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Published in final edited form as: Curr Opin Physiol. 2022 Jun 3;28:100561. doi: 10.1016/j.cophys.2022.100561

m6A RNA methylation: A dynamic regulator of cardiac muscle and extracellular matrix

Charles P Rabolli 1,2, Federica Accornero 1,2,*
PMCID: PMC10249538  NIHMSID: NIHMS1904218  PMID: 37304645

Abstract

Post-transcriptional modifications encompass a large group of RNA alterations that control gene expression. Methylation of the N6-Adenosine (m6A) of mRNA is a prevalent modification which alters the life cycle of transcripts. The roles that m6A play in regulating cardiac homeostasis and injury response are an active area of investigation, but it is clear that this chemical modification is a critical controller of fibroblast to myofibroblast transition, cardiomyocyte hypertrophy and division, and the structure and function of the extracellular matrix. Here we discuss the latest findings of m6A in cardiac muscle and matrix.

Introduction

Regulation of cardiac development, homeostasis, and stress responses requires functional coordination of multiple cell types in the heart. The extracellular matrix (ECM) is a critical mediator of cell communication. Every cardiac cell can contribute to the ECM composition to ultimately orchestrate the remodeling of the heart following challenges or growth stimuli. Changes in the cardiac extracellular matrix are largely mediated by gene expression regulation in both cardiomyocytes and non-myocytes. While transcription is a well-established regulatory step of cardiac gene expression, the role of post-transcriptional events for heart structure and function is a rapidly evolving field of study.

Post-transcriptional modifications contain a wide breadth of molecular diversity ranging from the well-known 7-methylguanosine cap at the 5’ end of mRNA and the poly(A) tail at its 3’ end, to splicing events that alter the sequence of mature transcripts. Among the lesser-known forms of post-transcriptional modifications, are the over 150 different types of internal chemical modifications that can occur within RNAs in Eukaryotic cells [1]. On mRNA specifically, these include N6-methyladenosine (m6A) [2–4], N1-methyladenosine [5], 5-methylcytosine [3, 4], Pseudouridine [6], and others [7]. Moreover, there are numerous alterations that can occur to noncoding RNAs (including tRNA, rRNA, lncRNA snRNA, miRNA and more), which further regulate gene expression [8]. Although each modification may play an important role in cardiac gene regulation, this review will focus on m6A as an established critical mRNA modification in the heart.

m6A has been implicated in the regulation of nearly all steps of mRNA metabolism, including decay, folding, maturation, transport, localization, and translation. It accomplishes these effects through dynamic processing by methyltransferases and demethylases, and by differential engagement of RNA binding proteins [9–13]. m6A formation is regulated by a methyltransferase-like 3 and 14 (METTL3/METTL14) heterodimer that serves as the catalytic component of a complex including Wilms Tumor 1 Associated Protein (WTAP) [7, 14] and Vir like m6A methyltransferase associated protein (VIRMA) [15]. Collectively, this complex catalyzes methylation of adenosines within the RRACH (R = G/A, H = A/C/U) motif [16]. The prevalence of this modification is counterbalanced by RNA demethylases which belong to the AlkB family of dioxygenases, namely fat-mass and obesity associated protein (FTO) [17] and AlkB Homolog 5 (ALKBH5) [18]. Finally, proteins most commonly from the YT521-B homology (YTH) domain family (YTHDF1–3, YTHDC1–2) preferentially recognize m6A-modified transcripts to exert downstream effects [10, 13, 19–23]. Despite sharing a conserved YTH RNA binding domain, the consequences of m6A recognition by each of these proteins vary significantly, which further contribute to the diverse and ever-growing list of m6A functions in mammalian cells [9].

Considering that m6A has been shown to exert context dependent effects, it is important to review its role in each specialized system of interest. Here we explore the current understanding of m6A and its regulation in the mammalian myocardium. As the role of m6A in cardiac ECM has been minimally studied, we will also provide a short description of m6A as it pertains to the ECM in other organ systems.

RNA methylation marks heart development and pathology

Biochemical and sequencing analyses showed that cardiac remodeling is accompanied by changes in m6A content in both human samples and animal models of heart failure. Increased levels of RNA methylation have been observed in ischemic [24•] and non-ischemic cardiomyopathy [24–27•] human heart samples, as well as pig [24•] and mouse [24•] myocardial infarction models, compared to their respective healthy-heart controls. These data raised interesting hypotheses on the link between m6A content and pathological cardiac remodeling. Berulava et al then showed that in healthy murine and human hearts, nearly 1 in 4 transcripts contains m6A methylation, furthering the credibility of m6A as a critical regulator of gene expression, even at baseline [27•].

As the relationship between increasing m6A levels and worsening cardiac function began to clear, some groups studied whether m6A could play a role in postnatal development. Investigation of the role of RNA methylation in the first week of rodent life led to discordant results. Yang et al found that in Sprague-Dawley rats, there were fewer m6A methylated mRNA transcripts at post-natal day 7 (P7) than at birth [28]. On the other hand, one study using C57BL/6J mice showed m6A peaks were lower at P1, increased through P7, and then were little changed through P28 [29]. This last finding was supported by Gong et al, who further found higher levels of METTL3 at P7 compared to P1 in the same mouse strain [30]. These contradictory findings highlight an extremely important, and presently unanswered, question of whether different species or even strains of the same species, may have different levels or patterns of RNA methylation. It is also important to note that multiple cell types can contribute to the overall changes in cardiac m6A levels. Regardless, it is evident that m6A is dynamically regulated in the heart and could therefore represent a point of coordination for stress responses by tuning gene expression. We will next discuss potential roles for both non-myocytes and cardiomyocytes in m6A-dependent cardiac biology with a particular emphasis on ECM regulation, as changes in its composition dictate cell-cell communication for the heart.

m6A in non-myocytes

Due to the small number of studies that have investigated the role of m6A in cardiac ECM composition, we will first briefly discuss the function of m6A as it relates to fibrosis and the ECM in other organ systems. In line with the findings in cardiac disease, m6A levels have been found to increase after lung injury, and this increase in methylation drives fibroblast activation and myofibroblast conversion [31]. Lowering m6A levels through silencing of METTL3 was sufficient to inhibit fibroblast to myofibroblast transition, both in vivo and in vitro [31], which further highlights the potential role of m6A methylation in regulating fibrosis post-injury. Myofibroblast formation was further shown to involve YTHDF1, emphasizing the importance of the m6A binding proteins in exerting downstream effects. Moreover, m6A levels were increased in animal models of bleomycin induced pulmonary fibrosis as well as in lung samples taken from patients with Idiopathic Pulmonary Fibrosis [31], thus showing a similar m6A injury-responsiveness as seen in cardiovascular diseases. In a model of pulmonary arterial hypertension, the increase in m6A content post injury was concomitant with decreased levels of the demethylases, FTO and ALKBH5, and increased levels of the core methyltransferase, METTL3 [32]. Intriguingly, when comparing pulmonary arterial hypertension tissue vs control tissue, one of the most upregulated pathways was “ECM-receptor interaction” [32] which is a pathway that is repeatedly shown to be differentially regulated in analyses involving m6A. More specifically, Collagen III alpha 1 (Col3a1), which is a key component of the ECM, has been identified as a target gene of METTL3 in triple negative breast cancer [33]. Increased methylation of Col3a1 transcripts in this condition serves to down-regulate the expression of the protein [33]. Finally, in a model of osteoarthritis, silencing of METTL3 was shown to decrease the expression of matrix metallopeptidase (MMP) 13 and Collagen X, while increasing the expression of Aggrecan and Collagen II [34]. Conversely, overexpressing METTL3 increased the levels of MMP1 and MMP3, and decreased expression of TIMP metallopeptidase inhibitor-1 (TIMP1) and TIMP-2 [35]. Taken together, these findings highlight the potential role that the m6A pathway may play in regulating fibrosis and ECM remodeling after injury (Figure 1).

Figure 1: Effects of m6A modifications in Fibroblasts.

Figure 1:

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(Left Panel) Elevated m6A levels in fibroblasts stimulate a transition towards myofibroblasts, increase the secretion of MMP3 and Collagens I and III and decrease the expression of TIMP1 and TIMP2. (Right Panel) Decreased m6A levels in fibroblasts inhibit the transition towards myofibroblasts, increase the expression of Aggrecan and Collagen II, and decrease the expression of MMP13 and Collagen X.

While m6A methylation is important for the life of messenger RNAs, it can target noncoding RNAs as well [36] with important consequences in the cardiovascular system. Methylation of pri-miR-19a was shown to promote maturation of miR-19a, thereby affecting proliferation and invasion of atherosclerotic vascular endothelial cells [37]. Moreover, METTL3 inhibited endothelial cell autophagy by promoting development of pri-miR-20b in an m6A dependent manner [38]. In fibroblasts, vasoactive microRNAs are frequently m6A methylated, and levels of microRNA methylation in fibroblasts increase after hypoxia [39], emphasizing the importance of m6A in fibroblast biology with potential consequences in stress-induced ECM remodeling. Furthermore, METTL3 dependent m6A methylation regulated levels of the long noncoding RNA (lncRNA) MALAT1, with implications for renal fibrosis [40]. One group also found that m6A on circular RNA may regulate silica-induced pulmonary fibrosis [41], showcasing the diversity of RNA species that can be m6A methylated as a means for controlling homeostasis and injury responses. Given the importance of m6A in the regulation of the ECM in various other organ systems, m6A may be a key component that regulates cardiac fibrosis post-injury.

m6A regulation of non-myocytes in the heart has been far less studied than m6A in cardiomyocytes, and to date there is only a single study, by Li et al, which investigated the effects of m6A on cardiac fibroblasts. They found that METTL3 protein was increased in fibrotic tissue after myocardial infarction (MI) [42•]. Further they showed that overexpression of METTL3 in cardiac fibroblasts in vitro led to an increase in Collagens I and III, stimulated fibroblast proliferation and their activation towards myofibroblasts, and that it does so at least partially through the SMAD pathway [42•]. SMAD proteins are a family of proteins that are best known for transducing the signal of Transforming Growth Factor-β to the nucleus to alter gene expression [43]. Notably, SMAD2 and SMAD3 have been found to interact with the m6A methyltransferase complex and to use mRNA methylation as a mechanism through which these SMAD proteins exert effects [44]. Inhibition of METTL3 in cardiac fibroblasts improved function in vivo after MI. Interestingly, after conducting RNA sequencing and m6A sequencing on control and METTL3 knockdown cardiac fibroblasts, the authors found differential expression of an “ECM-receptor interaction” pathway [42•]. This finding is noteworthy because this same pathway has appeared in multiple studies that have examined differences in m6A levels [29, 32]. In addition to the importance of METTL3 in m6A regulation of cardiac fibroblasts, another study found that activating these cells using Angiotensin II involved the FTO pathway through its regulation of Dickkopf WNT signaling pathway inhibitor 2 (DKK2) [45]. This finding gives further credence to the delicate balance that exists between the methylase and demethylase enzymes, and how both groups play important roles in homeostatic equilibrium and injury response mechanisms. While it is clear from these studies that m6A plays a part in the development of cardiac fibrosis through inducing the transition from fibroblasts to myofibroblasts, further work is needed to identify the specific functions of the m6A pathway with respect to the cardiac ECM. While non-myocytes are key to ECM remodeling, regulation of gene expression in cardiomyocytes themselves can also ultimately affect the extracellular environment, as discussed next.

m6A regulation in cardiomyocytes

Ex vivo approaches in isolated cell systems have highlighted the importance of m6A in cardiomyocyte biology [46]. m6A increases specifically in cardiomyocytes both following hypoxic stimulation [47, 48] and induction of hypertrophy [49•]. Attempting to elucidate the functions of these proteins in vivo, however, has proven to be more complicated. Dorn et al, through a cardiomyocyte specific deletion of METTL3, found that METTL3 itself is necessary to both prevent eccentric cardiomyocyte remodeling and to allow for a normal physiologic response to stress, as shown by accelerated heart failure following pressure overload in METTL3 knockout mice [49•]. Using a METTL3 overexpressing transgene system, they also showed that METTL3 overexpressing mice exhibit cardiac hypertrophy at baseline, but find that METTL3 overexpression does not accelerate dysfunction following overload stress [49•]. These findings, however, run counter to those shown by Kmietczyk et al, who found that METTL3 overexpression via an adeno-associated virus serotype 9 (AAV9) lessened dysfunction after pressure overload by attenuating pathological hypertrophy [25•]. A possible explanation for these different conclusions could be the difference in overexpression model, or the difference in mouse strain used, the latter of which could further solidify the idea that strain and species may have different m6A landscapes, as previously mentioned. Ultimately, the importance of METTL3 in regulating hypertrophic growth is clear and is also supported by Gao et al [50].

Further studies corroborated the importance of m6A in the heart by manipulating the level of its demethylases. Knockout of FTO led to worse cardiac function after pressure overload [27•], whereas overexpression of FTO improved cardiac function after MI [24•] and in a model of diabetic cardiomyopathy [51]. Furthermore, following endotoxemia, mice had increased m6A levels and decreased FTO levels, with a downregulation of FTO in the myocardium which led to a surge in inflammatory cytokines [52]. Shen et al also found that FTO levels were decreased following both pressure overload and doxorubicin induced heart failure, which was concomitant with an increase in m6A levels following both injuries [53]. All these studies, therefore, support the idea that decreasing m6A on FTO targets may be beneficial to heart function. Similar to the findings with FTO, wherein increased demethylase level impacts cardiac adaptation to stress, protein levels of the demethylase ALKBH5 increase in murine models of diabetic cardiomyopathy through a YTHDF2 dependent mechanism, ultimately resulting in decreased m6A levels and cardioprotection [54]. Moreover, overexpressing ALKBH5 in neonatal mouse ventricular cardiomyocytes via an adenovirus successfully inhibited apoptosis and increased autophagy in hypoxia/reoxygenation treated cells, further supporting the protective role of increased demethylase activity [46].

While the observed m6A-dependent changes in heart size and function may be due to changes in cardiomyocyte hypertrophy, an alternative hypothesis is that it could be due to cardiomyocyte hyperplasia. Turning attention away from a hypertrophic injury response to a regenerative focus, groups began to look at the first week postnatally, a time when murine cardiomyocytes maintain an ability to proliferate. Gong et al showed that a CRISPR/Cas9 knockdown of METTL3 in mice improved cardiomyocyte proliferation, both at baseline and after ischemia/reperfusion (I/R), through alterations in Yes-associated protein (YAP) signaling [30]. In line with the findings of a potential benefit from decreased METTL3, Su et al showed that global cardiac m6A levels and METTL3 levels decrease in both young and aged mice 2 hours after I/R injury, despite finding no age-related m6A content differences at baseline [55]. This decrease in m6A and METTL3 post I/R is particularly intriguing, as it conflicts with the increase in m6A and METTL3 reported by Song et al after I/R injury [46]. One potential explanation for this difference could be the short timeframe (2 hours) used by Su et al, whereas it is unclear how long after I/R Song et al waited to harvest the tissue. This point raises the interesting question of whether there is a transient decrease in both m6A and METTL3 following I/R, before a longer-term increase in both. In accordance with altered expression of the m6A methyltransferase, Wang et al have shown that knockdown of WTAP, a member of the m6A methylase complex, improved cardiac function after I/R injury, giving further credence to the potentially therapeutic benefits of decreasing m6A formation [48]. ALKBH5, an m6A demethylase, also appears to play a role in cardiomyocyte proliferation. After apical resection, ALKBH5 knockout mice have a decreased regenerative ability, whereas ALKBH5 overexpressing animals have an increased regenerative potential, both in neonatal mice after apical resection [29], and in adult mice after I/R, through YTHDF1 dependent translation of YAP [56]. Further, ALKBH5 may play a key role in the fate determination of human embryonic stem cells, where overexpression of ALKBH5 blocks their differentiation into cardiomyocytes [57].

While the roles of the methylase and demethylase proteins have been more clearly elucidated, the precise functions of the m6A binding proteins have yet to reach consensus. YTHDF2 increases in human and animal heart failure samples and may present a protective mechanism through modulating Myh7 mRNA [58]. YTHDF1 and YTHDF3, however, appear to have interrelated effects. YTHDF1 knockdown impairs the ability of embryonic stem cells to differentiate into cardiomyocytes, whereas loss of YTHDF3 accelerates this differentiation and does so partially through suppressing YTHDF1 [59]. Gao et al generated knockout mice for YTHDF1, YTHDF2, YTHDF3, and YTHDC1. Using these models, they found that deletion of YTHDC1 led to dilated cardiomyopathy through abnormal splicing of m6A modified Titin mRNA, whereas mice with deletion of YTHDF1, 2, and 3, did not have any baseline phenotypic defects [60].

As with non-myocytes, cardiomyocytes too have m6A sites not only on messengers but also on noncoding RNAs. Doxorubicin upregulated METTL14 which increased methylation on the lncRNA KCNQ1OT1, ultimately contributing to cardiomyocyte ferroptosis [61]. In a hypoxia/reperfusion experiment on mouse myocardial cells, Shen et al found that FTO reduced the m6A levels on Mhrt, which is a lncRNA transcribed from the antisense strand of Myh7 [53]. In cardiomyoblast (H9c2) cells, METTL3 and METTL14 have been shown to methylate the lncRNA H19 which impacts the cellular response to hypoxic reconditioning [62, 63]. Taken together, the m6A methylation of both coding and noncoding RNA play an important role in regulating the cardiomyocyte’s response to stress. While several molecular pathways have been highlighted as m6A-modulated in both cardiomyocytes and non-myocytes, the direct impact of these modifications on cardiac function and ECM remodeling is still an open question that will be an exciting area of research.

Conclusion

The regulation of m6A mRNA is a highly dynamic process that is controlled by a delicate balance between methyltransferases and demethylases. Levels of m6A mRNA play an important role in controlling cardiac phenotypes, through regulation of both non-myocyte and cardiomyocyte RNA. Increased levels of m6A in fibroblasts drive their transition into myofibroblasts, consequently affecting their secretion of ECM proteins. While the role of m6A in cardiac fibroblasts needs to be further studied, it is clear that, like cardiomyocytes, cardiac fibroblasts are dynamically regulated by m6A, and this connection may provide critical insight into the mechanisms of heart disease. In cardiomyocytes, m6A has been implicated in many different processes, ranging from neonatal cardiomyocyte regeneration to regulation of survival and hypertrophy in the adult heart. Given these findings, it is possible to envision the application of knowledge on m6A to a clinical setting. m6A levels could serve as a biomarker for disease detection or progression [64]. Moreover, future pharmacological alteration of m6A levels [65] or engineering novel approaches to affect the regulation of m6A-RNA [66] may prove beneficial in treating cardiovascular disease. Overall, the studies discussed here highlight the emerging importance of regulating cardiac m6A levels , and open exciting new therapeutic avenues for the treatment of heart disease.

Funding

This work was supported by the NIH under grants [R01 HL 136951 and R01 HL 154001 to F.A] and by the US-Israel Binational Science Foundation (BSF) grant number 2017094 to F.A.

Footnotes

Conflict of interest statement

Nothing declared.

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