3-NITROTYROSINE IN AGING OF SKELETAL MUSCLE AND HEART

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DESCRIPTION: (Adapted from the Application). The long-term goal of this project is to understand the origin of the prolonged intracellular calcium transients that accompany aging of skeletal and cardiac muscle. These investigators will focus on the sarcoplasmic reticulum (SR) Ca-ATPase which mediates the rate limiting re sequestration of calcium that reestablishes resting levels of cytosolic calcium for muscle relaxation. Therefore deficits in active calcium transport by the Ca-ATPase are likely responsible for the prolonged relaxation times observed for aged cardiac and skeletal muscle. Based on their recent observations, it is hypothesized that the multiple 3-nitrotyrosine (3NY) modifications of the SERCA2a isoform of the CaATPase that accumulate in aged skeletal muscle explain these defects in contractility. The appearance of 3NY modification of SERCA2a in the aged heart suggests effects on cardiac muscle as well. 3NY modification is a characteristic indicator of peroxynitrite (ONOO-) which is formed in vivo by the diffusion-limited reaction of superoxide and nitric oxide, both produced in abundance in muscle. 3NY formation and concomitant inactivation of the Ca-ATPase can be simulated by in vitro exposure of isolated SR membranes to ONOO-. The selectivity of 3NY formation to the SERCA2a isoform of the Ca-ATPase, both in vitro and in aged skeletal muscle, to the exclusion of the highly homologous SERCA1 isoform, implicate phospholamban, which is selectively expressed with SERCA2a, as a critical factor in oxidative stress to skeletal and cardiac muscle. Therefore, the specific aims include: (1) Identify the role of phospholamban in the selective nitration of the SERCA2a isofomm of the Ca-ATPase; (2) Compare the oxidative modification of SERCA2a in the heart with that in skeletal muscle during aging; (3) Identify the functional relevance of each in vivo and in vitro 3NY site and its conformational consequences; and (4) describe the conformational features of nitrated SERCA2a that create a substrate for the denitrase activity. Delineation of pathways resulting from oxidative stress that lead to defects in calcium regulation is relevant to human health as this understanding is a prerequisite for design of effective therapies for both mitigating bass of muscle function during aging and preventing the exacerbation of myocardial damage during surgery and other therapies involving oxidative stress.