Aim 1. To determine the time course, composition, and source of increased oxidant activity in unloaded muscle. Experiments will use mice conditioned by hindlimb unloading for up to two weeks. The antigravity muscle soleus will be studied to define the time course of oxidant dysregulation after unloading, to determine the relative contributions of reactive oxygen species vs. nitric oxide derivatives, and to test mitochondria as the primary source of increased oxidant activity.

Aim 2. To evaluate selected antioxidants as countermeasures for weakness in unloaded muscle. In these experiments, mice conditioned by hindlimb unloading will be treated with one of four interventions that oppose oxidant activity or oxidant-mediated signaling: allopurinol (xanthine oxidase inhibitor), curcumin (NF-kappaB inhibitor), Bowman-Birk inhibitor complex (protease inhibitor), or N-acetylcysteine (NAC; reduced thiol donor). Unloaded soleus will be tested for protection against oxidative stress, contractile dysfunction, and muscle atrophy.

Aim 3. To test NAC as a countermeasure for handgrip fatigue in humans. Experiments in healthy volunteers will define the appropriate preparation (solution vs. capsule) and dosage for oral NAC administration and will test NAC effects on handgrip strength and handgrip fatigue during concentric exercise.

Task 1. To determine the time course, composition, and source of increased oxidant activity in unloaded muscle. Experiments use mice conditioned by hindlimb unloading for up to three weeks. The antigravity muscle soleus was excised for functional and biochemical assays to assess changes in oxidant regulation. Experiments to address Task 1 are complete. Related studies of muscle gene expression during acute gravitational unloading were also completed this year. Homologous studies of gene expression in unloaded bone are in progress.

Task 2. To test selected antioxidants as possible countermeasures for weakness of unloaded muscle. Mice conditioned by hindlimb unloading were treated with compounds that oppose oxidant-mediated signaling via four distinct mechanisms: allopurinol, curcumin, Bowman-Birk inhibitor complex, or N-acetylcysteine (NAC). Soleus muscle atrophy and contractile function were monitored to assess countermeasure efficacy. Experiments to address Task 2 are complete.

Task 3. To evaluate NAC as a countermeasure for handgrip fatigue in humans. We previously showed that NAC inhibits human muscle fatigue. A final series of experiments in healthy volunteers have optimized the preparation and dosage of oral NAC administration for use in future countermeasure testing. A related study has identified the antioxidant OTC as a promising anti-fatigue agent and an alternative to NAC.

Experiments will address three specific aims:

Aim 1. To determine the time course, composition, and source of increased oxidant activity in unloaded muscle. Experiments will use mice conditioned by hindlimb unloading for up to two weeks. The antigravity muscle soleus will be studied to define the time course of oxidant dysregulation after unloading, to determine the relative contributions of reactive oxygen species vs. nitric oxide derivatives, and to test mitochondria as the primary source of increased oxidant activity.

Aim 2. To evaluate selected antioxidants as countermeasures for weakness in unloaded muscle. In these experiments, mice conditioned by hindlimb unloading will be treated with one of four interventions that oppose oxidant activity or oxidant-mediated signaling: allopurinol (xanthine oxidase inhibitor), curcumin (NF-kappaB inhibitor), Bowman-Birk inhibitor complex (protease inhibitor), or N-acetylcysteine (NAC; reduced thiol donor). Unloaded soleus will be tested for protection against oxidative stress, contractile dysfunction, and muscle atrophy.

Aim 3. To test NAC as a countermeasure for handgrip fatigue in humans. Experiments in healthy volunteers will define the appropriate preparation (solution vs. capsule) and dosage for oral NAC administration and will test NAC effects on handgrip strength and handgrip fatigue during concentric exercise.

Task 1. To determine the time course, composition, and source of increased oxidant activity in unloaded muscle. Experiments use mice conditioned by hindlimb unloading for up to three weeks. The antigravity muscle soleus was excised for functional and biochemical assays to assess changes in oxidant regulation. Experiments to address Task 1 are complete. Related studies of muscle gene expression during acute gravitational unloading were also completed this year. Homologous studies of gene expression in unloaded bone are in progress.

Task 2. To test selected antioxidants as possible countermeasures for weakness of unloaded muscle. Mice conditioned by hindlimb unloading were treated with compounds that oppose oxidant-mediated signaling via four distinct mechanisms: allopurinol, curcumin, Bowman-Birk inhibitor complex, or N-acetylcysteine (NAC). Soleus muscle atrophy and contractile function were monitored to assess countermeasure efficacy. Experiments to address Task 2 are complete. Collaborative plans to extend this work by testing additional nutritional compounds have stalled this year due to shifts in corporate priorities at Unilever.

Task 3. To evaluate NAC as a countermeasure for handgrip fatigue in humans. NAC has been shown to inhibit human muscle fatigue in a variety of published reports. Experiments in healthy volunteers will optimize the preparation and dosage of oral NAC administration and will test NAC effects on handgrip strength and handgrip fatigue during concentric exercise. Experiments to address Task 3 are in progress. A related study of the antioxidant OTC as an anti-fatigue agent is also in progress. Both studies will be completed in the coming year.

Experiments will address three specific aims:

Aim 1. To determine the time course, composition, and source of increased oxidant activity in unloaded muscle. Experiments will use mice conditioned by hindlimb unloading for up to two weeks. The antigravity muscle soleus will be studied to define the time course of oxidant dysregulation after unloading, to determine the relative contributions of reactive oxygen species vs. nitric oxide derivatives, and to test mitochondria as the primary source of increased oxidant activity.

Aim 2. To evaluate selected antioxidants as countermeasures for weakness in unloaded muscle. In these experiments, mice conditioned by hindlimb unloading will be treated with one of four interventions that oppose oxidant activity or oxidant-mediated signaling: allopurinol (xanthine oxidase inhibitor), curcumin (NF-kappaB inhibitor), Bowman-Birk inhibitor complex (protease inhibitor), or N-acetylcysteine (NAC; reduced thiol donor). Unloaded soleus will be tested for protection against oxidative stress, contractile dysfunction, and muscle atrophy.

Aim 3. To test NAC as a countermeasure for handgrip fatigue in humans. Experiments in healthy volunteers will define the appropriate preparation (solution vs. capsule) and dosage for oral NAC administration and will test NAC effects on handgrip strength and handgrip fatigue during concentric exercise.

Task 1. Experiments originally proposed for Aim 1 are complete. We are extending this line of research by a new series of studies that test for changes in muscle and bone gene expression during gravitational unloading. DNA microarray techniques are being used to screen for genes that regulate muscle and bone growth, catabolism, and redox homeostasis. We seek novel markers of tissue adaptation and new targets for therapeutic intervention.

Task 2. Our most recent study tested Bowman-Birk inhibitor complex (BBIC), a soybean-derived nutritional supplement, for effects on unloaded muscle. Soleus muscles of mice fed BBIC exhibited lower intracellular oxidant activity, less atrophy, and a smaller decrement in specific force. These data confirm previous findings by other investigators that BBIC protects unloaded muscle in mice and provide the first direct evidence that BBIC has antioxidant properties. In combination, the available data identify BBIC as an attractive compound for future tests in humans, i.e., during bedrest. Experiments proposed for original Aim 2 are now complete. To extend this work, we have begun collaborations with Unilever, a major international corporation in the field of food products and nutrition. We are working with scientists at Unilever to test novel nutritional interventions that may preserve muscle and bone during unloading.

Task 3. Our progress on Aim 3 has been hampered by the unexpected departures of two key personnel. Our study coordinator and medical advisor each left Kentucky for positions outside the state. We have subsequently recruited outstanding replacements. Dr. Leonardo Ferreira, a postdoctoral fellow trained in redox biology and muscle metabolism, is the new study coordinator. Our new medical advisor is Dr. Leigh Ann Callahan, a board certified subspecialist in pulmonary and critical care medicine. Both individuals have completed human studies training and are approved by the IRB to participate in our protocol. Dr. Ferreira is fully trained in use of the ergometer system and data collection software. He is now recruiting and training subjects to participate in NAC studies. Despite these considerable delays, we retain our original goal of completing all three study arms during the final year of the project.

1. To determine the time course, composition, and source of increased oxidant activity in unloaded muscle. Our experiments use mice conditioned by hindlimb unloading for up to three weeks. The antigravity muscle soleus is tested using functional and biochemical assays to determine any changes in free radical regulation by the muscle. Data from the previous year show that soleus muscles respond to hindlimb unloading by increasing the activity of NF-kappaB, a signaling molecule that promotes muscle atrophy. Also, soleus muscle mass decreases 43% after 10 days of unloading. This atrophy is biphasic, occurring rapidly over the first 3 days and then more slowly. Free radical activity in the muscle shows the opposite response, increasing during the first three days and then leveling off. These data suggest free radicals stimulate wasting of unloaded muscles.

2. To test selected antioxidants as possible countermeasures for weakness of unloaded muscle. Mice conditioned by hindlimb unloading are being treated with compounds that inhibit free radical activity. These include curcumin, N-acetylcysteine (NAC), allopurinol, and Bowman-Birk inhibitor concentrate (BBIC). Soleus muscle atrophy and contractile function are measured to determine the effectiveness of these possible countermeasures. We have found that antioxidant countermeasures vary in their effectiveness. Dietary supplementation with curcumin or NAC caused biochemical changes in the muscles that appeared promising. But the overall effects of these two compounds was unremarkable. Neither inhibited muscle atrophy or improved muscle function during unloading. In contrast, oral administration of allopurinol was protective. This compound lessened the weakness caused by muscle unloading despite the fact that muscle atrophy still occurred.

3. To evaluate NAC as a countermeasure for handgrip fatigue in humans. NAC has been shown to inhibit human muscle fatigue in several recent studies. Experiments in healthy volunteers are optimizing the preparation and dosage of oral NAC administration used to delay muscle fatigue during handgrip exercise. In the past year, we recruited a study coordinator and created a specially-designed device (‘ergometer’) to measure handgrip fatigue. The new ergometer overcomes several limitations of an earlier machine used in our previous research studies. We hope the new ergometer will provide more reliable measurements. We are now conducting dose-response studies of NAC capsules administered orally. To date we have screened 11 volunteers. Eight were unable to perform our fatiguing exercise protocol reproducibily and have been excused from the study. Three individuals have successfully completed the entire protocol and their blood samples are currently being analyzed to determine NAC effects.

Aim 1. To determine the time course, composition, and source of increased oxidant activity in unloaded muscle. Experiments will use mice conditioned by hindlimb unloading for up to two weeks. The antigravity muscle soleus will be studied to define the time course of oxidant dysregulation after unloading, to determine the relative contributions of reactive oxygen species vs. nitric oxide derivatives, and to test mitochondria as the primary source of increased oxidant activity.

Aim 2. To evaluate selected antioxidants as countermeasures for weakness in unloaded muscle. In these experiments, mice conditioned by hindlimb unloading will be treated with one of four interventions that oppose oxidant activity or oxidant-mediated signaling: allopurinol (xanthine oxidase inhibitor), curcumin (NF-kappaB inhibitor), Bowman-Birk inhibitor complex (protease inhibitor), or N-acetylcysteine (NAC; reduced thiol donor). Unloaded soleus will be tested for protection against oxidative stress, contractile dysfunction, and muscle atrophy.

Aim 3. To test NAC as a countermeasure for handgrip fatigue in humans. Experiments in healthy volunteers will define the appropriate preparation (solution vs. capsule) and dosage for oral NAC administration and will test NAC effects on handgrip strength and handgrip fatigue during concentric exercise.

[Ed. note: FY2005 record created in December 2010 when discovered missing; needed for statistical purposes]