The overall objective of this proposal is to elucidate the mechanism of the newly discovered phenomenon of """"""""late preconditioning."""""""" We will attempt to develop a unifying pathophysiologic paradigm applicable both to late preconditioning against myocardial stunning and to late preconditioning against myocardial infarction. Our fundamental hypothesis is that, in both cases, late preconditioning is triggered by the exposure to oxygen-derived free radicals during the preconditioning ischemia (""""""""oxyradical hypothesis of late preconditioning""""""""). We further propose that the protection afforded by late preconditioning is mediated by the synthesis of cardioprotective proteins, such as antioxidant enzymes and/or stress proteins. All studies will be conducted in conscious animals, i.e., in preparations that are as physiological as possible; specifically, we will employ a conscious pig model in which we have demonstrated that brief ischemia induces late preconditioning against stunning as well as infarction. The role of oxygen-derived free radicals will be directly investigated by in vivo detection and quantification with spin trapping. A broad multidisciplinary approach will be used that will combine diverse techniques (free radical chemistry and biochemistry, integrative physiology, spin trapping, mass spectrometry, measurement of infarct size and regional contractile function) and will integrate chemical information at the molecular level with physiological information at the conscious animal level. The first step will be to thoroughly define the time-course of late preconditioning against stunning and infarction. To elucidate whether late preconditioning results in decreased production of free radicals, free radical generation in the preconditioned myocardium will be directly measured and correlated with the protection observed. The oxyradical hypothesis of late preconditioning will be tested by determining whether free radical scavengers prevent late preconditioning and, conversely, whether a free radical-generating system induces late preconditioning. The molecular structure of the free radicals generated during the preconditioning ischemia will be identified by 14C-labeling, HPLC, EPR spectroscopy, and mass spectrometry. A comprehensive, in-depth analysis of the free-radical reactions associated with the preconditioning ischemia will be performed using a new generation of spin traps with unique diagnostic potential [(MO3)PBN, 4-POBN, alpha-13C PBN, 2,6-diffluoro PBN, deuterated PBN]. The role of antioxidant enzymes and stress proteins as mediators of late preconditioning will be initially explored by determining whether the changes in the myocardial levels of Mn-SOD, Cu,Zn-SOD, catalase, glutathione peroxidase, glutathione reductase and HSP70, 90, 60, 27, and 100 correlate with the changes in the severity of myocardial stunning and in infarct size. Then, to establish whether there is a cause-and-effect relationship between increases in these proteins and late preconditioning, antioxidant enzymes will be selectively inhibited and the effect of this inhibition on preconditioning against stunning and infarction will be assessed; similarly, the synthesis of stress proteins will be blocked and the effect of this intervention on preconditioning and of ischemia/reperfusion injury in general. Elucidation of the mechanism of late preconditioning should facilitate the development of drugs that duplicate its powerful protective effects.
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