Abstract 14824: Cardiomyopathy-associated RBM20-mutations Within the RS Domain Lead to Aberrant Splicing of TTN and RYR2 in Human Myocardium and to a Mislocalisation of the RBM20-protein
Introduction: Mutations in human RBM20 have previously been shown to cause dilated cardiomyopathy. Within the nucleus RBM20 partially colocalises with other splice factors and binds RNA. Deep sequencing of the cardiac transcriptome of a RBM20 deficient rat model revealed RBM20 dependent regulation of myocardial alternative splicing. We have identified the previously published RBM20 mutation p.P638L and the novel mutation p.P635C in two German families. The mutations are highly penetrant leading to terminal heart failure and/or to sudden cardiac death.
Hypothesis: Little is known on aberrant myocardial splicing in human RBM20-mutation carriers. Goal of this study was to gain insight into the mechanisms leading to cardiomyopathy by analysing ventricular myocardium of RBM20-mutation carriers. Additionally, we investigated the molecular defects of the RBM20 mutations on its subcellular distribution.
Methods: Cardiac tissue from patients with RBM20-mutations was obtained during heart transplantation or the implantation of a ventricular assist device, respectively. Ventricular myocardium was used for splicing analysis of TTN and RYR2 by real-time-PCR. The alternative myocardial splicing of Titin was additionally examined by Western Blot. Additionally the localization of RBM20-EYFP protein chimera was compared between mutants and wildtype.
Results: We identified aberrant RYR2 splicing in the RBM20 mutant myocardium characterised by an increased inclusion of an additional 24bp-exon. Furthermore we found a decrease of the major cardiac titin splice variant N2B by real-time PCR and Titin Western Blots in the RBM20 mutants. In contrast to the wild type RBM20-EYFP which localized to the nucleus of the analysed cells, RBM20-p.S635C and RBM20-P638L mutants showed no nuclear localisation. In contrast, RBM20-mutations outside of the conserved RS-domain did not abolish the nuclear localization of RBM20.
Conclusions: We present here first data showing that mutations affecting the conserved RS-domain of RBM20 lead to a subcellular mislocalisation of the protein. We assume that the absence of the mutant protein from the nucleus contributes to the aberrant splicing of RYR2 and TTN which we could observe in the explanted myocardium of mutation carriers.
Author Disclosures: A. Gaertner-Rommel: None. B. Klauke: None. W. Linke: None. D. Kececioglu: None. K. Laser: None. J. Gummert: None. H. Milting: None.
- © 2016 by American Heart Association, Inc.