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

Generation of iPSC-derived sympathetic neurons and their effect on iPSC-cardiomyocytes in a patient-specific induced pluripotent stem cell Takotsubo-model
B. Wenner1, D. Hübscher1, G. Hasenfuß1, K. Streckfuß-Bömeke2
1Herzzentrum, Klinik für Kardiologie und Pneumologie, Universitätsmedizin Göttingen, Göttingen; 2Institut für Pharmakologie und Toxikologie, Universitätsklinikum Würzburg, Würzburg;
Background/Purpose: Takotsubo syndrome (TTS) is characterized by an acute transient dysfunction of the left ventricle that presents itself with regional wall motion abnormalities. The pathomechanisms are still yet to be discovered in its entirety. TTS can be defined as Brain-heart syndrome (BHS), since the cardiac phenotype with metabolic disturbances and cardiac arrhythmias may be a result of a dysregulated Central Autonomic Network (CAN), an internal regulation system of the brain. Previous works identified a significantly higher catecholamine concentration in the blood of patients with acute TTS, most likely via a dysfunction of the CAN. In a former 2D and 3D study of TTS patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), our research group demonstrated enhanced β-adrenergic signaling and higher sensitivity to catecholamine-induced stress toxicity as mechanisms associated with TTS. Here, we aimed to establish a protocol for the differentiation of iPSCs into functional sympathetic neurons (iPSC-SymNs) to analyze their impact on iPSC-CMs and their contribution to the development of TTS.

Methods and Results: We established a 3-step protocol by using neuronal progenitor aggregates, which were plated at later time points for further maturation. We tested the stage specific expression on mRNA (qPCR) and on protein level (immunofluorescence staining (IF)) of differentiated cells in a time-dependent manner including SOX10 positive neural crest cells, from which symNs arise from. Our mature iPSC-symNs expressed the neuronal marker βIII-tubulin as well as the sympathetic markers tyrosine hydroxylase and dopamine-β-hydroxylase on mRNA as well as on protein level. We were able to generate iPSC-symNs with a measurable norepinephrine (NE) output and confirmed the expression of a characteristic ganglionic receptor profile (CHRNA3, CHRNA7, CHRNB4) on mRNA level. Furthermore, we generated functional iPSC-CMs and used these to establish a protocol for co-culture of iPSC-CMs and iPSC-symNs. Using the specific nicotinergic acetylcholine receptor agonist (-)-Cytisine, we stimulated the symN-CM co-culture to mimic a stress-triggered response from the brain to the symNs and therefore provoke an enhanced NE output of the co-cultured iPSC-symNs. To quantify the response of the co-cultured iPSC-CMs to this, we used a multielectrode array and found an enhanced beating frequency of the co-cultured iPSC-CMs in contrast to not-co-cultured iPSC-CMs stimulated under the same conditions.

Conclusion: We were able to establish an effective protocol for the differentiation of iPSC-symNs with the expression of characteristic sympathetic markers such as TH and DBH and a measurable NE output. Co-culture experiments of TTS-iPSC-CM and -SN confirmed the functional interaction between both cell types. The stimulation with neuronal-specific agonists resulted in increased beating frequency of the iPSC-CM. Therefore, with this work, we opened the door to identify the individual contribution of the heart or brain component to the pathogenesis of TTS.

https://dgk.org/kongress_programme/jt2022/aP800.html