Epigenetic role of N ⁶-methyladenosine (m⁶A) RNA methylation in the cardiovascular system

Abstract

As the most prevalent and abundant transcriptional modification in the eukaryotic genome, the continuous and dynamic regulation of N⁶-methyladenosine (m⁶A) has been shown to play a vital role in physiological and pathological processes of cardiovascular diseases (CVDs), such as ischemic heart failure (HF), myocardial hypertrophy, myocardial infarction (MI), and cardiomyogenesis. Regulation is achieved by modulating the expression of m⁶A enzymes and their downstream cardiac genes. In addition, this process has a major impact on different aspects of internal biological metabolism and several other external environmental effects associated with the development of CVDs. However, the exact molecular mechanism of m⁶A epigenetic regulation has not been fully elucidated. In this review, we outline recent advances and discuss potential therapeutic strategies for managing m⁶A in relation to several common CVD-related metabolic disorders and external environmental factors. Note that an appropriate understanding of the biological function of m⁶A in the cardiovascular system will pave the way towards exploring the mechanisms responsible for the development of other CVDs and their associated symptoms. Finally, it can provide new insights for the development of novel therapeutic agents for use in clinical practice.

1. Introduction

Epigenetics has attracted considerable attention in the biomedical field. Accumulating evidence has shown that epigenetic alterations can affect a variety of common pathological reactions, including ischemia, inflammation, aging, and tumorigenesis (He et al., 2013). Epigenetics can also have a vital impact on gene expression and function without altering the base sequence of DNA. Moreover, these effects are reversible, heritable and are influenced by the external environment (Gibney and Nolan, 2010; Mazzio and Soliman, 2012).

Current epigenetic mechanisms include mainly DNA methylation, post-translational histone modification, chromatin remodeling, and deployment of non-coding RNA. Recent advances in the field of epigenetics can aid our understanding of a series of complicated biological processes associated with aging (Rodriguez-Rodero et al., 2010), development (Smith et al., 2012), inflammation (Bayarsaihan, 2011), immunity (Fernández-Morera et al., 2010), stem cell biology (Calvanese and Fraga, 2012), and angiogenesis (Buysschaert et al., 2008).

The role of reversible RNA methylation has just begun to attract worldwide scientific attention compared with the processes of DNA methylation and histone modification which are now well-established in genetics and cell biology (He, 2010; Panneerdoss et al., 2018). As an important part of developing RNA epigenetics, the modification of N ⁶-methyladenosine (m⁶A) has recently been explored in the cardiovascular field (Liu and Pan, 2015). It has been shown that the m⁶A modification is the most universal and abundant internal modification of eukaryotic messenger RNA (mRNA) and long non-coding RNA (lncRNA) and occurs mainly in the 3'-untranslated regions (3'-UTRs) and nearby the stop codons of the mRNA molecules (Parashar et al., 2018; Gan et al., 2019). Furthermore, several independent lines of evidence have revealed that the continuous and dynamic regulation of m⁶A may have a far-reaching impact on modulating the expression of specific genes (Niu et al., 2013). In this review, we outline recent advances and discuss potential therapeutic strategies for managing m⁶A in relation to several common cardiovascular diseases (CVDs), related metabolic disorders, and external environmental factors.

2. Biological function of m⁶A modification

2.1. Methyltransferase and demethylases of m⁶A

It has been shown that m⁶A methylation is catalyzed by the multicomponent RNA methyltransferase complex, RNA demethylases, and m⁶A readers (Liu et al., 2014). The core components of the RNA methyltransferase complex include methyltransferase-like 3/14 (METTL3/14) and Wilms tumor 1 associated protein (WTAP) (Liu et al., 2014). Among them, the spliceosome-associated protein WTAP has recently been found to play its role by recruiting RNA METTL3/14 to sites of methylation in the nucleus (Liu et al., 2014). Although the methyltransferase complex of m⁶A methylation has not been fully elucidated, remarkable progress including the discovery of ALKB homolog 5 (ALKBH5) and fat mass-and obesity-related proteins (FTOs) has recently been made. The so-called erasers of specific markers operate on target transcripts via an α-ketoglutarate-and Fe²⁺-dependent way in the m⁶A and N ⁶,2'-O-dimethyladenosine (m⁶A_m) of mammalian poly-adenylated RNA (Gerken et al., 2007; Niu et al., 2013; Mauer et al., 2017). In addition to the methyltransferase complex and demethylase enzyme, m⁶A is also regulated by the activity of m⁶A readers, such as YT521-B homology (YTH) m⁶A RNA-binding proteins (YTHDF), which can regulate the stability and degradation of RNAs and affect the efficiency of target mRNA translation (Wang and He, 2014; Kennedy et al., 2016). Therefore, m⁶A regulates its effects mainly by recruiting a variety of proteins containing the YTH domains and via its assembly by methyltransferases that are located in relative consensus motifs following transcription (Wei and Moss, 1977; Patil et al., 2018). The m⁶A modification may not only affect the secondary structure of mRNA and the interactions between mRNA-protein and mRNA-small nuclear RNA (snRNA), but may also be involved in modulating translatability, RNA transport and splicing, and susceptibility to post-transcriptional silencing (Zhong et al., 2008).

2.2. m⁶A regulation in the eukaryotic genome

It is believed that m⁶A methylation plays an important role in the regulation of gene expression by affecting the stability, degradation, and translation of RNA (Fu et al., 2014; Meyer et al., 2015). Several studies have confirmed that alteration of m⁶A levels may have a significant impact on various biological processes in mammals, such as the maintenance and differentiation of embryonic stem cells (ESCs), transcriptional splicing, nuclear RNA export, protein translation control, cell fate determination, circadian rhythm modification, heat shock response, meiotic progression, and neuronal function (Guo et al., 2017; Roignant and Soller, 2017; Wei et al., 2017). Moreover, a lack of m⁶A modification can lead to the development of several diseases, such as obesity, cancer, type 2 diabetes mellitus (T2DM), infertility, and developmental arrest (Wei et al., 2017).

Therefore, attention has been focused on the role and importance of m⁶A regulation in the eukaryotic genome in relation to physiological and pathological processes. The m⁶A profiles of full transcriptomes have shown that m⁶A modifications occur in countless RNA transcripts with unique patterns of distribution. Although the role of m⁶A editors in the processing, synthesis, and degradation of mRNAs and proteins has been discovered (Patil et al., 2018), the functions and mechanisms of most RNA modifications found in mRNA molecules remain poorly understood, especially with regard to the development of CVDs.

3. m⁶A RNA modification in CVDs

Due to improvements in the standard of living and medical care, CVD mortality has declined markedly in certain high-income countries of Western Europe and North America (Libby et al., 2013). However, CVD remains the leading cause of death worldwide in both developed and developing countries (Iyen et al., 2019). Accumulating evidence has shown the contribution of several important but underappreciated issues, such as air pollution, to the genesis and development of CVDs (Cosselman et al., 2015; Peña and Rollins, 2017). Epigenetics plays an effective and leading regulatory role in modulating cardiovascular repair functions (Jin et al., 2019). Therefore, new concepts and therapeutic measures must be developed for myocardial repair and regeneration to improve cardiac function. However, although certain transcription factors and co-activators have been studied in heart failure (HF), post-transcriptional modifications that mediate specific mRNAs affecting the main protein expression and cardiac function have not been explored in detail.

As a key enzyme of m⁶A, FTO is widely expressed in cardiac ventricular tissues, with a high level of expression in humans (Boissel et al., 2009). Therefore, particular attention must be paid to the possible associations between m⁶A and the physiological and pathological processes of different CVDs. Emerging evidence has identified epigenetic regulation of m⁶A and suggested possible therapeutic strategies for treating the development of cardiac defects, including ventricular septal and atrio-ventricular defects (Boissel et al., 2009), arrhythmias (Carnevali et al., 2014), coronary heart disease (Gustavsson et al., 2014), and cardiomyocyte hypertrophy (Dorn et al., 2019) (Fig. 1).

Fig. 1.

Molecular roles of N ⁶-methyladenosine (m⁶A) enzymes in different cardiovascular diseases, related metabolic disorders, and external environmental factors

m⁶A methylation is catalyzed by the multicomponent RNA methyltransferase complex (methyltransferase-like 3/14 (METTL3/14), Wilms tumor 1 associated protein (WTAP), and KIAA1429), RNA demethylases (ALKB homolog 5 (ALKBH5) and fat mass-and obesity-related protein (FTO)), and m⁶A readers (YT521-B homology (YTH) domain family 1/2/3 (YTHDF1/2/3), YTH domain containing 1 (YTHDC1), heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1), and E74-like factor 3 (elF3)). Modification of m⁶A may not only affect the secondary structure of messenger RNA (mRNA) and the interactions between mRNA-protein and mRNA-small nuclear RNA (snRNA), but may also be involved in modulating translatability, RNA transport and splicing, and susceptibility to post-transcriptional silencing. Furthermore, emerging evidence has identified the epigenetic regulation of m⁶A and suggested possible therapeutic strategies for treating the development of cardiac defects and internal metabolic disorders, including heart failure, myocardial infarction, cardiomyocyte hypertrophy, obesity, and diabetics mellitus. TFEB: transcription factor EB; MAP3K6: mitogen-activated protein kinase (MAPK) kinase kinase 6; MAP4K5: MAPK kinase kinase kinase 5; Nppa: natriuretic peptide precursor (Npp)-A; MyD88S: splice variant of myeloid differentiation factor 88; SERCA2a: sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 2a; MYH7: β-myosin heavy chain 7; Ryr2: ryanodine receptor 2; Angptl4: angiopoietin-like 4; Per2: period 2; Arntl: aryl hydrocarbon receptor nuclear translocator like; FOXO1: forkhead box O1; G6PC: glucose-6-phosphatase (G6Pase) catalytic subunit; DGAT2: diacylglycerol acyltransferase 2; RBW15: RNA-binding motif protein 15; HuR: human antigen R; hnRN: heterogeneous nuclear ribonucleoprotein; CCR4-NOT: carbon catabolite repressor protein 4-negative on TATA

3.1. Ischemic heart diseases

By altering the oxygenation capacity of cardiomyocytes directly, ischemic heart disease caused by stenosis or occlusion of the myocardial coronary artery is considered one of the main causes of morbidity and mortality worldwide, including myocardial infarction (MI) and HF (Ge et al., 2019).

Several studies have shown that cardiomyocytes under chronic anoxic conditions, such as anemia, chronic intermittent hypoxia, and sleep apnea, can retain the homeostasis of heart contractile function and energy metabolism by triggering the corresponding regulation pathways and mediating cardiac metabolic remodeling via induction of transcription (Essop, 2007; Cole et al., 2016).

As one of the main drivers of metabolic disturbance in ischemic disease and MI, hypoxia is the result of multiple physiological and pathological stimuli (da Luz Sousa Fialho et al., 2019). Previous studies of cardiac ventricular biopsies from patients who had undergone coronary artery bypass surgery have shown that hypoxia inducible factor (HIF) is a major transcription factor with significantly increased activity in the early period of post-MI (Lee et al., 2000; Smith et al., 2008). It is well-established that HIF-1 activates most of the regulatory factors involved in oxygen homeostasis in mammals by binding to hypoxia response elements (HREs) and activating the transcription of multiple target genes which may play a critical role in the hypoxic response at both the cellular and the systemic levels. Subsequently, this process regulates the relevant signal transduction pathways and the corresponding microenvironments of the pathological blood vessels induced by hypoxia and ischemia (Ortiz-Barahona et al., 2010).

Due to the deficiency in oxygen availability, cellular metabolism of cardiomyocytes is one of the major changes that occur during chronic adaptation to hypoxia (Dang and Semenza, 1999). In addition to metabolism, the binding between HIF and HREs can be affected by epigenetic mechanisms, such as CpG methylation and oxidative DNA damage caused by hypoxia and reactive oxygen species (ROS) (Rodriguez et al., 1997). It has also been reported that HIF-1 is associated with hypoxia and may be regulated by histone methylation directly. With regard to certain common methylases, several studies have shown that hypoxia induces the expression of ALKBH5 in various types of breast cancer cells (Essop, 2007). In addition, el Azzouzi et al. (2013) confirmed the binding sites of FTO mRNA for several hypoxia-induced cardiac microRNAs (miRs) well-known to be up-regulated in HF, such as miR-21, miR-24, miR-488, miR-224, and miR-489. These results suggest that epigenetic regulation of hypoxia may play a significant role in the pathogenesis of CVDs.

In hypoxia/reoxygenation (H/R)-treated cardiomyocytes and ischemia/reperfusion (I/R)-treated mouse heart tissues, the modification of m⁶A was increased by METTL3, which is the main factor of abnormal m⁶A regulation. This was caused by promoting the translation of specific mRNAs (Lin et al., 2016; Song et al., 2019).

The silencing of METTL3 can increase autophagic flux and inhibit apoptosis in H/R-treated cardiomyocytes, suggesting that METTL3 is a negative regulator of autophagy, which determines the cellular and systemic function of H/R-treated cardiomyocytes (Song et al., 2019). METTL3 methylates transcription factor EB (TFEB), a key gene involved in lysosomal biogenesis during the autophagic process, at two m⁶A residues in the 3'-UTR. METTL3 enhances the binding of the protein heterogeneous nuclear ribonucleoprotein D (hnRNPD) to TFEB pre-mRNA, thereby decreasing the expression level of TFEB (Pastore et al., 2016). Further experiments have shown that the autophagic flux enhanced by METTL3 deficiency is TFEB-dependent. Therefore, it is reasoned that METTL3 knockdown can increase the levels of the green fluorescent protein (GFP)-TFEB protein by up-regulating the endogenous levels of TFEB via the adenosine monophosphate (AMP)-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway (Song et al., 2019).

3.1.1 Heart failure

Current evidence shows that dysregulation of m⁶A levels in RNA can lead to the development of several pathological mechanisms, such as HF, at the molecular, cellular, and organic levels (Jia et al., 2011; Meyer et al., 2012). Mathiyalagan et al. (2019) confirmed that the increased expression of m⁶A induced by ischemia and hypoxia may be reduced by altering the expression of FTOs. This evidence was reported in a study of different models of HF and hypoxic primary cardiomyocytes (Mathiyalagan et al., 2019). In addition to improving cardiomyocyte function and attenuating cardiac remodeling in HF by restoring the expression of relative proteins, such as sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 2a (SERCA2a), experimental evidence has shown that FTO overexpression decreases cardiac fibrosis and promotes angiogenesis in models of MI (Mathiyalagan et al., 2019). Furthermore, it has been shown that cardiac contractile transcripts associated with ischemia and hypoxia are demethylated selectively by FTO, which can increase the stability of mRNA and protein expression (Hess et al., 2013).

3.1.2 Myocardial infarction

MI is a heart attack caused by the formation of plaque in the interior walls of the arteries that reduces blood flow to the heart and damages the heart muscles due to tissue inflammation and scarring (Lu et al., 2015). Epicardial specificity in response to injury occurs by down-regulating a group of genes expressed differentially in the epicardium (Bochmann et al., 2010). This is considered one of the main mechanisms of MI development.

During embryogenesis, the proepicardium differentiates into cardiac vasculature and interstitial cells and eventually becomes a single epithelial cell layer covering the heart, which is essential for normal development (Reese et al., 2002; Cai et al., 2008; Zhou et al., 2008; van Wijk et al., 2009). However, it remains controversial whether the epicardium can differentiate into endothelial cells of the vasculature (Smart et al., 2007; Winter and Groot, 2007) and whether it can contribute directly to the formation of the myocardium (Zhou et al., 2008). Although previous study has shown that the epicardium can regulate cardiac regeneration following injury (noted in lower vertebrates), it has also been reported that epicardial cell genes may be a promising therapeutic target following the injury of left coronary artery (LCA) ligation by the application of transcriptomics (Bochmann et al., 2010). Therefore, these studies suggest that the involvement of epigenetics should be explored with regard to MI.

Mathiyalagan et al. (2019) demonstrated that the levels of FTO in mice were down-regulated within 4 h following MI induction by LCA. Furthermore, they examined cardiac remodeling soon after MI and demonstrated that maintenance of FTO expression for nearly one week could be helpful in protecting and repairing the function of cardiomyocytes by improving cardiac homeostasis. Overexpression of FTO was caused by adenoviral transfection. These data suggested the use of FTO or FTO mimics for therapeutic targeting.

3.2. Hypertrophy

Hypertrophic cardiomyopathy is one of the common causes of myocardial injury accompanied by pathological phenomena, such as myocardial hypertrophy (Hou et al., 2017; Liu et al., 2019).

In addition to other common stimuli, Dorn et al. (2019) confirmed that methylation regulated by METTL3 on N ⁶-adenosines is essential for the pathological process of hypertrophy in vivo and in vitro. Although the increase in the expression level of METTL3, which acts with METTL14, WTAP, or other regulatory subunits, may cause cardiomyocyte remodeling, it is notable that the inhibition of its activity can abrogate the ability of cardiomyocytes to cause cardiac compensatory hypertrophy in response to hypertrophic stimuli, such as aging and stress. Furthermore, cardiac-specific METTL3 knockout mice were shown to promote eccentric cardiomyocyte remodeling and dysfunction in vivo (Liu et al., 2014; Dorn et al., 2019). These data highlight that the dynamic methylation and modification mediated by METTL3 are necessary for regulating the myocyte growth response.

Since both METTL3 and FTO are localized in the nucleus of isolated cardiac myocytes, FTO knockdown was found to decrease the augmented size of neonatal rat cardiac myocytes (NRCMs) induced by α-adrenergic stimulation in vitro (Kmietczyk et al., 2019). Taken collectively, these results suggest that m⁶A methylation may be a novel and vital epigenetic mechanism that should be studied in relation to the maintenance of cardiac homeostasis.

In addition, due to the tissue specificity of different ALKBH proteins, it is still debatable whether they can be used with similar therapeutic effects as those of the METTL3-containing protein, which is expressed ubiquitously (Fedeles et al., 2015).

3.3. Abnormal cardiac development

The consensus sequence contained in m⁶A is highly homologous between human and mouse species, and their mRNA levels are regulated in a tissue-specific manner from the embryonic period to adulthood (Dominissini et al., 2012; Meyer et al., 2012). Higher m⁶A expression in embryogenesis than in adult tissues has suggested the critical importance of m⁶A during growth and development at the cellular and organic levels.

Mouse ESCs (mESCs) are well-known for their roles in embryonic development. Wang et al. (2014) reported that METTL3 and METTL14 can modify m⁶A to maintain and even improve the self-renewal capability of mESCs. Their enrichment analysis indicated that m⁶A editing might be associated with the stability of RNA, which was in turn negatively associated with regulatory proteins by blocking the binding of human antigen R (HuR)’s RNA, thereby retaining the biological function of mESCs (Wang et al., 2014).

Moreover, in a study of growth retardation and developmental delay, FTO and its homozygous mutant were shown to play a vital role in the early development of the human central nervous and cardiovascular systems by maintaining the stability of 2-oxoglutarate turnover and N-methyl-nucleoside demethylase activity (Daoud et al., 2016).

3.4. Downstream gene expression

Alteration in the expression levels of myocardial genes in response to different pathological stimuli plays a major role in the occurrence and progression of HF in several animal models (Tan et al., 2002). A recent study has highlighted the importance of specific modifications that occur in reversible mRNA sequences that control gene expression at the epigenetic level (Tan et al., 2002). As an increase in the levels of m⁶A methylation is associated with cardiomyopathy, it is meaningful to investigate the role of internal m⁶A modification on the expression of the associated genes. Dynamic m⁶A methylation can influence the stability of transcription by regulating the efficiency of mRNA translation in stressed cardiomyocytes. For example, in addition to the size and cardiac function of cardiomyocytes, the expression of the downstream markers natriuretic peptide precursor (Npp)-A (Nppa) and -B (Nppb) that are associated with hypertrophy can be influenced by the enzymatic activity of METTL3. This suggests that m⁶A modification plays an integrative role in the regulation of cardiac gene expression and cellular growth response in vivo and in vitro (Kmietczyk et al., 2019).

Overall, the engineering and manipulation of m⁶A enzymes or any other type of small molecule at specific sites may be a novel therapeutic avenue for several cardiac diseases caused by dysregulation of m⁶A, such as HF and MI (James et al., 2003).

4. m⁶A RNA modification is an internal metabolic disorder caused by external environmental factors

Different studies have attempted to understand the important and necessary role of m⁶A in relation to metabolism and environmental effects that can, in turn, lead to the development of pathophysiology and abnormal heart function noted in CVDs (Table 1).

Table 1.

Multiple functions exerted by m⁶A RNA methylation in various cardiovascular diseases

Disease	Molecule	Target gene	Function	Regulation	Mechanism	Reference
Ischemic heart diseases	METTL3	TFEB	Writer	Up-regulation	Silencing METTL3 enhances autophagic flux and inhibits apoptosis in H/R-treated cardiomyocytes by TFEB	Pastore et al., 2016
	ALKBH5	TFEB	Eraser	Down-regulation	ALKBH5 overexpression enhances autophagic flux and inhibits apoptosis in H/R-treated cardiomyocytes by TFEB	Pastore et al., 2016
Heart failure (HF)	FTO	SERCA2a	Eraser	Down-regulation	FTO overexpression improves cardiomyocyte function and attenuatis cardiac remodeling in HF by regulating SERCA2a	Mathiyalagan et al., 2019
Myocardial infarction (MI)	FTO	Nppa/MYH7/Ryr2/SERCA2a	Eraser	Down-regulation	FTO overexpression protects and repairs cardiomyocyte function through improving cardiac homeostasis after MI	Mathiyalagan et al., 2019
Hypertrophy	METTL3	MAP3K6/MAP4K5/MAPK14/Nppa/Nppb	Writer	Up-regulation	Silencing METTL3 maintains normal cardiac function in response to hypertrophic stimuli through stress-response mechanism	Dorn et al., 2019
	FTO		Eraser	Up-regulation	Silencing FTO blunts hypertrophy of NRCM in response to α-adrenergic stimulation	Kmietczyk et al., 2019
Abnormal cardiac development	METTL3/14	HuR	Writer	Down-regulation	The level of METTL3 expression maintains and even improves the self-renewal capability of mESCs	Wang et al., 2014
	FTO		Eraser	Mutation	FTO plays an important role in early development of human central nervous and cardiovascular systems	Daoud et al., 2016
Obesity	FTO	Angptl4	Eraser	Up-regulation	FTO influences triglyceride metabolism in adipocytes and by post-transcriptional regulation of Angptl4	Wang et al., 2015
	YTHDF2	PPARγ/C/EBPα	Reader	Down-regulation	Adipogenesis of porcine adipocytes can be led by a loss of m6A on FAM134B through m6A-YTHDF2-dependent way	Cai et al.,2019
Diabetes mellitus	FTO	FOXO1/G6PC/DGAT2	Eraser	Up-regulation	FTO expression is highly related tothe impairment of plasma glucose and even multiple complications by altering target genes	Yang et al., 2019
Abnormal circadian rhythm	METTL3	Per2/Arntl	Writer	Down-regulation	Silencing METTL3 can elicit circadian period elongation and RNA processing delay by distributing clock genes Per2 and Arntl	Fustin et al., 2013
Inflammation	METTL3	MyD88S	Writer	Up-regulation	Silencing METTL3 attenuates theinflammatory response via regulating MyD88S in LPS-induced hDPCs	Liu et al., 2019

m⁶A: N ⁶-methyladenosine; METTL3: methyltransferase-like 3; ALKBH5: ALKB homolog 5; FTO: fat mass-and obesity-related protein; YTHDF2: YT521-B homology (YTH) domain family 2; TFEB: transcription factor EB; SERCA2a: sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 2a; Nppa: natriuretic peptide precursor (Npp)-A; MYH7: β-myosin heavy chain 7; Ryr2: ryanodine receptor 2; MAP3K6: mitogen-activated protein kinase (MAPK) kinase kinase 6; MAP4K5: MAPK kinase kinase kinase 5; HuR: human antigen R; Angptl4: angiopoietin-like 4; PPARγ: peroxisome proliferator-activated receptor γ; C/EBPα: CCAAT enhancer-binding protein α; FOXO1: forkhead box O1; G6PC: glucose-6-phosphatase (G6Pase) catalytic subunit; DGAT2: diacylglycerol acyltransferase 2; Per2: period 2; Arntl: aryl hydrocarbon receptor nuclear translocator like; MyD88S: splice variant of myeloid differentiation factor 88; H/R: hypoxia/reoxygenation; NRCM: neonatal rat cardiac myocyte; mESCs: mouse embryonic stem cells; FAM134B: family with sequence similarity 134 member B; LPS: lipopolysaccharide; hDPCs: human dental pulp cells

4.1. Obesity

The improvement in the general standard of living has rendered obesity a sub-health symptom. Several clinical and basic research studies have shown that the increasing incidence and mortality of various CVDs and even cancers are closely associated with obesity, suggesting it contributes to the severe financial burden associated with the treatment of these conditions (Scuteri et al., 2007; Koliaki et al., 2019).

In addition to dietary habits, the FTO gene, which is closely linked to obesity, was identified by the present study as a positive regulator of energy homeostasis, for example via lipid metabolism, by modulating m⁶A levels in RNA and DNA sequences in humans (Bartosovic et al., 2017; Chen and Du, 2019). The physiological and pathological development of obesity may be associated with FTO variants, which carry the at-risk alleles of single-nucleotide polymorphisms (SNPs), including rs9939609, rs8050136, rs3751812, rs7202116, and rs9930506, in the first intron of the FTO gene (Church et al., 2010; Wang et al., 2015). Variants of FTO were reported to have enhanced FTO expression and/or activity in previous study performed on homozygous individuals carrying the at-risk alleles (Church et al., 2010). Moreover, most studies demonstrated that these at-risk SNPs appeared to promote energy intake and a preference for energy-dense foods (Cecil et al., 2008; Speakman et al., 2008; Timpson et al., 2008; Haupt et al., 2009), while in two independent genome-wide association studies, the FTO gene was shown to be a high risk factor contributing to early-onset and severe obesity of European and Indonesian subjects (Dina et al., 2007; Daya et al., 2019).

An analysis of different phenotypes of mice has shown that adipocyte-specific FTO can play an important role in influencing triglyceride metabolism in adipocytes and consequently body weight. This is caused partly by post-transcriptional regulation of downstream genes, such as Angptl4. This may decrease energy expenditure and promote fatty acid storage in adipose tissues, regardless of the intake from a high-fat or standard diet (Wang et al., 2015).

Furthermore, a loss of m⁶A on family with sequence similarity 134 member B (FAM134B) can lead to the adipogenesis of porcine adipocytes through the m⁶A-YTHDF2-dependent pathway (Cai et al., 2019).

The epigenetic modification of m⁶A can be a risk factor of CVDs related to obesity, although the exact molecular mechanism remains unclear.

4.2. Diabetes mellitus

Pathoglycemia induced by diabetes mellitus, including hypoglycemia and hyperglycemia, has been identified to be the main risk factor contributing to CVDs and all-cause mortality in diabetic patients. In addition to controlling blood glucose, an increase in insulin sensitivity has become a new auxiliary or main treatment strategy of diabetes, notably in cases with insulin resistance (Khunti et al., 2015; Abdul-Ghani et al., 2019).

Furthermore, hyperglycemia may lead to the down-regulation of m⁶A levels by enhancing the expression of FTO or other methyltransferases in patients with T2DM, suggesting an important epigenetic role of m⁶A in regulating glucose and lipid metabolism disorders (Gilbert and Liu, 2012; Yang et al., 2019). In addition to dysregulated oxidation of glucose, increased FTO expression induced by diabetes is highly associated with the impairment of plasma glucose and even multiple complications caused by altering the expression of target functional genes, such as forkhead box O1 (FOXO1), glucose-6-phosphatase (G6Pase) catalytic subunit (G6PC), and diacylglycerol acyltransferase 2 (DGAT2) (Souness et al., 1982; Kursawe et al., 2016; Li et al., 2017; Yang et al., 2019).

Recently, it has been shown that an altered content of m⁶A can be used as a novel biomarker of T2DM since significantly lower levels of m⁶A are noted in both T2DM patients and diabetic rats. At present, FTO expression is the only indicator found to be positively correlated with the risk of diabetes by correlation analysis (Shen et al., 2015).

Nevertheless, whether m⁶A modification in diabetes mellitus is locus-specific remains to be elucidated.

4.3. Abnormal circadian rhythm

Previous study has suggested that the circadian rhythm (day/night cycle) is one of the basic and vital features of the natural environment due to its contribution to the regulation of normal development and function of the cardiovascular system (Melkani and Panda, 2017). It affects the expression of related genes and the levels of hormones associated with cardiac function, such as angiotensin II, renin, aldosterone, growth hormone, and atrial natriuretic peptide (McNamara et al., 2001; Martino et al., 2004; Martino and Sole, 2009).

Moreover, it has been suggested that the occurrence and the severity of certain CVDs are highly associated with the circadian rhythm via specific intrinsic mechanisms that are currently unknown (Mukamal et al., 2000; Henriques et al., 2003).

The circadian clock may regulate metabolism in an epigenetic feedback loop by modifying the expression levels of certain genes coding for metabolic switches or other important components. This modification is caused at the level of transcription. The acetylation/deacetylation of histones occurs on the promoters related to protein complexes or on clock proteins directly. In addition to this process, methylation of histone proteins has been shown to play a vital role in maintaining the normal function of the circadian rhythm in vivo (Asher et al., 2008; Nakahata et al., 2008; Katada and Sassone-Corsi, 2010).

A recent study has shown that the transcriptional functions of several clock genes, such as period 2 (Per2) and aryl hydrocarbon receptor nuclear translocator like (Arntl), play an important role in extending the circadian rhythm period by inhibiting the m⁶A modification of methylase METTL3, thereby promoting the uncoupling between steady-state pre-mRNA and cytoplasmic mRNA rhythm (Fustin et al., 2013). It has also been predicted that the period of the circadian rhythm is positively associated with the efficiency of degradation and the nuclear import and export of Per2 mRNA, as determined by network model analysis (Wilkins et al., 2007; Miki et al., 2012). Moreover, Arntl is much more sensitive than Per2 with regard to the inhibition of METTL3 due to its higher mRNA nuclear accumulation and longer period of transcription (Fustin et al., 2013).

The decrease in RNA methylation induced by the pharmacological action of 3-deazaadenosine (3-DZA) might elongate the period of circadian rhythm in vivo and in vitro via the decreased hydrolysis of S-adenosylhomocysteine (SAH) (Fustin et al., 2013; Singhal et al., 2013; Lokody, 2014).

It has been suggested that m⁶A modification affects the circadian rhythm and may regulate cardiac function to a certain degree.

4.4. Inflammation

CVDs and diabetes mellitus are defined as metabolic syndromes and have recently been investigated as chronic inflammatory diseases triggered by long-term stimulation of abnormal levels of plasma glucose, free fatty acids, and blood pressure. These abnormal biochemical profiles are promoted via the oxidative stress pathway and the interaction between the cytokines and the renin-angiotensin system (Wong et al., 2008; Grandl and Wolfrum, 2018; Fiechter et al., 2019).

Inflammation, including innate and adaptive immunity, is one of the major and fundamental risk factors of all stages of atherosclerosis (Weber and Noels, 2011; Legein et al., 2015; Ketelhuth et al., 2019). Recent studies have shown that the pathological development of atherosclerosis is driven by immune reactions and is related mainly to immune effector mechanisms, which are involved in the infiltration and accumulation of lipoproteins in the arterial intima (Libby et al., 2011, 2013; Ketelhuth and Hansson, 2016).

In addition to the epigenetic regulation of SNPs that is related to different degrees of CVD risks, hyperacetylation at the 27th lysine residue of the histone H3 protein (H3K27ac) is linked to lower expression of proinflammatory genes in the reversible process of inflammation and aging. This may also explain certain unclear mechanisms in the link between inflammation and the progression of CVDs (Smith and Humphries, 2009; IL6R Genetics Consortium Emerging Risk Factors Collaboration, 2012; Xuan et al., 2016; Mitrokhin et al., 2017; Cheng et al., 2018).

Furthermore, telomere shortening and modulation of certain microRNAs associated with DNA damage are considered another epigenetic mechanism accounting for inflammation, since they can promote the secretion of proinflammatory cytokines through several proinflammatory pathways (Olivieri et al., 2015).

Since the specific mechanism of the association between inflammation and atherosclerosis has not been fully clarified, m⁶A has become a new focus for the development of novel clinical therapeutic strategies against CVD.

Recent studies have demonstrated that the loss of N ⁶-adenosine methyltransferase METTL3 can attenuate the increased expression levels of several inflammatory cytokines and various genes associated with the inflammatory response, mainly by changing the phosphorylation levels of related signaling pathways and by regulating the mRNA splicing of splice variant of myeloid differentiation factor 88 (MyD88S) in lipopolysaccharide (LPS)-induced human dental pulp cells (HDPCs). This suggests the possible role of epitranscriptomic modification of m⁶A during the process of inflammation in pathophysiological mechanisms leading to CVDs (Feng et al., 2018).

5. Conclusions

Several independent studies have confirmed that the continuous and dynamic regulation of m⁶A in different internal biological metabolic processes has a profound impact on modulating the expression of cardiac genes in the pathological development of CVDs. Moreover, these risk factors can be combined with several other environmental factors that affect the development of these diseases. This occurs by regulation of RNA transcription, splicing, processing, translation, and decay (Niu et al., 2013; Guo et al., 2017; Roignant and Soller, 2017; Wei et al., 2017). Therefore, certain m⁶A enzymes, including FTO and METTL3, have been shown to control epitranscriptomic regulation of specific cardiac defects caused by distinct mechanisms. However, the possible association between m⁶A and other CVDs and even between cardiovascular-associated diseases, such as high blood pressure and congenital heart disease, remains unexplored. Although the use of specific inhibitors in different models of animals or cardiocytes has established the cardioprotective role of RNA-modifying enzymes in m⁶A modification, additional therapeutic agents for CVDs should be assessed in future studies.