Currently, we do not have a full understanding of the complexities involved in oxidant production during disuse-induced skeletal muscle atrophy. Skeletal muscle wasting that occurs during pathological conditions, such as sepsis and chronic heart failure, has been associated with an overproduction of nitric oxide (NO) [2,55]. However, upregulation of iNOS, rather than nNOSμ, was cited as causal in cachectic muscle atrophy.

However, during disuse-induced atrophy, it is unclear whether excess NO production, source, or localization accelerates the atrophic process. In a study by Suzuki et al. [83], 14 days of hindlimb unloading was associated with a sarcoplasmic translocation of the mu (μ) – isoform of neuronal nitric oxide synthase (nNOSμ) from the sarcolemma-localized dystrophin glycoprotein complex (DGC) – and this translocation correlated with elevated levels of sarcoplasmic NO [83]. Their group followed this experiment up with genetic and pharmacological nNOS inhibition studies that indicated translocation of nNOSμ activity during hindlimb unloading as a critical event promoting atrophy [83]. Dystrophin remained attached to the DGC, while dysferlin, a multifunctional protein involved in membrane repair, remained bound to nNOSμ. Inhibition of nNOS prevented activation of FoxO3a and ubiquitin ligases [83]. Cytosolic levels of •NO were elevated as determined via EPR (electron paramagnetic resonance). We found that nNOSμ translocation is an early event in the mechanical unloading process, occurring within 3 days with the rat model [36]. In addition, Vitadello et al. [92] also found untethering of nNOSμ from the sarcolemma and translocation of nNOSμ with hindlimb unloading, linked to downregulation of grp94 [93]. Grp94 is an HSP90-like stress protein that chaperones protein folding [45]. Curcumin, the primary compound in the spice tumeric, with an antioxidant and anti-inflammatory properties mitigated nNOSμ translocation and atrophy [92].

In contrast, Lomonosova et al. [42] reported that L-arginine mitigated hindlimb unloading-induced reduction of nNOS, •NO, dystrophin, and HSP90 protein levels and mRNA transcripts gene expression. Heterogeneity in dystrophin sarcolemmal localization was found, suggesting disruption of the DGC. Furthermore, hindlimb unloading reduced levels of desmin [42], a Z-disc fiber protein that transmits loading to the DGC and surrounding extracellular matrix protein (e.g., laminin, collagen). Protein abundance was determined from serial frozen cross sections and thus assessed global levels, rather than site-specific changes. Efficacy of L-arginine may suggest a substrate limitation or decoupling of nNOSμ. In addition, a different spin trap was used for EPR in the Lomonosova study compared with Suzuki et al.: diethyl-dithiocarbamate (DETC) vs. N-methyl-D-glucamine-dithiocarbamate (MGD). Thus the global role of •NO and downstream nitrosative species remain uncertain in unloading-induced atrophy. However, we propose that (1) disruption of the DGC and membrane environment and untethering of nNOSμ from the sarcolemma, (2) subcellular location of nNOS and downstream effects on FoxO3a-ubiquitin ligase signaling, (3) substrate availability, (4) the role of other NOS isoforms, and (5) local •NO bioavailability may be more important than global •NO levels in regulating muscle fiber atrophy with mechanical unloading. However, additional research is crucial to unraveling these mysteries.

Interestingly, evidence indicates NOS and •NO are involved in the regulation of the type I MHC isoform [70, 82]. For instance, a recent report found that with 8 weeks of NOS inhibition, there was a significant decrease in the percentage of type I fibers and concomitant increase in the percentage of type IIa fibers in the soleus muscle [82]. Since disuse leads to a shift in fiber type from type I to type IIa, these data point toward the notion that a reduction in myofiber NO may be a critical event promoting the shift.

It is proposed that in healthy muscle, sarcolemma-localized nNOSμ is required for myofiber mechanotransduction. For example, muscle fiber hypertrophy with mechanical overloading was dependent upon intact sarcolemmal nNOSμ and NADPH oxidase 4 (Nox4) [27]. Genetic ablation or inhibition of nNOSμ early in the overloading process attenuated muscle hypertrophy by over 50% [27]. In support of these findings, the reduction in sarcolemma nNOSμ that occurs with unloading leaves the muscle fiber susceptible to sarcolemmal lysis by neutrophils upon reloading [54], thus slowing recovery, whereas mice expressing a muscle-specific nNOS transgene were significantly protected from sarcolemma damage and injury in response to reloading [54]. Clearly, nNOSμ plays a vital role as a load-sensitive molecule in skeletal muscle. Translocation of nNOSμ has been reported to occur in response to various forms of disuse: hindlimb unloading [36, 83, 90], spaceflight [66], denervation [83], bed rest [63], and intensive care-associated critical illness myopathy [40]. While some have observed disuse-induced alterations in nNOSμ synthesis and/or degradation, the mislocalization of nNOSμ in postural muscles is a common event associated with atrophy. Suzuki et al. [83] observed a causal link between nNOSμ untethering and proteolytic FoxO3a activation during hindlimb unloading. Our group demonstrated that administration of a superoxide dismutase (SOD)/catalase mimetic (EUK134) reduced the extent of nNOSμ untethering from the sarcolemma and translocation to the sarcoplasm following 54 h of hindlimb unloading [36]. This effect attenuated the activation of FoxO3a, subsequent atrophy, and shift in fiber type in the soleus muscle [36]. These data suggest that disuse-induced translocation of nNOSμ is due in part to a rise in oxidative stress.

Much remains to be learned concerning the many roles of nNOSμ during mechanical unloading. For example, further examination is needed to elucidate whether translocated nNOSμ leads to elevated localized NO within the sarcoplasm of the fibers and which signaling pathways are affected. Does nitrosylation of glutathione play a role in dephosphorylation of FoxO3a? What are the mechanisms of substrate limitation? What are the mechanisms underlying redox regulation of nNOSμ during unloading? A causal link between nNOSμ translocation that is directly associated with depressed protein synthesis is also of interest. In addition, identifying the molecular mechanisms guiding nNOSμ untethering from dystrophin and alpha-syntrophin in the DGC is of significant concern. In addition, are the effects of nNOS translocation fiber type dependent? Does nNOS translocation trigger fiber-type switch from slow to fast twitch with unloading?

In summary, disuse-induced skeletal muscle atrophy is due to elevation of ROS in the myofiber environment. Oxidative stress is implicated in the regulation of blunted rates of muscle protein synthesis and increased proteolysis (summarized in Fig. 11.2). Various myofiber oxidant sources participate in the production of ROS and are potentially involved in a ROS-induced ROS-release feedback mechanism. In addition, future studies are needed to determine the specific upstream signaling events that trigger the disuse-induced ROS production from each oxidant source. While the data remains unclear concerning NO production during disuse, there is a better understanding of the link between atrophying muscle and nNOSμ translocation. However, future studies are needed to elucidate the connections between oxidative stress, nNOSμ translocation, and the affected atrophic signaling cascades.