In-vitro-Charakterisierung der kardialen Elektrophysiologie beim Noonan- Syndrom und Noonan-Syndrom mit multiplen Lentiginosen anhand eines stammzell-basierten 3D-Modells

by Leonie Böhmker
Doctoral thesis
Date of Examination:2025-03-17
Date of issue:2025-02-27
Advisor:Prof. Dr. Bernhard Danner
Referee:Prof. Dr. Bernhard Danner
Referee:Prof. Dr. Thomas Meyer
Persistent Address: http://dx.doi.org/10.53846/goediss-11107

 

 

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Abstract

English

Noonan syndrome and Noonan syndrome with multiple lentigines are classified as RASopathies, resulting from mutations in the RAS/MAPK signaling pathway. Patients typically exhibit a range of symptoms, with cardiac abnormalities posing the highest health risks. Currently, no definitive causal therapy exists, although early treatments targeting the signaling pathway have shown promising results. Despite identifying the genetic mutations involved, the correlation between genotype and phenotype remains unclear. This research aimed to deepen the understanding of the cardiac phenotype in affected individuals by studying electrophysiology using miniaturized three-dimensional heart tissues. Induced pluripotent stem cells (iPSCs) derived from patients' skin or blood were differentiated into cardiomyocytes and combined with collagen and fibroblasts to create 3D bioartificial cardiac tissues that displayed spontaneous contractions, facilitating disease modeling. Electrophysiological measurements involved using microelectrode arrays to assess extracellular cardiac field potentials, which provide insights into action potentials and excitation propagation. Additionally, genetically encoded calcium indicators were employed to visualize intracellular calcium oscillations within cardiomyocytes through fluorescence microscopy. The study focused on two specific mutations in the RAS/MAPK pathway: SHP2Q510E/+ and RAF1S257L/+. These were compared against isogenic controls and partially treated with a pathway inhibitor. Findings revealed that SHP2-mutant tissues exhibited prolonged field potentials and calcium transients compared to corrected tissues, with the inhibitor partially restoring the normal phenotype. For RAF1-mutant tissues, those without calcium indicators showed shortened field potentials, while those with calcium indicators demonstrated prolonged potentials and altered calcium parameters. The presence of the calcium indicator appeared to have a more significant impact on the electrophysiological phenotype than the underlying mutation, raising concerns about its suitability. Overall, this research advances the understanding of cardiac electrophysiology in RASopathies by elucidating the underlying pathomechanisms and establishing methods for synchronized measurements of field potential, force development, and calcium oscillations in bioartificial heart tissues. These contributions not only enhance the comprehension of excitation-contraction coupling in cardiac cells but also highlight potential avenues for therapeutic intervention.
Keywords: Rasopathy; Noonan syndrome; Noonan syndrome with multiple lentigines; Induced pluripotent stem cells; iPSCs; 3D bioartificial cardiac tissues; electrophysiology
 
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