Purpose: Implantable cardiac vagus nerve stimulators are a promising new treatment for heart failure1, which may improve both quality of life and ejection fraction. Animal studies also suggest an anti-fibrillatory effect of stimulating the cardiac vagus nerve that may involve a nitric oxide (NO) dependent pathway2, although the exact site of action in the cardiac-neural axis is still debated. We investigated whether carbamylcholine (CCh), a stable analogue of the neurotransmitter acetylcholine, can mimic the effect of vagus nerve stimulation on ventricular fibrillation threshold (VFT), and whether this mechanism is dependent on muscarinic (MR) and/or nicotinic receptor (NR) stimulation and/or generation of NO.Methods: Hearts were isolated from adult male Sprague Dawley rats (300-350g) and Langendorff perfused in constant flow mode (baseline perfusion pressure 54.75±7.27mmHg, LV developed pressure 77.5±5.6mmHg, heart rate 282.8±3.4bpm, n=6). VFT was reproducibly determined by pacing at a fixed cycle length (150msec) for 20 beats followed by a 5sec 50Hz burst at increasing current amplitude (mA) until sustained VF was induced. VF was cardioverted to sinus rhythm by perfusion with 1ml of high concentration potassium chloride solution (50mmol/L). Electrical restitution (RT) was derived from optically mapped action potentials from hearts loaded with RH237 and blebbistatin (10μM, n=6). NO metabolites (NOx) were measured in snap frozen coronary effluent using an ozone chemiluminescence assay. All data are presented as mean±SEM with ANOVA/t-test where appropriate. Results: CCh (200nM, n=9) significantly (p<0.05) reduced baseline heart rate from 292±8 to 224±6bpm). Independent of this heart rate change, CCh also caused a significant increase in VFT in paced hearts that could be reversed with washout of the drug (control 1.5±0.25 vs. CCh 2.4±0.4 mA vs. wash out 1.14±0.18 mA). CCh modified cardiac repolarisation with flattening of the RT curve (control 0.36±0.1 vs CCh 0.15±0.03, n=6, p<0.05). The effect of CCh on VFT was completely abolished by the MR antagonist atropine (0.1μM, n=6) or the NR antagonist, mecamylamine (10μM, n=6). The specific nNOS inhibitor AAAN (10μM, n=6) or the soluble guanylate cyclase inhibitor ODQ (10μM, n=6) also prevented the rise in VFT with CCh. CCh perfusion significantly increased NOx content following the onset of bradycardia (pre bradycardia 0.001±0.02 vs post bradycardia 0.23±0.1μmol NOx, n=8). Conclusions: These data demonstrate that the protective effect of CCh on VFT is dependent upon muscarinic receptor and nicotinic receptor stimulation. Generation of NO appears to be involved in mediating this protective effect, via a nNOS, sGC dependent pathway.