The experimental protocols were conducted following the Guide for the Care and Use of Laboratory Animals (National Research Council Committee for the Update of the Guide for the, and A. Use of Laboratory Animals, 2011) and approved by the Institutional Animal Care and Use Committee of the Medical College of Georgia at Augusta University. Two to three month-old C57BL/6 female mice (The Jackson Laboratory) and 20 to 24 month-old C57BL/6 female mice (National Institute on Aging) were used in this study. To avoid the influence of gender, female mice were used in the present study. All mice were maintained under controlled environmental conditions and allowed to acclimate for at least 2 weeks in our facilities before use.

The surgical procedure used to induce myocardial I/R in mice was performed as previously described (Chen et al., 2019). After anesthetization with the intraperitoneal injection of 100 mg/kg body weight (BW) of ketamine and 10 mg/kg BW of xylazine, endotracheal intubation was performed with a 24-gauge catheter and mice were ventilated with room air at a rate of 195 breaths/min using a Harvard Rodent Ventilator (Model 55–7058, Holliston, MA, United Kingdom). A left thoracotomy was performed at the level of the fourth intercostal space, and the heart was briefly everted from the thoracic cavity by a pericardial incision. An 8–0 nylon suture was placed under the left coronary artery and then threaded through plastic PE10 tubing to form a snare for reversible left coronary artery occlusion. The thoracic cavity, pectoralis muscles, and skin were then closed in order using 6–0 nylon suture. Ischemia was induced by occlusion of the left coronary artery for 45 min, followed by removing the tubing to induce reperfusion for 2 hrs. Sham-operated control mice were subjected to the thoracotomy procedure without the ischemia and reperfusion (n = 5 per group). The chest was closed and the mice allowed to recover. Mice were sacrificed at 2 hours after reperfusion.

Total RNA was extracted by RNAzol (Molecular Research Center, Inc., Cincinnati, OH, United States) from the homogenates of the peri-ischemic areas of the heart tissues following the manufacturer’s protocol. RNA quantity and purity were assessed with a Nanodrop 2000 spectrophotometer (Thermo Scientific).

For quantification of m6A in total RNA, we used the EpiQuik m6A RNA Methylation Quantification Kit (Colorimetric, Epigentek) following the manufacture’s instructions. Briefly, 300 ng of RNA per sample was added to the assay well and incubated with a binding solution. Capture antibody, detection antibody, and enhancer solutions were then added to assay wells separately in a suitable diluted concentration as recommended by the manufacturer’s protocol. After that, the developer solution and stop solution were added. The m6A levels were quantified using the colorimetrical analysis by reading the absorbance at 450 nm, and then calculations were performed based on corresponding control samples.

Reverse transcription was performed with 1 μg total RNA using RevertAid First Strand cDNA Synthesis kits (Thermo Scientific). The cDNA was used to perform qRT-PCR on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories) using PowerUp SYBR Green Master Mix (Thermo Fisher). Amplification was performed at 50°C for 2 min, 95°C for 2 min, followed by 50 cycles of 95°C for 15 s, and 60°C for 1 min with the primers listed in Table 1. The relative gene expression levels were normalized to β-actin and subsequently calculated using the comparative cycle threshold method (ΔΔCt).

The protein component of the heart tissue was lysed with RIPA lysis buffer (Alfa Aesar). After quantifying by BCA protein kit (Thermo Scientific), equal amounts of protein were loaded on a 10% or 12% sodium dodecyl sulfate-polyacrylamide gel and separated by electrophoresis. Then, the protein was transferred to a nitrocellulose membrane. After blocking with Odyssey blocking buffer (LI-COR Biosciences), membranes were incubated with rabbit anti-Mettl3 (1:1,000, Proteintech), rabbit anti-Mettl14 (1:1,000, ABclonal), rabbit anti-WTAP (1:1,000, Cell Signaling Technology), rabbit anti-ALKBH5 (1:1,000, NOVUS Biologicals), rabbit anti-FTO (1:1,000, NOVUS Biologicals), and mouse anti-GAPDH (1:5,000, MilliporeSigma) overnight on a rocker at 4°C. Membranes were then incubated for 1 h at room temperature with IRDye 680 goat anti-rabbit IgG and IRDye 800 Goat anti-mouse IgG (1:10,000, LI-COR Biosciences). The probed blots were scanned using an Odyssey infrared imager (LI-COR Biosciences).

Mouse HL1 cardiomyocytes (a generous gift from Dr. Claycomb) were cultured in Claycomb medium (Sigma-Aldrich) containing 10% fetal bovine serum (Thermo Fisher Scientific), 100 μg/ml penicillin/streptomycin (Thermo Fisher Scientific), 0.1 mM norepinephrine (Sigma-Aldrich) and 2 mM GlutaMAX (Thermo Fisher Scientific). Cells were plated in 6-well cell culture plates pre-coated with 0.5% (ml/ml) fibronectin and 0.2% gelatin (Sigma-Aldrich).

To investigate specific targets of Mettl3 in cardiomyocyte cell lines following simulated I/R we constructed a pLV-EF1a-Flag-Mettl3-Neo plasmid by amplifying the full-length wild type human flag-Mettl3 cDNA from pcDNA3/Flag-METTL3 (a gift from Chuan He, Addgene plasmid # 53739) by PCR using the primers: forward 5′-GGT​GTC​GTG​AGG​ATC​GCC​ACC​ATG​GAT​TAC​AAG​GAT​GAC-3′, reverse 5′-CGC​CCT​CGA​GGA​ATT​CTA​TAA​ATT​CTT​AGG​TTT​AGA​GAT​GAT​ACC​ATC​TGG​GT-3’. The amplified DNA bands were then excised and purified from gel using QIAquick PCR gel purification kit (Qiagen). Purified DNA was in-framed cloned into the BamH1 and EcoR1 sites of pLV-EF1a-IRES-Neo (a gift from Tobias Meyer, Addgene plasmid # 85139) using Infusion Cloning kit (Clontech).

Lentiviral particles were packaged in HEK293FT cells (Thermo Fisher) by co-transfecting pLV-EF1a-Flag-Mettl3-IRES-Neo or empty pLV-EF1a-IRES-Neo plasmids and helper plasmids including pMD2. G (Addgene #12259) and psPAX2 (Addgene #12260) using lipofectamine 3000 reagents (Thermo Fisher). After 48 h, the viral supernatant was collected and filtered through a 0.45 μm filter to remove cells debris. The lentiviral vectors were purified by adding polyethylene glycol 6000 (PEG6000) (final concentration 8.5%) and NaCl (final concentration 0.4 M) to the supernatant as previously reported (Jin et al., 2020). When HL1 cells reached 80% confluence, they were infected with purified empty lentivirus and Mettl3 lentivirus in complete culture medium containing 8 μg/ml of polypropylene. Three days after lentiviral transduction, G418 (500 μg/ml) was added for cell selection to get HL1 ^ctrl (HL1 with empty lentivirus infection), and HL1 ^Mettl3 (HL1 with Mettl3 lentivirus infection).

To mimic the pathological process of ischemia/reperfusion in vitro, we developed a protocol in which HL1 cardiomyocytes were subjected to oxygen-glucose deprivation (0% O₂ for 45 min in serum-free, glucose-free DMEM media using an anoxia pouch) followed by application of oxidative stress (500 µM H₂O₂ for 2 h).

RNA was extracted from HL1 ^ctrl and HL1 ^Mettl3 with/without iH/R treatment using RNAzol®RT (Molecular Research Center, Inc.). RNA was then fragmented using NEBNext® Magnesium RNA Fragmentation Module (New England Biolabs). The m6A methylRNA immunoprecipitation (MeRIP) was performed using EpiMark® N6-Methyladenosine Enrichment Kit according to instructions with modification as follows. Briefly, 2 µl N6-Methyladenosine Antibody was added to protein G magnetic beads (Thermo Fisher) and incubated at 4°C for 2 h. Following two washes in reaction buffer, the RNA was added to the antibody–bead mixture containing RNasin Plus RNase Inhibitor (Promega) and incubated at 4°C for 2 h. Next, RIP-enriched RNAs were isolated from the antibody-immobilized protein G beads using Monarch RNA cleanup binding buffer from Monarch RNA Cleanup kit (New England Biolabs). RT-qPCR was performed to evaluate the relative abundance of specific mRNA in m6A RIP complexes, as well as input samples from HL1 ^ctrl and HL1 ^Mettl3 with/without iH/R treatment. We assessed the m6A level of Bcl2, Bax and PTEN, which are recognized as key regulators of cardiomyocyte apoptosis and survival (Shukla et al., 2011). We designed RT-qPCR primers according to m6A-Atlas (www.xjtlu.edu.cn/biologicalsciences/atlas), which presents a detailed analysis of the m6A epitranscriptome (Tang et al., 2021).

GraphPad Prism vision 8.0 software was used for the statistical analysis. Results were presented as the mean ± standard error of the mean (SEM). All results were analysed by one-way ANOVA with multiple comparisons, and Tukey's multiple comparison test was applied. The two-tailed Student’s t-test was performed to compare two groups. A p-value of less than 0.05 was considered statistically significant.

We quantified and compared m6A levels in total RNA isolated from young and aged mouse hearts under physiological conditions. No significant difference in levels of m6A RNA was observed between young hearts and aged hearts (Figure 1).

To investigate whether there is a difference in the expression of enzymes that control m6A RNA modifications, we examined the expression of “writers” (methyltransferases, including Mettl3, Mettl14 and WTAP) and “erasers” (demethylases, including Alkbh5 and FTO) in young and aged hearts. The expression of Mettl3, Mettl14, WTAP, Alkbh5 and FTO showed no significant difference at the mRNA level in aged hearts compared to young hearts (Figure 2A). At the protein level, there was also no significant difference in the expression of Mettl3, Mettl14, WTAP, Alkbh5 or FTO in young and aged hearts (Figures 2B,C). Collectively, these data suggest the expression of m6A RNA methylases and demethylases was stable in hearts during aging under physiological conditions.

To test the impact of aging on m6A RNA modifications in response to acute myocardial I/R injury, we examined the m6A levels in RNA extracted from young and aged hearts at 2 h after I/R injury. In young mice, I/R injury led to a 45% decrease of m6A RNA levels in comparison with sham control, while there was only a 16% decrease detected in aged mice under these conditions (Figures 3A,B). These results suggest that acute myocardial I/R injury-induced decrease of m6A RNA level occurs in both young and aged mice. Notably, however, the magnitude of reduction of m6A RNA is less in aged hearts following acute myocardial I/R injury.

To identify the expression pattern of m6A RNA methylases and demethylases in governing the age-related differences in m6A RNA methylation post-acute myocardial I/R injury, we measured the expression of three m6A RNA methylases (Mettl3, Mettl14, and WTAP) and two demethylases (Alkbh5 and FTO). The expression of Mettl14, WTAP and Alkbh5 (mRNA or protein) did not change significantly in young versus aged hearts in response to acute cardiac I/R injury. After acute cardiac I/R injury, both young and aged hearts exhibited reduced levels of Mettl3 mRNA and protein by qRT-PCR and Western blotting (Figure 4A–F), in accordance with reduced m6A RNA levels (Figures 3A,B). In addition, we only observed significant downregulation of FTO mRNA and protein levels in aged ischemic hearts (Figures 4B,D,F), suggesting that age-related downregulation of FTO may offset the effect of reduced Mettl3 on m6A RNA levels following acute I/R injury.

We selected Bcl2, Bax and PTEN to investigate the effects of iH/R on m6A methylation of key anti-apoptotic and pro-apoptotic genes, as public m6A databases show that they have m6A peaks near the stop codon or in their 3′UTR region (Chen et al., 2020; Yang et al., 2020). As shown in Figures 5A–C, iH/R stress significantly reduced m6A methylation of PTEN compared to sham-treated cardiomyocytes while having smaller and less consistent effects on the m6A level of Bax and Bcl2 mRNAs.

Next, we tested the effects of Mettl3 overexpression on regulating m6A methylation levels of the three key anti-apoptotic and pro-apoptotic genes in the absence and presence of iH/R. As shown in Figures 5A–C, HL1 ^Mettl3 did not exhibit differences in m6A levels of Bcl2, Bax, or PTEN levels compared to control HL1 ^ctrl in sham-treated cells (i.e., in the absence of iH/R). However, iH/R led to significantly increased m6A levels of both Bax and PTEN mRNA in HL1 ^Mettl3, indicating that Mettl3-mediated m6A methylation is selectively dependent on hypoxic stress.