A reproducible and physiologically relevant human iPSC-derived platform for in vitro modeling of the neurocardiac junction

The cardiac autonomic nervous system is central to various cardiac diseases, yet its regulation in the human heart remains poorly understood due to the lack of reliable models. Here, we report the development of a neurocardiac co-culture system using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and sympathetic neurons (hiPSC-SNs). Both cell types were characterized molecularly and electrophysiologically and subsequently used for the establishment of the co-culture model in a two-well chambers insert, forming a dense axonal network connected with hiPSC-CMs. The co-culture demonstrated robust functional interactions: hiPSC-CMs maintained a stable beating rate, while hiPSC-SNs exhibited significantly increased firing activity after 7 days. Nicotine stimulation enhanced hiPSC-SN activity, resulting in an increased beating rate in co-cultured hiPSC-CMs, an effect that is absent in monoculture. This rise in the beats was abolished by nicotinic acetylcholine receptor blockade through α-bungarotoxin on hiPSC-SNs in co-culture. The β-blocker propranolol mitigated isoproterenol or nicotine effects on hiPSC-CMs. Using a fluorescent tracer, functional exocytosis and norepinephrine release was confirmed in hiPSC-SNs in co-culture. This novel neurocardiac model replicates neuronal control of cardiomyocytes and provides a new and robust platform for studying neuro-cardiac interactions. It represents a promising tool for advancing disease modeling and pharmacologic research in cardiac pathophysiology.