Oxygen dependence of ROS generation in contracting myofibers

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A deleterious systemic effect of chronic obstructive pulmonary disease (COPD) is peripheral muscle dysfunction, a condition that contributes to the exercise intolerance and reduced quality of life of COPD patients. The causes of the peripheral muscle dysfunction have yet to be fully elucidated. It has recently become clear that the hypoxia of COPD, oxidative stress within active muscle, and circulating inflammatory cytokines (particularly TNF-a) are potential mechanisms that may play a role in the muscle myopathy found in many COPD patients. However, there remains a paucity of data at the cellular level concerning the manner in which O2 availability to contracting skeletal muscle affects the interactions between intracellular PO2 (PjO2) and cell homeostasis, metabolic state, reactive O2 species (ROS) generation, and contractile function. We now use an isolated, intact single skeletal muscle fiber model in which slow- or fast-twitch fibers can be isolated from mice. By utilizing this model, intrinsic properties of contracting isolated single myofibers can be investigated without confounding factors related to the microcirculation, fiber recruitment patterns, and fiber type heterogeneities seen in whole muscle. We are proposing to use single isolated myofibers, from both healthy control mice and a model of pulmonary inflammation, to elucidate the regulatory pathways that are affected by P|O2 and thereby alter cellular function. Specifically, we are proposing to conduct experiments related to the general hypothesis that several important cellular regulatory factors (i.e. pH, Ca2+ handling, PI,fuel preference, ROS generation, etc.) are modualted by P|O2 (even at levels that do not inhibit oxidative phophorylation) which thereby modifies contractile function of the muscle fiber in a manner that is fiber type dependent. Our experimental preparation will allow us to precisely control the extracellular milieu, metabolic and respiratory rate will be varied by electrical stimulation, intracellular PO2 measured, specific pharmacological blockades will be induced, and intracellular fluorescent imaging (pH, Ca2+, ROS, PI,etc) will be conducted. The originality and significance of the proposed experiments will be to investigate the O2 dependence of regulatory pathways that determine oxidative stress and single myofiber function, which has important implications related to health at the cellular, organ, and whole body level, particularly during hypoxia or disease states involving hypoxia such as COPD.