Hydroxyl radical and glutathione interactions alter calcium sensitivity and maximum force of the contractile apparatus in rat skeletal muscle fibres

摘要

Studies on intact muscle fibres indicate that reactive oxygen species (ROS) produced during muscle activity, or applied exogenously, can cause decreased force responses primarily by reducing the Ca²⁺ sensitivity of the contractile apparatus. Identification of the molecular basis of this effect is complicated by the fact that studies on skinned muscle fibres in general have not observed reduced contractile Ca²⁺ sensitivity when applying ROS, predominantly H2O2. Here, using skinned fibres from rat extensor digitorum longus (EDL) and soleus muscle, it is shown that although H2O2 (≥ 100 μm) has little effect by itself, when added in the presence of myoglobin it causes marked reduction in the Ca²⁺ sensitivity of the contractile apparatus, probably due to production of hydroxyl radicals (OH^•). Maximum force production is also reduced, but only with larger or more prolonged treatments. The effects are not prevented by tempol, a potent superoxide scavenger. Dithiotreitol (DTT) produces little reversal of the sensitivity change if applied afterwards, but it does substantially reverse all the changes if applied before the fibre undergoes an activation sequence. When glutathione (GSH, 5 mm) is present, exposure of EDL fibres to H2O2 and myoglobin causes an increase in Ca²⁺ sensitivity, with longer treatments causing a subsequent decrease, whereas in soleus fibres it causes only decreases in sensitivity and maximum force. The increased Ca²⁺ sensitivity in EDL fibres is evidently due to the summed actions of (i) a potentiating effect of glutathionylation, which can be reversed by DTT and only occurs in fast-twitch fibres, and (ii) a less reversible reduction in sensitivity. Western blotting showed that reductions in Ca²⁺ sensitivity were not due to loss of troponin-C. The present findings help provide a mechanistic basis for diverse findings on the effects of ROS in muscle fibres and implicate OH^• radicals and glutathione as likely mediators of the effects.