MiR‐199a‐5p promotes ferroptosis‐induced cardiomyocyte death responding to oxygen–glucose deprivation/reperfusion injury via inhibiting Akt/eNOS signaling pathway

Abstract

Myocardial ischemia/reperfusion (I/R) injury is associated with the poor outcome and higher mortality after myocardial infarction. Recent studies have revealed that miR‐199a‐5p participates in the process of myocardial I/R injury, but the precise roles and molecular mechanisms of miR‐199a‐5p in myocardial I/R injury remain not well‐studied. Ferroptosis has been proposed to promote cardiomyocyte death, closely associated with myocardial I/R injury. Herein, the present study aimed to explore the function and mechanisms by which miR‐199a‐5p regulates whether miR‐199a‐5p contributes to ferroptosis‐induced cardiomyocyte death responding to oxygen–glucose deprivation/reoxygenation (OGD/R) injury, an in vitro model of myocardial I/R injury focusing on Akt/eNOS signaling pathway. The results found that ferroptosis‐induced cardiomyocyte death occurs and is accompanied by an increase in miR‐199a‐5p level in OGD/R‐treated H9c2 cells. MiR‐199a‐5p inhibitor ameliorated ferroptosis‐induced cardiomyocyte death as evidenced by the increased cell viability, the reduced reactive oxygen species (ROS) generation, lactate dehydrogenase (LDH) activity, malondialdehyde (MDA) and Fe²⁺ contents, and the up‐regulated glutathione (GSH)/glutathione disulphide (GSSG) ratio as well as glutathione peroxidase 4 (Gpx4) protein expression in H9c2 cells‐exposed to OGD/R, while miR‐199a‐5p mimic had the opposite effects. In addition, OGD/R led to the inhibition of Akt/eNOS signaling pathway, which was also blocked by miR‐199a‐5p inhibitor and aggravated by miR‐199a‐5p mimic. Furthermore, LY294002, an inhibitor of Akt/eNOS signaling pathway, abrogated miR‐199a‐5p inhibitor‐induced the reduction of ferroptosis‐induced cardiomyocyte death. In summary, our findings demonstrated that miR‐199a‐5p plays a central role in stimulating ferroptosis‐induced cardiomyocyte death during ischemic/hypoxic injury via inhibiting Akt/eNOS signaling pathway.

INTRODUCTION

In the past decade, ischemic heart disease has become a major cause of morbidity and mortality worldwide.^1,2 Currently, reperfusion therapy, including thrombolysis, is the most effective approach for acute ischemic heart disease treatment,³ but, reperfusion itself results in the aggravation of ischemic injury, a process known as myocardial ischemia/reperfusion (I/R) injury.⁴ Multiple pathological mechanisms of myocardial I/R injury are associated with a series of molecular biology events including apoptosis, oxidative stress, calcium overload, inflammation, and mitochondrial dysfunction.^5,6 Nevertheless, due to its complexity, a clear mechanism underlying this pathology is still not fully understood. Therefore, the elucidation of the underlying molecular mechanisms of myocardial damage during I/R has great significance, which may provide a novel potential target for clinical treatment.

Ferroptosis is a novel form of regulated cell death namely characterized by intracellular the iron-dependent peroxidation of lipids which distinguishes itself from necrosis, apoptosis, pyroptosis, and autophagy.⁷ Biochemically, it was well established that intracellular glutathione (GSH) depletion, decreased GSH-dependent phospholipid hydroperoxiodase and glutathione peroxidase 4 (Gpx4) activity, increased lipid peroxides and ROS accumulation, and Fe²⁺ oxidizing lipids in a fenton-like manner are involved in the process of ferroptosis.⁸ In recent years, numerous studies have confirmed the important regulatory roles of ferroptosis in the pathogenesis of many diseases,⁹ and evidenced that ferroptosis is a key mechanism implied in myocardial I/R injury.^10,11 The induction of ferroptosis-induced cardiomyocyte injury leads to impaired myocardial function, and ferroptosis inhibitors effectively attenuate myocardial damage and cardiomyocyte death triggered by I/R,^12,13 indicating the potential role of ferroptosis-induced cardiomyocyte damage as the cause of myocardial I/R injury. Although the mechanisms underlying the regulation of ferroptosis received extensive attention, the molecular mechanism of ferroptosis-induced cardiomyocyte damage responding to myocardial I/R injury has not been fully clarified.

MicroRNAs (miRNAs) are small non-coding RNAs (containing ∼22 nucleotides) that negatively regulate gene expression at the posttranscriptional level, participating in major cellular processes, including cell proliferation, differentiation, apoptosis, and autophagy.¹⁴ Among them, miR-199a-5p attracted growing interest in the field of oncology, such as cervical cancer, non-small cell lung cancer, and papillary thyroid carcinoma in the last few years.^15,16 Recently, the researches on the involvement of miR-199a-5p in the control of cardiac physiology and in the pathogenesis of heart failure have been revealed.^17–19 Various studies found that myocardial miR-199-5p levels are increased under cardiac damage, which is responsible for pathophysiological alterations contributing to the development of heart diseases including myocardial I/R injury.^20,21 Although miR-199-5p is involved in the progression of I/R-induced myocardial injury, whether miR-199-5p synergistically functions in the ferroptosis-induced cardiomyocyte damage during I/R injury remains unknown.

While evidence revealed that the inhibition of AKT/eNOS activities contributes to the progression of myocardial I/R injury,^22,23 but the connections of AKT/eNOS signaling pathway in terms of their function with miR-199-5p/ferroptosis were not clear. Hence, the present study aimed at exploring whether miR-199-5p contributes to ferroptosis–induced cardiomyocyte death during I/R injury in vitro via regulating AKT/eNOS signaling pathway. We confirmed that miR-199-5p promotes ferroptosis-induced cardiomyocyte death via inhibiting AKT/eNOS signaling pathway, then contributing to OGD/R injury, which provides new insights into the molecular mechanisms of myocardial I/R injury and avenues for the development of future therapy.

MATERIALS AND METHODS Cell culture and treatment

The rat myocardial cell line H9c2 was purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and grown in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/ml)-streptomycin (100 g/ml). To establish an in vitro model of myocardial I/R injury, H9c2 cells were treated with oxygen–glucose deprivation/reoxygenation (OGD/R) process as described previously.²⁴ Briefly, H9c2 cells were cultured in serum and glucose-free DMEM in a hypoxic incubator containing 1% O₂, 5% CO₂, and 94% N₂ at 37 °C for 6 h. After hypoxia, the cells were incubated in complete culture medium under normal growth conditions (5% CO₂ and 95% air) for an additional 12 h. Control cells were cultured in complete DMEM under normal growth conditions. To explore ferroptosis occurs under OGD/R condition, H9c2 cells were pretreated with ferroptosis inhibitor (Ferrostatin-1, 60 nM; Medchemexpress, Shanghai, China) following by OGD (6 h)/R (12 h) treatment. To investigate the role of miR-199a-5p in ferroptosis–induced cardiomyocyte death during the OGD/R process, H9c2 cells were transfected with miR-199a-5p inhibitor to down-regulate miR-199a-5p level and miR-199a-5p mimic to up-regulate miR-199a-5p level, and then exposed to OGD (6 h)/R (12 h). To demonstrate the function of AKT/eNOS signaling pathway in the protection of miR-199a-5p inhibitor on ferroptosis-induced cardiomyocyte death during OGD/R condition, H9c2 cells were pretreated with AKT inhibitor (LY294002, 25 μM; Haoyuan Chemexpress Co. Ltd, Shanghai, China) for 1 h and then treated with transfected with miR-199a-5p inhibitor prior to OGD (6 h)/R (12 h).

Transfection of cells with miR-199a-5p inhibitor and mimic

MiR-199a-5p inhibitor, miR-199a-5p mimic and negative controls were synthesized by GenePharma (ShangHai, China). For testing miR-199a-5p loss- and gain-of-functions, H9c2 cells were transfected with miR-199a-5p inhibitor, inhibitor negative control (inhibitor-NC), miR-199a-5p mimic or mimic negative control (mimic-NC) at a concentration of 100 nm using the Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The sequences were as follows: hsa-miR-199a-5p inhibitor, 5′-GAACAGGUAGUCUGAACACUGGG-3′; inhibitor-NC, 5′-CAGUACUUUUGUGUAGUACAA-3′; hsa-miR-199a-5p mimic, Sense sequence 5′-CCCAGUGUUCAGACUACCUGUUC-3′, Antisense sequence 5′-ACAGGUAGUCUGAACACUGGGUU-3′; mimic-NC, Sense sequence 5′- UUCUCCGAACGUGUCACGUTT-3′, Antisense sequence 5′- ACGUGACACGUUCGGAGAATT-3′. The transfection efficiency was verified by qRT-PCR.

Cell viability and cytotoxicity assay

The H9c2 cells were seeded into a 96-well plate at a density of 1 × 10⁴ cells/well. After treatment, the cell viability was detected using the Cell Counting Kit-8 (CCK-8) kit (Beyotime Institute Biotechnology, Shanghai, China) according to the manufacturer's instructions. 10 μl of CCK-8 solution was added to each well, and after 3 h incubation at 37°C, the absorbance at 450 nm was measured on a microplate reader (Synergy HT, Bio-Tek, USA).

The cytotoxicity was assessed by determining the activity of LDH in cell culture supernatants using a LDH activity assay kit (NanJing JianCheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instruction. In brief, the culture supernatant was collected and incubated with reconstituted substrate mix for 30 min in the dark at room temperature, followed by the addition of the stop solution. The absorbance at 490 nm was determined using a microplate reader (Synergy HT).

Total RNA isolation and qRT-PCR analysis

Total RNA from H9c2 cells was isolated by a TRIzol reagent (Invitrogen, CA, USA), and then 1 μg of RNA was reversely transcribed to first cDNA using a PrimeScript RT reagent kit with gDNA Eraser (Takara, Tokyo, Japan). QRT-PCR was carried out using an SYBR green PCR kit (Takara) on an ABI PRISM® 7500 Sequence Detection System (Applied Biosystems, USA) following the manufacturer's instructions, with U6 served as an inner control. The PCR primer sequences were as follows: miR-199a-5p, Forward 5′-GCCAAGCCCAGTGTTCAGAC-3′ and Reverse 5′-GTGCAGGGTCCGAGGTATTC-3′; U6, Forward 5′-CTCGCTTCGGCAGCACA and Reverse 5′-AACGCTTCACGAATTTGCGT-3′. The relative expression of miR-199a-5p was calculated using the 2^−(ΔΔCt) method and normalized to U6.

Detection of ROS generation

To measure the intracellular ROS level, treated H9c2 cells were stained with 5 μM of DCFH-DA (Molecular Probes, Eugene, OR, USA) at 37°C for 20 min. After washing with PBS, cells were resuspended in PBS (500 μl) and the ROS level was detected by flow cytometric analysis (Becton-Dickinson, Mountain View, CA, USA).

Measurement of malondialdehyde (MDA) content and glutathione (GSH)/glutathione disulphide (GSSG) ratio

The lipid peroxidation product MDA concentration and GSH/GSSG ratio in cell lysates were assessed using a Lipid Peroxidation MDA Assay kit (Beyotime Institute of Biotechnology, shanghai, China) and a GSH Kit (Dojindo Laboratory, Kumamoto, Japan) in accordance with the provided instructions. Protein concentration was determined by a BCA assay kit (Beyotime Institute of Biotechnology). The absorbance at 532 nm for MDA content was analyzed by a microplate reader (Synergy HT). The GSH and GSSG levels were calculated according to a standard curve and normalized to total protein level.

Measurement of Fe²⁺ level

For the iron detection, H9c2 cells were collected and immediately homogenized with PBS. After centrifugation at 12,000 × g at 4°C for 10 min, the supernatant was performed to detect iron concentration (Fe²⁺ level) using the Iron Assay Kit (Abcam, Shanghai, China) according to the manufacturer's instruction. Briefly, standards (50 μl) or samples (50 μl) were mixed with quantichrom working reagent (200 μl) in a 96-well plate and incubated at room temperature overnight. The OD at a wavelength of 590 nm was measured using a microplate reader (Synergy HT).

Measurement of nitric oxide (NO) in culture supernatant

The amount of nitric oxide (NO) produced by H9c2 cells was measured with Griess reagent (Beyotime Institute of Biotechnology) according to the manufacturer's instruction. A volume of supernatant (50 μl) was collected and incubated with an equivalent volume of Griess reagent I and Griess reagent II. The absorbance at 540 nm was measured using a microwave reader (Synergy HT).

Western blotting analysis

Total protein from H9c2 cells was extracted using RIPA lysis buffer (Beyotime Institute Biotechnology). Protein concentration was determined using a BCA assay kit. The equal amounts of proteins were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes (PVDF; Millipore, Bedford, USA). After blocking in 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against Gpx4 (1:1000, Abcam, Cambridge, MA), P-Akt Ser⁴⁷³ (1:1000, Abcam), AKT (1:1000, Abcam), P-eNOS Ser¹¹⁷⁷ (1:1000, Cell Signaling Technology, Danvers, MA), eNOS (1:1000, Abcam), and GAPDH (1:2000, Cell Signaling Technology) at 4°C overnight. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, Abcam) for 1 h at room temperature. Finally, the protein bands were visualized via an enhanced chemiluminescence (ECL) substrate kit (Pierce Biotechnology, Rockford, IL). The expressions of proteins were quantified by gel imaging system (NIH Image, Bethesda, MD, USA) and normalized to GAPDH.

Statistical analysis

All data were expressed by mean ± standard deviation (SD) from at least three independent experiments. The statistical analysis was performed by SPSS 19 statistical software. Statistical significance was analyzed by Students' t test between two groups or by one-way ANOVA followed by Tukey's posttest among multiple groups. p < 0 05 was defined as a statistically significant difference.

RESULTS Ferroptosis-induced cardiomyocyte death occurs in H9c2 cells-exposed to OGD/R and is accompanied by the increased miR-199a-5p level

Ferroptosis has been implicated in the pathological process associated with myocardial ischemia–reperfusion (I/R) injury.^10,25 First, to evaluate ferroptosis-induced cardiomyocyte death after myocardial I/R injury, we determined the effects of ferroptosis inhibitor (ferrostatin-1; Fer-1) and ferroptosis activator (Erastin) on oxygen–glucose deprivation/reperfusion (OGD/R; 6 h ischemia and 12 h reperfusion)-induced cardiomyocyte injury and ferroptosis. Our results showed that Fer-1 treatment significantly reduced OGD/R-induced the down-regulation of cell viability (Figure 1A) and the up-regulation of LDH activity (Figure 1B). And, Erastin decreased cell viability (Figure 1A) and increased LDH release (Figure 1B), which had similar effects to OGD/R treatment. Reactive oxygen species (ROS), lipid peroxidation, glutathione (GSH) depletion, and redox-active iron accumulation (Fe²⁺) are the key events in the process of ferroptosis. Our results further found that OGD/R as well as Erastin treatment led to the increase of ROS generation (Figure 1C) and MDA content (Figure 1D), the reduction of GSF/GSSH ratio (Figure 1E), and the up-regulation of Fe²⁺ level (Figure 1F) in H9c2 cells, while the effects of OGD/R were blocked by Fer-1. GPx4, as a marker of ferroptosis, was also markedly decreased in OGD/R- and Erastin-treated H9c2 cells; unquestionably, Fer-1 increased the expression of Gpx4 in H9c2 cell-subjected with OGD/R (Figure 1G). In addition, OGD/R also significantly up-regulated the miR-199a-5p level in H9c2 cells (Figure 1H). Taken together, our findings confirmed the contribution of ferroptosis-induced cardiomyocyte death to OGD/R injury, and revealed that miR-199a-5p may participate in the regulation of ferroptosis-induced cardiomyocyte death under OGD/R.

FIGURE 1. Ferroptosis-induced cardiomyocyte death was activated and miR-199a-5p expression was increased in H9c2 cardiomyocytes injected with OGD/R. (A) the viability of cell was measured by CCK-8 assay. (B) the activity of LDH, (C) the generation of ROS, (D) the content of MDA, (E) the ratio of GSH/GSSH, and (F) the level of Fe2+ were evaluated using commercial kits, respectively. (G) the expression level of Gpx-4 was detected by Western blot analysis. (H) the level of miR-199a-5p was determined by qRT-PCR analysis. Data are expressed as mean ± SD (n = 3–4 per group). **p [less than] 0.01 versus control group; ##p [less than] 0.01 versus OGD/R group

MiR-199a-5p promotes ferroptosis-induced cardiomyocyte death during OGD/R process

To test the function of miR-199a-5p in ferroptosis-induced cardiomyocyte death under OGD/R condition, we performed loss of function and gain of function experiments with transfection of the miRNA inhibitor and miRNA mimic. The transfection efficiency was verified by qRT-PCR and the result revealed that the miR-199a-5p inhibitor transfection resulted in a significant lower level of miR-199a-5p compared with inhibitor-NC transfection, while miR-199a-5p mimic transfection led to obvious higher level of miR-199a-5p compared with mimic-NC transfection (Figure 2A). Subsequently, our results showed that miR-199a-5p inhibitor blocked OGD/R-induced a decrease in cell viability (Figure 2B) and an increase in LDH activity (Figure 2C), while miR-199a-5p mimic aggravated OGD/R-induced a decrease in cell viability (Figure 2B) and an increase in LDH release (Figure 2C). In addition, miR-199a-5p inhibitor reduced ROS production (Figure 2D) and MDA content (Figure 2E), increased GSF/GSSH ratio (Figure 2F), and decreased Fe²⁺ level (Figure 2G) compared with inhibitor-NC group in H9c2 cells-exposed OGD/R treatment, while miR-199a-5p mimic further increased ROS production (Figure 2D) and MDA content (Figure 2E), decreased GSF/GSSH ratio (Figure 2F), and increased Fe²⁺ level (Figure 2G) compared with mimic-NC group in H9c2 cells-exposed OGD/R treatment. Furthermore, OGD/R-induced the down-regulation of Gpx4 expression level was reversed by miR-199a-5p inhibitor and exacerbated by miR-199a-5p mimic (Figure 2H). These results indicated that miR-199a-5p promotes ferroptosis-induced cardiomyocyte death under OGD/R condition.

FIGURE 2. MiR-199a-5p contributed to ferroptosis-induced cardiomyocyte death under OGD/R condition. H9c2 cells were transfected with inhibitor-NC, miR-199a-5p inhibitor, mimic-NC or miR-199a-5p mimic, and then subjected to OGD/R. (A) the transfection efficiency was verified by qRT-PCR analysis. (B) the viability of cell was measured by CCK-8 assay. (C) the activity of LDH, (D) the generation of ROS, (E) the content of MDA, (F) the ratio of GSH/GSSH, and (G) the level of Fe2+, in H9c2 cells were evaluated using commercial kits, respectively. (H) the expression level of Gpx-4 was detected by Western blot analysis. Data are expressed as mean ± SD (n = 3–4 per group). **p [less than] 0.01 versus control group; ##p [less than] 0.01 versus OGD/R + inhibitor-NC group; $p [less than] 0.5; $$p [less than] 0.01 versus OGD/R + mimic-NC group

MiR-199a-5p contributes to the inhibition of AKT/eNOS signaling pathway in OGD/R-treated H9c2 cells

Researches confirm that Akt/eNOS signaling pathway is involved in the progression of myocardial I/R injury.^23,24 To better understand its underlying mechanism of myocardial I/R injury, the effects of miR-199a-5p on the expression patterns of Akt/eNOS signaling pathway in OGD/R-treated H9c2 cells were detected by Western blot analysis (Figure 3A), which demonstrated that OGD/R obviously reduced the P-Akt/Akt ratio (Figure 3B) and P-eNOS/eNOS ratio (Figure 3C). However, these effects were blocked by miR-199a-5p inhibitor and deteriorated by miR-199a-5p mimic. In addition, miR-199a-5p inhibitor increased NO level, while miR-199a-5p mimic decreased NO level, in H9c2 cells-exposed to OGD/R (Figure 3D). Taken together, these results suggested that miR-199a-5p participates in the inhibition of Akt/eNOS signaling pathway in OGD/R-treated H9c2 cells.

FIGURE 3. MiR-199a-5p was involved in the inhibition of Akt/eNOS signaling pathway in OGD/R-treated H9c2 cells. H9c2 cells were transfected with miR-199a-5p inhibitor or miR-199a-5p mimic for 48 h, and then subjected to OGD/R. (A) the protein expressions were detected by Western blot analysis. Quantitative analysis of relative levels of (B) P-Akt/Akt ratio and (C) P-eNOS/eNOS ratio. (D) the level of nitric oxide (NO) in culture supernatant was measured with Griess reagent. Data are expressed as mean ± SD (n = 3–4 per group). *p [less than] 0.05; **p [less than] 0.01 versus control group; #p [less than] 0.05; ##p [less than] 0.01 versus OGD/R group

Repression of the Akt/eNOS signaling pathway attenuated the protective effect of miR-199a-5p inhibitor on ferroptosis-induced cardiomyocyte death under OGD/R condition

Finally, to further elaborate on the role of Akt/eNOS signaling pathway in miR-199a-5p-promoted-ferroptotic cell death under OGD/R condition, the inhibitor LY294002 was used to suppress the activation of Akt/eNOS signaling pathway. Results showed that LY294002 attenuated miR-199a-5 inhibitor-induced the up-regulation of P-Akt and P-eNOS levels in OGD/R-treated H9c2 cells (Figure 4A), indicating that LY294002 successfully suppresses the activation of Akt/eNOS signaling pathway. Next, our results observed that LY294002 also blocked miR-199a-5p inhibitor-induced an increase in cell viability (Figure 4B) and a decrease in LDH activity (Figure 4C) under OGD/R condition. In addition, LY294002 blocked miR-199a-5p inhibitor-led to the down-regulation of ROS production (Figure 4D) and MDA (Figure 4E), the up-regulation of GSH/GSSH ratio (Figure 4F), the reduction of Fe²⁺ level (Figure 4G) and Gpx4 expression (Figure 4H) in OGD/R-exposed H9c2 cells. These results indicated that miR-199a-5p promotes ferroptosis-induced cardiomyocyte death via activating Akt/eNOS signaling pathway during OGD/R process.

FIGURE 4. LY294002 blocked miR-199a-5p inhibitor-promoted ferroptosis-induced cardiomyocyte death in OGD/R-treated H9c2 cells. H9c2 cells were pretreated with Akt inhibitor (LY294002, 25 μM) for 1 h and then transfected with miR-199a-5p inhibitor prior to OGD/R. (A) the protein expressions were detected by Western blot analysis. (B) the viability of cell was measured by CCK-8 assay. (C) the activity of LDH, (D) the generation of ROS, (E) the content of MDA, (F) the ratio of GSH/GSSH, and (G) the level of Fe2+ in H9c2 cells were evaluated using commercial kits, respectively. (H) the expression of Gpx-4 was detected by Western blot analysis. Data are expressed as mean ± SD (n = 3–4 per group). **p [less than] 0.01 versus OGD/R + inhibitor-NC group; #p [less than] 0.05; ##p [less than] 0.01 versus OGD/R + miR-199a-5p inhibitor group

DISCUSSION

In this study, we determined the contribution of miR-199a-5p to the ferroptosis-induced cardiomyocyte death during I/R injury in vitro, and explored a new undying mechanism focusing on Akt/eNOS signaling pathway, thus providing the new therapeutic targets for myocardial I/R injury. In this study, we found that ferroptosis-induced cardiomyocyte death was activated in OGD/R process. Then, we verified that miR-199a-5p inhibitor blocked ferroptosis-induced cardiomyocyte death, while miR-199a-5p mimic exacerbated ferroptosis-induced cardiomyocyte death, responding to OGD/R injury. In addition, miR-199a-5p inhibitor promoted Akt/eNOS signaling pathway in OGD/R-treated H9c2 cells, and inhibition of Akt/eNOS signaling pathway reversed miR-199a-5p inhibitor-induced the attenuation of ferroptotic cell death. Taken together, our results demonstrated that miR-199a-5p promotes ferroptosis-induced cardiomyocyte death induced by OGD/R via suppressing Akt/eNOS signaling pathway.

Ferroptosis is a newly an iron-dependent, nonapoptotic form of regulated cell death that has recently been discovered during the pathological process of myocardial I/R injury.^10,25 The occurrence of ferroptosis leads to cardiomyocyte death driven by iron-dependent lipid peroxidation, which can be antagonized by glutathione peroxidase 4 (GPX4, a key antioxidant enzyme in the process of ferroptosis directly detoxify lipid hydroperoxides generated by ROS), participates in the pathogenic mechanisms of myocardial I/R injury in vivo and in vitro.^12,13 Ferroptosis inhibitors, such as ferrostatin-1 (Fer-1), have successfully alleviated myocardial I/R injury.²⁶ Consistent with these studies, we found that OGD/R treatment led to ferroptotic cell death as evidenced by the down-regulation of cell viability, the up-regulation of LDH activity, MDA content and GSH/GSSG ratio, raised Fe²⁺ level as well as Gpx4 expression in H9c2 cells, while these effects were blocked by Fer-1. In addition, Erastin, a ferroptosis inducer, also induced ferroptotic cardiomyocyte injury. Taken together, these results indicated that ferroptosis-induced cardiomyocyte death contributes to the process of myocardial I/R injury, and inhibition of ferroptosis tremendously reduced the myocardial I/R injury in vitro.

Emerging evidence reveals that increased miR-199a-5 level is responsible for pathophysiological alterations contributing to the development of heart diseases including heart failure.^19,20 However, the underlying mechanisms of miR-199a-5 in myocardial I/R injury are not well-understood. Many studies have shown that miRNAs modulate ferroptosis in myocardial infarction.^27,28 MiR-190a-5p negatively regulates ferroptosis in rat cardiomyocyte H9c2 cells, resulting in down-regulation of ROS, MDA and Fe²⁺ levels, which can provide a potential therapeutic target for myocardial infarction.²⁹ MiR-15a-5p over-expression strengthens ferroptosis, then aggravated hypoxia-induced myocardial cell injury.²⁷ However, during myocardial I/R injury, the effects of miR-199a-5 on ferroptosis have not been reported. In present study, the results found that the occurrence of ferroptosis-induced cardiomyocyte death was accompanied by the increase of miR-199a-5 level in OGD/R-treated H9c2 cells. In addition, miR-199a-5 inhibitor attenuated ferroptosis-induced cardiomyocyte death, while miR-199a-5 mimic exacerbated ferroptosis-induced cardiomyocyte death, in OGD/R-treated H9c2 cells. These results indicated that miR-199a-5 participates in ferroptosis-induced cardiomyocyte death, which may contribute to myocardial I/R injury.

It has already been shown that Akt-induced the phosphorylation of eNOS with a subsequent increase in the production of NO is a vital downstream effector in cardiomyocyte survival signaling during myocardial I/R injury.^22,23,30 The activation of Akt/eNOS signaling pathway is involved in the cardioprotection against ischemia/reperfusion injury.^22,31 Research reveals that various miRNAs have been implicated in regulating the AKT/eNOS signaling pathway.^32,33 However, the effect of miR-199a-5 on AKT/eNOS signaling pathway has not been reported. Our findings first showed that miR-199a-5 inhibitor promoted the activation of Akt/eNOS signaling pathway, while miR-199a-5 mimic reduced the activation of Akt/eNOS signaling pathway, in OGD/R-treated H9c2 cells, indicating the potential role of Akt/eNOS signaling pathway in the function of miR-199a-5 during OGD/R process. Recently, studies have confirmed that the activation of Akt signaling suppresses ferroptosis via regulating multiple downstream factor.^34,35 Consistent with these studies, our results further found that the inhibition of Akt/eNOS signaling pathway induced by LY294002 blocked the protective effects of miR-199a-5 inhibitor on ferroptosis-induced cardiomyocyte death under OGD/R condition, thereby highlighting the promotion of miR-199a-5 to ferroptosis-induced cardiomyocyte death dependency on the inhibition of Akt/eNOS signaling pathway during OGD/R. Notably, a study demonstrated that HIF-1α-GSK3β-mPTP axis mediates the regulation of miR-199a-5 in cardiomyocyes.³⁶ Another study also confirmed that attenuating endoplasmic reticulum (ER) stress contributes to myocardial protection of down-regulation of miRNA-199a-5p.³⁷ Given the relationship between Akt/GSK-3β pathway and ferroptosis³⁴ and an inseparable link between ER stress and ferroptosis,³⁸ Akt/GSK-3β pathway or ER stress may also involve in the antiferroptosis of miR-199a-5 in OGD/R injury, which are worthy of further verification in future research.

There are some limitations to this study. Firstly, our experiments were only performed in vitro H9c2 cells model, which could not fully mimic myocardial I/R injury in vivo. Hence, animal and/or primary cardiomyocytes studies should be carried out for further investigation. Secondly, miRNAs suppress the expression of target genes at the post-transcriptional level,³⁹ and the luciferase reporter assay is certainly needed to identify a direct target of miR-199a-5 which may be involved in regulating AKT/eNOS signaling pathway in subsequent studies. Thirdly, LY294002 is also used to inhibit AKT/eNOS signaling pathway, siRNA-mediated the knockdown of AKT or eNOS can be added to observe the function of AKT/eNOS signaling pathway. Finally, based on previous studies, other target genes or signaling pathway, such as HIF-1α,³⁶ sirtuin 1 (Sirt1),¹⁸ and ERK, p38, and JNK signaling pathways⁴⁰ are the direct target genes/pathway of miR-199a-5, which is worth exploring whether these genes play a role in the antiferroptosis of miR-199a-5 during myocardial OGD/R injury.

All in all, our findings demonstrated that miR-199a-5 promotes ferroptosis-induced cardiomyocyte death via the inhibition of AKT/eNOS signaling pathway, thereby contributing to OGD/R injury, suggesting its potential as a target for the development of myocardial I/R injury and focusing on inhibition of miR-199a-5 may be beneficial for attenuating myocardial I/R injury.

CONFLICT OF INTEREST

All authors declare no conflict of interest.