Abstract 1353 ADAR2 represses circRNA formation in the human heart

Background & Purpose: Circular RNAs (circRNAs) are covalently closed RNAs formed by an alternative splicing process called back-splicing. The formation and deregulation of circRNAs have been linked to several cardiovascular diseases, such as acute myocardial infarction or heart failure (HF). The back-splicing process of circRNAs has been associated with the formation of double-stranded RNA (dsRNA) structures, which can be formed by primate-specific, inverted Alu repeats. These dsRNA structures are the primary target of the RNA editing enzyme ADAR2. We aimed to analyze the contribution of ADAR2 to circRNA formation in healthy and failing human hearts.

Methods & Results: We analyzed the circular transcriptome in the left ventricle of 20 HF patients and 10 controls using next generation sequencing and found 173 HF-regulated circRNAs. The majority of circRNAs (166) were upregulated in HF patients compared to controls. ADAR2 protein levels were reduced (-68%) in HF patients and enriched after MG-132 treatment, suggesting a proteasomal degradation in the failing human heart. Sequencing analysis of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) treated with shADAR2 revealed 2,654 differentially expressed genes. KEGG pathway analysis of the upregulated genes showed an enrichment of cardiomyopathy-associated genes, indicating a possible contribution of ADAR2 reduction to HF. Analysis of the circular transcriptome in shADAR2-treated hiPSC-CM showed 138 significantly regulated circRNAs, with the majority of circRNAs being upregulated (63%) suggesting a negative association of ADAR2 and circRNA expression in the failing heart. Knockdown of ADAR2 in hiPSC-CM led to the upregulation of 87 circRNAs. Detailed analysis in vitro showed an interaction of ADAR2 with Alu elements flanking the back-splice site of one exemplar circRNA and suppression of this circRNA formation through adenosine-to-inosine RNA editing of Alu elements. Overexpression of this circRNA resulted in an impairment of the sarcomere in hiPSC-CM.

Conclusion: The failing human myocardium is characterized by the upregulation of specific circular non-coding RNA and a reduction of the RNA editing enzyme ADAR2 and subsequent A-to-I RNA editing. Our findings support a mechanistic role of RNA editing in the pathogenesis of human heart failure.

Clin Res Cardiol (2023). https://doi.org/10.1007/s00392-023-02180-w
ADAR2 represses circRNA formation in the human heart
K. E. Kokot¹, J. M. Kneuer¹, D. John², S. Rebs³, M. Möbius-Winkler¹, S. Erbe¹, M. Müller⁴, M. Andritschke¹, S. Gaul¹, B. Sheikh⁵, J. Haas⁶, H. Thiele⁷, O. J. Müller⁸, S. Hille⁸, F. Leuschner⁶, S. Dimmeler², K. Streckfuß-Bömeke³, B. Meder⁶, U. Laufs¹, J.-N. Boeckel¹
¹Klinik und Poliklinik für Kardiologie, Universitätsklinikum Leipzig, Leipzig; ²Zentrum für Molekulare Medizin, Institut für Kardiovaskuläre Regeneration, Goethe Universität Frankfurt am Main, Frankfurt am Main; ³Institut für Pharmakologie und Toxikologie, Universitätsklinikum Würzburg, Würzburg; ⁴Agnes Wittenborg Institut für translationale Herz-Kreislaufforschung, Herz- und Diabeteszentrum NRW, Bad Oeynhausen; ⁵Helmholtz-Institut für Metabolismus-, Adipositas- und Gefäßforschung, Leipzig; ⁶Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie, Universitätsklinikum Heidelberg, Heidelberg; ⁷Klinik für Innere Medizin/Kardiologie, Herzzentrum Leipzig - Universität Leipzig, Leipzig; ⁸Klinik für Innere Medizin III, Schwerpunkt Kardiologie und Angiologie, Universitätsklinikum Schleswig-Holstein, Kiel;
Background & Purpose: Circular RNAs (circRNAs) are covalently closed RNAs formed by an alternative splicing process called back-splicing. The formation and deregulation of circRNAs have been linked to several cardiovascular diseases, such as acute myocardial infarction or heart failure (HF). The back-splicing process of circRNAs has been associated with the formation of double-stranded RNA (dsRNA) structures, which can be formed by primate-specific, inverted Alu repeats. These dsRNA structures are the primary target of the RNA editing enzyme ADAR2. We aimed to analyze the contribution of ADAR2 to circRNA formation in healthy and failing human hearts. Methods & Results: We analyzed the circular transcriptome in the left ventricle of 20 HF patients and 10 controls using next generation sequencing and found 173 HF-regulated circRNAs. The majority of circRNAs (166) were upregulated in HF patients compared to controls. ADAR2 protein levels were reduced (-68%) in HF patients and enriched after MG-132 treatment, suggesting a proteasomal degradation in the failing human heart. Sequencing analysis of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) treated with shADAR2 revealed 2,654 differentially expressed genes. KEGG pathway analysis of the upregulated genes showed an enrichment of cardiomyopathy-associated genes, indicating a possible contribution of ADAR2 reduction to HF. Analysis of the circular transcriptome in shADAR2-treated hiPSC-CM showed 138 significantly regulated circRNAs, with the majority of circRNAs being upregulated (63%) suggesting a negative association of ADAR2 and circRNA expression in the failing heart. Knockdown of ADAR2 in hiPSC-CM led to the upregulation of 87 circRNAs. Detailed analysis in vitro showed an interaction of ADAR2 with Alu elements flanking the back-splice site of one exemplar circRNA and suppression of this circRNA formation through adenosine-to-inosine RNA editing of Alu elements. Overexpression of this circRNA resulted in an impairment of the sarcomere in hiPSC-CM. Conclusion: The failing human myocardium is characterized by the upregulation of specific circular non-coding RNA and a reduction of the RNA editing enzyme ADAR2 and subsequent A-to-I RNA editing. Our findings support a mechanistic role of RNA editing in the pathogenesis of human heart failure.
https://dgk.org/kongress_programme/jt2023/aP1364.html