Epitranscriptomics of Ischemic Heart Disease-The IHD-EPITRAN Study Design and Objectives

Vilbert Sikorski, Pasi Karjalainen, Daria Blokhina, Kati Oksaharju, Jahangir Khan, Shintaro Katayama, Helena Rajala, Satu Suihko, Suvi Tuohinen, Kari Teittinen, Annu Nummi, Antti Nykänen, Arda Eskin, Christoffer Stark, Fausto Biancari, Jan Kiss, Jarmo Simpanen, Jussi Ropponen, Karl Lemström, Kimmo Savinainen, Maciej Lalowski, Markku Kaarne, Mikko Jormalainen, Outi Elomaa, Pertti Koivisto, Peter Raivio, Pia Bäckström, Sebastian Dahlbacka, Simo Syrjälä, Tiina Vainikka, Tommi Vähäsilta, Nurcan Tuncbag, Mati Karelson, Eero Mervaala, Tatu Juvonen, Mika Laine, Jari Laurikka, Antti Vento, Esko Kankuri, Vilbert Sikorski, Pasi Karjalainen, Daria Blokhina, Kati Oksaharju, Jahangir Khan, Shintaro Katayama, Helena Rajala, Satu Suihko, Suvi Tuohinen, Kari Teittinen, Annu Nummi, Antti Nykänen, Arda Eskin, Christoffer Stark, Fausto Biancari, Jan Kiss, Jarmo Simpanen, Jussi Ropponen, Karl Lemström, Kimmo Savinainen, Maciej Lalowski, Markku Kaarne, Mikko Jormalainen, Outi Elomaa, Pertti Koivisto, Peter Raivio, Pia Bäckström, Sebastian Dahlbacka, Simo Syrjälä, Tiina Vainikka, Tommi Vähäsilta, Nurcan Tuncbag, Mati Karelson, Eero Mervaala, Tatu Juvonen, Mika Laine, Jari Laurikka, Antti Vento, Esko Kankuri

Abstract

Epitranscriptomic modifications in RNA can dramatically alter the way our genetic code is deciphered. Cells utilize these modifications not only to maintain physiological processes, but also to respond to extracellular cues and various stressors. Most often, adenosine residues in RNA are targeted, and result in modifications including methylation and deamination. Such modified residues as N-6-methyl-adenosine (m6A) and inosine, respectively, have been associated with cardiovascular diseases, and contribute to disease pathologies. The Ischemic Heart Disease Epitranscriptomics and Biomarkers (IHD-EPITRAN) study aims to provide a more comprehensive understanding to their nature and role in cardiovascular pathology. The study hypothesis is that pathological features of IHD are mirrored in the blood epitranscriptome. The IHD-EPITRAN study focuses on m6A and A-to-I modifications of RNA. Patients are recruited from four cohorts: (I) patients with IHD and myocardial infarction undergoing urgent revascularization; (II) patients with stable IHD undergoing coronary artery bypass grafting; (III) controls without coronary obstructions undergoing valve replacement due to aortic stenosis and (IV) controls with healthy coronaries verified by computed tomography. The abundance and distribution of m6A and A-to-I modifications in blood RNA are charted by quantitative and qualitative methods. Selected other modified nucleosides as well as IHD candidate protein and metabolic biomarkers are measured for reference. The results of the IHD-EPITRAN study can be expected to enable identification of epitranscriptomic IHD biomarker candidates and potential drug targets.

Keywords: A-to-I; N6-methyladenosine; RNA modifications; adenosine-to-inosine; biomarkers; epitranscriptomics; ischemic heart disease; m6A.

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; or in the writing of the manuscript.

Figures

Figure 1
Figure 1
Overview of the RNA m6A modification and A-to-I RNA editing and their known writers, readers, and erasers. m6A modification and A-to-I editing occur in most RNA species. ADAD1-2, adenosine deaminase domain-containing protein 1-2; ADAR1-3, double-stranded RNA-specific adenosine deaminase 1-3; ADAT1-3, adenosine deaminases acting on tRNAs; ALKBH5, alkB homolog 5 RNA demethylase; A-to-I, adenosine-to-inosine RNA editing; circRNA, circular RNA; ENDOV, human endonuclease V; eIF3, eukaryotic initiation factor 3; EWSR1, Ewing sarcoma breakpoint region 1 protein; FMRP, fragile X retardation protein; FTO, fat mass and obesity associated protein; G3BP1, Ras GTPase-activating protein-binding protein 1; HAKAI, E3 ubiquitin-protein ligase Hakai; HNRNP-A2B1,-C,-G, heterogeneous nuclear ribonucleoprotein A2/B1 and C1/C2 and G; HuR, human antigen R; IGF2BP1-3; The insulin-like growth factor-2 mRNA-binding proteins 1, 2, and 3; LIN28A, Lin-28 homolog A; lncRNA, long non-coding RNA; METTL3,-14,-16, N6- adenosine-methyltransferase catalytic subunit/non-catalytic subunit/METTL16; METTL5, methyltransferase Like 5; mRNA, messenger RNA; miRNA, microRNA; Prcc2a, proline rich coiled-coil 2 A; RBM15, RNA binding motif protein 15; rRNA, ribosomal RNA; snoRNA, small nucleolar RNA; TRMT112, TRNA methyltransferase subunit 11-2; tRNA, transfer RNA; VIRMA, vir like m6A methyltransferase associated; WTAP, Wilm’s tumor associated protein; (YTH)DC1-2, YTH domain-containing protein 1 ja 2; (YTH)DF1-3, YTH N6-methyladenosine RNA binding protein 1-3; ZCCHC4, zinc finger CCHC-type containing 4; ZC3H13, zinc finger CCCH domain-containing protein 13; *, miRNAs can also derive from pre-mRNA introns.
Figure 2
Figure 2
IHD-EPITRAN hypotheses (A). Coronary plaques signal bone marrow residing HSCs to increase proliferation promoting the efflorescence of CH and extramedullary hematopoiesis (Ly-6Chigh monocytosis), which both seed epitranscriptomically distinct cells to the circulation. (B). Leukocytes and platelets patrolling in the proximity and inside the atherosclerotic plaques, ischemic myocardium, and stressed endothelium oscillate back and seed EVs to the circulation with detectable alterations in their m6A and A-to-I RNA signatures. (C). The ischemic myocardium prime patrolling leukocytes and secrete paracrine EVs encasing m6A and A-to-I modified RNA molecules, entering also to the circulation. A-to-I, adenosine-to-inosine; CH, clonal hematopoiesis; EV, extracellular vesicle; HSC, hematopoietic stem cell; Ly6C, lymphocyte antigen 6; m6A, N6-methyladenosine; RBCs, red blood cells; TCs, thrombocytes; WBCs, white blood cells.
Figure 3
Figure 3
Outline and sample collection in the IHD-EPITRAN study. The gray scale provides an arbitrary scale for disease severity across cohorts. STEMI, ST-elevation myocardial infarction; CABG, coronary artery bypass grafting; AVR, aortic valve replacement; CCTA, coronary computed tomography angiogram; ICA, invasive coronary angiography; RAA, right atrial appendage; IHD, ischemic heart disease.
Figure 4
Figure 4
Study samples with respective principal analysis methods of the IHD-EPITRAN study. CVD, cardiovascular disease; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; IHD, ischemic heart disease; meRIP seq, methylated RNA immunoprecipitation sequencing; MRM, multiple reaction monitoring; RAA, right atrial appendage; UHPLC-MS/MS; ultra-high-performance triple quadrupole liquid chromatography tandem mass spectrometry.
Figure 5
Figure 5
Principal study cohort comparisons in the IHD-EPITRAN study. Referred outcomes are listed in Table 3 (A). Venn diagram-based illustration of the preferred four-partite approach, due to its highest degree of adjustments, to acquire inter-cohort comparison-based outcomes. (B). Three-partite comparison scheme to enable comparisons even in the possible case of delay in one cohort recruitment, which is also the case with (C) depicting pairwise comparisons with lest adjustments for inter-cohort outcomes. Intracohort outcomes are achieved via pairwise prospective comparisons. (D). Timeline for the prospective outcome comparisons. Long-term follow-up of the study cohorts I-IV could provide dimensions of detecting incident and IHD exacerbations (Section 2.6 and Discussion). Abbreviations: AVR, aortic valve replacement cohort III; AVS, aortic valve stenosis; CABG, coronary artery bypass grafting cohort II; CCTA, coronary computed tomography angiogram cohort I; CVD, cardiovascular disease; IHD, ischemic heart disease, MACCE, major adverse cardiovascular and cerebrovascular event; PCI, percutaneous coronary intervention; STEMI, ST-elevation myocardial infarction cohort I.

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