Original Contribution
Intracellular generation of reactive oxygen species by contracting skeletal muscle cells

https://doi.org/10.1016/j.freeradbiomed.2005.04.010Get rights and content

Abstract

The aim of this work was to examine the intracellular generation of reactive oxygen species in skeletal muscle cells at rest and during and following a period of contractile activity. Intracellular generation of reactive oxygen species was examined directly in skeletal muscle myotubes using 2′,7′-dichlorodihydrofluorescein (DCFH) as an intracellular probe. Preliminary experiments confirmed that DCFH located to the myotubes but was readily photoxidizable during repeated intracellular fluorescence measurements and strategies to minimize this were developed. The rate of oxidation of DCFH did not change significantly over 30 min in resting myotubes, but was increased by approximately 4-fold during 10 min of repetitive, electrically stimulated contractile activity. This increased rate was maintained over 10 min following the end of the contraction protocol. DCF fluorescence was distributed evenly throughout the mytotube with no evidence of accumulation at any specific intracellular sites or localization to mitochondria. The rise in DCF fluorescence was effectively abolished by treatment of the myotubes with the intracellular superoxide scavenger, Tiron. Thus these data appear to represent the first direct demonstration of a rise in intracellular oxidant activity during contractile activity in skeletal muscle myotubes and indicate that superoxide, generated from intracellular sites, is the ultimate source of oxidant(s) responsible for the DCFH oxidation.

Introduction

Exercise has been associated with an increased generation of free radicals and reactive oxygen and nitrogen species (ROS) [1], [2] and a large number of studies have detected this by measurement of increased concentrations of the products of reactions of these species with lipids, proteins, and DNA in tissues and blood from animals and man following different exercise protocols (see [3] for reviews). Skeletal muscle appears to be an important source for generation of these species [2], [4]. Mitochondria have been claimed to be the major intracellular site for increased generation of reactive oxygen species during exercise [1] and several authors have assumed that the increased ROS generation that occurs during contractile activity is directly related to the elevated oxygen consumption that occurs with increased mitochondrial activity [1], [5], [6]. The precise relationship between the pattern of contractile activity and the extent and nature of the ROS generation is currently unclear with widely different exercise and contraction protocols reported to increase ROS activity [3].

Because of ready access to the extracellular compartment, most studies in this area have examined ROS that are released by skeletal muscle during contractile activity or are generated in the muscle extracellular space during contractions. The species that have been detected include superoxide [4], [7], hydroxyl radicals [8], [9], nitric oxide (NO) [10], and hydrogen peroxide [11]. Much initial work in this area investigated the potential generation of ROS during damaging contractile activity [3], but it has subsequently become evident that ROS are produced by skeletal muscle during nondamaging contractions [4] and that these substances may play roles in signaling adaptive responses to contractile activity [4], [12].

In order to evaluate the roles of ROS in mediating adaptations and/or damage to skeletal muscle during contractions it is necessary to understand the sites of generation of specific ROS by skeletal muscle cells. 2′,7′-Dichlorodihydrofluorescein-diacetate (DCFH-DA) has been widely used as a relatively nonspecific intracellular probe for ROS [13]. DCFH-DA is nonpolar and crosses cell membranes readily and within the cell it is hydrolyzed by cytosolic hydrolases to DCFH. This compound rapidly reacts with hydrogen peroxide in the presence of peroxidases and with some other ROS to form fluorescent dichlorofluorescein (DCF). In practice, use of DCFH-DA as an intracellular probe to directly image or measure ROS activities is limited by photo-oxidation [14] and only few studies have directly examined the oxidation of DCFH in situ in muscle fibers [7], [14], [15]. The effect of contractile activity on DCFH oxidation has been studied by homogenization prior to fluorescence measurements in myotubes [11] and skeletal muscle [16], but this approach may be susceptible to artifactual generation of ROS during the homogenization process [17].

In recent studies we have demonstrated that electrically stimulated skeletal muscle myotubes in culture can be used to examine the extracellular generation and release of ROS during contractile activity [18]. The aim of the current work was to use DCFH-DA as a nonspecific probe to examine intracellular ROS generation by skeletal muscle cells prior to, during, and following a demanding period of contractile activity. Our hypothesis was that the myotubes at rest would slowly oxidize DCFH and that this would increase rapidly during the period of contractile activity.

Section snippets

Myotube cultures

H-2kb muscle cells (a myoblast cell line established from the muscle of a neonatal H-2k-b-tsA58 mouse) [19] were maintained in a 5% CO2 humidified incubation in Dulbecco's modified Eagle's medium (DMEM) (Sigma Chemical Co, Poole, Dorset, UK) supplemented with 20% fetal calf serum (FCS), 10% horse serum (HS), 0.5% chick embryo extract (CEE) (Invitrogen Life Technologies, CA, USA). To maintain the cells in an undifferentiated state, they were grown at 33°C and the medium was supplemented with

Loading of cells with DCFH

In order to confirm that the oxidizable DCFH was only present within the myotubes, the DCF fluorescence was visualized and overlaid on a light microscopic image of the myotubes and this is shown in Fig. 1A. This demonstrates that the DCF is predominantly within the myotubes and not located between myotubes. Variability between preliminary measurements of DCF fluorescence from myotubes indicated potential differences in the amount of DCFH loaded into myotubes and a method for monitoring the

Discussion

DCFH-DA was found to accumulate readily in myotubes and the DCF fluorescence showed no preferential emission from any parts of the muscle cell (Fig. 2), as reported by Arbogast and Reid [15]. Preliminary data indicated that the DCFH was very susceptible to photo-oxidation during measurements of fluorescence emission. We therefore followed the procedures indicated by Murrant et al. [14] to control this potential artifact. These were to conduct the experiments in a darkened laboratory and to

Acknowledgments

The authors thank the Wellcome Trust (Grant 062955/Z/00) for financial support, Drs. J.E. Morgan and T.A. Partridge (MRC Cell Biology Group, Imperial College of Medicine, London) for providing the H-2kb muscle cells, and Dr. M.B. Reid (University of Kentucky) for helpful discussions.

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    Both of these authors made an equal contribution to this work.

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