Hypoxemia is both a causative agent for, and a result of, numerous human pathologies. The deleterious effects of hypoxemia on cellular function can be profound. In particular, the effect of hypoxia on carbohydrate utilization from both intra- and extracellular sources can be strongly influenced by the degree of tissue oxygenation, and these changes can have significant consequences on skeletal muscle function-even when the intracellular PO2 may be well above that necessary for adequate mitochondrial respiration. However, there is a paucity of information concerning the specific mechanisms that result in alterations in skeletal muscle metabolism and contractile function during conditions of reduced O2 availability in the various muscle fiber types. Understanding the mechanisms related to skeletal muscle dysfunction during hypoxia in the specific fiber types is significant because of the alterations in fiber type composition in whole muscle that occurs with certain pathologies (such as diabetes) and aging. The principle objective of this project is to examine on a cellular level a number of hypotheses related to the mechanisms of hypoxia-induced alterations in skeletal muscle metabolism and function using an isolated single skeletal muscle fiber model (slow- and fast-twitch fibers from frogs and mice) during high-intensity and/or endurance work. Using this model, the extracellular milieu can be precisely controlled, metabolic and respiratory rates can be varied by stimulation, contractile performance can monitored, intracellular PO2 and O2 uptake measured, and different types of fluorescent imaging can non-invasively monitor intracellular events (i.e. pH, Ca2+ handling, glucose uptake, etc.). A particularly exciting aspect of this proposal is the use of single fibers from transgenic mice to address some of the hypotheses. By using an isolated single skeletal muscle cell, intrinsic properties of the working cell can be investigated without confounding factors of microcirculation and fiber type heterogeneities. The originality and significance of the proposed experiments reside in the integration of several established and new techniques to determine the mechanisms by which hypoxia and glucose availability alters muscle metabolism and function on a cellular level. The proposed studies will provide a singular opportunity to address long-standing questions concerning the O2 dependence of cellular metabolism and function in the different skeletal muscle fiber types; which has direct implications concerning cell, organ, and organismal health, particularly during disease states involving metabolic disorders and O2 deprivation.
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