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.

Sudden cardiac death (SCD) is the most common mode of death outside of infancy in childhood hypertrophic cardiomyopathy (HCM),^1,2 with higher annual rates compared with those in adults with the disease.³ Current practice guidelines recommend primary prevention implantable cardioverter defibrillators (ICDs) in children based on the presence of clinical risk factors for SCD extrapolated mostly from observational adult studies,^4,5 but this approach poorly discriminates risk in both adult and pediatric populations.^6,7 A clinical risk tool (HCM Risk-SCD) that estimates the 5-year risk of SCD was developed⁸ and validated in adults with HCM.^9-12 The aim of this study was to develop a similar pediatric SCD risk model using a large, international cohort and compare its performance with the adult model.

The aim of this study was to determine the frequency of heterozygous truncating ALPK3 variants (ALPK3tv) in patients with hypertrophic cardiomyopathy (HCM) and confirm their pathogenicity using burden testing in independent cohorts and family co-segregation studies. In a discovery cohort of 770 index patients with HCM, 12 (1.56%) were heterozygous for ALPK3tv [odds ratio(OR) 16.11, 95% confidence interval (CI) 7.94–30.02, P = 8.05e−11] compared to the Genome Aggregation Database (gnomAD) population. In a validation cohort of 2047 HCM probands, 32 (1.56%) carried heterozygous ALPK3tv (OR 16.17, 95% CI 10.31–24.87, P < 2.2e−16, compared to gnomAD). Combined logarithm of odds score in seven families with ALPK3tv was 2.99. In comparison with a cohort of genotyped patients with HCM (n = 1679) with and without pathogenic sarcomere gene variants (SP+ and SP−), ALPK3tv carriers had a higher prevalence of apical/concentric patterns of hypertrophy (60%, P < 0.001) and of a short PR interval (10%, P = 0.009). Age at diagnosis and maximum left ventricular wall thickness were similar to SP− and left ventricular systolic impairment (6%) and non-sustained ventricular tachycardia (31%) at baseline similar to SP+. After 5.3 ± 5.7 years, 4 (9%) patients with ALPK3tv died of heart failure or had cardiac transplantation (log-rank P = 0.012 vs. SP− and P = 0.425 vs. SP+). Imaging and histopathology showed extensive myocardial fibrosis and myocyte vacuolation. Heterozygous ALPK3tv are pathogenic and segregate with a characteristic HCM phenotype. Truncating variants in ALPK3 are a cause of 1–2% of autosomal dominant hypertrophic cardiomyopathy and are associated with a phenotype characterized by extensive fibrosis and a predominantly concentric or apical pattern of left ventricular hypertrophy without left ventricular outflow tract obstruction. (A) Main phenotype characteristics and outcomes. ALPK3tv, alpha-protein kinase 3-truncating variants; CK, creatine kinase; HCM, hypertrophic cardiomyopathy; ICD, implantable cardioverter–defibrillator; LVH, left ventricular hypertrophy; LVSD, left ventricular systolic dysfunction; SCD, sudden cardiac death. (B) Cardiac magnetic resonance imaging showing the prevalent phenotype of severe mid to apical hypertrophy and extensive late gadolinium enhancement in three patients. Left to right: 4-chamber view end-diastole cine image, 4-chamber view late gadolinium enhancement image, short axis late gadolinium enhancement image. (C) Kaplan–Meier analysis comparing incidence of an outcome of heart failure death and transplant between ALP3tv patients, sarcomere positive and sarcomere negative. SARC+: sarcomere positive; SARC−: sarcomere negative.

Aims Mutations in the RS-domain of RNA-binding motif protein 20 (RBM20) have recently been identified to segregate with aggressive forms of familial dilated cardiomyopathy (DCM). Loss of RBM20 in rats results in missplicing of the sarcomeric gene titin ( TTN ). The functional and physiological consequences of RBM20 mutations outside the mutational hotspot of RBM20 have not been explored to date. In this study we investigated the pathomechanism of DCM caused by a novel RBM20 mutation in human cardiomyocytes. Methods and Results We identified a family with DCM carrying a mutation (RBM20E913K/+) in a glutamate-rich region of RBM20. Western blot analysis of endogenous RBM20 protein revealed strongly reduced protein levels in the heart of a RBM20E913K/+ carrier. RNA deep-sequencing demonstrated massive inclusion of exons coding for the spring region of titin in the RBM20E913K/+ carrier. Titin isoform analysis revealed a dramatic shift from the less compliant N2B towards the highly compliant N2BA isoforms in RBM20E913K/+ heart. Moreover, an increased sarcomere resting-length was observed in single cardiomyocytes and isometric force measurements revealed an attenuated Frank-Starling mechanism (FSM), which was rescued by protein kinase A treatment. Conclusions A mutation outside the mutational hotspot of RBM20 results in haploinsufficiency of RBM20. This leads to disturbed alternative splicing of TTN , resulting in a dramatic shift to highly compliant titin isoforms and an impaired FSM. These effects may contribute to the early onset, and malignant course of DCM caused by RBM20 mutations. Altogether, our results demonstrate that heterozygous loss of RBM20 suffices to profoundly impair myocyte biomechanics by its disturbance of TTN splicing.

Objective: To explore the onset and progression of cardiac involvement in juvenile neuronal ceroid lipofuscinosis (JNCL). Methods: The study population comprised an unselected group of 29 children and adolescents with genetically verified JNCL. We focused on T-wave abnormalities on an EKG, cardiac hypertrophy, and left ventricular systolic function on echocardiography, and heart rates and heart rate variability (HRV) on 24-hour EKG recordings. The surviving patients were observed for 7½ years. The 24-hour EKG recording was repeated after 3 years. Results: Abnormally deeply inverted T waves were present in one-third of the initial EKG recordings and were reported as early as 14 years of age. We found coherence between the presence of repolarization disturbances of the ventricular myocardium at the initial recordings and risk of death during the observation period. At increasing age, heart rate and HRV, expressed as the vagal index (number of adjacent RR intervals deviating more than 6%), were significantly reduced, suggesting an age-dependent bidirectional effect of JNCL on heart rate: one through decreasing parasympathetic activity on the heart and the other through a direct negative influence on sinus node automaticity. Coherence between bradycardia and arrhythmia and occurrence of sinus arrests and atrial flutter with increasing age indicated an age-dependent decrease in sinus node activity also. In the early 20s, a high frequency of ventricular hypertrophy occurred. Conclusions: Progressive cardiac involvement with repolarization disturbances, ventricular hypertrophy, and sinus node dysfunction occur in JNCL. We recommend that the attention on heart involvement in JNCL and other neuronal ceroid lipofuscinosis subtypes should be intensified.