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 FLBER 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 FOX03A 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 DG^ 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 OFTHE LIMB-GIRDLE MUSCIDAR DYSTROPHIES, PATHOLOGIES INVOLVING MUTATIONS TO DGC AND OTHER SARCOLEMMAL PROTEINS. BECAUSE MDS ANTL MICROGRAVITY BOTH INCREASES SUSCEPTIBILITY OF MUSCLES TO DAMAGE, OXIDATIVE STRESS, AND PRO-INFLAMMATORY SIGNALING, A SHARED ROOT CAUSE IS SUSPECTED: DISRUPTION OFTHE 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 STIMIDATING 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 FLBER-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. SPECIFLC AIM 1 WILL IDENTIFY NOX-DEPENDENT SIGNALING ON NNOS TRANSLOCATION AND MUSCLE FLBER ATROPHY WITH HINDLIMB UNLOADING. SPECIFLC 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. 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) REGULATOIY MECHANISMS THAT GOVERN ALTERATIONS IN SKELETAL MUSCLE, ALL PROGRAMMATIC EMPHASES AND GOALS OFTHE CURRENT SPACE BIOLOGY NRA.