Sciences within European Young Researcher Community272The neuro-cardiac interaction defines an extracellular microdomain required for neurotrophic signaling273Gut microbiota is important in the development of angiotensin II driven arterial hypertension and vascular dysfunction in mice274Role of the mitochondrial protein Opa1 in the regulation of the cardiac sympathetic neuron physiology (2016)

Purpose: Sympathetic neurons (SNs) innervate the myocardium with a defined topology that allows physiological modulation of cardiac activity. Limiting amounts of neurotrophins released by cardiac cells control SN viability and myocardial distribution, whose impairment has been described in a number of heart diseases (e.g. myocardial infarction, heart failure). Therefore, the fine control of cardiac innervation is crucial to ensure the physiological sympathetic function. It has been demonstrated that SNs directly interact with cardiomyocytes (CMs). Although it has been proposed that these contact sites may have a role in adrenergic stimulation of the CMs, whether direct interaction is needed for efficient neurotrophic signalling and correct innervation pattern is not known. Methods and Results: Electron microscopy and immunofluorescence on mouse heart slices and SN/CM co-cultures showed close association between neurons and CMs and enrichment of the NGF receptor (TrkA) at the contact site, suggesting that specialized and locally organized signalling domains exist (neuro-cardiac junction, NCJ). In addition, silencing of NGF expression by CMs in cocultures led to 66% decrease of neuronal density, supporting that NGF released by CMs sustains SN viability. We tested whether SN/CM interactions are required for NGF-mediated pro-survival signalling to the neuron and correct myocardial innervation. Cultured neurons in contact with CMs showed fast TrkA activation, NGF uptake, bigger synaptic boutons and survived NGF withdrawal, whereas CM-conditioned medium did not sustain neuronal viability because of the very low NGF concentration (1.61 pg/mL). Altogether, these results support that the NCJ is essential for intercellular neurotrophin signalling. Consistently, NGF concentration at the contact site was estimated by using the TrkA inhibitor K252a and resulted about 1000-fold higher (1.75 ng/mL) when compared to that in CM conditioned medium. Dystrophin accumulation on CM membrane contacted by SNs was observed in mouse cardiac slices, supporting a role for dystrophin at the NCJ. Consistently, hearts from mdx mice showed 74.36% decrease of innervation, with no significant changes of NGF expression. In the absence of exogenous neurotrophin in the culture medium, reduced TrkA activation and neuronal survival was observed in mixed co-cultures between wild type SNs and mdx CMs when compared to the wild type controls. Collectively, these results support that loss of dystrophin at the CM membrane impairs neurotrophinmediated signalling to the neurons and cardiac sympathetic innervation. Conclusions: Taken together, our results suggest that NGF-dependent pro-survival signalling to the neurons requires the direct interaction with myocytes, facilitating TrkA activation thanks to the development of an isolated microdomain characterized by high NGF concentration.