Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

Investigation of RBM20-mutation based cardiomyopathy in atrial cardiomyocytes

T. A. Buchwald1, S. Rebs2, F. Sedaghat-Hamedani3, E. Kayvanpour3, B. Meder3, G. Hasenfuß1, K. Streckfuß-Bömeke2, für die Studiengruppe: DZHK

1Herzzentrum, Klinik für Kardiologie und Pneumologie, Universitätsmedizin Göttingen, Göttingen; 2Institut für Pharmakologie und Toxikologie, Universitätsklinikum Würzburg, Würzburg; 3Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie, Universitätsklinikum Heidelberg, Heidelberg;

Background: Mutations in the splice factor RBM20 account for up to 6% of genetic cardiomyopathies. Mutations at position R634 in the hotspot RS-domain were found to cause dilative cardiomyopathy (DCM) (R634W) or left ventricular non-compaction cardiomyopathy (LVNC) (R634L). Furthermore, RBM20 cardiomyopathy was defined as a highly penetrant and arrhythmogenic cardiomyopathy. While these diseases have been extensively studied in ventricular cardiomyocytes, the functional consequences and possible pathogenicity of RBM20-mutations in atrial cardiomyocytes is still an unmet need. Purpose: Here, we aimed to identify whether the distinct RBM20 mutations from DCM and LVNC patients cause a disease phenotype in atrial cardiomyocytes in comparison to the ventricular counterparts in a RBM20-patient-specific stem cell model of atrial cardiomyocytes (iPSC-aCM). Methods: We generated iPSC-aCM of a DCM- and an LVNC-patient harboring the RBM20-mutations R634W (DCM) and R634L (LVNC) and investigated RBM20-dependent alternative splicing by quantitative PCR and Ca2+ homeostasis with confocal microscopy and Fluo-4 dyes in iPSC-aCM. To investigate the direct impact of the RBM20-mutations at p.R634, rescue lines with wildtype RBM20 were generated by CRISPR/Cas9 technology and used as isogenic control lines. Results: We were able to demonstrate successful differentiation of both patient- and isogenic rescue lines into atrial iPSC-CM expressing typical atrial marker such as MLC2a or PITX2. In contrast to ventricular DCM and LVNC-iPSC-CM, showing shared and differential missplicing of sarcomeric (TTN, LDB3) and Ca2+ handling (RYR2, CaMK2d, TRDN) RBM20 target genes, , only TTN was misspliced in both DCM and LVNC iPSC-aCM, which was reversed in the isogenic rescue-lines. None of the other known RBM20 splicing targets showed any differential missplicing in the atrial cells. We analyzed the impact of the RBM20 mutations p.R624W/L on the calcium homeostasis and found that LVNC-aCM show a significantly fastened transient rise time compared to control-, isogenic rescue- and DCM-aCM, similar to their ventricular counterparts. In contrast, the significant calcium leakage previously observed in ventricular DCM-CM is not present in DCM-aCM. Conclusion: We show the first iPSC-model of splice-defect associated RBM20-dependent LVNC and DCM in atrial cardiomyocytes. Our results suggest that the RBM20-dependent alternative splicing machinery differs between atrial and ventricular cardiomyocytes. In summary, the RBM20-R634L-LVNC-aCM exhibit structural and calcium handling aberrations, whereas RBM20-R634W-DCM show none of these pathologies in the aCM.

https://dgk.org/kongress_programme/jt2022/aP1886.html