Our long-term goal is to understand the role of protein and lipid oxidation in age-related modifications of calcium regulation in the heart. Our focus will be on the sarcoplasmic reticulum (SR) Ca-ATPase, an active transport protein that is central to the maintenance of low resting levels of intracellular calcium and normal myocardial relaxation. Effects of oxidation, aging, and susceptibility to oxidative stress will be examined for the protein and lipid components of SR membranes. Much of this work will rely on comparison with results from similar studies with skeletal SR, where the skeletal Ca-ATPase isoform is more abundant and the membrane composition is simpler. Our three specific aims are: FIRST, to identify oxidative modifications of the Ca-ATPase, its regulatory protein, phospholamban, and SR lipids, initiated by exposure of isolated SR vesicles to reactive oxygen species. We will emphasize reactive oxygen species whose chemistry is simple and allows quantification. These free radicals will be targeted to either the lipid bilayer or to the aqueous medium, in order to specifically oxidize membrane-spanning or soluble peptides. In this way we seek insight into mechanisms of oxidative damage to cellular proteins. The relationship between reactive oxygen species and function is not available from biologically aged samples where a large number of reactions, coupled with the presence of cellular repair mechanisms complicate our understanding of processes leading to protein damage. SECOND, we will define the role of the cellular environment in modifying the susceptibility of SR membranes to oxidative conditions. This approach, utilizing primary cultures of myocytes, will provide an opportunity to examine the effect of oxidative conditions on protein and lipid turnover. Using the knowledge gained from these model systems, we will (THIRDLY), identify age-related oxidative modifications, and their associated structural and functional consequences. Work with both model and biologically aged systems will emphasize quantitative identification of the products of free radical-mediated modifications of proteins and lipids. The combined use of fluorescence and spin-label electron paramagnetic resonance spectroscopies will provide information regarding protein conformation and bilayer dynamics. An increased understanding of age-related molecular defects of calcium regulation in cardiac muscle is relevant to human health, since oxidation of biomolecules is thought to play a major role in aging and myocardial infarction. This understanding is necessary for the design of effective therapies for delaying the onset and progress of decreased myocardial function and susceptibility to oxidative stress.
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