M utations in the RNA-binding motif protein 20 (RBM20) have been shown to cause a clinically aggressive form of dilated cardiomyopathy (DCM).1 A next-generation sequencing study in a large cohort of idiopathic DCM patients revealed that RBM20 belongs to the most frequently affected genes in DCM.2 RBM20 is essential for proper splicing of a large number of genes, and loss of RBM20 induces splicing defects in, for example, titin.3 These splicing defects in titin are thought to be an important reason why these mutations in RBM20 cause DCM. 3,4 However, the pathophysiological role of RBM20 mutations may not be limited to abnormal splicing. Given its essential role in splicing, we hypothesized that RBM20 may also regulate other splicing-dependent processes, like the formation of circular RNAs (circRNAs). If so, this would be of importance because it adds a novel potential disease mechanism. Editorial, see p 966 In This Issue, see p 965Despite their discovery over 20 years ago, circRNAs have only recently been recognized as a novel class of noncoding RNA molecules. Because of their unusual properties, they were presumed to be by-products of aberrant RNA splicing.5 Decades later, next-generation sequencing has revealed that thousands of endogenous circRNAs are expressed in mammals, including the cardiovascular system, 6 and that some of these circRNAs are even more abundant than their linear counterparts. Rationale: 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.Objective: To establish the role of RBM20 in the formation of circRNAs in the heart. Methods and Results: 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. Conclusions: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 circRNAs. Khan et al circRNAs in the Human Heart 997CircRNAs are produced by the canonical spliceosome machinery, by back-splicing of e...

We show that loss of Rbm20 disturbs Ca handling and leads to more proarrhythmic Ca releases from the sarcoplasmic reticulum. Patients that carry a pathogenic RBM20 mutation have more ventricular arrhythmias despite a similar left ventricular function, in comparison with patients with a TTN mutation. Our experimental data suggest that RBM20 mutation carriers may benefit from treatment with an I blocker to reduce their arrhythmia burden.

Peroxisome proliferator-activated receptor δ (PPARδ) is a critical regulator of energy metabolism in the heart. Here, we propose a mechanism that integrates two deleterious characteristics of heart failure, hypoxia and a metabolic shift toward glycolysis, involving the microRNA cluster miR-199a∼214 and PPARδ. We demonstrate that under hemodynamic stress, cardiac hypoxia activates DNM3os, a noncoding transcript that harbors the microRNA cluster miR-199a∼214, which shares PPARδ as common target. To address the significance of miR-199a∼214 induction and concomitant PPARδ repression, we performed antagomir-based silencing of both microRNAs and subjected mice to biomechanical stress to induce heart failure. Remarkably, antagomir-treated animals displayed improved cardiac function and restored mitochondrial fatty acid oxidation. Taken together, our data suggest a mechanism whereby miR-199a∼214 actively represses cardiac PPARδ expression, facilitating a metabolic shift from predominant reliance on fatty acid utilization in the healthy myocardium toward increased reliance on glucose metabolism at the onset of heart failure.

We identified the miR-15 family as a novel regulator of cardiac hypertrophy and fibrosis acting by inhibition of the TGFβ-pathway.