Abstract Background: N6-methyladenosine (m^6A) modification is one of the most common chemical modifications of eukaryotic mRNAs, which play an important role in tumors and cardiovascular disease through regulating mRNA stability, splicing and translation. However, the changes of m^6A mRNA and m^6A-related enzymes in pulmonary arterial hypertension (PAH) remain largely unexplored. Methods: MeRIP-seq was used to identify m^6A methylation in lung tissues from control and MCT-PAH rats. Western blot and immunofluorescence were used to evaluate expression of m^6A-related enzymes. Results: Compared with control group, m^6A methylation was mainly increased in lung tissues from MCT-PAH rats. The up-methylated coding genes in MCT-PAH rats were primarily enriched in processes associated with inflammation, glycolysis, ECM-receptor interaction and PDGF signal pathway, while genes with down-methylation were enriched in processes associated with TGF-β family receptor members. The expression of FTO and ALKBH5 downregulated, METTL3 and YTHDF1 increased and other methylation modification-related proteins was not significantly changed in MCT-PAH rats lung tissues. Immunofluorescence indicated that expression of FTO decreased and YTHDF1 increased in small pulmonary arteries of MCT-PAH rats. Conclusion: m^6A levels and the expression of methylation-related enzymes were altered in PAH rats, in which FTO and YTHDF1 may play a crucial role in m^6A modification. Keywords: pulmonary arterial hypertension, N6-methyladenosine, FTO, YTHDF1 INTRODUCTION Pulmonary arterial hypertension (PAH) is a progressive disease with unfavorable treatment outcomes and poor prognosis [[44]1]. The pathogenic mechanisms of PAH primarily include inflammation, immune abnormalities, oxidative stress and epigenetic changes, which cause pulmonary vasoconstriction and vascular remodeling [[45]2]. Typical pathological characteristics of PAH are intimal hyperplasia resulting in cavity stenosis or occlusion, muscular thickening in the small pulmonary arterioles, adventitial fibrosis and in situ thrombosis, which is related to abnormalities in the proliferation and apoptosis of endothelial cells and pulmonary arterial smooth muscle cells and extracellular matrix remodeling [[46]2, [47]3]. Currently available drugs aim mainly to decrease pulmonary vasoconstriction rather than reverse vascular remodeling [[48]4]. However, end-stage PAH is caused mainly by vascular remodeling [[49]5, [50]6], thus, the need to identify a new approach to treat vascular remodeling is extremely urgent. Numerous studies have indicated that epigenetic modifications, including DNA methylation, histone modifications and microRNA dysregulation, play important roles in regulating PAH [[51]7]. RNA, an intermediate in the flow of genetic information from DNA to proteins, is an important part of the central dogma of molecular biology, and its various chemical modifications mediate the regulation of many biological processes [[52]8]. N6-methyladenosine (m^6A) modification of RNA transcripts is the most prevalent modification in many classes of RNA [[53]9]. m^6A modification is a critical regulator of mRNA stability, protein expression, and several other cellular processes [[54]10]. Recently, a transcriptome-wide map of the m^6A modification of circular RNAs (circRNAs) in hypoxia-mediated pulmonary hypertension (HPH) was constructed, and the level of m^6A circRNAs was found to be decreased in HPH [[55]11]. However, changes in m^6A mRNA methylation and the expression levels of m^6A-related enzymes in PAH lung tissues remain largely unexplored. m^6A modification is one of the most abundant and prevalent internal modifications of mRNA, and like DNA methylation, it is dynamically regulated by various m^6A-related enzymes including writers, erasers and readers [[56]12]. The installation of m^6A is catalyzed by “writers”, such as the multicomponent methyltransferase complex consisting of Methyltransferase Like 3 and 14 (METTL3, METTL14) [[57]13, [58]14]. “Erasers”, including fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5), are responsible for catalyzing the removal of m^6A methylation [[59]15, [60]16]. “Readers”, such as the YT521-B homology (YTH) domain-containing protein family, which includes YTHDF (YTHDF1, YTHDF2, YTHDF3), YTHDC1, and YTHDC2, specifically recognize m^6A and regulate the splicing, localization, degradation and translation of RNA [[61]17, [62]18]. In this study, we used methylated RNA immunoprecipitation sequencing (MeRIP-seq) to establish the transcriptome-wide m^6A methylome profile of lung tissue from rats with monocrotaline (MCT)-induced PAH. Then, western blot and immunofluorescence were used to detect methylation modification-related enzymes. Moreover, we screened potential target transcripts involved in PAH. RESULTS Hemodynamic test The pulmonary artery velocity profile in the PAH group was dagger-shaped, and the pulmonary artery blood flow acceleration time (PAAT) was correspondingly reduced compared to that in the control group (19.60 ± 2.54 vs 27.88 ± 2.71 ms, p < 0.0001). In addition, the ventricular septum was significantly shifted to the left ventricle, and the right ventricle was enlarged in the PAH group compared to the control group (4.74 ± 0.69 vs 3.34 ± 0.17 mm, p < 0.0001). Compared to that in the control group, the tricuspid annular plane systolic excursion (TAPSE) was greatly reduced in the PAH group (1.36 ± 0.07 vs 1.51 ± 0.12 mm, p < 0.01) ([63]Figure 1A). Four weeks after MCT injection, the right ventricular systolic pressure (RVSP) in the PAH group was elevated (45.97 ± 4.25 mmHg vs 25.74 ± 0.73 mmHg, P < 0.0001), and the RV/ (LV + S) value was increased in the PAH group compared with the control group (0.51± 0.05 vs 0.26 ± 0.03 g, p < 0.0001) ([64]Figure 1B). Figure 1. [65]Figure 1 [66]Open in a new tab (A) Changes of echocardiography in rats after intraperitoneal injection of monocrotaline (MCT) for 4 weeks. (B) The hemodynamic test results of the two groups. Compared with the control group, the right ventricular systolic pressure was significantly increased in the PAH group, and RV/ (LV + S) also increased in the PAH group. (C) Pulmonary artery HE staining images of the control group and PAH group were obtained under microscopy. Pulmonary artery remodeling was observed in PAH group compared with control group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Hematoxylin-eosin (HE) staining The results of HE staining showed that the medial muscle of the pulmonary artery was significantly thickened (74.67 ± 3.94% vs 19.93 ± 5.63%, P < 0.0001) and that the vascular lumen was reduced in the PAH group compared with the control group. Moreover, in the control group, 67.67 ± 5.25% of the arterioles were non-muscularized (NM) vessels, and 12.33 ± 4.92% were fully muscularized (FM) vessels. In contrast, partially muscularized vessels (PM) and FM vessels showed a greater proportion (57.67 ± 6.60% and 22.33 ± 2.49%) in MCT-PAH rats, while NM vessels occupied a lower proportion (20.00 ± 5.72%) ([67]Figure 1C). The results of hemodynamic analysis and HE staining indicated that the rat model of PAH had been successfully established. Methylation profile of lung tissue from rats with PAH Lung tissues were collected from the two groups. MeRIP-seq analysis identified 922 nonoverlapping m^6A sites in the control group, 9059 nonoverlapping m^6A sites in the PAH group, and 18655 overlapping m^6A sites between the two groups ([68]Figure 2A). Nearly one-third of the m^6A sites were found exclusively in the PAH group, suggesting that methylation modification plays an important role in PAH. Figure 2. [69]Figure 2 [70]Open in a new tab Overview of N6-methyladenosine methylation within mRNAs in the control and MCT groups. (A) Venn diagram showing the overlap of m^6A peaks within mRNAs in two groups. (B) Pie charts showing the percentage of m^6A peaks in five non-overlapping segments of transcripts. Both the control group and the PAH group had the most abundant m^6A peak in the coding sequence. (C) Distributions of fold enrichment of m^6A peaks in five segments. The mean fold enrichment in the stop codon segments was the largest in the control group, while that value in 3' UTR was the largest in the PAH group. Error bars represent the standard error of the mean. (D) Proportion of genes harboring different numbers of m^6A peaks in two groups. Most genes have only one m^6A peak. The enrichment peaks were annotated to the nearest gene by bioinformatic analysis approaches, thus mapping the genome through the use of annotation information. We systematically classified these m^6A sites into five transcript regions—5’UTRs, 3’UTRs, stop codons, start codons and coding sequences (CDs)—and found that the m^6A sites were distributed mostly in CDs, stop codons, and start codons in both groups ([71]Figure 2B). Notably, the mean fold enrichment was largest in the stop codon segments in the control group but the 3’UTR regions in the PAH group ([72]Figure 2C), indicating that there are different methylation patterns between normal and disease. Meanwhile, the distribution pattern of methylated modifications was similar to previous studies [[73]19, [74]20]. Notably, 47.7% of the m^6A-modified coding genes in the control group and 36.2% of those in the PAH group contained only one m^6A peak, consistent with a single m^6A site or a cluster of adjacent m^6A residues. The next highest percentage contained two m^6A peaks, while a relatively small percentage contained three or more peaks ([75]Figure 2D), which is agree with the trend of the proportions previously reported for the pig liver [[76]21] and mouse heart [[77]22]. Next, differentially methylated m^6A sites (DMMSs) between the groups were identified by diffReps with the following default screening criteria: an FDR ≤ 0.0001 and a fold change ≥ 2. We selected 3298 DMMSs between the two groups. A total of 777 m^6A sites exhibited decreased methylation, and 2521 exhibited increased methylation. On average, 23.6% and 76.4% of the m^6A sites exhibited prominently decreased and increased methylation, respectively, in PAH lung tissues relative to control lung tissues ([78]Table 1). [79]Tables 2, [80]3 show the top ten genes with increased and decreased methylation. Table 1. Total numbers of differentially methylated N6-methyladenosine peaks and associated gene. Item Hypermethylated peak Hypermethylated gene Hypomethylated peak Hypomethylated gene mRNA 2521 1261 777 568 [81]Open in a new tab Table 2. Top ten up-regulated genes. chrom txStart txEnd GeneName Foldchange chr10 14521318 14521596 Tpsab1 1643.7 chr2 124777380 124777456 Cpa3 852.7 chr6 39260307 39260379 LOC257642 528.3304473 chr15 38492079 38492215 Cma1 523.6 chr1 235026074 235026209 Ms4a2 454.3 chr10 14519180 14519530 Tpsab1 422.8 chr15 38920068 38920123 Mcpt2 399.3 chr6 147108214 147108505 AABR06046430.3 375.8 chr6 148133714 148134006 Ighg 324.7 chr15 38499889 38500287 Mcpt1l1 317.4916885 [82]Open in a new tab Table 3. Top ten down-regulated genes. chrom txStart txEnd GeneName Foldchange chr13 94156781 94157040 Mpz 144.4 chr15 37540300 37540399 Myh7 127.2 chr8 118499941 118500097 Als2cl 111.7 chr1 32944021 32944397 Zfp72 110.5 chr2 230629248 230629515 Mybphl 93.6 chr17 67605795 67605900 Ryr2 89.8 chr17 17414465 17414480 Iars 88.2 chr2 227847959 227848030 Wdr77 87.9 chr5 163485561 16485644 Arhgef19 83.9 chr2 228004667 228004738 Wdr77 82.9 [83]Open in a new tab To assess their distribution profiles, all DMMSs within mRNAs were mapped to chromosomes ([84]Figure 3A). The five chromosomes harboring the most DMMSs were chromosomes 1, 2, 4, 3 and 5. However, when the number of DMMSs harbored by a chromosome was normalized by the length of that chromosome, chromosomes 10, 20, 3, 13 and 16 were the five chromosomes with the highest relative densities of DMMSs ([85]Figure 3B). We further investigated the regions with DMMSs in mRNA and found that most were located in CDs ([86]Figure 3C). Moreover, most m^6A sites in the stop codons with upregulated methylation showed the highest fold change, however, most m^6A sites in the 5’UTRs with downregulated methylation showed the highest fold change ([87]Figure 3D). [88]Figure 3B, [89]3D suggesting the preferences of methylation