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Rationale: The heart is mainly innervated by the sympathetic nervous system that is involved in the fight or flight response. Sympathetic neurons (SNs), whose cell bodies are placed in the stellate and superior cervical ganglia, mediate the main physiological mechanism increasing the frequency and force of cardiac contraction through release of norepinephrine. Recently, we have reported that SNs regulate heart trophism through stimulation of β2 adrenergic receptors and repression of muscle specific ubiquitin ligases (i.e. Murf1 and Atrogin1) but not much is known about the effects of SNs on sarcomeres. Nerve growth factor (NGF) released by the myocardium controls cardiac innervation by SNs after binding to its receptor (TrkA) and is required for neuronal survival. Thus, bidirectional coupling between SGNs and the heart takes place: the heart needs to be coupled to the SNs to receive norepinephrine stimulation for an efficient increase in heart contraction, and conversely, SNs are coupled to the heart for neurotrophic stimulation that is required for neuronal viability. However, whether a cell-cell interaction occurs in the SN-heart coupling is not known. An interaction between the muscle and the neuron that has been well described both in terms of function and structure, is the neuro-muscular junction (NMJ), characterized by membrane thickenings, acetylcholinergic receptor clustering, reduced intermembrane space (70-50 nm) and neurotrophin release by the postsynaptic myocyte (e.g. NT3, NT4). Considering the interaction between the SNs and the heart (neuro-cardiac junction, NCJ), this study aims i) to evaluate the effects of anterograde SN stimulation on sarcomeric structures, ii) to determine whether specific cellular structures are present at the SN/Cardiomyocyte (CM) contact site, iiI) to investigate the role of SGN/CM contact in NGF-mediated signaling. Results: To analyze changes in sarcomere structure, cultured CMs were treated with adrenergic stimuli (clenbuterol, phenylephrine and norepinephrine) or nutrient/serum deprived by HBSS incubation. Since starvation and sympathetic denervation share common targets (e.g. ubiquitin ligases), we can make a parallelism between the two pro-atrophic stimuli for alterations in the sarcomeres. Incubation with adrenergic agonists did not cause significant changes, whereas starvation caused a 41.86% decrease in sarcomere area, suggesting that sarcomere degradation is faster than its synthesis. This result was confirmed by experiments of in live imaging performed on CMs transfected with a construct encoding for the z-line localized RFP-zasp. To understand whether all sarcomeric proteins share the same fate during HBSS treatment, immunofluorescence (IF) and western blot (WB) analyses were performed on different proteins localized in the sarcomeres. While α actinin and cardiac tropoinin (cTn) I showed delocalization and degradation, no significant changes were measured for cTnT upon HBSS treatment, suggesting that sarcomeric proteins are degraded in different ways. To understand which protein degradation system is involved in sarcomere disassembly, we considered the autophagy-lysosome and ubiquitin proteasome systems (UPS). WB and IF analyses supported the activation of both systems in cells treated with HBSS. In live imaging of CMs co-transfected with constructs encoding for RFP-zasp and EGFP-LC3 showed LC3 enrichment near sarcomeres in nutrient/serum deprived cells, suggesting that autophagy may be involved in sarcomere degradation. Moreover, IF staining showed ubiquitin marked sites near the M-line of sarcomeres in cells incubated with HBSS, suggesting that ubiquitin ligases may be involved in sarcomere disassembly. Since Murf1 is a muscle specific ubiquitin ligase, localized in the M-line of sarcomeres and upregulated upon nutrient/serum deprivation, we evaluated its role. Its overexpression caused a 88.57% decrease in the sarcomere area, when compared to controls, whereas its silencing in starved CMs did not prevent sarcomere degradation (446.19 ±35.65 vs 144.91 ±26.25 μm2 of sarcomeric area in controls and silenced CMs respectively). These results were confirmed by in live imaging on RFP-zasp transfected CMs, and suggest that UPS and in particular Murf1 are involved in HBSS induced sarcomeric disassembly and that Murf1-mediated degradation is not the only process. Considering the analysis of the neuro-cardiac interaction, IF staining on rat heart cryosections showed dense innervation of the heart by sympathetic neurons that mainly interact with CMs when compared to other cardiac cell types that are well represented in the heart (e.g. cardiac fibroblasts, CFs). Electron microscopy on mouse heart slices and rat SN/CM co-cultures showed a close association between SNs and CMs (intermembrane distance around 70 nm), neurotransmitter vesicle accumulation and increased membrane protein density. These data support that a direct interaction between the sympathetic neurons and the CMs exists. To analyze such interaction, SN/CM co-cultures were developed by isolating sympathetic ganglia neurons (SGN) from the superior cervical ganglia and CMs from the hearts of neonatal rats. Both cell types were characterized using IF staining for dopamine β-hydroxylase, a maker for noradrenergic neurons, and for α actinin, a sarcomeric protein. Moreover, IF staining showed an enrichment of cell-to-cell adhesion molecules including β-catenin and cadherin at the contact sites between processes and CMs. Such enrichment developed after 2 weeks of co-culture, suggesting that SGN/CM co-cultures are subjected to time dependent maturation. In spotted co-cultures allowing to identify processes on wither CMs or non CM cardiac cells (mainly fibroblasts), a higher area occupied by processes was measured on CMs when compared to the other cardiac cells after NGF withdrawal (67.11 ±12.36% vs 3.79 ±1.12% of area occupied by processes respectively), supporting the preferential interaction of SGNs with CMs. This idea is further supported by the observation that SGNs develop larger contact sites on CMs than in other cardiac cells (82.88 ±1.3% decrease in contact area on non CM cardiac cells when compared to CMs). Taken together, all these data suggest that SGNs establish a direct and stable interaction with CMs and not other cardiac cells. Since the myocardium is known to produce NGF that is required for SN viability, the functional role of the NCJ was assessed considering NGF signaling. This neurotrophin is synthesized by CMs, as detected by the western blot analysis. Transfection of CMs with siRNA against NGF caused a 72.91% decrease of the neurotrophin expression, reducing neuronal density in SGN/CM co-cultures (65.72 ±9.33% decrease in mean neuronal density when compared to the scramble siRNA). This effect was abolished by the addition of NGF in the culture medium and supports that SGNs are dependent on CM derived NGF. NGF binding to its receptor enables TrkA activation, endocytosis and retrograde transport to the neuronal soma. TrkA retrograde movements were assessed by monitoring transport velocity, using imaging in transiently TrkA-DsRed2 transfected SGNs. The speed of retrograde TrkA-DsRed2 movements depended on the presence of NGF (0.32 ±0.06 vs 0.19 ±0.03μm/s in presence or absence of NGF). In co-cultures, retrograde movements were higher and faster in processes contacting CMs than those contacting other cardiac cells (0.24 ±0.05 vs 0.11 ±0.02μm/s respectively), supporting the idea that TrkA is activated on CMs and not on the other cardiac cells and that SGN survival requires CM derived NGF. Since SGNs interact with CMs and depend on CM released NGF, we tested the hypothesis that the NCJ is necessary for neuronal survival. IF on mouse heart slices showed TrkA enrichment at SGN/CM contact site, suggesting that NGF signaling may be involved in the NCJ. Moreover, CM-conditioned medium did not prevent neuronal death (58.21 ±10.42% decrease in mean neuronal density when compared to SGN/CM co-cultures), suggesting that NGF in the medium is not sufficient for neuronal survival. Consistently, we measured NGF concentration in CM-conditioned medium and it was a 1000-fold lower than the minimal dose required for neuronal survival (0.13 ±0.08pM). To evaluate whether a single cell-to-single cell NGF signaling occurs between SGNs and CMs, co-cultures were co-transfected with siRNA against NGF and a plasmid encoding for the GFP that allows the identification of NGF silenced CMs. Sympathetic processes on NGF-silenced CMs showed a 19.56 ±4.01% decrease in the neuro-cardiac contact area when compared to those on untransfected CMs of the same co-culture, supporting that direct cell-cell NGF mediated signaling is present. Moreover, co-cultures were transfected with a construct encoding NGF in order to detect NGF accumulation in processes using the IF. Only processes in contact with transfected CM contained NGF puncta, while those in contact with untransfected cells of the same co-culture did not contain NGF (43.43 ±10.77 vs 4.17 ± 4.1% of processes on transfected or un-transfected CMs). Taken together, these data suggest that the establishment of a neuro-cardiac interaction is necessary to allow NGF signaling. In the end of this work, we interfered with NGF signaling using different strategies. First, we used an anti-NGF antibody to sequester NGF. Second, since from TEM analysis we detected sites of cell-to-cell distance of 10nm, we used the smaller TrkA antagonist c(92-96). Third, we used k252a that has a size comparable to that of c(92-96) and that is membrane permeable. Whereas every approach worked on SGNs alone leading to a significant reduction in neuronal density, only k252a was able to reduce neuronal density in co-cultures (73.24 ±4.18% decrease in mean neuronal density when compared to the control), suggesting that the NCJ is an isolated microenvironment protected from diffusion. Since k252a led to neuronal loss in co-cultures, we used this inhibitor to estimate NGF concentration at the contact site, incubating SGNs alone with k252a and NGF at increasing concentrations. The estimated concentration was 1.4 ±0.03nM, 3.5 times higher than the minimal dose required for neuronal survival, supporting that the NCJ is characterized by high NGF concentration. Conclusions: Taken together, our results suggest that sympathetic neurons establish a direct interaction with CMs and that they are dependent on CM derived NGF. Morever, NGF-dependent pro-survival signal to the SGN needs this direct interaction that facilitates NGF activation of TrkA thanks to the development of an isolated microdomain characterized by a high NGF concentration and TrkA enrichment. Finally, sarcomere dismantlement during atrophic remodeling involves the activation of protein degradation systems and in particular of the ubiquitin ligase Murf1, whose regulation by SNs may affect sarcomere structure