Interaction between stem-cell-derived sympathetic neurons and cardiomyocytes in a patient–specific Takotsubo syndrome model

Takotsubo syndrome (TTS), also known as stress-induced cardiomyopathy, is characterized by an acute left ventricular dysfunction, typically without the presence of stenosis. Since it is believed that a malfunctioning central autonomic nervous system, leading to excessive sympathetic activity, plays a role in the condition's development, TTS can be described as a brain-heart syndrome. Previous studies identified a significantly higher catecholamine concentration in patients with acute TTS. Our previous work showed enhanced sensitivity to catecholamine-induced stress toxicity and a genetic predisposition as mechanisms associated with TTS by using a TTS patient-specific induced pluripotent cardiac stem cell (iPSC) model. Here, we aim to investigate whether iPSC-derived TTS sympathetic neurons (SN) impact iPSC-cardiomyocytes (CM) and whether they also contribute to the development of TTS.

TTS-iPSC lines with various genetic variants were utilized to develop a 3-step protocol, which includes stages of neuronal progenitor aggregates and 2D monolayers.Immunohistochemical staining and qPCR data showed SN-specific marker expression (PHOX2B, TH, and DBH) increase with a characteristic ganglionic receptor profile (CHRNA3, CHRNA7 and CHRNB4) throughout the differentiation. Quantitative analyses by FLOW cytometry of 25-day-old SN indicated ~80% of the population's autonomic progenitors (PHOX2B+ cells) and ~10% TH+ cells. Finally, prominent basal and KCl-stimulated noradrenaline secretion proved SN functionality. In the next step, we established a protocol for CM and SN co-culturing. We demonstrated crosstalk of both cell types by mimicking a stress-triggered response from the SNs using the nicotinic acetylcholine receptor agonists Cytisine and Nicotine. Enhanced beating frequency of co-cultured iPSC-CM was demonstrated using a multielectrode array. When blocking postsynaptic beta-adenoreceptors with Propranolol, no change in beating frequency was observed after stimulation with Cytisine and Nicotine. In a mix-and-match approach, the neuronal TTS mutagenic influence on the cardiac phenotype will be further analyzed. In a next step, TTS cell lines will be used in the established innervated human myocardium model (iEHM) to analyze TTS neuronal and cardiac crosstalk in a 3D environment.

In conclusion, we established an effective protocol for the differentiation of functional iPSC-SNs that can be used in co-culture of TTS-iPSC-CM and -SN to confirm the functional interaction between both cell types. Investigating the impact of TTS genetic predisposition on sympathetic neurons next to cardiomyocytes is crucial to identifying potential drug targets to treat TTS.