The expression and regulation of circRNAs has been associated with chronic systolic heart failure (HF). Adenosine-to-Inosine (A-to-I) RNA editing is the most abundant form of RNA editing in humans. It is mediated by the editing enzymes ADAR1 and ADAR2. The extent of A-to-I editing and the resulting effects on circular RNA formation in the healthy and diseased human heart are not known.

We analyzed RNA editing events and circRNA expression in human control (n=10) and failing left ventricles (n=20) using Next Generation Sequencing (NGS) and subsequent processing with the RNAEditor tool. Most RNA editing events were located in intronic regions of protein coding genes (72.6%), here, primarily in Alu elements. In HF patients, RNA editing was significantly reduced in intronic regions of protein coding genes. We identified A-G (42.9%) and T-C (41.2%) nucleotide switches to be the most prominent RNA modifications, both coding for adenosine-to-inosine editing (A-to-I) and being reduced in heart failure patients compared to controls. A significant reduction of RNA editing was found in reported HF-associated genes, such as RYR2 (66.4% reduction), PLN (87.4% reduction) and AKAP13 (70.5% reduction) compared to controls. Out of 6,601 circRNAs detected in the human heart, 165 were significantly upregulated in HF patients. A total of 109 of these circRNAs were independently regulated from their host genes suggesting an alternative splicing mechanism. Among those, we identified two circular transcripts of the AKAP13 gene. In the AKAP13 mRNA, A-to-I editing was also significantly reduced in Alu elements located in introns flanking the circAKAP13 backsplice-site (BSS). Interestingly, ADAR2 protein expression was significantly reduced in the failing heart (69% reduction). We found ADAR2 to interact with the flanking Alu elements using RNA IP. Reduction of ADAR2 led to a significant elevation of circAKAP13 (1.88-fold), while editing in the flanking Alu elements was reduced. Vice versa, ADAR2 overexpression resulted in reduced circAKAP13 levels (39.7% reduction). Finally, we designed an AKAP13 mini-gene containing exons 15-19 as well as different intronic Alu elements flanking the circAKAP13 BSS. In accordance with our previous findings, ADAR2 overexpression resulted in reduced circAKAP13 levels (45.4% reduction), while editing in the flanking Alu elements was increased (6.3-fold).

In summary, we determined the extent of RNA editing in the healthy and the failing human heart for the first time. We found an association of reduced A-to-I RNA editing in intronic Alu elements and increased circRNA formation, potentially mediated by ADAR2. Our findings suggest an ADAR2-mediated mechanism controlling circRNA formation in the human heart.