Cardiac circRNAs Arise Mainly From Constitutive Exons Rather Than Alternatively Spliced Exons

Circular RNAs (circRNAs) are a relatively new class of RNA molecules, and knowledge about their biogenesis and function is still in its infancy. It was recently shown that alternative splicing underlies the formation of circular RNAs (circRNA) arising from the Titin (TTN) gene. Since the main mechanism by which circRNAs are formed is still unclear, we hypothesized that alternative splicing, and in particular exon skipping, is a major driver of circRNA production. We performed RNA sequencing on human and mouse hearts, mapped alternative splicing events, and overlaid these with expressed circRNAs at exon-level resolution. In addition, we performed RNA sequencing on hearts of Rbm20 KO mice to address how important Rbm20-mediated alternative splicing is in the production of cardiac circRNAs. In human and mouse hearts, we show that cardiac circRNAs are mostly (~90%) produced from constitutive exons and less (~10%) from alternatively spliced exons. In Rbm20 KO hearts, we identified 38 differentially expressed circRNAs of which 12 were produced from the Ttn gene. Even though Ttn appeared the most prominent target of Rbm20 for circularization, we also detected Rbm20-dependent circRNAs arising from other genes including Fan1, Stk39, Xdh, Bcl2l13, and Sorbs1. Interestingly, only Ttn circRNAs seemed to arise from Rbm20-mediated skipped exons. In conclusion, cardiac circRNAs are mostly derived from constitutive exons, suggesting that these circRNAs are generated at the expense of their linear counterpart and that circRNA production impacts the accumulation of the linear mRNA

SCN5A mutation type and topology are associated with the risk of ventricular arrhythmia by sodium channel blockers

Background: Ventricular fibrillation in patients with Brugada syndrome (BrS) is often initiated by premature ventricular contractions (PVCs). Presence of SCN5A mutation increases the risk of PVCs upon exposure to sodium channel blockers (SCB) in patients with baseline type-1 ECG. In patients without baseline type-1 ECG, however, the effect of SCN5A mutation on the risk of SCB-induced arrhythmia is unknown. We aimed to establish whether presence/absence, type, and topology of SCN5A mutation correlates with PVC occurrence during ajmaline infusion. Methods and results: We investigated 416 patients without baseline type-1 ECG who underwent ajmaline testing and SCN5A mutation analysis. A SCN5A mutation was identified in 88 patients (S+). Ajmaline-induced PVCs occurred more often in patients with non-missense mutations (Snon-missense) or missense mutations in transmembrane or pore regions of SCN5A-encoded channel protein (Smissense-TP) than patients with missense mutations in intra-/extracellular channel regions (Smissense-IE) and patients without SCN5A mutation (S−) (29%, 24%, 9%, and 3%, respectively; P < 0.001). The proportion of patients with ajmaline-induced BrS was similar in different mutation groups but lower in S− (71% Snon-missense, 63% Smissense-TP, 70% Smissense-IE, and 34% S−; P < 0.001). Logistic regression indicated Snon-missense and Smissense-TP as predictors of ajmaline-induced PVCs. Conclusions: SCN5A mutation is associated with an increased risk of drug-induced ventricular arrhythmia in patients without baseline type-1 ECG. In particular, Snon-missense and Smissense-TP are at high risk

Circular RNAs open a new chapter in cardiovascular biology

Circular RNAs (circRNAs) are emerging as a new class of non-coding RNA molecules. This unusual class of RNA species is generated by a back-splicing event of one or two exons, resulting in a covalently closed circRNA molecule. Owing to their circular form, circRNAs are protected from degradation by exonucleases and have greater stability than linear RNA. Advances in computational analysis of RNA sequencing have revealed that thousands of different circRNAs are expressed in a wide range of mammalian tissues, including the cardiovascular system. Moreover, numerous circRNAs are expressed in a disease-specific manner. A great deal of progress has been made in understanding the biogenesis and function of these circRNAs. In this Review, we discuss the current understanding of circRNA biogenesis and function, with a particular emphasis on the cardiovascular system

circRNAprofiler: an R-based computational framework for the downstream analysis of circular RNAs

BACKGROUND: Circular RNAs (circRNAs) are a newly appreciated class of non-coding RNA molecules. Numerous tools have been developed for the detection of circRNAs, however computational tools to perform downstream functional analysis of circRNAs are scarce. RESULTS: We present circRNAprofiler, an R-based computational framework that runs after circRNAs have been identified. It allows to combine circRNAs detected by multiple publicly available annotation-based circRNA detection tools and to analyze their expression, genomic context, evolutionary conservation, biogenesis and putative functions. CONCLUSIONS: Overall, the circRNA analysis workflow implemented by circRNAprofiler is highly automated and customizable, and the results of the analyses can be used as starting point for further investigation in the role of specific circRNAs in any physiological or pathological condition

A comparison of myocardial magnetic resonance extracellular volume mapping at 3 T against histology of tissue collagen in severe aortic valve stenosis and obstructive hypertrophic cardiomyopathy

Objective: Quantitative extracellular volume fraction (ECV) mapping with MRI is commonly used to investigate in vivo diffuse myocardial fibrosis. This study aimed to validate ECV measurements against ex vivo histology of myocardial tissue samples from patients with aortic valve stenosis or hypertrophic cardiomyopathy. Materials and methods: Sixteen patients underwent MRI examination at 3 T to acquire native T1 maps and post-contrast T1 maps after gadobutrol administration, from which hematocrit-corrected ECV maps were estimated. Intra-operatively obtained myocardial tissue samples from the same patients were stained with picrosirius red for quantitative histology of myocardial interstitial fibrosis. Correlations between in vivo ECV and ex vivo myocardial collagen content were evaluated with regression analyses. Results: Septal ECV was 30.3% ± 4.6% and correlated strongly (n = 16, r = 0.70; p = 0.003) with myocardial collagen content. Myocardial native T1 values (1206 ± 36 ms) did not correlate with septal ECV (r = 0.41; p = 0.111) or with myocardial collagen content (r = 0.32; p = 0.227). Discussion: We compared myocardial ECV mapping at 3 T against ex vivo histology of myocardial collagen content, adding evidence to the notion that ECV mapping is a surrogate marker for in vivo diffuse myocardial fibrosis

RBM20 Regulates Circular RNA Production From the Titin Gene

RNA-binding motif protein 20 (RBM20) is essential for normal splicing of many cardiac genes, and loss of RBM20 causes dilated cardiomyopathy. Given its role in splicing, we hypothesized an important role for RBM20 in forming circular RNAs (circRNAs), a novel class of noncoding RNA molecules. To establish the role of RBM20 in the formation of circRNAs in the heart. Here, we performed circRNA profiling on ribosomal depleted RNA from human hearts and identified the expression of thousands of circRNAs, with some of them regulated in disease. Interestingly, we identified 80 circRNAs to be expressed from the titin gene, a gene that is known to undergo highly complex alternative splicing. We show that some of these circRNAs are dynamically regulated in dilated cardiomyopathy but not in hypertrophic cardiomyopathy. We generated RBM20-null mice and show that they completely lack these titin circRNAs. In addition, in a cardiac sample from an RBM20 mutation carrier, titin circRNA production was severely altered. Interestingly, the loss of RBM20 caused only a specific subset of titin circRNAs to be lost. These circRNAs originated from the RBM20-regulated I-band region of the titin transcript. We show that RBM20 is crucial for the formation of a subset of circRNAs that originate from the I-band of the titin gene. We propose that RBM20, by excluding specific exons from the pre-mRNA, provides the substrate to form this class of RBM20-dependent circRNA

Titin Circular RNAs Create a Back-Splice Motif Essential for SRSF10 Splicing

BACKGROUND: TTN (Titin), the largest protein in humans, forms the molecular spring that spans half of the sarcomere to provide passive elasticity to the cardiomyocyte. Mutations that disrupt the TTN transcript are the most frequent cause of hereditary heart failure. We showed before that TTN produces a class of circular RNAs (circRNAs) that depend on RBM20 to be formed. In this study, we show that the back-splice junction formed by this class of circRNAs creates a unique motif that binds SRSF10 to enable it to regulate splicing. Furthermore, we show that one of these circRNAs (cTTN1) distorts both localization of and splicing by RBM20. METHODS: We calculated genetic constraint of the identified motif in 125 748 exomes collected from the gnomAD database. Furthermore, we focused on the highest expressed RBM20-dependent circRNA in the human heart, which we named cTTN1. We used shRNAs directed to the back-splice junction to induce selective loss of cTTN1 in human induced pluripotent stem cell-derived cardiomyocytes. RESULTS: Human genetics suggests reduced genetic tolerance of the generated motif, indicating that mutations in this motif might lead to disease. RNA immunoprecipitation confirmed binding of circRNAs with this motif to SRSF10. Selective loss of cTTN1 in human induced pluripotent stem cell-derived cardiomyocytes induced structural abnormalities, apoptosis, and reduced contractile force in engineered heart tissue. In line with its SRSF10 binding, loss of cTTN1 caused abnormal splicing of important cardiomyocyte SRSF10 targets such as MEF2A and CASQ2. Strikingly, loss of cTTN1 also caused abnormal splicing of TTN itself. Mechanistically, we show that loss of cTTN1 distorts both localization of and splicing by RBM20. CONCLUSIONS: We demonstrate that circRNAs formed from the TTN transcript are essential for normal splicing of key muscle genes by enabling splice regulators RBM20 and SRSF10. This shows that the TTN transcript also has regulatory roles, besides its well-known signaling and structural function. In addition, we demonstrate that the specific sequence created by the back-splice junction of these circRNAs has important functions. This highlights the existence of functionally important sequences that cannot be recognized as such in the human genome but provides an as-yet unrecognized source for functional sequence variation