ROS-Mediated Decline in Maximum Ca2+-Activated Force in Rat Skeletal Muscle Fibers following In Vitro and In Vivo Stimulation

We hypothesised that normal skeletal muscle stimulated intensely either in vitro or in situ would exhibit reactive oxygen species (ROS)-mediated contractile apparatus changes common to many pathophysiological conditions. Isolated soleus (SOL) and extensor digitorum longus (EDL) muscles of the rat were bubbled with 95% O-2 and stimulated in vitro at 31 degrees C to give isometric tetani (50 Hz for 0.5 s every 2 s) until maximum force declined to <= 30%. Skinned superficial slow-twitch fibers from the SOL muscles displayed a large reduction (similar to 41%) in maximum Ca2+-activated specific force (F-max), with Ca2+-sensitivity unchanged. Fibers from EDL muscles were less affected. The decrease in F-max in SOL fibers was evidently due to oxidation effects on cysteine residues because it was reversed if the reducing agent DTT was applied prior to activating the fiber. The GSH:GSSG ratio was similar to 3-fold lower in the cytoplasm of superficial fibers from stimulated muscle compared to control, confirming increased oxidant levels. The presence of Tempol and L-NAME during in vitro stimulation prevented reduction in F-max. Skinned fibers from SOL muscles stimulated in vivo at 37 degrees C with intact blood supply also displayed reduction in F-max, though to a much smaller extent (similar to 12%). Thus, fibers from muscles stimulated even with putatively adequate O-2 supply display a reversible oxidation-induced decrease in F-max without change in Ca2+-sensitivity, consistent with action of peroxynitrite (or possibly superoxide) on cysteine residues of the contractile apparatus. Significantly, the changes closely resemble the contractile deficits observed in a range of pathophysiological conditions. These findings highlight how readily muscle experiences ROS-related deficits, and also point to potential difficulties when defining muscle performance and fatigue.