Abstract 162 Arrhythmias in the Takotsubo Syndrome: Investigating genetic predisposition using a patient-specific atrial iPSC-cardiomyocyte model

Background and Aims:

The Takotsubo syndrome (TTS) is characterized by an acute left ventricular dysfunction, usually in the absence of stenosis. Although the disease affects mainly the left ventricle, transient impairments in left atrium function with a prevalence of atrial fibrillation of around 18% are observed. This study was performed to identify the role of genetic predispositions for the development of arrhythmias and how arrhythmic events could contribute to TTS. Analysis of pharmacological drugs should demonstrate their suitability as patient-specific treatment option in vitro.

Methods and Results:

We generated induced pluripotent stem cell-derived atrial cardiomyocytes from TTS patients (TTS-iPSC-aCMs), in which genetic variants in AHNAK (Cav1.2 regulating gene) and CASQ2 (calcium buffering gene in the sarcoplasmic reticulum (SR)) were found. These variants were incorporated into control lines (Ctrl-AHNAK and Ctrl-CASQ2) and rescued in the TTS line (TTS-res-AHNAK) using CRISPR/Cas9 genome editing. To mimic a TTS event aCMs were treated with catecholamines. We analyzed the activity of phosphodiesterases (PDEs) and observed that after β-AR stimulation, the strong effects of PDE4 in atrial controls were significantly decreased in aTTS-CMs. We could demonstrate this decreased PDE4 activity by using the PDE4 inhibitor Rolipram with decreased Rolipram-induced increase in cAMP-FRET measurements as well as lowered systolic Ca2+ transient amplitude (epifluorescence microscopy) after catecholamine treatment in TTS cells compared with controls. This effect was previously described in human atrial myocardium of AF patients, and we were able to completely rescue PDE4 activity after application of PDE4 activator MR-L2. Furthermore, an isoprenaline-induced increase in systolic Ca2+ transient amplitude was more pronounced in TTS-aCMs than in control cells and could be rescued by the β-blocker Metoprolol. Caffeine applications showed increased SERCA2 activity as well as increased fractional Ca2+ release from SR in TTS-aCMs compared with control aCMs, which is caused solely by the identified CASQ2 variant. We performed confocal Ca2+ measurements and observed an increased diastolic Ca2+ leak in the SR and a higher number of arrhythmic events in TTS patients after Isoprenaline treatment in comparison to controls. Since we could demonstrate that the increased Ca2+ leak was completely rescued in TTS-res-AHNAK-aCMs the described AHNAK mutation was defined as the underlying genetic cause for catecholamine-induced TTS- arrhythmic events. These effects were rescued by Metoprolol as well as after application of PDE4 activator MR-L2. In addition, TTS patients displayed faster Ca2+ kinetics and an elevated beating frequency compared with controls, which was underlined on a molecular level by increased phospholamban phosphorylation in TTS-aCM. Subsequent treatment with Metoprolol rescued the Ca2+ kinetic parameters in all cell lines.

Conclusion:

In conclusion, we were able to demonstrate the considerable role of the atrium in the development of arrhythmias in TTS. We found TTS-patient-specific differences with reduced PDE4 activity, elevated arrhythmic events, altered calcium homeostasis, and enhanced sensitivity to catecholamines and could identify specific cardiac mutations partially as the underlying causes. Applications of both ß-blocker Metoprolol and PDE4 activator MR-L2 were proven to have a therapeutic benefit for TTS in vitro.

Clin Res Cardiol (2023). https://doi.org/10.1007/s00392-023-02302-4
Arrhythmias in the Takotsubo Syndrome: Investigating genetic predisposition using a patient-specific atrial iPSC-cardiomyocyte model
C. Zinke¹, C. Brand¹, G. Syed Ali¹, D. Hübscher¹, K. Ajmail¹, W. Maurer¹, V. Nikolaev², A. Zibat³, G. Hasenfuß¹, B. Wollnik³, K. Streckfuß-Bömeke⁴
¹Herzzentrum, Klinik für Kardiologie und Pneumologie, Universitätsmedizin Göttingen, Göttingen; ²Zentrum für Experimentelle Medizin, Experimentelle Herz-Kreislaufforschung, Universitätsklinikum Hamburg-Eppendorf, Hamburg; ³Humangenetik, Universitätsmedizin Göttingen, Göttingen; ⁴Institut für Pharmakologie und Toxikologie, Universitätsklinikum Würzburg, Würzburg;
Background and Aims: The Takotsubo syndrome (TTS) is characterized by an acute left ventricular dysfunction, usually in the absence of stenosis. Although the disease affects mainly the left ventricle, transient impairments in left atrium function with a prevalence of atrial fibrillation of around 18% are observed. This study was performed to identify the role of genetic predispositions for the development of arrhythmias and how arrhythmic events could contribute to TTS. Analysis of pharmacological drugs should demonstrate their suitability as patient-specific treatment option in vitro. Methods and Results: We generated induced pluripotent stem cell-derived atrial cardiomyocytes from TTS patients (TTS-iPSC-aCMs), in which genetic variants in AHNAK (Cav1.2 regulating gene) and CASQ2 (calcium buffering gene in the sarcoplasmic reticulum (SR)) were found. These variants were incorporated into control lines (Ctrl-AHNAK and Ctrl-CASQ2) and rescued in the TTS line (TTS-res-AHNAK) using CRISPR/Cas9 genome editing. To mimic a TTS event aCMs were treated with catecholamines. We analyzed the activity of phosphodiesterases (PDEs) and observed that after β-AR stimulation, the strong effects of PDE4 in atrial controls were significantly decreased in aTTS-CMs. We could demonstrate this decreased PDE4 activity by using the PDE4 inhibitor Rolipram with decreased Rolipram-induced increase in cAMP-FRET measurements as well as lowered systolic Ca2+ transient amplitude (epifluorescence microscopy) after catecholamine treatment in TTS cells compared with controls. This effect was previously described in human atrial myocardium of AF patients, and we were able to completely rescue PDE4 activity after application of PDE4 activator MR-L2. Furthermore, an isoprenaline-induced increase in systolic Ca2+ transient amplitude was more pronounced in TTS-aCMs than in control cells and could be rescued by the β-blocker Metoprolol. Caffeine applications showed increased SERCA2 activity as well as increased fractional Ca2+ release from SR in TTS-aCMs compared with control aCMs, which is caused solely by the identified CASQ2 variant. We performed confocal Ca2+ measurements and observed an increased diastolic Ca2+ leak in the SR and a higher number of arrhythmic events in TTS patients after Isoprenaline treatment in comparison to controls. Since we could demonstrate that the increased Ca2+ leak was completely rescued in TTS-res-AHNAK-aCMs the described AHNAK mutation was defined as the underlying genetic cause for catecholamine-induced TTS- arrhythmic events. These effects were rescued by Metoprolol as well as after application of PDE4 activator MR-L2. In addition, TTS patients displayed faster Ca2+ kinetics and an elevated beating frequency compared with controls, which was underlined on a molecular level by increased phospholamban phosphorylation in TTS-aCM. Subsequent treatment with Metoprolol rescued the Ca2+ kinetic parameters in all cell lines. Conclusion: In conclusion, we were able to demonstrate the considerable role of the atrium in the development of arrhythmias in TTS. We found TTS-patient-specific differences with reduced PDE4 activity, elevated arrhythmic events, altered calcium homeostasis, and enhanced sensitivity to catecholamines and could identify specific cardiac mutations partially as the underlying causes. Applications of both ß-blocker Metoprolol and PDE4 activator MR-L2 were proven to have a therapeutic benefit for TTS in vitro.
https://dgk.org/kongress_programme/ht2023/aBS138.html