In recent years, great technological leaps in sequencing technologies have enabled the in-depth investigation of the genetic basis of a multitude of human disorders, and they have paved the way for a new era of personalized medicine (1,2). Such breakthrough developments in sequencing technologies have substantiated the deep complexity of associations between genotype and phenotype and revealed unexpected cases, such as those of identical twins carrying the same disease mutations, but exhibiting different clinical features, such as balance problems and the development of blindness (3). Such discrepancies can be attributed to epigenetic and/or epitranscriptomic differences. The term epigenetics, first introduced by C.H. Waddington in 1942, refers to the study of the mechanisms and molecules that can perpetuate variable gene activity states in the context of the same DNA sequence (4). Epigenetic mechanisms include DNA methylation, chromatin remodeling, histone modifications, gene activity regulation by non-coding RNA (ncRNA) molecules and protein-protein interactions (5). These mechanisms, which include a vast array of different molecules and pathways, regulate genomic structure and transcriptional activity in response to the ever-changing profiles of cell-intrinsic, cell-cell and cell-extrinsic signals (6).

Epigenetic regulation has been increasingly gaining interest due to its strong relationship with environmental adaptation. As new insights are gained, novel distinctions are also formed, leading to the emerging field of epitranscriptomics. Instead of encompassing all epigenetic regulation, epitranscriptomics focuses on modifications at the RNA level (7). Due to the vast array of effects that coding and ncRNAs exert in regulating the differential response of organisms to environmental stimuli, as well as homeostasis maintenance, epitranscriptomics has turned into an explosive field of research. Several different RNA modification databases have been established throughout the years in an effort to catalogue the plethora of RNA modifications that are continuously being detected. These include databases such as Modomics (https://iimcb.genesilico.pl/modomics/) (8-11), RMBase v2.0 (http://rna.sysu.edu.cn/rmbase/) (12), DARNED (https://darned.ucc.ie/) (13), the RNA Modification Database (https://mods.rna.albany.edu/) (14) and REDIportal v2.0 (http://srv00.recas.ba.infn.it/atlas/) (15), encompassing >172 RNA modifications to date.

Epitranscriptomic changes induced by such mechanisms have been implicated in various diseases and most of them display reversible chemistry, making epitranscriptomics a promising candidate for providing novel therapeutics (16). As such, a number of reviews have already been published discussing the ever-expanding field of epitranscriptomic modifications, with a limited number focusing on their effects under the prism of cardiovascular disease (CVD). Most notable reviews have focused on N6-methyladenosine (m⁶A) modifications, as the most prevalent epitranscriptomic modification and its role in CVD (17,18). Although, Kumari et al (17) featured a section about m⁶A readers, Chen et al (18) also discussed the potential for m⁶A modification to influence CVD risk factors. Focusing more on clinical trials investigating epigenetic-sensitive drugs for heart failure (HF), Napoli et al (19) also outlined the discovery of epigenetic biomarkers and signatures of cardiac remodeling. On the other hand, Fischer and Vondriska (20) focused their discussion on epigenetic changes occurring in CVD, but did not expand into RNA modifications, as was also the case for Schiano et al (21), who discussed epigenetic mechanisms underlying the various pathologies encompassed by the CVD umbrella-term. Although the authors mentioned CVD epitranscriptomics as an emerging layer of epigenetic regulation in CVD, they also highlighted the need for further research that covers this subject matter.

In the present review, the most prevalent epitranscriptomic modifications that have been shown to be involved in the field of CVD have been outlined (22), in an effort to extensively cover the area of RNA modifications, without focusing on a single one. This study also briefly discussed the mode of action of each modification and then explored their respective effect on both coding and ncRNAs, including microRNAs (miRNAs/miRs) and long ncRNAs (lncRNAs), in the context of CVD. Furthermore, the current methods of RNA modification detection that have been on the forefront of epitranscriptomic research were also explored in brief. Finally, available data on genetic associations of RNA modifications, as well as therapeutic implications of epitranscriptomic approaches, in the heart were discussed.

CVD is currently the leading cause of death worldwide, accounting for almost half the total number of deaths (23). CVD encompasses a wide array of heart and vessel-related pathologies, including, but not limited to HF, coronary heart disease, hypertension, hypertrophic and dilated cardiomyopathy, as well as congenital heart disease (24). Accumulating data have shown that cardiovascular risk factors may alter epigenomic patterns and that several cardiovascular biomarkers are associated with epigenetic modifications (25). DNA methylation appears to contribute to processes underlying CVDs, such as atherosclerosis, hypertension and inflammation (26-28). Moreover, epidemiological studies imply that methylation of repetitive sequences such as long-interspersed nucleotide repetitive elements-1 (LINE-1) and Alu elements are associated with CVD (26). Specifically, patients with prevalent ischemic heart disease (IHD) and stroke displayed lower blood LINE-1 methylation, while elevated methylation of Alu elements was associated with CVD and obesity in Chinese individuals (26). Histone modifications have also been implicated in processes, such as hypertension and atherosclerosis, while histone deacetylase 4 overexpression following myocardial infarction (MI) has been shown to increase myocardial fibrosis and cardiac hypertrophy, eventually leading to cardiac dysfunction (29). Although epigenetic regulation has been the focus of attention, RNA modifications have only recently started becoming the focus of CVD researchers.

Epitranscriptomic regulation manifests through the action of different enzymes. Enzymes that modify the RNA itself are called 'writers', while the ones that recognize and remove modifications are termed 'erasers'. Finally, 'readers' are the group of enzymes that bind the modifications themselves (30,31). These different modifications are classified into groups based on their different characteristics. These groups include classification into reversible and non-reversible (where erasers are lacking), substitutional and non-substitutional (32), cap (where the modifications happen to the 5′-end of the RNAs) or internal modifications [where the modifications occur within the 5′- or 3′-untranslated regions (UTRs) or within transcript introns] (33), and finally, modifications on coding or ncRNAs (34). NcRNAs have now been studied extensively and have been proven to have important regulatory effects in both physiological and pathological conditions. The term ncRNAs encompasses a large array of RNA molecules, including, but not limited to the major classes, such as miRNAs, lncRNAs and circular RNAs (circRNAs), as well as transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs) and others (35). ncRNA regulatory roles extend from interacting with RNA/DNA-binding proteins, being part of complex structures, interacting with messenger RNA (mRNA) molecules to participating in translation and guiding chemical modifications (35). Through post-transcriptional modifications, ncRNAs display discrete temporal and spatial expression patterns, reflecting a precise regulation of their expression (36).

Epitranscriptomic editing of ncRNAs is quite prevalent during physiological, but also pathological conditions (37). miRNA editing is capable of creating alternative miRNAs, known as isomiRs (38). isomiR Bank, a database integrating >300,000 detected isomiRs (https://mcg.ustc.edu.cn/bsc/isomir/) (39), gives an estimate of the extent of additional layers of regulation that this editing process can generate. Although, isomiRs were first dismissed as artifacts, follow-up research has shown that almost half of miRNA transcripts are edited, and these edited transcripts can be loaded into the RNA-induced silencing complex and exert their regulatory activity (40). miRNA modifications happen either in the 3′-end or in the 5′-end sequences. Although, 3′-end editing is more prevalent (41), it mostly influences miRNA stability and activity. 5′-end editing, on the other hand, introduces modifications in the seed sequence, altering the target set of the miRNA and regulating new pathways (42-45). Moreover, circRNA efficiency and translation have been shown to be subject to regulation by distinct RNA modifications, such as m⁶A, 5-methylcytosine (m⁵C) and pseudouridylation (Ψ) modifications (46), as was also the case for numerous lncRNAs, which have been found to have roles in various CVD-related pathways, such as atherosclerosis and pulmonary hypertension (47).

In the present study, the epitranscriptomic modifications are classified into three major categories. The most prevalent form of epitranscriptomic modification, as in epigenetics, is RNA methylation (Fig. 1), which can affect adenosines in different positions [N¹-methyladenosine (m¹A), m⁶A, 2′-O-methylation (Nm)], cytosines [m⁵C, 5-hydroxymethylcytosine (hm⁵C)] or guanosines [7-methylguanosine (m⁷G)] (48). The second group encompasses substitutional modifications (Fig. 2), which include A-to-I and C-to-U RNA editing (49,50). Finally, the third group of modifications includes all epitranscriptomic changes that do not fall into any of the previous two categories [such as Ψ (51) and 8-oxoguanine (8-OxoG)], but nevertheless, have a proven or implied role in CVD (52) (Fig. 3).

In the past decade, dramatic advances in the development of powerful sequencing technologies have facilitated transcriptomic investigation in a faster, more efficient and more in-depth manner than ever before. Such advances have also assisted greatly in the study of epigenetics and epitranscriptomics. The use of RNases constitutes one of the earliest methods of mapping mRNA modifications and still exhibits the highest sensitivity for m⁶A mapping (208). In the same manner, the more recent MAZTER-seq (209) and m⁶A-REF-seq (210) technologies exploit the discovery of methylation-blocked endoRNases. Another method, termed site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography, also known as SCARLET, utilizes site-specific cleavage and splint ligation and has also been extensively used to detect m⁶A modifications in both coding RNA and lncRNAs (211). Furthermore, antibody incorporating techniques have been established for the detection of RNA modifications. These include the m⁶A-LAIC-seq or m⁶A-level and isoform-characterization sequencing method, which uses immunoprecipitation in total RNA samples (56), as well as the widely used MeRIP-Seq technology, which maps m⁶A-methylated RNA through the use of m⁶A-specific antibodies (212). Combining the aforementioned technique with Ab cross-linking, allowed the enhancement of the resolution of the technique, giving rise to methylation individual-nucleotide resolution UV cross-linking and immunoprecipitation (124). Applications of the same principle of cross-linking include PA-m⁶A-seq (213), but also m¹A-MAP (110) in the case of m¹A modification mapping.

Alternative methods for the detection of RNA modifications take advantage of chemical reactions that are limited to a certain type of RNA modification, combining them with short-read sequencing. RNA-BisSeq, aimed toward the mapping of m⁵C, involves the chemical deamination of cytidines except for m⁵C (118). Modern library preparation protocols, yield RNA fragments with nucleotide modifications at the 5′- or 3′-end, which can be used for the enrichment of the RNA fragments in RNA seq libraries (214,215). The Nm-Seq and RibOxi-Seq techniques used to map internal Nm modifications entail the treatment of RNA fragments with NaIO₄ oxidation, which along with additional steps, leads to the enrichment of 3′-Nm-containing fragments and improvement of the final transcriptome-wide RNA analysis (166,216). Using the same principle, RiboMethSeq is based on the protection of the phosphodiester bond in RNA when Nm occurs at the 5′-neighboring ribose (217). Following alkaline hydrolysis, library preparation and 5′- and 3′-extremity counting, the aforementioned protection is translated into a signal.

Mass spectrometry (MS) has also been an invaluable tool for RNA modification analysis. State-of-the-art MS methods are being employed for the detection and quantification of chemical modifications in RNA, yielding different types of information based on the type of MS analysis (218). Top-down MS analysis can identify and localize mass-altering RNA modifications in undigested RNA, while also allowing de novo sequencing to be performed. Nevertheless, non-altering mass modifications, such as m¹A, m⁶A and mass-silent modifications, such as pseudouridine, remain a major challenge (219,220). Bottom-up MS is conducted for the mass mapping of partially hydrolyzed RNA, and MS approaches can generate oligonucleotides and sequencing ladders that can be subsequently interpreted into RNA sequences and localization of the modifications (221). Another MS-based method is nucleoside MS, which is performed on complete RNA hydrolysates, followed by liquid chromatography separation of the nucleoside mixtures. While highly accurate for the detection of chemical modifications, it cannot provide sequence information or localization of the modification (222). Still, each method's advantages can be combined to overcome limitations and drawbacks on high-throughput RNA modification mapping, while appropriate software for MS data processing should always be incorporated (223).

A-to-I modifications are either investigated via the traditional method of screening for A-to-G mismatches in reverse transcribed RNAs (224), by the use of the more recently developed inosine chemical erasing (ICE) methods, or by the use of transgenic mice where ADAR knockdown is followed by deep-sequencing. In the case of ICE, reverse transcription is blocked by the formation of N¹-cyanoethylinosine after acrylonitrile processing. This method combined with deep-sequencing gave rise to ICE-seq, for high-throughput investigation of A-to-I modifications (225). In the case of Ψ modification profiling, several high-throughput sequencing techniques are utilized, wherein treatment with N-cyclohe xyl-N′-(2-morpholinoethyl)-carbodiimide-metho-p-toluenesulfonate specifically modifies Ψ, G and U residues on RNA. Although the G and U modifications are later removed, the chemically induced modification on Ψ is stable and blocks reverse transcription (226). Such methods include Ψ-seq (227), PSI-seq (228), Pseudo-seq (229) and CeU-seq (230).

Novel sequencing approaches enable direct RNA sequencing without amplification or cDNA conversion. The rapidly developing technology of nanopore sequencers, such as the one created by Oxford Nanopore Technologies (ONT), includes the use of a synthetic membrane with embedded nanopores in an ionic solution (231). As an ionic current passes through the nanopore, an individual read is recorded by a sensor and the corresponding data is acquired by the sequencer's implemented software. Characteristic changes in the current reads during the movement of a nucleic acid strand, as it traverses the nanopore from one chamber to the other, enable the identification of the strand's nucleic acid sequence, in a process known as 'base-calling' (232). Nucleotide modifications in ONT reads can be determined with the use of specialized software, such as Nanopolish and the ONT integrated CpG-methylation calling software (233).

High-throughput sequencing techniques have not only promoted the field of epitranscriptomic profiling, but have, through Genome-wide Association Studies (GWAS), facilitated the identification of single nucleotide polymorphisms (SNPs) in a variety of diseases, including CVD (234). These studies have led to the identification of >5,000 associations with CVD (https://www.ebi.ac.uk/gwas/) (235), exhibiting the importance of SNPs in CVD emergence. Several databases have also been developed in an effort to catalogue disease-associated polymorphisms that affect epitranscriptomic modifications. These databases include m⁶Avar (236) and m⁶ASNP (237), both of which catalogue m⁶A-related polymorphisms, m⁷GHub (238) focusing on m⁷G-related SNPs, RMDisease encompassing >200,000 human SNPs that affect m⁶A, m¹A, m⁶Am, m⁵U, m⁷G, Ψ and Nm modifications (239) and the RNA Framework, which is a rounded toolkit for the analysis of post-transcriptional modifications (240).

In terms of epitranscriptomic genetic variation, research remains at an early stage. As expected due to the greater emphasis given so far on m⁶A-related modifications, in the context of CVD, a number of m⁶A-related SNPs have been recognized as genetic variants associated with CVD. Multiple GWAS studies by Mo et al (241) have paved the way in this field and associated m⁶A-SNPs with a variety of CVD factors. More specifically, m⁶A-SNPs were shown to be associated with coronary artery disease (241) and have a potential role in the regulation of blood pressure (242), as well as in the regulation of lipid metabolism (243). Furthermore, several m⁶A-related SNPs were found to affect the expression of multiple disease-causing genes, with potential adverse effects for ischemic stroke in humans (244). In this context, the genetic variant rs12286, which is strongly associated with coronary artery disease, was shown to be able to affect ADAMTS7 expression, by regulating the upstream m⁶A methylation (241). Ali et al (113) analyzed the levels of m¹A/G methylation in mitochondrial-encoded RNA across multiple tissue types, followed by the identification of overlaps between peak associated nuclear variants and disease-associated variants with significance on a genome-wide level. Nuclear genetic variants (rs13874, rs1084535), which are associated with inferred methylation levels at mt-RNR2 and several mt-tRNA P9 sites, were in linkage disequilibrium (LD) with rs34080181, which has been linked to atrial fibrillation (245). Furthermore, the intronic variant in polyribonucleotide nucleotidyltransferase 1 mitochondrial (rs2627773) that is associated with inferred methylation levels of mt-RNR2, is in LD with rs1975487, which is associated with diastolic pressure (246). Franzén et al (247) mapped A-to-I RNA editing quantitative trait loci (edQTLs) in order to identify clinical features associated with RNA editing. Subsequently, they evaluated the disease relevance of RNA editing by intersecting the edQTLs with GWAS data (247). More specifically, the authors intersected edSNPs with lead SNPs from published GWAS data. Of note, the rs10847434 SNP, which is associated with coronary artery disease (248) had an edQTL with an editing site in the 3′ exon of apolipoprotein C1 pseudogene 1, a locus that has been linked to coronary artery disease (249). Additionally, the SNP rs4739066, a polymorphism associated with MI (250), was also found to have two edQTLs with editing sites in the 3′-UTR of the α-tocopherol transfer protein gene, a gene associated with the level of severity of atherosclerotic lesions in the area of the proximal aorta (251). Taken together, the aforementioned studies suggest that there is still a large unexplored area of genetic variation related to CVD pathogenesis, especially in regards to epitranscriptomic modifications.

Although the field of epitranscriptomics is still in its infancy, there are already efforts being made to utilize such new regulatory knowledge for the development of novel therapeutic approaches, both for epigenetic and epitranscriptomic modifications in the context of CVD (19). As previously discussed, most epitranscriptomic research in CVD has so far been focused on m⁶A modifications and, as such, methods have focused on identifying ways to manipulate m⁶A methylation levels in the context of various therapeutic approaches. In a seminal study by Lu et al (252), it was established that curcumin was able to attenuate the effects of lipid metabolism disorder and increase total cholesterol in the liver, via the increase of m⁶A methylation, suggesting a protective role for this modification against hyperlipidemia. Recently, a large scale epitranscriptomic study has been established to identify IHD biomarkers in circulation, termed the IHD-EPITRAN study. This study is expected to include 200 patients, split into two cohorts of IHD and non-IHD patients, focusing on the identification of m⁶A and A-to-I modification biomarkers (253). Additionally, limited approaches have also been taken in an effort to modulate m⁶A demethylation. Inhibition of demethylation was selectively blocked by the use of meclofenamic acid (MA) in vitro (254), while using the recently developed CRISPR-Cas13b technology, Li et al (255) attempted to manipulate m⁶A modified transcripts and specifically demethylate m⁶A marks. In a similar manner, Cox et al (256) developed the RNA Editing for Programmable A to I Replacement system, also utilizing CRISPR-Cas13b technology, to address disease-causing mutations. Although such tools are still outside the reach of clinical practice, as multiple technical and ethical concerns remain unaddressed, they pave the way for the development of future personalized CVD therapeutics.

The field of epitranscriptomics has been rapidly emerging, as the focus regarding disease development, environmental adaptation and homeostasis maintenance, shifts from the rigid genomic structure to the much more dynamic transcriptomic landscape. Although there have been major advances in transcriptomic profiling, understanding the mechanisms in which the transcriptome itself is differentially regulated through modifications, will allow for the development of novel and precise pharmacological interventions. The additional level of regulatory sensitivity that epitranscriptomic modifications are shown to offer, corresponds to the increased level of specificity required for any successful therapeutic intervention. To date, epitranscriptomic modifications are nearing 200, but not all of them have been thoroughly evaluated, nor do they all appear with equal frequency. Although epitranscriptomic research progresses rapidly in the fields of cancer and neurodegenerative disorders (257,258), in the context of CVD the number of modifications that have a significant impact are just beginning to be elucidated (Table I). However, their biological and clinical significance cannot be denied, as shown by the plethora of studies published in the past couple of years, showing the effect of RNA modifications in CVDs (Fig. 4).

Regarding RNA methylations, undoubtedly m⁶A has garnered the most attention. Although coding RNA modifications have been the focus of most m⁶A studies in the heart, a number of publications have emerged, pinpointing the importance of various RNA methylations in multiple levels of non-coding regulation in the heart. These events occur in multiple miRNA maturation stages, including primary-miRNA, pre-miR and mature miRNA levels. Coupled with recently emerging implications regarding methylation modifications in circRNAs and lncRNAs, such as XIST, in the context of CVD, it is becoming evident that this type of epitranscriptomic modification is paramount for physiological non-coding regulation and offers an additional regulatory level of gene expression, sensitive to environmental factors. Nevertheless, methylations are only one of the available modifications in the RNA modification toolset. Substitutional modifications have also been gaining attention in the cardiovascular field, especially with the emergence of recent studies exhibiting the importance of ADAR1 for cardiac development, homeostasis, as well as physiological cardiac function in adult mice (169,170,259). What is noteworthy is that the ADAR1-mediated modification, in this case, involved miR-199a-5p, exhibiting the intricate and dynamic regulation that such modifications offer in tandem with ncRNA regulation. This fact is further highlighted by studies by van der Kwast et al (44,260), where A-to-I editing of miR-478b-3p created an isomiR with a completely different targetome and extensive angiogenic pathway effects, further establishing the relationship between epitranscriptomic modification and miRNA-mediated regulation. Alongside A-to-U editing events, C-to-U editing offers a much more delicate regulation network, with increased specificity. Although cardiac-specific effects in vivo have yet to be reported in relation to C-to-U editing, the expression of the tissue-specific APOBEC-2 deaminase in the heart, coupled with the capacity of APOBEC3A to edit hypertension genes under hypoxic conditions in vitro, point to still unexplored events, similar to the editing of apolipoprotein in the liver, in a tissue-specific manner.

In this context, cardiac aging has recently emerged as an exciting new field, exploring among others, the possible connection between RNA modifications and the various morphological and biomolecular changes that take place during the cardiac aging process. Increased cardiac fibrosis, left ventricular hypertrophy and valvular degeneration are just some of the main physiological changes that occur during human cardiac aging (261). Of note, cardiac fibrosis emergence has been linked to changes in RNA modifications, such as m⁶A and inosine (262), while cardiomyocyte aging has been found to be affected by RNA methylation (263). Cardiomyocyte hypertrophy, responsible for the thickening of the left ventricle walls during cardiac aging, has also been linked to m⁶A methylation. More specifically, in vitro and in vivo experiments have shown that m⁶A RNA methylase METTL3 could promote cardiomyocyte hypertrophy, whereas METTL3 inhibition inhibited the hypertrophic potential of cardiomyocytes (78). All of the above provide just a glimpse of the still unexplored effects that epitranscriptomic regulation may potentially have during cardiac aging.

Finally, a large number of modifications have also been detected in mitochondria. Due to the importance of mitochondria for physiological cardiac function, these editing events can have severe implications for both homeostasis and disease emergence. HF, despite its various complications, has historically been studied as a left ventricular disease. As such, m¹A modifications in mitochondrial 16s rRNA, as well as tRNAs, but also components of the mitochondrial complex I (such as mt-ND5) in the left ventricle, can have severe implications for both HF and various other CVDs. Additionally, NSUN4-mediated mitochondrial m⁵C methylation is required for physiological function, as shown by the emergence of cardiomyopathy, after NSUN4 cardiac-specific deletion. While examining mitochondrial RNA methylation, Van Haute et al (264) demonstrated NSUN3 as a novel human m⁵C RNA methyltransferase, specializing in mitochondrial tRNA^Met. Mutations of NSUN3 caused reduced methylation and absence of formulation of cytosine residues at position 34 of the mitochondrial tRNA^Met, leading to reduced mitochondrial translation and the development of mitochondrial disease. Thus, mitochondrial RNA methylation seems to affect mitochondrial function as well as the translation of mitochondrial proteins, leading to the emergence of pathology (264). Even though other mitochondrial RNA modifications have not been implicated in CVD, it is safe to assume that we are only starting to scratch the surface, as various more mitochondrial modifications have been described, such as Ψ modifications. As already reviewed by Bohnsack and Sloan (265), the mitochondrial epitranscriptome is rapidly gaining interest as a key regulator of dynamic, efficient and accurate responses to metabolic needs. Mutations in mitochondrial RNase P protein 2, a mitochondrial RNase P subcomplex cofactor, participating in m¹A and m1G mt-tRNA modifications, were shown to cause cardiomyopathy (106,266). Last, but not least, a few RNA modifications, such as C-to-U editing by APOBEC3A or m⁶A modification of the FTO protein were shown to be manifesting during hypoxic conditions. Taking into account the extensive role of hypoxia in metabolic regulation, mitochondrial biogenesis and cardiac remodeling, such modifications further cement the role of epitranscriptomic regulation in the adaptation to ever-changing environmental stimuli both in physiological, but also in pathological conditions.

This new knowledge is now paving the way towards a new chapter in personalized medicine (267), where an in-depth understanding of epitranscriptomic modifications could not only enable more accurate patient classification based on epitranscriptomic 'profiles' or specific epitranscriptomic biomarkers, but, more importantly, allow for early predictions of response to treatment. Early evidence in this direction stems from the area of oncology, where specific RNA modifications (e.g. m⁶A) appear to be associated with therapeutic response and/or resistance (268). Significant promise also lies in the development of novel targeted epitranscriptomic therapies against CVD. The fine mapping of the role of epitranscriptomic changes in different aspects of CVD development, is likely to unveil a multitude of promising therapeutic targets, that could subsequently be modulated by targeted approaches, such as small molecule inhibitors. A number of such approaches are currently being pursued against FTO in the milieu of cancer (269). For example, the US Food and Drug Administration-approved nonsteroidal anti-inflammatory drug ethyl ester form of MA, MA2, was found to be an FTO inhibitor, which led to elevated levels of m⁶A modification in mRNAs in glioblastoma cells, suppressing tumor progression and prolonging the lifespan of glioblastoma stem cell-grafted mice (270). MO-I-500 was developed to selectively inhibit the m⁶A demethylase activity of FTO and was found to successfully inhibit the survival and/or colony formation of a triple-negative inflammatory breast cancer cells (271,272). R-2HG was found to bind directly to FTO and inhibit m⁶A demethylase activity leading to the inhibition of leukemic cell growth/survival and leukemia progression (273). FB23-2 was also effective in inhibiting the progression of human AML in xenotransplantation mice, by achieving the potent inhibition of FTO (274). In an effort to expedite the discovery of such inhibitors different predictive in silico approaches are also employed (275-279). The majority of the aforementioned approaches are based on conventional pipelines on databases' data management (280,281). However, at present in the post-genomic era, state-of-the-art approaches based on artificial intelligence are being employed, thus providing novel and radical solutions for the management and analysis of high amounts of data, where algorithms and convolutional networks not only decipher information by removing noise and reducing dimensionality, but also produce new knowledge and associations (282). The tremendous clinical potential of these advancements is supported by the early establishment of multiple companies focusing on epitranscriptomics, such as Accent, Gotham and Storm Therapeutics. It is only a matter of time before similar avenues are explored in the field of CVD, as evidenced by recent studies on epitranscriptomic modification-based therapy solutions.

All of the above serve to show we currently stand at the shore of cardiac epitranscriptomic research, where a vast ocean of information still remains unexplored. We still have to understand the relationship between the number of modifications that each coding or ncRNA carries, to their respective effect, or the methods of action for tissue-specific RNA modifications in response to physiological or pathological stimuli. As we delve deeper into CVD epitranscriptomics, we are sure to come closer to the 'holy grail' of personalized medicine and targeted therapeutics.