Few processes are more important for the maintenance of cellular integrity as the availability and utilization of O2 for cellular respiration. As critical as O2 is for maintaining cellular, and ultimately organismal homeostasis, there is little specific information that quantifles the relationship between intracellular O2 levels and cell function. The objective of this project is to determine the O2 dependence of cell respiration, metabolism, and function in isolated single skeletal muscle fibers. Specifically, we will test the hypothesis that cellular function is affected, both directly and indirectly, by intracellular [O2] levels well above those considered rate limiting for isolated mitochondria. Single, membrane intact, skeletal muscle fibers will be isolated from the iliofibularis muscle of Xenopus laevis and placed into a glass capillary chamber in which the extracellular milieu can be precisely controlled, metabolic and respiratory rate can be varied by stimulation, O2 uptake measured, and optical imaging can be conducted. An optical imaging system will be employed to measure various intracellular processes under altered states of cell oxygenation. To test our primary hypothesis, intracellular PO2 will be measured under varied conditions and the relationships between cell PO2 and respiration, glycolytic rate, the regulators of oxidative phosphorylation, and the regulation of contractile function will be determined. Because three different fiber types, with low, intermediate, and high mitochondrial content, can be isolated from this muscle, the relationship between mitochondrial density (and distribution) and the processes listed above will be examined. In addition, experiments are proposed that test hypotheses concerning the importance of myoglobin in intracellular O2 transport, potential intracellular O2 sensors, and causes of cell damage related to inadequate oxygenation. By utilizing an isolated single skeletal muscle cell, intrinsic properties of the working cell can be investigated without confounding factors of microcirculation and fiber- to-fiber heterogeneities. The originality and significance of the proposed experiments reside in the integration of several established techniques to investigate single cell function as it is affected by [O2]. In addition, the unique research expertise of the personnel assembled in this application provides a singular opportunity to study these processes. The proposed studies will provide valuable information defining the O2 dependence of cellular function; which has direct implications concerning cell, organ, and organismal health, particularly during disease states induced by O2 deprivation.
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