ABSTRACT
BACKGROUND: We tested recent evidences that IP triggers selective activation of protein kinase C (PKC) isozymes using isolated Langendorff-perfused rabbit heart with PKC activator, phorbol ester (PMA, 0.01 nM) or inhibitor (calphostin C, 200 nM). METHODS: After stabilization of baseline hemodynamics, the hearts were subjected to 45 min global ischemia (I) followed by 120 min reperfusion (R) with IP (IP group, n=18) or without IP (ischemic control group, n=16). IP was induced by single episode of 5 min I and 10 min R. In the PMA-treated group (n=19) and calphostin C-treated preconditioned group (n=15), PMA and calphostin C was given for 5 and 15 min before 45 min I, respectively. Myocardial cytosolic and membrane PKC activities were measured by 32P- -ATP incorporation into PKC-specific pepetide: PKC isozymes were analyzed by Western blot with monoclonal antibodies. RESULTS: IP significantly increased the recovery of the LV function including LVDP and coronary flow (p <0.05):however, enhancement of the functional recovery disappeared by calphostin C or PMA treatment. Cytosolic PKC activity decreased to 82-76% in the IP and PMA-treated group (p <0.05): membrane PKC activity increased to 218-272% (p <0.01). However, both fraction of PKC activity was not changed in the calphostin C-treated preconditioned group. In addition, Western blot revealed that PKC- alpha and epsilon, especially epsilon, were selectively translocated during subsequent sustained ischemia after IP or PMA administration. IP and PMA also reduced infarct size (frim 38 to 10-20%, p <0.05). However, calphostin C blocked infarct reduction effect of IP. CONCLUSION: These results indicate that in isolated rabbit heart model, cardioprotective effect of IP may be related, at least in part, to trigger selective translocation of PKC, especially epsilon isotype.
Subject(s)
Antibodies, Monoclonal , Blotting, Western , Cytosol , Heart , Hemodynamics , Ischemia , Ischemic Preconditioning , Isoenzymes , Membranes , Protein Kinase C , Protein Kinases , ReperfusionABSTRACT
BACKGROUND: Single or multiple episodes of brief period of ischemia and reperfusion(ischemic preconditioning, IP) have been shown to limit infarct size after a subsequent longer period of ischemia. A considerable number of possible mechanisms has been proposed, however, controversies still remain. Accordingly, we evaluated the effect of four cycles of 5 minutes ischemia and 5 minutes reperfusion(IP) followed by subsequent 30 minutes ischemia(ISCH) and 60 minutes reperfusion using isolated Langendorff-Perfused rabbit hearts. Methods and RESULTS: After a 50-minute recovery phase, parameters of the left ventricular function(LVF) including left ventricular developed pressure(LVDP), contractility and the heart rate were recorded, and ultrastructure was examined. Myosin ATPase activity was determined by measurement of inorganic phosphorus and isozymes of the myosin heavy chain were examined by polyacrylamide gel electrophoresis containing pyrophosphate buffer. The ISCH hearts showed severe to irreversible change of the cardiac myocytes homogenously in contrast to the IP hearts in which changes were not homogenous and irreversible injury was only focal. However, parameters of the LVF were not significantly different between the IP and the ISHC hearts during reperfusion. Myosin ATPase activities were also not significantly different(0.67+/-0.123 micromol/mg protein/h in the IP hearts, 0.56+/-0.172 micromol/mg protein/h in the ISCH hearts, and 0.76+/-0.239 micromol/mg protein/h in the control hearts). Band patterns of the myofibrillar proteins, separated by sodium ddodecyl sulfate-polyacrylamide gel electrophoresis, revealed no differences between the IP, ISCH and the control hearts. Myosin heavy chains in the IP and the ISCH hearts were separated into 3 isozymes, V1,V2and V3in pyrophosphate gel electrophoresis in contrast that the control hearts revealed two isozymes, V1and V2. However, there were no differences in the protein composition and electrophoretic motility between the IP and the ISCH hearts. CONCLUSION: These results indicate that IP could not attenuate the changes in LVF, myosin ATPase activity and myosin isozymes on reperfusion, however, it could attenuate the ultrastructural changes of the cardiac myocytes.
Subject(s)
Adenosine Triphosphatases , Electrophoresis , Electrophoresis, Polyacrylamide Gel , Heart Rate , Heart , Ischemia , Ischemic Preconditioning , Isoenzymes , Myocytes, Cardiac , Myosin Heavy Chains , Myosins , Phosphorus , Reperfusion , Sodium , Ventricular Function, LeftABSTRACT
An experimental model of isolated rabbit working hearts was used to investigate whether supplementing cardioplegic solution with oxygen free radical scavengers exerts protective effect on ischemic and reperfusad heart.Animal hearts wero randomly divided into two groups : simple cardioplegic solution was used in the control group ; cardioplegic solution was supplemented with superoxide dismutase plus catalase in the experimental group.Following 120 min of ischemic arrest and 30 min of reperfusion, recoveries of LVPSP, LVEDP, LV + dp/dt max and LV-dp/dt max, expressed as percentage of preischemic control valu-S, and tissue ATP contjnt w-r significantly better (P