Muscle Unloading and Immobilization Exhibit Different Type-Specificity in Myofiber Atrophy and Neuromuscular Junction Degeneration

Introduction: Skeletal muscle fibers are generally classified as fast- and slow-twitch fibers, each innervated by distinct motor neurons at the neuromuscular junction (NMJ). Muscle atrophy progresses in a type-specific manner during disuse, which may be associated with NMJ degeneration. Muscle disuse involves a reduction in both mechanical load and neural input onto myofibers, but their respective influences on the NMJ and myofibers are poorly understood. In this study, we aimed to elucidate the association between NMJ degeneration and myofiber atrophy by comparing two different disuse models in mice. Methods: Male C57BL/6J mice (12-13 weeks old) were divided into untreated control (CON), tail suspension (TS), and cast immobilization (IM) groups. TS mice were suspended by their tails to reduce the mechanical load on the hindlimb muscles but were allowed free movement. IM mice were suspended with their hindlimbs immobilized in casts to restrict neuromuscular activity. After 20 days of intervention, the neuromuscular transmission index (the ratio of the 10th to 1st compound muscle action potential amplitude during repetitive sciatic nerve stimulation), the wet weights of lower hindlimb muscles, myofiber size, NMJ morphology, and the expression of proteins and mRNAs related to protein metabolism and NMJ formation were analyzed. Additionally, adeno-associated virus-mediated overexpression of Dok7 and Neurotrophin-4 (NT4), proteins associated with NMJ formation and maintenance, was performed to determine their roles in preventing NMJ degeneration and myofiber atrophy. Results: The wet weight of the gastrocnemius muscle was significantly higher in TS than in IM, whereas that of the soleus was significantly higher in IM than in TS. Type I and IIa fibers in the soleus were significantly smaller in TS than in IM, while type IIb fibers in the plantaris were significantly smaller in IM than in TS. The neuromuscular transmission index was significantly lower in IM than in TS. TS had a significantly lower acetylcholine receptor (AChR) density in type I than in type II fibers in the soleus, whereas IM exhibited a significantly smaller presynaptic nerve terminal area in type II than in type I fibers. The phosphorylation level of RPS6 was significantly lower in TS than in IM, while mRNA expression of atrogin-1 and MuRF-1 was significantly lower in IM than in TS. The protein expression of Dok7, which is involved in AChR clustering, was significantly lower in TS than in IM in the soleus, whereas mRNA expression of NT4 was significantly lower in IM than in TS in the plantaris. Dok7 overexpression had no significant effect on myofiber size but prevented the impairment of neuromuscular transmission and reduction in AChR density in TS. NT4 overexpression prevented the atrophy of type IIb fibers in the plantaris and the degeneration of presynaptic nerve terminals in IM. Conclusions: Our results suggest that reduced mechanical load on myofibers during TS causes degeneration of postsynaptic AChRs and myofiber atrophy in type I fibers. Dok7 overexpression prevented the AChRs degeneration but not myofiber atrophy, which implies that AChRs degeneration does not directly lead to muscle atrophy in this model. Our results also suggest that reduced neural activity during IM causes degeneration of presynaptic nerve terminals and myofiber atrophy in type II fibers. As NT4 overexpression prevented both nerve terminal degeneration and myofiber atrophy, NT4 is a potential key factor in preventing NMJ degeneration and muscle atrophy. Taken together, we found evidence that muscle atrophy induced by different disuse models exhibits different fiber type-specific characteristics, in which neuromuscular interactions are deeply involved. This study was supported by Japan Society for the Promotion of Science and by Yamaha Motor Foundation for Sports. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.