Submitted on November 29, 2006
Accepted on July 8, 2007
PKC co-immunoprecipitates with cytochrome c oxidase subunit IV and is associated with improved cytochrome c oxidase activity and cardio-protection
DeHuang Guo1, Tiffany Nguyen1, Mourad Ogbi1, Huda Tawfik1, Guochun Ma1, Qilin Yu2, Robert William Caldwell3, and John A. Johnson3*
1 Augusta, Georgia, United States; Pharmacology & Toxicology, Medical College of Georgia School of Medicine, Augusta, Georgia, United States
2 Pharmacology & Toxicology, Medical College of Georgia, Augusta, Georgia, United States
3 Pharmacology & Toxicology, Medical College of Georgia School of Medicine, Augusta, Georgia, United States
* To whom correspondence should be addressed. E-mail: jjohnson{at}mail.mcg.edu.
We have utilized an in situ rat coronary ligation model to establish an 
PKC-cytochrome oxidase subunit IV (COIV) co-immunoprecipitation (co-IP) 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 (cTnI). Homogenates prepared from the regions at risk (RAR) and not at risk (RNAR) for I/R injury were fractionated into cell soluble (S), 600 x g low speed centrifugation (L), Percoll / Optiprep density gradient-purified mitochondrial (M) and 100,000 x g particulate (P) fractions. COIV immunoreactivity and cytochrome c oxidase (CO) 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 
, , and 
PKC isozymes in the M fraction under basal conditions. Following PC, we observed a 61% increase in PKC levels in the RAR M fraction when compared to the RNAR M fraction. In RAR mitochondria we also observed a 2.8-fold increase in epsilon PKC serine 729 phospho-immunoreactivity (autophosphorylation) indicating the presence of activated epsilon PKC in mitochondria following PC. PC administered prior to prolonged I/R induced a 1.9-fold increase in the co-immunoprecipitation (co-IP) of COIV with anti- PKC antisera and a 2-fold enhancement of CO activity. Our results suggest that epsilon PKC 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.
