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These data suggest that postcon modulates PKC during early reperfusion by increasing PKCepsilon expression and translocation to a site other than the outer mitochondrial membrane, and limits translocation of PKCdelta to mitochondria and associated deleterious signalling.

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Oxidative stress-stimulated nuclear factor-kappa B (NF-kappa B) activation has been associated with rapid transcription of TNF-alpha and induction of apoptosis. This study tested the hypothesis that postconditioning (Postcon) reduces myocardial apoptosis and inhibits translocation of NF-kappa B and release of TNF-alpha secondary to an attenuation of oxidant generation during reperfusion. Anesthetized rats were subjected to 30 min of ischemia and 3 h of reperfusion and divided randomly to Control or Postcon (three cycles of 10-s reperfusion and 10-s reocclusion applied at the onset of reperfusion) group, respectively. Relative to Control, Postcon reduced the plasma malondialdehyde (1.21 +/- 0.08 vs. 0.8 +/- 0.06* microM/mL) and decreased the generation of superoxide radical in area at risk myocardium (dihydroethidium staining). Compared with Control, Postcon also inhibited translocation of NF-kappa B to nuclei (167% +/- 21% vs. 142% +/- 18%*), decreased the level of plasma TNF-alpha (1,994 +/- 447 vs. 667 +/- 130* pg/mL), and inhibited caspase-3 activity (0.57% +/- 0.1% vs. 0.21% +/- 0.1%*). The number of apoptotic cells (percent total nuclei) in ischemic myocardium was reduced (20% +/- 1% vs. 11% +/- 2%*), consistent with reduced appearance of DNA fragmentation. To support whether oxidant generation is important in the triggering of cytokine release and apoptosis, N-acetylcysteine (NAC), a potent antioxidant agent, was administered before ischemia and at reperfusion. Treatment with NAC inhibited superoxide radical generation and decreased plasma malondialdehyde to a comparable level to that in Postcon, concomitant with an inhibition of NF-kappa B expression (42% +/- 8%*) and reduction of release of TNF-alpha (231 +/- 72* pg/mL). Caspase-3 activity (0.33% +/- 0.1%*) and apoptotic cells (12% +/- 1%*) were also comparably reduced by NAC. These data suggest that Postcon attenuates myocardial apoptosis, reduces caspase-3 activity, and is potentially mediated by inhibiting oxidant-activated NF-kappa B-TNF-alpha signaling pathway. *P < 0.05 Postcon and NAC vs. Control.

no intervention at R; Postcon: three cycles of 30 s R alternating with 30 s re-occlusion were applied at R; 5-hydroxydecanoate (5-HD): the mitochondrial K(ATP) channel blocker was infused 5 min before Postcon; HMR1098: the sarcolemmal K(ATP) channel blocker was administered 5 min before Postcon. After 24 h of R, infarct size was smaller in Postcon relative to CONTROL (27 +/- 4%* Vs. 39 +/- 2% of area at risk), consistent with a reduction in CK activity (66 +/- 7* Vs. 105 +/- 7 IU/g). The infarct-sparing effect of Postcon was blocked by 5-HD (48 +/- 5%(dagger)), but was not altered by HMR1098 (29 +/- 3%*), consistent with the change in CK activity (102 +/- 8(dagger) in 5-HD and 71 +/- 6* IU/g in HMR1098). In H9c2 cells exposed to 8 h hypoxia and 3 h of reoxygenation, Postcon up-regulated expression of mito-K(ATP) channel Kir6.1 protein, maintained mitochondrial membrane potential and inhibited mitochondrial permeability transition pore (mPTP) opening evidenced by preserved fluorescent TMRE and calcein staining. The protective effects were blocked by 5-HD, but not by HMR1098. These data suggest that in a clinically relevant model of ischemia-reperfusion (1) Postcon reduces infarct size and decreases CK activity after prolonged reperfusion; (2) protection by Postcon is achieved by opening mitochondrial K(ATP) channels and inhibiting mPTP opening. *P < 0.05 Vs. CONTROL; P < 0.05 Vs. Postcon.