Quantifizierung spontaner Calcium-Freisetzungen in humanen atrialen Kardiomyozyten aus induzierten pluripotenten Stammzellen (iPSC)
by Marie Catharin Klopp née Klopp
Date of Examination:2025-03-11
Date of issue:2025-03-13
Advisor:Prof. Dr. Niels Voigt
Referee:Prof. Dr. Niels Voigt
Referee:Prof. Dr. Samuel T. Sossalla
Referee:Prof. Dr. Ralf Dressel
Persistent Address:
http://dx.doi.org/10.53846/goediss-11146
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Abstract
English
Atrial fibrillation (AF) is a cardiac disorder characterized by disorganized electrical excitation of the atria. AF is the most common cardiac arrhythmia in clinical practice and is associated with increased cardiovascular mortality and morbidity. It has been shown that increased spontaneous diastolic Ca²⁺ releases from the sarcoplasmic reticulum (SR), known as the Ca²⁺ leak, play a significant role in the onset and maintenance of AF. A major challenge in AF research and the development of therapeutic approaches is the lack of an appropriate model. Recent studies have therefore proposed the use of cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs). This study evaluated the Ca²⁺ leak in the form of Ca²⁺ sparks to answer questions regarding the Ca²⁺ homeostasis of iPSC-CMs and to further establish this cell model. For this purpose, iPSC-CMs were loaded with the fluorescent Ca²⁺ indicator Fluo-4-acetoxymethyl ester (Fluo-4 AM) and analyzed using confocal microscopy. The resulting images were used to qualitatively and quantitatively assess Ca²⁺ sparks with the software ImageJ®. A frequently discussed characteristic of iPSC-CMs is their developmental stage and lack of maturity. Therefore, this study examined changes in the Ca²⁺ leak during their maturation process. The Ca²⁺ leak of atrial and ventricular iPSC-CMs was measured and compared over a period of 150 days, starting from differentiation. The study demonstrated that spontaneous diastolic Ca²⁺ leakage decreased over time in both atrial and ventricular iPSC-CMs. These findings support the hypothesis that iPSC-CMs develop a more mature electromechanical coupling mechanism with age. Another part of the study investigated whether in vitro tachypacing could induce changes in spontaneous diastolic Ca²⁺ release from the SR, similar to those observed in persistent AF. To this end, atrial and ventricular iPSC-CMs were electrically stimulated for 24 hours at frequencies of 1 and 3 Hz, after which the Ca²⁺ leak was measured. The results showed that in both atrial and ventricular iPSC-CMs, spontaneous diastolic Ca²⁺ leakage decreased with increasing pacing frequency. The reasons for this decrease despite higher pacing frequencies are discussed in this study. It is suggested that tachypacing may have initiated a maturation process in iPSC-CMs. Furthermore, the duration of tachypacing may not have been sufficient to observe the expected changes in the Ca²⁺ leak. To mimic the pathophysiology of AF, iPSC-CMs were subjected to arrhythmic stimulation in further experiments. The arrhythmia was examined separately from tachycardia at the molecular and electrophysiological levels and led to a significant increase in the Ca²⁺ leak in ventricular iPSC-CMs. However, this effect was not observed in atrial iPSC-CMs. Additional experiments in this study investigated the effects of Ivabradine on iPSC-CMs. The aim was to analyze how Ivabradine affects the Ca²⁺ leak and the spontaneous contraction frequency of iPSC-CMs. In atrial iPSC-CMs, Ivabradine treatment did not alter spontaneous contraction frequency or Ca²⁺ leakage. In contrast, Ivabradine appeared to have an arrhythmogenic effect in ventricular iPSC-CMs. This observation cannot be explained by the molecular mechanism of Ivabradine. The observed effects may be due to the immaturity of the cells or an unspecific action of Ivabradine. To examine how the Ca²⁺ leak can be modulated by interventions in Ca²⁺ homeostasis, further experiments involved the inhibition of protein kinases CaMKII and PKA using Carbachol (CCh) and H-89. CCh significantly reduced diastolic Ca²⁺ leakage in both atrial and ventricular iPSC-CMs, leading to a pronounced antiarrhythmic effect. H-89 treatment also reduced diastolic Ca²⁺ leakage in atrial iPSC-CMs. However, no effect was observed in ventricular iPSC-CMs. These experiments demonstrate that the Ca²⁺ homeostasis of iPSC-CMs can be modulated in a similar manner to that of adult primary human cells. The fact that both cell types rely on the same mechanisms highlights their similarities. This represents a significant opportunity for cardiovascular disease research using the iPSC-CM model. Although iPSC-CMs resemble primary human cardiomyocytes, they are not entirely comparable. While iPSC-CMs exhibit significant similarities to adult cells, their immature characteristics mean that not all findings can be directly extrapolated. Overall, this study contributes to a deeper understanding of the mechanisms involved in the Ca²⁺ homeostasis of iPSC-CMs and provides a valuable foundation for future research into cardiac Ca²⁺ homeostasis. Furthermore, this work enhances the electrophysiological characterization of iPSC-CMs and supports their establishment as a model for AF research
Keywords: Atrial fibrillation; Calcium Sparks; IPS-cardiomyozytes; Calcium leak; Calcium handling
