An inhibitor of the δPKC interaction with the d subunit of F1Fo ATP synthase reduces cardiac troponin I release from ischemic rat hearts: utility of a novel ammonium sulfate precipitation technique.
We have previously reported protection against hypoxic injury by a cell-permeable, mitochondrially-targeted δPKC-d subunit of F1Fo ATPase (dF1Fo) interaction inhibitor [NH2-YGRKKRRQRRRMLA TRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK-COOH] in neonatal cardiac myo-cytes. In the present work we demonstrate the partitioning of this peptide to the inner membrane and matrix of mitochondria when it is perfused into isolated rat hearts. We also used ammonium sulfate ((NH4)2SO4) and chloroform/methanol precipitation of heart effluents to demonstrate reduced card-iac troponin I (cTnI) release from ischemic rat hearts perfused with this inhibitor. 50% (NH4)2SO4 saturation of perfusates collected from Langendorff rat heart preparations optimally precipitated cTnI, allowing its detection in Western blots. In hearts receiving 20 min of ischemia followed by 30, or 60 min of reperfusion, the Mean±S.E. (n=5) percentage of maximal cTnI release was 30 ± 7 and 60 ± 17, respectively, with additional cTnI release occurring after 150 min of reperfusion. Perfusion of hearts with the δPKC-dF1Fo interaction inhibitor, prior to 20 min of ischemia and 60-150 min of reperfusion, reduced cTnI release by 80%. Additionally, we found that when soybean trypsin inhibitor (SBTI), was added to rat heart effluents, it could also be precipitated using (NH4)2SO4 and detected in western blots. This provided a convenient method for normalizing protein recoveries between groups. Our results support the further development of the δPKC-dF1Fo inhibitor as a potential therapeutic for combating cardiac ischemic injury. In addition, we have developed an improved method for the detection of cTnI release from perfused rat hearts.
Department of Pharmacology & Toxicology and Program in Regenerative Medicine, Institute of Molecular Medicine and Genetics, Georgia Regents University, Augusta, Georgia, USA.
Mourad
M
Ogbi
Ijeoma
I
Obi
John A
JA
Johnson
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000737:chemistry*; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D011232:Chemical Precipitation; D002725:Chloroform / Q000737:chemistry; D008297:Male; D000432:Methanol / Q000737:chemistry; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D017202:Myocardial Ischemia / Q000378:metabolism* / Q000473:pathology; D009206:Myocardium / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D010477:Perfusion; D011485:Protein Binding / Q000187:drug effects; D051745:Protein Kinase C-delta / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013025:Glycine max / Q000737:chemistry; D019210:Troponin I / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000494:pharmacology
D015415:Biomarkers; D010455:Peptides; D021122:Protein Subunits; D019210:Troponin I; D014361:Trypsin Inhibitors; D002725:Chloroform; D051745:Protein Kinase C-delta; D006180:Proton-Translocating ATPases; D000645:Ammonium Sulfate; D000432:Methanol