Programme Contents

Stretch-induced oxidative damage to mdx muscle: the role of NADPH oxidase

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by the absence of the protein dystrophin, which connects the cytoskeleton to the surface membrane. In the mdx mouse, an animal model of DMD,we have shown that increased stretch-activated channel (SAC) activity is the main source of Ca²⁺ influx following stretched contractions (Yeung et al., 2005). Canonical transient receptor potential 1 (TRPC1) is the putative SAC protein in mammalian cells (Maroto et al., 2005). It is known that some TRPC channels are activated by reactive oxygen species (ROS) and given that mdx muscles show evidence of oxidative stress, we postulated that stretch-induced ROS might activate SACs in dystrophic muscles. NADPH oxidase produces ROS in smooth muscle subjected to stretch (Grote et al., 2003), and so we also investigated whether this was a source of the stretch-induced ROS production in mdx skeletal muscle. The two hypotheses tested through these experiments were: 1) Stretch-induced ROS mediate Ca²⁺ influx through SACs and cause muscle damage; 2) NADPH oxidase is the main source of the stretch-induced ROS production in mdx muscle.

In the first experiments, we investigated whether the antioxidant N-acetylcysteine (NAC) could reduce stretch-induced muscle damage in mdx muscle. mdx and wild type (C57Bl/10ScSn) mice were euthanased and the extensor digitorum longu muscles removed. Muscles were perfused with or without 20 mM NAC. Solutions also contained 0.02% Evans Blue Dye (EBD) for assessment of membrane permeability. Muscles underwent 3 stretched (eccentric) contractions at 35°C. Tetanic force was measured before and 60 min after eccentric contractions and then muscles were frozen and sectioned for EBD uptake. Following the stretched contractions, force fell to 35 ± 3% for mdx muscles and NAC significantly improved force to 51 ± 2% (p < 0.01). As expected, force was much greater for wild-type muscles (69 ± 5%) and NAC had no additional effect. The area of EBD uptake was 8.6 ± 1.8% in mdx muscle cross-sections and this was significantly reduced by NAC to 2.6 ± 0.8% (p < 0.01). Wild-type muscles had a value of 1.8 ± 0.7%, which was not significantly different to NAC.

Secondly, we tested if ROS could activate SACs. mdx and wild type mice were killed by cervical dislocation and single muscle fibres from the flexor digitorum brevis were dissected. Fibres were loaded with the fluorescent Ca²⁺ probe Fluo-4 AM and subjected to 10 stretched contractions. Following the stretched contractions, there was a significant increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) up to 30 min for mdx fibres (p < 0.001) but not for wild type fibres. The increased [Ca²⁺]_i in mdx fibres was prevented by the antioxidant tiron (5 mM), which also reduced the force deficit. We then showed, in resting mdx fibres, that 10 μM hydrogen peroxide (H₂O₂), also increased [Ca²⁺]_i, which could be returned to baseline levels by the SAC blocker, streptomycin. Taken together, these findings suggested that increased ROS production during stretched contractions activate SACs and allow Ca²⁺ entry in mdx muscle.

Finally, we explored whether NADPH oxidase was the main source of the enhanced ROS production during stretch. As with tiron, the increased [Ca²⁺]_i in mdx fibres following stretched contractions was significantly inhibited (p < 0.01) by 1 μM diphenyleneiodium (DPI), an NADPH oxidase inhibitor, and this was also accompanied by increased muscle force. In preliminary experiments, another NADPH oxidase inhibitor, apocynin, also decreased the [Ca²⁺]_i after stretched contractions.

The results of these experiments show that ROS are an important cause of stretch-induced damage to mdx muscle. As well as deleterious effects on proteins and muscle membranes, ROS also activate SACs, causing an influx of Ca²⁺ and the activation of Ca²⁺-dependent damage pathways, such as calpains. We also provide evidence that NADPH oxidase is a primary source of ROS in stretched mdx muscle. We now aim to investigate why SACs are more sensitive to ROS in mdx muscle and to determine the key proteins targeted by ROS, which impair muscle function and contribute to dystrophic muscle damage.

Grote K, Flach I, Luchtefeld M, Akin E, Holland SM, Drexler H & Schieffer B. (2003) Circulation Research, 92: e80-e86.

Maroto R, Raso A, Wood TG, Kurosky A, Martinac B & Hamill OP. (2005) Nature Cell Biology, 7: 179-85.

Yeung EW, Whitehead NP, Suchyna TM, Gottlieb PA, Sachs F & Allen DG. (2005) Journal of Physiology, 562:367-80.