A mitochondrial-targeted antioxidant improves myofilament Ca2+ sensitivity during prolonged low frequency force depression at low PO2.

KEY POINTS: Skeletal muscle contractile activity is associated with an enhanced reactive oxygen species (ROS) generation. At very low PO2, ROS generation by mitochondria can be elevated in intact cells. An elevated intracellular oxidant activity may affect muscle force development and recovery from fatigue. We treated intact single muscle fibres with a mitochondrial antioxidant and stimulated the fibres to contract at a low extracellular PO2 that is similar to the intracellular PO2 that is observed during moderate to intense exercise in vivo. The mitochondrial antioxidant prevented a sustained decrease in the myofibrillar Ca2+ sensitivity and improved muscle submaximal force development after fatigue at low extracellular PO2. ABSTRACT: 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 O2 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 [Ca2+ ] 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 [Ca2+ ] transients (peak [Ca2+ ]c ) were equally decreased in both groups compared to pre-fatigue values. The combined force and peak [Ca2+ ]c data demonstrated that myofibrillar Ca2+ sensitivity was diminished in the untreated fibres 30 min after fatigue compared to pre-fatigue values, but Ca2+ 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 Ca2+ sensitivity, which alleviates the fatigue induced PLFFD.

Ca²âº-pumping impairment during repetitive fatiguing contractions in single myofibers: role of cross-bridge cycling.

The energy cost of contractions in skeletal muscle involves activation of both actomyosin and sarcoplasmic reticulum (SR) Ca²âº-pump (SERCA) ATPases, which together determine the overall ATP demand. During repetitive contractions leading to fatigue, the relaxation rate and Ca²âº pumping become slowed, possibly because of intracellular metabolite accumulation. The role of the energy cost of cross-bridge cycling during contractile activity on Ca²âº-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 Ca²âº pumping is impaired during fatiguing contractions. Fibers were stimulated in the absence (control) and presence of BTS and cytosolic calcium ([Ca²âº]c) transients or intracellular pH (pHi) changes were measured. BTS treatment allowed normal [Ca²âº]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 [Ca²âº]c and the increase in basal [Ca²âº]c did not occur with BTS incubation. The progressively slower Ca²âº 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 + cyanide), there was no further slowing in SR Ca²âº pumping during contractions compared with the BTS-alone condition. Furthermore, the fall in pHi was significantly less during the BTS + 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 Ca²âº pumping rate during fatiguing contractions.