Purpose: Genome wide association studies have implicated neuronal nitric oxide synthase adaptor protein (NOS1-AP/CAPON) as a potential molecular marker of both QT abnormalities on the ECG and sudden cardiac death (SCD). SCD is often triggered by cardiac sympathetic stimulation which is also able to modulate the QT interval. Interestingly, NO generated by NOS-1 reduces noradrenaline (NA) release, but this pathway is down-regulated in animals with hypertension or following myocardial infarction. To establish if this adaptor protein plays a role in sympathetic neurotransmission we investigated whether endogenous NOS1-AP was present in cardiac sympathetic neurons. We also tested the hypothesis that enhancing neuronal NOS1-AP expression via adenoviral gene transfer decreases sympathetic induced NA release by altering the regulation of intracellular calcium handling. Methods and Results: Immunohistochemistry demonstrated that NOS1-AP resides in cardiac sympathetic neurons in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) controls, but the expression of NOS1-AP is significantly (P<0.05) reduced in the SHR (n=6) compared to the WKY (n=6). The peak [Ca2+]i transient of isolated stellate neurons was significantly enhanced in the SHR (n=12) compared to the WKY (n=11). A novel noradrenergic cell specific vector (Ad.PRSx8-NOS1-AP/mCherry) or its control vector were transfected into sympathetic stellate neurons in vitro. Western blotting showed NOS1-AP expression was up-regulated following gene transfer in the SHR (n=6 v empty virus n=6). The peak [Ca2+]i transient (n=19) was also reduced compared with Ad.PRSx8-mCherry alone (n=16). NOS1 inhibition (AAAN, 10µM) significantly increased the [Ca2+]i transient after Ad.PRSx8-NOS1-AP/mCherry transfection. Electrophysiological studies showed that the peak calcium current (ICa) density in SHR neurons overexpressing NOS1-AP (-17.8 ± 1.2 pA/pF, n=6) was significantly reduced compared with that of the empty vector control in SHR neurons (-25.2 ± 3.1 pA/pF, n=7). Moreover, Ad.PRSx8-NOS1-AP/mCherry reduced 3H-NA release from SHR atria (n=7) compared with empty vector controls (n=6). Conclusions: Artificial up-regulation of cardiac sympathetic NOS1-AP via targeted gene transfer can directly attenuate intracellular Ca2+ and suppress ICa, resulting in decreased NA release in the SHR. This may provide a novel method for decreasing enhanced cardiac sympathetic neurotransmission in disease states where excessive NA release might trigger sudden cardiac death in patients with QT abnormalities.