We have established the hindlimb suspension mouse model in our lab based on the literature. In a modified cage, the tail of the mouse was unloaded with a 30 degree angle between the torso and the floor of the cage. The mouse had easy access to the food and the water and freedom to move within the cage. The distance between the mice and the food was adjusted so that the mice could not step on the food with their hind legs. The mice adapted to the system within 2-3 days.
Using the suspension apparatus we have built, we tested the effect of NMN administration on muscle weight during hindlimb unloading. 6-month-old male C57BL/6J mice, which are equivalent to 20-30-year-old humans, were randomly allocated into six groups. Three groups were ambulatory (Ctrl), and three groups were hindlimb suspended (HS) for 14 days. Both Ctrl groups and HS groups were treated with vehicle (Ctrl-0 and HS-0) or NMN at 400 mg/kg/day (Ctrl-400 and HS-400) or 800 mg/kg/day (Ctrl-800 and HS-800) in drinking water 3 days before and during the two weeks of hindlimb suspension. We observed similar levels of muscle atrophy as previously published in hindlimb muscles of the HS-0 mice compared to the Ctrl-0 mice, including soleus, gastrocnemius, tibialis anterior (TA), extensor digitorum longus (EDL), and quadriceps, validating our animal model.
To quantify the effect of NMN administration on preventing unloading-induced muscle atrophy, we calculated the relative muscle weight compared to the control groups after hindlimb suspension. 400 mg/kg/day of NMN treatment reduced muscle weight loss in soleus, gastrocnemius, and EDL. However, due to a higher variation in the vehicle-treated group, the difference was not statistically significant in soleus and gastrocnemius. The reasons for the higher variation in the HS-0 group remain unclear. We will increase the n value in the future experiments to increase statistical power. 800 mg/kg/day of NMN treatment did not alleviate muscle atrophy except in quadriceps, suggesting that 800 mg/kg/day NMN is beyond the dose to confer beneficial effect, at least in the hindlimb suspension model.
Bone mineral loss is also a major health threat to the astronauts during spaceflight. To assess the effect of NMN administration on bone mineral density and structure, we dissected and fixed the tibias and femurs to analyze bone mineral density and morphology. The samples are currently under investigation using micro-CT and immunofluorescence staining.
Slow-to-fast fiber type transition significantly decreases fatigue resistance of the slow-twitch muscles, which impairs the ability of muscles to sustain continuous work. At the molecular level, we detected a significant downregulation of a slow-twitch fiber gene Myl2, and an upregulation of a fast-twitch fiber gene Myh1 in the soleus after hindlimb suspension. We are currently quantifying the expression of the slow- and fast-twitch fiber genes in the NMN-treated mice. Considering that SIRT1 deacetylates and activates PGC-1a, a governor of the formation of slow-twitch fibers, we expect that activating SIRT1 by NMN administration will alleviate slow-to-fast transition and may preserve the fatigue resistance of the slow-twitch muscles.