EUK-134 ameliorates nNOSμ translocation and skeletal muscle fiber atrophy during short-term mechanical unloading

Abstract

. EUK-134 ameliorates nNOS translocation and skeletal muscle fiber atrophy during short-term mechanical unloading. Am J Physiol Regul Integr Comp Physiol 306: R470-R482, 2014. First published January 29, 2014 doi:10.1152/ajpregu.00371.2013.-Reduced mechanical loading during bedrest, spaceflight, and casting, causes rapid morphological changes in skeletal muscle: fiber atrophy and reduction of slow-twitch fibers. An emerging signaling event in response to unloading is the translocation of neuronal nitric oxide synthase (nNOS) from the sarcolemma to the cytosol. We used EUK-134, a cellpermeable mimetic of superoxide dismutase and catalase, to test the role of redox signaling in nNOS translocation and muscle fiber atrophy as a result of short-term (54 h) hindlimb unloading. Fischer-344 rats were divided into ambulatory control, hindlimb-unloaded (HU), and hindlimb-unloaded ϩ EUK-134 (HU-EUK) groups. EUK-134 mitigated the unloading-induced phenotype, including muscle fiber atrophy and muscle fiber-type shift from slow to fast. nNOS immunolocalization at the sarcolemma of the soleus was reduced with HU, while nNOS protein content in the cytosol increased with unloading. Translocation of nNOS from the sarcolemma to cytosol was virtually abolished by EUK-134. EUK-134 also mitigated dephosphorylation at Thr-32 of FoxO3a during HU. Hindlimb unloading elevated oxidative stress (4-hydroxynonenal) and increased sarcolemmal localization of Nox2 subunits gp91phox (Nox2) and p47phox, effects normalized by EUK-134. Thus, our findings are consistent with the hypothesis that oxidative stress triggers nNOS translocation from the sarcolemma and FoxO3a dephosphorylation as an early event during mechanical unloading. Thus, redox signaling may serve as a biological switch for nNOS to initiate morphological changes in skeletal muscle fibers. atrophy; disuse; skeletal muscle; nNOS; oxidative stress; FoxO3a SKELETAL MUSCLE IS A HIGHLY specialized and adaptable mesodermic tissue capable of rapid remodeling in response to changes in mechanical loading and stretch (28, 38). Transmittance and detection of loading in skeletal muscle are paramount in regulating differentiation, cell growth, and protein turnover. The ability to sense and relay loading (i.e., mechanotransduction) is, in part, performed by proteins adjacent to and spanning cell membranes (i.e., sarcolemma) in skeletal muscle (47). Mechanical unloading that occurs with limb casting, splinting, bed rest, and spaceflight, elicits a substantial loss of forcegenerating capacity, linked to a diminishment in muscle fiber cross-sectional area or atrophy (12, 23). The unloading phenotype also includes a shift of a portion of skeletal muscle fibers from slow-twitch to fast-twitch (12). Muscle atrophy that occurs with unloading is coupled to a net loss of contractile proteins, a function of increased protein degradation combined with a decrease in protein synthesis (12,23). Recent studies emphasize the importance of proteolytic pathways, including ubiquitin proteasome, calpains, autophagy...

From physical inactivity to immobilization: Dissecting the role of oxidative stress in skeletal muscle insulin resistance and atrophy

“…These results have been confirmed by using antioxidant agents such as EUK-134 (SOD and catalase mimetic) [71] and SS-31 (mitochondria-targeted antioxidant) [54,121]. The inhibition of mitochondrial ROS production prevented soleus atrophy and UPS/autophagy activation induced by casting [54], whereas EUK-134 limited skeletal muscle atrophy and FOXO3a activation induced by hindlimb unloading [71]. Taken together, these data support that RONS activate UPS and autophagy in immobilization-induced skeletal muscle atrophy.…”

“…A more restricted number of studies focused on the effects of immobilization on α,β-unsaturated aldehydes (e.g., 4-HNE, MDA), markers of lipid peroxidation. 4-HNE content increased in rat soleus after 8 days of hindlimb unloading [64,71], whereas elevations in MDA and TBARS contents were reported after 10 and 14 days of hindlimb unloading, respectively [61,69]. All together, these results suggest that immobilization first induced lipid peroxidation and later protein carbonylation in skeletal muscle.…”

Age-related alterations in the sarcolemmal environment are attenuated by lifelong caloric restriction and voluntary exercise

“…Sarcolemmal nNOS was drastically mislocalized in aged plantaris muscle fibers, where it was observed predominately in the sarcoplasm. Previously, our group (Lawler and others 2014) and Suzuki et al (Suzuki and others 2007) found that mechanical unloading and denervation cause translocation of nNOSµ away from the sarcolemma. Mislocalization of nNOS is also noted in Duchenne muscular dystrophy (Brenman and others 1995) and other types of neuromuscular disorders (Finanger Hedderick and others 2011), further emphasizing the physiological importance of the protein for proper muscle function to occur.…”

Redox control of skeletal muscle atrophy

“…Rather, Suzuki and colleagues report that muscle inactivity is associated with a dissociation of neuronal NO synthase (nNOS) from the dystroglycan complex and propose that the release of nNOS from the membrane results in nNOS activation and increased production of NO [7]. The finding that muscle disuse results in increased cytosolic nNOS has been confirmed by others [8, 27, 28]. Nonetheless, it is important to note that there are no published reports documenting higher NO production by non-membrane bound nNOS [29].…”

“…Indeed, it is widely accepted that oxidative stress contributes to skeletal muscle atrophy due to both disease and prolonged muscle inactivity [3-6]. Further, evidence also links nitrosative stress to skeletal muscle atrophy resulting from chronic disease or prolonged muscle disuse [7, 8]. …”