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Modeling Takotsubo syndrome with patient-specific induced pluripotent stem cell-derived cardiomyocytes

dc.contributor.advisorStreckfuß-Bömeke, Katrin PD Dr.
dc.contributor.authorBorchert, Thomas
dc.titleModeling Takotsubo syndrome with patient-specific induced pluripotent stem cell-derived cardiomyocytesde
dc.contributor.refereeStreckfuß-Bömeke, Katrin PD Dr.
dc.description.abstractengTakotsubo syndrome (TTS) is a severe acute heart disease presumably caused by high catecholamine levels resulting in wall motion abnormalities of the left ventricle. Despite the severe phenotype of TTS, the exact disease mechanisms remain elusive. This project aims to model TTS with the help of induced pluripotent stem cell (iPSC) derived cardiomyocytes (iPSC-CMs) on a patient-specific level. To achieve this goal, 4 TTS patients and 3 control probands were recruited. Human fibroblasts or peripheral blood mononuclear cells (PBMCs) were reprogrammed to iPSCs using integration-free methods. The resulting iPSCs exhibited typical iPSC-morphology and expressed specific pluripotency-related genes on messenger RNA (mRNA) and protein level. The cells could be readily differentiated into cells from all three different germ-layers in vitro and in vivo. IPSCs were successfully differentiated to iPSC-CMs with purity over 80 % and expressing cardiac markers like cTnT and α-actinin. To mimic a TTS event, iPSC-CMs were treated with increasing concentrations of catecholamines such as epinephrine (Epi) or isoproterenol (Iso). The used high catecholamine levels did not induce significant levels of apoptosis, therefore excluding acute cytotoxicity. To test electrophysiological outcomes in the iPSC-CMs, beating rate and regularity were analyzed by using the multi-electrode array (MEA) system. MEA measurements uncovered electrical stunning for iPSC-CMs stimulated with rising levels of catecholamines. TTS-iPSC-CMs were more vulnerable to this effect. The remaining TTS-iPSC-CMs showed a stronger frequency increase when stimulated with Iso than CTRL-iPSC-CMs. Functioning calcium (Ca2+) cycling is of utmost importance in cardiomyocytes since Ca2+ is a key player in excitation-contraction coupling and Ca2+ abnormalities are involved in many heart diseases. TTS-iPSC-CMs demonstrated differences in Ca2+ cycling compared to CTRL-iPSC-CMs on multiple levels: Under basal conditions and after Iso stimulation, times to 50% decay (T50) were shorter and rise times were longer in TTS-iPSC-CMs in comparison to CTRL-iPSC-CMs. CTRL-iPSC-CMs showed a higher spark frequency at higher Iso levels (1 μM), but overall, there was no difference in total Ca2+ leakage. To analyze TTS on the tissue level, engineered heart muscles (EHM) were generated and used as a 3D-model. When challenged with Iso, the TTS-iPSC-EHMs showed a significantly higher sensitivity compared to CTRL-iPSC-EHMs as shown by a lower half-maximal effective concentration (EC50). After 24 h Iso pre-stimulation, the CTRL-iPSC-EHMs exhibiting a stronger EC50 increase than TTS-iPSC-EHM, thereby exhibiting stronger receptor desensitization. Additionally, beating frequency changes after 24 h Iso stimulation were higher in TTS-iPSC-EHM compared to CTRL-iPSC-EHM, which is in line with the reported frequency differences in the 2D MEA measurements. Interestingly, TTS-iPSC-EHM showed a lower force development than CTRL-iPSC-EHM without catecholamine stimulation. Since adrenergic stimulation results in positive inotropic and chronotropic effects of the heart and thereby regulates lipid metabolism in cardiomyocytes, lipid droplets were quantified using Oil Red O. The TTS-iPSC-CMs exhibited higher lipid accumulation in comparison to CTRL-iPSC-CMs, suggesting a role of lipotoxicity in the disease. The selective β-adrenergic receptor (β-AR) blockers CGP (β1-AR blocker) and ICI (β2-AR blocker) were used to uncover the roles of different β-ARs in CTRL- and TTS-iPSC-CMs. In CTRL-iPSC-CMs, β2-AR blocker alone was enough to reduce an Iso-dependent frequency increase and an Iso-dependent T50 and rise time decrease, thus underpinning the role of the β2-ARs in CTRL-iPSC-CMs. In TTS-iPSC-CMs, β1-AR blockage abolished Iso-dependent T50 and rise time reduction, which was not the case for β2-AR blockers and therefore underpins an important role of β1-AR in TTS-iPSC-CMs. In conclusion, these results demonstrate that CTRL- and TTS-iPSC-CMs behave differently with respect to catecholamine sensitivity, Ca2+ handling, lipid accumulation, electrophysiology, and β-AR preferences. This behavior hints towards a genetic predisposition in TTS, lowering the threshold for a TTS
dc.contributor.coRefereeMeyer, Thomas Prof. Dr.
dc.subject.engTakotsubo cardiomyopathyde
dc.subject.engβ-adrenergic signalingde
dc.subject.engDisease modelingde
dc.affiliation.instituteMedizinische Fakultät
dc.subject.gokfullMedizin (PPN619874732)de

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