Time course of early and delayed myocardial protection induced by vagal nerve stimulation preconditioning

We previously reported that pre-ischemic vagus nerve stimulation (VNS) protects against myocardial ischemia, resembling classical ischemic preconditioning (cPC). This study investigates the time course and mechanisms underlying VNS-induced cardioprotection. Male FVB/N mice (3-5 months) underwent 30 min regional myocardial ischemia followed by 120 min reperfusion (IR). Ten-minute right-sided cervical VNS was delivered at defined intervals before ischemia to assess early and delayed protective windows. Risk area (RA) and infarct size (IS) were quantified using Evans Blue/tetrazolium staining. Myocardial phosphorylation of protein kinase B (Akt), glycogen synthase kinase-3β (GSK-3β), inducible nitric oxide synthase (iNOS), and mitochondrial respiration were evaluated. IR controls exhibited an IS of 57 ± 7% of the RA. VNS elicited a biphasic cardioprotective response. Early protection was observed when ischemia occurred 5 min, 3 h, or 6 h after VNS, with IS reduced to 44 ± 8%, 34 ± 7%, and 36 ± 9%, respectively (p ≤ 0.0001 vs. IR). This phase depended on muscarinic acetylcholine receptor (mAChR) activation, involved Akt/GSK-3β/NOS signaling, and was associated with preserved mitochondrial respiration. The protective effect was abolished by atropine, NG-nitro-L-arginine methyl ester (L-NAME), or the mitoK_ATP channel blocker 5-hydroxydecanoate (5HD). Delayed protection emerged 72 h after VNS, reducing IS to 42 ± 7% (p = 0.0001 vs. IR). This phase was independent of mitochondrial respiration preservation and required mAChR and iNOS signaling, as it was abolished by atropine, L-NAME, or the selective iNOS inhibitor 1400W, but not by 5HD. These findings identify pre-ischemic VNS as a biphasic cardioprotective strategy with distinct phase-specific mechanisms, highlighting its potential therapeutic relevance in limiting IR injury.