Proteomic investigation of human skeletal muscle before and after 70 days of head down bed rest with or without exercise and testosterone countermeasures

IntroductionLong-term head-down bed rest (HDBR) results in musculoskeletal losses similar to those observed during long-term space flight. Agents such as testosterone, in addition to regular exercise, are effective countermeasures for reducing loss of skeletal muscle mass and function.ObjectiveWe investigated the skeletal muscle proteome of healthy men in response to long term HDBR alone (CON) and to HDBR with exercise (PEX) or exercise plus testosterone (TEX) countermeasures.MethodBiopsies were performed on the vastus lateralis before (pre) HDBR and on HDBR days 32 (mid) and 64 (post). Extracted proteins from these skeletal muscle biopsies were subjected to 2-dimensional gel electrophoresis (2DE), stained for phosphoproteins (Pro-Q Diamond dye) and total proteins (Sypro Ruby dye). Proteins showing significant fold differences (t-test p ≤ 0.05) in abundance or phosphorylation state at mid or post were identified by mass spectroscopy (MS).ResultsFrom a total of 932 protein spots, 130 spots were identified as potentially altered in terms of total protein or phosphoprotein levels due to HDBR and/or countermeasures, and 59 unique molecules emerged from MS analysis. Top canonical pathways identified through IPA included calcium signaling, actin cytoskeleton signaling, integrin linked kinase (ILK) signaling, and epithelial adherens junction signaling. Data from the pre-HDBR proteome supported the potential for predicting physiological post-HDBR responses such as the individual’s potential for loss vs. maintenance of muscle mass and strength.ConclusionsHDBR resulted in alterations to skeletal muscle abundances and phosphorylation of several structural and metabolic proteins. Inclusion of exercise alone or in combination with testosterone treatment modulated the proteomic responses towards cellular reorganization and hypertrophy, respectively. Finally, the baseline proteome may aid in the development of personalized countermeasures to mitigate health risks in astronauts as related to loss of muscle mass and function.引言：长期头低位卧床（head-down bed rest, HDBR）可诱发与长期太空飞行相似的肌肉骨骼系统退行性流失。除常规运动外，睾酮等干预措施可有效缓解骨骼肌质量与功能的丢失。 研究目的：本研究旨在探究健康男性分别接受单纯长期头低位卧床（CON）、卧床联合运动（PEX）以及卧床联合运动与睾酮（TEX）干预时的骨骼肌蛋白质组响应特征。 研究方法：分别于头低位卧床前（pre）、卧床第32天（中期，mid）及第64天（后期，post）采集受试者股外侧肌活检样本。提取上述样本中的蛋白质，进行二维凝胶电泳（2-dimensional gel electrophoresis, 2DE），随后分别采用磷酸化蛋白染色剂（Pro-Q Diamond染料）与总蛋白染色剂（Sypro Ruby染料）进行染色。针对中期或后期样本中丰度或磷酸化水平存在显著倍数变化（t检验p≤0.05）的蛋白质，采用质谱（mass spectroscopy, MS）进行鉴定。 研究结果：本次实验共检出932个蛋白斑点，其中130个斑点的总蛋白或磷酸化蛋白水平因头低位卧床及/或干预措施发生潜在改变，经质谱分析最终鉴定出59种独特蛋白质分子。通过IPA分析鉴定得到的核心经典通路包括钙信号通路、肌动蛋白细胞骨架信号通路、整合素连接激酶（integrin linked kinase, ILK）信号通路及上皮黏着连接信号通路。头低位卧床前的蛋白质组数据表明，其可用于预测个体卧床后的生理响应，例如预测个体肌肉质量与力量的流失或维持潜能。 研究结论：头低位卧床可导致多种结构蛋白与代谢蛋白的骨骼肌丰度及磷酸化水平发生改变。单独采用运动干预或运动联合睾酮治疗，可分别将蛋白质组响应调控至细胞重组织与肌肉肥大方向。基线蛋白质组可助力开发个性化对抗措施，以减轻宇航员因肌肉质量与功能流失所面临的健康风险。