Abstract
ROS serve as important signaling molecules and interact with multiple cell signaling and regulatory pathways to modulate changes in gene expression. The aim was to examine the effect of nNOS overexpression on ROS activities in skeletal muscle fibres at rest and following a period of contractile activity. To elucidate the roles of ROS, we examined their intracellular activities by using fluorescence microscopy. Mice overexpressing nNOS and age‐matched wild type (WT) mice were used in this study. Flexor digitorum brevis muscles were dissected and incubated in collagenase to isolate single muscle fibres. Fibres were plated on culture dishes and were loaded with DAF‐FM DA, DHE or DCFH DA, fluorescent probes for the assessment of nitric oxide (NO·) superoxide (O2·) and a general probe for ROS respectively. Contractile activity was induced by electrical stimulation. Control non‐stimulated fibres from the nNOS mice showed a higher rate of increase in DAF‐FM fluorescence compared with the WT group implying a higher NO· formation. Fibres from nNOS overexpressors showed a reduced ethidium fluorescence compared with fibres from WT mice at rest indicating a lower O2· generation. No differences were observed between groups following contractions. These results suggest that overexpression of nNOS can increase NO· ‘bioavailability’ in skeletal muscle cells and reduce O2· toxicity. Supported by the A Onassis Foundation (GR).
Published Version
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