Inhibition of S‐nitrosoglutathione Reductase During Contractions Slows Recovery of Low‐Frequency Force in Isolated Fast‐twitch Muscle and in Intact Single Myofibers

Abstract

Nitric oxide (NO) is a free radical produced during contractions in myofibers and has been shown to be involved in the regulation of contractility, glucose uptake and myofiber repair. NO can form S‐nitrosoglutathione (GSNO), a by‐product that is metabolized by GSNO reductase (GSNOR). However, it is not known whether GSNO is produced during fatiguing contractions. The ongoing research tested the hypothesis that GSNOR scavenges the GSNO produced during repetitive contractions to protect contractile and calcium handling proteins from being S‐nitrosylated. To test this hypothesis, the effects of acute inhibition of GSNOR (GSNORi; SPL‐334, 10 μM) on contractility during and after fatiguing contractions in fast‐twitch muscle were examined. Extensor digitorum longus (EDL) muscles or flexor digitorum brevis (FDB) myofibers from both hind limbs of mice (C57BL6/J; 8–9 weeks old) were placed in an experimental chamber for isometric force measurements during electrical stimulation. During muscle recovery from fatigue in the EDL treated with GSNORi (n=6), force developed at low frequencies of stimulation was lower than the force developed in the control EDL (55–70% vs. 80% of pre‐fatigue values after 120 min of recovery, respectively, P<0.05). For FDB myofibers, contractile measurements were taken before and after treatment with GSNORi. The contractile paradigm in single myofibers (n=7) produced a higher decrease in force at submaximal frequencies of stimulation at 120 min recovery (~49%) compared to EDL muscle when GSNORi was absent. When GSNORi was present, the force at submaximal frequencies was not further changed by GSNORi incubation (~51%). Contractile and metabolic S‐nitrosylated proteins were detected in muscles following fatiguing contractions in the presence and absence of GSNORi by using the S‐nitrosothiol resin‐assisted capture (SNO‐RAC) method. Together, these results indicate that GSNOR activity has an important role in protecting contractile function during stimulation patterns that lead to muscle fatigue in EDL muscle.