One of the most important cytosolic Ca2+ buffers present in mouse fast-twitch myofibers, but not in human myofibers, is parvalbumin (PV). Previous work using conventional PV knockout mice suggests that lifelong PV ablation increases fatigue resistance, possibly due to compensations in mitochondrial volume. In this work, PV gene ablation was induced only in adult mice (PV-KO), and contractile and cytosolic Ca2+ responses during fatigue were studied in isolated muscle and intact single myofibers. Results were compared to control littermates (PV-Ctr). We hypothesized that the reduced myofiber cytosolic Ca2+ buffering developed only in adult PV-KO mice leads to a larger cytosolic free Ca2+ concentration ([Ca2+]c) during repetitive contractions, increasing myofiber fatigue resistance. Extensor digitorum longus (EDL) muscles from PV-KO mice had higher force in unfused stimulations (~50%, P<0.05) and slowed relaxation (~46% higher relaxation time, P<0.05) vs PV-Ctr, but muscle fatigue resistance or fatigue-induced changes in relaxation were not different between genotypes (P>0.05). In intact single myofibers from flexor digitorum brevis (FDB) muscles, basal and tetanic [Ca2+]c during fatiguing contractions were higher in PV-KO (P<0.05), accompanied by a greater slowing in estimated sarcoplasmic reticulum (SR) Ca2+ pumping vs PV-Ctr myofibers (~84% reduction, P<0.05), but myofiber fatigue resistance was not different between genotypes (P>0.05). Our results demonstrate that although the estimated SR Ca2+ uptake was accelerated in PV-KO, the total energy demand by the major energy consumers in myofibers, the cross-bridges and SR Ca2+ ATPase, were not altered enough to affect the energy supply for contractions, and therefore fatigue resistance remained unaffected.

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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.

During fatiguing contractions, skeletal muscle fibers produce nitric oxide (NO) (and possibly S‐nitrosoglutathione (GSNO)) due to the activation of calcium‐calmodulin dependent NO‐synthases (NOS). Myofibers contain GSNO reductase (GSNOR), an enzyme that consumes GSNO, thereby controlling intracellular protein S‐nitrosylation. The present investigation 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 from both hindlimbs of mice (C57BL6/J; 8–9 weeks old) were dissected and placed in an experimental chamber for ex‐vivo isometric force measurements during electrical stimulation. For each mouse, one EDL was treated with the GSNORi, or L‐NMMA (L‐N‐monomethyl Arginine, a NOS inhibitor; 400 μM), or both, while the other EDL served as an untreated control. Muscles were repetitively stimulated at increasing frequencies until force was decreased to 30% of initial force. Contractile recovery from fatigue was determined at submaximal and maximal frequencies of stimulation for 120 min post‐fatigue. The treatment with either GSNORi or L‐NMMA did not change maximal or submaximal force development. Acute GSNORi incubation significantly decreased the force development during fatiguing contractions (~30% less force) compared to untreated muscles. During muscle recovery from fatigue in the untreated control muscle, force developed at low frequencies of stimulation was ~80% of pre‐fatigue values after 120 min of recovery. However, when GSNORi was present during fatigue and during recovery post‐fatigue, the force developed at submaximal and maximal frequencies of stimulation were both significantly impaired compared to control (to 55–70% of pre‐fatigue values after 120 min, P<0.05 vs control). The NO‐dependent effects of GSNORi incubation on post‐fatigue recovery were tested when L‐NMMA was present to block NO production. L‐NMMA completely abolished the delayed contractile recovery detected with GSNORi. These data suggest that fatiguing contractions increase NO production thereby enhancing the intracellular amounts of GSNO, which can negatively affect muscle recovery post‐fatigue when GSNOR is inhibited. These results indicate that GSNOR activity has an important role in protecting contractile function during stimulation patterns that lead to muscle fatigue.Support or Funding InformationNIH (AR069577), CNPq (Universal)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.