Chronic smokers present lower limb muscle dysfunction prior to the onset of overt respiratory limitations. Skeletal muscle dysfunction induced by cigarette smoke (CS) includes muscle weakness, loss of muscle mass, and decreased fatigue resistance, which directly lead to exercise intolerance. Smokers and those who develop COPD have higher incidence of muscle injury and show poor muscle regenerative capacity. The mechanism by which muscle regeneration may be affected following injury in chronic smokers is not well investigated. Therefore, we hypothesized that chronic CS exposure delays the trajectory of muscle regeneration following an eccentric exercise-induced muscle injury (EEIMI). We aim to investigate the consequences of short- (2 month) and long- (4 month) term CS exposure in mice on muscle regeneration following EEIMI via lengthening contraction procedure (LCP). Our investigation evaluated changes in ex-vivo force contractility, myofiber cross-sectional area (CSA), and the presence of central nucleated myofibers (CNM) 7 days following LCP, during the muscle reconstruction phase. We found the CS exposure leads to a small decrease in EDL force production, a reduction in myofiber CSA, and increase of CNM in injured legs, suggesting regeneration is delayed. We aimed further to investigate the time course of muscle satellite cell (SC) fusion during the regenerative process in Pax7CreERTdTomato mice to understand better the extent of damage induced by the LCP in the TA and EDL muscles. SC fusion was only detected after 5 days recovery in TA and EDL. Together, the data suggest that CS exposure delays early stages of muscle regeneration process.

Nitric oxide (NO) is an important signaling molecule in the process of muscle repair. It is involved in inflammatory signaling as well as satellite cell signaling– specifically satellite cell proliferation, differentiation and maturation. Previous studies have shown that NO impacts exercise performance and fatigue resistance, but there are less studies on the impact of NO after exercise, during muscle repair. We investigated the role that nitric oxide modulation may play in muscle repair with a GSNOR inhibitor (SPL-334), ISDN (isosorbide dinitrate–NO donor), and L-NIL (iNOS inhibitor). We found that GSNORi attenuated reduction of force after exercise-induced muscle damage (EIMD) in a lengthening-contractions model in EDL (extensor digitorum longus) muscle but did not affect the number of Tdt+ myofibers or cross-sectional area of the Tdt+ myofibers in the TA (tibialis anterior) muscle. ISDN had no effect on force production but decreased the number of Tdt+ myofibers and increased the CSA (cross-sectional area) of Tdt+ myofibers. L-NIL did not affect the force reduction but did increase the total number of Tdt+ myofibers post-EIMD. These data suggest that excess NO via ISDN may reduce quiescent satellite cell activation, iNOS inhibition may increase regeneration via macrophage signaling, and GSNORi may not impact muscle regeneration but instead affect the force produced post-LCP via ryanodine receptor hypernitrosylation.

Skeletal muscle fibers produce nitric oxide (NO) and possibly the S-nitrosylated form of glutathione, S-nitrosoglutathione (GSNO), due to Ca2+ transients during fatigue. GSNO reductase (GSNOR), an enzyme that denitrosylates S-nitrosothiols, scavenges GSNO to protect proteins from being S-nitrosylated. Prior studies have revealed GSNOR as a protective enzyme against excessive S-nitrosylation by GSNO in many tissues, but it has not been shown to directly regulate proteins during fatigue in skeletal muscle. We investigated the role of GSNOR during fatiguing contractions, particularly the acute effects of a pharmacological GSNOR inhibitor (GSNORi), on fast-twitch muscle contractility and fatigue resistance using whole extensor digitorum longus (EDL) muscles and intact single flexor digitorum brevis (FDB) myofibers from mice. GSNORi did not result in any changes in contractile function under non-fatiguing conditions. EDL exposed to GSNORi were less fatigue tolerant and less capable of recovering after fatigue. NO synthase (NOS) inhibitor NG-Methyl-L-arginine (L-NMMA) blocked GSNORi’s effects on fatigue tolerance and recovery. GSNORi treated myofibers exhibited depressed recovery of contractile force post- fatigue compared to controls treated with dimethyl sulfoxide (DMSO), the vehicle used to solubilize GSNORi, matching the EDL data. Myofibers showed no changes in Ca2+-handling or the force-Ca2+ relationship, nor in time to fatigue, thereby contradicting the results of our EDL experiments. These data suggest that NO produced during muscle fatigue leads to an increase in GSNO, which may subsequently lead to an increase in intracellular S-nitrosothiols that specifically alters myofibrillar rather than Ca2+- handling protein function, and that GSNOR regulates against excessive S-nitrosylation of these proteins.