Methyltransferase (m6A “writers”) are protein components that catalyze m6A methylation of RNAs. m6A modification is achieved by a large methyltransferase complex (MTC). The m6A MTC mainly consists of the methyltransferase-like proteins, such as METTL3 and METTL14, and various protein factors indispensable for their proper catalysis, including WTAP, RBM15, ZC3H13, and RBM14. METTL3 is responsible for the installation of m6A on RNA. According to a phylogenetic study, METTL14 is a homolog of METTL3, with 43% similarity. The METTL3/METTL14 form a stable heterodimer core complex exhibits far more m6A methyltransferase activity than either component alone. The METTL3/METTL14 complex preferentially methylates RNA on sites of RRACH (R = A/G, H = A/U/C) in vitro, in accordance with the RRACH motif distribution of m6A transcriptome-wide (14). WTAP, as the scaffold protein of m6A-MTC, exhibits an influence on the METTL3/METTL14 compound structure and substrate composition (15). Additionally, several proteins have been shown to involved in substrate binding, catalytic efficiency, stability, and localization in addition to the core of m6A complex. RBM15, ZC3H13, HAKAI, and VIRMA, for example, have been identified as WTAP components, and this association offers a scaffold for METTL3/METTL14 recruitment for methylation (16).

Demethylases (m6A “erasers”) are enzymes that remove the methyl group from RNA. m6A is one of the few intrinsic RNA modifications that may be reversed. The identification of RNA demethylase FTO, was the crucial breakthrough that revived the m6A function. FTO catalyzes oxidative demethylation of m6A in an Fe(II)- and α-KG-dependent manner which preferentially bind to pre-mRNA strands in intronic regions, in the proximity of alternatively spliced exons and poly(A) sites (17). FTO is a member of the ALKB family. Another family member, ALKBH5, has also been discovered as a demethylase. Both ALKBH5 and FTO are confined to nucleus and located by nuclear speckles, which show the splicing effects of methylation (18). Instead of the FTO oxidative demethylation, ALKBH5 catalytic reaction directly removes the methyl group from the m6A methylated adenosine (19). ALKBH5 found as m6A mRNA demethylase employing Fe²⁺ and 2OG cofactor, which work together to remove the methyl group in m6A containing substrates.

The biological function of m6A modification is regulated by m6A-binding proteins, also known as the “readers,” including YTHDF1/2/3, YTHDC1/2, IGF2BP1/2/3, HNRNPA2B1 and eIF3. Reader proteins that act as functional mediators, selectively identity target m6A-modified mRNA and regulate varies of RNA metabolism processes such as RNA splicing, transport, translocation, translation and degradation (20). YTHDC1 is a nuclear protein involved in gene splicing. However, YTHDF1-3 are cytoplasmic m6A readers. YTHDF1 and YTHDF3 collaborate to influence the translation of m6A-containing mRNAs, whereas YTHDF2 speeds up mRNA decay and YTHDC1 influences the nuclear processing of its targets (21). Moreover, a number of recent papers claim to have discovered the presence of additional sorts of readers. IGF2BPs (IGF2BP1-3), HNRNPA2B1 and eIF3 can affect the splicing, translation, stability and degradation of mRNA (21). IGF2BPs belong to a conserved family of RNA-binding, and associate with target mRNAs in cytoplasmic ribonucleoprotein complexes, and enhance the stability and storage of their target mRNAs, therefore regulating the output of gene expression (22). HNRNPA2B1 can directly binds nuclear transcripts to induces alternative splicing effects and boosting microRNA processing (23). eIF3 plays an important role in recruitment of the pre-initiation complex PIC to mRNA. Moreover, YTHDF1 relocates from the cytoplasm to the nucleus and regulates translation may depends on interaction with eIF3 (24).

Cardiac hypertrophy is one of the predominant components of cardiac remodeling. During the early stages of stress, cardiac hypertrophy serves as a compensatory mechanism occurred in cardiomyocytes, manifested as myocytes hypertrophy, and progressive thickening of the ventricular wall. Sustained pathological hypertrophy, on the other hand, is a major cause of cardiomyocytes remodeling and HF. In addition to the transcriptional control of gene expression during hypertrophy, posttranscriptional regulation of protein expression is increasingly recognized as a vital mechanism for hypertrophic control (26) and m6A methyltransferase METTL3 is one of them (Table 1). Investigation showed that the level of m6A modification was significantly increased in isolated neonatal rat ventricular cardiomyocytes responded to hypertrophic stimulation by using m6A immunoprecipitation followed by RNA sequencing (27). In order to further investigate the possibility that m6A is involved in the regulation of cardiomyocyte homeostasis and hypertrophy in vivo, METTL3-overexpressing mice were studied and showed significant cardiac hypertrophy when exposed to pressure overload stress. However, results did not show accelerated cardiac dysfunction. METTL3 knockout mice, on the other hand, revealed indications of failure on both morphological and functional levels (27). These evidences implied that the METTL3-m6A pathway could be a novel critical regulator of cardiac homeostasis. Interestingly, opposite findings were revealed by Kmietczyk et al. (28). In compared with healthy myocardium, their investigation indicated a higher METTL3 activity and a predominance of RNA transcripts enriched in m6A in human failing heart and dilated cardiomyopathy. Additionally, overexpression and knockdown of METTL3 expression affected the cellular size and cardiomyocytes remodeling both in vitro and in vivo. Mechanically, according to Dorn et al. (27), m6A peaks were selectively abundant in mRNAs encoding protein kinases and modifiers, such as mitogen-activated protein kinases (MAPKs) family, resulting in the significant increase of cardiomyocyte size. On the other side, Kmietczyk et al. (28) suggested the mRNA methylation is highly dynamic when exposed to stress conditions, which in turn to regulate translational efficiency by modifying transcript stability. These two findings appear to be contradictory, and previous researches have revealed that the genetic background of mice may influence the phenotypic outcome of cardiac disorders post-intervention. It is possible that variances in methodologies and mice strain backgrounds which may explain some of the disparities in the data. However, this also highlights that the underlying mechanisms are highly uncertain, and further investigations would be necessary.

As for FTO, a well-known m6A demethylase protein, has been linked to cardiac hypertrophy and muscular contraction in cardiac remodeling (Table 1). Gan et al. revealed that nuclear FTO expression in cardiomyocytes was considerably increased during an adipokine induced cardiomyocyte hypertrophic response in newborn rat cardiomyocytes, leptin-induced FTO upregulation in cardiomyocytes via JAK2/STAT3-dependent CUX1 upregulation (29). The researchers also discovered that FTO knockdown reduced the hypertrophic response in vitro, indicating that FTO plays an essential regulatory function in cardiac hypertrophy.

Recently, the role of apoptosis in the heart during ischemic and non-ischemic cardiomyopathies has been explored to involved in cardiac remodeling and heart failure. Structural or functional alterations in the myocardium may be intimately associated with myocardial ischemia. Alterations in blood supply that occur over time may result in changes in cardiac tissue which may have impacts on both short- and long-term cardiovascular function and prognosis. Myocardial infarction causes widespread cardiac damage via ischemic and ischemia/reperfusion injury, resulting in a deleterious effect on cardiomyocytes and cardiac functions. Cardiomyocyte apoptosis and necrosis, is a hallmark characteristic features of myocardial infarction and cardiac remodeling. Autophagy is triggered during ischemic stress to protect cardiomyocytes from ischemic or ischemia/reperfusion injury. However, excessive autophagy activation may have a deleterious effect on the heart in reperfusion and other stress circumstances, revealing the controversial nature of autophagy in myocardial ischemia. In numerous animal models, blocking apoptosis or regulating autophagy process could effectively reduce cardiac remodeling and heart failure (30, 31).

Song et al. (32) identified that in hypoxia/reoxygenation (H/R)-treated cardiomyocytes as well as the ischemia/reperfusion (I/R)-treated mice heart, the m6A methylations were increased owing to the upregulation of METTL3. Silencing METTL3 enhanced autophagic flux and inhibited apoptosis in H/R-treated cardiomyocytes. Whereas the RNA demethylase ALKBH5 acted in a opposite manner during myocardial I/R (32). Mechanistically, METTL3 methylates the transcription factor EB (TFEB), a critical regulator of lysosomal biogenesis and also stimulated the expression of autophagy genes (33). Silencing of METTL3 promoted TFEB expression and its nuclear translocation. Furthermore, TFEB can reduce METTL3 expression by decreasing the stability of the METTL3 mRNA. TFEB knockdown eliminated the enhanced autophagy driven by METTL3 downregulation, demonstrating that METTL3 mediates autophagic flux in a TFEB-dependent manner (32). Although authors did not evaluate changes to the overall m6A levels following ALKBH5 dysregulated, they demonstrated a vital role for m6A in autophagy regulation in H/R cardiomyocytes (Table 1).

Besides, Mathiyalagan et al. (34) demonstrated the m6A modifications were increased in failing human (both ischemic and non-ischemic), post-MI pig and mouse hearts and hypoxic cardiomyocytes, resulting in a decrease in contractile performance. To identify the m6A regulators in the ischemic myocardium, the expressions of the major methylases, demethylases, and reader proteins were measured. They found FTO expression was significantly decreased in failing mammalian hearts and hypoxic cardiomyocytes. It is remarkable that loss of FTO exhibited a significantly increased number of arrhythmic events which are mediated primarily by cardiac contractile genes regulation, such as SERCA2A, MYH6/7 and RYR2, resulting in contractile dysfunction in cardiomyocytes. In vivo, FTO overexpression improved cardiac function remarkably during the chronic stage of post-myocardial infarction (34). FTO prevented cardiac contractile transcription deterioration and enhanced protein expression by selective demethylation under ischemic circumstances, thereby safeguarded cardiomyocyte contractile function by regulating intracellular Ca²⁺ dynamics (34).

Cardiac fibrosis, defined by pathological activation of cardiac fibroblasts and excessive buildup of extracellular matrix (ECM) in the afflicted tissue. Cardiac fibroblasts are activated and transform into myofibroblasts which produce a large amount of matrix metalloproteinases (MMPs), leading to accumulation and composition alteration of ECM. In the cardiac remodeling, cardiac fibrosis contributes to the dysfunction of failing hearts by making the heart stiffer and reducing its function due to its inability to contract or conduct electric impulses required for the transmission of the contraction. The activation and differentiation of cardiac fibroblasts are also influenced by m6A methylation. Increased METTL3 expression was shown in cardiac fibroblasts treated with TGF-β1 and in the murine hearts suffered from chronic myocardial infarction (35). METTL3 overexpression promoted cardiac fibrosis, fibroblast-to-myofibroblast transition, and enhanced extracellular matrix production and accumulation, whereas METTL3 silencing had the opposite effect. In vivo, silencing METLL3 significantly suppressed cardiac fibrosis progression. Smad2/3 are key regulators of the fibrogenesis process in cardiac fibroblasts, whereas silencing METTL3 reduced the upregulation of Smad2/3 induced by TGF-β1, suggesting that METTL3 regulated cardiac fibrosis at least partially through Smad-mediated pathway (35). Consistent with these results, Kmietczyk et al. (28) carefully analyzed and revealed that AAV9-mediated METTL3 overexpression following TAC operation significantly decreased fibrosis and collagen transcription in mice hearts. In addition to modifying cardiomyocyte hypertrophy, AAV9-mediated METTL3 overexpression may partially modulate the cardiac fibroblast phenotype. These evidences indicated METTL3-mediated m6A methylation played a critical role in cardiac fibrosis regulation (Table 1).

In addition, along with the positively regulating contractile protein expression, FTO-dependent demethylation was also indicated to affected critical non-contractile processes involving tissue morphogenesis, angiogenesis, extracellular matrix organization, fibrosis, and cell proliferation and differentiation in murine MI hearts (34). In FTO-overexpressing mice post-MI models, fibrosis in mice overexpressing FTO was significantly decreased compared with their respective controls, as measured by scar size (%) (34).

Vascular remodeling refers to alterations in the structure of resistance vessels contributing to elevated systemic vascular resistance. It is a dynamic structural change that involves alterations to numerous cellular including endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and fibroblasts and non-cellular components, which may lead to vascular stiffness. During vascular remodeling and heart failure, abnormalities in ECs and VSMCs proliferation and migration, endothelial dysfunction, inflammatory processes, and the production or breakdown of extracellular matrix components are present and predominant (36, 37). METTL3 and FTO have been shown to be involved in the regulation of vascular or cardiac dysfunction under stress conditions (Table 1). Under hypoxia stimulations, METTL3 can stimulate the differentiation of adipose-derived stem cells into vascular VSMCs (38). Also, evidences showed that overexpression of FTO in the ischemic myocardium could reduce cardiac fibrosis and increase angiogenesis (34). For angiogenesis, anti-CD31 immunostaining was performed by Mathiyalagan et al. (34). They measured the angiogenic response by detecting CD31-positive endothelial cells in the infarct border zone of murine hearts 4 weeks after MI, and discovered FTO-overexpressing hearts had a much higher proportion of CD31-positive cells than their respective control group (34). METTL14, as a methylase, was discovered to stimulate inflammatory response in ECs and promote atherosclerotic plaque formation by interacting with forkhead box O1 (FOXO1). Through YTHDF1 recognition, METTL14 enhanced the translation of FOXO1 mRNA, therefore increased the expression of the endothelial adhesion molecules VCAM-1 and ICAM-1, which promoting endothelial adhesion (39). In addition, researchers revealed that METTL14 mediated the m6A modification of pri-miR-19a, promoted its processing and maturation, which promoting the invasion and proliferation of cardiovascular endothelial cells (8).

Numerous investigations have revealed that long non-coding RNAs played critical regulatory roles under stressful situation: lncRNAs Mhrt, XIST, MIAT, Chast, CHRF, ROR, H19, and Plscr4 are involved in myocardial hypertrophy, whereas XIST, MALAT1, GAS5, Neat1, AK139328, APF, CAIF, AK088388, CARL, HOTAIR, and NRF are involved in cardiomyocytes apoptosis and necrosis. Moreover, MIAT, MALAT1, Wisper, MEG3, and H19 are involved in extracellular matrix reconstruction. In addition, MALAT1, GAS5, H19, TUG1, AK098656, TRPV1, Giver, and Lnc-Ang362 have been implicated in vascular remodeling (11, 40). m6A modification has been identified in long non-coding RNAs by methylated RNA immunoprecipitation sequencing (MeRIP-seq) m6A on lncRNAs accounts for ~12% of the total m6A peaks (41). The m6A methylation of lncRNAs has been demonstrated to modify their structure (42), stability (43), transport and destruction (44), hence altering the biological processes or the interaction with other RNA molecules (45–47). However, only a few of m6A modification on lncRNA have been reported in cardiac diseases. In this section, we will focus on the potential function of m6A of lncRNAs in cardiac remodeling (Table 2).

LncRNA myosin heavy chain associated RNA transcript (Mhrt) have been proven in regulating the cardiac remodeling. Mhrt is related to heart failure due to its ability to modulate cardiac hypertrophy. Mhrt increased the expression of KLF4 through direct binding to miR-145a-5p or inhibiting phosphorylation of KLF4, to prevent ERK and KLF4 interaction, hence inhibiting myocardin expression and the development of cardiac hypertrophy (48). Besides, Mhrt could protect cardiomyocytes from apoptosis against the H₂O₂ or H/R exposure (49). Shen et al. (50) observed that the expression of FTO and Mhrt were downregulated in heart failure mouse model. FTO overexpression inhibited the m6A modification of Mhrt, hence increased the expression of Mhrt, which inhibited the apoptosis of cardiomyocytes induced by the H/R. These results indicated that m6A modification of Mhrt participates in the development of cardiac disease (50).

LncRNA X-inactive specific transcript (XIST), plays a critical role in the regulation throughout the whole spectrum of human diseases and can be utilized as a novel diagnostic and prognostic biomarker for human disease (51, 52). XIST was investigated positively regulates S100B expression and enhance TLR2 expression, thereby modulates the progression of cardiomyocyte hypertrophy by miR-330-3p/S100B pathway and miR-101/TLR2 axis (53, 54). XIST was up-regulated in cardiomyocytes after infarction. Overexpression of XIST might promote cardiomyocyte apoptosis and inhibit proliferation by mediating PDE4D expression via targeting miR-130a-3p (55). In acute myocardial infarction, XIST protected hypoxia-induced cardiomyocyte injury and repressed myocardial apoptosis by interacting directly with various miRNAs and positively regulated expression of anti-apoptotic biomarkers such as Bax, hexokianse 2, and Notch1 (56, 57). Researchers have shown XIST is highly methylated with at least 78 m6A residues. m6A methylation of XIST creates a multiprotein complex with RBM15/RBM15B, WTAP, and METTL3, which in turn recruits the silencing complex, thereby promotes XIST-mediated transcriptional repression (45).

LncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is widely expressed and highly conserved in human tissues (58). MALAT1 has been reported to increase cardiomyocytes proliferation or apoptosis in rats with myocardial I/R induced injury through activating canonical signaling pathways, such as PI3K/Akt and β-catenin signaling pathways (59, 60). Also, MALAT1 was discovered to promote cardiomyocyte apoptosis of HL-1 or H9C2 cells under H/R conditions via interacting with microRNAs (61, 62). LncRNA MALAT1 profoundly induced by stimuli such as hypoxia, cytokine and oxidative stress in ECs, and it could regulate various pathophysiological processes of cardiac remodeling regarding to vascular remodeling (63, 64). For example, ox-LDL is thought to be a critical factor in the initiation and progression of endothelial dysfunction. MALAT1 induced by ox-LDL was also found to protect against endothelial injury by sponging the miR-22-3p via activation of AKT pathway (65). Recently, several m6A-deposition sites of MALAT1 have been identified, by the method which can accurately determines m6A status at any site in mRNA/lncRNA, named SCARLET. It has been verified that lncRNA MALAT1 was also highly modified by m6A and contained several m6A motifs (A2515, A2577, A2611, and A2720) (66). m6A modifications on site A2577 and A2515 of MALAT1 destabilize the RNA hairpin of MALAT1, releasing the poly-U tract and increasing the binding with HNRNPC and HNRNPG (42, 67), indicating MALAT1 could be m6A modified to regulate the development of various diseases including cardiac remodeling.

LncRNA GAS5 was found to promote myocardial I/R injury by sponging miR-532-5p in myocardial cells (68). LncRNA GAS5 was also confirmed to participate in the vascular remodeling. Wang et al. showed that lncRNA GAS5 regulated ECs and VSMCs function, GAS5 knockdown would aggravate microvascular dysfunction by altering β-catenin signaling activity, thus increased neovascularization and capillary leakage (69). The m6A modifications of GAS5 have been found. ALKBH5 inhibited m6A modification of GAS5 to its stability. Besides this, m6A promoted the degradation of GAS5 in a YTHDF2-dependent manner (70). Additionally, it was discovered that silencing m6A “reader” YTHDF3 enhances the degradation of GAS5 (44). These findings suggests that m6A-modified GAS5 may play a role in the development of cardiac remodeling.

In recent decades, lncRNA H19, a potential serum marker for coronary heart disease, is involved in the regulation of vascular remodeling. For example, in I/R condition, lncRNA H19 is reported to largely participated and promoted myocardial apoptosis (71). Researchers have investigated that H19 overexpression promoted VSMCs proliferation and inhibited its apoptosis (72). The m6A modified-H19 has been revealed involving in the development of cardiac diseases. In H₂O₂-induced senescence, lncRNA H19 expression decreased and m6A modification increased following H/R, ALKBH5 regulated the expression of H19 by mediating its m6A modification levels (73).