Reduced oxygen intermediates have been implicated in the initiation and progression of various disease states such as ischemia/reperfusion injury, the inflammatory response of myocardium and the toxic action of drugs (e.g., adriamycin). We have hypothesized that the major target organelles injured by free radical attack are the sarcolemma and sarcoplasmic reticulum which are the key regulators of myocardial contractility. The purpose of this project is therefore to define the underlying biochemical, physiological and morphological changes that occur in these organelles as a result of attack by oxygen free radicals and neutrophil derived oxidants.
The specific aim of this project is to answer the following three hypotheses. A combination of in vitro and in vivo models will be used to evaluate these hypotheses. The primary hypothesis is if hydrogen peroxide, hydroxyl radicals and neutrophil derived oxidants such as hypochlorous acid, monochloramine and taurine monochloramine cause dysfunctions of isolated sarcolemma and sarcoplasmic reticulum. We will characterize the important biochemical parameters of sarcolemma such as Na+-Ca2+ exchange, Na+, K+-ATPase, muscarinic and adrenergic receptors and (Ca2+-Mg2+)-ATPase, calcium transport of sarcoplasmic reticulum, and morphological changes in these isolated organelles following in vitro attack by oxidizing species. The second hypothesis is that oxygen free radicals and oxidants inactivate proteins by modification of amino acid residues or initiation of oxidation of sulfhydryl groups which would result in alteration of conformation or cleavage of molecules. We will test this hypothesis on purified (Ca2+-Mg2+)- ATPase of sarcoplasmic reticulum and determine amino acid residues by Pico-tag HPLC in control and oxidants treated enzyme. The third hypothesis is if oxygen radicals bring about biochemical dysfunctions in isolated rat heart. We will perfuse the isolated working heart with free radicals and oxidants and study the myocardial contractility, Na+-Ca2+ exchange, Na+, K+- ATPase and receptor functions in isolated sarcolemma and (Ca2+- Mg2+)-ATPase and calcium uptakes in homogenates. We will also study the in vivo lipid peroxidation following these interventions by HPLC detection of diene-conjugates either in perfusate or muscle extract. This integrated approach should result in more meaningful understanding of our knowledge of free radicals and neutrophil derived oxidants attack on cardiac muscle and function. A better understanding of the mechanism of damage to these membranes is important because it may suggest rational therapies to improve contractility following reperfusion.
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