Fat mass and obesity-associated gene overexpression ameliorates endothelial dysfunction after myocardial infarction by mediating N6-methyladenosine modification of ATP-binding cassette subfamily F member 1

Figure 1:. The levels of m6A modification and FTO were detected in myocardial tissues from MI mice and OGD-treated HUVEC. (a) Identification of m6A-related genes was differentially expressed in GSE66360 dataset. (b) The m6A methylation quantification analysis was determined in the myocardial tissues from MI and Sham (control) mice (n = 3 for each group). (c) Western blotting determined the level of FTO in the myocardial tissues from MI mice and controls (n = 3 for each group). (d) The m6A methylation quantification analysis was determined in OGD-treated HUVEC and untreated HUVEC. (e) Western blotting determined the level of FTO in OGD-treated HUVEC and untreated HUVEC. ✶✶P < 0.01, FTO: Fat mass and obesity-associated protein, MI: Myocardial infarction, m6A: N6-methyladenosine, OGD: Oxygen–glucose deprivation, HUVEC: Human umbilical vein endothelial cell.

Figure 2:. FTO alleviated OGD induces HUVEC dysfunction. (a) The FTO level in HUVECs following transfection with OE-FTO lentivirus or OE-NC lentivirus. (b) CCK8 analyzes the viability ability of OGD-induced HUVECs following transfection with OE-FTO lentivirus or OE-NC lentivirus for 24 h. (c) Wound healing assay analysis the migration rate of OGD-induced HUVECs following transfection with OE-FTO lentivirus or OE-NC lentivirus. (d) Transwell analysis of the migration rate of OGD-induced HUVECs transfected with OE-FTO lentivirus or OE-NC lentivirus. (e) Tube formation analysis of the angiogenesis rate of OGD-induced HUVECs transfected with OE-FTO lentivirus or OE-NC lentivirus. Scale bar: 100 μm. Magnification, ×200. n = 3, ✶✶P < 0.01. FTO: Fat mass and obesity-associated protein, OGD: Oxygen–glucose deprivation, HUVEC: Human umbilical vein endothelial cell, OE-NC: The control, OE-FTO: FTO overexpression.

Figure 3:. FTO-mediated m6A modification in OGD-treated HUVEC. (a) The number of peaks in the OE-FTO and OE-NC groups. (b and c) Distribution of differentially methylated m6A peaks in the OE-FTO and OE-NC groups. (d) The sequence motif of the m6A peak regions. (e) GO enrichment analysis revealed the enriched differentially m6A methylated mRNAs. (f) KEGG enrichment analysis revealed the enriched differentially m6A methylated mRNAs. n = 3. OGD: Oxygen–glucose deprivation, HUVEC: Human umbilical vein endothelial cell, m6A: N6-methyladenosine, CDS: Coding sequences, UTR: Untranslated region, Exon: Expressed region, OE-NC: The control, OE-FTO: FTO overexpression, GO: Gene ontology, KEGG: Kyoto encyclopedia of genes and genomes.

Figure 4:. FTO mediated differential transcriptome in OGD treated HUVEC. (a) Heatmap plots for differentially expressed mRNAs. (b) Volcano plots for differentially expressed mRNAs. (c) GO enrichment analysis indicated the enriched differential mRNAs. (d) KEGG enrichment analysis indicated the enriched differential mRNAs. (e) Four-quadrant graph for m6A methylation regulated the mRNAs. (f) The ABCF1 mRNAs level was detected in OGD-treated HUVEC and untreated HUVEC. n = 3. ✶✶P < 0.01. OGD: Oxygen–glucose deprivation, HUVEC: Human umbilical vein endothelial cell, GO: Gene ontology, KEGG: Kyoto encyclopedia of genes and genomes, m6A: N6-methyladenosine, Hypo-Up: hypomethylated and upregulated, Hyper-Up: Hypermethylated and upregulated, Hypo-Down: Hypomethylated and downregulated, Hyper-Down: Hypermethylated and downregulated.

Figure 5:. FTO-mediated m6A modification of ABCF1 mRNA in OGD-treated HUVEC. (a) IGV tracks showed the m6A methylation abundance of ABCF1 in HUVECs. In the red-boxed region, a distinct m6A peak was clearly observed in the negative control (NC) group. In contrast, the height of this peak was markedly reduced in the FTO overexpression group. These observations indicate that overexpression of FTO significantly decreased the m6A methylation level of ABCF1 at this specific locus. (b) RIP-qPCR assays assessed the ABCF1 mRNA enrichment by the anti-FTO antibody. (c) MeRIP-qPCR indicated the m6A level of ABCF1 in HUVECs. (d) The mRNA stability assays showed the mRNA level of ABCF1 in HUVECs. (e) Western blotting assays indicated the protein level of ABCF1 in HUVECs. n = 3. ✶✶P < 0.01. FTO: Fat mass and obesity-associated protein, ABCF1: ATP-binding cassette subfamily F member 1, OGD: Oxygen-glucose deprivation, HUVEC: Human umbilical vein endothelial cell, RIP: RNA immunoprecipitation, MeRIP: Methylated RNA immunoprecipitation.

Figure 6:. FTO overexpression reduced myocardial pathological injury after MI in vivo. (a) H&E-stained myocardial tissues. Scale bar: 500 μm. (b) Masson-stained myocardial tissues. Scale bar: 500 μm. (c) EF and (d) FS were measured to evaluate the cardiac function of mice. (e) LDH, (f) CK-MB and (g) cTnI levels in myocardial tissues. (h) Protein levels of FTO and ABCF1 in myocardial tissues, shown by western blotting. FTO: Fat mass and obesity-associated protein, MI: Myocardial infarction, LDH: Lactate dehydrogenase, CK-MB: Creatine kinase MB isoenzyme, cTnI: Cardiac troponin I, EF: Ejection fraction, FS: Fractional shortening, H&E: Hematoxylin-eosin. n = 6 for each group. (a-d) ✶✶P < 0.01 between Sham and MI, ##P < 0.01 between MI + OE-NC and MI + OE-FTO. (e-h) ✶✶P < 0.01 between MI + OE-NC and MI + OE-FTO.