Introduction: Sympathetic hyper-responsiveness is an early hallmark of hypertension and may even precede the overt clinical signs of the disease itself. Metabolic and oxidative stress linked to impaired NO bioavailability has been implicated as a putative cellular determinant underpinning enhanced sympathetic gain (1,2). Recent studies have shown neuronal release of noradrenaline in animal models of hypertension (3) is related to greater intracellular calcium transients ([Ca2+]i) (4), associated with impaired nNOS-cGMP signalling (5). However, the molecular links to abnormal [Ca2+]i regulation are poorly understood.Methods: In this study, we used label-free quantitative (LFQ) proteomics to compare cultured cardiac sympathetic stellate neurons from pro-hypertensive spontaneously hypertensive rat (SHR) with normotensive Wistar-Kyoto rat (WKY) pups. We then performed adenovirus transduction of neuronal nitric oxide synthase (nNOS) in these SHR (termed SHRnNOS) and WKY (termed WKYnNOS) cultured neurons. Canonical protein interaction pathways and networks were generated using Ingenuity Pathway Analysis (IPA) to study protein regulation. In order to evidence patterns of protein enrichment or depletion at a systemic level we also carried out functional network analyses of the proteins returned by the LFQ study using network association data from the STRING database, followed by clustering and functional enrichment analysis using data from the Gene Ontology. Results: Multiple regulatory changes of proteins involved in metabolic processes, stress responses, scaffolding, tissue architecture and cell migration were observed in the SHR compared to the WKY neurons. IPA analysis indicated that pathways closely related to energy production, cellular organisation and metabolic function were perturbed in SHR neurons. Unexpectedly we found abnormally high levels of non-muscle Myosin 9/Myosin IIA in pro-hypertensive neurons. We also detected down regulation of mitochondrial complexes. The artificial up-regulation of nitric oxide via viral nNOS transduction rescued these changes observed in the SHR neurons. Discussion: These results indicate that pre-programming of genes involved in regulating Ca2+ handling proteins in sympathetic neurons occur even before the phenotype of sympathetic hyper-responsiveness is apparent. Enhancing nNOS expression rescues several signalling pathways coupled to metabolic, cytoskeletal and calcium signalling that are all linked to abnormal neurotransmission in this genetic model of hypertension.