RNA epigenetic modifications: a new field of research in calcific aortic valve disease

This editorial refers to ‘The N6-methyladenosine demethylase ALKBH5 is a novel epigenetic regulator of aortic valve calcification’ by Y. Wang et al., https://doi.org/10.1093/cvr/cvae253. Calcific aortic valve disease (CAVD) is a frequent heart valve disorder characterized by a thickening and mineralization of aortic valve leaflets (AVLs). Analyses of surgically explanted AVLs have underlined that mineralization of the AVLs is associated with inflammatory cell infiltrates. The production of different cytokines by inflammatory cells provides cues that promote the transition of valve interstitial cells (VICs) into activated fibroblast, which produce extra-cellular matrix (ECM), and osteoblast-like cells involved in the mineralization process. For instance, interleukin-6 (IL6) and interleukin-1 (IL1) promote the osteogenic transition of VICs and the development of CAVD.1 Also, studies have consistently underlined that activation of the transforming growth factor beta 1 (TGFB1) pathway is involved in the activation of VICs and the production of ECM. Together these factors promote cell fate transition of VICs by altering gene regulatory networks through key transcription factors such as RUNX2, MSX2, MRTF, and SMAD2/3.2 Growing evidence support that epigenetic alterations are involved in cell fate determination of VICs and are actively involved in the development of CAVD. Epigenome modifications are known to affect various critical cell functions and are involved in the development and cell fate determination. Epigenomic modifications are under the control of group of molecules including writers, erasers, and readers. These molecules control chemical modifications of histones, DNA, and RNA and exert profound control on gene expression. DNA methylation has been shown to be involved in the regulation of PLPP33 and long noncoding RNA H19,4 which are involved in lysophosphatidic acid signalling and expression of NOTCH1, two important cell fate regulators of VICs. In recent years, methylation of RNA has been shown to be involved in mRNA splicing, stability, nuclear export, and translation. Furthermore, evidence suggest that N6-methyladenosine (m6A) of RNA controls group of transcripts with developmental and cell fate implications.5 RNA methyl transferases including METTL3 and METTL14 add m6A to RNA, whereas erasers consisting in demethylases such as FTO and ALKBH5 remove these modifications. As such a delicate balance control cellular m6A status. Different readers of m6A including YTH domain containing proteins impact cell response to m6A. Wang et al.6 report intriguing findings on the role of m6A and the TGFB-SMAD2 axis in VICs. Targeted approach showed that expression of ALKBH5 was decreased in mineralized portions of surgically explanted AVLs when compared with non-mineralized area of the same valve. These data were corroborated when using diseased and control non-mineralized AVLs. In a series of functional investigation with isolated human primary VICs, the authors showed that knockdown of ALKBH5 promoted the mineralization of cell cultures, whereas the overexpression provided a partial rescue in decreasing inorganic phosphate-induced mineralization. Of note, silencing ALKBH5 in VICs resulted in increased expression of IL1B and TGFBR2, which encodes for a receptor involved in TGB1 signalling. By using meRIP-qPCR, the authors confirmed that silencing of ALKBH5 increased m6A on transcripts encoding for TGFBR2. Next, data using actinomycin-D, a blocker of transcription, showed that downregulation of ALKBH5 in VICs increased the stability of mRNA encoding for TGFBR2. Consistently, experiments showed that overexpression of ALKBH5 decreased the phosphorylation of SMAD2, a transcription factor downstream to TGFBR2. Further experiments showed that SMAD2 was involved in the mineralization of VIC cultures promoted by the knockdown of ALKBH5. Among the YTH domain containing proteins, the silencing of YTHDF1 reduced the mineralization of VIC cultures promoted by the knockdown of ALKBH5. Furthermore, the reduction of YTHBF1 attenuated the impact of lower ALKBH5 on expression of TGFBR2. Taken together, these data provide the evidence that ALKBH5 controls TGFB1-SMAD2 pathway through the m6A reader YTHBF1. This study, the first-of-its-kind in CAVD, highlighting the role of RNA epigenome modifications on osteogenic transition of VICs needs to be commented. The present work is likely just scratching the surface as open-ended approaches such as unbiased m6A-seq and CLIP-seq7 could be used to provide a broader picture regarding the implication of m6A in CAVD. Several unresolved questions remain to be answered, which may shed crucial light on how epigenome alterations participate to CAVD by modifying gene regulatory networks in VICs and valve endothelial cells (VECs), two crucial cell types involved in heart valve functions. To this effect, what are the driving factors that promote alteration of m6A in VICs and VECs during CAVD and how RNA binding proteins (RBPs) modulate mRNA fate in cell- and disease-context conditions? Recent investigations by the GTEx consortium showed that genetic variants impacting m6A as quantitative trait loci (m6A-QTLs) were largely tissue specific and had poor overlap with expression QTLs, but colocalized with genome-wide association study risk loci for several disorders.8 Also, analysis of CLIP-seq data, a technique documenting the interactions of RBPs with RNAs, identified several allele-specific bindings at heterozygous sites.9 Recently, Cas13-directed methyltransferase allows the empirical evaluation of m6A modification on mRNA stability and alternative splicing.10,11 Also, the development of single-cell m6A profiling has provided an additional tool to assess cell populations at different stages of differentiation and ultimately may provide sufficient granularity to assess how m6A may be involved in cell fate transition, a process of key importance in the development of CAVD. Hence, a logical next step will be to interrogate m6A at genome-wide scale in disease-relevant cells such as VICs and VECs and to assess the functional impact of these modifications in large-scale screens. Adding this layer of understanding could be transformative for the field and provide key mechanistic insights about the complex processes involved in the development of CAVD (Figure 1). ALKBH5, an RNA demethylase, is decreased in calcified area of aortic valves vs. non-calcified area. ALKBH5 is also decreased in CAVD vs. control valves. Functional assays show that a knockdown of ALKBH5 in VICs induces a global increase in m6A, notably on genes such as TGFBR2, BMP2, and MSX2, leading to an increase in their expression. Overall, ALKBH5 depletion in the aortic valve leads to increased inflammation trough IL1-β and osteoblastic differentiation of VICs. This work offers new research perspectives on the role of epigenetic RNA modifications in the context of CAVD. Concerning the m6A modification, further work is required to understand the role of this mark on the transcriptome of different valve cell types. Unbiased approaches such as meRIP-seq, m6A-QTL, scm6A-seq coupled with CRISPR-based technologies are required to elucidate the role of m6A in the development of CAVD. CAVD, calcific aortic valve disease; m6A, N6-methyladenosine; meRIP-seq, methylated RNA immunoprecipitation sequencing; m6A-QTL, N6-methyladenosine quantitative trait loci; scm6A-seq, single-cell N6-methyladenosine sequencing. Authors' work is supported by the Canadian Institutes of Health Research project grants PJT191807 (P.M.), PJT159697 (P.M.) and the Quebec Heart and Lung Institute Fund (P.M.). M.B. Is the recipient of a student grant from the Fonds de Recherche du Québec; P.M. is the recipient of the Joseph C. Edwards Foundation granted to Université Laval.

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