Investigation of RBM20-mutation based LVNC and DCM in an iPSC model: Novel insights into Ca2+ handling, affection of the atria and crosstalk between cardiomyocytes and endothelial cells

by Tjark Alexander Buchwald
Doctoral thesis
Date of Examination:2025-03-31
Date of issue:2025-03-27
Advisor:Prof. Dr. Katrin Streckfuß-Bömeke
Referee:Prof. Dr. Katrin Streckfuß-Bömeke
Referee:Prof. Dr. Ralf Dressel
Sponsor:Promotionskolleg für Medizinstudierende der UMG (Else-Kröner-Fresenius-Stiftung)
Persistent Address: http://dx.doi.org/10.53846/goediss-11184

 

 

Files in this item

Name:Dissertation Tjark Alexander Buchwald.pdf
Size:2.59Mb
Format:PDF

This file will be freely accessible after 2026-03-31.

Abstract

English

In this study, RBM20-mutation based LVNC and DCM were investigated based on an already established in-vitro platform with iPSC generated from two patients: One had LVNC and the RBM20 mutation p.R634L, the other had DCM and the RBM20 mutation p.R634W. Isogenic rescue iPSC-lines with RBM20-wt had been created for both patients as well. Starting out from the results of a previous doctoral thesis, first, the pluripotency of an isogenic rescue iPSC-line from the LVNC patient was confirmed and complemented. Next, further investigations into the exact pathological mechanisms of both diseases were undertaken concerning expression of major Ca2+ cycling proteins, interaction of iPSC-CM and -EC in the case of LVNC and alternative splicing and sarcomeric regularity in iPSC- aCM. In the first part of this project, after successful cardiac differentiation of the patient-, isogenic rescue- and healthy unrelated control iPSC into iPSC-CM, expression of Ca2+ handling proteins was analysed. Here, the DCM-CM showed no alterations. Meanwhile, the LVNC-CM showed reduced expression of SERCA2a and PLN, while NCX and CamK2δ showed no differences. Furthermore, Cx43 and RBM20 showed no differences in their expression while KCNH2 was downregulated only in the LVNC-CM. These results unravel another part of the complex disturbances in cellular ion handling that occur in both LVNC and DCM. Yet, even more investigations are necessary to fully elucidate these complex pathologies, especially concerning the exact impact of the sometimes counteracting alterations on the final outcome of cellular ion currents and concentrations. In the second part of this project, iPSC-EC were differentiated to investigate EC-CM interplay in the context of LVNC. An in-vitro scratch assay revealed reduction of iPSC-EC wound healing capacity by LVNC-CM supernatant. iPSC-CM sarcomeric regularity under EC influence could not be analysed successfully. Co-cultivation of iPSC-EC and -CM in organoids revealed no effect of iPSC-EC RBM20-mutation status on alternative splicing of RBM20 targets in the CM. This is the first clue that EC-CM interaction is involved in the pathogenesis of RBM20-mutation based LVNC. The main limitation of the findings in this study is that no endocardial cells were used, which should become standard for future experiments into this topic. For the third part of this project, iPSC-aCM of both patients and the three controls were differentiated successfully. They showed no downregulation of RBM20-mRNA and no missplicing of RBM20 targets except for TTN. Analysis of sarcomeric regularity was attempted but only delivered conflicting results. This is the first investigation of RBM20- mutation based cardiomyopathies in iPSC-aCM. The results from the analysis suggest a milder affection of the atria in RBM20-mutation based cardiomyopathy, especially in combination with data from other studies. Further investigation into ion handling, RNP granule formation, sarcomeric regularity, alternative splicing machinery as well as transferring the approach to other models and other genetic cardiomyopathies are warranted.
Keywords: RBM20; iPSC; Alternative Splicing; Calcium Handling; iPS-endothelial cells; Cardiomyopathy