m⁶A reader YTHDF1 promotes cardiac fibrosis by enhancing AXL translation

Abstract

Cardiac fibrosis caused by ventricular remodeling and dysfunction such as post-myocardial infarction (MI) can lead to heart failure. RNA N⁶-methyladenosine (m⁶A) methylation has been shown to play a pivotal role in the occurrence and development of many illnesses. In investigating the biological function of the m⁶A reader YTHDF1 in cardiac fibrosis, adeno-associated virus 9 was used to knock down or overexpress the YTHDF1 gene in mouse hearts, and MI surgery in vivo and transforming growth factor-β (TGF-β)-activated cardiac fibroblasts in vitro were performed to establish fibrosis models. Our results demonstrated that silencing YTHDF1 in mouse hearts can significantly restore impaired cardiac function and attenuate myocardial fibrosis, whereas YTHDF1 overexpression could further enhance cardiac dysfunction and aggravate the occurrence of ventricular pathological remodeling and fibrotic development. Mechanistically, zinc finger BED-type containing 6 mediated the transcriptional function of the YTHDF1 gene promoter. YTHDF1 augmented AXL translation and activated the TGF-β-Smad2/3 signaling pathway, thereby aggravating the occurrence and development of cardiac dysfunction and myocardial fibrosis. Consistently, our data indicated that YTHDF1 was involved in activation, proliferation, and migration to participate in cardiac fibrosis in vitro. Our results revealed that YTHDF1 could serve as a potential therapeutic target for myocardial fibrosis.

Keywords

cardiac fibrosis / YTHDF1 / AXL / ZBED6 / heart failure

1 Introduction

Polytype cardiovascular diseases result in high morbidity and mortality rates because of adverse cardiac structural and cardiac insufficiency, leading to heart failure (HF) [1,2]. Myocardial fibrosis is a common pathological consequence of myocardial infarction (MI), which serves as the primary risk factor for HF [3–5]. Cardiac fibrosis results from the increased synthesis of fibrogenic proteins and excessive extracellular matrix (ECM) protein deposition after the activation of cardiac fibroblasts (CFs) [6,7]. The progression of cardiac fibrosis is irreversible on account of the limited power of regeneration of adult hearts, necessitating the quick formation of a stable scar to prevent ventricular wall rupture [8,9]. Myofibroblasts are the key cell types involved in ECM formation after MI injury, and harnessing this regenerative process to modify infarction scar properties shows great potential as a new therapeutic avenue for treating HF [5,10]. However, despite that limited surgical remedies options exist, the application of antifibrotic therapy in HF remains unknown.

Research on cardiac fibrosis stemming from MI injury often focuses on several transcription factors and transcriptional auxiliary activators, while the potential effects of post-transcriptional regulation on critical cardiac protein expression and cardiac function remain largely unexplored. N⁶-methyladenosine (m⁶A) methylation plays a crucial role in regulating RNA processing, translation, splicing, and stability [11]. m⁶A methylation plays an important role in various cardiac diseases, and it has been well established [12,13]. The m⁶A reader YTHDF1 is involved in translational regulation, and it energetically promotes controlled protein synthesis [14]. However, the function of YTHDF1 in cardiac fibrosis remains unexplored. Recently, the m⁶A demethylase ALKBH5 was reported to inhibit the occurrence and development of non-small cell lung cancer by decreasing YAP expression regulated by YTHDFs and repressing YAP activity mediated by miR-107/LATS2 [15]. Moreover, the survival rate of patients with breast cancer decreased when YTHDF1 was overexpressed [16], highlighting the importance of YTHDF1 in promoting cell growth and proliferation. Whether YTHDF1 contributes to adverse left ventricular remodeling through protective or pathological effects remains unclear.

AXL participates in various cell biological processes, and it has been reported to be relevant to the invasion and metastasis of multiple human cancers, including lung cancer, breast cancer, and melanomas [17–19]. Moreover, AXL appears to have critical effects on the mechanism of HF, as indicated by its high expression levels in patients with HF [20–22]. Deberge et al. demonstrated that AXL can accelerate the glycolytic metabolism of cardiac macrophages and IL-1β secretion, resulting in enhanced myocardial inflammation and adverse ventricular remodeling [22]. Given the importance and ambiguous effects of AXL on cardiac diseases, the mechanism of AXL in myocardial fibrosis is worth investigating. Several available reports have elucidated the signaling pathways involved in cardiac fibrosis, including PI3K/Akt, PAFR/YAP1, and JAK2/STAT3 [23–25]. Transforming growth factor-β (TGF-β) is an important initiator of fibrosis in many organs [26–28], and it plays a critical role in cardiac repair and fibroblast function [29]. After TGF-β activation, Smad2/3 proteins enter the nucleus to play a transcriptional role, thereby activating fibrosis development [30]. Therefore, further research on TGF-β/Smad2/3 signaling in cardiac fibrosis is warranted. In the present study, we carried out MI surgery and TGF-β induced CF activation to provide evidence supporting the inducing effects of YTHDF1 on cardiac fibrosis in vivo and in vitro. The transcriptional repressor zinc finger BED-type containing 6 (ZBED6) was found to decrease the expression level of YTHDF1. In addition, YTHDF1 recognizes the methylation sites on AXL and promotes the transcription and translation of AXL mRNA, leading to the activation of the TGF-β/Smad2/3 signaling pathway and exacerbation of myocardial fibrosis. Overall, our experimental results confirm that YTHDF1 may provide insights into novel therapeutic approaches for treating cardiac fibrosis.

2 Materials and methods

2.1 Animals

C57BL/6 mice were raised at 25 °C with a 12 h light/12 h dark cycle and provided with standard water and diet. The mice were euthanized by intraperitoneal injection of avertin (200 mg/kg, Sigma Aldrich Corporation, USA). This investigation was conducted with the approval of the Institutional Animal Care and Use Committee of Harbin Medical University (approval number: IRB3027722) and in compliance with the guidelines outlined in the NIH Guide for the Care and Use of Laboratory Animals. Peri-infarct tissue of the left ventricle of mice was used in subsequent experiments.

2.2 Adeno-associated virus serotype 9 (AAV9) and AdV viral vector establishment

C57BL/6 mice were intravenously injected with AAV9 carrying a specific short hairpin RNA fragment targeting YTHDF1 (shYTHDF1-V and Postn-shYTHDF1-V) or a negative control (shNC-V and Postn-shNC-V), as well as AAV9 carrying YTHDF1 (YTHDF1-V) or an empty vector-carrying virus (NC-V) as a control. This intervention aimed to modulate the cardiac-specific expression of the YTHDF1 gene via tail vein injection and infected mice with 1.5 × 10¹¹ AAV9 YTHDF1 vector genomes 2 weeks prior to MI surgery. An adenoviral vector encoding the mouse YTHDF1 gene was subcloned and inserted into an adenoviral vector (YTHDF1-adenovirus (AdV)) along with an empty vector control (NC-AdV), both of which were constructed by Hanbio Biotechnology Co., Ltd. (Shanghai, China).

2.3 Mouse model of MI

The detailed procedure for establishing an MI model in mice followed slight modifications as described previously [31]. In brief, 8-week-old male C57BL/6 mice were narcotized by avertin (200 mg/kg) via intraperitoneal injection, intubated, and connected to a rodent respirator. A 7-0 nylon suture was used to ligate the left anterior descending coronary artery (LAD). In the sham group, the LAD was only passed through sutures without ligation. Subsequently, functional echocardiography of mouse hearts was performed to determine whether the MI model was successfully established.

2.4 Echocardiographic measurement

The mice underwent echocardiography using the M-mode echocardiography setting with the Vevo2100 echocardiographic system (Visualsonics, Toronto, Ontario, Canada). The ultrasound machine was equipped with a 10.0 MHz phase-array transducer to assess cardiac function. At least three consecutive cardiac cycles were recorded and averaged for analysis.

2.5 Masson trichrome staining

Heart tissues obtained from each group were fixed with 4% paraformaldehyde and then embedded in paraffin blocks. Following the manufacturer’s recommendations, a Masson trichromatic staining kit (Solarbio, China) was used to stain paraffin sections. The entire heart image was analyzed using ImageJ to measure the fibrotic area, and the percentage of fibrotic area relative to the total area was calculated and marked using image analysis software.

2.6 Cell culture

Hearts of 1- to 3-day-old mice were excised, rinsed with cold PBS, and then digested on a 4 °C shaker overnight using trypsin (Solarbio, Beijing, China) and Dulbecco’s modified Eagle’s medium (Biological Industries, Haemek, Israel). The obtained cardiac tissue was further diverted in type II collagenase (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and then collected until all heart pieces were digested. After centrifugation, the cells were incubated in an incubator (95% air–5% CO₂) for 1.5 h. Nonadherent myocardium cells were dislodged from the medium to obtain pure CFs.

For the preparation of adult mouse CFs, the left ventricle of C57BL/6 mice was first cut into 1 mm slices. Then, these slices were digested with 50% (w/v) type II collagenase and 100% (w/v) trypsin (Solarbio, Beijing, China) at 37 °C. Afterward, digestion was terminated, and the process was repeated until no visible tissue remained. The cells were then scattered in a culture medium and incubated in a 95% air–5% CO₂ incubator for 1.5 h to allow the collection of CFs for subsequent experiments.

2.7 Cell transfection and treatment

CFs were transfected with 50 nmol/L siRNAs or plasmids for 48 h using X-treme gene siRNA transfection reagent (Roche, Basel, Switzerland) or Lipofectamine 3000 reagents (Invitrogen, Carlsbad, USA) following the manufacturer’s instructions. YTHDF1-specific small interfering RNA (si-YTHDF1), AXL-specific siRNA (si-AXL), ZBED6-specific siRNA (si-ZBED6), and negative control siRNA (si-NC) were purchased from RiboBio (Guangzhou, China), and the plasmids were purchased from Ibsbio (Shanghai, China). The mouse siRNA sequences were as follows:

si-YTHDF1:

sense 5′-GCACACAACCUCUAUCUUUTT-3′

antisense 5′-AAAGAUAGAGGUUGUGUGCTT-3′;

si-AXL:

sense 5′-UAUCACAGGUGCCAGAGGATT-3′

antisense 5′-UCCUCUGGCACCUGUGAUATT-3′;

si-ZBED6:

sense 5′-CCGUACAACUAUUUCUCAATT-3′

antisense 5′-UUGAGAAAUAGUUGUACGGTT-3′.

After transfection, TGF-β (10 ng/mL; PeproTech, Rocky Hill, NJ, USA) was added to the culture medium for 48 h.

2.8 CCK 8 assay

CFs (2×10⁴) were seeded into 96-well plates per well and cultivated with CCK-8 (Beijing Labgic Technology Co., Ltd., Beijing, China) for 1.5–2 h at 37 °C in a light-protected environment. Subsequently, cell viability was assessed by using a colorimetric microplate reader.

2.9 EdU (5-ethynyl-2′-deoxyuridine) staining

EdU staining was performed using the Cell-Light EdU Apollo 567 In Vitro Kit (RiboBio, CAT#C10310-1). In brief, CFs were seeded in a 24-well plate, followed by transfection and TGF-β treatment. Subsequently, the cells were incubated in 50 μmol/L EdU reagent from the EdU kit (RiboBio, Guangzhou, China) for 2 h. After rinsing the CFs with cold PBS, they were fixed and decolored, followed by permeabilization using 300 μL of 0.5% Triton X-100. Then, the CFs were treated with Apollo and Hoechst, and a fluorescence microscope was used to observe and capture images.

2.10 Wound healing assay

After CFs were seeded in a six-well plate, the cells were scraped using a sterilized white tip in three straight lines, and the floating cells were washed with PBS. Photographs were taken using a digital single-lens reflex camera at 0 and 48 h after experimental treatment, and the cell migration area was analyzed by ImageJ.

2.11 Cycloheximide (CHX) treatment

After 48 h of CF transfection, CHX at 50 μg/mL (Sigma-Aldrich, #C7698) was added at 0 and 3 h to inhibit protein synthesis prior to cell harvesting [32]. Protein was extracted for immunoblot analysis.

2.12 Immunofluorescence

For heart tissues, subsequent experiments were conducted using the abovementioned paraffin sections. At the cellular level, CFs were seeded onto glass coverslips in 24-well plates, and further experiments were conducted after appropriate treatment. In brief, the prepared samples were permeabilized with Triton X-100 and blocked with bovine serum albumin in PBS in a light-protected environment for 30–40 min. After blocking with goat serum at 37 °C for 1 h, primary antibodies against YTHDF1 (#17479-1-AP, Proteintech, Wuhan, China, 1:500), AXL (#13196-1-AP, Proteintech, Wuhan, China, 1:500), Col-1 (#GTX26308, Gene Tex, TX, USA, 1:500), and α-SMA (α-smooth muscle actin) (#AF1032, Affinity Biosciences, Changzhou, China, 1:500) were incubated overnight at 4 °C. Subsequently, a fluorescein-594 secondary antibody (1:300) or fluorescein-488 secondary antibody (1:300) was applied under dark conditions for 1 h. Nuclei were dyed with DAPI (Beyotime Institute of Biotechnology, Shanghai, China), and images were acquired under a laser scanning confocal microscope.

2.13 RNA isolation, cDNA synthesis, and qRT-PCR

According to the manufacturer’s instructions, mRNA was extracted from the peri-infarct tissue of the left ventricle of mice and treated with TRIzol (Invitrogen, Carlsbad, CA, USA). RNA sample concentrations were measured using the NanoDrop ND-8000 (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, a reverse transcription kit (Toyobo, Japan) was adopted to synthesize cDNA with 500 ng of RNA per sample. The expression levels of YTHDF1, α-SMA, Col1a1, Col3a1, CTGF (connective tissue growth factor), AXL, and ZBED6 mRNAs were quantified on the ABI 7900HT fast real-time PCR System (Applied Biosystems, Foster City, CA, USA) by using SYBR Green Master Mix (Toyobo, Japan) and normalized to the expression level of GAPDH or 18S mRNA as an internal reference. Gene expression was calculated using the 2^−△△Ct method. The primer sequences are shown in Table S1.

2.14 Western blot

Peri-infarct tissue of the left ventricle of mice and cells were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing 1% protease inhibitor (Roche, Switzerland) and phosphatase inhibitor (Roche, Switzerland). A bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) was used to determine the protein concentration. The proteins were then separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The membrane was incubated overnight with the following primary antibodies: GAPDH (#TA-08, ZsBio, Beijing, China, 1:1000), YTHDF1 (#220162, Abcam, Cambridge, UK, 1:1000), phospho-Smad2/3 (#WL02305, Wanleibio, Shenyang, China, 1:500), Smad2/3 (#WL01520, Wanleibio, Shenyang, China, 1:500), AXL (#13196-1-AP, Proteintech, Wuhan, China, 1:1000), ZBED6 (#HPA068807, Atlas Antibodies, Sweden, 1:1000), and TGF-β1 (#WL02998, Wanleibio, Shenyang, China, 1:500) at 4 °C. Subsequently, the membranes were incubated with a secondary antibody for 60 min at room temperature. The blotted membranes were scanned by using the Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA). Band densitometry was quantified using Image Studio.

2.15 RNA-binding protein immunoprecipitation (RIP) assay

The Magna RIP RNA-binding Protein Immunoprecipitation Kit (Millipore, MA, USA) was used to perform the RIP assay. In brief, the isolated mouse hearts were homogenized in complete RIP lysis buffer and then incubated with 50 μL of Protein-A/G agarose beads (Roche, USA) and 5 μg of YTHDF1 (#17479-1-AP, Proteintech, Wuhan, China) or m⁶A (#202003, Synaptic Systems, Goettingen, Germany) antibodies overnight. Subsequently, the immune complexes were stained and washed six times. The immunoprecipitated RNA was purified and then subjected to qRT-PCR analysis to detect the final products.

2.16 Proteomic analysis

The mouse heart tissue was placed in lysis buffer and then treated with a high-intensity ultrasonic processor (Scientz). After trypsin digestion and purification, a TMT kit was used for treatment in accordance with the manufacturer’s scheme. Finally, the released peptides were identified and quantified by LC–MS/MS using a Thermo Scientific LTQ Orbitrap XL with a Finnigan Nanospray II electrospray ionization source. Thermographic analysis was performed as described by PTM Biology.

2.17 Luciferase reporter assay

The plasmids containing a YTHDF1 promoter were transfected into cells. In accordance with the manufacturer’s instructions, cell lysates were subjected to luciferase assays with a Dual-Luciferase Reporter Assay System (Promega, Wisconsin, USA). The luciferase activity was determined by measuring the ratio between firefly and Renilla luciferase activities.

2.18 Chromatin immunoprecipitation (ChIP)

ChIP assays were performed using the Pierce Agarose ChIP Kit (Thermo Scientific, Carlsbad, CA, USA) following the manufacturer’s protocol. In brief, the cells were crosslinked with formaldehyde and lysed with lysis buffer. Then, the chromatin was sonicated to obtain fragmented pieces ranging from 150 to 900 bp. The resulting chromatin was incubated at 4 °C with the ZBED6 antibody or an isotype-matched control IgG overnight. Subsequently, the immune complexes were eluted using elution buffer and purified, and DNA was isolated for further analysis by qRT-PCR.

2.19 Electrophoretic mobility shift assay (EMSA)

The NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Fisher Scientific Company, Lot# 78833) was used to extract nuclear and cytoplasmic proteins. A LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific Company, Lot# 20148) was used on the basis of the protocol. The nucleus extract was hatched with binding buffer and labeled probe, poly (dI. dC), at 20 °C for 30 min. In the binding competition experiment, excessive unlabeled cold competition probes or mutant probes were added. The conjunctive complex was decomposed on a polyacrylamide gel and then crosslinked and detected by chemiluminescence. The double-stranded biotin-labeled probe sequence was as follows: 5′-GGCTAGGAGGCCAGCCCAGCAGCC-biotin-3′. The mutant probe sequence was as follows: 5′-GGCTAGTCTTAACTAAACGCAGCC-3′.

2.20 Statistical analysis

The data were statistically analyzed using GraphPad Prism 7.0. The data were expressed as the mean ± SEM. Student’s t test was used to compare two groups, while one-way ANOVA with Dunnett’s correction was used to perform multigroup analysis. A P value of < 0.05 was considered statistically significant.

3 Results

3.1 Upregulation of YTHDF1 in response to MI injury in CFs

In investigating the regulation of m⁶A readers in MI-induced cardiac fibrosis, we measured the mRNA expression levels of several known genes associated with m⁶A methylation, including YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2, in mouse hearts following MI. Our data revealed a significant upregulation of YTHDF1 expression in post-MI mice (Fig.1 and 1B, S1A). In determining the cellular localization of YTHDF1, we analyzed the human protein ATLAS database and performed qRT-PCR analysis, which both indicated the high expression of YTHDF1 in CFs (Fig. S1B and S1C). Consistent with the in vivo results, the YTHDF1 expression level was robustly elevated in TGF-β-induced CFs isolated from neonatal and adult mice (Fig.1–1E, S1D). These findings collectively indicate that the upregulation of YTHDF1 following MI may serve as a crucial molecular marker of cardiac damage, providing further evidence of m⁶A involvement in cardiac fibrosis.

3.2 Attenuation of cardiac dysfunction and pathological ventricular remodeling through YTHDF1 inhibition post-MI injury

In exploring the function of YTHDF1 in cardiac fibrosis following MI injury, we utilized AAV9 carrying shYTHDF1-V to silence endogenous YTHDF1 in mouse hearts. The successful knockdown of shYTHDF1 was first verified by qRT-PCR (Fig. S2A) and Western blot analysis (Fig.2). Then, the YTHDF1 protein expression level was measured in each group of mouse hearts, and YTHDF1 silencing reversed the increased YTHDF1 level in mice with MI (Fig.2). Subsequently, echocardiography revealed partially restored cardiac function in MI-shYTHDF1 mice compared with MI-shNC-V mice following MI surgery. This phenomenon was evident from the significantly increased percentages of EF (ejection fraction) and FS (fractional shortening) and decreased LVID;d (left ventricular internal dimension at end-diastole) and LVID;s (left ventricular internal dimension at systole) in MI-shYTHDF1 mice compared with mice with MI (Fig.2–2G). Moreover, the cardiac function of normal mice remained unaffected by YTHDF1 knockdown (Fig. S2B–S2F). Four weeks after MI, the heart fibrotic area and heart-to-body weight ratio were reduced in MI-shYTHDF1 mice (Fig.2–2J). In addition, the aberrant upregulation of mesenchymal phenotype markers, including α-SMA, Col1al, Col3a1, and CTGF, was attenuated upon YTHDF1 silencing (Fig.2–2M). Furthermore, we used AAV9 carrying a periostin promoter (Postn-shNC-V and Postn-shYTHDF1-V) targeting fibroblasts for functional verification and found that Postn-shYTHDF1-V restored impaired cardiac function, reduced HW/BW, lessened infarct size, and decreased mRNA levels of fibrotic markers in mice with MI (Fig. S3). Collectively, these results demonstrate that YTHDF1 knockdown can improve cardiac function and ameliorate cardiac fibrosis post-MI injury in vivo.

3.3 Adverse effects of YTHDF1 overexpression on cardiac function and cardiac fibrosis following MI injury

In validating the detrimental role of YTHDF1 in cardiac fibrosis in vivo, we used AAV9 carrying YTHDF1-V to overexpress YTHDF1 in animal experiments. The successful delivery of YTHDF1 was verified at the mRNA and protein levels (Fig.3, S4A), and the overexpression of YTHDF1 further increased the YTHDF1 protein expression level compared with that in mice with MI (Fig.3). Remarkably, YTHDF1 overexpression exacerbated left ventricular dysfunction in mice following MI, as evidenced by further decreases in EF and FS, along with increased LVID;d and LVID;s (Fig.3–3G). However, no significant changes were observed in normal mice upon YTHDF1 overexpression (Fig. S4B–S4F). Moreover, the heart-to-body weight ratio was significantly increased with YTHDF1-V treatment (Fig.3). Furthermore, Masson staining revealed a substantial enlargement of the fibrotic area in the hearts overexpressing YTHDF1 compared with those of the model group (Fig.3 and 3J). Consistent with these findings, an elevated fluorescence intensity of α-SMA (Fig.3 and 3L) and increased mRNA levels of fibrotic markers were observed in the injured mouse hearts following YTHDF1 overexpression (Fig.3). Collectively, these data strongly support the detrimental effect of YTHDF1 on cardiac remodeling after MI.

3.4 YTHDF1 modulates TGF-β-induced activation of CFs in vitro

For further analysis of the role of YTHDF1 in mediating TGF-β-induced activation of myofibroblasts in vitro, CFs were cultured from neonatal mice and effectively transfected with si-YTHDF1 (Fig.4 and 4B). YTHDF1 was significantly downregulated after silencing YTHDF1 in TGF-β-induced CFs (Fig.4). Notably, the knockdown of YTHDF1 attenuated the enhanced cell viability, proliferation, and migration caused by TGF-β activation in vitro (Fig.4–4H). Consistent with the in vivo observations, TGF-β stimulation resulted in a significant increase in the fluorescence intensity of α-SMA, whereas YTHDF1 knockdown notably attenuated α-SMA expression (Fig.4 and 4J). Furthermore, YTHDF1 silencing restrained the fibroblast-to-myofibroblast transition, as evidenced by decreased mRNA expression levels of fibrotic markers (Fig.4). These findings provide compelling evidence for the involvement of YTHDF1 in regulating cardiac fibrosis by impeding the aberrant proliferation and migration of CFs in vitro.

3.5 ZBED6 suppresses YTHDF1 transcription in CFs

Our findings revealed an increase in YTHDF1 mRNA and protein expression in mice with MI, prompting us to investigate potential transcription factors involved in the regulation of YTHDF1. Using the Animal Transcription Factor Database, we identified ZBED6 as a potential transcriptional repressor of YTHDF1, which can inhibit its expression (Fig. S5A). In proving this hypothesis, we tested the expression of ZBED6 in mice with MI and fibroblasts treated with TGF-β and found significant downregulation of ZBED6 mRNA and protein expression (Fig.5–5F). Subsequently, we used ZBED6-specific small-interfering RNA (si-ZBED6) to knockdown endogenous ZBED6 expression (Fig.5–5I) and increase YTHDF1 expression (Fig.5–5L). Conversely, upon confirming the overexpression efficiency of ZBED6 (Fig.5–5O), we observed that ZBED6 could inhibit the transcription of YTHDF1 (Fig.5–5R). In addition, the luciferase assay results demonstrated reduced activity in the reporter vector containing the wild-type ZBED6 sequence (Fig.5). Subsequent ChIP assays and EMSAs using designed primers confirmed the direct interaction between ZBED6 and the YTHDF1 promoter (Fig.5 and 5U). Therefore, our findings support the notion that ZBED6 negatively regulates YTHDF1 expression.

3.6 YTHDF1 promotes AXL translation and activates TGF-β-mediated Smad2/3 signaling to regulate fibrotic signaling

In elucidating the molecular mechanism underlying the role of YTHDF1 in cardiac fibrosis regulation, we conducted proteomics analysis to identify differentially expressed genes between the MI + shNC-V and MI + shYTHDF1-V groups (Fig. S6A). Among these genes, AXL was of particular interest because of its reported association with myocardial fibrosis [33]. We investigated the impact of YTHDF1 loss or gain on AXL expression in cardiac fibrosis. As observed, the increase in AXL protein expression was reversed or enhanced upon the knockdown or overexpression of YTHDF1, respectively (Fig.6 and 6B). Moreover, si-YTHDF1 led to decreased AXL protein expression (Fig.6–6E). The exogenous overexpression of YTHDF1 resulted in an immediate and notable increase in the protein expression level of AXL (Fig.6–6H). In identifying the m⁶A-methylated targets of AXL in the transcriptome, we performed MeRIP from mouse tissues. RIP analysis was used to confirm the interaction between AXL mRNA and m⁶A (Fig.6) or YTHDF1 (Fig.6). Considering the well-established function of YTHDF1 in promoting translation [14], we hypothesized that YTHDF1 might regulate the protein stability or translation efficiency of AXL. Consistent with this hypothesis, CHX treatment revealed that YTHDF1 deficiency in CFs significantly reduced the level of AXL protein, whereas YTHDF1 overexpression remarkably stabilized AXL protein levels (Fig.6 and 6L). Based on previous reports, AXL knockdown using AXL siRNA downregulates the expression level of TGF-β1 and genes involved in its signaling pathway [34]. Consistent with these findings, the downregulation or exogenous overexpression of YTHDF1 led to a decrease or further increase in TGF-β1 and phosphorylated Smad2/3 to total Smad2/3 ratio levels in vivo and in vitro (Fig.6, 6N, S6B). In brief, these data demonstrate that YTHDF1 regulates AXL expression levels by promoting translation and activating downstream TGF-β/Smad2/3 signaling to modulate the fibrotic response.

3.7 YTHDF1 targets AXL to promote myofibroblast proliferation and migration via the TGF-β-Smad2/3 signaling pathway in vitro

Based on our previous research demonstrating the involvement of YTHDF1 in cardiac fibrosis in vivo, we aimed to confirm the role of YTHDF1 in mediating the AXL-induced proliferation and migration of CFs. Thus, we constructed an adenovirus overexpressing YTHDF1 and successfully confirmed its transfection efficiency (Fig.7, S7A). We measured the YTHDF1 and AXL protein expression levels in four groups of CFs and found that YTHDF1 protein expression was stably upregulated after the overexpression of YTHDF1 in the last three groups. However, AXL silencing had no effect on the YTHDF1 protein level, but it decreased the expression level of the AXL protein (Fig. S8A and S8B). The knockdown of AXL (Fig.7, S7B) counteracted the CF activation induced by YTHDF1-AdV, as evidenced by a reversal of the trends in cell viability, proliferation, and migration (Fig.7–7G). Furthermore, in investigating whether the observed interplay between YTHDF1 and AXL in fibroblast proliferation and migration affected fibrotic signaling, we performed immunofluorescence and qRT-PCR. The results showed that the increase in fibrotic markers induced by YTHDF1-AdV could be reversed by AXL silencing (Fig.7 and 7J). Notably, our data also revealed that AXL knockdown modulated the stimulation of the YTHDF1-mediated TGF-β/Smad2/3 signaling pathway (Fig.7). Our findings further show that YTHDF1 targets AXL, enhancing the migration, proliferation, and collagen deposition in CFs, partly through the activation of the TGF-β/Smad2/3 signaling pathway.

4 Discussion

In the current research, we uncovered compelling in vivo and in vitro evidence highlighting the crucial role of the m⁶A methylation reader YTHDF1 in CFs and cardiac function under pathological conditions. These findings prompted us to use a novel molecular mechanism underlying cardiac fibrosis: MI injury → ZBED6↓ → YTHDF1↑ → AXL↑ → TGF-β↑ → phosphorylated Smad2/3↑ (Fig.8). Overall, our research highlights the intricate molecular interactions and pathways involved in cardiac fibrosis, providing valuable insights into potential therapeutic targets for mitigating cardiac dysfunction in pathological conditions.

Despite major advancements in preventive measures and treatment strategies for MI, HF results in the main causes of mortality and morbidity for cardiovascular diseases [35,36]. Cardiac fibrosis and the associated cardiac dysfunction are prominent features of cardiovascular diseases, including HF [37]. Hence, regulators involved in fibrosis following MI could substantially enhance the development of clinical treatment approaches. m⁶A methylation has emerged as a critical player in the pathogenesis and development of various heart diseases, such as HF, atherosclerosis, and cardiac remodeling [13,38]. In our research, we investigated the translational control mechanism of the m⁶A reader YTHDF1 in cardiac fibrosis (Fig.8).

Based on our findings, we provided a comprehensive demonstration for the first time that YTHDF1 plays a crucial role in the progression of cardiac fibrosis by promoting translation. Interestingly, we observed a significant increase in YTHDF1 expression level in activated CFs and mice with MI. This observation is compelling, as YTHDF1 has often been described as a transcription-promoting factor driving proliferation. Recently, an extensive study highlighted the crucial role of YTHDF1 in various cancers, where it interacts with initiation factors to enhance translation efficiency [31,37]. In our study, we provided evidence demonstrating that fibroblast-derived YTHDF1 significantly contributes to cardiac fibrosis in vivo and in vitro. The loss of YTHDF1 function following MI attenuated cardiac remodeling, resulting in improved cardiac function and reduced fibrosis compared with those of mice with MI. Conversely, gain-of-function experiments with YTHDF1 exacerbated left ventricular remodeling, leading to increased infarct size, enhanced collagen deposition, and decreased cardiac function. These results strongly indicate that YTHDF1 may serve as an important pro-proliferative gene in the heart and may participate in cardiac remodeling, providing substantial evidence for the detrimental role of YTHDF1 in heart injury.

ZBED6 plays a crucial role as a transcription factor in placental mammals, influencing growth, development, and proliferation [39–41]. Notably, ZBED6 can repress the transcription of insulin-like growth factor 2 through complex regulatory mechanisms involving multiple promoters, providing strong evidence for its essential role in muscle metabolism [42]. Although ZBED6 has been implicated in the regulation of the transcription of multiple genes, its precise functional target in myocardial fibrosis remains largely unexplored. In this study, we utilized the Animal Transcription Factor Database to predict the regulatory element of YTHDF1 and successfully identified ZBED6 as a direct inhibitor of YTHDF1 transcription. The interaction between ZBED6 and YTHDF1 was further confirmed through luciferase reporter assays and ChIP assays. These results provide compelling evidence for the pathological role of YTHDF1 in driving adverse cardiac fibrosis. ZBED6 may participate in cardiac injury by differentially regulating gene transcription, although it remains uncertain whether YTHDF1 is influenced by additional factors. Thus, further investigations are warranted to elucidate the complete regulatory network involving ZBED6 and its impact on cardiac fibrosis.

AXL is encoded by 20 exons located on chromosome 19q13.2 [43]. Previous studies have reported the participation of AXL in liver fibrosis, pulmonary fibrosis, and intestinal fibrosis [44–46]. Moreover, knocking down AXL was reported to reduce inflammation and cardiac fibrosis in Trypanosoma cruzi-infected mouse hearts, providing strong evidence for the role of AXL in myocardial fibrosis [47]. However, the potential mechanism of action of AXL in cardiac fibrosis remains unclear. In addition, previous research has indicated that YTHDF1 enhances EIF3 translation, thereby accelerating the occurrence and metastasis of ovarian cancer [28]. Furthermore, the abundant distribution of m⁶A sites in the 3′-UTR of AXL has been implicated in ovarian cancer [48]. This finding may partially explain the increased AXL expression regulated by YTHDF1 in promoting translation in cardiac fibrosis. Testosterone replacement therapy has been shown to ameliorate aging-related cardiac remodeling by enhancing Gas6 expression, but the therapeutic effect is nullified by the loss of downstream AXL, highlighting the importance of AXL as a mechanistic signal in cardiac fibrosis [33]. In our study, through cointervention of YTHDF1 with si-AXL, we demonstrated that silencing AXL alleviated the detrimental effects induced by YTHDF1, as evidenced by decreased proliferation, migration, and collagen deposition in vitro. Our findings further show that the cardiac damage mechanism of YTHDF1 is mediated by the selective promotion of AXL translation during cardiac fibrogenesis, leading to increased AXL mRNA stability and protein expression. This finding reveals the deleterious role of AXL in the progression of cardiac fibrosis. The TGF-β signaling pathway is involved in many aging-related diseases and is often associated with the occurrence of fibrosis [49–51]. AXL plays a critical role in testosterone-mediated treatment of HF and fibrosis [33]. Furthermore, the components of the TGF-β signaling pathway, including the TGF-β receptor, Smads, and AXL, are interconnected [52]. Therefore, we examined whether the canonical fibrotic signaling pathway TGF-β/Smad2/3 is involved downstream of the YTHDF1-AXL axis. Our results confirmed the role of the YTHDF1-AXL axis in the development of cardiac fibrosis following MI by activating the TGF-β/Smad2/3 signaling pathway. Furthermore, the malignant association between YTHDF1 and the fibrosis signaling pathway may serve as the pathological basis for cardiac fibrosis following MI injury and subsequent HF. However, several outstanding questions remain unanswered that await further investigation. Our results illustrate that YTHDF1 may interact with other genes involved in regulating myocardial fibrosis in addition to AXL, and whether YTHDF1 could directly target TGF-β1 or Smad2/3 remains to be further explored.

Our research has revealed the novel role of YTHDF1 in promoting pathological cardiac fibrosis following MI injury. We identified YTHDF1 as a potential biomarker that promotes myofibroblast proliferation and contributes to myocardial fibrosis. Mechanistically, YTHDF1 facilitates AXL translation and protein expression, a process regulated by ZBED6 transcription control. This activation subsequently triggers the TGF-β/Smad2/3 signaling pathway, which is involved in pathological remodeling and cardiac fibrosis post-MI injury. These findings help us elucidate the function of m⁶A in cardiac MI injury and indicate that targeting YTHDF1 may be a potential therapeutic strategy for cardiovascular diseases.

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