Low Po₂ conditions induce reactive oxygen species formation during contractions in single skeletal muscle fibers

Am J Physiol Regul Integr Comp Physiol. 2013 Jun 1;304(11):R1009-16. doi: 10.1152/ajpregu.00563.2012. Epub 2013 Apr 10.

Abstract

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 μM) 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 < 0.05). ROS accumulated at a faster rate in low vs. high Po₂. Force was reduced >30% for each condition except low Po₂ with ebselen, which only decreased ~15%. We concluded that single myofibers under low Po₂ conditions develop accelerated and more oxidative stress than at Po₂ = 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.

Keywords: confocal; ebselen; hypoxia; myofiber; reactive oxygen species.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Analysis of Variance
  • Animals
  • Antioxidants / pharmacology
  • Azoles / pharmacology
  • Electric Stimulation
  • Female
  • Hypoxia / metabolism*
  • In Vitro Techniques
  • Isoindoles
  • Microscopy, Confocal
  • Muscle Contraction / physiology*
  • Muscle Fatigue / physiology
  • Muscle Fibers, Skeletal / metabolism*
  • Muscle, Skeletal / physiology
  • Organoselenium Compounds / pharmacology
  • Reactive Oxygen Species / metabolism*
  • Signal Transduction / drug effects
  • Signal Transduction / physiology
  • Xenopus laevis

Substances

  • Antioxidants
  • Azoles
  • Isoindoles
  • Organoselenium Compounds
  • Reactive Oxygen Species
  • ebselen