Both skeletal and cardiac muscle sarcoplasmic reticulum (SR) are sensitive to oxidative stress induced directly by free radicals, and indirectly by an increase in the cellular redox potential. Oxidative stress results from cardiac ischemia and reperfusion, and in skeletal muscle results in muscle fatigue. The molecular mechanism by which the SR controls and responds to changes in its redox environment, and how this influences Ca2+ homeostasis is the main goal of this research proposal.The major site of oxidative damage to SR appears to be highly reactive sulfhydryl groups, which under mildly oxidative conditions, oxidize to disulfides. This causes the Ca2+ release channel from SR to open and the intracellular Ca2+ concentration to increase. It is our goal to measure the redox potential of these thiols in both cardiac and skeletal muscle, and to determine how physiologically relevant components in the cellular environment control the redox potential of the ryanodine receptor. Using single channel measurements, we will also determine how the Ca2+ release channel responds to alterations in the redox potential on both its cytoplasmic and lumenal sides of the SR membrane. Moreover, rapid changes in the local redox potential will enable us to determine how quickly the receptor responds to changes in its redox environment.Not only does this proposal focus on how the ryanodine receptor responds to its redox environment, but it also identifies for the first time an endogenous NAD (P) H dependent oxidase from SR, which produces superoxide, and which stimulates the ryanodine receptor. It is our goal to characterize this oxidase, to understand how its activity is influenced by changes that occur to the muscle during increased activity, and to determine its role in skeletal muscle fatigue.
