YTHDF2 Promotes Cardiac Ferroptosis via Degradation of SLC7A11 in Cardiac Ischemia

Abstract

Aims:

Myocardial ischemia–reperfusion (I/R) injury facilitates cardiomyocyte death and endangers human health. N6-methyladenosine (m6A) methylation plays a critical role in cardiovascular diseases. The m6A reader YTHDF2 identifies m6A-modified RNA and promotes target RNA degradation. Hence, we hypothesized that YTHDF2 affects I/R injury by regulating RNA stability.

Results:

Both messenger RNA (mRNA) and protein levels of YTHDF2 were upregulated in I/R mice and hypoxia-reoxygenation (H/R)-induced cardiomyocytes. Silencing endogenous YTHDF2 abrogated cardiac dysfunction and lowered the infarct size in I/R mice, and the forced expression of YTHDF2 aggravated these adverse pathological processes. Consistently, the protective effect of silencing YTHDF2 occurred in cardiomyocytes exposed to H/R and erastin. Further, RNA-Seq and RNA-binding protein immunoprecipitation (RIP) revealed that YTHDF2 recognized the m6A modification sites of the ferroptosis-related gene solute carrier family 7 member 11 (SLC7A11) mRNA to promote its degradation both in vivo and in vitro. Inhibition of SLC7A11 impaired cardiac function, increased infarct size, and the release of lactate dehydrogenase (LDH) in I/R mice after silencing YTHDF2. The beneficial effects of si-YTHDF2 on H/R injury were reversed by co-transfection with SLC7A11-specific siRNA (si-SLC7A11), which substantially exacerbated ferroptosis and the production of reactive oxygen species.

Innovation and Conclusion:

The cardioprotective effects of silencing YTHDF2 are accomplished by increasing SLC7A11 stability and expression, reducing ferroptosis, and providing novel potential therapeutic targets for treating ischemic cardiac diseases.

Introduction

Cardiovascular diseases threaten human life and significantly influence global health load (Chouchani et al., 2014). Myocardial ischemia–reperfusion (I/R) injury is caused by oxidative stress damage to cardiomyocytes following coronary artery recanalization (Chen et al., 2020). During I/R injury, oxidative stress can severely impair cardiac function (Gumpper-Fedus et al., 2022; Guo et al., 2020a).

Excessive mitochondrial reactive oxygen species (ROS) can affect the permeability of mitochondria and cause oxidative damage to specific internal molecules, which is the main driving factor leading to I/R (Cai et al., 2022). Cardiac I/R injury induces various inflammatory responses during cardiomyocyte necrosis and apoptosis, leading to cardiac systolic and diastolic dysfunctions (Heusch, 2015). Cardiomyocytes are characterized by terminal differentiation and non-regeneration; hence, there is an urgent need to mitigate irreversible lesions in cardiomyocytes to cure cardiac diseases.

Emerging evidence has revealed that other processes, in addition to apoptosis and necrosis, participate in myocardial cell death, including pyroptosis, autophagy, and ferroptosis (Del Re et al., 2019). Ferroptosis, an iron-dependent form of regulated programmed cell death that was newly discovered in 2012, is accompanied by an increase in intracellular iron levels and engendering of oxidative hydroxyl radicals, as distinguishing factors from the other cell death modes (Dixon et al., 2012; Wu et al., 2020).

Innovation

Myocardial ischemia–reperfusion (I/R) injury is caused by oxidative stress damage to cardiomyocytes after coronary artery recanalization, which endangers human health (Chen et al., 2020). The main aim of this study was to explore the potential regulatory mechanism of the N6-methyladenosine (m6A) reader YTHDF2 in I/R injury. Our findings showed that the cardioprotective effects of silencing YTHDF2 were accomplished by enhancing the stability and expression of solute carrier family 7 member 11 (SLC7A11). We propose that the downregulation of SLC7A11 affects the transport of glutamate and cysteine and increases the synthesis of reactive oxygen species, which may provide novel potential therapeutic targets for treating ischemic cardiac diseases.

Ferroptosis affects lipid peroxidation and increases ROS levels (Dixon et al., 2012). Moreover, a ruptured mitochondrial outer membrane, increased membrane density, decreased mitochondrial volume, and significant morphological modifications arise owing to ferroptosis (Yao et al., 2021). Cells have a similar array of compounds against ferroptosis; solute carrier family 7 member 11 (SLC7A11) transduces extracellular cystine into glutathione (GSH), and then glutathione peroxidase 4 (GPX4) transforms toxic lipid hydroperoxides to nontoxic lipid alcohols with GSH to synergistically restrain ferroptosis (Mao et al., 2022; Zhang et al., 2021a).

Further, ferroptosis participates in numerous pathological processes, such as those involved in multiple sclerosis and nonalcoholic steatohepatitis (Feng et al., 2022). Ferroptosis is an important factor in cardiac impairment (Fang et al., 2020). Consequently, deciphering the connection between ferroptosis and cardiac I/R injury is crucial for the development of innovative therapies for angiocardiopathy.

Epigenetic regulation is conducive to adjusting and controlling the restored functions of angiogenesis (Shi et al., 2021). N6-methyladenosine (m6A), a dynamic reversible methylation modification, occurs on the sixth nitrogen atom of adenine in RNA (Gilbert et al., 2016). Moreover, m6A requires the joint participation of m6A methyltransferase complexes (writers), m6A demethylases (erasers), and m6A recognition proteins (readers) to modulate RNA metabolic processes associated with alternative splicing, localization, stabilization, and decay (Zhang et al., 2020; Zhao et al., 2021). Particular m6A “readers,” such as the YT521-B homology (YTH) domain family, exploit the functions of the whole (Lu et al., 2021).

YTHDF2, an m6A reader that promotes the degradation of m6A-modified messenger RNA (mRNA) (Wang et al., 2014), is associated with neoplastic diseases, such as prostate cancer, hepatocellular carcinoma, and pancreatic cancer (Guo et al., 2020b; Hou et al., 2019; Li et al., 2020). However, the relationship between YTHDF2 and cardiac I/R injury remains unclear.

Thus, this study was designed to investigate the effect of YTHDF2 on myocardial impairment after I/R injury and clarify the mechanism by which YTHDF2 regulates myocardial I/R injury in mice. These results may provide potential therapeutic targets for the treatment of ischemic cardiac diseases.

Results

Upregulation of YTHDF2 in I/R mouse hearts and hypoxia-reoxygenation cardiomyocytes

m6A methylation modification plays a critical role in cardiovascular diseases. We initially quantified the mRNA levels of the m6A methyltransferases METTL3, METTL14, and WTAP; demethylases FTO and ALKBH5; and m6A-binding proteins YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2 in ischemic hearts after I/R injury to determine the pattern of m6A methylation. mRNA and protein expression levels of the m6A reader YTHDF2 were seven- and two-fold higher than in the sham control, respectively (Fig. 1A, B).

FIG. 1.

YTHDF2 expression was upregulated in I/R mouse hearts. (A) The mRNA expression levels of m6A-modified writers, erasers, and readers in I/R mouse hearts. n = 5. *p < 0.05, **p < 0.01 versus Sham. (B) YTHDF2 protein expression level in I/R myocardium. n = 6. **p < 0.01 versus Sham. (C) Immunofluorescence analysis of YTHDF2 (green) and α-actinin (red) in Sham and I/R heart tissues. DAPI (blue) was used to stain nuclei. Scale bar = 20 μm. n = 3. *p < 0.05 versus Sham. (D) Western blot analysis of YTHDF2 in H/R myocardial cells. n = 6. **p < 0.01 versus Ctrl. Data are expressed as mean ± SEM. H/R, hypoxia-reoxygenation; I/R, ischemia–reperfusion; m6A, N6-methyladenosine; mRNA, messenger RNA; SEM, standard error of measurement.

Immunofluorescence staining revealed that YTHDF2 expression increased after I/R injury (Fig. 1C). Consistent with the in vivo results, mRNA and protein levels of YTHDF2 were elevated in neonatal mouse ventricular cardiomyocytes (NMVCs) subjected to hypoxia-reoxygenation (H/R) treatment (Supplementary Fig. S1 and Fig. 1D). Therefore, we assumed that the m6A reader YTHDF2 is generally upregulated and may be linked to the pathological process of I/R.

Inhibition of YTHDF2 attenuates I/R-induced cardiac injury in mice

To determine whether YTHDF2 is involved in I/R, we conducted loss-of-function studies using shYTHDF2-V to silence YTHDF2 via tail vein injection (Fig. 2A). Successful delivery and knockdown of shYTHDF2-V were verified by GFP staining, Western blotting, and polymerase chain reaction (PCR) (Supplementary Fig. S2A–D) in mouse hearts. YTHDF2 upregulation in I/R mice was significantly reversed by sh-YTHDF2 (Fig. 2B).

FIG. 2.

Knockdown of YTHDF2 alleviates cardiac injury in I/R mice. (A) Schematic diagram of I/R after AAV9 vector carrying an NC-shRNA or YTHDF2-shRNA fragment was injected into the mouse heart via tail vein and then harvested at the corresponding time point. (B) Western blot analysis of YTHDF2 protein level in mice. n = 4. **p < 0.01 versus Sham+shNC-V; ^## p < 0.01 versus I/R+shNC-V. shNC-V or shYTHDF2-V represents the AAV9 vector carrying an NC-shRNA or YTHDF2-shRNA fragment. (C–E) Representative images of echocardiographs and statistics of EF and FS. Sham+shNC-V, I/R+shYTHDF2-V n = 7, I/R+shNC-V n = 8. **p < 0.01 versus Sham+shNC-V; ^# p < 0.05 versus I/R+shNC-V. (F, G) Evans blue/TTC staining showed the infarct size of myocardial tissue in I/R mice. Scale bar = 2 mm. I/R+shNC-V n = 4, I/R+shYTHDF2-V n = 6. **p < 0.01 versus I/R+shNC-V. (H) Detection of LDH release in mouse serum. n = 6. *p < 0.05 versus Sham+shNC-V; ^# p < 0.05 versus I/R+shNC-V. (I) Serum CK-MB level in mice. n = 3. **p < 0.01 versus Sham+shNC-V; ^# p < 0.05 versus I/R+shNC-V. (J) Detection of cTNT level in mouse serum. n = 5. **p < 0.01 versus Sham+shNC-V; ^## p < 0.01 versus I/R+shNC-V. (K) Representative H&E staining images of the mouse hearts. Scale bar = 20 μm. n = 4. Data are expressed as mean ± SEM. AAV9, adeno-associated virus 9; CK-MB, creatine kinase-MB; cTNT, cardiac troponin-T; EF, ejection fraction; FS, fractional shortening; H&E, hematoxylin and eosin; LDH, lactate dehydrogenase; NC, negative control; shRNA, short hairpin RNA; TTC, 2,3,5-triphenyl tetrazolium chloride.

To assess the biological function of YTHDF2 in mice with cardiac injury, we measured cardiac function and found that YTHDF2 deficiency had no effect on normal mice (Supplementary Fig. S2E, F) but rescued the impaired ejection fraction (EF), fractional shortening (FS), and left ventricular internal dimension at systole (LVID; s) in the hearts of I/R mice (Fig. 2C–E and Supplementary Fig. S2G). Evans blue/2,3,5-triphenyl tetrazolium chloride (TTC) staining showed that the infarct size was reduced by shYTHDF2 in I/R-injured mice (Fig. 2F, G).

Lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), and cardiac troponin-T (cTNT) are biomarkers of cardiac injury (Zhang et al., 2021b); consistent with expectations, the upregulated levels of LDH, CK-MB (Xiao et al., 2022; Yang et al., 2023; Zhang et al., 2021b), and cTNT post-I/R were significantly reversed by silencing YTHDF2 (Fig. 2H–J).

Hematoxylin and eosin (H&E) staining revealed that the downregulation of YTHDF2 alleviated I/R-induced disorder in cardiomyocyte arrangement (Fig. 2K). Together, these data indicated that decreased YTHDF2 levels have a cardioprotective effect against I/R injury.

Forced expression of YTHDF2 aggravates cardiac I/R injury

To demonstrate whether YTHDF2 is detrimental to the myocardium, we performed gain-of-function studies by overexpressing YTHDF2 with YTHDF2-V for our subsequent experiments (Fig. 3A). GFP fluorescence intensity was significantly enhanced (Supplementary Fig. S3A, B), with higher protein and mRNA levels of YTHDF2 than those in the NC-V group in mouse hearts (Supplementary Fig. S3C, D). YTHDF2 upregulation in I/R mice was significantly enhanced by YTHDF2-V treatment (Fig. 3B).

FIG. 3.

Overexpression of YTHDF2 further aggravates I/R damage. (A) Schematic diagram of I/R after AAV9 vector carrying an NC or YTHDF2 fragment was injected into the mouse heart via tail vein and then harvested at the corresponding time point. (B) Western blot analysis of YTHDF2 protein level in mice. n = 4. **p < 0.01 versus Sham+NC-V; ^## p < 0.01 versus I/R+NC-V. NC-V or YTHDF2-V represents the AAV9 vector carrying the NC or YTHDF2 gene. (C–E) Representative echocardiographs and statistics of EF (%) and FS (%). Sham+NC-V, I/R+YTHDF2-V n = 7, I/R+NC-V n = 8. **p < 0.01 versus Sham+NC-V; ^# p < 0.05, ^## p < 0.01 versus I/R+NC-V. (F, G) Infarct size of I/R hearts stained with Evans blue/TTC. Scale bar = 2 mm. I/R+NC-V n = 4, I/R+YTHDF2-V n = 5. **p < 0.01 versus I/R+NC-V. (H) Serum LDH release levels in mice. n = 6. **p < 0.01 versus Sham+NC-V; ^# p < 0.05 versus I/R+NC-V. (I) Serum CK-MB level in mice. n = 4. **p < 0.01 versus Sham+NC-V; ^## p < 0.01 versus I/R+NC-V. (J) Detection of cTNT level in mouse serum. n = 6. **p < 0.01 versus Sham+NC-V; ^## p < 0.01 versus I/R+NC-V. (K) Representative H&E staining images of mouse hearts after I/R for 24 h. Scale bar = 20 μm. n = 4. Data are expressed as mean ± SEM.

We investigated whether YTHDF2 caused hazardous effects similar to those of I/R injury in normal mice. Echocardiography confirmed that YTHDF2 did not affect EF (%) or FS (%) compared with those in the NC-V group (Supplementary Fig. S3E, F). YTHDF2 impaired cardiac function (Fig. 3C–E and Supplementary Fig. S3G) and increased the infarct size (Fig. 3F, G) and levels of LDH, CK-MB, and cTNT (Fig. 3H–J) post-I/R injury. Consistently, the pernicious histological effects of I/R on the myocardial tissue were aggravated by YTHDF2 (Fig. 3K). These results showed that the forced expression of YTHDF2 facilitates cardiac I/R injury.

YTHDF2 mediates ferroptosis and regulates cardiac injury induced by I/R

To explore the molecular mechanism by which YTHDF2 regulates cardiac I/R injury, we performed RNA-Seq using I/R+shNC-V and I/R+shYTHDF2-V myocardia. According to the RNA-Seq results, the expression levels of 439 genes, including 213 upregulated and 226 downregulated genes, were significantly altered compared with those in the I/R+shNC-V group (Supplementary Fig. S4).

Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed a strong correlation between ferroptosis and shYTHDF2 post-I/R (Fig. 4A). The morphological characteristics of ferroptosis are mainly manifested in the mitochondria, including an increase in membrane density and a decrease or even disappearance of the mitochondrial cristae (Dixon et al., 2012; Yagoda et al., 2007; Yang and Stockwell, 2008).

FIG. 4.

YTHDF2 mediates the ferroptosis signaling pathway and regulates I/R myocardial injury. (A) KEGG enrichment analysis of different signaling pathways in I/R mouse hearts. (B) Electron microscopy was used to assess mitochondrial ultrastructural architecture. Scale bar = 1 and 5 μm. n = 3. (C) Total iron levels were measured in heart tissues. n = 5. *p < 0.05 versus Sham+shNC-V; ^## p < 0.01 versus I/R+shNC-V. (D) PCR analysis of the mRNA levels of TFR1, FPN, Ptgs2 and ACSL4 in mice. n = 4–6. **p < 0.01 versus Sham+shNC-V; ^## p < 0.01 versus I/R+shNC-V. (E) Western blot analysis of TFR1, FPN and GPX4 protein levels in mice. n = 4. *p < 0.05, **p < 0.01 versus Sham+shNC-V; ^# p < 0.05, ^## p < 0.01 versus I/R+shNC-V. (F, G) Serum GSH/GSSG ratio and MDA levels in mice. n = 4–6. *p < 0.05, **p < 0.01 versus Sham+shNC-V; ^# p < 0.05, ^## p < 0.01 versus I/R+shNC-V. (H) Mitochondrial ultrastructural architecture assessed by electron microscopy. Scale bar = 1, 5 μm. n = 3. (I) Total tissue iron levels were measured in I/R mice after YTHDF2 or NC virus treatment. n = 5. *p < 0.05 versus I/R+NC-V. (J) The mRNA levels of ferroptosis-related genes in I/R-NC-V and I/R-YTHDF2-V. n = 4–6. *p < 0.05, **p < 0.01 versus I/R+NC-V. (K) Protein expression levels of TFR1, FPN, and GPX4 in mice. n = 4. *p < 0.05, **p < 0.01 versus I/R+NC-V. (L, M) Serum GSH/GSSG ratio and MDA release in mice. n = 4–6. *p < 0.05 versus I/R+NC-V. Data are expressed as mean ± SEM. ACSL4, acyl-CoA synthetase long-chain family member; FPN, ferroportin; GPX4, glutathione peroxidase 4; GSH, glutathione; GSSG, oxidized glutathione; KEGG, Kyoto Encyclopedia of Genes and Genomes; MDA, malondialdehyde; PCR, polymerase chain reaction; Ptgs2, prostaglandin-endoperoxide synthase 2; TFR1, transferrin receptor 1.

Therefore, we used electron microscopy to assess the mitochondrial ultrastructural architecture. Promiscuous mitochondria and reduced cristae density in I/R mice were abolished by shYTHDF2 (Fig. 4B). YTHDF2 deficiency reversed the increase in iron content in I/R heart tissue (Fig. 4C). mRNA and protein levels of transferrin receptor 1 (TFR1), which imports iron from the extracellular space into cells (Yang and Stockwell, 2008), and iron exporter ferroportin (FPN), which transfers iron out of the cell (Donovan et al., 2005), were reversed by shYTHDF2 in I/R mice (Fig. 4D, E).

We then measured the expression levels of acyl-CoA synthetase long-chain family member (ACSL4) and prostaglandin-endoperoxide synthase 2 (Ptgs2), two biomarkers of ferroptosis (Yang et al., 2014), and found that the deletion of YTHDF2 downregulated ACSL4 and Ptgs2 mRNA levels in I/R hearts (Fig. 4D). Consistently, the protein expression of GPX4, an endogenous lipid peroxidation inhibitor, was inhibited by cardiac I/R injury and upregulated by shYTHDF2 (Fig. 4E).

Decreased GSH levels result in ferroptosis, and we found that the ratio of GSH/oxidized glutathione (GSSG) was reduced in I/R mice and returned to normal levels by silencing YTHDF2 (Fig. 4F). Aberrant levels of the lipid peroxide malondialdehyde (MDA) in I/R mice were reversed by shYTHDF2 (Fig. 4G).

YTHDF2 overexpression enhanced mitochondrial disarrangement and iron accumulation (Fig. 4H, I). Further, changes in ferroptosis-related genes at the mRNA and protein levels were altered by YTHDF2 in I/R mice (Fig. 4J, K). Abnormal levels of GSH/GSSG and MDA were intensified by YTHDF2 (Fig. 4L, M). Collectively, these data revealed that YTHDF2 participates in regulating ferroptosis and aggravating cardiac I/R injury.

Deficiency of YTHDF2 alleviates cardiomyocyte injury after H/R treatment through ferroptosis

These results raise the question of whether inhibiting YTHDF2 in NMVCs exposed to H/R produces the same results in vivo. To clarify this, we performed a loss-of-function experiment using siRNAs. mRNA and protein levels of YTHDF2 were lower after transfection with si-YTHDF2 than in the negative control (NC) group (Supplementary Fig. S5A and Fig. 5A).

FIG. 5.

Silencing of YTHDF2 alleviates cardiomyocyte injury after H/R processing. (A) Transfection of 50 nM si-YTHDF2 for 48 h in cardiomyocytes verified the interference efficiency of YTHDF2. n = 8. **p < 0.01 versus NC. (B) Cell viability was detected by CCK-8 in cardiomyocytes. n = 7. **p < 0.01 versus NC; ^# p < 0.05 versus H/R+NC. (C–E) Live and death ratios were measured in H/R cardiomyocytes after treatment with si-YTHDF2. n = 6. Scale bar = 50 μm. **p < 0.01 versus NC; ^## p < 0.01 versus H/R+NC. (F) Concentrations of LDH in cardiomyocytes. n = 5. *p < 0.05 versus NC; ^# p < 0.05 versus H/R+NC. (G, H) The ROS level was detected by a flow cytometer to detect the production of ROS. n = 6. **p < 0.01 versus NC; ^## p < 0.01 versus H/R+NC. (I) The mRNA expression levels of TFR1, FPN, Ptgs2 and ACSL4 in cardiomyocytes. n = 3–6. **p < 0.01 versus NC; ^# p < 0.05, ^## p < 0.01 versus H/R+NC. (J) Determination of TFR1, FPN, and GPX4 protein expression relative levels in cardiomyocytes. n = 4–5. **p < 0.01 versus NC; ^## p < 0.01 versus H/R+NC. Data are expressed as mean ± SEM. CCK-8, cell counting kit-8; ROS, reactive oxygen species.

Moreover, the detrimental effects, including decreased cell viability accompanied by high levels of LDH after H/R damage, were countered by si-YTHDF2 (Fig. 5B–F). Further experiments revealed that YTHDF2 knockdown attenuated the H/R-induced production of ROS, a characteristic of ferroptosis (Fig. 5G, H, and Supplementary Fig. S5B, C).

Consistent with the in vivo results, the effects of H/R on TFR1, FPN, Ptgs2, and ACSL4 mRNA levels were significantly altered on YTHDF2 silencing (Fig. 5I). Moreover, it abolished the abnormal protein expression levels of TFR1, FPN, and GPX4 in H/R-treated cardiomyocytes (Fig. 5J).

Silencing YTHDF2 represses erastin-induced cardiomyocyte ferroptosis

To investigate whether the downregulation of YTHDF2 protects against cardiac I/R injury in response to ferroptosis, erastin, a ferroptosis activator that targets the cystine/glutamate antiporter (system X_c ⁻), was used in further experiments (Dixon et al., 2012). Similar to what was observed in H/R-induced cardiomyocytes, the inhibition of YTHDF2 had a protective effect against erastin-induced injury, including reduced cell viability, increased LDH levels, and ROS accumulation (Fig. 6A–G and Supplementary Fig. S5D, E).

FIG. 6.

Consumption of YTHDF2 inhibits erastin-induced cardiomyocyte injury. (A) Cardiomyocytes were treated with si-YTHDF2 for 48 h and 5 μM erastin for 24 h. Cell viability was measured by CCK-8 assay. n = 7. **p < 0.01 versus NC; ^# p < 0.05 versus erastin+NC. (B–D) Cell viability was detected to assess the ratio of live to dead cardiomyocytes. Scale bar = 50 μm. n = 4. **p < 0.01 versus NC; ^# p < 0.05 versus erastin+NC. (E) The release of LDH in cardiomyocytes was measured. n = 5. **p < 0.01 versus NC; ^## p < 0.01 versus erastin+NC. (F, G) The production of ROS was detected by a flow cytometer. n = 6. **p < 0.01 versus NC; ^## p < 0.01 versus erastin+NC. (H) The mRNA levels of TFR1, FPN, Ptgs2, and ACSL4 were determined in cardiomyocytes. n = 5–6. *p < 0.05, **p < 0.01 versus NC; ^# p < 0.05, ^## p < 0.01 versus erastin+NC. (I, J) Determination of TFR1, FPN, and GPX4 protein expression relative levels in cardiomyocytes processed with erastin and si-YTHDF2. n = 5. **p < 0.01 versus NC; ^## p < 0.01 versus erastin+NC. Data are expressed as mean ± SEM. SLC7A11, solute carrier family 7 member 11.

The inhibition of YTHDF2 reversed the abnormal mRNA levels of TFR1, FPN, Ptgs2, and ACSL4 after treatment with erastin (Fig. 6H). Western blotting showed that the abnormal protein expression levels of TFR1, FPN, and GPX4 were reversed by YTHDF2 inhibition (Fig. 6I, J). These results indicated that YTHDF2 knockdown mitigates erastin-induced ferroptosis in cardiomyocytes.

YTHDF2 facilitates the degradation of SLC7A11 mRNA

To uncover the downstream mechanism of the regulation of cardiac ferroptosis by YTHDF2, we used a heatmap to analyze the differentially expressed genes in the I/R+shNC-V and I/R+shYTHDF2-V myocardium (Fig. 7A). The differential expression of SLC7A11 protein and mRNA in I/R mice and H/R cardiomyocytes was reversed by the overexpression or inhibition of YTHDF2 (Fig. 7B–D and Supplementary Fig. S6A–C).

FIG. 7.

SLC7A11 mRNA can be degraded by YTHDF2. (A) The heatmap shows differentially expressed genes in I/R+NC-V and I/R+shYTHDF2-V in the myocardium. n = 3. The red arrow points to SLC7A11. (B) The protein expression level of SLC7A11 was detected by Western blot in mice. n = 3. **p < 0.01 versus Sham+shNC-V; ^# p < 0.05 versus I/R+shNC-V. (C) SLC7A11 protein expression level in I/R mice after overexpression of YTHDF2. n = 6. **p < 0.01 versus I/R+NC-V. (D) The expression level of SLC7A11 protein in cardiomyocytes. n = 6. **p < 0.01 versus NC; ^# p < 0.05 versus H/R+NC. (E, F) RIP followed by PCR showed the binding of SLC7A11 with anti- RNA m6A or anti-YTHDF2 in mice or cardiomyocytes. The IgG group was an NC to preclude nonspecific binding. The Y axis represents the percent of input for each IP sample according to the formula: %Input = 1/10 × 2^{Ct [IP] − Ct [input]}. n = 3. *p < 0.05 versus Sham or Ctrl.^† (G) After transfection with si-YTHDF2 for 48 h, cardiomyocytes were treated with Act D (5 μg/mL) to inhibit gene transcription, and the percentage of SLC7A11 mRNA in cardiomyocytes was quantified using PCR at the indicated time point. n = 6. **p < 0.01 versus Act D+NC. Data are expressed as mean ± SEM. Act D, actinomycin D; IP, immunoprecipitation; RIP, RNA-binding protein immunoprecipitation.

Next, we verified that there were many m6A modification sites on SLC7A11 mRNA using SRAMP, and RNA immunoprecipitation results confirmed that the binding of m6A to SLC7A11 was enhanced by I/R injury (Supplementary Fig. S7A and Fig. 7E). YTHDF2 accelerates the degradation of target mRNAs by recognizing m6A modification sites (Wang et al., 2014).

The RNA immunoprecipitation data confirmed that SLC7A11 combined with YTHDF2 in mouse hearts and was more strongly identified by YTHDF2 in H/R-induced cardiomyocytes (Supplementary Fig. S7B and Fig. 7F). To examine whether YTHDF2 influences SLC7A11 mRNA stability, actinomycin D (Act D) was used to inhibit RNA transcription. PCR showed that silencing YTHDF2 significantly repressed the degradation of SLC7A11 mRNA in cardiomyocytes treated with Act D (Fig. 7G).

YTHDF2 targets SLC7A11 to regulate ferroptosis in I/R mice and H/R cardiomyocytes

We hypothesized that the beneficial effect of sh-YTHDF2 in I/R mice is mediated by SLC7A11. To clarify this, we silenced SLC7A11 using shSLC7A11-V in YTHDF2 knockdown mice and found that silencing SLC7A11 did not affect EF (%) and FS (%) (Supplementary Fig. S8A, B). Western blotting and PCR (Supplementary Fig. S8C, D) confirmed the successful knockdown of SLC7A11 in mouse hearts.

The beneficial effect of YTHDF2 inhibition on cardiac function (Fig. 8A–C) was reversed by silencing SLC7A11. Suppression of SLC7A11 also abrogated the decrease in infarct size and LDH level (Fig. 8D–F). siRNAs were designed to suppress the expression of SLC7A11 (Supplementary Fig. S8E, F) in vitro. Co-transfection with si-YTHDF2 and SLC7A11-specific siRNA (si-SLC7A11) intensified H/R-induced cardiomyocyte injury, which promoted cell death and increased the release of LDH and ROS compared with si-YTHDF2 (Fig. 8G–L and Supplementary Fig. S8G).

FIG. 8.

YTHDF2 targets SLC7A11 to regulate ferroptosis in H/R cardiomyocytes. (A–C) Representative echocardiographs and statistics of EF (%) and FS (%). n = 7. *p < 0.05 versus I/R+shYTHDF2-V+shNC-V. (D, E) Infarct size of mouse hearts stained with Evans blue/TTC. Scale bar = 2 mm. I/R+shYTHDF2-V, I/R+shYTHDF2-V+shNC-V n = 5, I/R+ shYTHDF2-V+shSLC7A11-V n = 7. *p < 0.05 versus I/R+shYTHDF2-V+shNC-V. (F) Serum LDH release levels in mice. n = 6. *p < 0.05 versus I/R+shYTHDF2-V+shNC-V. (G) Cell viability was examined by CCK-8 in cardiomyocytes cotransfected with si-YTHDF2 and si-SLC7A11 after H/R. n = 6. **p < 0.01 versus H/R+si-YTHDF2+si-NC. (H–J) Live and dead cell staining was used to examine the viability of cardiomyocytes. Scale bar = 50 μm. n = 4. *p < 0.05 versus H/R+si-YTHDF2+si-NC. (K) Detection of LDH release in cardiomyocytes. n = 6. *p < 0.05 versus H/R+si-YTHDF2+si-NC. (L) The production of ROS was detected by a flow cytometer. n = 6. **p < 0.01 versus H/R+si-YTHDF2+si-NC. (M) TFR1, FPN, and ACSL4 mRNA expression levels in cardiomyocytes. n = 4–5. *p < 0.05, **p < 0.01 versus H/R+si-YTHDF2+si-NC. (N) Protein expression levels of TFR1, FPN, and GPX4 in cardiomyocytes. n = 4–5. **p < 0.01 versus H/R+si-YTHDF2+si-NC. Data are expressed as mean ± SEM. si-SLC7A11, SLC7A11-specific siRNA.

Further, the disadvantageous expressions of ferroptosis-related genes at mRNA and protein levels were aggravated after co-transfection with SLC7A11 and YTHDF2 siRNAs (Fig. 8M, N). Taken together, the deficiency of YTHDF2 attenuates cardiac ferroptosis by stabilizing SLC7A11 m6A modification.

Discussion

The aim of this study was to elucidate the regulatory role and potential mechanisms of YTHDF2 in cardiac ferroptosis and I/R injury. Initially, we found that protein and mRNA levels of YTHDF2 increased and exerted detrimental effects on I/R-treated hearts and H/R-induced cardiomyocytes. Forced expression of YTHDF2 resulted in infarct size enlargement and cardiac dysfunction, whereas knockdown of YTHDF2 reduced these effects after cardiac I/R injury.

Extensive research has demonstrated that the cardioprotective effects of silencing YTHDF2 partly occur by recognizing the m6A modification sites of SLC7A11 and inhibiting its mRNA degradation and subsequent ferroptosis. Briefly, these findings prompted us to delineate this process as follows: I/R or H/R → YTHDF2 ↑ → SLC7A11 stability ↓ → ROS ↑ and iron overload → cardiac ferroptosis → aggravated cardiac injury, suggesting therapeutic targets for treating ischemic cardiac diseases.

The targeting of mRNA using m6A modification relies on reading protein, which comprises the YTH domain family (YTHDF1–3, YTHDC1–2) and IGF2 mRNA-binding protein family (Frye et al., 2018). Multiple studies have shown that the abnormal expression of m6A modification-reading proteins participates in some physiological and pathological processes, such as those involved in biological individual growth and development, sperm development and maturation, DNA damage repair, and various types of disease (Deng et al., 2018; Frye et al., 2018).

Recent studies have demonstrated that m6A methylation plays a critical role in cardiovascular diseases. For example, METTL3 regulates cardiac homeostasis and hypertrophy and FTO mitigates cardiac function during remodeling and repair (Dorn et al., 2019; Mathiyalagan et al., 2019). However, the effect of YTHDF2 on myocardial I/R injury remains unclear. In the present study, we explored the role and regulatory mechanisms of YTHDF2 in heart damage.

We found that the mRNA and protein expression of YTHDF2 increased during cardiac I/R injury, which steadily worsened cardiac function. In addition, the results demonstrated that the inhibition of YTHDF2 ameliorated impaired EF (%) and FS (%) and increased infarct size, LDH release, and CK-MB and cTNT levels post-I/R, whereas YTHDF2 aggravated cardiac damage.

To investigate the underlying mechanism, we used RNA-Seq and KEGG analysis. An additional consequence of this observation is the involvement of ferroptosis in YTHDF2-mediated myocardial injury. There is evidence to suggest that ferroptosis plays an essential role in tumors (Kim et al., 2016), neurodegenerative diseases (Ishii et al., 2019), renal failure (Adedoyin et al., 2018), and cardiovascular diseases (Fang et al., 2019).

In one study, in which the differentially expressed genes between treatment and control groups in a doxorubicin-induced mouse cardiomyopathy model were analyzed, Hmox1 was upregulated, which mediated the release of iron ions in heme to induce ferroptosis and even heart failure owing to iron overload (Fang et al., 2019). In addition, rapamycin (mTOR) inhibits ferroptosis in mouse cardiomyocytes during I/R injury; this may be because mTOR inhibits ROS production, although the underlying mechanism remains to be elucidated (Baba et al., 2018).

Another study revealed that fer-1 (an iron death inhibitor) reduces cardiomyocyte death in heart transplantation and myocardial I/R models, while improving left ventricular systolic function and ventricular remodeling in I/R models (Li et al., 2019). Consistent with these studies, we demonstrated that ferroptosis was activated by cardiac I/R injury, as evidenced by changes in morphological characteristics and levels of ferroptosis markers.

We found that YTHDF2 knockdown impeded promiscuous mitochondria and reduced cristae density, reversing ferroptosis in I/R mice and H/R cardiomyocytes, whereas YTHDF2 overexpression facilitated these harmful effects.

Iron homeostasis regulates multiple iron transport-related proteins. The Andrews Laboratory of Duke University in the United States reported for the first time that the cardiac-specific knockout of TFR1 induces a lethal phenotype, which was attributed to mitochondrial dysfunction caused by severe iron deficiency in the myocardium (Xu et al., 2015).

FPN1, the only cellular iron efflux protein found in mammals, has been specifically knocked out in mouse hearts, which induces cardiac iron metabolism imbalances and cardiac insufficiency (Lakhal-Littleton et al., 2015). Disorders in cardiac FPN1 can increase iron efflux, which accounts for fatal iron deficiency (Gao et al., 2015).

Iron homeostasis plays a vital role in cardiovascular disease. Iron overload triggers ferroptosis by increasing ROS accumulation, which leads to cardiovascular diseases. Our data showed that silencing YTHDF2 reversed iron overload in I/R mice, as evidenced by increased iron content and TFR1 expression and decreased FPN expression. Consistently, YTHDF2 deficiency mitigated erastin-induced ferroptosis in cardiomyocytes. The overexpression of YTHDF2 facilitates these harmful effects. Overall, our gain- and loss-of-function experiments demonstrated that YTHDF2 regulates cardiomyocyte injury via ferroptosis.

Many studies have indicated that YTHDF2 induces the degradation of target mRNA and that differentiation-related mRNAs with the m6A modification in neurons undergo degradation, impairing the self-renewal of neural stem/progenitor cells and development of neurons, resulting in neurodevelopmental defects (Li et al., 2018; Wang et al., 2014).

In hepatocellular carcinoma, YTHDF2 reduces the stability of EGFR mRNA by binding to the m6A site of the 3′UTR (untranslated region) of EGFR and inhibits the ERK/MAPK signaling pathway, thereby exerting a tumor suppressor effect (Zhong et al., 2019). YTHDF2 also reduces the level of UBXN1 by recognizing the m6A modification of UBXN1 mRNA mediated by METTL3, which activates NF-κB, facilitating target gene transcription and the development of glioma (Chai et al., 2021).

Moreover, YTHDF2 recognizes the METTL3-mediated m6A modification of PTEN mRNA and promotes the degradation of PTEN, which leads to excessive PASMC proliferation via the activation of the PI3K/Akt signaling pathway (Qin et al., 2021). Consistent with this, YTHDF2 is upregulated in cardiac hypertrophy. However, YTHDF2 alleviates this injury by promoting the degradation of Myh7 mRNA.

The pathological process of myocardial hypertrophy involves enlarged cardiomyocytes and the proliferation of interstitial cells and connective tissue, which reduces myocardial contractility, obstructs the blood supply, and increases oxygen consumption and heart failure (HF) (Xu et al., 2021).

Myocardial I/R-induced cell death is primarily caused by peroxidation, inflammation, and intracellular calcium overload (Talukder et al., 2009). Therefore, we speculated that YTHDF2 recognizes various m6A modification sites and plays different roles in HF and I/R injury to facilitate mRNA degradation.

Volcano plot analysis showed that SLC7A11 was upregulated by shYTHDF2-V in I/R mice. Ferroptosis, a newly discovered form of iron-dependent cell death, is caused by the inhibition of the cystine/glutamate antiporter (system X_c ⁻) (Dixon et al., 2012). Deferoxamine reduces ferroptosis by inhibiting glutamine metabolism in an isolated mouse heart I/R model (Gao et al., 2015).

SLC7A11 inhibits iron overload-induced ferroptosis by influencing cysteine uptake, decreasing ROS production, and conferring protection against ferroptosis during iron overload (Wang et al., 2017). YTHDC2 destabilizes SLC7A11 mRNA through its m6A reading YTH domain, restraining cystine uptake and downstream antioxidant programs and inhibiting pulmonary adenocarcinoma effects (Ma et al., 2021).

We found that YTHDF2 inhibition significantly increased the stability of SLC7A11 mRNA by recognizing multiple m6A binding sites and relieving cardiomyocyte damage. Moreover, the inhibition of SLC7A11 counterbalanced the beneficial effects of YTHDF2 knockdown on I/R and H/R injury.

In conclusion, our initial observations revealed a cardinal regulatory relationship between YTHDF2 and ferroptosis during cardiac I/R injury. YTHDF2 can inhibit SLC7A11, affecting the transport of glutamate and cystine and reducing the synthesis of GPX4.

However, this can cause iron overload. The synergistic effects of these two factors trigger ferroptosis and aggravate myocardial ischemia–reperfusion injury. Consequently, YTHDF2 targeting SLC7A11 to regulate ferroptosis has novel therapeutic value for myocardial I/R injury and provides potential applications for clinical treatment.

This study has the following shortcomings: (1) The reason for the upregulation of YTHDF2 is currently unclear, and we speculate that there may be transcription factors or noncoding RNAs that regulate the expression of YTHDF2 in cardiac I/R mice. (2) In addition to SLC7A11, YTHDF2 may affect the stability of other RNA that regulate myocardial I/R injury. However, these questions require further investigation.

Materials and Methods

Mouse model of cardiac I/R injury

This study was approved by the Institutional Animal Care and Use Committee of Harbin Medical University. All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Eight-week-old male C57BL/6 mice weighing 20–22 g were intraperitoneally injected with a solution containing avertin to achieve anesthesia (0.2 g/kg; Sigma–Aldrich Corporation).

After intubation of the trachea, the left descending coronary artery (LAD) was ligated with 7-0 nylon for 45 min, and blood perfusion was gently restored for 24 h to establish a mouse model of cardiac I/R injury. The sutures were passed through the LAD of other mice without ligation, as in the sham group. Echocardiography was performed to evaluate the heart function, followed by dissection to obtain tissue samples encompassing the infarcted regions for other experiments.

Gene delivery in mice

C57BL/6 mice were injected with the virus solution adeno-associated virus 9 (AAV9) carrying YTHDF2 and SLC7A11 special short hairpin RNA (shRNA) or YTHDF2 (1 × 10¹¹ virus particles; shYTHDF2-V, shSLC7A11-V, shNC-V, YTHDF2-V, or NC-V) via tail vein injection, and a cTNT promoter conjugated with the GFP gene was constructed (Hanbio Biotechnology). After 4 weeks, I/R surgery was performed.

The sequences of shYTHDF2-V were: sense 5′-CUAGAGAACAACGAGAAUATT-3′ and antisense 5′-UAUUCUCGUUGUUCUCUAGTT-3′. The sequences of shSLC7A11-V were: sense 5′-UGACAAACGUGGCCUAUUUTT-3′ and antisense 5′-AAAUAGGCCACGUUUGUCATT-3′. The expression of YTHDF2 and SLC7A11 was determined using PCR and Western blotting.

Echocardiographic analysis

Mice were anesthetized with 2% avertin 24 h after reperfusion, followed by M-mode echocardiography using a Vevo2100 echocardiographic system (Visualsonics, Toronto, Canada) at a probe frequency of 10 MHz to assess left ventricle (LV) function. The LV of the mice was vertically shot with a noninvasive ultrasound beam, followed by measurement of the LV internal dimensions at EF, FS and LVID for an average of at least three consecutive cardiac cycles.

Evans blue and TTC staining

After 24 h of reperfusion, the LAD ligature was removed and 2% Evans blue dye was injected into the mouse abdominal aorta. Whole hearts obtained from euthanized mice were frozen in a −80°C refrigerator. After hardening, the hearts were cut into four slices with a thickness of 1 mm and stained with 2% TTC dyeing buffer (Solarbio, Beijing, China) at 37°C for 20 min.

Finally, the sections were photographed and ImageJ (NIH) was used to calculate the infarcted area of the hearts.

Electron microscopy

A patch of the heart obtained from C57BL/6 mice was fixed in 2.5% glutaraldehyde and washed with Tris-buffered saline. After fixation using 1% osmium tetroxide at 4°C, the tissues were stained with 1% uranyl acetate. Ethanol was used for dehydration, and epoxy resin was used to embed the samples. Finally, the samples were stained with uranyl acetate and lead citrate, and photographs were captured using a JEM-1200 electron microscope (FEI, Hillsboro, OR).

Immunofluorescence

The heart sections were permeabilized with 0.5% Triton X-100 at room temperature for 30–40 min in the dark and subsequently blocked with 10% normal goat serum at 37°C for 1 h. Afterward, the primary antibody YTHDF2 or α-actinin was added to each slide, which were incubated overnight at 4°C, and then incubated with the corresponding secondary antibody for 1 h in the dark.

After rinsing three times with phosphate buffered saline (PBS), the nuclei were stained with DAPI (Beyotime Institute of Biotechnology, Shanghai, China) for 5–8 min, and immunofluorescence was analyzed using a laser scanning confocal microscope.

H&E staining

The heart sections were dewaxed and dehydrated with xylene and alcohol. Sections were stained with hematoxylin and eosin (Solarbio) according to the manufacturer's instructions.

Measurement of labile iron level

Iron concentrations were measured using an iron assay kit (No. ab83366; Abcam, Cambridge, MA) according to the manufacturer's instructions. Iron buffer or iron reducer was added to the heart samples to detect iron or total iron, which were incubated with the iron probe for 60 min. Finally, the absorbance was measured using a colorimetric microplate reader at OD 593 nm to determine the iron concentration.

Assay kits

LDH, GSH, GSSG, and MDA levels were measured using LDH, GSH, GSSG, and MDA detection assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). CK-MB and cTNT levels were measured using CK-MB and cTNT enzyme-linked immunosorbent assay (ELISA) kits (Elabscience, Wuhan, China), as previously described (Zhang et al., 2021b).

Primary cardiomyocyte culture

NMVCs from 1- to 3-day-old mice were digested in a solution containing trypsin (Solarbio) and Dulbecco's modified Eagle's medium (Biological Industries, Haemek, Israel) for 12 h at 4°C with rotation. The tissues were then digested with collagenase Type II (Thermo Fisher Scientific, Waltham, MA) until the hearts disappeared, and the lysates were collected and centrifuged at 1500 rpm for 10 min. The precipitated cells were resuspended in the culture medium. Dissociated cardiomyocytes were seeded onto plates for follow-up experiments.

Cell transfection and treatment

YTHDF2-specific small interfering RNA (si-YTHDF2), si-SLC7A11, and negative control siRNA (si-NC) were purchased from RiboBio (Guangzhou, China). Transfection with siRNAs at a final concentration of 50 nM was performed using the X-treme GENE Transfection Reagent (Roche, Basel, Switzerland). The sequences of siRNAs for mouse si-YTHDF2 were: sense 5′-CUAGAGAACAACGAGAAUATT-3′ and antisense 5′-UAUUCUCGUUGUUCUCUAGTT-3′.

The sequences of the siRNAs for mouse si-SLC7A11 were: sense 5′-UGACAAACGUGGCCUAUUUTT-3′ and antisense 5′-AAAUAGGCCACGUUUGUCA-3′. Forty-eight hours after transfection, cardiomyocytes were cultured under hypoxic conditions (5% CO₂ and 95% N₂) for 12 h, followed by reoxygenation for 24 h to establish the H/R model.

Cell viability by cell counting kit-8 assay

Cardiomyocytes were incubated with cell counting kit-8 (CCK-8) solution (Beijing Labgic Technology Co., Ltd., Beijing, China) for 1.5 h in the dark in a 37°C incubator. Absorbance was measured at 450 nm using a colorimetric microplate reader to evaluate cell viability.

Live/dead and ROS staining

The viability and ROS content of the cardiomyocytes were assessed using a live/dead assay kit (No. L3224; Life Technologies) and a ROS assay kit, respectively, according to the manufacturer's instructions. The live and dead cells were stained green and red, respectively.

The NMVCs were incubated with 10 μM DCFH-DA (No. S0033S-1, Beyotime Institute of Biotechnology) for ROS staining at 37°C for 30–60 min. Finally, the fluorescence intensity of ROS was measured using a laser scanning confocal microscope or flow cytometer, as previously described (Anselmino et al., 2020; Valerio et al., 2022; Zhu et al., 2021).

Western blotting

A bicinchoninic acid protein kit (Beyotime Institute of Biotechnology) was used to measure the protein concentration. Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes.

The membranes were blocked in confining liquid (A mixed with B), followed by incubation overnight at 4°C with primary antibodies against YTHDF2 (No. 24744-1-AP, 1:500; Proteintech, Wuhan, China), GPX4 (No. 67763-1-Ig, 1:500; Proteintech), FPN (No. 26601-1-AP, 1:1000; Proteintech), TFR1 (No. 13-6800, 1:1000; Invitrogen, Carlsbad, CA), SLC7A11 (No. 175186, 1:1000; Abcam, Cambridge, United Kingdom), and GAPDH (No. TA-08, 1:1000; ZsBio, Beijing, China). Next, horseradish peroxidase-labeled anti-rabbit/mouse IgG (1:10,000; LI-COR Bioscience, Lincoln, NE) was incubated for 1 h at room temperature in the dark.

The membranes were washed three times with Tris-buffered saline and Tween-20 for 8 min each at the beginning and end of secondary antibody incubation. The membranes were scanned and analyzed using an Odyssey infrared imaging system (LI-COR Bioscience), and protein bands were assessed and quantified. GAPDH was used as an internal control.

RNA isolation and quantitative real-time RT-PCR

Total RNA was extracted from the cardiac tissues and cardiomyocytes using TRIzol reagent (Invitrogen). The RNA concentration was measured using a NanoDrop ND-8000 (Thermo Fisher Scientific). The total RNA mass was controlled at 500 ng, and DNA was synthesized using a reverse transcription kit (Toyobo). Real-time PCR was performed using SYBR Green Master Mix (Toyobo) to quantify YTHDF2, SLC7A11, FPN, TFR1, Ptgs2, and ACSL4 levels on an ABI 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA).

Expression was normalized to that of GAPDH or 18S as an internal control. The 2^−△△Ct method was used to determine the relative RNA levels. The primers used in this study were: GAPDH-F 5′-AAGAAGGTGGTGAAGCAGGC-3′, GAPDH-R 5′-TCCACCACCCTGTTGCTGTA-3′, 18S-F 5′-CCTGGATACCGCAGCTAGGA-3′, 18S-R 5′-GCGGCGCAATACGAATGCCCC-3′, SLC7A11-F 5′-TCAAAAGCTTGGCCATCTGC-3′, SLC7A11-R 5′-CTGTGAGCTTGCCTCACTGTA-3′, ACSL4-F 5′-CCACACTTATGGCCGCTGTT-3′, ACSL4-R 5′-GGGCGTCATAGCCTTTCTTG-3′, Ptgs2-F 5′-CTGCGCCTTTTCAAGGATGG-3′, Ptgs2-R 5′-GGGGATACACCTCTCCACCA-3′, FPN-F 5′-GTGGAGTACTTCTTGCTCTGG-3′, FPN-R 5′-CTGCTTCAGTTCTGACTCCTC-3′, YTHDF2-F 5′-TAGCCAACTGCGACACATTC-3′, YTHDF2-R 5′-CACGACCTTGACGTTCCTTT-3′, TFR1-F 5′-GGAGCATGCCGAGAAACTGA-3′, and TFR1-R 5′-CTCTCCCAGTCATCACGGTC-3′.

Act D treatment

The NMVCs were plated in 6-well plates at a density of 1 × 10⁶ cells/well. After transfection for 48 h, the cells were treated with Act D (5 g/mL; MedChemExpress) and dimethyl sulfoxide (DMSO; BioFroxx) to inhibit transcription, followed by collection at different time points.

RNA-binding protein immunoprecipitation assay

N6-methyladenine RNA-binding protein immunoprecipitation (Me-RIP) and YTHDF2-RNA-binding protein immunoprecipitation (RIP) were performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer's protocol.

In summary, fresh hearts were homogenized in RIP lysis buffer, incubated overnight with 50 μL of protein-A/G agarose beads (Roche, USA) and antibodies against m6A (No. 202003, 5 μg; Synaptic Systems, Goettingen, Germany) or YTHDF2 (No. 24744-1-AP, 5 μg; Proteintech), transiently stained, and washed six times with RIP washing buffer. Finally, the immunoprecipitated RNA was purified and subjected to PCR.

RNA-Seq

Hearts were delivered to a company that constructed the mRNA library and sequenced RNA using LC Bio (Hangzhou, China). KEGG enrichment was used to assess the expression changes in the quantified genes under different conditions to screen for differentially expressed genes. KEGG enrichment analysis, heatmaps, and volcano plots were constructed as described by LC Bio.

Statistical analysis

All data are presented as the means ± standard error of measurement of at least three independent experiments and were statistically analyzed using GraphPad Prism 7.0. Student's t-test was used to analyze two groups, and one-way analysis of variance with Dunnett's correction was applied for multiple-group comparisons. A p value of <0.05 indicated a significant difference.

An electronic laboratory notebook was not used.

Data Availability

The datasets used and/or analyzed to support the findings of this study are available in this article or in the Supplementary Figures S1–S8. All the raw data supporting the results of this study are available from the corresponding author on request.

Footnotes

Authors' Contributions

B.Y., N.W., and Y.B. supervised and designed the study. P.P., W.S., H.W., J.J., K.L., Y.J., H.D., L.Z., W.J., Y.Y., and Y.X. performed all experiments. Y.B., P.P., W.S., and H.W. wrote the article. X.K., Z.Z., F.Z., J.S., and C.W. collected and analyzed the data. All the authors approved the final version of the article.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by the National Natural Science Foundation of China (Nos. 82104168 and U21A20339), China Postdoctoral Science Foundation (No. 2021M693832), and Heilongjiang Province Postdoctoral Science Foundation (No. LBH-Z20174).

Supplementary Material

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Figure S4

Supplementary Figure S5

Supplementary Figure S6

Supplementary Figure S7

Supplementary Figure S8

Abbreviations Used

References

Adedoyin

, Boddu

, Traylor

, et al. Heme oxygenase-1 mitigates ferroptosis in renal proximal tubule cells. Am J Physiol Renal Physiol, 2018; 314(5):F702–F714; doi: 10.1152/ajprenal.00044.2017

Anselmino

, Starbuck

, Labanca

, et al. Heme oxygenase-1 is a pivotal modulator of bone turnover and remodeling: Molecular implications for prostate cancer bone metastasis. Antioxid Redox Signal, 2020; 32(17):1243–1258; doi: 10.1089/ars.2019.7879

Baba

, Higa

, Shimada

, et al. Protective effects of the mechanistic target of rapamycin against excess iron and ferroptosis in cardiomyocytes. Am J Physiol Heart Circ Physiol, 2018; 314(3):H659–H668; doi: 10.1152/ajpheart.00452.2017

Cai

, Guo

, Chang

, et al. Empagliflozin attenuates cardiac microvascular ischemia/reperfusion through activating the AMPKalpha1/ULK1/FUNDC1/mitophagy pathway. Redox Biol, 2022; 52:102288; doi: 10.1016/j.redox.2022.102288

Chai

, Chang

, et al. YTHDF2 facilitates UBXN1 mRNA decay by recognizing METTL3-mediated m(6)A modification to activate NF-kappaB and promote the malignant progression of glioma. J Hematol Oncol, 2021; 14(1):109; doi: 10.1186/s13045-021-01124-z

Chen

, Chen

, Niu

, et al. Poly(I:C) preconditioning protects the heart against myocardial ischemia/reperfusion injury through TLR3/PI3K/Akt-dependent pathway. Signal Transduct Target Ther, 2020; 5(1):216; doi: 10.1038/s41392-020-00257-w

Chouchani

, Pell

, Gaude

, et al. Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS. Nature, 2014; 515(7527):431–435; doi: 10.1038/nature13909

Del Re

, Amgalan

, Linker

, et al. Fundamental mechanisms of regulated cell death and implications for heart disease. Physiol Rev, 2019; 99(4):1765–1817; doi: 10.1152/physrev.00022.2018

Deng

, Su

, Weng

, et al. RNA N(6)-methyladenosine modification in cancers: Current status and perspectives. Cell Res, 2018; 28(5):507–517; doi: 10.1038/s41422-018-0034-6

10.

Dixon

, Lemberg

, Lamprecht

, et al. Ferroptosis: An iron-dependent form of nonapoptotic cell death. Cell, 2012; 149(5):1060–1072; doi: 10.1016/j.cell.2012.03.042

11.

Donovan

, Lima

, Pinkus

, et al. The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis. Cell Metab, 2005; 1(3):191–200; doi: 10.1016/j.cmet.2005.01.003

12.

Dorn

, Lasman

, Chen

, et al. The N(6)-methyladenosine mRNA methylase METTL3 controls cardiac homeostasis and hypertrophy. Circulation, 2019; 139(4):533–545; doi: 10.1161/CIRCULATIONAHA.118.036146

13.

Fang

, Cai

, Wang

, et al. Loss of cardiac ferritin H facilitates cardiomyopathy via Slc7a11-mediated ferroptosis. Circ Res, 2020; 127(4):486–501; doi: 10.1161/CIRCRESAHA.120.316509

14.

Fang

, Wang

, Han

, et al. Ferroptosis as a target for protection against cardiomyopathy. Proc Natl Acad Sci U S A, 2019; 116(7):2672–2680; doi: 10.1073/pnas.1821022116

15.

Feng

, Byrne

, Targher

, et al. Ferroptosis and metabolic dysfunction-associated fatty liver disease: Is there a link?. Liver Int, 2022; 42(7):1496–1502; doi: 10.1111/liv.15163

16.

Frye

, Harada

, Behm

, et al. RNA modifications modulate gene expression during development. Science, 2018; 361(6409):1346–1349; doi: 10.1126/science.aau1646

17.

Gao

, Monian

, Quadri

, et al. Glutaminolysis and transferrin regulate ferroptosis. Mol Cell, 2015; 59(2):298–308; doi: 10.1016/j.molcel.2015.06.011

18.

Gilbert

, Bell

, Schaening

. Messenger RNA modifications: Form, distribution, and function. Science, 2016; 352(6292):1408–1412; doi: 10.1126/science.aad8711

19.

Gumpper-Fedus

, Park

, Ma

, et al. MG53 preserves mitochondrial integrity of cardiomyocytes during ischemia reperfusion-induced oxidative stress. Redox Biol, 2022; 54:102357; doi: 10.1016/j.redox.2022.102357

20.

Guo

, Hong

, He

, et al. NFkappaB promotes oxidative stress-induced necrosis and ischemia/reperfusion injury by inhibiting Nrf2-ARE pathway. Free Radic Biol Med, 2020a;159:125–135; doi: 10.1016/j.freeradbiomed.2020.07.031

21.

Guo

, Li

, Jiang

, et al. RNA demethylase ALKBH5 prevents pancreatic cancer progression by posttranscriptional activation of PER1 in an m6A-YTHDF2-dependent er. Mol Cancer, 2020b;19(1):91; doi: 10.1186/s12943-020-01158-w

22.

Heusch

. Molecular basis of cardioprotection: Signal transduction in ischemic pre-, post-, and remote conditioning. Circ Res, 2015; 116(4):674–699; doi: 10.1161/CIRCRESAHA.116.305348

23.

Hou

, Zhang

, Liu

, et al. YTHDF2 reduction fuels inflammation and vascular abnormalization in hepatocellular carcinoma. Mol Cancer, 2019; 18(1):163; doi: 10.1186/s12943-019-1082-3

24.

Ishii

, Warabi

, Mann

. Circadian control of BDNF-mediated Nrf2 activation in astrocytes protects dopaminergic neurons from ferroptosis. Free Radic Biol Med, 2019; 133:169–178; doi: 10.1016/j.freeradbiomed.2018.09.002

25.

Kim

, Zhang

, Ma

, et al. Ultrasmall nanoparticles induce ferroptosis in nutrient-deprived cancer cells and suppress tumour growth. Nat Nanotechnol, 2016; 11(11):977–985; doi: 10.1038/nnano.2016.164

26.

Lakhal-Littleton

, Wolna

, Carr

, et al. Cardiac ferroportin regulates cellular iron homeostasis and is important for cardiac function. Proc Natl Acad Sci U S A, 2015; 112(10):3164–3169; doi: 10.1073/pnas.1422373112

27.

, Xie

, Ying

, et al. YTHDF2 mediates the mRNA degradation of the tumor suppressors to induce AKT phosphorylation in N6-methyladenosine-dependent way in prostate cancer. Mol Cancer, 2020; 19(1):152; doi: 10.1186/s12943-020-01267-6

28.

, Zhao

, Wang

, et al. Ythdf2-mediated m(6)A mRNA clearance modulates neural development in mice. Genome Biol, 2018; 19(1):69; doi: 10.1186/s13059-018-1436-y

29.

, Feng

, Gauthier

, et al. Ferroptotic cell death and TLR4/Trif signaling initiate neutrophil recruitment after heart transplantation. J Clin Invest, 2019; 129(6):2293–2304; doi: 10.1172/JCI126428

30.

, Han

, Hu

, et al. N6-methyladenosine reader IMP2 stabilizes the ZFAS1/OLA1 axis and activates the Warburg effect: Implication in colorectal cancer. J Hematol Oncol, 2021; 14(1):188; doi: 10.1186/s13045-021-01204-0

31.

, Chen

, Zhang

, et al. The m(6)A reader YTHDC2 inhibits lung adenocarcinoma tumorigenesis by suppressing SLC7A11-dependent antioxidant function. Redox Biol, 2021; 38:101801; doi: 10.1016/j.redox.2020.101801

32.

Mao

, Lei

, Zhuang

, et al. Ferroptosis as an important driver of lupus. Protein Cell, 2022; 13(5):313–315; doi: 10.1007/s13238-021-00892-1

33.

Mathiyalagan

, Adamiak

, Mayourian

, et al. FTO-dependent N(6)-methyladenosine regulates cardiac function during remodeling and repair. Circulation, 2019; 139(4):518–532; doi: 10.1161/CIRCULATIONAHA.118.033794

34.

Qin

, Qiao

, Li

, et al. The m(6)A methyltransferase METTL3 promotes hypoxic pulmonary arterial hypertension. Life Sci, 2021; 274:119366; doi: 10.1016/j.lfs.2021.119366

35.

Shi

, Cao

, Zhang

, et al. Comprehensive analysis of N6-methyladenosine RNA methylation regulators expression identify distinct molecular subtypes of myocardial infarction. Front Cell Dev Biol, 2021; 9:756483; doi: 10.3389/fcell.2021.756483

36.

Talukder

, Zweier

, Periasamy

. Targeting calcium transport in ischaemic heart disease. Cardiovasc Res, 2009; 84(3):345–352; doi: 10.1093/cvr/cvp264

37.

Valerio

, Keceli

, Moschetta

, et al. Enduring reactive oxygen species emission causes aberrant protein S-glutathionylation transitioning human aortic valve cells from a sclerotic to a stenotic phenotype. Antioxid Redox Signal, 2022; 37(13–15):1051–1071; doi: 10.1089/ars.2021.0133

38.

Wang

, An

, Xie

, et al. Characterization of ferroptosis in murine models of hemochromatosis. Hepatology, 2017; 66(2):449–465; doi: 10.1002/hep.29117

39.

Wang

, Lu

, Gomez

, et al. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature, 2014; 505(7481):117–120; doi: 10.1038/nature12730

40.

, Zhang

, et al. Cathelicidin aggravates myocardial ischemia/reperfusion injury via activating TLR4 signaling and P2X7R/NLRP3 inflammasome. J Mol Cell Cardiol, 2020; 139:75–86; doi: 10.1016/j.yjmcc.2019.12.011

41.

Xiao

, Zhang

, Wu

, et al. CIRKIL exacerbates cardiac ischemia/reperfusion injury by interacting with Ku70. Circ Res, 2022; 130(5):e3–e17; doi: 10.1161/CIRCRESAHA.121.318992

42.

, Wang

, Chen

, et al. YTHDF2 alleviates cardiac hypertrophy via regulating Myh7 mRNA decoy. Cell Biosci, 2021; 11(1):132; doi: 10.1186/s13578-021-00649-7

43.

, Barrientos

, Mao

, et al. Lethal cardiomyopathy in mice lacking transferrin receptor in the heart. Cell Rep, 2015; 13(3):533–545; doi: 10.1016/j.celrep.2015.09.023

44.

Yagoda

, von Rechenberg

, Zaganjor

, et al. RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels. Nature, 2007; 447(7146):864–868; doi: 10.1038/nature05859

45.

Yang

, SriRamaratnam

, Welsch

, et al. Regulation of ferroptotic cancer cell death by GPX4. Cell, 2014; 156(1–2):317–331; doi: 10.1016/j.cell.2013.12.010

46.

Yang

, Stockwell

. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem Biol, 2008; 15(3):234–245; doi: 10.1016/j.chembiol.2008.02.010

47.

Yang

, Zhang

, Yang

, et al. Interdependent nuclear co-trafficking of ASPP1 and p53 aggravates cardiac ischemia/reperfusion injury. Circ Res, 2023; 132(2):208–222; doi: 10.1161/CIRCRESAHA.122.321153

48.

Yao

, Li

, Fang, et al. Emerging roles of energy metabolism in ferroptosis regulation of tumor cells. Adv Sci (Weinh), 2021; 8(22):e2100997; doi: 10.1002/advs.202100997

49.

Zhang

, Sun

, Bai

, et al. RBMS1 regulates lung cancer ferroptosis through translational control of SLC7A11. J Clin Invest, 2021a;131(22):e152067; doi: 10.1172/JCI152067

50.

Zhang

, Zhang

, Cai

, et al. The long noncoding RNA lncCIRBIL disrupts the nuclear translocation of Bclaf1 alleviating cardiac ischemia-reperfusion injury. Nat Commun, 2021b;12(1):522; doi: 10.1038/s41467-020-20844-3

51.

Zhang

, Luo

, Zou

, et al. Genetic analyses support the contribution of mRNA N(6)-methyladenosine (m(6)A) modification to human disease heritability. Nat Genet, 2020; 52(9):939–949; doi: 10.1038/s41588-020-0644-z

52.

Zhao

, Hu

, Sun

, et al. Loss of m6A demethylase ALKBH5 promotes post-ischemic angiogenesis via post-transcriptional stabilization of WNT5A. Clin Transl Med, 2021; 11(5):e402; doi: 10.1002/ctm2.402

53.

Zhong

, Liao

, Zhang

, et al. YTHDF2 suppresses cell proliferation and growth via destabilizing the EGFR mRNA in hepatocellular carcinoma. Cancer Lett, 2019; 442:252–261; doi: 10.1016/j.canlet.2018.11.006

54.

Zhu

, Tian

, Yang

, et al. Procyanidin B2 promotes intestinal injury repair and attenuates colitis-associated tumorigenesis via suppression of oxidative stress in mice. Antioxid Redox Signal, 2021; 35(2):75–92; doi: 10.1089/ars.2019.7911

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.82 MB

YTHDF2 Promotes Cardiac Ferroptosis via Degradation of SLC7A11 in Cardiac Ischemia–Reperfusion Injury

Abstract

Aims:

Results:

Innovation and Conclusion:

Introduction

Innovation

Results

Upregulation of YTHDF2 in I/R mouse hearts and hypoxia-reoxygenation cardiomyocytes

Inhibition of YTHDF2 attenuates I/R-induced cardiac injury in mice

Forced expression of YTHDF2 aggravates cardiac I/R injury

YTHDF2 mediates ferroptosis and regulates cardiac injury induced by I/R

Deficiency of YTHDF2 alleviates cardiomyocyte injury after H/R treatment through ferroptosis

Silencing YTHDF2 represses erastin-induced cardiomyocyte ferroptosis

YTHDF2 facilitates the degradation of SLC7A11 mRNA

YTHDF2 targets SLC7A11 to regulate ferroptosis in I/R mice and H/R cardiomyocytes

Discussion

Materials and Methods

Mouse model of cardiac I/R injury

Gene delivery in mice

Echocardiographic analysis

Evans blue and TTC staining

Electron microscopy

Immunofluorescence

H&E staining

Measurement of labile iron level

Assay kits

Primary cardiomyocyte culture

Cell transfection and treatment

Cell viability by cell counting kit-8 assay

Live/dead and ROS staining

Western blotting

RNA isolation and quantitative real-time RT-PCR

Act D treatment

RNA-binding protein immunoprecipitation assay

RNA-Seq

Statistical analysis

Data Availability

Footnotes

Authors' Contributions

Author Disclosure Statement

Funding Information

Supplementary Material

Abbreviations Used

References

Supplementary Material