Cardiac extracellular vesicles aggravate cardiomyocyte ferroptosis in myocardial ischemia-reperfusion injury via miR-155-5p-Nfe2l2 signaling

doi:10.21203/rs.3.rs-4903592/v1

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Cardiac extracellular vesicles aggravate cardiomyocyte ferroptosis in myocardial ischemia-reperfusion injury via miR-155-5p-Nfe2l2 signaling

https://doi.org/10.21203/rs.3.rs-4903592/v1

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Ischemia-reperfusion (IR) injury represents a major cause of cell death post myocardial infarction. Ferroptosis is a newly discovered form of regulated cell death (RCD) dependent on iron and reactive oxygen species (ROS). We recently confirmed that cardiac IR triggers the increased release of extracellular vesicles (EVs) which aggravates cardiac dysfunction. Whether and how these EVs contribute to cardiac ferroptosis during myocardial IR injury remain elusive. Murine myocardial IR models were established by ligation of the left anterior descending coronary artery for 45 minutes and then reperfusion. Then EVs from the heart subjected to IR (IR-EVs) were isolated. Adoptive transfer of IR-EVs and EVs inhibition experiments confirmed that IR-EVs act as a vital factor that contributes to the cardiomyocyte ferroptosis during cardiac IR, with increased Ptgs2 expression and malondialdehyde (MDA) production, as well as decreased NADPH level. Moreover, miR-155-5p enriched in IR-EVs can be delivered into cardiomyocytes and promoted the ferroptosis of cardiomyocytes in the peroxidation injury. Nfe2l2 was further confirmed as the target gene of miR-155-5p by luciferase reporter assay. Consistently, molecules targeting Nfe2l2 modulated the H₂O₂or oxygen glucose deprivation/reoxygenation (OGD/R) induced ferroptosis, involving the downstream antioxidant response elements (AREs) of the Nfe2l2 pathway including Nqo1, HO1, Fth1, and Slc7a11. In conclusion, the present results provide a novel EV-based ferroptosis regulation mechanism in cardiac IR injury. Strategies targeting the IR-EVs-miR-155-5p-Nfe2l2 axis may be of therapeutic potential to prevent cardiac ferroptosis and dysfunction after myocardial IR.

Extracellular vesicles

Myocardial ischemia-reperfusion injury

miRNA

Ferroptosis

Ischemic heart disease is still a leading cause of death worldwide. Despite the recent advances in acute myocardial infarction treatment such as percutaneous coronary intervention and coronary artery bypass grafting, myocardial ischemia-reperfusion (IR) injury remains a common issue in clinical settings, which lead to aggravated cardiac dysfunction and eventually heart failure.

Previous studies found that the pathways leading to major cell death including apoptosis, necroptosis, necrosis and ferroptosis, are responsible for cardiac IR injury^1,2. Strategies to prevent the cell death in peri-infarct zone have been confirmed to preserve cardiac function³. Therefore, it is of great importance to investigate the cell death mechanisms during myocardial IR injury.

As a newly identified regulated cell death (RCD), ferroptosis is characterized by the iron-dependent accumulation of lipid hydroperoxides and can be caused by the impairment of the glutathione peroxidase 4 (GPX4)⁴. Biochemical changes closely related to ferroptosis include lipid peroxidation⁵, high expression of Ptgs2, which was considered as a marker to assess ferroptosis⁶ and low level of NADPH⁷. Emerging evidence indicated that ferroptosis is involved in IR injuries of the liver⁶ and heart^8,9. The ferroptosis inhibitors, such as iron chelator and glutaminases inhibitor can protect the murine heart from IR injury¹⁰. However, the precise mechanisms in which cardiac ferroptosis are triggered and regulated remain poorly understood. Elucidation of the molecular mechanisms of IR-induced ferroptosis may provide new targets for clinical interventions.

Extracellular vesicles (EVs) are nanosized cell-derived membranous vesicles that exist in various biological fluids¹¹. By transferring multiple molecules such as proteins, lipids, and RNAs¹², EVs are recognized as key players in cell-to-cell communication across our body. It has been demonstrated that these highly heterogeneous nanosized vesicles contribute to the pathophysiology of the heart. We recently disclosed that intracardiac EVs induced by IR (IR-EVs) contribute to the proinflammatory responses in heart and these EVs possess specific miRNAs¹³. It is reported that miRNAs play key roles in regulation of different cell death. Previous studies showed that EVs secreted by cardiomyocytes after hypoxia stimulation contain specific miRNAs, which can induce apoptosis and autophagy of the surrounding cells^14,15. Other studies confirmed the involvement of miRNAs in the ferroptotic injury of various pathological processes^16,17. Therefore, we hypothesized that IR-EVs may regulate the ferroptosis of cardiomyocytes via transferring their miRNA cargo following IR.

Here we discovered a novel EV-based ferroptosis regulation mechanism in cardiac IR injury. Our in vitro and in vivo studies demonstrated that IR-EVs can deliver miR-155-5p into cardiomyocytes and facilitate IR-induced ferroptosis by targeting the Nfe2l2 pathway. The present study provides some new insights into the pathogenic mechanism and indicates the potential therapeutic strategies targeting the IR-EVs-miR-155-5p-Nfe2l2 axis during IR injury in clinical settings.

Animals

Male C57BL/6 mice aged 10–12 weeks were purchased from SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Mice were housed in a specific-pathogen-free room with constant temperature (23–24°C), humidity (55 ± 5%), and light (12–12 h light-dark schedule). All the mice had free access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee of Tongji University (Number: TJLAC-019-148) and conformed to the European Directive 63/2010 on the protection of animals used for scientific purposes.

Establishment of the myocardial IR models and grouping

Computer-generated random numbers were used for mice grouping. The myocardial IR models were established as previously described¹⁸. Briefly, an intraperitoneal injection of 1.5% pentobarbital (50 mg/kg body weight, Sigma, USA) was performed for mouse anesthesia. After endotracheal intubation and mechanical ventilation, the chest cavity was opened in the 4th intercostal space. The left anterior descending coronary artery was ligated with an 8 − 0 silk suture. The suture was then removed 45 minutes later. Once removing the silk suture, intracardiac injection of 50µL IR-EVs (2×10⁸/µl) or PBS was performed through the left ventricle. After the operation, the mice were placed on a heated blanket until recovered from anesthesia. Intraperitoneal injection of GW4869 (2.5 mg/kg, Sigma-Aldrich) was performed 1 h before IR surgery in the GW4869 group. 24 h later, mice were sacrificed by cervical dislocation and plasma and heart samples were collected for further evaluation.

Echocardiography analysis

Cardiac function was evaluated with a Vevo2100 ultrasound system (Visual Sonics, Canada) at 24 h after IR surgery. Briefly, mice were mildly anesthetized with 1.25% isoflurane. The left ventricle internal diameters at end-systole (LVESD) and end-diastole (LVEDD) were measured in two-dimensional long-axis views. The left ventricular ejection fraction (EF) and fraction shortening (FS) were calculated for cardiac function assessment.

Myocardial enzymes detection

Murine blood was collected in heparin-containing collection tubes 24h after IR surgery. The plasma was isolated by centrifugation (2000g, 4˚C, 10min) to detect aspartate transaminase (AST) and creatine kinase (CK) levels using Beckmann AU680 (Beckman Coulter, Inc.) according to manufacturer's instructions.

EV isolation and identification

24h after surgery, the cardiac tissue was harvested and IR-EVs was isolated as previously described¹⁸. Briefly, after phosphate-buffered saline (PBS) perfusion, left ventricle tissues subjected to IR injury were removed and cut into small pieces. The heart tissues were incubated in 4 ml of 0.1% type II collagenase (Sigma, USA) at 37°C for 30 min. The digested tissue was centrifuged at 300 g for 5 min at 4°C to remove the tissues and cells. The supernatant was centrifuged at 2000 g for 10 min, and then at 10000 g for 10 min at 4°C. The supernatant was collected and then centrifuged at 120,000 g for 2h at 4°C (Optima L-100XP Ultracentrifuge, Beckman Coulter). EVs were obtained after one wash with PBS.

The concentration of EVs was tested using the ZetaView® Nanoparticle tracking analysis (NTA) technique (Particle Metrix, Germany) with 3 replicates. The fresh-isolated EVs were fixed with 2.5% glutaraldehyde and loaded onto 200 Mesh carbon-coated formvar grids for 5 min. Next, 2% phosphotungstic acid was used for EV-staining (5 min at room temperature) and the transmission electron microscope (TEM; Hitachi, HT7700) for EVs detection.

Primary cardiomyocyte isolation and cell culture

Neonatal mice aged 1 to 3 days were sacrificed by cervical dislocation and used for the primary cardiomyocyte isolation. Briefly, the heart was cut and washed with ice-cold PBS. Ventricle tissues were shredded and incubated in 0.1% type II collagenase (Sigma, USA) for 4-8h at 4°C. Tissues were then digested with 0.1% type II collagenase 5 min per time at 37°C for 3–5 times. The supernatants were collected and transferred to an equal volume of Dulbecco's Modified Eagle Medium (DMEM, Gibco; Thermo Fisher Scientific, USA) supplemented with 50% fetal bovine serum (FBS, Gibco; Thermo Fisher Scientific, USA). After digestion, single-cell suspension was obtained. Cells were pelleted, and resuspended in DMEM containing 10% FBS, 1% penicillin (100 U/ml)/streptomycin (100 µg/ml). After 2 h incubation, the non-adherent cells were collected and cultured in DMEM supplemented with 10% exosome-depleted FBS (Gibco; Thermo Fisher Scientific, USA), and 1% penicillin/streptomycin. On the next day, cells were washed with PBS, cultured in DMEM complete medium with 0.1mM BrdU (Sigma, USA) and treated with different conditions. The mouse cardiac myocytes (MCM) cell line was purchased from the American Type Culture Collection (ATCC, Gaithersburg, MD, USA) and cultured in DMEM containing 10% exosome-depleted FBS and 1% penicillin/streptomycin and subjected to different treatments.

Cell models and treatments

To simulate in vivo myocardial IR injury, two cell injury models were used in this study: (1) H₂O₂ induced cell model: MCM cells were stimulated with 5 mM H₂O₂ for 2 hours and then collected for detection; (2) Oxygen glucose deprivation/reoxygenation (OGD/R) model: the primary cardiomyocytes were cultured in serum-free/glucose-free DMEM (Gibco; Thermo Fisher Scientific, USA) at 37 ℃, 5% CO2 in hypoxia (< 1%) incubator for 8 hours, and then replaced with high glucose DMEM medium containing 10% exosome-depleted FBS at 37 ℃, 5% CO2 in normoxia incubator for 2 hours. Cells incubated under normoxic conditions were served as controls. As for IR-EVs treatment, cells were cocultured with IR-EVs (10¹⁰/mL) for 24h before H₂O₂ or OGD/R treatment. The treatments of different kinds of RCD inhibitors (1µM Ferrostatin-1, 10µM necrostatin-1, and 10µM Z-VAD(OMe)-FMK respectively) were performed 2h before H₂O₂ stimulation. TBHQ (5µM) and ML385 (5µM) were used in the indicated group. All these inhibitors are from MCE (USA).

Cell viability and cytotoxicity assay

According to the manufacturer's instructions, cell viability was estimated using the Cell Counting Kit-8 (CCK-8) (Yeasen Biotech, China). Absorbance at 450 nm is proportional to the number of living cells.

For cytotoxicity assessment, the CytoTox 96 Assay(Promega, USA) was used to measure lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis. The amount of released LDH in culture supernatants is proportional to the number of lysed cells.

Flow cytometry apoptosis assay

Cell apoptosis was detected using the Annexin V FITC apoptosis detection kit (BD Pharmingen, USA) according to the manufacturer’s instructions on the BD FACSVerse™ System.

Malondialdehyde (MDA) assay

The relative MDA concentration in cell and tissue lysates was detected using a Lipid Peroxidation Assay Kit (Beyotime, China) according to the manufacturer’s directions. Briefly, the MDA in the sample reacted with thiobarbituric acid (TBA) to generate an MDA-TBA adduct. The MDA-TBA adduct was quantified colorimetrically (OD = 532 nm).

NADP/NADPH assay

NADP/NADPH Assay Kit (Abcam, USA) was used to measure the relative NADP and NADPH concentration in the cell and tissue lysates according to the manufacturer’s instructions. Results are quantified using a plate reader at OD450 nm.

Iron assay

According to the manufacturer's instructions, the intracellular iron level was determined using the iron assay kit (Sigma, USA). Briefly, cells were washed in cold PBS and then homogenized on ice. Released iron in the supernatant is reacted with a chromagen resulting in a colorimetric (593 nm) product, proportional to the iron present.

Luciferase reporter assay

Nfe2l2-3’UTR-wt/mut was sub-cloned into the pmirGLO dual-luciferase plasmid (Promega, USA). The pmirGLO- nfe2l2-3’UTR-wt/mut plasmid was then co-transfected into 293 T cells with miR-155-5p mimic or NC mimic. According to the manufacturer's instructions, luciferase activities were examined via Dual-Luciferase Reporter Assay System (Promega, USA).

Histology

Hearts were perfused and fixed in 4% paraformaldehyde. Midventricular short-axis heart sections (5 µm) were stained with Prussian blue (free iron stain) (Sigma, USA) to examine iron deposition. Immunohistochemical staining were performed by using Rabbit polyclonal anti-4-hydroxynonenal (4-HNE) antibody (Abcam, USA) and HRP anti-rabbit IgG (Abcom, USA) according to the manufacturer's instructions.

Transfection of miRNA mimics and inhibitors

Cells were transfected with negative control miRNA, miR-155-5p mimic, or miR‐155-5p inhibitor (synthesized commercially by Sangon Biotech) using riboFECT™ CP Transfection Regent (RiboBio R10035.3, China) according to the manufacturer’s instructions.

Real-time quantitative polymerase chain reaction (RT-qPCR)

TRIzol reagent (15596–018, Invitrogen, USA) RNA was used to isolate the total RNA. For mRNA, RNAs were transcribed into complementary DNAs (cDNAs) using the PrimeScript RT reagent kit (Takara Bio, Inc., Japan). For miRNA, reverse transcription was performed using the microRNA SReverse Transcription Kit (Tiangen Biotech, China). RT-qPCR was performed using SYBR Green MasterMix (Applied Biosystems; Thermo Fisher Scientifc, Inc., USA). The miRNA and mRNA expression levels were respectively normalized to the endogenous control U6 small nuclear RNA and β-actin. The relative expression of each RNA was calculated based on the 2^-∆∆CT method. The primers used in the study were presented in Supplementary Table 1.

Western Blot

After indicated treatments, the samples were lysed with RIPA Buffer. Proteins were extracted and loaded on 10% SDS-PAGE gels, then transferred to PVDF membranes. After blocking for 1h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies directed to: CD9 (Abcam, UK, 1:1000 dilution), TSG101 (Abcam, UK, 1:1000 dilution), Nfe2l2 (Abcam, UK, 1:1000 dilution), Caspase3/cleaved Caspase3 (CST, USA, 1:1000 dilution), GPX4 (CST 52455, 1:000 dilution), NQO1/HO1 (Abcam, ab80588/ab68477, 1:000 dilution), Ferritin heavy chain (FHC/FTH1;Abcam, ab183781, 1:000 dilution), SLC7A11 (Abcam, ab175186, 1:000 dilution) and β-actin (CST 3700, 1:5000 dilution). After TBST washing, the membranes were incubated with corresponding secondary antibodies (CST 7074 or 7076, 1:1000 dilution) for 1 h, and the bands were detected by chemiluminescence (Pierce, USA).

Statistical Analysis

The data were presented as the mean ± Standard Error of Mean (SEM) and analyzed using Prism 8.0 (GraphPad Software Inc.). The student’s t-test was used for comparison between the two groups. The comparison of multiple groups was performed with the one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test. A P-value ≤ 0.05 was considered statistically significant.

IR-EVs enhanced the cardiac ferroptosis during cardiac IR injury

We recently reported that administration of IR-EVs significantly aggravated cardiac inflammation and dysfunction in murine cardiac IR models¹³. To demonstrate the role of IR-EVs on cardiac ferroptosis in vivo, we isolated IR-EVs and identified their typical characteristics by TEM, NTA, and western blot, respectively (Sup Fig. 1A-C). Next, 50µL IR-EVs (2×10⁸/µL) or PBS were intracardially injected into IR mice or the sham. Consistent with our previous report, IR-EVs treatment markedly decreased the cardiac function (Fig. 1A-C) and aggravated myocardial injury (Fig. 1D) compared to the PBS group in mice subjected to IR. Then, we found much higher Ptgs2 expression and MDA production in the heart of mice treated with IR-EVs than that with PBS (Fig. 1E and F). Furthermore, total NADP and NADPH levels were significantly reduced (Fig. 1G and H), and both 4-HNE production (Fig. 1I), and iron levels (Fig. 1J), were markedly elevated in the IR + IR-EVs group, compared with the IR + PBS group. In addition, IR-EVs treatment significantly inhibited the expression of GPX4 in the heart subjected to IR (Fig. 1K-M). Although in sham mice, administration of IR-EVs did not significantly affect the cardiac function, it did decrease both productions of NADP and NADPH (Fig. 1G and H), and the mRNA level of GPX4 in heart (Fig. 1K). Prussian blue staining also showed the increase of iron deposition in the sham heart receiving IR-EVs (Fig. 1J). Conversely, EV inhibition by GW4869^19,20 increased the expression of GPX4 (Fig. 1N) and significantly relieved the ferroptotic injury in the IR-injured heart (Fig. 1O-R). These results indicated that IR-EVs act as a vital factor that contributes to the ferroptotic injury during cardiac IR.

IR-EVs aggravated H₂O₂-induced ferroptosis of cardiomyocytes

Previous studies demonstrated that ferroptosis is initiated by oxidative stress²¹, and the latter is one of the most important pathological mechanisms in cardiac reperfusion injury²². To investigate the role of IR-EVs on myocardial ferroptosis during IR injury in vitro, we constructed an H₂O₂-induced cell death model using the MCM cell line which was tested by different kinds of RCD inhibitors including ferrostatin-1 (a ferroptosis inhibitor), necrostatin-1 (a potent necroptosis inhibitor that targets receptor-interacting protein 1) and ZVAD-FMK (a potent apoptosis inhibitor). We found that the decrease of cell viability induced by H₂O₂ (5 mM) could be reversed by ferrostatin-1 and necrostatin-1 (Sup Fig. 2). Further flow cytometry analysis showed that H₂O₂ induced a large number of cell death, while there was only a small part of apoptotic cells (Fig. 2A and B). Addition of Ferrostatin-1 or Necrostatin-1, rather than ZVAD-FMK significantly reduced the ratio of dead cells, compared to control H₂O₂ treated cells (Fig. 2A and C). Of all these RCD inhibitors, Ferrostatin-1 showed the best protective effect, which suggested that ferroptosis represents as a major form of cell death in the H₂O₂-induced cell injury models.

Then, we evaluated the role of IR-EVs on cardiomyocyte ferroptosis. Compared with H₂O₂ treated control MCM cells, addition of IR-EVs increased the cell death (PI⁺ cells in Fig. 2D) and reduced their viability (Fig. 2E). In addition, we found that IR-EVs treatment significantly decreased the ferroptosis-suppressor protein GPX4 (Fig. 2F and H) rather than significantly influenced the expression of caspase3 (Fig. 2G) in H₂O₂-induced cells. Ferroptosis of MCM cells induced by H₂O₂ was further determined by the widely used indicators of ferroptosis including Ptgs2, MDA, and NADPH levels (Fig. 2I-L). Compared with the H₂O₂ group, addition of IR-EVs markedly increased the expression of Ptgs2 (Fig. 2I) and the production of MDA (Fig. 2J), while reduced the total NADP and NADPH levels (Fig. 2K and L). These results suggested that IR-EVs aggravated ferroptosis in H₂O₂ induced cell injury.

IR-EVs delivered miR-155-5p into cardiomyocytes

As an important mediator for intercellular communication, EVs can regulate the function of target cells by transferring their miRNA cargos. Next, IR-EVs were labeled with red fluorescent dye Dil, and then cultured with MCM cells for 24h. Confocal microscope revealed that IR-EVs were transferred into MCM cells (Fig. 3A). We previous identified a series of high-expressed miRNAs in IR-EVs, and the top 3 enriched miRNAs included miR-novel-chr7_40384, miR-155-5p, and miR-484¹³. To determine whether these miRNAs can be transferred into cardiomyocytes, we detected their expressions in the MCM cells after co-culture with IR-EVs. The expression of miR-155-5p (Fig. 3B) rather than pri-mir155 (Fig. 3C) was significantly increased in MCM cells after co-culture with IR-EVs. Consistently, further confocal microscope confirmed the up-taken of IR-EVs (Fig. 3D) and qPCR results showed the highly enriched miR-155-5p (Fig. 3E) in primary cardiomyocytes co-cultured with IR-EVs. Meanwhile, there was no significant difference of pri-mir155 expression in primary cardiomyocytes with and without IR-EVs treatment (Fig. 3F). Therefore, miR-155-5p in IR-EVs may act as a potential candidate to regulate cardiomyocytes ferroptosis.

IR-EVs enriched miR-155-5p contributed to the ferroptosis of cardiomyocytes

To further explore the effect of miR-155-5p on cardiomyocyte ferroptosis, miR-155-5p mimics and inhibitors were synthesized. We found that miR-155-5p overexpression aggravated the cytotoxicity of H₂O₂ stimulated MCM cells (Fig. 4A). Moreover, compared with the H₂O₂ + miR-Sc group, the H₂O₂ + miR-155-5p mimics group showed enhanced expressions of Ptgs2 (Fig. 4B), Acsl4 (Sup Fig. 3A), and Atp5g3 (Sup Fig. 3B), and elevated production of MDA (Fig. 4C). While addition of miR-155-5p mimics reduced the levels of the total NADP and NADPH in H₂O₂ treated MCM cells (Fig. 4D and E). Moreover, inhibition of miR-155-5p alleviated ferroptosis in H₂O₂ treated MCMs with decreased expression of Ptgs2, Acsl4, and Atp5g3 (Sup Fig. 4). Consistently, miR-155-5p mimics promoted cell injury (Fig. 4F) and decreased cell viability (Fig. 4G) in the primary murine cardiomyocytes undergoing OGD/R. Addition of miR-155-5p mimics also facilitated the OGD/R induced ferroptotic injury, manifested as increased MDA production (Fig. 4H), and decreased levels of the total NADP and NADPH (Fig. 4I and J). Interestingly, H₂O₂ treatment increased the activation of the caspase3 pathway which was significantly reduced in cells treated with miR-155-5p mimic (Sup Fig. 5A and B). The expression of GPX4 was decreased in the H₂O₂ induced MCMs and was much lower in MCMs transfected with miR-155-5p mimics than those transfected with miRNA scramble (Fig. 4K and L). In addition, miR-155-5p overexpression reduced the viability of H₂O₂ stimulated MCM cells, which was counteracted by ferroptosis inhibitor Ferrostatin-1 (Fig. 4M).

Then, we sought to determine whether IR-EVs exerted their role in ferroptosis of cardiomyocytes via miR-155-5p. As shown by Fig. 4N, IR-EVs aggravated H₂O₂ induced cytotoxicity which could be enhanced by transfection of miR-155-5p mimics and abolished by transfection of miR-155-5p inhibitors into IR-EVs. Similarly, IR-EVs treatment increased the iron level in H₂O₂ stimulated MCMs, which could be enhanced by transfection of miR-155-5p mimics and abolished by transfection of miR-155-5p inhibitors into IR-EVs (Fig. 4O). These findings indicated that miR-155-5p in IR-EVs promoted the ferroptosis of cardiomyocytes in the peroxidation injury.

miR-155-5p in IR-EVs can target Nfe2l2 pathway in cardiomyocyte

Next, we predicted the target genes of miR-155-5p using TargetScan and Miranda databases. The top 10 predicted genes were presented in Supplementary Table 2. Among these candidate genes, Nfe2l2 was known as an antioxidant gene²³, which was further confirmed as the target of miR-155-5p by luciferase reporter assay (Fig. 5A and B). Moreover, the top 10 target genes of other high-expressed miRNAs were also predicted (Sup Fig. 6). While there were no relevant reports showing their roles in ferroptosis regulation. IR-EVs pretreatment reduced both mRNA and protein levels of Nfe2l2 in H₂O₂ stimulated MCM cells (Fig. 5C-E). Consistently, miR-155-5p overexpression decreased the expression of Nfe2l2 in MCMs treated with H₂O₂ (Fig. 5F-H). In addition, intro-cardiac injection of IR-EVs decreased the expression of Nfe2l2 in the IR-injured heart (Fig. 5I-K). While EV-inhibition by GW4869 increased the level of Nfe2l2 in the heart following IR (Fig. 5L-N).

In the OGD/R treated primary cardiomyocytes, overexpression of miR-155-5p reduced the expressions of Nfe2l2, Nqo1, Ho1, Fth1, Slc7a11, and GPX4 (Fig. 5O). Transfection of miR-155-5p inhibitor increased the expression of Nfe2l2 pathway genes in the primary cells receiving OGD/R treatment (Sup Fig. 7). Similarly, in the H₂O₂-stimulated MCM model, the expressions of cellular protective genes (AREs) were decreased in the miR-155-5p overexpressed cells (Fig. 5P), while increased in the miR-155-5p inhibited ones (Sup Fig. 8A-D). Overexpression of miR-155-5p also suppressed the protein levels of Ho1, Fth1, and Slc7a11 in MCMs treated with H₂O₂ (Fig. 5Q). Compared to the IR injured mice, those treated with IR-EVs showed higher levels of Nqo1, Ho1, Fth1, and Slc7a11 in the heart (Sup Fig. 9). IR-EVs inhibition by GW4869 resulted in increased expression of Nfe2l2 pathway genes in the murine heart post IR (Sup Fig. 10). These results demonstrated that IR-EVs as well as their miR-155-5p cargo inhibit Nfe2l2 pathway during myocardial IR injury.

miR-155-5p contribute to cardiomyocyte ferroptosis via Nfe2l2 pathway

In order to investigate the role of Nfe2l2 on cardiomyocyte ferroptosis during IR injury, TBHQ (Nfe2l2 activator) and ML385 (Nfe2l2 inhibitor) were applied in both MCM and primary cardiomyocyte injury models. Firstly, we found that TBHQ elevated the mRNA expressions of Nfe2l2 (Fig. 6A) and the downstream AREs including Nqo1 (Fig. 6B), HO1 (Fig. 6C), and Fth1 (Fig. 6D) in the H₂O₂ stimulated MCMs. As a transcriptional target of Nfe2l2, Slc7a11 was also increased in the H₂O₂ + TBHQ group compared with the H₂O₂ + DMSO group (Fig. 6E). While ML385 diminished the expression of Nfe2l2 and Ho1, rather than Nqo1, Fth1, and Slc7a11 in H₂O₂ induced MCMs (Sup Fig. 11). Further, TBHQ treatment protected the MCM cells from H₂O₂ induced injury with elevated cell vitality (Sup Fig. 12) and reduced cell death (Fig. 6F and G). However, ML385 treatment increased the percentage of dead cells in H₂O₂-induced MCMs (Sup Fig. 12, Fig. 6F and G), however, interestingly reduced the ratio of apoptotic cells (Fig. 6F and H). Nfe2l2 activation by TBHQ alleviated H₂O₂-induced ferroptosis with decreased MDA production (Fig. 6I). On the contrary, the production of MDA was increased in H₂O₂-induced MCM cells treated with ML385 compared to the control group (Fig. 6I). More importantly, overexpression of miR-155-5p increased the MDA levels in H₂O₂-induced MCMs, which was alleviated by TBHQ and further aggravated by ML385 (Fig. 6J).

In primary cardiomyocytes receiving OGD/R treatment, addition of miR-155-5p mimics decreased the cell viability while addition of TBHQ reversed this effect (Fig. 6K). In addition, ML385 treatment further decreased the cell viability and increased MDA levels in miR-155-5p transfected cardiomyocytes (Fig. 6K and L). Collectively, these results suggested that miR-155-5p in IR-EVs aggravated IR-induced ferroptosis via targeting the Nfe2l2 pathway.

Restoring cardiac blood flow after the coronary obstruction is the dominant treatment strategy, but the secondary myocardial cell death and cardiac function decline after reperfusion remain intractable. Protecting non-renewable cardiomyocytes from cell injury/death is essential to preserve heart function during ischemic injury²⁴. Recent study confirmed the involvement of RCDs in cardiomyocytes loss after IR¹. The well-known RCDs such as caspase-dependent apoptosis have been studied as an important type of myocardial cell death during heart injury. Recently, other forms of cell death have been revealed to participate in heart diseases²⁵. A previous study confirmed that the newly identified ferroptosis played a vital role in cardiac injury induced by doxorubicin and was considered to be an important target for myocardial protection²⁶. Recent study revealed ferroptosis as the predominant type of cardiomyocytes death in the mice 24 hours after IR and inhibition of cardiomyocyte ferroptosis protected the heart from IR injury and dysfunction²⁷. Here we firstly demonstrated that IR-EVs represented as an important factor triggering the cardiomyocyte ferroptosis post IR, and suppression of EV production may become a promising strategy to prevent the cardiomyocyte from IR induced ferroptosis. Our previous report showed that IR-EVs not only aggravate the local cardiac inflammation, they also promote a systemic inflammatory response in other non-ischemic organs¹³. The present results indicated the mild effect of IR-EVs on the murine heart including the decreased NADP and increased iron deposition. Whether there is a local dose-dependent effect between the IR-EVs and cardiac ferroptosis and whether IR-EVs promote ferroptosis in other organs need further determination.

GPX4 is known as one of the most important proteins protecting cells against ferroptotic injury²⁸. Previous studies revealed that inactivation of GPX4 lead to ferroptosis^29–31. In this study, IR-EVs treatment markedly reduced GPX4 protein expression in the H₂O₂ treated cardiomyocytes but did not significantly affect their expression of Caspase3, a key protein in the apoptosis pathway. Therefore, in the H₂O₂-induced cell injury model, ferroptosis may be a major form of cell death, and IR-EVs mainly contributed to ferroptosis rather than apoptosis.

Recent studies highlighted the role of EVs as cell-to-cell communication vehicles in different pathophysiological processes. The function of EVs largely depends on their contents. We previously confirmed the enrichment of miR-155-5p in IR-EVs. The circulating miR-155-5p was significantly elevated in the patients with heart failure³². The detrimental role of miR-155-5p was disclosed in cerebral IR injury via targeting DUSP14³³. In addition, miR-155 participates in various cell death processes in cancer cells including apoptosis^34–36 and autophagy³⁷. In mesenchymal stem cells, miR-155-5p induces ROS generation by targeting the transcription factor C/ebpβ³⁸. All these findings imply a close relation between miR-155-5p and IR-induced cell injury. The present study for the first time confirmed that miR-155-5p enriched in IR-EVs significantly promotes the ferroptosis of cardiomyocytes by inhibiting the Nfe2l2 pathway during IR injury.

As an important antioxidative transcriptional factor, Nfe2l2 regulates the expression of various cytoprotective genes involved in detoxification and antioxidant. Increasing evidence disclosed the central role of Nfe2l2 against multiple cellular defenses^39–41. Physical and chemical insults involving oxidative stress can activate Nfe2l2 and induce the expression of a series of AREs to resist the injury²³. Previous studies demonstrated an important effect of Nfe2l2 in heart diseases^42–44 as well as in the development of ferroptosis^45–47. MiR-155 can regulate Nfe2l2 pathway in lung cancer cells⁴⁸ and human bronchial epithelial cells⁴⁹, but the mechanism is not clear. In the present study, we confirmed that miR-155-5p could reduce the antioxidant activity of cardiomyocytes through direct targeting Nfe2l2 gene. It was reported that suppression of the AREs promotes ferroptosis in the hepatocellular carcinoma cells treated with erastin and sorafenib⁵⁰. SLC7A11 is a vital subunit of the glutamate-cystine antiporter system Xc⁻ which typically mediates the exchange of extracellular L-cystine and intracellular L-glutamate. SLC7A11 inhibition results in GSH depletion and enhanced ferroptosis following erastin treatment⁵¹. Deletion of Slc7a11 induced tumor-selective ferroptosis and inhibited the growth of pancreatic ductal adenocarcinoma⁵². Our study further confirmed that miR-155-5p, as well as Nfe2l2 activator and inhibitor, regulated the expression of AREs during H₂O₂ injury, which suggested that Nfe2l2-mediated anti-ferroptosis activity was associated with the induction of Nqo1, Ho1, Fth1, and SLC7A11.

IR injury involves various forms of cell death including apoptosis and ferroptosis. Consistent with previous reports in which downregulation of miR-155-5p induced apoptosis in breast cancer cells³⁵ and increase of miR-155-5p lead to less apoptosis in human endothelial cells³⁶, our current results revealed that addition of miRNA-155 mimic inhibited the activation of caspase3 pathway in H₂O₂ treated MCM. Inhibition of Nfe2l2 by ML385 also decreased the H₂O₂ induced apoptosis but enhanced the ferroptosis in MCM cells. These results suggested that the Nfe2l2-mediated mechanism may play a vital role in modulating the transition between cell apoptosis and ferroptosis in the condition of IR injury.

This study still has some limitations. Firstly, IR-EVs are released by diverse cells in the injured heart, such as cardiomyocytes, endothelial cells, fibroblasts and inflammatory cells. How IR-EVs with different origins exert their functions during IR injury need further exploration. In addition, EVs are reported to possess multiple kinds of cargos, how other contents in IR-EVs play their roles are still elusive. Finally, the relationship between ferroptosis and apoptosis as well as other forms of cell death is largely unknown. Although our results suggested the possible role of Nfe2l2-mediated mechanism in the transition between cardiomyocyte apoptosis and ferroptosis post IR, the exactly biological function, and the value of this transition in disease control warrant in-depth investigation.

In summary, the present study newly demonstrated an EV-based ferroptosis regulation mechanism in cardiac IR injury. IR-EVs can deliver miR-155-5p into cardiomyocytes and facilitate IR-induced ferroptosis via restraining Nfe2l2 antioxidative pathway. Therefore, inhibition of ferroptosis via targeting IR-EVs/miR-155-5p may be exploited to achieve desirable therapeutic ends.

IR: Ischemia-reperfusion

RCD: Regulated cell death

EVs: Extracellular vesicles

MDA: Malondialdehyde

OGD/R: Oxygen glucose deprivation/reoxygenation

AREs: Antioxidant response elements

GPX4: Glutathione peroxidase 4

LVESD: Left ventricle internal diameters at end-systole

LVEDD: Left ventricle internal diameters at end-diastole

EF: Ejection fraction

FS: Fraction shortening

AST: Aspartate transaminase

CK: Creatine kinase

PBS: Phosphate-buffered saline

DMEM: Dulbecco's Modified Eagle Medium

MCM: Mouse cardiac myocytes

CCK-8: Cell Counting Kit-8

LDH: Lactate dehydrogenase

4-HNE: 4-hydroxynonenal

SEM: Standard Error of Mean

NTA: Nanoparticle tracking analysis

TEM: Transmission electron microscope

Supplementary materials

Supplementary material is available online.

Authors contribution

XZ and WH: designed the study; XG: performed experiments and collected the data for analysis; JL, QM and EW: supervised the animal experiments. XL, SS and XG: performed in vitro assays. XZ and ZL: project administration. XZ, WH and XG: Funding acquisition. XG: wrote the manuscript; XZ: revised the manuscript. All authors reviewed and approved the final version of the manuscript.

Funding

The present study was supported by the program of Science and Technology Commission of Shanghai Municipality (STCSM; grant no. 23ZR1452500), the National Natural Science Foundation of China (NSFC; grant nos. 81670458 and 82100350) and Key Discipline Construction Project of Shanghai Pudong New Area Health Commission (Grant No. PWZxk-2022-20).

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author. The miRNA sequencing data are available in Gene Expression Omnibus, accession GSE189888.

Ethics approval and consent to participate

All animal procedures in this study were approved by the Institutional Animal Care and Use Committee of Tongji University (Number: TJLAC-019-148).

Consent for publication

Not applicable.

Acknowledgements

None.

Competing interests

The authors declare that they have no competing interests.

Author details

¹Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China;

²Shanghai Heart Failure Research Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China;

³Department of Thoracic Surgery, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China;

⁴Department of Heart Failure, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China;

⁵Department of Cardiovascular Surgery, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200120, China

⁶Shanghai Institute of Stem Cell Research and Clinical Translation, Shanghai 200120, China

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Cardiac extracellular vesicles aggravate cardiomyocyte ferroptosis in myocardial ischemia-reperfusion injury via miR-155-5p-Nfe2l2 signaling

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Abstract

Figures

Introduction

Materials and methods

Animals

Establishment of the myocardial IR models and grouping

Echocardiography analysis

Myocardial enzymes detection

EV isolation and identification

Primary cardiomyocyte isolation and cell culture

Cell models and treatments

Cell viability and cytotoxicity assay

Flow cytometry apoptosis assay

Malondialdehyde (MDA) assay

NADP/NADPH assay

Iron assay

Luciferase reporter assay

Histology

Transfection of miRNA mimics and inhibitors

Real-time quantitative polymerase chain reaction (RT-qPCR)

Western Blot

Statistical Analysis

Results

IR-EVs enhanced the cardiac ferroptosis during cardiac IR injury

IR-EVs aggravated H2O2-induced ferroptosis of cardiomyocytes

IR-EVs delivered miR-155-5p into cardiomyocytes

IR-EVs enriched miR-155-5p contributed to the ferroptosis of cardiomyocytes

miR-155-5p in IR-EVs can target Nfe2l2 pathway in cardiomyocyte

miR-155-5p contribute to cardiomyocyte ferroptosis via Nfe2l2 pathway

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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IR-EVs aggravated H₂O₂-induced ferroptosis of cardiomyocytes