Our long term goal is to understand the cellular processes that are responsible for the accumulation of oxidized calcium regulatory proteins in senescent muscle and how these related to their prolonged contraction and relaxation times. Here, we focus on the ryanodine receptor (RyR) and the sarcoplasmic reticulum (SR) Ca-ATPase, two calcium regulatory proteins that, together, play a major role in eliciting intracellular calcium transients in muscle. Their respective roles consist of calcium release to initiate muscle contraction, and the rate-limiting active resequestration of cytosolic calcium into the SR lumen to allow relaxation. Our previous work has shown specific and substantive levels of 3-nitrotyrosine (3Ngamma) modification of the SERCA2a isoform of the Ca-ATPase in heart and skeletal muscle which increases with age of the animal. 3NY is a characteristic product of peroxynitrite (ONOO-), and suggests the simultaneous generation of superoxide (O2.-) and nitric oxide (NO.) in myocytes. We hypothesize that accumulation of 3Ngamma modification of SERCA2a during aging is due to defects in cellular mechanisms designed to minimize reactive oxygen species (ROS), or those involved in the degradation of oxidized proteins. We therefore propose to focus on gaining a detailed understanding of degradation pathways or whether increased levels of ROS overwhelm the normal functioning of these pathways. We further hypothesize that the presence of the strong thiol oxidant, ONOO-, in myocytes will functionally alter the cysteine-rich RyR and its methionine rich regulator, calmodulin. The latter protein has been previously shown to be oxidized in aging. In addition, ONOO-, formation may deplete NO> which regulates the RyR., both directly and through the FK binding protein. Therefore, we propose the following specific aims: (1) Identify cellular pathways for the degradation of SERCA2a, and possible defects in aging; and (2) Characterize the regulation of the RyR by oxidized CaM, and other regulatory molecules in senescent muscle. Knowledge of cellular mechanisms that lead to accumulation, and altered regulation by oxidized proteins will be essential for the design of therapeutic approaches for maintenance of optimal heart and skeletal muscle function during aging.
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