Abstract
The cardiac autonomic nervous system is known to play a key role in many cardiac disorders. However, many details of the neuronal regulation of the human heart remain elusive due to the lack of reliable experimental models. Here, we describe the generation of an in vitro neurocardiac model based on human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) and sympathetic neurons (iPSC-SNs). iPSC-SNs in monoculture expressed MAP-2 (neuronal marker), TH, DBH (adrenergic lineage markers), and peripherin (peripheral nervous system marker) as evaluated by flow cytometry, immunofluorescence and western blot analyses. Furthermore, iPSC-SNs exhibited spontaneous firing and burst activity, measured using the Maestro Edge Multi-Electrode Array (MEA). iPSC-CMs and iPSC-SNs were co-cultured in two chambers separated by a silicon insert, and after insert removal, iPSC-SNs formed axons projecting towards iPSC-CMs. While the beat rate of iPSC-CMs was stable after 7 days of co-culture, iPSC-SNs significantly increased their firing activity after 7 days of co-culture. Nicotine treatment was used to trigger iPSC-SN activity. A significant increase in the beat rate of iPSC-CMs in co-culture was observed after nicotine treatment, which had no effect on iPSC-CMs in mono-culture. The increased beat rate of iPSC-CMs in response to nicotine stimulation was reduced upon administration of the β-blocker Propranolol, while it remained unaffected by α-bungarotoxin, which blocks nicotinic receptors. After 7 days of co-culture, a significant decrease in FFN270 (a fluorescent tracer of norepinephrine)-stained vesicles was observed in nicotine-treated cells, indicating effective neurotransmitter release and functional exocytosis. Collectively, these data confirm the ability of iPSC-SNs to establish functional connections with iPSC-CMs. The proposed co-culture system represents a valuable model to study diseases with compromised neuro-cardiac interactions, facilitating both disease modeling and pharmacological testing.
Funding:
Interreg: ITAT1047