Variants of the Nitric Oxide Synthase 1 Adaptor Protein (NOS1AP) locus are strongly related to QT interval prolongation and sudden cardiac death (SCD) in human. Neurogenic cardiac damage due to subarachnoid hemorrhage, stroke, epilepsy and myocardial infarction is known to contribute to sudden death in most cases. Our aim was to study the role of NOS1AP in the neurogenic cardiac damage by silencing NOS1AP expression in the Nucleus Tractus Solitarii (NTS) area of the brainstem using lentiviral vector-mediated NOS1AP shRNA (Lv-NOS1AP-shRNA). Real time PCR data showed NOS1AP mRNA levels were expressed in the NTS 3-fold higher than other organs such as kidney and heart in Sprague Dawley (SD) rats. Microinjection of Lv-NOS1AP-shRNA in the NTS caused significant reduction in NOS1AP expression in SD rats. NOS1AP knockdown in NTS did not alter blood pressure (BP), heart rate (HR) recorded by radiotelemetry. However, ECG analysis revealed heart rate variability (HRV) was significantly reduced (SDNN, 51.2±5.6 vs 5.0±1.3ms, P< 0.001, n=6) and QTc interval was markedly prolonged (72.4±4 vs 105±11 ms, P< 0.05, n=6) in NOS1AP knockdown rats. Myocardial damage was also observed with the downregulation of NOS1AP in the NTS of SD rats due to the presence of contraction band necrosis. To study the cellular mechanisms underlying NOS1AP action, we investigated the effect of NOS1AP knockdown on NMDA-induced neurotoxicity in primary cultured neuronal cells from the brainstem. Treatment of cells with Lv-NOS1AP-shRNA significantly reduced NOS1AP expression, and was associated with increased NO production and NMDA-induced neurotoxicity, suggesting a protective effect of NOS1AP. Co-immunoprecipitation studies revealed that the association between neuronal Nitric Oxide Synthase (nNOS) and NMDA Receptor (NMDAR) was significantly increased in neurons treated with Lv-NOS1AP-shRNA, suggesting that NOS1AP might compete with NMDAR in binding to nNOS. Therefore, knockdown NOS1AP expression results in increased association between NMDAR and nNOS, leading to elevated glutamate-induced NO production and neurotoxicity. In summary, all results indicate that NOS1AP plays an important role in the protection of neurons from glutamate-induced neurotoxicity. NOS1AP dysfunction in the NTS might increase the risk of neurogenic cardiac damage, leading to QT interval prolongation, even sudden cardiac death.