Nerve Growth Factor transfer from cardiomyocytes to innervating sympathetic neurons activates TrkA receptors at the neuro-cardiac junction.

The integration of ex vivo and in vitro data, described in this manuscript, together with our previous demonstration that sympathetic neurons (SNs) contact target cardiomyocytes (CMs) at the neuro-cardiac junction (NCJ), which underlies intercellular synaptic communication (Prando et al., 47), demonstrate that: CMs are the cell source of Nerve Growth Factor (NGF), required to sustain innervating cardiac SNs; NCJ is the place of the intimate liaison, between SNs and CMs, allowing on the one hand neurons to peremptorily control CM activity, and on the other, CMs to adequately sustain the contacting, everchanging, neuronal actuators; alterations in NCJ integrity may compromise the efficiency of 'CM-to-SN' signaling, thus representing a potentially novel mechanism of sympathetic denervation in cardiac diseases. ABSTRACT: Background Sympathetic neurons densely innervate the myocardium with non-random topology and establish structured contacts (i.e. neuro-cardiac junctions, NCJ) with cardiomyocytes, allowing synaptic intercellular communication. Establishment of heart innervation is regulated by molecular mediators released by myocardial cells. The mechanisms underlying maintenance of cardiac innervation in the fully developed heart, are, however, less clear. Notably, several cardiac diseases, primarily affecting cardiomyocytes, are associated to sympathetic denervation, supporting that retrograde 'cardiomyocyte-to-sympathetic neuron' communication is essential for heart cellular homeostasis. Objective We aimed to determine whether cardiomyocytes provide Nerve Growth Factor (NGF) to sympathetic neurons, and the role of the NCJ in supporting such retrograde neurotrophic signaling. Methods and Results Immunofluorescence on murine and human heart slices shows that NGF and its receptor, Tropomyosin-receptor-kinase-A, accumulate respectively in the pre- and post-junctional sides of the NCJ. Confocal immunofluorescence, scanning ion conductance microscopy and molecular analyses, in co-cultures, demonstrate that cardiomyocytes feed NGF to sympathetic neurons, and that such mechanism requires a stable intercellular contact at the NCJ. Consistently, cardiac fibroblasts, devoid of NCJ, are unable to sustain SN viability. ELISA assay and competition binding experiments suggest that this depends on the NCJ being an insulated microenvironment, characterized by high [NGF]. In further support, real-time imaging of Tropomyosin-receptor-kinase-A-vesicle movements demonstrate that efficiency of neurotrophic signaling parallels the maturation of such structured intercellular contacts. Conclusions Altogether, our results demonstrate the mechanisms which link sympathetic neuron survival to neurotrophin release by directly innervated cardiomyocytes, conceptualizing sympathetic neurons as cardiomyocyte-driven heart drivers. Abstract figure legend Sympathetic neuron (SN, green) varicosities establish synaptic contacts with target cardiomyocytes (CMs, pink), which we previously called Neuro-Cardiac Junction (NCJ, Prando et al. J Physiol 47). At NCJs, CMs release selectively NGF, which by activating TrkA signaling, is key to sustain neuronal survival. This article is protected by copyright. All rights reserved