Prolonged spaceflight is associated with significant skeletal muscle atrophy and loss of force generating capacity. Consequentially, the risk of injury for astronauts is elevated during extra-vehicular activities as well as following reentry to Earth and movement across extraterrestrial terrain. Recent evidence suggests that increased oxidative stress is involved in muscle wasting during mechanical unloading in a microgravity environment. In addition, reloading following an unloading perturbation results in oxidative-induced protein degradation and subsequent skeletal muscle injury. Therefore, for this project, we intend to use animal models to test the therapeutic effectiveness of novel, targeted countermeasures to prevent these detrimental effects of oxidative stress and improve physical performance and health of astronauts.
Overview
Developing Novel,Targeted Countermeasures to Reduce Oxidative Stress and Skeletal Muscle Atrophy During Microgravity (First Award Fellowship)
Principal Investigator:
James M. Kuczmarski, Ph.D.
Organization:
Texas A&M University
Technical Summary
Skeletal muscle is a highly specialized tissue that adapts rapidly to changes in mechanical loading by altering its cross-sectional area and mass. The reduced mechanical loading of spaceflight causes postural muscles in the lower extremities and flexors in the upper extremities to lose fiber cross-sectional area and force-generating capacity. Unloading of skeletal muscle also increases susceptibility to damage upon reloading with reentry to Earth and movement across extraterrestrial terrain. Consequentially, skeletal muscle atrophy and impaired contractile function are critical limitations to astronaut physical performance during spaceflight missions and increase their susceptibility to injury. It is therefore imperative to develop novel treatment strategies to prevent these detrimental muscular alterations with spaceflight.
Skeletal muscle atrophy and weakness experienced during microgravity, and accepted ground models of weightlessness, are a function of increased contractile protein degradation coupled with decreased protein synthesis. Recent studies from our lab and others indicate that oxidative stress plays a key role in this process. Oxidative-related effects are elicited directly as well as by inducing translocation of the mu-splice variant of nitric oxide synthase-I away from the sarcolemma and subsequent stimulation of proteolysis. Despite these established effects of oxidative stress, studies utilizing antioxidant supplementation have yielded mixed results, largely through their lack of specificity of the mechanisms leading to oxidative stress. In this study, we plan to develop novel and more targeted countermeasures to reduce oxidative stress and associated skeletal muscle effects of microgravity. This includes utilizing different agents that either: (1) elevate chaperone-proteins, such as heat shock protein-70 (HSP70), and limit oxidative-induced protein degradation; or (2) inhibit upstream agonists of reactive oxygen species production, including the renin-angiotensin system (RAS). In doing so, we will elicit pharmacological interventions in a hind limb unloaded (HLU) rodent model of microgravity. Our global study hypothesis is that oxidative stress is an underlying mechanism of skeletal muscle atrophy and dysfunction with spaceflight, and as such, targeted countermeasures of oxidative stress will effectively limit muscle wasting and improve function.
Objectives:
1. Demonstrate skeletal muscle atrophy and dysfunction with simulated microgravity.
2. Further characterize the pro-oxidative milieu in skeletal muscle as well as delineate potential pathways involving oxidative stress that are responsible for atrophy and dysfunction during unloading and disuse.
3. Implement countermeasures that target HSP70 as well as RAS in order to prevent the effects of oxidative stress.
4. Determine whether the targeted countermeasures of oxidative stress effectively limit muscle wasting and improve function.
The effects of HLU on skeletal muscle with and without pharmacological interventions will be determined with:
• Western blotting, following differential centrifugation, to measure protein expression in various subcellular fractions. This will include proteins related to oxidative stress, nitric oxide signaling, and proteolysis.
• Immunohistochemistry for fiber type analysis, assessment of various oxidative stress markers (4-hydroxynonenal), and protein expression/localization determination.
• Immunofluorescence on fresh and/or frozen hindlimb muscle samples for characterization of the oxidative environment and apoptosis using a luminometer and/or fluorescent microscopy.
• ELISA assays for measurement of apoptosis and NF-kappa B activity.
• An isolated muscle fiber bundle preparation to assess contractile function.
During the first year of this project, we plan to assess the efficacy of countermeasures that raise HSP70 against the effects of oxidative stress and unloading-induced muscle atrophy. In the second year, we will move forward to identify the contribution of RAS to oxidative stress as well as muscle atrophy and inhibit its effects through an angiotensin II receptor blocker.
Earth Applications
The findings of our study will not only have implications for space life science research but also translate to benefit global human health on Earth. Many of the mechanisms responsible for muscle wasting with spaceflight parallel those of various muscular disorders on Earth including muscular dystrophy, age-related sarcopenia, as well as disuse associated with a sedentary lifestyle, spinal denervation, or chronic immobility. Therefore, if these countermeasures are effective in this model of microgravity, future investigation could focus on their efficacy in other muscular diseases. This could ultimately lead to therapeutic strategies that improve the quality of terrestrial life.