Abstract
Background: The cardiac autonomic nervous system is pivotal in several cardiac diseases, yet many aspects of neuronal regulation in the human heart remain unclear due to the lack of reliable experimental models.
Methods: Commercial hiPSCs were differentiated into hiPSC-derived cardiomyocytes (hiPSC-CMs) and sympathetic neurons (hiPSC-SNs) and both cell types were characterized for molecular and electrophysiological aspects. The neurocardiac model was established by separately plating hiPSC-CMs and hiPSC-SNs in a two-chamber silicone insert. The newly generated neurocardiac model was then analyzed through the use of immunofluorescence and gene expression techniques. Immunofluorescence, gene expression techniques, and microelectrode arrays assessed the co-culture’s functionality. Results were analyzed by t-test or ANOVA. Additionally, neuronal control over cardiomyocytes was evaluated using various drugs, demonstrating the model's full functionality.
Results: Successful differentiations of hiPSC-CMs and hiPSC-SNs were confirmed through positivity for lineage markers and electrophysiological profiling. In the newly created co-culture the removal of the insert signs the beginning of the axonal projections towards hiPSC-CMs, resulting in a dense axonal network connected with hiPSC-CMs. The beating rate of hiPSC-CMs was stable after 7 days of co-culture, whereas hiPSC-SNs significantly increased their firing activity after 7 days of co-culture. Nicotine treatment was used to induce hiPSC-SN activity, resulting in a significant increase in the beat rate of hiPSC-CMs in co-culture, which had no effect on hiPSC-CMs in monoculture. The beat frequency of hiPSC-CMs in co-culture was unchanged after nicotinic acetylcholine receptor blockade with α-bungarotoxin and further nicotine administration. However, the β-blocker propranolol was able to reduce the beating rate of hiPSC-CMs after isoproterenol or nicotine treatment. Using a fluorescent tracer for norepinephrine, we observed a significant neurotransmitter release and a functional exocytosis from hiPSC-SNs in co-culture as a result of nicotine triggering.
Conclusions: Here we demonstrate the generation of a novel neurocardiac co-culture model. The data collected confirm the ability of hiPSC-SNs to control the beating rate of hiPSC-CMs and to establish functional electrochemical connections with them. The proposed human co-culture system provides a valuable model to study diseases with impaired neuro-cardiac interactions, facilitating both disease modelling and pharmacological testing