leupeptins and Myocardial-Ischemia

The aim of this study was to investigate the role of secondary free radicals and calpain, a calcium-activated cysteine protease, in the development of reperfusion injury in the heart. The time course of radical generation was assessed directly by Electron Paramagnetic Resonance (EPR) and spin trapping with N-ter butyl-alpha-phenylnitrone (PBN), in isolated perfused rat heart subjected to 30 minutes of global ischemia and 30 minutes of reperfusion. The effect of leupeptin, a calpain inhibitor, was assessed on postischemic dysfunction. The antioxidant properties of leupeptin were also investigated by using allophycocyanin, a fluorescent protein sensitive to oxidative stress generated by the H2O2 + Cu++ system. Moreover, we measured the capacities of leupeptin to scavenge hydroxyl (.OH) and superoxide (O2-.) radicals using EPR technique. Our results show that myocardial reperfusion is associated with an increase of alkyl, alkoxyl free radicals release; the administration of catalase 5.10(5) UI/L significantly reduces this release, but didn't improve the postischemic contractile function of the heart. In our study leupeptin 50 microM possess, in vitro, antioxidant properties and scavenging abilities against .OH and O2-., in return leupeptin does not influence the cardiac functions during reperfusion period. In conclusion, our results confirm that myocardial reperfusion induces an important production of secondary free radicals associated with contractile dysfunction. The role of calpain in myocardial ischemia-reperfusion injury remains to be clarified 1) by assessing the activities of calpain and calpastain, its main endogenous inhibitor, during these periods, 2) by measuring the ability of leupeptin in inhibiting the calpain dependent proteolysis.

Topics: Animals; Antioxidants; Calcium-Binding Proteins; Calpain; Catalase; Cathepsins; Cyclic N-Oxides; Cysteine Proteinase Inhibitors; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Free Radicals; Hydroxyl Radical; Leupeptins; Magnetic Resonance Spectroscopy; Male; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion Injury; Nitrogen Oxides; Oxidative Stress; Phycocyanin; Rats; Rats, Wistar; Spin Labels; Superoxides; Time Factors

Ankyrin links cytoskeleton and integral membrane proteins and is proteolyzed in vitro by calpain, a Ca2+-dependent protease. In the present study, we examined the localization of two ankyrin isoforms, erythrocyte (red blood cell)-type (ankyrin(R)) and brain-type (ankyrin(B)), and their proteolysis after ischemia-reperfusion in the subcellular fractions of perfused rat heart by immunoblotting and by immunohistochemistry using specific antibodies. Both isoforms were observed to be distributed chiefly in the myofibril-nucleus (1,OOOx g pellet: P1) fraction, while ankyrin(R) was located substantially in the membrane (100,000x g pellet: P2) fraction. Reperfusion after 10 min or more of global ischemia induced preferential proteolysis of ankyrin(R) in the P2 fraction and ankyrin(B) in the P1 fraction. The proteolysis of ankyrin(R), but not ankyrin(B), was effectively inhibited by the synthetic calpain inhibitor acethyl-leucyl-leucyl-norleucinal. The immunohistochemical examination showed that anti-ankyrin(R) delineated striations, sarcolemma and nuclei, and the staining was decreased after ischemia-reperfusion, while anti-ankyrin(B) showed diffuse staining. The proteolysis of ankyrin(R) may interfere with force conduction through disruption of the linkage between integral membrane proteins and the myofibril-cytoskeleton.

Topics: Animals; Ankyrins; Brain; Calpain; Cell Fractionation; Cysteine Proteinase Inhibitors; Erythrocytes; In Vitro Techniques; Leupeptins; Male; Molecular Weight; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Peptide Fragments; Rats; Rats, Wistar