Modeling sympathetic neuro-cardiac interactions in a hiPSC-based microphysiological system

The cardiac autonomic nervous system is a key driver of various cardiac disorders and arrhythmias. However, investigating neuronal regulation of the human heart has proven difficult due to the limited availability of reliable experimental models. Here, we present a novel microphysiological system using a compartmentalized microfluidic device (MFD) to integrate co-cultured human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) and sympathetic neurons (hiPSC-SNs). MFD is composed of two wide-open chambers separated by microfluidic microchannels. Patch-clamp electrophysiology, RT-qPCR, immunofluorescence, and video-motion recording were used to characterize the phenotype of hiPSC-SNs and their functional coupling with hiPSC-CMs in the MDF. Immunofluorescence and RT-qPCR demonstrated significantly increased expression of the sympathetic neuronal markers peripherin, tyrosine hydroxylase, βIII-tubulin, dopamine β-hydroxylase, and nicotinic acetylcholine receptors, confirming successful differentiation into a sympathetic neuronal phenotype. Patch-clamp electrophysiology further demonstrated functional maturation, with hiPSC-SNs exhibiting spontaneous action potentials and nicotine-triggered membrane depolarization in response to 1 μM nicotine. Co-culture of hiPSC-CMs and hiPSC-SNs within the MFD promoted axonal projection into the cardiomyocyte chamber, establishing a physical connection between the two cell types. After 10 days of co-culture, functional integration was confirmed by a significant increase in hiPSC-CMs action potential frequency and beating rate, as recorded by patch-clamp and video motion tracking, respectively. Furthermore, selective nicotine stimulation of the neuronal chamber increased both action potential frequency and beating rate in hiPSC-CMs, whereas β-adrenergic blockade with 5 μM propranolol significantly reduced the beating rate. Collectively, these data demonstrate the feasibility of differentiating hiPSCs into functional sympathetic neurons and establishing a robust neuro-cardiac interface. This microphysiological system represents a powerful platform for investigating disorders characterized by impaired neuro-cardiac interactions, offering a valuable tool for both disease modeling and pharmacological screening.