The neuro-cardiac interaction defines an extracellular microdomain required for neurotrophic signaling

Purpose: Cardiac activity is tuned by sympathetic neurons (SNs), whose survival depends on limiting amounts of neurotrophins released by the myocardium. This study aims i) to determine whether specific cellular structures are present at the SN/cardiomyocyte (CM) contact site, ii) to investigate the role of SN/CM interaction in NGF-mediated signaling. Methods and results: Electron microscopy and immunofluorescence on mouse heart slices and rat SN/CM co-cultures showed close association between SNs and CMs and enrichment of the NGF receptor (TrkA) at the contact site. These data support that specialized and locally organized signaling domains exist (neuro-cardiac junction, NCJ). We tested the functional role of the NCJ in sustaining neuronal survival. Silencing of NGF expression by CMs in co-cultures led to 66% decrease of neuronal density, supporting that SN viability depends on NGF released by CMs. SNs cultured on NGF-silenced CMs showed 20% decrease in the NCJ area when compared to those on wild type CMs of the same culture. Moreover, NGF uptake was observed only in processes contacting NGF- overexpressing CMs, supporting that the NCJ is central to neurotrophin-mediated signaling. Consistently, cultured SNs in contact with CMs survived NGF withdrawal, whereas neurons alone treated with CM- conditioned medium did not survive because of the very low NGF concentration (1.61 pg/mL). Conversely, NGF concentration at the contact site was estimated by using the TrkA inhibitor K252a and resulted about 1000-fold higher (1.75 ng/mL), supporting that the NCJ allows amplification of intercellular NGF signaling. Dystrophin accumulation on CM membrane contacted by SNs was observed in mouse cardiac slices. Consistently, hearts from mdx mice showed 74.36% decrease of innervation, with no significant changes of NGF expression, supporting that ablation of dystrophin impairs cardiac SNs. Conclusions: Taken together, our results suggest that NGF-dependent signaling to the neurons requires a direct and specialized interaction with myocytes