Changes in the intracellular concentration of ROS and/or NO induced by passive stretching in skeletal muscle fibres from aged and dystrophic mice

Resumen

Skeletal muscle constantly produces reactive oxygen species (ROS) and nitric oxide (NO) which may play an important role in signalling and regulatory pathways. Isolated muscles in vitro release NO to the extracellular space [66] and passive stretching of muscle increases the release of NO from rat skeletal muscle in vitro [67]. Recently, we have developed a model to study the generation of ROS and NO in real time in isolated single muscle fibres subjected to a protocol of passive stretching [68].The aim of this study was to evaluate the effect of passive stretching on the intracellular concentration of ROS and NO in single muscle fibres from young and old mice and in an animal model of Duchenne muscular dystrophy, the mdx mouse. We used young (2–4 month-old) and old (26–28 month-old) C57BL/6 mice and mdx mice. Muscle fibres were isolated from the Flexor Digitorus Brevis and attached to a flexible silicone membrane which had been previously coated with a collagen Matrigel? matrix. Fibres were loaded with different fluorophore probes: DCFH (general detector of ROS), hydroethidine (detector of superoxide) and DAF-FM (detector of NO). The passive stretching protocol was applied to fibres using the FX-4000? Flexercell® system. Using fluorescence microscopy, fluorescence emission from fibres at different time points was quantified by image analysis in order to detect the intracellular concentration of ROS and/or NO.Results from positive control experiments indicated that the technique is able to detect changes in the intracellular concentration of ROS and/or NO.However, the changes of fluorescence observed in the fibres subjected to the protocol of passive stretching were lower and less evident, which indicates that the generation of ROS and NO induced by passive stretching is relatively low.