A critical limitation to astronaut performance during spaceflight missions including those to the International Space Station and Mars is the impaired contractile ability of skeletal muscles. Skeletal muscle is a highly specialized tissue that rapidly adapts to dynamic changes in mechanical loading by modifying its ultrastructure and mass. Detection and transmittance of loading are vital to muscle integrity and protein turnover, with a nexus associated with the subsarcolemmal cytoskeleton that includes the dystrophin-glycoprotein complex (DGC) and caveolae. This interface between myocytes and the extracellular matrix confers mechanical integrity and activates cell signaling pathways that regulate protein turnover, mitochondrial, muscle fiber cross-sectional area, and fiber phenotype. Mechanical unloading experienced in the microgravity of spaceflight elicits large decrements in force generating capacity via reduction in muscle fiber cross sectional area or atrophy. While unloading-induced atrophy is a function of reduced protein synthesis coupled with increased protein degradation, activation of the transcription factors FoxO3a and nuclear factor-kappaB have drawn significant attention as mediators. Although the upstream triggers are not fully understood, translocation of neuronal nitric oxide synthase (nNOS) from the DGC and sarcolemma to the sarcoplasm is a novel signaling event that stimulates proteolysis, and thus atrophy. New clues can be gleaned from Duchenne and many of the limb-girdle muscular dystrophies, pathologies involving mutations to DGC and other sarcolemmal proteins. Because MDs and microgravity both increases susceptibility of muscles to damage, oxidative stress, and pro-inflammatory signaling, a shared root cause is suspected: disruption of the subsarcolemmal cytoskeleton. New studies and Preliminary Data indicate the importance of NAD(P)H oxidase (NOX), a sarcolemmal source of oxidative stress, and caveolin-3 in stimulating myopathy in muscular dystrophy models. NOX may be activated upstream by angiotensin II and c-abl, a tyrosine kinase. Further, our pilot data show that unloading-induced alterations in caveolin-3 and nNOS are redox dependent and linked to atrophy and shift in muscle fiber-type. Our central hypothesis is that oxidative stress produced by NOX pathways and mitochondria directly contribute to a sarcolemmal loss of nNOS during mechanical unloading, enhancing proteolysis and suppressing protein synthesis. The rodent hindlimb unloading model and novel fractional synthesis and degradation will be used to test our hypotheses.
In short, our primary objective is to identify the trigger by which skeletal muscles switch from being an anabolic to a catabolic machine during spaceflight, causing muscle atrophy and weakness. We propose that oxidative stress produced by a NOX pathway and mitochondria move the master switch protein (nNOS) away from the cell membrane, which slows protein synthesis and accelerates breakdown of protein. Our study would directly address high-priority gaps in Recapturing a Future for Space Exploration including the role of reactive oxygen species in protein balance and maintaining the slow contractile phenotype. Our grant proposal would also forward our understanding of (1) alterations in the subsarcolemmal cytoskeleton and membrane domains, (2) sensing and transduction of loading and gravity, (3) protein turnover, and (4) regulatory mechanisms that govern alterations in skeletal muscle, all programmatic emphases and goals of the current Space Biology NRA.
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Specific Aim 1 will identify NOX-dependent signaling on nNOS translocation and muscle fiber atrophy with hindlimb unloading. Specific Aim 2 will determine directly the role of mitochondrial and cytosolic oxidative stress in movement of nNOS away from the muscle cell membrane and disruption of DGC with mechanical unloading.
We have made significant progress on both Specific Aims 1 & 2, including the completion of the following experiments:
(1) "Administration of the superoxide dismutase/catalase mimetic, Eukarion-134, prevents short-duration (54hr) unloading induced atrophy."
(2) "Administration of EUK-134 prevents 7-day unloading induced atrophy." We continued experimentation with EUK-134 to include a longer duration 7-day unloading protocol to determine the effectiveness of EUK-134 at ameliorating soleus muscle atrophy following 7-days of hindlimb unloading and compare with the 2-day unloading protocol. For this subset of 7- day EUK-134 treated animals, we also measured expression of proteins involved in protein synthesis including protein kinase b (Akt) and mTOR.
We have recently published a paper in the American Journal of Physiology: Regulatory, Integrative, and Comparative Physiology entitled, “EUK-134 ameliorates nNOSµ translocation and skeletal muscle fiber atrophy during short-term mechanical unloading.” In this paper, we demonstrate that administration of the cell permeable synthetic superoxide dismutase (SOD)/catalase mimetic, EUK-134, prevents a reduction in Type I fiber cross sectional area following a 2-day (~54 hour) unloading protocol in the absence of changes in soleus muscle mass. In conjunction with a reduction of Type I fiber atrophy, EUK-134 prevented a shift in fiber type from type I to type II, an effect commonly observed with mechanical unloading. In addition, EUK-134 was associated with reduced expression of NOX-2 subunits, ROS production (dihydroethidium assay), and reduced ROS-related signaling/damage (4-hydroxynonenal). The positive effects of EUK-134 may be also related to a reduction in nNOS translocation from the sarcolemma to the cytosol and inactivation of the proteolytic signaling molecule, Fox-O3a.
Research will continue under the "Upstream Regulation of Nos2 and Skeletal Muscle Atophy During Microgravity and Countermeasure Development" investigation. Additional data and results can be found in supported publication.
Kwak HB, Lee Y, Kim JH, Van Remmen H, Richardson AG, Lawler JM. MnSOD Overexpression Reduces Fibrosis and Pro-Apoptotic Signaling in the Aging Mouse Heart. J Gerontol A Biol Sci Med Sci 2015 May;70(5):533-44.
Lawler JM, Kunst M, Hord JM, Lee Y, Joshi K, Botchlett RE, Ramirez A, Martinez DA. "EUK-134 ameliorates nNOSµ translocation and skeletal muscle fiber atrophy during short-term mechanical unloading." Am J Physiol Regul Integr Comp Physiol. 2014 Apr 1;306(7):R470-82. [DOI]
Kuczmarski JM, Hord JM, Lee Y, Guzzoni V, Rodriguez D, Lawler MS, Garcia-Villatoro EL, Holly D, Ryan P, Falcon K, Garcia M, Janini Gomes M, Fluckey JD, Lawler JM. Effect of EUK-134 on Akt-mTOR signaling in the rat soleus during 7 days of mechanical unloading. Exp Physiol. 2018 Jan 8. [DOI]
Lawler JM, Garcia-Villatoro EL, Guzzoni V, Hord JM, Botchlett R, Holly D, Lawler MS, Gomes MJ, Ryan P, Rodriguez D, Kuczmarski JM, Fluckey JD, Talcott S. Effect of combined fish oil & curcumin on murine skeletal muscle morphology and stress response proteins during mechanical unloading. Nutr Res. 2019 Jan 7.[DOI]