The overall objective of this research is to test the ability of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) to inhibit skeletal muscle atrophy, weakness and changes in gene expression due to simulated weightlessness in rats using the hindlimb-suspension model.

The specific aims for this proposal are to:

1. Determine if high-dose aspirin treatment inhibits soleus and plantaris fiber atrophy, deficits in maximum force and the activation of NF-kappa B seen after seven days of hindlimb unloading. The inhibition of cyclooxygenase activity by NSAID treatment will also be assessed in muscles;
2. Determine if curcumin or naproxen treatment inhibits soleus and plantaris fiber atrophy, deficits in maximum force and NF-kappa B or cyclooxygenase activity due to seven days of unloading, and;
3. Determine if the expression of genes known to be upregulated with seven days of unloading are normalized by treatment with any of the NSAIDs.

The ineffectiveness of curcumin in vivo may be due to the low bioavailability. To determine if aspirin inhibits NF-kappa B activity, C2C12 mouse myotubes were treated with ten ngrams/ml TNF-alpha for three hours either in the presence or absence of 20mM aspirin. TNF-alpha caused an 11-fold increase in NF-kappa B activity and aspirin abolished this increase. In unloaded animals, injection of aspirin did not attenuate the increase in NF-kappa B activity, but it did attenuate the unloading-induced muscle atrophy. Therefore, this dose of aspirin may be working by the inhibition of other molecules, which we have yet to test.

Although the NSAIDs outlined above have pleiotropic effects, both aspirin and curcumin inhibit NF-kappa B activity by inhibiting I kappa B kinase. Therefore, to gain further insight into other components of the NF-kappa B pathway that are necessary for unloading-induced atrophy, a fourth and fifth aim were added using genetic approaches to focus on the I kappa B alpha protein (which is downstream of I kappa B kinase) and the c-rel gene. By determining the involvement of particular proteins and genes in the atrophy process, specific pharmacological countermeasures may be tested in subsequent studies.

Aim 4: To determine if I kappa B alpha is required for unloading-induced NF-kappa B activity, muscle atrophy and upregulation of atrophy-related genes. For this aim, an I kappa B alpha-dominant negative plasmid (known as a superrepressor) was injected into the soleus muscle of rats. This superrepressor is resistant to phosphorylation by I kappa B kinase and therefore resistant to ubiquitination and subsequent degradation. NF-kappa B activity was increased five-fold with hindlimb unloading, but completely abolished in the unloaded muscles injected with the superrepressor. Furthermore, soleus muscle fiber cross-sectional area was decreased by 40 percent following seven days of unloading, but was attenuated by 40 percent in the unloaded muscles injected with the superrepressor. In addition, the increase in gene expression of atrogin-1/MAFbx, Cathepsin L, Nedd4, IEX-1, 4E-BP1 and FOXO3a with unloading was significantly attenuated in the unloaded muscles injected with the superrepressor.

Aim 5: To determine if c-rel is required for unloading-induced NF-kappa B activity and muscle atrophy. For this aim, we unloaded wild type (WT) and c-rel knockout (-/-) mice. NF-kappa B activity was increased eight-fold with unloading in the soleus muscle of WT mice and remained equally elevated with unloading in the c-rel -/- mice. Moreover, muscle atrophy was the same in WT unloaded and c-rel -/- unloaded. This eliminates c-rel as a contributor to the atrophy process, and therefore, as a therapeutic target.

Ongoing experiments will test whether naproxen and possibly celecoxib inhibit skeletal muscle atrophy, deficits in maximum force and NF-kappa B or cyclooxygenase activity due to seven days of unloading.

Earth-based Applications of Research Project
By identifying specific proteins and genes involved in skeletal muscle atrophy and testing nutritional and/or pharmacological countermeasures to them, we will be in a much better position to combat the deleterious changes in muscle function due to any type of disuse.

Project Description
NASA Task Book Entry