upregulated compared with the Sham group (Figure 1E); in the cell model, the expression of miR-138-5p in cells in the ISO group was higher compared to the Control group (Figure 1F). In summary, we showed that miR-138-5p expression was enhanced in HF. This result implies that miR-138-5p may have a significant impact on the onset and progression of HF.

Figure 1.

Figure 1

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The expression of miR-138-5p is upregulated in the myocardial tissue of rats with HF. (A-B) Echocardiography confirmed the successful construction of the acute myocardial infarction rat model (6 animals per group); (C) Increased LDH secretion levels were detected following ISO-induced H9c2 cell injury (four independent experiments); (D) Flow cytometry detected apoptosis in H9c2 cells after ISO treatment (four independent experiments); (E) RT-qPCR detected the expression level of miR-138-5p in the myocardial tissue of rats with HF, with the Sham group serving as the control (6 animals per group); (F) RT-qPCR detected the expression level of miR-138-5p in H9c2 cells after injury, with the Control group serving as the control (four independent experiments); data are presented as mean ± SD. FITC, fluorescein isothiocyanate; HF, heart failure; ISO, isoproterenol; LDH, lactate dehydrogenase; LVEF, left ventricular ejection fraction; miR-138-5p, microRNA-138-5p; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SD, standard deviation.

Cardiac dysfunction was rescued by miR-138-5p inhibition in the rats with HF

This study investigated the role of miR-138-5p in HF-induced cardiac dysfunction. In animal experiments, the rats with HF were randomly separated into an NC group (NC antagomir) and miR-138-5p silencing group (miR-138-5p antagomir). In cell experiments, ISO-treated H9c2 cells were grouped into an NC inhibitor group and miR-138-5p inhibitor group. RT-qPCR was used to measure the expression levels of miR-138-5p. The results showed that after silencing miR-138-5p, the expression of miR-138-5p in both rat myocardial tissue and cardiomyocytes was diminished (Figure 2A-B), confirming the effectiveness of the silencing.

Figure 2.

Figure 2

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Cardiac dysfunction is rescued by miR-138-5p inhibition. (A) Successfully constructed rats with HF were randomly divided into an NC group (NC antagomir) and a miR-138-5p silencing group (miR-138-5p antagomir), with RT-qPCR detecting the silencing efficiency of miR-138-5p (6 animals per group); (B) ISO-treated H9c2 cells were divided into an NC inhibitor group and a miR-138-5p inhibitor group, with RT-qPCR detecting the silencing efficiency of miR-138-5p (four independent experiments); (C-D) Echocardiography was performed to assess cardiac function indicators in rats (6 animals per group); (E) RT-qPCR was utilized to test the mRNA expression levels of apoptosis-related genes (four independent experiments); (F) Flow cytometry was performed to assess the apoptosis level of H9c2 cells (four independent experiments); (G) LDH secretion level of H9c2 cells was detected (four independent experiments); data are presented as mean ± SD. HF, heart failure; LDH, lactate dehydrogenase; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; miR-138-5p, microRNA-138-5p; NC, negative control; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SD, standard deviation.

We then measured the LVEDD, LVESD, LVEF, and LVFS of the rats. The results showed that after silencing miR-138-5p, the LVEDD and LVESD decreased, while the LVEF and LVFS increased (Figure 2C-D), suggesting that silencing miR-138-5p improved cardiac dysfunction and alleviated HF.

After treating ISO-induced injured H9c2 cells with an inhibitor to silence miR-138-5p, RT-qPCR was used to assess the expressions of the apoptosis-related genes Bax, Bcl-2, and Caspase-3. The results showed that after silencing miR-138-5p, the expressions of Bax and Caspase-3 in the cells was downregulated, while the expression of the anti-apoptotic gene Bcl-2 was upregulated (Figure 2E). Flow cytometry analysis demonstrated that the apoptosis rate of cells in the miR-138-5p inhibitor group was lower than that in the NC inhibitor group (Figure 2F). Moreover, compared to the NC inhibitor group, the LDH secretion level in the miR-138-5p inhibitor group was diminished (Figure 2G). These results indicated that silencing miR-138-5p reduced ISO-induced H9c2 cell injury.

In summary, the results showed that silencing miR-138-5p could effectively ameliorate cardiac dysfunction in the rats with HF and suppress cardiomyocyte apoptosis. This result implies that miR-138-5p may be a potential target for HF treatment.

EZH2 is a downstream target gene of miR-138-5p

To further elucidate the mechanism by which miR-138-5p affects HF, we used TargetScan (https://www.targetscan.org/vert_80/) to predict possible downstream target genes of miR-138-5p, and EZH2 was identified as a potential direct target (Figure 3A). Previous studies have shown that EZH2 can inhibit cardiomyocyte apoptosis,35 so we hypothesized that miR-138-5p may affect cardiomyocyte apoptosis by regulating the expression of EZH2.

Figure 3.

Figure 3

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EZH2 is a downstream target gene of miR-138-5p. (A) Bioinformatics prediction of miR-138-5p binding to the EZH2 3′UTR sequence; (B) RT-qPCR detection of EZH2 mRNA expression levels in myocardial tissue from rats with HF (6 animals per group); (C) RT-qPCR detection of EZH2 mRNA expression levels after ISO-induced H9c2 cell injury (four independent experiments); (D-E) miR-138-5p and EZH2 expression levels after transfection of H9c2 cells with miR-138-5p mimic and control NC mimic (four independent experiments); (F) Luciferase reporter assay validating the targeting of miR-138-5p to the EZH2 3′UTR (four independent experiments); data are presented as mean ± SD. EZH2, enhancer of zeste homolog 2; EZH2-Mut, mutant-type EZH2; EZH2-WT, wild-type EZH2; miR-138-5p, microRNA-138-5p; NC, negative control; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SD, standard deviation; 3′UTR, 3′-untranslated region.

To verify this hypothesis, RT-qPCR was performed to analyze the expression of EZH2 in HF myocardial tissue and ISO-induced H9c2 cells. The results demonstrated that the expression levels of EZH2 in HF myocardial tissue and ISO-induced injured H9c2 cells were downregulated (Figure 3B-C). After transfecting miR-138-5p mimic and its control NC mimic into H9c2 cardiomyocytes, the results showed that versus the NC mimic group, the expression of miR-138-5p was elevated in the miR-138-5p mimic group, while the expression of EZH2 was diminished (Figure 3D-E), confirming that miR-138-5p negatively regulated the expression of EZH2.

To verify the direct targeting relationship between miR-138-5p and EZH2, we conducted a dual-luciferase reporter gene assay. The constructed EZH2 fragment was subcloned downstream of the luciferase reporter gene, and the luciferase reporter gene was then cotransfected with miR-138-5p mimic into H9c2 cells. The results showed that the wild-type 3′-UTR of EZH2 had a reduced translation level in the presence of the miR-138-5p mimic. In addition, the mutated 3′-UTR failed to show a significant response to miR-138-5p (Figure 3F), indicating that miR-138-5p specifically targeted the EZH2 3′UTR. Taken together, these findings imply that EZH2 is a downstream target gene of miR-138-5p, and that miR-138-5p can target and inhibit the expression of EZH2.

miR-138-5p facilitates cardiomyocyte apoptosis by targeting and inhibiting EZH2

We divided the cells in the ISO-induced H9c2 injury model into three groups: NC mimic + oe-NC group, miR-138-5p mimic + oe-NC group, and miR-138-5p mimic + oe-EZH2 group. The expression levels of miR-138-5p and EZH2 in the cells of each group were measured by RT-qPCR. The results showed that versus the NC mimic + oe-NC group, the expression of miR-138-5p was upregulated in the miR-138-5p mimic + oe-NC group, while the expression of EZH2 was downregulated. In the miR-138-5p mimic + oe-EZH2 group, the oe of EZH2 partially restored the expression level of EZH2 (Figure 4A-B).

Figure 4.

Figure 4

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miR-138-5p facilitates cardiomyocyte apoptosis by targeting and inhibiting EZH2. (A-B) The ISO-induced H9c2 cell injury model was divided into three groups: NC mimic + oe-NC group, miR-138-5p mimic + oe-NC group, and miR-138-5p mimic + oe-EZH2 group. The mRNA expression levels of miR-138-5p and EZH2 in the cells of each group were detected by RT-qPCR (four independent experiments); (C) RT-qPCR analysis of the mRNA expression levels of apoptosis-related genes in the cells (four independent experiments); (D) Flow cytometry detection of the apoptosis level of H9c2 cells (four independent experiments); Data are presented as mean ± SD. EZH2, enhancer of zeste homolog 2; FITC, fluorescein isothiocyanate; ISO, isoproterenol; miR-138-5p, microRNA-138-5p; NC, negative control; oe, overexpression; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SD, standard deviation.

Further RT-qPCR analysis of the expressions of apoptosis-related genes in the cells of each group showed that versus the NC mimic + oe-NC group, the expressions of Bax and Caspase-3 were upregulated, while the expression of Bcl-2 was downregulated in the miR-138-5p mimic + oe-NC group. In the miR-138-5p mimic + oe-EZH2 group, the oe of EZH2 reversed these changes (Figure 4C). Meanwhile, flow cytometry was used to test the apoptosis rate, and the results showed that compared with the NC mimic + oe-NC group, the cardiomyocyte apoptosis rate was enhanced in the miR-138-5p mimic + oe-NC group, while it was reduced in the miR-138-5p mimic + oe-EZH2 group, further validating the protective role of EZH2 against miR-138-5p-induced cardiomyocyte apoptosis (Figure 4D).

In summary, the results showed that miR-138-5p facilitated cardiomyocyte apoptosis by targeting and suppressing the expression of EZH2, and that the oe of EZH2 partially counteracted the apoptotic-inducing effect of miR-138-5p.

EZH2 inhibits cardiomyocyte apoptosis by promoting H3K27 histone methylation in the Myd88 promoter region

EZH2 modulates the expression of downstream genes through histone methylation.36 Previous studies have shown that Myd88 promotes cardiomyocyte apoptosis in cardiovascular diseases.37 The aim of the current study was to investigate the potential of EZH2 to suppress cardiomyocyte apoptosis by enhancing H3K27 histone methylation specifically within the promoter region of the Myd88 gene. In the mechanistic study, the cardiomyocyte model was categorized into an oe-NC group and an oe-EZH2 group. RT-qPCR and Western blotting were used to assess the expression levels of EZH2 and Myd88 in the cells of each group. The findings showed that, in contrast to the oe-NC group, EZH2 expression was upregulated and Myd88 expression was downregulated in the oe-EZH2 group (Figure 5A). Further ChIP experiments were conducted to assess enrichment of H3K27me3 in the Myd88 promoter region, and the results showed that enrichment of H3K27me3 in the Myd88 promoter region was elevated in the oe-EZH2 group (Figure 5B), suggesting that EZH2 inhibited the transcriptional activity of Myd88 through H3K27 methylation.

Figure 5.

Figure 5

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EZH2 inhibits cardiomyocyte apoptosis by promoting H3K27 histone methylation in the Myd88 promoter region. (A) EZH2 was overexpressed by transfecting oe-EZH2 plasmid into H9c2 cells, and the expression level of Myd88 was detected (four independent experiments); (B) CHIP-qPCR detection of the amount of H3K27me3 enriched in the Myd88 gene promoter (four independent experiments); (C) The ISO-induced H9c2 cell injury model was divided into groups: control group, oe-NC group, oe-EZH2 + oe-NC group, and oe-EZH2 + oe-Myd88 group. The mRNA expression level of Myd88 in the cells of each group was detected by RT-qPCR (four independent experiments); (D) RT-qPCR analysis of the mRNA expression levels of apoptosis-related genes in the cells (four independent experiments); (E) Flow cytometry detection of the apoptosis level of H9c2 cells (four independent experiments). Data are presented as mean ± SD. EZH2, enhancer of zeste homolog 2; FITC, fluorescein isothiocyanate; ISO, isoproterenol; Myd88, myeloid differentiation primary response gene 88; NC, negative control; oe, overexpression; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SD, standard deviation.

To further validate the role of Myd88 in EZH2-regulated cardiomyocyte apoptosis, the ISO-induced H9c2 cell injury model was separated into the oe-NC group, oe-EZH2 + oe-NC group, and oe-EZH2 + oe-Myd88 group, as well as a control group. RT-qPCR was used to quantify the expression level of Myd88 in the cells of each group. The results showed that compared to the oe-NC group, the expression of Myd88 was downregulated in the oe-EZH2 + oe-NC group, while it was restored in the oe-EZH2 + oe-Myd88 group (Figure 5C). RT-qPCR was then used to assess the expression levels of apoptosis-related genes in the cells of each group. The results showed that after oe of EZH2, the expressions of Bax and Caspase-3 were downregulated, while the expression of the anti-apoptotic gene Bcl-2 was upregulated. In the oe-EZH2 + oe-Myd88 group, these changes were partially reversed (Figure 5D). Finally, flow cytometry was used to measure the apoptosis rate, and the results showed that after oe of EZH2, the apoptosis rate was reduced, but that it was enhanced in the oe-EZH2 + oe-Myd88 group (Figure 5E). These results further confirmed that EZH2 suppresses the expression of Myd88 through H3K27 histone methylation, thereby inhibiting cardiomyocyte apoptosis, and that the oe of Myd88 can reverse this inhibitory effect.

DISCUSSION

HF is a condition in which the heart fails to pump adequate blood to fulfill the body’s requirements for blood and oxygen, making it a serious illness with high rates of morbidity and mortality.38 HF exacerbates weakness and diminishes the quality of life, potentially resulting in the need for hospitalization and even fatality.39 Despite significant advances in medical and device therapies for HF, there remains a considerable amount of residual risk.38 Our results demonstrated that miR-138-5p promotes HF in rats by inhibiting EZH2 and reducing H3K27me3 methylation at the MyD88 promoter.

Specifically, we found that the expression of miR-138-5p was increased in the myocardial tissue of rats with HF. This implies that miR-138-5p may play a role in the development and progression of HF. Furthermore, when miR-138-5p was silenced, the cardiac insufficiency in the HF rats was effectively improved, and cardiomyocyte apoptosis was also inhibited. This indicates that decreasing the expression of miR-138-5p may have therapeutic potential for HF. Xu et al. demonstrated the upregulation of miR-138-5p under simulated microgravity conditions, and that this inhibited osteoblast proliferation and induced osteoblast apoptosis. In addition, silencing miR-138-5p partially alleviated the impact on apoptosis and proliferation in MC3T3-E1 cells.40 On the other hand, Mao et al. reported that reduced levels of miR-138-5p were a predictive factor for the development of acute cerebral infarction, and that they were correlated with poorer patient outcomes.41

We used bioinformatics tools to predict possible downstream target genes of miR-138-5p, and EZH2 was identified as a potential direct target. Our results showed that miR-138-5p could target and inhibit the expression of EZH2, suggesting a potential molecular mechanism through which miR-138-5p affects HF. We also found that by targeting and inhibiting the expression of EZH2, miR-138-5p promoted cardiomyocyte apoptosis. This suggests that during the process of HF, miR-138-5p may exacerbate cardiomyocyte damage and apoptosis by inhibiting EZH2. In addition, the oe of EZH2 partially reversed the pro-apoptotic effect of miR-138-5p. This further confirms the important role of EZH2 in regulating cardiomyocyte apoptosis and suggests that modulating the expression of EZH2 may improve HF. A previous study showed that circ_SMG6 competitively binds to miR-138-5p resulting in an increase in EGR1 expression, and that this exacerbated myocardial ischemia/reperfusion injury in mice and hypoxia/reoxygenation-induced cell damage.42 Various non-coding RNAs and signaling pathways are known to be involved in modulating EZH2 function. This novel epigenetic mechanism influences target gene expression, impacting physiological and pathological processes in cardiovascular diseases such as ischemic heart disease, cardiac fibrosis, atherosclerosis, heart development, and cardiomyocyte regeneration and hypertrophy.13 A previous study found that EZH2 was present in epicardial cells during the development of both human and mouse hearts. Deletion of EZH2 in these cells led to disrupted epicardial cell migration, myocardial hypoplasia, and abnormalities in coronary plexus development, ultimately causing embryonic lethality.43

Tao et al. investigated the possible function of miR-152-3p in the toll-like receptor-induced inflammatory response observed in systemic lupus erythematosus, and the results showed that DNMT1 may accumulate at the MyD88 promoter, and that miR-152-3p could prevent the methylation of MyD88 by directly targeting DNMT1. Moreover, suppressing the expression of miR-152-3p led to a decrease in MyD88 expression.44 Our study also demonstrated that EZH2 suppressed the expression of Myd88 by increasing the level of H3K27 histone methylation. This indicates that EZH2 not only directly participates in the regulation of cardiomyocyte apoptosis, but also indirectly affects the progression of HF by influencing the expressions of other genes, such as Myd88. In addition, our results showed that the oe of Myd88 reversed the inhibitory effect of EZH2 on cardiomyocyte apoptosis. This further emphasizes the importance of Myd88 in HF, and suggests that modulating the expression of Myd88 may also be a strategy for treating HF.

Mechanistically, miR-138-5p targets and represses EZH2, decreasing H3K27me3 at the MyD88 promoter to enhance its expression, thereby promoting cardiomyocyte apoptosis and exacerbating HF. Previous studies have demonstrated that miR-138-5p can target genes such as KDM6B45 and SIRT18 to regulate macrophage polarization or cardiomyocyte apoptosis. Our study is the first to clearly establish that miR-138-5p regulates Myd88 expression through EZH2. This targeting specificity may stem from the unique epigenetic regulatory pattern of EZH2 in cardiomyocytes; for example, EZH2 modulates H3K27me3 levels at specific gene promoter regions in cardiomyocytes, thereby influencing the downstream effects of miR-138-5p. This represents a novel finding. Furthermore, distinct from previously reported roles of EZH2 in other cardiovascular diseases, our study is the first to report that EZH2 influences cardiomyocyte apoptosis in HF by regulating Myd88 expression. For example, in vascular smooth muscle cells, increased EZH2 expression mediates phenotypic transformation under injury conditions by altering H3K27me3 methylation levels and regulating SM22α expression, thereby accelerating atherosclerosis progression;14 in diabetic cardiac fibrosis, EZH2 directly suppresses the expression of the anti-fibrotic gene PPAR-γ by catalyzing H3K27 trimethylation.27 As previously mentioned, the function of MyD88 varies depending on specific cardiovascular disease contexts and cell types.17 While prior research has shown that MyD88 in T cells inhibits inflammatory responses by suppressing TCR signaling, our study found that enhanced MyD88 expression in cardiomyocytes promoted apoptosis. This functional difference likely originates from cell-type specificity: in T cells, MyD88 primarily participates in immune signal transduction; in cardiomyocytes, MyD88 may directly induce cell death by activating apoptosis-related signaling pathways (e.g., FasL/Caspase-8). This further strengthens the novelty of our research.

Of note, although our study indicates the important role of the miR-138-5p/EZH2/Myd88 axis in HF, potential alternative mechanisms should be considered when interpreting these findings. For example, EZH2 may affect HF progression through MyD88-independent pathways. Previous studies have shown that EZH2 can directly regulate epigenetic modifications of cardiac fibrosis-related genes46 or suppress the transcription of pro-remodeling genes via H3K27me3 methylation.47 In addition, miR-138-5p may simultaneously target multiple genes. Besides EZH2, its known targets such as SIRT18 and AMPK48 are all involved in myocardial energy metabolism regulation, and these parallel pathways may collectively contribute to HF phenotypes.

In summary, our results demonstrate the role and mechanism through which a high expression of miR-138-5p promotes HF in rats. By targeting and inhibiting the expression of the EZH2 gene, miR-138-5p reduces the level of H3K27me3 methylation in the Myd88 promoter region, leading to an increased expression of Myd88, promotion of cardiomyocyte apoptosis, and exacerbation of HF (Figure 6). This study used an innovative approach to explore the specific mechanism of action of miR-138-5p in HF, and revealed the important role of the miR-138-5p/EZH2/Myd88 axis in HF, providing new directions and targets for the research and treatment of HF. However, this study has several limitations. First, while we used injections of miR-138-5p antagomir to validate the role of the miR-138-5p/EZH2/Myd88 axis in HF, we did not systematically evaluate the systemic physiological impacts of this intervention in the rats. For example, the miRNA antagomir may have caused off-target effects in non-cardiac tissues (e.g., liver, adipose tissue), potentially leading to body weight loss, metabolic disturbances, or reduced locomotor activity. Second, although we confirmed the direct targeting of EZH2 by miR-138-5p, we cannot completely exclude potential confounding effects from its regulation of other target genes. In addition, EZH2 may participate in HF progression by methylating other genes, and the contributions of these parallel pathways require further investigation. Finally, substantial translational challenges remain before clinical application can be considered, necessitating additional preclinical and clinical studies to validate the therapeutic potential of this regulatory axis.

Figure 6.

Figure 6

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A diagram for the molecular mechanism. miR-138-5p targets and inhibits the expression of EZH2 gene, then enhanced Myd88 expression, promoting cardiomyocyte apoptosis, and exacerbating HF. EZH2, enhancer of zeste homolog 2; HF, heart failure; miR-138-5p, microRNA-138-5p; Myd88, myeloid differentiation primary response gene 88.

DECLARATION OF CONFLICT OF INTEREST

The authors declared that they have no conflicts of interest regarding this work.

FUNDING

This work was supported by the Medical Health Science and Technology Project of Zhejiang Province (No. 2025KY1418).

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