miR-486 attenuates myocardial ischaemia/reperfusion injury by inhibiting apoptosis and autophagy activation via the FOXO1/PTEN/AKT/mTOR axis

Abstract

Background

Effective therapeutic interventions for myocardial ischaemia/reperfusion injury (MIRI) are lacking. The expression of miR-486, a cardiac-enriched microRNA, is downregulated in ischaemic myocardium; however, its functional role and underlying molecular mechanisms in MIRI remain unclear.

Methods

To overexpress miR-486 in vivo, MIRI rat models were administered miR-486 AAV. Cardiac function was assessed using echocardiography, infarct size using 2,3,5-triphenyltetrazolium chloride staining, and collagen deposition using haematoxylin and eosin and Masson’s trichrome staining. In vitro, H9c2 cardiomyocytes were exposed to oxygen-glucose deprivation/reperfusion (OGD/R) and transfected with a miR-486 mimic, miR-486 inhibitor, or respective negative controls. Dual-luciferase reporter assays were conducted in HEK-293T cells to validate FOXO1 and PTEN as direct targets of miR-486. Apoptosis- and autophagy-related protein expression was analysed using western blotting. siRNA-mediated silencing of FOXO1 or PTEN and pharmacological inhibition of AKT (MK2206) or mTOR (rapamycin) were used to establish the underlying signalling axis.

Results

AAV-mediated overexpression of miR-486 significantly improved cardiac function, reduced infarct size, and attenuated myocardial histopathological injury in MIRI rats. In H9c2 cells, the miR-486 mimic suppressed OGD/R-induced elevation of FOXO1 and PTEN protein levels, whereas the miR-486 inhibitor further augmented their expression. MiR-486 directly targeted FOXO1 and PTEN via their respective 3′-UTRs in dual-luciferase reporter assays. MiR-486 overexpression markedly reduced the Bax/Bcl-2 and cleaved caspase-3/caspase-3 ratios. Additionally, it decreased Beclin 1 and LC3-II/GAPDH and increased p62 expression, indicating concurrent suppression of apoptosis and excessive autophagy. Notably, these effects were reversed with miR-486 inhibition. Consistent findings occurred in vivo. siRNA-mediated silencing of either FOXO1 or PTEN partially reversed the pro-apoptotic and pro-autophagic effects induced by miR-486 inhibition. Under OGD/R conditions, the phosphorylation levels of AKT and mTORC1 downstream targets, including S6K and 4EBP1, were restored with miR-486 overexpression. The contribution of AKT/mTOR signalling to the protective effects mediated by the miR-486 mimic was confirmed with AKT or mTOR inhibition.

Conclusions

MiR-486 attenuates MIRI by directly targeting FOXO1 and PTEN, thereby facilitating AKT/mTOR pathway activation to co-ordinately suppress cardiomyocyte apoptosis and excessive autophagy. These findings support the involvement of the miR-486/FOXO1/PTEN/AKT/mTOR axis as an important cardioprotective mechanism and identify miR-486 as a potential therapeutic target for MIRI.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1007/s11033-026-12913-5.

Introduction

Ischaemic heart disease, the leading cause of global morbidity and mortality, is primarily triggered by obstructed coronary blood flow resulting from the rupture or erosion of atherosclerotic plaques [1]. Timely and effective myocardial reperfusion is critical for improving clinical outcomes in affected patients. Current reperfusion strategies include primary percutaneous coronary intervention, coronary artery bypass grafting, and thrombolytic therapy [2]. Although reperfusion is essential for salvaging the ischaemic myocardium, the process itself paradoxically initiates a cascade of cellular and molecular events that exacerbate myocardial injury and promote cardiomyocyte death, a phenomenon known as myocardial ischaemia/reperfusion injury (MIRI) [3, 4]. Despite substantial advances in interventional cardiology, MIRI remains a major clinical challenge because effective therapeutic strategies for this condition are lacking. Consequently, MIRI substantially attenuates the benefits of timely reperfusion therapy in acute myocardial infarction. The underlying molecular mechanisms of MIRI should be elucidated, and novel therapeutic targets for mitigating reperfusion-associated myocardial damage are urgently required.

Apoptosis and autophagy in cardiomyocytes are two central and potentially interrelated processes in the pathogenesis of MIRI [5]. Apoptosis substantially contributes to myocardial cell death during acute myocardial infarction [6]. Inhibiting the apoptotic pathway is a key therapeutic strategy for mitigating MIRI, primarily aimed at reducing infarct size [7]. Autophagy plays a dual role in myocardial injury: at basal levels, it serves a protective function that clears damaged organelles and supplies energy substrates, thereby maintaining cellular quality control. However, during pathological conditions, such as MIRI, excessive or dysregulated autophagy can become detrimental and exacerbate cardiac damage [8, 9]. Additionally, metabolic modifications, such as histone lactylation, may regulate autophagy or mitophagy and cardiac remodelling. Therefore, metabolic regulation may represent an additional layer of complexity in MIRI pathogenesis [10]. Apoptosis and excessive autophagy are not independent events; rather, they are intricately co-ordinated through shared upstream signalling networks [11]. However, the molecular regulators responsible for co-ordinating these processes during MIRI remain unclear.

MicroRNAs (miRNAs) are small non-coding RNAs that modulate gene expression at the post-transcriptional level and play essential roles in cardiovascular pathophysiology [12, 13]. MiR-486 is a cardiac-enriched miRNA implicated in cell survival, metabolism, and stress responses [13]. Previous studies have suggested that miR-486 may exert cardioprotective effects, primarily through its anti-apoptotic actions [14]. However, whether miR-486 simultaneously regulates cardiomyocyte apoptosis and excessive autophagy and whether this regulation involves the FOXO signalling pathway remains unknown.

In the present study, we investigated the role and mechanism of miR-486 in MIRI using an in vivo rat model with adeno-associated virus AAV-mediated miR-486 overexpression and in vitro H9c2 oxygen-glucose deprivation/reperfusion (OGD/R) model with gain- and loss-of-function approaches. We hypothesised that miR-486 alleviates MIRI by directly targeting FOXO1 and PTEN, thereby restoring AKT/mTOR-dependent pro-survival signalling activation and ultimately inhibiting cardiomyocyte apoptosis and excessive autophagy activation. To test this hypothesis, we combined bioinformatic prediction, dual-luciferase reporter assays, transfection-based gain- and loss-of-function experiments; siRNA-mediated rescue studies, and pharmacological inhibition of AKT and mTOR.

Materials and methods

Animals and experimental design

Adult male Sprague–Dawley rats (220–250 g) were used in this study. All animals were housed under specific pathogen-free conditions under a 12-h light/dark cycle with free access to food and water. After 1 week of acclimatisation, the rats were randomly assigned to four groups: (G1) Sham + AAV-negative control (NC), (G2) MIRI + AAV-NC, (G3) Sham + miR-486 AAV, and (G4) MIRI + miR-486 AAV.

For in vivo overexpression of miR-486, rats in the miR-486 AAV groups received a single tail vein injection of AAV9 expressing miR-486, whereas rats in the AAV-NC groups received an equivalent dose of NC AAV9 via the same route. Three weeks later, the MIRI modelling and sham operation were conducted.

The MIRI rat model was established as previously described with minor modifications. Rats were fasted for 24 h before surgery with free access to water. Anaesthesia was induced via intraperitoneal injection of 20% urethane (0.5 mL/100 g body weight, freshly prepared in saline). Subsequently, the rats were placed in the supine position and lead II electrocardiography was continuously monitored. After tracheal intubation, mechanical ventilation was initiated. A left thoracotomy was performed at the third to fourth intercostal space, and the pericardium was opened to expose the heart. The left anterior descending coronary artery was identified and ligated approximately 2 mm distal to its origin using a 6 − 0 silk suture. Myocardial ischaemia was confirmed based on regional cyanosis of the myocardium, blanching of the distal ventricular wall, and ST-segment elevation on electrocardiography. After 30 min of ischaemia, the ligature was released to allow reperfusion for 120 min. Reperfusion was verified based on partial recovery of myocardial colour and resolution of ST-segment elevation. In the sham groups, the same procedure was performed without ligation of the coronary artery.

Transthoracic echocardiography was performed using a high-resolution ultrasound imaging system to evaluate cardiac function after MIRI or sham operation. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured according to the manufacturer’s instructions.

For infarct size assessment, fresh heart tissues were collected and briefly frozen to facilitate sectioning. The hearts were cut into 2–3-mm transverse slices and incubated in 2,3,5-triphenyltetrazolium chloride (TTC) solution at 37 °C in the dark. Subsequently, the sections were photographed, and the infarct area was quantified using ImageJ software. Infarct size was expressed as the percentage of infarcted area relative to the total left ventricular area.

For histological analysis, myocardial tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. After deparaffinisation and rehydration, the sections were stained with haematoxylin and eosin (H&E) according to standard procedures. Masson’s trichrome staining was performed using a commercial kit according to the manufacturer’s instructions. Histopathological changes and collagen deposition were observed under a light microscope, and the collagen-positive area was quantified using ImageJ software.

At 120 min after reperfusion, transthoracic echocardiography was performed in one rat per group using a high-resolution ultrasound imaging system to confirm successful establishment of the model, after which the rats were returned to routine housing. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured according to the manufacturer’s instructions.

The rats were then allocated into two independent experimental cohorts, with five rats per group in each cohort. In one cohort, rats were euthanised at 24 h after reperfusion, and their hearts were collected for TTC staining. In the other cohort, rats underwent transthoracic echocardiography at 72 h after reperfusion and were subsequently euthanised; ; the hearts were harvested, and a portion of the left ventricular anterior wall was snap-frozen in liquid nitrogen and stored at − 80 °C for western blotting, while an adjacent portion was fixed in 4% paraformaldehyde and processed for H&E staining and Masson’s trichrome staining.

All animal procedures were approved by the Medical Ethics Committee of the Affiliated Hospital of Chengde Medical University (approval no. CYIFYLL2020244) and performed in accordance with the relevant institutional and national guidelines for the care and use of laboratory animals.

Cell culture and treatments

The rat cardiomyoblast cell line, H9c2, was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere containing 5% CO₂.

For oxygen-glucose deprivation/reperfusion (OGD/R), cells were incubated in glucose-free DMEM under hypoxic conditions (95% N₂ and 5% CO₂) for 8 h, followed by reoxygenation in complete medium under normoxic conditions for 4 h.

For gain- and loss-of-function experiments, H9c2 cells were transfected with miR-486 mimic, mimic NC, miR-486 inhibitor, inhibitor NC, siRNA targeting FOXO1 (si-FOXO1), siRNA targeting PTEN (si-PTEN), or the corresponding siRNA NC using Lipofectamine 3000 transfection reagent (Invitrogen, USA) according to the manufacturer’s instructions. After transfection, the cells were subjected to OGD/R treatment for subsequent experiments. All oligonucleotides were synthesised by Guangzhou RiboBio Co. Ltd. (Guangzhou, China).

For pathway inhibition experiments, H9c2 cells were treated with MK2206 (10 µM) or rapamycin (100 nM) for 24 h under OGD/R conditions. Vehicle-treated cells served as controls.

RNA extraction and reverse transcription and quantitative PCR (RT-qPCR)

Total RNA was extracted from H9c2 cells or myocardial tissues using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RT-qPCR for miR-486 were performed using Bulge-Loop™ miRNA-specific kits (RiboBio, Guangzhou, China). U6 small nuclear RNA was used as the internal reference. Relative miR-486 expression levels were calculated using the 2^−ΔΔCt(ΔCt=Ct_miRNAs-Ct_U6) method.

Western blotting

Total protein was extracted from H9c2 cells and myocardial tissues using RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined using a bicinchoninic acid assay kit. Equal amounts of protein were separated using SDS-PAGE and transferred onto PVDF membranes. After blocking, the membranes were incubated overnight at 4 °C with primary antibodies against FOXO1, PTEN, Bax, Bcl-2, cleaved caspase-3, caspase-3, Beclin1, LC3, p62, AKT, p-AKT, S6K, p-S6K, 4EBP1, p-4EBP1, GAPDH, and β-tubulin. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, and protein bands were visualised using an enhanced chemiluminescence detection system. A densitometric analysis was performed using ImageJ software. All protein expression levels were normalised to GAPDH, except for cleaved caspase-3 and caspase-3, which were normalised to β-tubulin. The Bax/Bcl-2, cleaved caspase-3/caspase-3, and LC3-II/GAPDH ratio were calculated for quantitative analysis. To evaluate autophagic flux, H9c2 cells were treated with bafilomycin A1 (BafA1; 100 nM) for the final 2 h before protein extraction. BafA1 was used to inhibit the lysosomal degradation of LC3-II, and the accumulation of LC3-II in the presence or absence of BafA1 was analysed using western blotting to assess autophagic flux.

Dual-luciferase reporter assay

HEK-293T cells were cultured in DMEM supplemented with 10% foetal bovine serum and antibiotics at 37 °C in a humidified atmosphere containing 5% CO₂. Cells were co-transfected with miR-486 mimic or mimic NC together with luciferase reporter plasmids containing either the wild-type FOXO1 3′-UTR (FOXO1-WT), mutant FOXO1 3′-UTR (FOXO1-MUT), wild-type PTEN 3′-UTR (PTEN-WT), or mutant PTEN 3′-UTR (PTEN-MUT).The FOXO1-WT, FOXO1-MUT, PTEN-WT, and PTEN-MUT reporter constructs were used to determine whether miR-486 directly interacts with the predicted binding sites within the 3′-UTRs of FOXO1 and PTEN. Luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega, USA) according to the manufacturer’s instructions. Firefly luciferase activity was normalised to Renilla luciferase activity.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 10.4.1 (GraphPad Software, USA) and SPSS 26.0 (IBM, USA). Data are presented as mean ± SD. For animal experiments, five rats per group were included in each independent experimental cohort. For cell-based experiments, data were obtained from three independent experiments. Comparisons between two groups were performed using an unpaired two-tailed Student’s t-test. Comparisons among multiple groups were performed using one-way ANOVA followed by Tukey’s post-hoc test. P < 0.05 was considered statistically significant. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Results

MiR-486 overexpression attenuates myocardial ischaemia/reperfusion injury (MIRI) in vivo

In our preliminary studies, the expression of miR-486 was significantly reduced in heart tissues of rats subjected to MIRI [15]. To further investigate the functional role of miR-486 in vivo, we established an overexpression model via tail-vein injection of an AAV vector expressing miR-486 and subsequently established a MIRI model. Electrocardiograms (ECGs) were recorded during model induction. Sham-operated rats showed normal ECGs tracings, whereas MIRI rats all exhibited marked ST-segment elevation, indicating successful induction of myocardial ischaemia (Fig. 1A). Echocardiographic analyses performed 72 h after reperfusion showed that, following MIRI, rats treated with miR-486 AAV exhibited significantly improved LVEF and LVFS compared with rats in the AAV-NC group (Fig. 1B, C).TTC staining revealed extensive myocardial infarction in the post-MIRI AAV-NC group, whereas the infarct size was significantly reduced in rats treated with miR-486 AAV9 (Fig. 1D-E). Furthermore, H&E and Masson’s trichrome staining demonstrated reduced myocardial histopathological injury and attenuated collagen deposition in miR-486 AAV-treated rats compared with those in the AAV-NC group (Fig. 1F, G). Collectively, these findings indicate that miR-486 confers marked cardioprotection against MIRI in vivo.

Fig. 1.

MiR-486 overexpression attenuates MIRI in vivo. (A) Representative echocardiogram traces showing successful establishment of the MIRI model. (B,C) Echocardiographic analysis of left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) at 72 h after reperfusion. (D,E) Representative TTC staining images and quantification of infarct size at 24 h after reperfusion. (F,G) Representative haematoxylin and eosin and Masson’s trichrome staining images and quantification of collagen deposition. Data are presented as mean ± SD (For echocardiographic assessment, TTC staining, and histological analyses, n = 5 rats per group in the corresponding experimental cohort.). Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. AAV, adeno-associated virus; MIRI, myocardial ischaemia/reperfusion injury; NC, negative control

Validation of miR-486 mimic and inhibitor transfection efficiency

Based on our previous finding that miR-486 expression was significantly downregulated in H9c2 cells subjected to OGD/R [15], we transfected H9c2 cells with miR-486 mimic, miR-486 inhibitor, and their corresponding NCs (mimic NC and inhibitor NC, respectively), to verify transfection efficiency. RT-qPCR analysis showed that miR-486 expression was significantly increased in the miR-486 mimic group compared with that in the mimic NC group (Fig. 2A), whereas it was significantly decreased in the miR-486 inhibitor group compared with that in the inhibitor NC group (Fig. 2B). These results indicate that both miR-486 mimic and miR-486 inhibitor effectively modulated miR-486 expression in H9c2 cells, confirming satisfactory transfection efficiency for the subsequent experiments.

Fig. 2.

Validation of the transfection efficiency of miR-486 mimic and inhibitor in H9c2 cells. (A) RT-qPCR analysis of miR-486 expression in H9c2 cells following transfection with miR-486 mimic or mimic NC. (B) RT-qPCR analysis of miR-486 expression in H9c2 cells following transfection with miR-486 inhibitor or inhibitor NC. Data are presented as mean ± SD from three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. **P < 0.01, ***P < 0.001. NC, negative control

MiR-486 directly targets FOXO1 and PTEN and negatively regulates their expression

Putative miR-486-binding sites within the FOXO1 and PTEN 3′-UTRs were identified using bioinformatics analyses (Fig. 3A). To determine whether FOXO1 and PTEN are direct targets of miR-486, dual-luciferase reporter assays were performed in HEK-293T cells. Compared with the mimic NC group, miR-486 mimic significantly reduced the luciferase activity of the FOXO1-WT and PTEN-WT reporters, whereas no significant changes were observed in the FOXO1-MUT and PTEN-MUT reporters (Fig. 3B). These findings indicate that miR-486 directly binds to the 3′-UTRs of both FOXO1 and PTEN. Western blot analysis demonstrated that OGD/R stimulation significantly increased FOXO1 and PTEN protein expression compared with those in the Control group (Fig. 3C). Under OGD/R conditions, FOXO1 and PTEN expression levels were significantly reduced after miR-486 mimic transfection compared with the OGD/R+mimic NC group, whereas miR-486 inhibition significantly increased FOXO1 and PTEN expression compared with the OGD/R+inhibitor NC group (Fig. 3D, E). The quantitative analyses confirmed these significant differences across experimental groups. Together, these results demonstrate that FOXO1 and PTEN are direct downstream targets of miR-486 and are negatively regulated by miR-486 signalling.

Fig. 3.

MiR-486 directly targets FOXO1 and PTEN and regulates their expression in H9c2 cells. (A) Schematic showing the predicted binding sites of miR-486 within the FOXO1 and PTEN 3′-UTRs and the corresponding mutant sequences used for luciferase reporter assays. (B) Dual-luciferase reporter assay in 293T cells co-transfected with miR-486 mimic or mimic NC together with FOXO1-WT/FOXO1-MUT or PTEN-WT/PTEN-MUT reporter constructs. (C) Representative western blot images and quantitative analysis of FOXO1 and PTEN protein expression in H9c2 cells under control conditions or after OGD/R treatment. (D) Representative Western blot images and quantitative analysis of FOXO1 and PTEN protein expression in H9c2 cells transfected with miR-486 mimic or mimic NC under OGD/R conditions. (E) Representative western blot images and quantitative analysis of FOXO1 and PTEN protein expression in H9c2 cells transfected with miR-486 inhibitor or inhibitor NC under OGD/R conditions. Data are presented as mean ± SD from three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. Statistical comparisons between the indicated groups are directly shown in the graphs.*P < 0.05, **P < 0.01, ***P < 0.001. MUT, mutation; NC, negative control; OGD/R, oxygen-glucose deprivation/reperfusion; WT, wild-type

MiR-486 inhibits cardiomyocyte apoptosis in vitro and in vivo

Given that both FOXO1 and PTEN are associated with apoptosis, we further investigated the effect of miR-486 on cardiomyocyte apoptosis. Western blot analysis showed that OGD/R stimulation significantly increased Bax/Bcl-2 and cleaved caspase-3/caspase-3 ratio compared with those in the Control group (Fig. 4A), confirming OGD/R-induced apoptotic activation. Additionally, miR-486 mimic significantly attenuated OGD/R-induced apoptotic changes, as evidenced by reduced Bax/Bcl-2 and cleaved caspase-3/caspase-3 ratios compared with those in the OGD/R+mimic NC group (Fig. 4B). The statistical differences among the indicated groups are presented in the corresponding graphs. Conversely, miR-486 inhibition further aggravated OGD/R-induced apoptotic responses, as demonstrated by increased Bax/Bcl-2 and cleaved caspase-3/caspase-3 ratios compared with those in the OGD/R+inhibitor NC group (Fig. 4C). These findings indicate that miR-486 negatively regulates cardiomyocyte apoptosis under ischaemic stress conditions. These findings were further confirmed in vivo. Compared with the Sham group, MIRI rats exhibited significantly increased Bax/Bcl-2 and cleaved caspase-3/caspase-3 ratios, whereas miR-486 AAV administration significantly reversed these apoptotic alterations compared with those in the MIRI + AAV-NC group (Fig. 4D).These results indicate that miR-486 exerts anti-apoptotic effects in vitro and in vivo and may contribute to the attenuation of MIRI.

Fig. 4.

MiR-486 inhibits cardiomyocyte apoptosis in vitro and in vivo. (A) Representative western blot images and quantitative analysis of the Bax/Bcl-2 ratio and cleaved caspase-3/caspase-3 ratio in H9c2 cells under control conditions or after OGD/R treatment. (B) Representative Western blot images and quantitative analysis of the Bax/Bcl-2 ratio and cleaved caspase-3/caspase-3 ratio in H9c2 cells transfected with miR-486 mimic or mimic NC under OGD/R conditions. (C) Representative western blot images and quantitative analysis of the Bax/Bcl-2 ratio and cleaved caspase-3/caspase-3 ratio in H9c2 cells transfected with miR-486 inhibitor or inhibitor NC under OGD/R conditions. (D) Representative western blot images and quantitative analysis of the Bax/Bcl-2 ratio and cleaved caspase-3/caspase-3 ratio in rat myocardial tissues from the indicated groups. Data are presented as mean ± SD. For in vivo experiments, n = 5 per group. For in vitro experiments, data were obtained from three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. Statistical comparisons between the indicated groups are directly shown in the graphs.*P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001. AAV, adeno-associated virus; NC, negative control; OGD/R, oxygen-glucose deprivation/reperfusion

MiR-486 suppresses excessive autophagy responses and regulates autophagic flux in cardiomyocytes

As both FOXO1 and PTEN are also involved in autophagy-related signalling pathways, we further investigated the effect of miR-486 on cardiomyocyte autophagy. Western blot analysis showed that OGD/R stimulation significantly increased Beclin1 expression and the LC3-II/GAPDH ratio while decreasing p62 expression compared with those in the Control group (Fig. 5A), suggesting enhanced autophagic activity under ischaemic stress. Additionally, miR-486 mimic significantly reversed OGD/R-induced autophagy-related alterations, including reduced Beclin1 expression and LC3-II/GAPDH ratio while restoring p62 expression compared with those in the OGD/R+mimic NC group (Fig. 5B). Conversely, miR-486 inhibition further enhanced OGD/R–induced autophagy activation, as demonstrated by the increased Beclin1 expression and LC3-II/GAPDH ratio and decreased p62 expression compared with those in the OGD/R+inhibitor NC group (Fig. 5C).To further determine whether the altered LC3-II levels reflected increased autophagosome formation or impaired autophagic degradation, an autophagic flux assay was performed using BafA1. Under OGD/R conditions, LC3-II accumulation was further increased in the presence of BafA1, indicating ongoing LC3-II turnover and the presence of active autophagic flux. Compared with OGD/R+mimic NC-transfected cells, miR-486 mimic significantly reduced LC3-II levels under OGD/R conditions, and this inhibitory effect remained evident following BafA1 treatment (Fig. 6A, B). These findings indicate that miR-486 attenuates excessive autophagy activation under OGD/R conditions rather than causing LC3-II accumulation through impaired autophagosome degradation. These findings were further confirmed in vivo. Compared with the Sham group, MIRI rats exhibited increased Beclin1 expression and LC3-II/GAPDH ratio and decreased p62 expression. Conversely, miR-486 AAV administration significantly reversed these autophagy-related alterations compared with those in the MIRI + AAV-NC group (Fig. 5D). Collectively, these results indicate that miR-486 suppresses excessive autophagy responses in cardiomyocytes under ischaemic stress and may contribute to the attenuation of MIRI.

Fig. 5.

MiR-486 suppresses cardiomyocyte autophagy activation in vitro and in vivo. (A) Representative western blot images and quantitative analysis of Beclin1, LC3-II/GAPDH, and p62 expression in H9c2 cells under control conditions or after OGD/R treatment. (B) Representative western blot images and quantitative analysis of Beclin1, LC3-II/GAPDH, and p62 expression in H9c2 cells transfected with miR-486 mimic or mimic NC under OGD/R conditions. (C) Representative western blot images and quantitative analysis of Beclin1, LC3-II/GAPDH, and p62 expression in H9c2 cells transfected with miR-486 inhibitor or inhibitor NC under OGD/R conditions. (D) Representative western blot images and quantitative analysis of Beclin1, LC3-II/GAPDH, and p62 expression in rat myocardial tissues from the indicated groups. Data are presented as mean ± SD. For in vivo experiments, n = 5 per group. For in vitro experiments, data were obtained from three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. Statistical comparisons between the indicated groups are directly shown in the graphs. *P < 0.05,***P < 0.001, ****P < 0.0001. AAV adeno-associated virus; NC, negative control; OGD/R, oxygen-glucose deprivation/reperfusion

Fig. 6.

MiR-486 attenuates excessive autophagy activation under OGD/R conditions. (A) Representative western blot images showing LC3-II expression in H9c2 cells subjected to OGD/R with or without miR-486 mimic and bafilomycin A1 (BafA1) treatment. BafA1 was used to inhibit lysosomal degradation of LC3-II and evaluate autophagic flux.(B) Quantitative analysis of LC3-II/GAPDH levels under the indicated conditions. OGD/R increased LC3-II accumulation, and BafA1 further enhanced LC3-II levels, indicating ongoing LC3-II turnover and the presence of active autophagic flux. Compared with mimic NC-transfected cells, miR-486 mimic reduced LC3-II accumulation under OGD/R conditions.The maintained difference between groups after BafA1 treatment indicates that miR-486 attenuates excessive autophagy activation rather than causing LC3-II accumulation through impaired degradation. Data are presented as mean ± SD from three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. Statistical comparisons between the indicated groups are directly shown in the graphs. *P < 0.05, **P < 0.01

FOXO1 and PTEN mediate the effects of miR-486 on OGD/R-induced apoptosis and autophagy activation in H9c2 cells

To further determine whether FOXO1 and PTEN contribute to the effects of miR-486 on OGD/R-induced apoptosis and autophagy activation in H9c2 cells, rescue experiments were performed under OGD/R conditions by co-transfecting H9c2 cells with the miR-486 inhibitor and siRNAs targeting FOXO1 or PTEN. Western blot analysis showed that, upon siRNA-mediated FOXO1 knockdown, cells co-transfected with si-FOXO1 and inhibitor NC exhibited significantly decreased Bax/Bcl-2 and cleaved caspase-3/caspase-3 ratios, reduced Beclin1 expression and LC3-II/GAPDH ratio, and increased p62 expression compared with cells co-transfected with the si-NC and inhibitor NC group. This indicates that FOXO1 knockdown alone attenuated OGD/R-induced apoptosis and autophagy activation. Furthermore, compared with cells co-transfected with si-NC and miR-486 inhibitor, cells co-transfected with si-FOXO1 and miR-486 inhibitor showed significant attenuation of the miR-486 inhibitor-induced changes in apoptosis- and autophagy-related markers (Fig. 7A), as evidenced by decreased Bax/Bcl-2 and cleaved caspase-3/caspase-3 ratios, reduced Beclin1 expression and LC3-II/GAPDH ratio, and increased p62 expression. These findings suggest that FOXO1 knockdown partially rescued the pro-apoptotic and pro-autophagic effects induced by miR-486 inhibition, with statistical differences among the indicated experimental groups directly presented in Fig. 7A. Similarly, upon siRNA-mediated PTEN knockdown, cells co-transfected with si-PTEN and inhibitor NC exhibited significantly decreased Bax/Bcl-2 and cleaved caspase-3/caspase-3 ratios, reduced Beclin1 expression and LC3-II/GAPDH ratio, and increased p62 expression compared with cells co-transfected with si-NC and inhibitor NC, indicating that PTEN knockdown alone also attenuated OGD/R-induced apoptosis and autophagy activation. Moreover, compared with cells co-transfected with si-NC and miR-486 inhibitor, cells co-transfected with si-PTEN and miR-486 inhibitor showed significant attenuation of the miR-486 inhibitor-induced changes in apoptosis- and autophagy-related markers (Fig. 7B), as evidenced by decreased Bax/Bcl-2 and cleaved caspase-3/caspase-3 ratios, reduced Beclin1 expression and LC3-II/GAPDH ratio, and increased p62 expression. These findings suggest that PTEN knockdown likewise partially rescued the pro-apoptotic and pro-autophagic effects induced by miR-486 inhibition.

Fig. 7.

FOXO1 and PTEN mediate the regulatory effects of miR-486 on OGD/R-induced apoptosis and autophagy activation in H9c2 cells. (A) Representative Western blot images and quantitative analysis of the Bax/Bcl-2 ratio, cleaved caspase-3/caspase-3 ratio, Beclin1, LC3-II/GAPDH, and p62 expression in H9c2 cells co-transfected with si-FOXO1 or siRNA NC and miR-486 inhibitor or inhibitor NC under OGD/R conditions. (B) Representative western blot images and quantitative analysis of the Bax/Bcl-2 ratio, cleaved caspase-3/caspase-3 ratio, Beclin1, LC3-II/GAPDH, and p62 expression in H9c2 cells co-transfected with si-PTEN or siRNA NC and miR-486 inhibitor or inhibitor NC under OGD/R conditions. Data are presented as mean ± SD from three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. Statistical comparisons between the indicated groups are directly shown in the graphs.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NC negative control; OGD/R, oxygen-glucose deprivation/reperfusion

Collectively, these results indicate that FOXO1 and PTEN are involved in mediating the effects of miR-486 on OGD/R-induced apoptosis and autophagy activation in H9c2 cells, suggesting that miR-486 exerts its protective effects through mechanisms involving FOXO1 and PTEN.

MiR-486 promotes AKT/mTOR signalling activation under OGD/R conditions

The AKT/mTOR signalling pathway is an important regulator of cardiomyocyte survival and autophagy-related responses. To further determine whether miR-486 regulates AKT/mTOR pathway activation, we examined the phosphorylation levels of AKT and the downstream mTORC1 targets S6K and 4EBP1 under OGD/R conditions.

Western blot analysis demonstrated that the phosphorylation levels of AKT (p-AKT/AKT), S6K (p-S6K/S6K), and 4EBP1 (p-4EBP1/4EBP1) were significantly reduced under OGD/R stimulation compared with those Control cells (Fig. 8A–C). Restoration of miR-486 expression significantly increased p-AKT/AKT, p-S6K/S6K, and p-4EBP1/4EBP1 levels compared with those in the OGD/R+mimic NC group, whereas inhibition of miR-486 further decreased phosphorylation levels compared with those in the OGD/R+inhibitor NC group (Fig. 8A–C). These results confirm that miR-486 positively regulates AKT/mTOR signalling activation during OGD/R injury and further support the involvement of the AKT/mTOR pathway in mediating the cardioprotective effects of miR-486.

Fig. 8.

MiR-486 promotes AKT/mTOR signalling activation under OGD/R conditions in H9c2 cells. (A) Representative western blot images and quantitative analysis of p-AKT/AKT expression in H9c2 cells under control conditions or after OGD/R treatment. (B) Representative western blot images and quantitative analysis of p-S6K/S6K expression in H9c2 cells under the indicated conditions. (C) Representative western blot images and quantitative analysis of p-4EBP1/4EBP1 expression in H9c2 cells under the indicated conditions. Compared with OGD/R or mimic NC groups, miR-486 mimic significantly increased phosphorylation levels of AKT, S6K, and 4EBP1, whereas the miR-486 inhibitor further reduced their phosphorylation levels. Data are presented as mean ± SD from three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. Statistical comparisons between the indicated groups are directly shown in the graphs.*P < 0.05, **P < 0.01, ***P < 0.001. NC, negative control; OGD/R, oxygen-glucose deprivation/reperfusion

Given the functional association of FOXO1 with Akt and mTOR in the regulation of apoptosis and autophagy, pharmacological inhibition experiments were performed under OGD/R conditions using the Akt inhibitor, MK2206, and mTOR inhibitor, rapamycin. These experiments were designed to determine whether Akt and mTOR are involved in mediating the effects of miR-486 on OGD/R-induced apoptosis and autophagy activation in H9c2 cells. Western blot analysis showed that, upon treatment with the Akt inhibitor MK2206, cells transfected with mimic NC exhibited significantly increased Bax/Bcl-2 and cleaved caspase-3/caspase-3 ratios, increased Beclin1 expression and LC3-II/GAPDH ratio, and decreased p62 expression compared with those in vehicle-treated cells transfected with mimic NC, indicating that Akt inhibition exacerbated OGD/R-induced apoptosis and autophagy activation. Furthermore, compared with vehicle-treated cells transfected with miR-486 mimic, cells treated with MK2206 and transfected with miR-486 mimic showed significant attenuation of miR-486-mediated protection against OGD/R-induced apoptosis and autophagy activation (Fig. 9A), as evidenced by increased Bax/Bcl-2 and cleaved caspase-3/caspase-3 ratios, increased Beclin1 expression and LC3-II/GAPDH ratio, and decreased p62 expression. These findings suggest that pharmacological inhibition of Akt with MK2206 partially abolished the protective effects of miR-486 against OGD/R-induced apoptosis and autophagy activation.

Fig. 9.

Akt and mTOR are involved in mediating the effects of miR-486 on OGD/R-induced apoptosis and autophagy activation in H9c2 cells. (A) Representative western blot images and quantitative analysis of the Bax/Bcl-2 ratio, cleaved caspase-3/caspase-3 ratio, Beclin1, LC3-II/GAPDH, and p62 expression in H9c2 cells treated with MK2206 or vehicle and transfected with miR-486 mimic or mimic NC under OGD/R conditions. (B) Representative western blot images and quantitative analysis of the Bax/Bcl-2 ratio, cleaved caspase-3/caspase-3 ratio, Beclin1, LC3-II/GAPDH, and p62 expression in H9c2 cells treated with rapamycin or vehicle and transfected with miR-486 mimic or mimic NC under OGD/R conditions. Data are presented as mean ± SD from three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. Statistical comparisons between the indicated groups are directly shown in the graphs.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NC negative control; OGD/R, oxygen-glucose deprivation/reperfusion

Similarly, upon treatment with the mTOR inhibitor rapamycin, cells transfected with mimic NC exhibited significantly increased Bax/Bcl-2 and cleaved caspase-3/caspase-3 ratios, increased Beclin1 expression and LC3-II/GAPDH ratio, and decreased p62 expression compared with those in vehicle-treated cells transfected with mimic NC, indicating that mTOR inhibition similarly exacerbated OGD/R-induced apoptosis and autophagy activation. Moreover, compared with vehicle-treated cells transfected with miR-486 mimic, cells treated with rapamycin and transfected with miR-486 mimic showed significant attenuation of miR-486-mediated protection against OGD/R-induced apoptosis and autophagy activation (Fig. 9B), as evidenced by increased Bax/Bcl-2 and cleaved caspase-3/caspase-3 ratios, increased Beclin1 expression and LC3-II/GAPDH ratio, and decreased p62 expression. Similarly, mTOR inhibition by rapamycin partially abolished the protective effects mediated by miR-486 against OGD/R-induced apoptosis and autophagy activation.

Collectively, these results indicate that Akt and mTOR are involved in mediating the effects of miR-486 on OGD/R-induced apoptosis and autophagy activation in H9c2 cells, as pharmacological inhibition of either Akt or mTOR significantly attenuated the protective effects of miR-486 (Table 1).

Table 1.

Primary antibodies used in this study

Target protein	Manufacturer/supplier	Catalogue number	Apparent molecular weight (kDa)
FOXO1	Proteintech	18592-1-AP	70–80
PTEN	Cell Signalling Technology	9552	54
Bax	Abcam	ab32503	21
Bcl-2	Abcam	ab182858	26
Caspase 3	Cell Signalling Technology	9662	35
Cleaved-Caspase 3	Cell Signalling Technology	9662	17, 19
LC3-II	Abcam	ab192890	14–17
p62	Abcam	ab109012	62
β-tubulin	Proteintech	10094-1-AP	50–55
GAPDH	Abcam	ab9485	36
Beclin1	Proteintech	11306-1-AP	52–60
AKT	Proteintech	88532-1-RR	52–62
p-AKT	Proteintech	66444-1-1 g	60–62
S6K	Huabio	HA722520	59
p-S6K	Huabio	HA721803	59
4EBP1	Huabio	ET1701-83	13
p-4EBP1	Huabio	HA723222	13

Discussion

In this study, miR-486 was downregulated in the myocardium after MIRI and in H9c2 cells subjected to OGD/R. Restoration of miR-486 expression alleviated MIRI in vivo, as evidenced by improved cardiac function, reduced infarct size, and attenuated myocardial histopathological damage. In vitro, miR-486 suppressed OGD/R-induced apoptosis and autophagy activation, whereas miR-486 inhibition produced the opposite effects. Mechanistically, FOXO1 was identified as a direct target of miR-486, PTEN expression was negatively regulated by miR-486, and rescue as well as pharmacological inhibition experiments indicated that the protective effects of miR-486 are mediated, at least in part, through the FOXO1/PTEN/AKT/mTOR axis.

The alleviated MIRI after miR-486 overexpression in vivo highlights the biological relevance of this miRNA in the ischaemic heart. These findings are consistent with our data showing reduced endogenous miR-486 expression after MIRI and OGD/R, suggesting that loss of miR-486 may contribute to the vulnerability of cardiomyocytes to ischaemia/reperfusion stress. Although miR-486 has been implicated in cytoprotective responses in other pathological settings, its role in MIRI remains incompletely defined. Our results extend the current understanding of miR-486 by demonstrating that restoration of its expression is sufficient to confer measurable cardioprotection in vivo. Notably, cardiac function and histological assessments were performed at 72 h after reperfusion, whereas infarct size was evaluated at 24 h. This dual-endpoint design was adopted because myocardial stunning may confound functional measurements at early time points, while infarct size is commonly assessed at 24 h after reperfusion.

Apoptosis is a major determinant of cardiomyocyte loss during reperfusion, while autophagy plays a context-dependent role in ischaemic heart disease. Basal or moderate autophagy is generally considered adaptive; however, sustained or excessive autophagy activation during reperfusion may aggravate myocardial injury. In this study, OGD/R increased the Bax/Bcl-2 ratio and the cleaved caspase-3/caspase-3 ratio, which was accompanied by increased Beclin1 expression, elevated LC3-II/GAPDH levels, and reduced p62 expression. Furthermore, BafA1-mediated autophagic flux analysis demonstrated that these changes reflected enhanced autophagic activity rather than impaired LC3-II degradation. These alterations were markedly reversed by miR-486 overexpression and exacerbated by miR-486 inhibition, indicating that miR-486 protects cardiomyocytes by co-ordinately limiting apoptosis and maladaptive autophagy-related responses under ischaemic stress.

Myocardial injury is commonly followed by extracellular matrix remodelling, in which excessive collagen deposition contributes to adverse structural changes over time. In this study, Masson’s trichrome staining at 72 h after reperfusion revealed increased interstitial collagen deposition in the MIRI group, which was significantly attenuated by miR-486 overexpression. Mature fibrosis requires weeks to develop, therefore, the findings at this time point should be interpreted as early interstitial collagen deposition rather than established fibrosis. The reduction in collagen deposition observed in the miR-486 AAV group may reflect, at least in part, the attenuated myocardial injury and subsequent inflammatory response rather than a direct antifibrotic effect of miR-486. Nevertheless, these results suggest that miR-486 may restrain the early initiation of adverse matrix remodelling, which could influence long-term cardiac repair.

FOXO1 emerged as a key downstream effector of miR-486 in this study. FOXO1 is a stress-responsive transcription factor that regulates genes involved in apoptosis, oxidative stress, and autophagy. We confirmed that FOXO1 is a direct target of miR-486 and miR-486 overexpression reduced FOXO1 protein levels in OGD/R-treated H9c2 cells. These findings provide a mechanistic basis for the anti-apoptotic and anti-autophagic effects of miR-486. In addition, PTEN was confirmed as a direct target of miR-486; PTEN-WT reporter activity was significantly suppressed by miR-486 mimic, whereas mutation of the predicted miR-486 binding site abolished this inhibitory effect. These findings indicate that miR-486 directly regulates PTEN expression and PTEN is an important downstream mediator of miR-486 signalling in cardiomyocytes.

The functional relevance of FOXO1 and PTEN was further supported by the rescue experiments. Silencing either FOXO1 or PTEN attenuated the pro-apoptotic and pro-autophagic effects induced by miR-486 inhibition under OGD/R conditions. Therefore, FOXO1 and PTEN are not merely associated with the downstream response to miR-486, but participate directly in mediating its effects on cardiomyocyte survival. FOXO1 and PTEN are established negative regulators of pro-survival signalling, therefore, their concurrent suppression by miR-486 may be an important molecular basis for the observed cardioprotective phenotype.

The AKT/mTOR pathway is a central regulator of cardiomyocyte survival and autophagy and an important downstream signalling pathway regulated by FOXO1 and PTEN. In this study, we directly evaluated AKT/mTOR pathway activation by assessing the phosphorylation levels of AKT and the downstream mTORC1 targets, S6K and 4EBP1. OGD/R significantly suppressed the phosphorylation levels of these signalling molecules, whereas miR-486 overexpression restored their phosphorylation status. Conversely, inhibition of miR-486 further reduced AKT/mTOR pathway activation.

These findings confirm that miR-486 promotes AKT/mTOR signalling activation under ischaemic stress conditions. Consistently, pharmacological inhibition of AKT with MK2206 or mTOR with rapamycin significantly attenuated the protective effects of miR-486 mimic against OGD/R-induced apoptosis and autophagy activation. Together, these results demonstrate that activation of the AKT/mTOR pathway is an essential component of the cardioprotective mechanism mediated by miR-486.

Together with the rescue data, our results support a model in which miR-486 alleviates OGD/R-induced injury by repressing FOXO1 and reducing PTEN expression, thereby favouring AKT/mTOR-dependent pro-survival signalling. Consistently, the reductions in the Bax/Bcl-2 ratio and cleaved caspase-3/caspase-3 ratios, along with normalisation of Beclin1, LC3-II/GAPDH, and p62, are consistent with co-ordinated suppression of apoptosis and autophagy activation.

Several limitations of this study should be acknowledged. First, although PTEN was validated as a direct target of miR-486 through dual-luciferase reporter assays, additional regulatory mechanisms contributing to PTEN modulation under ischaemic stress conditions require further investigation. Second, although we demonstrated that miR-486 regulates AKT/mTOR pathway activation by assessing phosphorylation levels of AKT and downstream mTORC1 targets, further studies using genetic approaches targeting specific pathway components will be required to further define the precise molecular mechanisms involved. Third, although autophagic flux was evaluated using BafA1-mediated LC3-II accumulation analysis in the present study, further validation using complementary approaches, such as tandem mRFP-GFP-LC3 reporter assays, may provide additional insights into the dynamic regulation of autophagy by miR-486. Fourth, the mechanistic studies were performed in H9c2 cells; validation in primary cardiomyocytes would further strengthen the translational relevance of the findings. Additionally, Masson’s trichrome staining was performed at 72 h after reperfusion; therefore, it reflects early interstitial collagen deposition rather than established fibrosis. Whether miR-486 limits long-term fibrotic and adverse ventricular remodelling requires evaluation at later time points. Furthermore, only miR-486 gain-of-function was examined in vivo; complementary loss-of-function studies and longer-term evaluation after reperfusion will be important in future work. Finally, as miRNAs commonly regulate multiple targets, other downstream effectors of miR-486 may also contribute to its cardioprotective actions.‌ The complexity of MIRI was previously acknowledged, implicating novel cell death pathways, such as cuproptosis and the role of neural-immune interactions [16]. Future investigations should involve confirming whether miR-486-mediated protection intersects with these broader signalling networks or specific cell types, such as endothelial cells or neutrophils, to provide a more comprehensive therapeutic strategy against MIRI.

In conclusion, miR-486 is a cardioprotective miRNA in MIRI. MiR-486 attenuated myocardial injury and suppressed OGD/R-induced apoptosis and excessive autophagy activation by directly targeting FOXO1 and PTEN and restoring AKT/mTOR signalling activation. These findings provide new insight into the molecular basis of miRNA-mediated cardioprotection and suggest that the miR-486/FOXO1/PTEN/AKT/mTOR regulatory network may be a potential therapeutic target for MIRI. Further studies are warranted to clarify additional regulatory mechanisms contributing to PTEN modulation under ischaemic stress, define the upstream and downstream signalling events more precisely as well as the translational potential of miR-486-based interventions.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

L.W.X.* and W.T.X.* performed all experiments and wrote the main manuscript text. G.M.L. ²supplemented follow-up experiments and revised the manuscript.Q.Y.S.# acquired research funding and supervised the whole study. All authors reviewed the manuscript.*These authors contributed equally to this work and should be considered co-first authors.²This author should be considered the second author for her contributions to this study.#Corresponding authorAll authors reviewed the manuscript.

Funding

1. Natural Science Foundation Project of Hebei Province (project number H2022406047). 2. Medical Science Research Project of Hebei Health Commission (project number20231399).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare no competing interests.

Ethics Statement

This study did not involve human participants, human recruitment, or the collection of identifiable human data. All animal experiments were approved by the Medical Ethics Committee of the Affiliated Hospital of Chengde Medical University (approval no. CYIFYLL2020244) and conducted in accordance with relevant institutional and national guidelines for the care and use of laboratory animals.

Competing interests

The authors declare no competing interests.