(Circulation. 1995;92:1546-1557.)
© 1995 American Heart Association, Inc.
Articles |
Does Ischemic Preconditioning Trigger Translocation of Protein Kinase C in the Canine Model?
From the Heart Institute, Hospital of the Good Samaritan (K.P., B.Z.S., R.A.K.), and the Departments of Medicine (Section of Cardiology) (K.P., R.A.K.) and Biochemistry and Molecular Genetics (M.A.S.), University of Southern California, Los Angeles.
Correspondence to Karin Przyklenk, PhD, Heart Institute/Research, Hospital of the Good Samaritan, 616 S Witmer St, Los Angeles, CA 90017.
Background Brief episodes of ischemia protect or "precondition" the heart and reduce the size of infarcts caused by subsequent sustained coronary artery occlusion, yet the mechanisms responsible for this cardioprotection remain unresolved. We tested the theory that translocation of protein kinase C (PKC) to the myocyte membranes, initiated in response to brief preconditioning ischemia and manifest during the initial minutes of the sustained occlusion, mediates this phenomenon by attempting to (1) blunt the cardioprotective effects of preconditioning by administration of the PKC inhibitors H-7 and polymyxin B, (2) visualize by fluorescence staining and confocal microscopy changes in the amount or location of PKC, and (3) quantify by incorporation of 32P into PKC-specific peptide changes in the subcellular distribution of PKC in preconditioned versus control hearts.
Methods and Results In the first three limbs of this study, anesthetized open-chest dogs underwent four 5-minute episodes of preconditioning ischemia or a comparable control period before 1 hour of sustained occlusion and 4 to 5 hours of reperfusion. Collateral blood flow was assessed with radioactive microspheres; area at risk (AR) was delineated by injection of blue dye; and the area of necrosis (AN) was measured by tetrazolium staining. AN/AR was smaller in preconditioned versus control dogs that received no treatment (6±2% versus 19±3%, P<.01), H-7 (2±2% versus 14±5%, P<.02), or polymyxin B (10±3% versus 29±5%, P<.01) during the preconditioning or control period. Additional dogs underwent four 5-minute episodes of ischemia, with biopsies obtained at baseline and after the first and fourth occlusions. Frozen sections were stained with a fluorescent probe for active PKC and viewed with confocal microscopy. No differences in the intensity or distribution of fluorescence staining were observed after brief ischemia compared with baseline. Finally, myocardial samples were obtained from dogs subjected to four 5-minute episodes of preconditioning ischemia and time-matched sham-operated controls. Incorporation of 32P into PKC-specific peptide revealed no quantitative difference in the subcellular distribution of PKC between control and preconditioned cohorts.
Conclusions H-7 and polymyxin B did not blunt the reduction in infarct size achieved with ischemic preconditioning. Neither fluorescence staining and confocal microscopy nor biochemical quantification revealed evidence of preconditioning-induced translocation of PKC to the cell membranes. These results fail to support the hypothesis that translocation of PKC, triggered by preconditioning ischemia, is an important mechanism for the reduction in infarct size seen with preconditioning in the dog model.
Key Words: H-7 • polymyxin B • ischemia • infarction
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