Abstract
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.
Highlights
Cardiac ischemia/reperfusion (IR) injury is responsible for more annual deaths than any other medical condition
We anticipated that the amount of cardiac troponin I (cTnI) released from our Langendorff heart preparations might be at the lower limit of detection and that there could be poor recovery of low amounts of cTnI following the Chl/MeOH extractions
Since the ‘‘d’’ subunit of F1Fo ATP synthase is found in the mitochondrial matrix our results suggest that following perfusion of hearts with the dPKC-dF1Fo inhibitor, sufficient FLAG epitope-tagged inhibitor peptide is delivered to the matrix to bind dPKC and prevent its interaction with dF1Fo
Summary
Cardiac ischemia/reperfusion (IR) injury is responsible for more annual deaths than any other medical condition. It occurs as a consequence of a heart attack when one or more of the coronary arteries become occluded, typically by atherosclerosis or thrombosis. CTnI is a myofibrillar protein which complexes with tropomyosin and troponins T and C to minimize Ca++ binding to troponin C, and prevent subsequent crossbridge formation between the actin and myosin myofilaments [7]. It plays a crucial role in the diastolic relaxation. In prolonged ischemia or IR, cTnI is released into serum, mostly as a consequence of cardiac myocyte necrotic cell death [1,2,3]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
