Cell-to-cell communication in cardiomyocytes is mediated by specific signalosomes, protein complexes that play essential roles in regulating cellular responses. Cardiomyocytes are highly structured cells, and one key example of microdomain organization is the formation of a functional complex between β2-adrenergic receptors (β₂ARs) and L-type Ca²⁺ channels (LTCCs) on the plasma membrane. However, the functional consequences of this microdomain localization remain incompletely understood. We found that LTCC activity is regulated by proximity coupling mechanisms exclusively via β₂ARs—not β₁ARs—highlighting a specific role for β₂ARs in modulating LTCC response to adrenergic stimulation under healthy conditions. This coupling is lost in heart failure, both in rodent models and in humans, and is dependent on caveolin-3.

Cardiomyocytes also release nerve growth factor (NGF), which guides sympathetic neurons (SN) to form specialized contact sites with cardiomyocytes, termed neuro-cardiac junctions (NCJs). The formation of these NCJ microdomains induces significant physiological changes in cardiac cells. Using a co-culture model of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and SN, we demonstrated that SNs promote functional maturation of hPSC-CMs in vitro. Co-culture resulted in increased structural elongation, enhanced sarcomere organization, and improved force-frequency relationships—key features of mature cardiomyocyte function. Nicotine stimulation elicited a norepinephrine-dependent βAR response from SNs, leading to elevated cAMP production and enhanced contraction and Ca²⁺ transient amplitudes at baseline. Notably, this functional maturation did not extend to metabolic pathways.

Furthermore, SN co-culture increased action potential amplitude and depolarization velocity in hPSC-CMs, supporting a role for SNs in promoting electrophysiological maturation. Our findings suggest that neurons may influence cardiomyocyte expression of channels and receptors, including βARs, LTCCs, and Na⁺ channels (NaCh). We developed a novel patch-clamp technique that enables recording of voltage-gated channels in the cell-attached configuration on specific membrane regions, including the NCJ. Our data show that NCJs are enriched in NaCh-ankyrin G complexes, likely serving as anchoring structures between neurons and cardiomyocytes. The presence of NCJs also increased the number of LTCC/caveolin-3 complexes, suggesting that innervation actively reshapes the functional and electrophysiological landscape of cardiomyocytes.

In conclusion, our work underscores the critical role of submembrane protein complexes in both healthy and pathological cardiac states and highlights how NCJ formation drives functional changes mediated by these complexes.