Abstract 19992: Delta PKC (dPKC) Binding to the “d” Subunit of F1Fo ATP Synthase (dF1Fo) Impairs Mitochondrial Energetics and Exacerbates Ischemia / Reperfusion Injury in Rats
We have previously demonstrated delta PKC (dPKC) involvement in cardiac myocyte energy deprivation via its interaction with dF1Fo and have developed a peptide inhibitor [NH2-YGRKKRRQRRRMLATRALSLIGKRAISTSVC-COOH] of this interaction, which targets to the mitochondrial matrix / inner membrane. In the present study, a 10 min ligation of the left anterior descending coronary artery (LAD) increased dPKC-dF1Fo co-immunoprecipitation (co-IP) by 5-fold and elevated dPKC-phosphoserine 643 immunoreactivity (a dPKC autophosphorylation site 56 +/- 17% (n=7, p<0.01) in mitochondria isolated from the left ventricular (LV) region at risk for ischemic injury (RAR). Intravenous (I.V.) injection of the dPKC-dF1Fo inhibitor (5 ug/kg) 20 min beforehand attenuated the co-IP by 71 +/- 13% (p<0.001, n=8). Blockage of the IP correlated with transient increases in cytochrome oxidase subunit IV (1.6-fold (p<0.05, n=8) and beta-tubulin (1.4-fold, (p<0.001, n=8), without changes in autophagy marker proteins (LC3II and p62) or mitochondria release of cytochrome c. The dPKC-dF1Fo inhibitor given I.V. prior to 30 min of ischemia by coronary ligation, and 3 hr of reperfusion, reduced 2,3,5-triphenyltetrazolium chloride-monitored infarct size by 29 +/- 7 % (n=6, p<0.001). This protection was correlated with a 1.7 +/- 0.2-fold (n=6, p<0.02) enhancement of ATP levels, a 2-fold reduction in tissue t-carbonyls / oxidative stress markers and a 61 +/- 12% (p<0.001, n=8)) reduction in Ca++-induced mitochondrial swelling. The inhibitor peptide also improved ADP (state 3) (1.5 +/- 0.2-fold (p<0.05, n=6) and maximal (FCCP)-stimulated (1.6 +/- 0.1-fold (p<0.03, n=6)) respiration and JC-1 monitored mitochondrial membrane potential (1.6 +/- 0.1-fold (n=6, p<0.03) following IR injury. This mitochondrially-targeted peptide therefore, protects against ATP loss, oxidative stress, mitochondrial swelling and infarction in vivo, which correlates with improved mitochondrial energetics. We propose it has potential as a first-in-class therapeutic for the treatment of cardiac IR injury.
Author Disclosures: M. Walker: None. R.W. Caldwell: None. Y. Yoon: None. J.A. Johnson: Research Grant; Significant; This research is currently supported by an AHA Greater Southeast Affiliate Grant-in-Aid #13GRNT17080109. Ownership Interest; Modest; A patent has been approved for the peptide discussed in the abstract. No financial relationships exist at present, but they could be pursued..
- © 2015 by American Heart Association, Inc.