The overall objective of this proposal is to conclusively test the hypothesis that postischemic myocardial dysfunction (myocardial """"""""stunning"""""""") is caused by oxygen-derived free radicals, and to develop useful antioxidant therapies. the oxyradical hypothesis will be directly investigated by in vivo detection and quantification of free radicals with two techniques: aromatic hydroxylation of phenylalanine and spin trapping with electron paramagnetic resonance (EPR) spectroscopy. The combination of these two independent but complementary techniques will enable measurement of both primary and secondary radicals, thereby providing a comprehensive assessment of the cascade of free radical reactions. All studies will be conducted in conscious animals, i.e., in preparations that are as physiological as possible. A multidisciplinary approach will be used that will combine diverse techniques (integrated physiology, free radical chemistry, spin trapping, and mass spectrometry) and will integrate chemical information at the molecular level with physiological information at the conscious animal level. To establish whether there is a cause-and-effect relationship between generation of free radicals in the stunned myocardium and contractile dysfunction, the ability of antioxidants to suppress PBN adduct production will be correlated with their ability to enhance recovery of contractility. The pathogenetic role of the OH radical in stunning will be elucidated by measuring OH production with aromatic hydroxylation and by determining whether the effects of antioxidants on OH correlate with their effects on contractile dysfunction. The importance of the nitric oxide- peroxynitrite pathway in the generation of OH will be assessed by measuring the effect of inhibitors of nitric oxide synthesis on OH production and on contractile dysfunction. The molecular structure of the trapped radicals will be identified by 14C-labeling, solvent separation, HPLC, electrochemical detection, EPR spectroscopy, and mass spectrometry. Furthermore, a comprehensive, in-depth analysis of free radical reactions in vivo will be performed by using different spin traps with complementary properties [PBN, (MO)3 PBN, 4-POBN] and a new generation of spin traps with unique diagnostic potential (alpha-13C PBN, 2,6-difluoro PBN, deuterated PBN). To determine whether the oxyradical hypothesis is applicable to species with little or no myocardial xanthine oxidoreductase, the effects of antioxidants on production of radicals and recovery of contractility will be compared in conscious pigs and in conscious dogs. To test the hypothesis that oxyradical generation is a common pathogenetic mechanism underlying the entire spectrum of postischemic dysfunction, studies will be performed in two different settings of stunning, i.e., after a single reversible ischemic insult (15-min coronary occlusion) and after multiple cumulative ischemic insults (twenty 5-min occlusions). This proposal should produce important new insights into the mechanism of myocardial stunning and of ischemia/reperfusion injury in general. The results should provide cogent evidence to either accept or reject the oxyradical hypothesis and should be relevant to several clinical situations in which transient ischemia may precipitate LV dysfunction, namely, unstable or variant angina, open-heart surgery, cardiac transplantation, and thrombolysis in acute myocardial infarction.
Showing the most recent 10 out of 125 publications
