Alkbh5 promotes Ythdf1 expression through demethylation thereby facilitating Fth1 translation to inhibit ferroptosis of myocardial infarction

Abstract

Background

Myocardial infarction (MI) is a leading cause of global mortality. Ferroptosis, an iron-dependent form of programmed cell death, has recently emerged as a critical player in cardiovascular diseases. N6-methyladenosine (m6A), the most prevalent RNA methylation modification in eukaryotic cells, has been implicated in various pathological processes; however, its regulatory role in MI through ferroptosis remains poorly understood. This study aimed to elucidate the mechanism by which m6A methylation mediates MI via ferroptosis.

Methods

A hypoxia/reoxygenation (H/R) model was established using H9C2 cells to simulate myocardial injury. RNA methylation levels were quantified via dot blot assay. Ferroptosis was evaluated by measuring lactate dehydrogenase (LDH) release, Fe²⁺ levels, glutathione (GSH), lipid reactive oxygen species (ROS), malondialdehyde (MDA), and apoptosis. The underlying molecular mechanisms were investigated using western blotting, quantitative real-time PCR (qPCR), methylated RNA immunoprecipitation (MeRIP), and RIP. Findings were further validated in a myocardial ischemia/reperfusion injury (MIRI) rat model.

Results

The results revealed that m6A levels were significantly elevated in the H/R cell model, accompanied by reduced expression of Alkbh5 mRNA. Moreover, Alkbh5 overexpression inhibited ferroptosis increased in the H/R model. Mechanistically, Alkbh5 overexpression decreased m6A levels of Ythdf1 and H9C2 cells while promoting Fth1 translation by enhancing Ythdf1 mRNA expression. Knockdown of Ythdf1 restored ferroptosis in the H/R model, counteracting the effects of Alkbh5 overexpression. Furthermore, Alkbh5 overexpression alleviated myocardial injury in the MIRI rat model, upregulated Ythdf1 mRNA expression, and increased Fth1 protein levels.

Conclusion

This study demonstrates that Alkbh5 ameliorates MI by inhibiting ferroptosis through m6A demethylation of Fth1. These findings provide novel insights into the molecular mechanisms underlying MI and highlight potential therapeutic targets for MI treatment.

Introduction

Myocardial infarction (MI), a leading cause of global mortality, is pathologically characterized by cardiomyocyte death resulting from prolonged ischemia, with clinical manifestations including acute chest pain, arrhythmias, and systemic complications that significantly threaten patient survival [1,2,3]. Despite significant advances in understanding the pathogenesis of MI, the molecular mechanisms underlying cardiomyocyte death remain incompletely elucidated. Emerging evidence highlights ferroptosis—an iron-dependent form of cell death driven by lethal lipid peroxidation—as a critical contributor to MI progression [4, 5]. Sustained elevation of reactive oxygen species (ROS) plays a pivotal role in MI development, while glutathione peroxidase 4 (GPX4), a key suppressor of ferroptosis, effectively eliminates ROS in cardiomyocytes [6]. Notably, Park et al. [7] demonstrated that GPX4 downregulation during MI leads to the accumulation of lipid peroxidation, resulting in ferroptotic cell death in H9C2 cardiomyocytes. Additionally, pharmacological inhibition of ferroptosis has been shown to attenuate myocardial injury in MI models [8]. These findings collectively underscore ferroptosis as a promising therapeutic target; however, the upstream regulatory mechanisms governing this process remain poorly understood.

RNA methylation is a post-transcriptional modification process in which methyl groups are selectively added to adenosine residues in RNA, catalyzed by methyltransferases. Among these modifications, N6-methyladenosine (m6A) stands out as the most prevalent and abundant methylation mark in eukaryotic cells [9]. m6A modification is a dynamic and reversible process regulated by three key components: methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers) [10]. Notably, the demethylase Alkbh5 (an eraser) and the m6A-binding protein Ythdf1 (a reader) have been identified as critical regulators in cardiovascular diseases. Alkbh5 removes m6A modifications to modulate RNA stability and translation, while Ythdf1 enhances the translation efficiency of m6A-modified transcripts [11, 12] Recent studies have implicated dysregulation of m6A in the pathogenesis of MI, with aberrant methylation patterns linked to processes such as apoptosis and angiogenesis [13]. For example, Alkbh5 has been shown to promote the transformation of fibroblasts into myofibroblasts under hypoxic conditions, thereby protecting against cardiac rupture post-MI [14]. Additionally, METTL14-mediated m6A methylation of pri-miR-5099 has been reported to facilitate cardiomyocyte pyroptosis in MI [15]. Despite these advances, the role of m6A modification in regulating ferroptosis during MI, particularly through Alkbh5, remains unexplored.

In this study, we aimed to elucidate the mechanism by which m6A methylation regulates MI through ferroptosis. Utilizing a hypoxia/reoxygenation (H/R) cell model and validating the findings in a myocardial ischemia/reperfusion injury (MIRI) rat model, we investigated the role of m6A modification in ferroptosis during MI. We hypothesized that m6A modification modulates ferroptosis by regulating the stability and expression of key target genes, thereby influencing MI progression. Our findings may provide novel insights into the molecular mechanisms underlying MI and offer a new theoretical foundation for the development of therapeutic strategies targeting ferroptosis in MI.

Methods

Cell culture and treatment

H9C2 cells were obtained from Procell (Wuhan, China). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Cat. No. 12491015, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Cat. No. 26140111, Gibco) and maintained at 37 °C in a humidified atmosphere containing 5% CO₂. Cells between passages 2 and 4 were utilized for all experiments. To establish the hypoxia/reoxygenation (H/R) model, cells were incubated in a hypoxia chamber with 0.1% O₂, 5% CO₂, and 95% N₂ for 6 h at 37 °C, followed by reoxygenation in a normoxic environment (95% air and 5% CO₂) for 12 h at 37 °C. To verify the successful establishment of the H/R model, cell viability was assessed using a Cell Counting Kit-8 (CCK-8; Cat. No. 40203ES60, Yeasen, Shanghai, China) according to the manufacturer’s instructions.

Cell transfection

Short hairpin RNA (shRNA) targeting Alkbh5, shRNA negative control (shNC), Alkbh5 overexpression plasmids, and the empty vector (pcDNA3.1) were provided by GenePharma (Shanghai, China). For transfection, 2 µg of each plasmid was introduced into H9C2 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The transfected cells were harvested 48 h post-transfection for subsequent analysis.

Animal study

The animal study was approved by the Ethics Committee of MDKN Biotechnology Co., Lt (Approval No. MDKN-2024-048) and conducted in accordance with institutional guidelines. Male Sprague Dawley rats (8–12 weeks old, 200–280 g) were obtained from GENET-MED (Jilin, China). The rats were housed under controlled conditions at 20–25 °C with a 12-hour light/dark cycle. One day prior to the establishment of the MIRI model, 12 rats were randomly divided into two groups (n = 6 per group) and injected via the tail vein with lentiviral vectors carrying either a negative control overexpression plasmid or an Alkbh5 overexpression plasmid. From the remaining rats, 6 were randomly assigned as the sham group, while the rest, along with the lentiviral-injected rats, were used to construct the MIRI model.

The MIRI rat model was established following the method described by Chen et al. [16]. Briefly, rats were anesthetized by intraperitoneal injection of 0.1% sodium pentobarbital (Cat. No. P3761, Sigma-Aldrich, Saint Louis, MO, USA) and mechanically ventilated using an animal ventilator after endotracheal intubation. A thoracotomy was performed to expose the heart, and the left anterior descending coronary artery was ligated with a 6 − 0 silk suture at its root. After 30 min of ischemia, the suture was released to allow reperfusion for 2 h. Successful induction of ischemia was confirmed by visible whitening of the left ventricular apex and anterior wall, while successful reperfusion was indicated by the restoration of red coloration in these regions. For the sham group, the same surgical procedure was performed, including left thoracotomy and exposure of the heart and left anterior descending artery. However, the coronary artery was not ligated; instead, a 6 − 0 silk suture was gently passed around the artery without tying. The chest was kept open for 30 min to simulate the ischemia duration, after which it was closed, and the rats were allowed to recover. At the end of the reperfusion period, arterial blood samples were collected and centrifuged at 1,000 × g and 4 °C for 10 min to isolate serum. The serum was aliquoted and stored at − 20 °C for subsequent analysis. The cardiac tissues were preserved in 10% paraformaldehyde and − 80 °C for further use, respectively.

Hematoxylin and Eosin (HE) staining

Rat cardiac tissues were stained using a HE staining kit (Beyotime, Beijing, China) to assess myocardial injury. Briefly, heart tissues were fixed in 10% paraformaldehyde, embedded in paraffin, and sectioned into 5-µm-thick slices. The sections were then stained with hematoxylin and eosin according to the manufacturer’s instructions. Finally, the stained sections were examined under an optical microscope for histological analysis.

Masson’s trichrome staining

Masson’s trichrome staining was conducted using a Masson’s trichrome staining kit (Beyotime). Paraffin sections were deparaffinized and sequentially treated with different concentrations of ethanol, followed by a 2-minute wash in distilled water. Sections were then stained with hematoxylin for 5 min, differentiated with acidic solution for 30 s, and rinsed under tap water for 10 min to return to blue. Next, ponceau-acid fuchsin staining was performed for 10 min, briefly rinsed, and differentiated with phosphomolybdic acid for 2 min. Light green SF yellowish staining was applied for 1 min, followed by differentiation with acidic solution for 1 min. After dehydration through ethanol series and clearing in xylene, sections were mounted and examined under a microscope.

Assessment of myocardial injury markers of rats

The levels of myocardial injury markers, including lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), and cardiac troponin I (cTNI), in rat serum were quantified using a commercial LDH activity assay kit, a rat CK-MB enzyme-linked immunosorbent assay (ELISA) kit, and a rat cTNI ELISA kit (all purchased from Solarbio, Beijing, China). All experiments were performed in strict accordance with the manufacturer’s protocols.

Detection of superoxide dismutase (SOD)

The SOD levels of cardiac tissues of rat were measured using a SOD assay kit (Beyotime). Tissue samples were homogenized in SOD sample preparation solution and centrifuged at 12,000 × g for 4 °C, 5 min to collect the supernatant. Next, 160 µL of WST-8/Enzyme Working Solution, 20 µL of sample, and 20 µL of Reaction Start Solution were mixed and incubated at 37 °C for 30 min. The absorbance was measured at 450 nm using a microplate reader.

Quantitative real-time PCR (qPCR)

Total RNA was extracted from H9C2 cells and rat sera using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Subsequently, the RNA was reverse-transcribed into cDNA using the HiScript II 1st Strand cDNA Synthesis Kit (Cat. No. R211, Vazyme, Nanjing, China). qPCR was performed on a CFX96 system (Bio-Rad, Hercules, CA, USA) using SYBR Green Master Mix (Cat. No. 4309155, Thermo Fisher Scientific, Waltham, MA, USA). The relative mRNA expression levels were calculated using the 2^−ΔΔCt method, normalized to the internal control gene GAPDH. The sequences of the primers used for qPCR are listed in Table 1.

Table 1 The primers for qPCR

Measurement of LDH release

The release of LDH from H9C2 cells in different groups was measured using an LDH Release Assay Kit (Beyotime, Shanghai, China). Briefly, H9C2 cells were seeded into 96-well plates. When the cells reached approximately 90% confluence, the culture medium was replaced with DMEM containing 1% fetal bovine serum. The cells were then treated according to the experimental design and grouped as described in the manufacturer’s protocol. For the control group, the LDH release reagent was added to the corresponding wells. Following incubation, the plates were centrifuged at 400×g for 5 min at room temperature. The supernatant was carefully collected and transferred to a new 96-well plate, followed by the addition of 60 µL of LDH detection solution to each well. The mixture was incubated for 30 min at room temperature, protected from light. Finally, the absorbance was measured at 490 nm using a microplate reader.

Measurement of Fe²⁺ levels

The Fe²⁺ levels of H9C2 cells and cardiac tissues in different groups were measured using a ferrous ion content assay kit (Solarbio). The cardiac tissue was homogenized in an ice bath and then centrifuged at 10,000×g for 10 min at 4 °C to collect the supernatant. Cells were broken up through ice bath ultrasound with reagent I and centrifuged at 10,000×g and 4℃ for 10 min to collect supernatant. The experiments were conducted according to the manufacturer’s protocol, and the absorbance was measured at 593 nm.

Measurement of glutathione (GSH) levels

The GSH levels of H9C2 cells in different groups were measured using a GSH content assay kit (Solarbio). Cells were resuspended in PBS and centrifuged at 600×g for 10 min. Next, cells were broken up through ice bath ultrasound and centrifuged at 8000×g and 4℃ for 10 min to collect supernatant. The experiments were conducted according to the manufacturer’s protocol, and the absorbance was measured at 412 nm.

Measurement of lipid ROS

Cells were seeded in a 6-well plate and incubated in serum-free medium containing 2 µmol/L C11-BODIPY (Cat. No. D3861, Thermo Fisher Scientific) for 30 min, protected from light, for lipid ROS staining. Subsequently, the cells were dissociated, resuspended in 400 µL of PBS, and subjected to flow cytometry analysis. Fluorescence was detected using an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The fluorescence intensity measured for each group was used as an indicator of lipid ROS levels. The ROS levels of and cardiac tissues were measured using a tissue ROS assay kit (Baiaolaibo, Beijing, China) according to the manufacturer’s protocol.

Measurement of malondialdehyde (MDA) level

The levels of MDA of H9C2 cells and cardiac tissues were measured using a lipid peroxidation MDA assay kit (Beyotime). Cells and cardiac tissues were lyzed using lysis buffer and centrifuged at 10,000×g and for 10 min to collect supernatant. The experiments were conducted according to the manufacturer’s protocol, and the absorbance was measured at 532 nm.

Measurement of apoptosis

An one step TUNEL apoptosis assay kit (Beyotime) was performed to measure the apoptosis in H9C2 cells of different groups. Cells were washed with PBS and fixed in 4% paraformaldehyde for 30 min and transferred to PBS containing 0.3% Triton X-100 for 5 min of incubation. Next, cells were washed twice with PBS and protected from light for 60 min at 37℃ with 50 µl TUNEL detection solution. The nucleus was stained by 4’,6-diamidino-2-phenylindole (DAPI; Beyotime). Apoptosis was observed using a fluorescence microscope.

Dot blot assay

Isolated total RNA of H9C2 cells was mixed in three times volume of incubation buffer and denatured at 65℃ for 5 min. Next, samples dissolved in SSC buffer were deposited on an Amersham Hybond-N + membrane (GE Healthcare, USA) for 5 min of UV crosslinked. Then, samples were washed with PBST and stained with 0.02% Methylene blue (Sangon Biotech, China). Input RNA content was displayed by scanning the blue dot. The membrane was incubated with anti-m5C, anti-m6A and anti-m7G overnight at 4℃. Dot blots were visualized by the imaging system after incubation with secondary antibody.

Western blot assay

Total protein was extracted from H9C2 cells and rat sera using radioimmunoprecipitation assay buffer (Cat. No. P0013B, Beyotime, Shanghai, China). The protein samples were loaded on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with 5% skim milk for 1 h, the membranes were incubated with primary antibodies overnight at 4℃. Next, samples were incubated with secondary antibodies (1/10000, ab6721, Abcam, Cambridge, UK) for 2 h at room temperature. The membranes were wash with 1×TBST for three times and visualized using the enhanced chemiluminescence reagent (Thermo Scientific, Waltham, MA, USA). The primary antibodies used in this study were as follows: anti-Alkbh5 (1/1000, ab195377, Abcam), anti-Tfrc (1/1000, ab214039, Abcam), anti-Acsl4 (1/10000, ab155282, Abcam), anti-Ftl (1/1000, ab69090, Abcam), anti-Gpx4 (1/1000, ab125066, Abcam), anti-Scl7a11 (1/1000, PA5-19207, Thermo Scientific), anti-Fth1 (1/1000, ab183781, Abcam) and anti-Gapdh (1/10000, ab181602, Abcam).

Methylated RNA Immunoprecipitation (MeRIP)

The m6A level of Ythdf1 was measured using a GenSeq^® m6A MeRIP kit (Cat. No. GS-EG-001 A, Cloudseq, Shanghai, China). Briefly, magnetic beads were incubated with 2 µl m6A antibodies or IgG antibodies for 1 h on a rotator and washed with IP buffer. Then, 250 µl of mixture containing fragmented RNA, nuclease-free water and 5×IP buffer were added to the prepared beads for 1 h incubation on a rotator at 4℃. RNA was purified according to the instructions and the mRNA expression of Ythdf1 was measured by qPCR.

Bioinformatic analysis

The potential m6A modification site of Ythdf1 was predicted using the SRAMP database (http://www.cuilab.cn/sramp).

Detection of RNA stability

Ythdf1 mRNA stability was measured using qPCR after H9C2 cells were treated with 5 µg/mL actinomycin D (Merck, Darmstadt, Germany) for 0, 4, 8 and 12 h.

RNA Immunoprecipitation (RIP)

The binding between Ythdf1 and Fth1 was detected by RIP assay using an imprint RNA immunoprecipitation kit (Sigma-Aldrich, St. Louis, MO, USA). H9C2 cells were lysed in RIP lysis buffer. Then, the lysate was incubated with anti-Fth1 or anti-IgG coated magnetic protein A/G beads overnight at 4℃. After purification, the mRNA expression of Fth1 was measured by qPCR.

Statistical analysis

All data were analyzed and processed using SPSS 22.0 software. Results were expressed as mean ± standard deviation. All cell experiments were conducted in triplicate to ensure reproducibility. Comparisons between two groups were performed using the Student’s t-test, while comparisons among three or more groups were conducted using one-way analysis of variance (ANOVA). P < 0.05 was recognized as statistically significant.

Results

Downregulated Alkbh5 mRNA expression leads to increased m6A methylation level in H/R cell model

To investigate the role of RNA methylation in MI, we established H/R cell models and measured various RNA methylation levels in these models. Compared with the control group, the levels of m5C (P < 0.05) and m6A (P < 0.01) were significantly increased in the H/R cell model, with the increase in m6A being more pronounced. In contrast, the m7G level showed no significant difference between the control group and the H/R cell model (Fig. 1A). Subsequently, we analyzed the mRNA expression levels of several methylases in H9C2 cells, and the results are presented in a heatmap. Notably, the mRNA expression of Alkbh5 was most significantly decreased in the H/R cell model (Fig. 1B). Taken together, these findings demonstrate that the m6A methylation level is elevated in the H/R cell model, potentially due to the reduced expression of the demethylase Alkbh5.

Fig. 1

Downregulated Alkbh5 mRNA expression leads to increased m6A methylation level in H/R cell model. (A) RNA m6A methylation was significantly increased in the H/R cell model. (B) The mRNA expression of several methylases was measured by qPCR

Alkbh5 overexpression alleviates ferroptosis in the H/R cell model

Ferroptosis has been shown to play a critical role in the progression of MI [7]. To investigate the role of Alkbh5 in ferroptosis, we upregulated the expression of Alkbh5 mRNA to elucidate its function. First, we measured the expression of Alkbh5 mRNA in H9C2 cells and found that it was significantly downregulated in the H/R cell model. However, overexpression of Alkbh5 in the H/R cell model partially restored its mRNA expression (P < 0.01, Fig. 2A). Next, we evaluated several ferroptosis-related indicators in H9C2 cells. Compared with the control group, the levels of LDH release, Fe²⁺, lipid ROS, and MDA were significantly increased in the H/R cell model (P < 0.01). These increases were partially reversed by Alkbh5 overexpression. Conversely, the level of GSH, which was reduced in the H/R cell model, was partially restored upon Alkbh5 overexpression (P < 0.01, Fig. 2B-F). Additionally, we assessed apoptosis in H9C2 cells across different groups. The results showed that the number of TUNEL-positive cells was markedly elevated in the H/R cell model but was significantly reduced upon Alkbh5 overexpression, indicating that Alkbh5 overexpression partially inhibited apoptosis induced by the H/R treatment (P < 0.01, Fig. 2G). In conclusion, these findings demonstrate that Alkbh5 overexpression alleviates ferroptosis and reduces apoptosis in the H/R cell model, suggesting a protective role of Alkbh5 in myocardial injury.

Fig. 2

Alkbh5 overexpression alleviates ferroptosis in the H/R cell model. (A) qPCR was performed to measure the expression of Alkbh5 in different groups of H9C2 cells. (B) LDH release was measured using a LDH release assay kit. (C) The Fe²⁺ level was measured using a ferrous ion content assay kit. (D) The GSH level was measured using a GSH content assay kit. (E) The lipid ROS level was measured by C11-BODIPY. (F) The MDA level was measured using a lipid peroxidation MDA assay kit. (G) Apoptosis was assessed by TUNEL assay

Alkbh5 mediates translation but not mRNA expression of Fth1

Next, we further investigated the mechanism by which Alkbh5 mediates ferroptosis. First, we measured the m6A methylation level in H9C2 cells and found that it was significantly inhibited upon Alkbh5 overexpression (Fig. 3A). Subsequently, we examined the effect of Alkbh5 overexpression on several ferroptosis-related proteins. The results revealed that Alkbh5 overexpression specifically upregulated the protein level of Fth1, while no significant changes were observed for other proteins (Fig. 3B). Based on these findings, we hypothesized that Fth1 might be a target of Alkbh5. To test this hypothesis, we measured the mRNA expression of Fth1. Interestingly, Alkbh5 overexpression did not affect Fth1 mRNA expression (Fig. 3C). To further validate the relationship between Alkbh5 and Fth1, we knocked down Alkbh5 mRNA expression in H9C2 cells (P < 0.01, Fig. 3D). Consistent with our previous findings, Alkbh5 knockdown had no effect on Fth1 mRNA expression but significantly downregulated the protein level of Fth1 (Fig. 3E, F). In summary, these results indicate that Alkbh5 regulates the translation of Fth1 without affecting its mRNA expression, suggesting a potential role of Alkbh5-mediated m6A modification in post-transcriptional regulation of Fth1 during ferroptosis.

Fig. 3

Alkbh5 mediated translation but not mRNA expression of Fth1. (A) The m6A level of in H9C2 cells was measured by dot blot assay. (B) The protein levels of ferroptosis-related proteins were measured by western blot assay. (C-E) The expression of Fth1 and Alkbh5 was measured by qPCR. (F) Western blot assay was performed to measure the protein levels of Alkbh5 and Fth1

Alkbh5-mediated m6A demethylation promotes the expression of Ythdf1 mRNA

As described above, Alkbh5 overexpression promotes the translation but not the mRNA expression of Fth1. YTHDF1, a reader protein known to enhance mRNA translation efficiency [17], was identified as a potential mediator of this process. To determine whether Alkbh5 promotes Fth1 translation through YTHDF1, we conducted a series of experiments. MeRIP analysis revealed that Alkbh5 significantly reduced the m6A methylation level of Ythdf1 mRNA (P < 0.01, Fig. 4A). Furthermore, Alkbh5 overexpression markedly increased the mRNA expression of Ythdf1, whereas Alkbh5 knockdown decreased its mRNA expression (P < 0.01, Fig. 4B, C). Next, we predicted that position 383 might represent the potential m6A modification site within Ythdf1 mRNA (Fig. 4D). An RNA stability assay demonstrated that Alkbh5 overexpression inhibited the degradation of Ythdf1 mRNA, thereby enhancing its stability (P < 0.01, Fig. 4E). Consistent with these findings, Ythdf1 mRNA expression was significantly downregulated in the H/R cell model (Fig. 4F). RNA RIP analysis further confirmed that Fth1 directly interacts with YTHDF1 (Fig. 4G). Taken together, these results demonstrate that Alkbh5-mediated m6A demethylation enhances the stability of YTHDF1 mRNA, leading to increased YTHDF1 expression and subsequent promotion of Fth1 translation.

Fig. 4

Alkbh5-mediated m6A demethylation promotes the expression of Ythdf1. (A) The m6A level of Ythdf1 was measured by MeRIP. (B and C) The expression of Ythdf1 was measured by qPCR. (D) The potential m6A site in Ythdf1 was predicted using the SRAMP database. (E) The stability of Ythdf1 mRNA was measured by qPCR after H9C2 cells treated with 5 µg/mL actinomycin D treatment for 0, 4, 8 and 12 h. (F) Ythdf1 expression in H9C2 cells was measured by qPCR. (G) The binding between Fth1 and Ythdf1 was measured by RIP

Ythdf1 knockdown promotes ferroptosis in the H/R cell model improved by Alkbh5 overexpression

Next, we performed rescue experiments to verify the role of YTHDF1 in ferroptosis. The mRNA expression of Ythdf1 was significantly reduced upon YTHDF1 knockdown (P < 0.01, Fig. 5A). Western blot analysis revealed that Alkbh5 overexpression upregulated the protein level of Fth1 in the H/R cell model, an effect that was partially reversed by YTHDF1 knockdown (P < 0.01, Fig. 5B). Furthermore, the protective effects of Alkbh5 overexpression on ferroptosis-related indicators were partially abrogated by YTHDF1 knockdown. Specifically, the reductions in LDH release, Fe²⁺ levels, lipid ROS levels, and MDA levels induced by Alkbh5 overexpression in the H/R cell model were partially restored upon YTHDF1 knockdown. Additionally, the increase in GSH levels mediated by Alkbh5 overexpression was also inhibited by YTHDF1 knockdown (P < 0.01, Fig. 5C-G). Apoptosis analysis further demonstrated that YTHDF1 knockdown reversed the inhibitory effect of Alkbh5 overexpression on apoptosis in the H/R cell model (P < 0.01, Fig. 5H). In conclusion, these results indicate that YTHDF1 knockdown promotes ferroptosis and apoptosis in the H/R cell model, thereby counteracting the protective effects of Alkbh5 overexpression.

Fig. 5

Ythdf1 knockdown promoted ferroptosis in the H/R cell model improved by Alkbh5 overexpression. (A) Ythdf1 expression was measured by qPCR. (B) Western blot was performed to measure the protein level of Fth1 in H9C2 cells of different groups. (C) LDH release was measured using a LDH release assay kit. (D) The Fe²⁺ level was measured using a ferrous ion content assay kit. (E) The GSH level was measured using a GSH content assay kit. (F) The lipid ROS level was measured by C11-BODIPY. (G) The MDA level was measured using a lipid peroxidation MDA assay kit. (H) Apoptosis was assessed by TUNEL assay

Alkbh5 alleviates myocardial injury and ferroptosis in the MIRI rat model through promoting Fth1 translation by increasing Ythdf1 mRNA expression

Finally, we validated the function of Alkbh5 in MI through animal experiments. HE and Masson’s trichrome staining revealed that myocardial injury was significantly exacerbated in the MIRI rat model but was partially alleviated by Alkbh5 overexpression (Fig. 6A). The increased fibrotic area in the MIRI group was significantly reduced by Alkbh5 overexpression (Fig. 6B). Furthermore, the levels of myocardial injury markers, including LDH activity, CK-MB, and cTnI, were markedly elevated in the MIRI rat model. These increases were partially reversed by Alkbh5 overexpression, indicating that Alkbh5 overexpression mitigated myocardial injury in the MIRI rat model (P < 0.01, Fig. 6C-E). Next, we assessed the expression of Ythdf1 mRNA in different groups of rats. The results showed that Ythdf1 mRNA expression was significantly downregulated in the MIRI rat model but was restored upon Alkbh5 overexpression (P < 0.01, Fig. 6F). Additionally, both the mRNA expression and protein level of Fth1 were significantly inhibited in the MIRI rat model (P < 0.01). Notably, Alkbh5 overexpression did not affect Fth1 mRNA expression but markedly upregulated its protein level (Fig. 6G and H). Additionally, we assessed the impact of Alkbh5 on ferroptosis in the MIRI rat model. Our results indicated that SOD level was decreased, whereas MDA, Fe²⁺, and lipid ROS were significantly elevated in the MIRI group compared to controls. Notably, these alterations were reversed by Alkbh5 overexpression (Fig. 6I-L). Moreover, the downregulated GPX4 mRNA and the increased ACSL4 mRNA in the MIRI rat model were partially restored by Alkbh5 overexpression (Fig. 6M and N). In summary, our findings confirm that Alkbh5 improves myocardial injury in the MIRI rat model by promoting Fth1 translation via increased Ythdf1 mRNA expression.

Fig. 6

Alkbh5 improved myocardial injury in the MIRI rat model through promoting Fth1 translation by increasing Ythdf1 expression. (A) The myocardial injury of rats in different groups was assessed by HE and Masson’s trichrome staining. (B) The quantitative results of myocardial fibrosis evaluated by Masson’s trichrome staining. (C-E) Myocardial injury was assessed by the levels of myocardial injury markers LDH activity, CK-MB and cTnI. (F and G) The expression of Ythdf1 and Fth1 was measured by qPCR. (H) The protein level of Fth1 was measured by western blot. (I-J) Ferroptosis of cardiac tissues of rats were evaluated by measuring SOD, MDA, Fe²⁺ and ROS levels. (M and N) qPCR was performed to measure GPX4 and ACSL4 mRNA expression

Discussion

Ferroptosis, a newly discovered mode of cell death in recent years, has garnered significant attention due to its profound impact on human health. Numerous studies have elucidated its roles in various diseases, including cancer, neurological disorders, and digestive system conditions [18]. Recently, the mechanism by which ferroptosis contributes to MI has also begun to emerge. Park et al. [7] were the first to demonstrate that GPX4 expression is downregulated during the early and middle stages of MI, thereby promoting ferroptosis in cardiomyocytes. Consistent with this finding, several studies have confirmed that GPX4 protein levels are lower in MI tissues compared to normal tissues, and the use of the GPX4 inhibitor RSL3 induces ferroptosis in H9c2 cells [7, 19]. Furthermore, Jiang et al. [20] identified that the expression of adaptor protein HIP-55 is upregulated in MI, and cardiac-specific overexpression of HIP-55 significantly alleviates both cardiomyocyte ferroptosis and MI-induced injury. Additionally, Li et al. [21] revealed that the downregulation of miR-26b-5p in MI-exo leads to increased expression of SLC7A11, which inhibits ferroptosis following acute MI and mitigates myocardial damage. Based on these findings, multiple studies have reported that pharmacological interventions targeting ferroptosis inhibition, such as dexmedetomidine, epigallocatechin gallate, and idebenone, can improve cardiac function and reduce MI-induced injury [22,23,24]. In line with these observations, our study demonstrates that ferroptosis and myocardial injury are significantly increased in H/R-induced H9C2 cells and the MIRI rat model, respectively. These results suggest that ferroptosis may play a critical role in exacerbating myocardial injury during MI. Collectively, these findings underscore the potential of targeting ferroptosis as a therapeutic strategy for attenuating MI-related pathologies.

In recent years, the modification of m6A methylation has emerged as a highly active research area. While most studies on m6A have focused on its role in cancer, its effects on cardiovascular diseases, including MI, are increasingly being reported. However, the regulation of m6A in MI remains relatively underexplored. Zhang et al. [25] demonstrated that m6A methylation levels are significantly upregulated in both in vivo and in vitro models of MI and identified that Hadh, Tet1, and Kcnn1 influence apoptosis and angiogenesis during MI. Yang et al. [14] further revealed that global knockdown of Alkbh5 reduces infarct size and improves cardiac function post-MI. Huang et al. [26] showed that inhibiting METTL3 during the early phase of MI effectively rescues dying cardiomyocytes. Collectively, these findings confirm that modulating m6A methylation via methylases can improve outcomes in MI. Moreover, m6A modification has been shown to mediate disease progression by regulating ferroptosis. For instance, Wu et al. [27] demonstrated that inhibiting METTL3 alleviates doxorubicin-induced cardiotoxicity by suppressing ferroptosis through TFRC m6A modification. Similarly, Qiao et al. [28] reported that FTO protects colorectal cancer (CRC) cells from ferroptosis, thereby promoting CRC tumorigenesis via the SLC7A11/GPX4 pathway. Additionally, several studies have investigated the role of m6A modification in ferroptosis-related mechanisms in MI. Tang et al. [29] revealed that knocking down METTL3 inhibits ferroptosis in cardiomyocytes by suppressing SLC7A11 m6A modification. Fang et al. [30] demonstrated that WTAP-mediated m6A modification of KLF6 exacerbates H/R-induced ferroptosis in human cardiomyocytes. Taken together, these studies highlight the critical role of m6A modification in MI; however, the involvement of m6A-regulated ferroptosis in MI remains poorly understood. In the present study, we observed that m6A levels were significantly upregulated in H9C2 cells subjected to H/R, while the expression of Alkbh5 mRNA was downregulated. Furthermore, overexpression of Alkbh5 effectively suppressed ferroptosis in the H/R cell model and alleviated myocardial injury in mice with MIRI. Mechanistically, Alkbh5 overexpression enhanced the translation of Fth1 by promoting Ythdf1 expression through demethylation. Collectively, these findings demonstrate that Alkbh5 modulates ferroptosis in MI via regulation of Fth1.

Fth1 is a gene that encodes ferritin heavy chain, a protein primarily responsible for storing and regulating iron ions within the body [31]. FHC plays a critical role in maintaining iron homeostasis, preventing iron overload, and protecting cells from oxidative stress [32]. Several studies have reported differential expression of Fth1 in MI. For example, Yang et al. [33] demonstrated that cinnamaldehyde inhibits ferroptosis and upregulates Fth1 expression in myocardial tissue in an acute MI rat model. Similarly, Yu et al. [23] showed that epigallocatechin gallate reduces ferroptosis induced by acute MI and alleviates myocardial ischemic injury by upregulating Fth1 expression. In the present study, we elucidated for the first time the role of the Alkbh5-YTHDF1-Fth1 axis in MI. Specifically, we revealed that Alkbh5 promotes Fth1 translation by enhancing YTHDF1 expression through m6A demethylation. This finding provides novel insights into the regulatory mechanisms underlying Fth1-mediated ferroptosis in MI. Collectively, our results highlight the potential therapeutic implications of targeting the Alkbh5-YTHDF1-Fth1 axis to mitigate ferroptosis and improve outcomes in MI.

However, several limitations remain in this study. First, although a MIRI rat model was employed, the physiological differences between rats and humans may limit the direct translation of these findings to clinical settings. Second, the potential dose-dependent protective effects of Alkbh5 overexpression on ferroptosis were not investigated. Future studies should explore this dose-response relationship to identify the optimal therapeutic dose for inhibiting ferroptosis and mitigating myocardial injury. Additionally, integrating multi-omics approaches could provide deeper insights into the role of m6A modification in MI pathogenesis. Furthermore, validating the therapeutic efficacy of Alkbh5 in larger animal models and clinical trials is essential to advance these findings toward clinical applications, potentially opening new avenues for MI treatment.

In conclusion, this study is the first to demonstrate that Alkbh5 promotes Ythdf1 expression through demethylation, thereby enhancing Fth1 translation and inhibiting ferroptosis in MI. These findings suggest that Alkbh5 could serve as a novel therapeutic target for MI by suppressing ferroptosis and reducing myocardial injury. Moreover, m6A modification, along with ferroptosis-related molecules Ythdf1 and Fth1, may represent potential diagnostic biomarkers for MI.