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

# 272 The neuro-cardiac interaction defines an extracellular microdomain required for neurotrophic signaling {#article-title-2} 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 co-cultures 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 neurotrophin-mediated 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. # 273 Gut microbiota is important in the development of angiotensin II driven arterial hypertension and vascular dysfunction in mice {#article-title-3} Background: The presence of gut microbiota is essential for proper function of the immune cells. Its impact on the blood pressure regulation and systemic vascular function - processes that are described to be immune cell regulated - is unknown. Methods and Results: There were no differences in endothelial function (measured by organ chamber experiments) or oxidative stress levels (measured by L012 chemiluminescence) in unchallenged germfree mice (GF) Swiss Webster mice compared to conventionally-reared (CONV-R) Swiss Webster mice. Angiotensin II (ATII, 1mg/kg/d for 7d)-infused GF C57BL/6 mice showed attentuated systemic and vascular oxidative stress levels (dihydroxyethidium staining and L0-12 enhanced chemiluminescence) compared to ATII infused CONV-R mice. There was also an attenuated vascular expression of monocyte chemo-attractant protein-1, NOX-2 and iNOS, as well as a reduced up-regulation the retinoic-acidreceptor-related orphan receptor-gt (ROR-gt) in the aorta and protection from endothelial dysfunction in ATII-treated GF C57BL/6 mice compared to ATII-treated CONV-R C57BL/6 mice. We also found attenuated infiltration of CD11b+ myelomonocytic cells, Ly6G+ neutrophils and Ly6C+ monocytes into the aortic vessel wall (FACS analysis) in the ATII-treated GF C57BL/6 mice compared to ATII-treated CONV-R C57BL/6 mice. This was accompanied by an attenuation of arterial hypertension (measured by tail-cuff method)in the ATII-treated GF mice. Besides, the ATII induced cardiac hypertrophy and hypercontractility (measured by echocardiography) and the cardiomyocyte hypertrophy and fibrosis (assessed by immunohistochemistry) were attenuated in GF mice compared to CONV-R control mice. Conclusion: Gut microbiota seem to facilitate Angiotensin II induced hypertension by supporting the infiltration of Ly6G+ neutrophils into the vessel wall. # 274 Role of the mitochondrial protein Opa1 in the regulation of the cardiac sympathetic neuron physiology {#article-title-4} Background: Cardiac sympathetic neurons (cSNs) are essential for cardiac homeostasis, as they mediate heart adaptation to stress conditions, and their basal activity is required to modulate cardiomyocyte trophism (1). cSN viability and activity depend on the correct mitochondrial function. The Optic Atrophy-1 protein (Opa1) controls mitochondrial fusion, ATP production and apoptosis, and its mutation is associated to Autosomal Dominant Optic Atrophy (ADOA), a neurodegenerative disease, characterized by ‘retinal ganglion cell’ death, myopathy and peripheral neuropathy. While the key role of Opa1 in the physiology of central neurons has been provided, less is known on its role in peripheral neurons, including cSNs. The aim of this study is thus to assess the role of Opa1 in cSN function and viability. Methods: IF, morphometry, ECHO and telemetry-based ECG recordings, both at baseline and during moderate exercise, were performed to characterize the effects of Opa1 haploinsufficiency on cSN physiology, during aging. Results: We generated a mouse model haploinsufficient for Opa1, in cSNs, by crossing mice expressing Cre-recombinase, under the control of tyrosine hydroxylase (TOH) promoter, with loxP-Opa1 transgenic mice (TOHcre-Opa1+/-). These mice were analysed at different ages from 3 to 18 mo. Reduction in the transcriptional level of Opa1 (about 50%) causes, already in the adulthood (3 mo.), a 25% reduction in the density of cSNs, which display fragmentation, reduction in the size of active releasing sites, and alterations in the innervation patterning, as compared to sex- and aged-matched controls. Such alterations progresses during aging, a condition associated to dysfunctional sympathetic function and innervation. The reduction in cSNs density, in TOH-Opa1+/- mice, resulted in a dysfunctional extrinsic control of the cardiac rhythm. Indeed, the heart rate variability was reduced already in adult TOH-Opa1+/- mice (SDNN, Control: 6.17±0,36 vs TOH-Opa1+/-: 3,62±0,46, in msec) and the reduction was more pronounced in older mice (SDNN, Control: 3,86±0,60 vs TOH-Opa1+/-: 1.17±0,19, in msec). Consistently, TOHcre-Opa1+/- displayed reduced adaptation to exercise. Conclusions: Our data demonstrate that the Opa1 is essential for cSN homeostasis, and indicate that Opa1 haploinsufficiency leads to precocious cSN aging. The mechanisms responsible for Opa1 haploinsufficiency-dependent cSN degeneration will be assessed in vitro, with a focus on the NGF signalling. To translate our findings to the human pathology, we will analyse SN phenotype in skin biopsies from ADOA patients. (1) Zaglia, et al Cardiovasc Res 2013