We have utilized an in situ rat coronary ligation model to establish a PKC-ε cytochrome oxidase subunit IV (COIV) coimmunoprecipitation in myocardium exposed to ischemic preconditioning (PC). Ischemia-reperfusion (I/R) damage and PC protection were confirmed using tetrazolium-based staining methods and serum levels of cardiac troponin I. Homogenates prepared from the regions at risk (RAR) and not at risk (RNAR) for I/R injury were fractionated into cell-soluble (S), 600 g low-speed centrifugation (L), Percoll/Optiprep density gradient-purified mitochondrial (M), and 100,000 g particulate (P) fractions. COIV immunoreactivity and cytochrome-c oxidase activity measurements estimated the percentages of cellular mitochondria in S, L, M, and P fractions to be 0, 55, 29, and 16%, respectively. We observed 18, 3, and 3% of PKC-δ, -ε, and -ζ isozymes in the M fraction under basal conditions. Following PC, we observed a 61% increase in PKC-ε levels in the RAR M fraction compared with the RNAR M fraction. In RAR mitochondria, we also observed a 2.8-fold increase in PKC-ε serine 729 phosphoimmunoreactivity (autophosphorylation), indicating the presence of activated PKC-ε in mitochondria following PC. PC administered before prolonged I/R induced a 1.9-fold increase in the coimmunoprecipitation of COIV, with anti-PKC-ε antisera and a twofold enhancement of cytochrome-c oxidase activity. Our results suggest that PKC-ε may interact with COIV as a component of the cardioprotection in PC. Induction of this interaction may provide a novel therapeutic target for protecting the heart from I/R damage.
|Journal||American Journal of Physiology - Heart and Circulatory Physiology|
|State||Published - Oct 2007|
- Coronary ligation
- Oxidative phosphorylation
- Protein kinase C