Isoflurane postconditioning prevents opening of the mitochondrial permeability transition pore through inhibition of glycogen synthase kinase 3β

Background: Postischemic administration of volatile anesthetics activates reperfusion injury salvage kinases and decreases myocardial damage. However, the mechanisms underlying anesthetic postconditioning are unclear. Methods: isolated perfused rat hearts were exposed to 40 min of ischemia followed by 1 h of reperfusion. Anesthetic postconditioning was induced by 15 min of 2.1 vol% isoflurane (1.5 minimum alveolar concentration) administered at the onset of reperfusion. In some experiments, atractyloside (10 pm), a mitochondrial permeability transition pore (mPTP) opener, and LY294002 (15 mu m), a phosphatidylinositol 3-kinase Inhibitor, were coadministered with isoflurane. Western blot analysis was used to determine phosphorylation of protein kinase B/Akt and its downstream target glycogen synthase kinase 3 beta after 15 min of reperfusion. Myocardial tissue content of nicotinamide adenine dinucleotide served as a marker for mPTP opening. Accumulation of MitoTracker Red 580 (Molecular Probes, Invitrogen, Basel, Switzerland) was used to visualize mitochondrial Junction. Results: Anesthetic postconditioning significantly improved functional recovery and decreased infarct size (36 +/- 1% in unprotected hearts vs. 3 +/- 2% in anesthetic postconditioning; P < 0.05). Isoflurane-mediated protection was abolished by atractyloside and LY294002. LY294002 inhibited isoflurane-mduced phosphorylation of protein kinase B/Akt and glycogen synthase kinase 313 and opened mPTP as determined by nicotinamide adenine dinucleotide measurements. Atractyloside, a direct opener of the mPTP, did not inhibit phosphorylation of protein kinase B/Akt and glycogen synthase kinase 316 by isoflurane but reversed isoflurane-mediated cytoprotection. Microscopy showed accumulation of the mitochondrial tracker in isoflurane-protected functional mitochondria but no staining in mitochondria of unprotected hearts. Conclusions: Anesthetic postconditioning by isoflurane effectively protects against repertlusion damage by preventing opening of the mPTP through inhibition of glycogen synthase kinase 3 beta.