An Inhibitor of the δPKC Interaction with the d Subunit of F(1)Fo 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 F(1)Fo ATPase (dF(1)Fo) interaction inhibitor [NH(2)-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 ((NH(4))(2)SO(4)) 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% (NH(4))(2)SO(4) 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-dF(1)Fo 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 (NH(4))(2)SO(4) 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-dF(1)Fo 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.