-Contractions in whole skeletal muscle during hypoxia are known to generate reactive oxygen species (ROS); however, identification of real-time ROS formation within isolated single skeletal muscle fibers has been challenging. Consequently, there is no convincing evidence showing increased ROS production in intact contracting fibers under low PO2 conditions. Therefore, we hypothesized that intracellular ROS generation in single contracting skeletal myofibers increases during low PO2 compared with a value approximating normal resting PO2. Dihydrofluorescein was loaded into single frog (Xenopus) fibers, and fluorescence was used to monitor ROS using confocal microscopy. Myofibers were exposed to two maximal tetanic contractile periods (1 contraction/3 s for 2 min, separated by a 60-min rest period), each consisting of one of the following treatments: high PO2 (30 Torr), low PO2 (3-5 Torr), high PO2 with ebselen (antioxidant), or low PO2 with ebselen. Ebselen (10 M) was administered before the designated contractile period. ROS formation during low PO2 treatment was greater than during high PO2 treatment, and ebselen decreased ROS generation in both low-and high-PO2 conditions (P Ͻ 0.05). ROS accumulated at a faster rate in low vs. high PO2. Force was reduced Ͼ30% for each condition except low PO2 with ebselen, which only decreased ϳ15%. We concluded that single myofibers under low PO2 conditions develop accelerated and more oxidative stress than at PO2 ϭ 30 Torr (normal human resting PO2). Ebselen decreases ROS formation in both low and high PO2, but only mitigates skeletal muscle fatigue during reduced PO2 conditions. hypoxia; confocal; reactive oxygen species; ebselen; myofiber REACTIVE OXYGEN SPECIES (ROS) play important roles in biological systems (1,44,46,47,49,50). ROS have been documented as a general response to ischemia-reperfusion injury (26, 43), muscle stimulation (29), and heat stress (44). Excessive ROS disrupt nearly all physiological systems (1,44,46,47,49,50). However, the underlying mechanism of ROS formation in hypoxic skeletal muscle has not been fully elucidated. Moreover, there has been little investigation of ROS generation within single skeletal muscle fibers under low PO 2 conditions using real-time measurements, which eliminates some of the problems associated with these measurements that have been made in whole animal or whole muscle preparations (46). Previous research suggests that in human skeletal muscle, intracellular PO 2 drops from ϳ30 Torr at rest to 3-5 Torr during exercise (40). Thus, it is of interest to investigate intracellular ROS formation during these conditions of low PO 2 in single contracting myocytes.Hypoxia causes a significant accumulation of reducing agents in the mitochondria, such as NADH and FADH 2 . Abrupt exposure to O 2 can promote immediate formation of superoxide anion (O 2 ·Ϫ ) in the mitochondrial electron transport chain (2, 37), thus initiating oxidative stress. Moreover, substantial data point to intracellular ROS formation in cardiac tissue during hypo...

Skeletal muscle can develop a prolonged low frequency-stimulation force depression (PLFFD) following fatigue-inducing contractions. Increased levels of reactive oxygen species (ROS) have been implicated in the development of PLFFD. During exercise the skeletal muscle intracellular PO2 decreases to relatively low levels, and can be further decreased when there is an impairment in O diffusion or availability, such as in certain chronic diseases and during exercise at high altitude. Since ROS generation by mitochondria is elevated at very low PO2 in cells, we tested the hypothesis that treatment of muscle fibres with a mitochondrial-targeted antioxidant at a very low, near hypoxic, PO2 can attenuate PLFFD. We treated intact single fibres from mice with the mitochondrial-specific antioxidant SS31, and measured force development and intracellular [Ca ] 30 min after fatigue at an extracellular PO2 of ∼5 Torr. After 30 min following the end of the fatiguing contractions, fibres treated with SS31 showed significantly less impairment in force development compared to untreated fibres at submaximal frequencies of stimulation. The cytosolic peak [Ca ] transients (peak [Ca ] ) were equally decreased in both groups compared to pre-fatigue values. The combined force and peak [Ca ] data demonstrated that myofibrillar Ca sensitivity was diminished in the untreated fibres 30 min after fatigue compared to pre-fatigue values, but Ca sensitivity was unaltered in the SS31 treated fibres. These results demonstrate that at a very low PO2, treatment of skeletal muscle fibres with a mitochondrial antioxidant prevents a decrease in the myofibrillar Ca sensitivity, which alleviates the fatigue induced PLFFD.

Nogueira L, Shiah AA, Gandra PG, Hogan MC. Ca 2ϩ -pumping impairment during repetitive fatiguing contractions in single myofibers: role of cross-bridge cycling. Am J Physiol Regul Integr Comp Physiol 305: R118 -R125, 2013. First published May 15, 2013 doi:10.1152/ajpregu.00178.2013.-The energy cost of contractions in skeletal muscle involves activation of both actomyosin and sarcoplasmic reticulum (SR) Ca 2ϩ -pump (SERCA) ATPases, which together determine the overall ATP demand. During repetitive contractions leading to fatigue, the relaxation rate and Ca 2ϩ pumping become slowed, possibly because of intracellular metabolite accumulation. The role of the energy cost of cross-bridge cycling during contractile activity on Ca 2ϩ -pumping properties has not been investigated. Therefore, we inhibited cross-bridge cycling by incubating isolated Xenopus single fibers with N-benzyl-p-toluene sulfonamide (BTS) to study the mechanisms by which SR Ca 2ϩ pumping is impaired during fatiguing contractions. Fibers were stimulated in the absence (control) and presence of BTS and cytosolic calcium ([Ca 2ϩ ]c) transients or intracellular pH (pHi) changes were measured. BTS treatment allowed normal [Ca 2ϩ ]c transients during stimulation without cross-bridge activation. At the time point that tension was reduced to 50% in the control condition, the fall in the peak [Ca 2ϩ ]c and the increase in basal [Ca 2ϩ ]c did not occur with BTS incubation. The progressively slower Ca 2ϩ pumping rate and the fall in pHi during repetitive contractions were reduced during BTS conditions. However, when mitochondrial ATP supply was blocked during contractions with BTS present (BTS ϩ cyanide), there was no further slowing in SR Ca 2ϩ pumping during contractions compared with the BTS-alone condition. Furthermore, the fall in pHi was significantly less during the BTS ϩ cyanide condition than in the control conditions. These results demonstrate that factors related to the energetic cost of cross-bridge cycling, possibly the accumulation of metabolites, inhibit the Ca 2ϩ pumping rate during fatiguing contractions. Ca 2ϩ uptake rate; skeletal muscle; cross-bridge cycling; fatigue; glycolysis; oxidative phophorylation DURING THE TRANSITION FROM rest to exercise in skeletal muscle, the demand for ATP increases several hundredfold, primarily because of the activation of both actomyosin (i.e., cross-bridge cycling) and sarcoplasmic reticulum (SR) Ca 2ϩ ATPase (SERCA) pumps (6). After membrane depolarization, cytosolic Ca 2ϩ concentration is transiently increased, thereby elevating actomyosin and SERCA ATPase activities. Although the Na ϩ -K ϩ -ATPase activity has a high contribution in the energy cost at rest [ϳ40% of total; (27)], it is quite small during contractions [ϳ1.5-7%; (6)]. Conversely, during contractions, actomyosin ATPase and SERCA account for ϳ60% and 40% of the total ATP demand, respectively (5,6,22,30). However, the energy cost of Ca 2ϩ handling by SERCA during contractions may be as high as 80% of the total energy cost in type IIB...

Hypoxia has been associated with an increased reactive oxygen species (ROS) production in contracting skeletal muscle. However, it is not known whether hypoxia can modulate the muscle contractility, particularly at physiological temperature. We hypothesized that a decrease in intracellular PO2 would regulate force in skeletal muscle through ROS. Intact single fast‐twitch fibers from mouse muscle (n=20) were stimulated (1–200 Hz) at ambient PO2 (~150 Torr) and under hypoxia (2–4 Torr) at 22, 32 and 35ºC. At both 22 and 32ºC, hypoxia did not affect contractility. However, at 35ºC under hypoxia, there was a significant (P<0.05) decrease in submaximal tension (28 ± 11%) compared to ambient PO2. Treatment of single myofibers with tiron (1 mM) and N‐acetyl‐cysteine (NAC; 0.5 mM) at low PO2 did not alter the decrease in submaximal force seen during hypoxia. However, with H2O2 (100 μM), submaximal tension increased after 2–5 min (51 ± 4%), and then decreased thereafter. These results suggest that hypoxia at physiological temperature diminishes submaximal force in single fast‐twitch fibers, but not in a ROS dependent manner. Supported by NIH (AR040155‐17) and APS Undergraduate Summer Fellowship.