We have previously proven that vagal stimulation (VS) decreases infarct size (IS) when applied before ischemia, thus mimicking classic preconditioning. Our objective was to study the chronology and mechanisms of the protective effect of VS, and to assess whether the protection is comprised of a first and second window. Mice were randomly assigned to different protocols (n=5-7 per group). The control group had 30 min of regional myocardial ischemia and 2 h of reperfusion (I/R). The other groups consisted of 10 min of pre-ischemic VS (pVS) and different times of recovery between VS and I/R protocol: 5 min, 3-6-12-24-48-72-96 h. The 5 min, 3 h and

72 h groups were redone to administer a muscarinic receptor inhibitor (atropine). pVS- 5min and pVS-72h were given a mitochondrial K+ ATP channel blocker (5-HD) and a nitric oxide synthase inhibitor (L-NAME). All animals were catheterized to measure ventricular function. Hearts were dyed with Evans Blue and incubated in TTC to
measure IS. pVS-5min had smaller IS in comparison to I/R (44±3% and 57±2%, respectively; p<0.05). pVS-3hs and pVS-6hs had a deeper IS reduction (34±3% and 34±3%, respectively; p<0.05 vs pVS-5min). Cardioprotection was lost at 12, 24 and 48 h post-VS (56±4%, 53±2%, 56±2%, respectively; p=NS vs I/R). Later at 72 h post-VS, cardioprotection reappeared (42±4%; p<0.05 vs pVS-48h) but was lost at 96 h post-VS (56±3%; p=NS vs I/R). The IS-reducing effect of pVS-5min, 3 and 72 h was abolished by atropine (56±2%, 56±3% and 56±3%; p=NS vs I/R), 5-HD in pVS- 5min and 72 h (55±2% and 62±5%, p=NS vs I/R) and L-NAME in pVS-5min and pVS-72h (56±3% and 57±2%, p=NS vs I/R). In conclusion, VS has two protection windows similar to classic preconditioning. The first window lasts 6 h and the second window appears at 72 h and is lost at 96 h. The mechanisms underlying the cardioprotective effect include muscarinic receptors, mitochondrial K+ ATP channels and nitric oxide synthase.