Abstract
Background and Aims: The Takotsubo syndrome (TTS) is characterised by an acute left ventricular dysfunction without exhibiting signs of stenosis. TTS affects mainly the left ventricle, however, its pathophysiology comprises transient impairments in left atrium functions with a prevalence of atrial fibrillation (AF) of around 18% correlating with higher ventricular heart rhythm disorders. In this study, we aimed to prove the hypothesis that molecular and cellular arrhythmic events contribute to the development of TTS under catecholamine stress and to test if the currently clinically used β-blockers (Metoprolol) are suitable for Takotsubo cardiomyopathy in vitro. Methods and Results: We generated induced pluripotent stem cell-derived atrial cardiomyocytes (TTS-iPSC- a CMs) from TTS patients, confirmed atrial marker expression (MLC2a, PItX2, NR2F2), and treated the cells with catecholamines to mimic TTS-phenotype. Using a cytosolic Förster resonance energy transfer (FRET) based cAMP sensor, we tested the activity of phosphodiesterases (PDEs) in TTS-iPSC-aCMs, which are besides β-ARs main players involved in cAMP signaling in CMs. We observed that after β-AR stimulation, the strong effects of the PDE4 family in the cytosol of atrial controls were significantly decreased in aCMs of the TTS patients. This effect was completely rescued after application of PDE4 activator MR-L2 and is in line with the previously described downregulation of PDE4 in human atrial myocardium of AF patients. In functional studies, isoprenaline-induced increase in systolic Ca 2+ transient amplitude was more pronounced in TTS iPSC-aCM compared to controls. These effects were rescued by both, the clinically approved β-blocker Metoprolol as well as the PDE4 activator MR-L2. To analyse arrhythmic events in atrial TTS CMs, we performed confocal Ca 2+ measurements and demonstrated that the diastolic sarcoplasmic reticulum Ca 2+ leak was increased in iPSC aCMs of TTS patients compared to control under basal conditions and after Iso-treatment. In addition, TTS patients displayed faster Ca 2+ kinetics compared to control cells, already under basal conditions. These results were underlined on a molecular level by increased phospholamban phosphorylation in TTS iPSC-aCM .Subsequent treatment with the beta-blocker Metoprolol rescued the Ca 2+ kinetic parameters in all cell lines. Conclusion: In conclusion, we were able to draw a comprehensive picture on the role of the arrhythmia in the development of TTS. We found TTS-specific differences with reduced PDE4 activity, elevated arrhythmic events and enhanced reactions to catecholamines, which could be rescued by the clinically approved drug Metoprolol and partly by the PDE4 activator MR-L2. Therefore, Metoprolol as well as MR-L2 demonstrate promising effects as patient-specific therapeutic target for TTS.
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