Brief ambulatory reloading elevates muscle protein synthesis but does not prevent disuse atrophy in hindlimb-unloaded rats

Disuse muscle atrophy remains a major challenge in contexts such as prolonged bed rest or microgravity. Here, we investigated whether brief bouts of ambulatory reloading could attenuate skeletal muscle atrophy caused by five days of hindlimb unloading (HU) in rats. Using a deuterium oxide tracer, we measured integrative protein synthesis (fractional synthesis rate, FSR) in the soleus, plantaris, and gastrocnemius muscles, including distinct portions of the muscle that are composed mostly of red, white, and mixed fibers. HU significantly reduced both muscle mass and FSR in the predominantly slow-twitch soleus and in the predominantly fast gastrocnemius. Intermittent ambulatory reloading (HU + AR) partially restored FSR in the soleus and gastrocnemius but did not recover soleus or gastrocnemius mass to control levels. The plantaris muscle showed no differences in mass or FSR among groups, suggesting muscle-specific responses to unloading and reloading. Fiber-type analyses revealed that portions of the gastrocnemius that are mostly red fibers had higher baseline FSR than mixed or white portions, while HU consistently depressed protein synthesis across all fiber types. In conclusion, although intermittent ambulation increased protein synthesis during HU, it was not sufficient to prevent overall muscle mass loss. These findings emphasize the importance of both the duration and intensity of loading in preserving skeletal muscle during periods of disuse.