Aim 1: Examining GSK3 activation in muscle and bone samples collected from male C57BL/6 mice from the RR9 mission
Aim 2: Examining GSK3 activation in muscle samples collected from female C57BL/6 from the RR1 mission
Aim 2 Experimental groups: RR1 female C57BL/6 (3-4 month old) mice were flown onboard the SpaceX4 Dragon capsule to the International Space Station. The flight group (FLT) mice were in orbit for 35 days. A Vivarium control and Ground control were housed on earth at the same time. Similar to Aim 1, we estimate a minimum of number of 4-10 samples per group across all measures in muscle. According to the Life Sciences Data Archive the left and right soleus are both available and stored in RNA later. For the purposes of our experiments we are only requesting right soleus muscles from each experimental group (n = 9-10 per group).
GSK3 activation will be assessed via an enzyme-linked GSK3 activity assay developed in our lab, as well as Western blotting for serine phosphorylation. Increased serine phosphorylation of GSK3 inactivates the kinase and thus an reduction in serine phosphorylation may be indicative of enhanced GSK3 activity. These assays will be completed on muscle (soleus and tibialis anterior) and bone (femur) samples.
Measures of muscle oxidative phenotype will be assessed via Western blotting for proteins that are abundant in oxidative fiber types including: myosin heavy chain I, PGC-1alpha, and other mitochondrial markers. Western blotting will also be conducted to examine muscle proteuloysis, specifically the ubiquitin proteasomal degradation pathway with total ubiquitinylation and the expression of E3 ligases MuRF1 and Atrogin-1. These assays will be completed on muscle (soleus and tibialis anterior) samples.
We will examine bone structure of right tibia's of male mice from the RR9 mission using µCT. Trabecular structural measurements assessed using 3D analyses will include: percent bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp). Cortical structural measurements using 2D analyses will include: cortical area fraction (Ct.Ar/Tt.Ar), cortical thickness (Ct.Th), periosteal perimeter (Ps.Pm), endocortical perimeter (Ec.Pm), and medullary area (Ma.Ar).
This experiment is currently in progress. Results will be available at the conclusion of the study.
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