Sympathetic neurons can modify the intrinsic structural and functional properties of human pluripotent stem...

The sympathetic nervous system densely innervates all cardiac chambers and is a key player in cardiac control, yet this relationship has scarcely been investigated using a stem cell-based model. This study investigates the effects that sympathetic neurons (SNs) have on human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in vitro, and whether they induce any degree of functional or structural maturity in these conventionally immature cells. SNs were isolated from neonatal rat pups, and cocultured with hPSC-CMs for up to 15 days. Structural changes in hPSC-CMs were analysed by microscopy techniques. Fluorescence resonance energy transfer was used to measure second messenger molecule cAMP production and β-adrenergic receptor (βAR) response. Contractile and Ca²⁺ transient activity was measured using CytoCypher. These cocultures promoted hPSC-CM structural elongation and increased sarcomere organization. Furthermore, the βAR response of cocultured hiPSC-CMs was larger, indicated by increased cAMP production upon neuronal nicotinic stimulation. Faster contraction and ratiometric Ca²⁺ transient peak height and kinetic parameters strongly indicate increased chronotropic response in coculture. Coculture with SNs also elicited an increase in action potential amplitude and depolarization velocity, further confirming that SNs contribute to hiPSC-CM functional maturation. Overall, we have found that SNs modulate hPSC-CMs in vitro, inducing a more mature functional response. As an in vitro tool, these cocultures could serve as a model of sympathoadrenergic disease, enabling new discovery avenues. KEY POINTS: The sympathetic nervous system controls the involuntary 'fight-or-flight' response, with the heart being one of key target organs. In certain neuro-cardiac diseases, the input from the sympathetic nervous system is hyperregulated, and can lead to increased speed or force of the heart's contraction. Human induced pluripotent stem cells (hiPSCs) represent a rapidly evolving field which allow us to create a cell of interest and model its structural and functional activity in a dish. Here we have created hiPSC-derived cardiomyocytes (hiPSC-CMs) and cocultured them with sympathetic neurons (SNs). We found that SNs are able to modulate structure of the hiPSC-CMs by reducing their circularity and increasing sarcomeric organization, and can significantly increase the speed of contraction and Ca²⁺ handling. Together, our data provide a platform to investigate the neuro-cardiac relationship in vitro, which could be used for patient-specific disease modelling in future.