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

Functional consequences of Calsequestrin mutation p.F189L in a Takotsubo Syndrom stem cell model

G. Syed Ali1, N. Dybkova1, Y. Li2, W. Maurer1, D. Hübscher1, J.-R. Ghadri3, G. Hasenfuß1, C. Templin3, S. T. Sossalla4, B. Wollnik2, K. Streckfuß-Bömeke5, für die Studiengruppe: DZHK

1Herzzentrum, Klinik für Kardiologie und Pneumologie, Universitätsmedizin Göttingen, Göttingen; 2Institut für Humangenetik, Universitätsmedizin Göttingen, Göttingen; 3Universitäres Herzzentrum, UniversitätsSpital Zürich, Zürich, CH; 4Klinik und Poliklinik für Innere Med. II, Kardiologie, Universitätsklinikum Regensburg, Regensburg; 5Institut für Pharmakologie und Toxikologie, Universitätsklinikum Würzburg, Würzburg;

Background and Aims: Takotsubo Syndrome (TTS) is characterized by acute transient left ventricular dysfunction in the absence of obstructive coronary lesions. We identified a higher sensitivity to catecholamine-induced stress toxicity as mechanism associated with the TTS phenotype. Although, a genetic predisposition is proposed for TTS, the pathomechanism remains to be clarified. In this work, we aimed to analyze the functional consequences of the mutation p.F189L in the calcium buffering protein Calsequestrin 2 (CasQ2), which we identified in one of our TTS patients, and its contribution to the development of TTS in a patient-specific stem cell model. Methods and Results: We used induced pluripotent stem cells-derived cardiomyocytes (iPSC-CM) of a TTS patient harboring the mutation p.F189L for functional analysis. In order to generate isogenic iPSC control lines, we performed CRISPR/Cas9 genome editing to rescue the identified heterozygous CasQ2 variant (C567>G; p.F189L) in the TTS-iPSCs to wild type (p.F189F, WT-TTS), or we introduced this heterozygous variant in control iPSC-lines (p.F189L+/-, het-CTRL). These cell lines were additionally compared to healthy control cell lines (CTRL). Pluripotency of all CRISPR/Cas9-generated iPSC lines was confirmed via gene/protein expression of pluripotency markers, in vitro differentiation capacity, STR-Analysis for pedigree proof and geno- and karyotyping. High cTNT positive iPSC-CM of over 90 % indicated successful cardiac differentiation of two to three month old iPSC-CMs for functional read-outs. The influence of CasQ2-F189L on sarcomeric reticulum (SR) calcium parameters was analyzed by epifluorescence microscopy using FURA2 and caffeine-applications. We found significantly decreased SR calcium content with an increased fractional release during systole as well as increased SERCA activity in TTS-iPSC-CMs as well as in the generated het-CRTL iPSC-CM harboring the same CasQ2 mutation p.F189L. This suggests an impact of p.F189L on SR Ca2+ content and kinetic. In contrast, of the described parameters only increased SERCA activity was rescued to wild type level in isogenic rescue WT-TTS iPSC-CM (p.F189F). Additionally, we observed a decreased Ca2+ transient amplitude in TTS-iPSC-CMs, which was completely rescued in the isogenic rescued WT-TTS iPSC-CM (p.F189F) cells. Furthermore, we analyzed the influence of CasQ2-F189L on the field potential duration (FPD) using the Multielectrode Array (MEA) system and observed a significant decrease in the FPD after Isoprenaline in the TTS-iPSC-CMs compared to CRTL-CM, which was significantly rescued in the isogenic rescue WT-TTS iPSC-CM (p.F189F). Conclusion: For the first time we established a gene-edited Takotsubo syndrome iPSC-CMs rescue stem cell model via CRISPR/Cas9. Our data demonstrated that the heterozygous p.F189L CasQ2 mutation has a major impact on intracellular Ca2+ handling, especially on the SR Ca2+load and kinetics, qualifying this CasQ2 as a potential target in a CRISPR/Cas9 or pharmacological based therapy strategy for TTS. Nevertheless, the rescue of this mutation in the TTS background did not completely reverse all TTS patient’s pathologies to restitutio ad integrum and therefore, additional mutations in this TTS patient are currently under investigation.

https://dgk.org/kongress_programme/jt2022/aP798.html