Roberson Paul A, Shimkus Kevin L, Welles Jaclyn E, Xu Dandan, Whitsell Abigale L, Kimball Eric M, Jefferson Leonard S, Kimball Scot R
Pennsylvania State University, Department of Cellular and Molecular Physiology, College of Medicine, Hershey, Pennsylvania.
J Appl Physiol (1985). 2020 Jul 1;129(1):36-46. doi: 10.1152/japplphysiol.00155.2020. Epub 2020 May 14.
Skeletal muscle atrophy is associated with disease, aging, and disuse. Hindlimb unloading (HU) in animals provides an experimental model to study muscle atrophy. A comprehensive time course for how HU affects biomarkers of protein synthesis and degradation acutely and chronically and the associated resistance to an anabolic stimulus following disuse remain undocumented. Sixteen-week-old C57BL/6 mice underwent 0, 1, 12, 24, 72, 168, or 336 h of HU. Following 336 h of HU, mice were reloaded for 1, 24, or 72 h. Another group of mice underwent 120 h of HU, were fasted or refed, and were then compared with similar condition control animals (CTL). Protein content and phosphorylation of biomarkers of protein synthesis, degradation, and autophagy were assessed in the soleus muscle. Gastrocnemius, soleus, and plantaris muscles atrophied within 120 h of HU. Protein synthesis trended toward decrease following 24 h of HU. p70S6K phosphorylation and protein synthesis increased with reloading. Following HU, changes in MAFbx and DEPTOR expression and DEPTOR phosphorylation were consistent with development of a catabolic state. DEPTOR expression recovered following reloading. Animals that underwent 120 h of HU exhibited attenuation of refeeding-induced p70S6K phosphorylation compared with CTL counterparts. Following 120 h of HU, protein synthesis, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation, and DEPTOR, MAFbx, and Sestrin1 expression indicated a catabolic state. Following 120 h of HU, autophagy markers, including p62 expression, REDD1 expression, LC3 ratio, and Unc-51-like autophagy-activating kinase 1 (ULK1) phosphorylation, indicated impaired autophagy. HU promotes a deleterious balance between protein synthesis and degradation. The time course herein provides scientists information about when the associated biomarkers become affected. Hindlimb unloading causes significant skeletal muscle atrophy by adversely affecting the balance between protein synthesis and breakdown. This study demonstrates a more complete time course for changes in biomarkers associated with protein synthesis and breakdown and investigates the associated anabolic resistance to an anabolic stimulus following hindlimb unloading. These data in concert with information from other studies provide a basis for designing future experiments to optimally interrogate a desired cellular biomarker or pathway.
骨骼肌萎缩与疾病、衰老和废用有关。动物后肢卸载（HU）提供了一个研究肌肉萎缩的实验模型。关于HU如何急性和慢性影响蛋白质合成与降解的生物标志物，以及废用后对合成代谢刺激的相关抵抗，目前尚无全面的时间进程记录。16周龄的C57BL/6小鼠接受0、1、12、24、72、168或336小时的HU处理。在336小时的HU处理后，小鼠重新加载1、24或72小时。另一组小鼠接受120小时的HU处理，禁食或重新喂食，然后与相似条件的对照动物（CTL）进行比较。评估比目鱼肌中蛋白质合成、降解和自噬生物标志物的蛋白质含量和磷酸化情况。腓肠肌、比目鱼肌和跖肌在HU处理120小时内发生萎缩。HU处理24小时后蛋白质合成呈下降趋势。重新加载后p70S6K磷酸化和蛋白质合成增加。HU处理后，MAFbx和DEPTOR表达及DEPTOR磷酸化的变化与分解代谢状态的发展一致。重新加载后DEPTOR表达恢复。与CTL组相比，接受120小时HU处理的动物在重新喂食诱导的p70S6K磷酸化方面表现出减弱。在120小时的HU处理后，蛋白质合成、真核翻译起始因子4E结合蛋白1（4E-BP1）磷酸化以及DEPTOR、MAFbx和Sestrin1表达表明处于分解代谢状态。在120小时的HU处理后，自噬标志物，包括p62表达、REDD1表达、LC3比率和Unc-51样自噬激活激酶1（ULK1）磷酸化，表明自噬受损。HU促进了蛋白质合成与降解之间的有害平衡。本文的时间进程为科学家提供了相关生物标志物何时受到影响的信息。后肢卸载通过不利地影响蛋白质合成与分解之间的平衡导致显著的骨骼肌萎缩。本研究展示了与蛋白质合成和分解相关的生物标志物变化的更完整时间进程，并研究了后肢卸载后对合成代谢刺激的相关合成代谢抵抗。这些数据与其他研究的信息共同为设计未来实验以最佳地探究所需的细胞生物标志物或途径提供了基础。
