Abstract

I. Background UNC45B, a myosin chaperone, affects myosin expression in C.elegans and has been reported to decrease with age in rats and humans. Therefore, it is thought that the decrease in UNC45B in the elderly may lead to lowering myosin level and the inability to regulate muscle quality, resulting in sarcopenia, an age-related loss of skeletal muscle mass. However, the mechanism by which UNC45B decreases with age remains unclear. In sarcopenia, atrophy occurs mainly in fast-twitch muscle fibers. Similarly, denervation leads to muscle atrophy mainly in fast-twitch muscle fibers, and therefore, it has been used as a model for sarcopenia. If UNC45B is reduced by muscle inactivity due to aging, it would be also reduced by inactivity due to denervation. Thus, we examined UNC45B expression in denervated muscle. II. Methods The right hindlimb muscles of 13-week-old male C57BL/6J mice were denervated by cutting and removing a 2 mm segment of the sciatic nerve, while the left hindlimb muscles served as sham-operated control. Two weeks after denervation, the gastrocnemius muscle was removed and weighed. Then the solution containing the homogenized muscle was centrifuged at 10000 g and the supernatant was used for Western blotting. Ⅲ. Results The muscle weight of the gastrocnemius muscle was decreased by about 50% and that myosin level was also significantly decreased by denervation (p<0.01), but the amount of UNC45B protein did not differ from that of the control leg. Although muscle protein synthesis measured by the SUnSET method was not changed by denervation, the amount of ubiquitinated protein was increased (p<0.001). Total and phosphorylation levels of Akt (an upstream regulator of the mechanistic target of rapamycin complex 1 (mTORC1) that facilitates protein synthesis), 4EBP1, and rpS6 (downstream targets of mTORC1) were increased by denervation (p<0.01). p62, LC3-I, and LC3-II, markers of autophagy, were increased in the denervated muscle (p62: p<0.05, LC3-I, LC3-II: p<0.01). Heat shock proteins that also act as chaperones for myosin, HSP90 and HSP73 were increased by denervation (HSP90: p<0.001, HSP73: p<0.05), while HSP70 was decreased (p<0.05). IV. Discussion Two weeks of denervation resulted in a significant gastrocnemius muscle weight decrease. However, no change had observed in UNC45B levels, indicating that UNC45B may not be involved in muscle atrophy caused by denervation, and the age-related decrease in UNC45B may also be not due to a decreased muscle activity. Denervation stimulated the protein degradation system while it did not affect muscle protein synthesis, suggesting that denervation-induced muscle atrophy may cause by enhancement of the degradation system. V. Conclusion In this study, there was no decrease in UNC45B after 2 weeks of denervation, suggesting that the decrease in UNC45B due to aging is not directly related to the decrease in muscle activity.

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