Ethical Considerations
All mice used in the study were housed at Zhongshan Hospital Fudan University. The animal experiments were conducted in accordance with the practices defined in the Guide for the Care and Use of Laboratory Animals which were approved and overseen by Zhongshan Hospital Fudan University institutional animal care and use committee.
Patients and samples
Human blood samples were collected in Zhongshan Hospital Fudan University. Selection criteria for observational groups (n=390) and control groups (n=390) were in accordance with the standard procedures as shown in Supplementary Table 11. Written informed consent was obtained from all subjects, and the study protocol was approved by the Clinical Research Ethics Committee of the Zhongshan Hospital Fudan University.
Cardiac-specific Mettl3 knockout mouse model
Mouse Genome Informatics (MGI) showed that homozygous mice with Mettl3 gene-knockout exhibited embryonic lethality between E3.5 and E8.5. Therefore, we used CRISPER/Cas9 technology to construct CKO mice2. The general process was divided into in vitro and in vivo stages. The in vitro stage included the following: 1) sgRNA design and construction; 2) donor carrier design and construction. The in vivo stage included the following: 1) microinjection and transplantation of fertilized eggs; 2) birth and identification of F0 generation mice; 3) breeding positive F0 generation mice; 4) birth and identification of F1 generation mice. Given that the METTL3 gene has 15 transcripts, based on the structure of the METTL3 gene, the exon 2–4 (ENSMUST0000022767.15) transcript of METTL3-201, which contains a 799-bp coding sequence, was identified as a knockout region. Knocking out this region should result in the interruption of protein function. During the construction process, we used CRISPR/CAS9 technology to modify the METTL3 gene. First, in vitro transcription of sgRNA was performed to construct a donor vector. Trace amounts of Cas9, sgRNA, and donors were injected into the fertilized eggs of C57BL/6JGpt mice. Positive F0 generation mice were obtained by fertilized egg transfer and verified through PCR and sequencing. By mating F0 generation positive mice with C57BL/6JGpt mice, stable F1 generation flox mice were obtained, followed by mating flox mice with CRE-ERT2 mice. Finally, flox/flox CRE-ERT2 mice and control flox/flox were obtained, and induced with tamoxifen to obtain conditional knockout (CKO) mice. Cardiomyocyte-specific inducible Mettl3 knockout mice (Mettl3CKO) were obtained from GemPharmatechTM (Nanjing, China). All experimental procedures were approved by the Animal Ethics Committee of Zhongshan Hospital Fudan University.
Reagents
Puromycin was bought from Merck/Millipore (Darmstadt, Germany). The DNA transfection reagent PolyJet was purchased from Signagen Laboratories (Rockville, USA). Actinomycin D was purchased from Sigma-Aldrich (City of Saint Louis, USA). The dual luciferase reporter assay system was purchased from Promega (Madison, USA). ClonExpress II One Step Cloning Kit was purchased from Vazyme (Nanjing, China).
Antibodies
The mouse monoclonal antibody against Flag (F3165, 1:5,000) was purchased from Sigma-Aldrich (City of Saint Louis, USA). The mouse monoclonal antibody against SPP1 (22952-1-AP, 1:2,000), FOS (66590-1-lg, 1:500), β-actin (20536-1-AP, 1:10,000), His tag (66005-1, 1:3,000) were obtained from Protein Tech (Rosemont, USA). The mouse monoclonal antibody against Cleaved-caspase3 (#9664, 1:1,000), rabbit monoclonal antibodies against haemagglutinin (HA) (3724, 1:2,000) were obtained from Cell Signaling Technology (Danvers, MA, USA). The rabbit polyclonal antibodies against METTL3 (ab195352, 1:1,000), HuR (ab196495, 1:1,000), Bcl2 (ab196495; 1:1,000), Bax (ab32503, 1:2,000), γ-H2AX (ab81299, 1:5,000), Caspase3 (ab13847,1:500), the goat anti-rabbit antibody (ab6721, 1:10,000), anti-mouse (ab6789, 1:10,000) were purchased from Abcam (Cambridge, CB2 0AX, UK).
Plasmids
SPP1, FOS, METTL3, and HuR were PCR-amplified and cloned into pCDH-Flag, pCDH-HA, pCDH, and pCMV-Flag vectors. The ORF and 3′ UTR of SPP1 and FOS were cloned into pCDH-SFB. 6×His-METTL3 was constructed by cloning METTL3 cDNAs into pET-28a (+) vector. Various mutations of SPP1 (ΔARE1, ΔARE2, ΔARE3), and FOS (ΔARE1, ΔARE2, ΔARE3, ΔARE4, and ΔARE5), SPP1 point mutation (A18T), and FOS point mutation(A786T) were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) in accordance with the manufacturer’s instructions. Primer sequences are described in Supplementary Table 2.
Cell culture and transfection
H9c2, AC16, 293T, and HL1 cells were obtained from the cell library of the Chinese Academy of Sciences and maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). All cell lines tested negative for mycoplasma. NRVMs were maintained in DMEM-F12 with 10% FBS. The cells were seeded in 35-mm, 60-mm or 100-mm plates and transfected with the indicated plasmids using PolyJet in accordance with the manufacturer’s instructions.
Mouse model of MI
After undergoing 2% isoflurane anesthesia, 8-week-old male C57BL/6 mice were selected. The skin was cut longitudinally through the chest and carefully separated from the chest wall, pectoralis major, and pectoralis minor muscles. Mosquito-like hemostatic forceps were used to expand the chest cavity between the pectoralis major and pectoralis minor muscles in the fourth intercostal space, gently exposing the location of the heart. The pleura and the pericardium were separated layer by layer. Then, 6 − 0 silk thread was used to ligate the anterior descending branch 2 mm below the left atrial appendage, and the heart was returned to its original position, Finally, we used 4 − 0 silk thread to suture the skin, and monitored the electrocardiogram of small animals to determine whether the mouse AMI model has been successfully established.
Mettl3-overexpression mouse model
METTL3 overexpression was achieved by in situ injection of AAV9 into the hearts of C57BL/6 mice, with a viral titer of 1.8×1012 after 3 w, stable overexpression of Mettl3 can be achieved. Overexpression AAV9 was purchased from Hanbio (Zhejiang, China), and the constructed vector was HBAAV2/9-CMV-r-METTL3-3xflag Null.
STM2457 treatment
STM2457 (MedChemExpress, Monmouth Junction, NJ, USA) was dissolved in a 20% 2-hydroxypropyl-beta-cyclodextrin (Glpbio, Montclair, CA, USA) vehicle as the working solution with a concentration of 5 mg/mL. Then 50 mg/kg of STM2457 or the respective vehicle was delivered to the mice via intraperitoneal (i.p.) injection once for a total of 14 days.
Subcellular fractionation analyses
H9c2 cells were collected and washed three times with cold Phosphate buffered saline (PBS). Nuclear or cytosolic fractions were prepared using the Nuclear Extract Kit (Active Motif). Subcellular fractionation analyses were constructed in line with standard procedures.
Generation of cell line
Mettl3- and HuR-knockout H9c2 cell lines were established using CRISPR/Cas9 as previously described. The guide RNAs (gRNAs) targeting the METTL3 or HuR gene were designed using the CRISPR design platform (https://zlab.bio/guide-design-resources), and the dual gRNA knockout method was used. The gRNA was cloned into the pENTR-spCAS9-T2A-EGFP vector, and CRISPR/Cas-9 and gRNA plasmids were cotransfected into H9c2 cells using X-tremeGENE 9 DNA Transfection Reagent (Roche, 6365787001). After transfection, GFP + cells were sorted by FACSAria II cell sorter (BD Biosciences) and seeded on feeder cells. Single H9c2 clones were picked and genotyped by PCR.
Lentivirus production and infection
Lentiviral packaging and infection were performed following previously established protocols49. In brief, HEK293T cells were cotransfected with pLKO.l or pLEX constructs along with the packaging plasmids psPAX2 and pMD2.G. After 5 h, the media was replaced with fresh DMEM containing 10% FBS. The virus-containing medium was collected 48 h later and filtered using a 0.45-um membrane (Merck Millipore). To infect the target cells, the virus-containing medium was supplemented with Polybrene (10 µg/ml). Infected cells were selected with puromycin for 48 h before being harvested or used in subsequent experiments.
RNA isolation, cDNA synthesis, and RT-PCR
Total RNAs were extracted from the cells or tissues using UNIQ-10 Column Trizol RNA Isolation Kit (Sangon Biotech, B511321-0100) and purified with the RNeasy mini kit (Qiagen, 74106). Total RNAs were reverse transcribed into complementary DNA using the Hifair® AdvanceFast One-step RT-gDNA Digestion SuperMix Kit (Yeasen Biotechnology, 11151ES60). RT–qPCR was performed on a QuantStudio 6 Flex system (Applied Biosystems) using Hieff® qPCR SYBR Green Master Mix (Yeasen Biotechnology, 11203ES08). Results were analyzed using the 2-ΔΔCt method and normalized to the expression of ACTB gene. Primer sequences are described in Supplementary Table 3.
Bioinformatic analysis
First, an m6A library was constructed. In brief, after total RNA extraction, Oligo-dT magnetic beads were used to enrich mRNA with polyA tails. We enriched the corresponding magnetic beads, and added fragment reagents dropwise to the EP tube to fragment the complete mRNA. Subsequently, the fragmented mRNA was divided into two groups and placed in different EP tubes. In one group, m6A antibody beads were added to enrich m6A methylated mRNA. After the corresponding mRNA was recovered, a sequencing library called m6A seq library (IP) was constructed. The other group was set as the control group, and the constructed library was a conventional transcriptome sequencing library, namely the RNA seq library (input). Illumina NovaseqTM 6000 as the platform was used, and 150 PE was corresponding mode. Second, the analysis process was operated. Briefly, we eliminated the sequencer connector sequence and low-quality or contaminated sequences, and the remaining sequences were CleanData. Then, we used FastQC software for quality control analysis. The default parameters of HISATA were used to match reads to the reference genome (Rattus norvegicus, genome version: Rnor_6.0). Subsequently, exomePeak and CHIP seeker were used for corresponding bioinformatics analysis (such as Peak calling analysis and Peak annotation). Finally, MEME and HOMER were used to analyze the corresponding motifs.
Flow cytometry assay
Harvested H9c2 cells were washed with PBS and incubated at 4°C for 45 min away from light. We then added 0.5 mL of prepared propidium iodide staining solution to each cell sample, gently mixed and resuspended the cells. All samples were incubated in dark at 37°C for 30 min, and then examined on BD AccuriTM C6 Plus flow cytometer for detection at the excitation wavelength of 488 nm. Finally, all data were analyzed in FlowJo 10.6.2 (Ashland, OR, USA).
Immunofluorescence staining
Cells were fixed and incubated with primary antibodies, Alexa Fluor dye-conjugated secondary antibodies and DAPI according to standard protocols. Cell imaging was performed on a Leica TCS SP8 WLL confocal laser scanning microscope (Leica).
TUNEL staining
TUNEL staining was performed following the manufacturer’s protocol of In Situ Cell Death Detection Kit (TMR red) (Roche, 12156792910). In brief, after immunofluorescent staining with anti-cTnT antibody (1:200, Thermo Fisher Scientific, MS-295-P), heart tissue sections or cell samples were permeabilized with permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice and washed with PBS twice. The samples were then stained with TUNEL reaction mixture at 37°C for 1 h. After washing with PBS three times for 5 min each, the samples were mounted with DAPI Fluoromount-G medium (SouthernBiotech, 0100 − 20) for imaging.
Immunoprecipitation
First, plasmid transfection was performed, and the protease inhibitor MG132 (10 µM) was processed for 12 h. Then we gently scraped off the cells, transferred them to a new EP tube, and incubated them at 4°C for 30 min; Subsequently, centrifuged them at 4°C and 13200 rpm for 10 min, transferred the supernatant with a pipette to another new EP tube, and left 50% of the supernatant protein in each centrifuge tube as whole cell lysate (WCL). The remaining protein sample was incubated with HA-beads or FLAG-beads at 4°C for 3 h. Then we placed the sample in a centrifuge at 300 g for 1 minute to remove buffer, and finally added 30 µL Protein loading. Finally, western blot was used to detect the expression level of the target protein.
Quantitative analysis of m6A by LC-MS/MS
A total of 100 ng non-ribosomal RNA, polyadenylated RNA or RIP RNA was digested by 1 U nuclease P1 (Sigma) in 30 µL of buffer containing 20 mM NH4OAc for 2 h at 42°C. Subsequently, 1× FastAP Buffer and 1 U FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific) were added, and the sample was incubated at 37°C for an additional 2 h. The samples were then centrifuged through filters (0.22 µm pore size, 4 mm diameter, Millipore) and injected into a reverse-phase ultraperformance liquid chromatography (C18 column) coupled to Triple Quad 6500 System (AB SCIEX). Nucleosides were detected in positive electrospray ionization mode. The quantification of nucleosides was based on the nucleoside-to-base ion mass transitions of 282 to 150 for m6A, and 268 to 136 for A by using standard curves generated in the same batch of samples with pure nucleosides. The ratio of m6A to A was calculated on the basis of the calibrated concentrations.
Methylated RNA immunoprecipitation sequencing (MeRIP-seq)
After precipitation and concentration determination, RNA was divided into approximately 200-nt fragments by incubation at 70°C for 7 min in a water bath. Then one-tenth of the aforementioned RNA fragments was saved as the input control for RNA-seq, and the rest was incubated with m6A (D9D9W) Rabbit mAb (Cell Signaling Technology) at 4°C for 2 h and then mixed with DynabeadsTM Protein A for Immunoprecipitation (Thermo Fisher Scientific) at 4°C for 2 h to obtain methylated RNA complex. The m6A antibody was digested with Proteinase K digestion buffer. Finally, the methylated RNA was purified for MeRIP-seq by CLOUDSEQ (Shanghai, China).
mRNA decay assay
The mRNA decay assay was performed as previously described50. In brief, cells were pretreated with actinomycin D at the indicated times. Total RNA was extracted with an RNA high-purity total RNA rapid extraction kit (Qiagen). cDNA was prepared using a GoScript reverse transcription system (Promega). Real-time PCR (RT-PCR) analysis was performed using 2× SYBR RT-PCR premixture (Promega) with the following conditions: 5 min at 95°C followed by 40 cycles at 95°C for 30 s, 60°C for 40 s and 72°C for 1 min using an ABI 7500 fast system. Data were normalized to expression of a control gene β-actin for each experiment.
Luciferase reporter assay
H9c2 cells were transfected with a pGL2-SPP1-promoter reporter or pGL2-FOS-promoter reporter. Luciferase activities were measured using a dual luciferase reporter assay kit in accordance with the manufacturer’s instructions.
RNA immunoprecipitation
The RNA immunoprecipitation assay was performed as previously described51. In brief, H9c2 cells were lysed and immunoprecipitated using anti-HuR antibody with normal mouse IgG as a negative control. The mRNA precipitated by anti-HuR or normal IgG was converted to cDNA and analyzed by RT-PCR analyses. The amounts of the precipitated mRNAs were normalized to the input RNA fractions to eliminate possible differences in RNA sample preparation.
Biotin-ARE1 pull-down assays
Pull-down assays were carried out by incubating biotin–ARE1 (0.1 µg; 5’-GCAAUUUAAGCAAUUUAAGCAAUUUAA-3’) with the cell lysates of H9c2 cells or with purified recombinant His–RRM1/2 (2 µg) for 4 h. Complexes were precipitated using Pierce streptavidin agarose beads, followed by immunoblotting analyses with the indicated antibodies.
Echocardiography
The mice were anaesthetized with 2% isoflurane, and their chest hairs were shaved. The mice were placed on a heated platform. Pre-heated ultrasound coupling gel was applied to the chest area, and a linear array transducer (18–23 MHz) was positioned to obtain one-dimensional M-mode images or two-dimensional B-mode parasternal long- and short-axis views on a VisualSonics Vevo 2100 Imaging System. FS, EF, LVIDd, LVIDs were calculated from the M-mode measurements using Vevo LAB ultrasound analysis software (FujiFilm, VisualSonics, Inc.).
PET/CT imaging
The mice were fasted for 8 h before injection of 200 µCi 18F-FDG via tail vein. During the uptake period (40–60 min), all of the mice were anesthetized under 1.5% isoflurane. Then the data of PET imaging were recorded, followed by 10-min CT-scan, using a microPET/CT scanner (Inveon; Siemens, Germany). All data were reconstructed using the microQ viewer software (Version 1.7.0.6; Siemens, Germany). Finally, region of interest (ROI) delineating the heart was drawn, and the mean standard uptake value (SUVmean) of the heart was obtained for 18F-FDG uptake.
Hematoxylin and eosin staining and Masson staining
The whole heart specimens from mouse models were paraffin-embedded and sliced into tissue sections with a thickness of 3–4 µm. After dewaxing, all sections were stained with hematoxylin dye (Servicebio) for 5 min and differentiated with 1% hydrochloric acid alcohol, and cytoplasmic staining was performed by immersing sections in eosin dye (Servicebio) for 3 min. Finally, the sections were sealed with gum, digitally scanned, and analyzed after dehydration and drying. Masson’s trichrome staining was performed in accordance with the standard procedures.
TTC staining
After 1 day of myocardial ischemia, the heart was quickly removed and frozen for 10 min. Four circular slices of myocardial tissue were obtained with a thickness of approximately 1 mm by conducting a transverse section of the heart. The cardiac slices were immersed in a 1% 2,3,5-triphenylte-trazolium chloride (TTC; MilliporeSigma, Burlington, MA, USA)-phosphate buffer saline (PBS) solution at 37°C for 15 min. The length of the entire left ventricular circumference and segment of the left ventricular circumference of the infarct area were traced manually in the digital images and measured using ImageJ software (National Institutes of Health). Finally, the infarct size was expressed as a percentage of the entire left-ventricular circumference.
Statistical Analysis
All experiments were repeated as indicated; n indicates the number of independent biological repeats. Data are expressed as mean ± standard error of the mean (SEM). GraphPad PRISM9 software was applied for statistical analysis and data presentation. Statistical significance was determined using unpaired two-tailed Student’s t-test, 1-way analysis of variance (ANOVA) with Tukey test, or 2-way ANOVA with Tukey multiple comparisons test. Data distribution was assumed to be normal, but was nor formally tested. In cases where statistics was derived, sample size was n = 3 or more biological replicates. Unless stated otherwise, the experiments were not randomized. Data collection and analysis were not performed blind to the conditions of the experiments.