Skeletal muscle function is dependent on its highly regular structure. In studies of dystrophic (dy/dy) mice, the proportion of malformed myofibers decreases after prolonged whole muscle stimulation, suggesting that the malformed myofibers are more prone to injury. The aim of this study was to assess morphology and to measure excitation-contraction (EC) coupling (Ca(2+) transients) and susceptibility to osmotic stress (Ca(2+) sparks) of enzymatically isolated muscle fibers of the extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles from young (2-3 mo) and old (8-9 mo) mdx and age-matched control mice (C57BL10). In young mdx EDL, 6% of the myofibers had visible malformations (i.e., interfiber splitting, branched ends, midfiber appendages). In contrast, 65% of myofibers in old mdx EDL contained visible malformations. In the mdx FDB, malformation occurred in only 5% of young myofibers and 11% of old myofibers. Age-matched control mice did not display the altered morphology of mdx muscles. The membrane-associated and cytoplasmic cytoskeletal structures appeared normal in the malformed mdx myofibers. In mdx FDBs with significantly branched ends, an assessment of global, electrically evoked Ca(2+) signals (indo-1PE-AM) revealed an EC coupling deficit in myofibers with significant branching. Interestingly, peak amplitude of electrically evoked Ca(2+) release in the branch of the bifurcated mdx myofiber was significantly decreased compared with the trunk of the same myofiber. No alteration in the basal myoplasmic Ca(2+) concentration (i.e., indo ratio) was seen in malformed vs. normal mdx myofibers. Finally, osmotic stress induced the occurrence of Ca(2+) sparks to a greater extent in the malformed portions of myofibers, which is consistent with deficits in EC coupling control. In summary, our data show that aging mdx myofibers develop morphological malformations. These malformations are not associated with gross disruptions in cytoskeletal or t-tubule structure; however, alterations in myofiber Ca(2+) signaling are evident.

Dantrolene is the drug of choice for the treatment of malignant hyperthermia (MH) and is also useful for treatment of spasticity or muscle spasms associated with several clinical conditions. The current study examines the mechanisms of dantrolene's action on skeletal muscle and shows that one of dantrolene's mechanisms of action is to block excitation-coupled calcium entry (ECCE) in both adult mouse flexor digitorum brevis fibers and primary myotubes. A second important new finding is that myotubes isolated from mice heterozygous and homozygous for the ryanodine receptor type 1 R163C MH susceptibility mutation show significantly enhanced ECCE rates that could be restored to those measured in wild-type cells after exposure to clinical concentrations of dantrolene. We propose that this gain of ECCE function is an important etiological component of MH susceptibility and possibly contributes to the fulminant MH episode. The inhibitory potency of dantrolene on ECCE found in wild-type and MH-susceptible muscle is consistent with the drug's clinical potency for reversing the MH syndrome and is incomplete as predicted by its efficacy as a muscle relaxant.

Introduction While the production of reactive oxygen species (ROS) during muscle contractile activity has been linked to both positive and negative adaptive responses, the sites for ROS generation within working muscle are not clearly defined. Methods We assessed cytosolic ROS production and mitochondrial redox potential with a targeted redox sensitive green fluorescent protein during repetitive field stimulation of single mature myofibers. Results Cytosolic ROS production increased by 94%, an effect that was abolished by pre-treatment with the reducing agent dithiothreitol. Mitochondrial redox potential was not altered during muscle contraction. In contrast, activity-dependent ROS production was ablated by an inhibitor of NADPH oxidase. Discussion We provide the first report on dynamic ROS production from mitochondria in single living myofibers and suggest that the mitochondria are not the major source of ROS during skeletal muscle contraction. Alternatively, our data support a role for NADPH oxidase-derived ROS during contractile activity.

The health of skeletal muscle is promoted by optimal nutrition and activity/exercise through the activation of molecular signaling pathways. Reactive oxygen species (ROS) or reactive nitrogen species (RNS) have been shown to modulate numerous biochemical processes including glucose uptake, gene expression, calcium signaling and contractility. In pathological conditions, ROS/RNS signaling excess or dysfunction contributes to contractile dysfunction and myopathy in skeletal muscle. Here we provide a brief review of ROS/RNS chemistry and discuss concepts of ROS/RNS signaling and its role in physiological and pathophysiological processes within striated muscle.