Role and mechanism of PKC in ischemic preconditioning of pig skeletal muscle against infarction

Protein kinase C (PKC) inhibitors, chelerythrine (Chel, 0.6 mg) and polymyx in B (Poly B, 1.0 mg), and PKC activators, phorbol 12-myristate 13-acetate (PMA, 0.05 mg) and 1-oleoyl-2- acetyl glycerol (OAG, 0.1 mg), were used as probes to investigate the role of PKC in mediation of ischemic precondition ing (IPC) of noncontracting pig latissimus dorsi (LD) muscles against infar ction in vivo. These drugs were delivered to each LD muscle flap (8 x 12 cm ) by 10 min of local intra-arterial infusion. It was observed that LD muscl e flaps sustained 43 +/- 5% infarction when subjected to 4 h of global isch emia and 24 h of reperfusion. IPC with three cycles of 10 min ischemia-repe rfusion reduced muscle infarction to 25 +/- 3% (P< 0.05). This anti-infarct ion effect of IPC was blocked by Chel (42 +/- 7%) and Poly B (37 +/- 2%) an d mimicked by PMA (19 +/- 10%) and OAG (14 +/- 5%) treatments (P< 0.05), gi ven 10 min before 4 h of ischemia. In addition, the ATP-sensitive K+ (K-ATP ) channel antagonist sodium 5-hydroxydecanoate attenuated (P< 0.05) the ant i-infarction effect of IPC (37 +/- 2%), PMA (44 +/- 17%), and OAG (46 +/- 9 %). IPC, OAG, and Chel treatment alone did not affect mean arterial blood p ressure or muscle blood flow assessed by 15-mu m radioactive microspheres. Western blot analysis of muscle biopsies obtained before (baseline) and aft er IPC demonstrated seven cytosol-associated isoforms, with nPKC epsilon al one demonstrating progressive cytosol-to-membrane translocation within 10 m in after the final ischemia period of IPC. Using differential fractionation , it was observed that nPKCe translocated to a membrane compartment other t han the sarcolemma and/or sarcoplasmic reticulum. Furthermore, IPC and prei schemic OAG but not postischemic OAG treatment reduced (P< 0.05) muscle mye loperoxidase activity compared with time-matched ischemic controls during 1 6 h of reperfusion after 4 h of ischemia. Taken together, these observation s indicate that PKC plays a central role in the anti-infarction effect of I PC in pig LD muscles, most likely through a PKC-K-ATP channel-linked signal -transduction pathway.