P94The neuro-cardiac interaction defines an extracellular microdomain required for neurotrophic signaling

Abstract

Purpose: Sympathetic ganglia neurons (SGNs) innervate the myocardium and tune heart rate and contractility. Development and viability of cardiac neurons depend on neurotrophins that are released in low amounts by the myocardium. This study aims to understand whether the direct cell-to-cell contact plays a role in NGF-mediated signaling between cardiomyocytes (CMs) and SGNs. Methods and results: Electron microscopy and immunofluorescence on mouse heart slices and rat SGN/CM co-cultures showed a close association between SGNs and CMs, neurotransmitter vesicle accumulation, increased membrane protein density 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, hereafter). We tested the functional role of the NCJ in NGF-mediated prosurvival signaling. NGF expression by CMs was assessed by western blot analysis and its silencing in co-cultures caused a 66% decrease of neuronal density, suggesting that neurons depend on NGF released by CMs. NGF binding to its receptor triggers TrkA retrograde transport to neuronal soma that was monitored using fluorescently labeled TrkA. TrkA-RFP retrograde movements were faster in processes contacting CMs than other cardiac cells, further supporting that neuronal NGF signaling is activated by the coupled CMs. SGNs cultured on NGF-silenced CMs showed a 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 neurotrophin mediated signaling is involved at the SGN/CM interaction. Consistently, cultured SGNs 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 (<5pg/mL). An anti-NGF antibody, a TrkA antagonist (c(92-96)) and inhibitor (k252a) were used to antagonize receptor activation by NGF. Only the small membrane permeable k252a reduced neuronal density, suggesting that the NCJ is an isolated microdomain. K252a was used to estimate NGF concentration at the contact site, which resulted about 1.5nM, four orders of magnitude higher than that in CM-conditioned medium, supporting that the NCJ allows amplification of intercellular NGF signaling. Conclusions: Taken together, our results suggest that the NGF-dependent pro-survival signal to the SGN needs a direct interaction with the CM that facilitates NGF activation of TrkA thanks to the development of an isolated microdomain characterized by a high NGF concentration.