Fernández-Rhodes María, Buchan Emma, Gagnon Stephanie D, Qian Jiani, Gethings Lee, Lees Rebecca, Peacock Ben, Capel Andrew J, Martin Neil R W, Oppenheimer Pola Goldberg, Lewis Mark P, Davies Owen G
School of Sport Exercise and Health Sciences, Loughborough University Loughborough UK.
School of Chemical Engineering, Advanced Nanomaterials Structures and Applications Laboratories, College of Engineering and Physical Sciences University of Birmingham Birmingham UK.
J Extracell Biol. 2024 Aug 21;3(8):e171. doi: 10.1002/jex2.171. eCollection 2024 Aug.
Skeletal muscle (SM) acts as a secretory organ, capable of releasing myokines and extracellular vesicles (SM-EVs) that impact myogenesis and homeostasis. While age-related changes have been previously reported in murine SM-EVs, no study has comprehensively profiled SM-EV in human models. To this end, we provide the first comprehensive comparison of SM-EVs from young and old human primary skeletal muscle cells (HPMCs) to map changes associated with SM ageing. HPMCs, isolated from young (24 ± 1.7 years old) and older (69 ± 2.6 years old) participants, were immunomagnetically sorted based on the presence of the myogenic marker CD56 (N-CAM) and cultured as pure (100% CD56) or mixed populations (MP: 90% CD56). SM-EVs were isolated using an optimised protocol combining ultrafiltration and size exclusion chromatography (UF + SEC) and their biological content was extensively characterised using Raman spectroscopy (RS) and liquid chromatography mass spectrometry (LC-MS). Minimal variations in basic EV parameters (particle number, size, protein markers) were observed between young and old populations. However, biochemical fingerprinting by RS highlighted increased protein (amide I), lipid (phospholipids and phosphatidylcholine) and hypoxanthine signatures for older SM-EVs. Through LC-MS, we identified 84 shared proteins with functions principally related to cell homeostasis, muscle maintenance and transcriptional regulation. Significantly, SM-EVs from older participants were comparatively enriched in proteins involved in oxidative stress and DNA/RNA mutagenesis, such as E3 ubiquitin-protein ligase TTC3 (TTC3), little elongation complex subunit 1 (ICE1) and Acetyl-CoA carboxylase 1 (ACACA). These data suggest SM-EVs could provide an alternative pathway for homeostasis and detoxification during SM ageing.
骨骼肌（SM）作为一个分泌器官，能够释放影响肌生成和体内平衡的肌动蛋白和细胞外囊泡（SM-EVs）。虽然之前有报道称小鼠SM-EVs存在与年龄相关的变化，但尚无研究对人类模型中的SM-EV进行全面分析。为此，我们首次对来自年轻和老年人类原代骨骼肌细胞（HPMCs）的SM-EV进行了全面比较，以明确与SM衰老相关的变化。从年轻（24±1.7岁）和老年（69±2.6岁）参与者中分离出的HPMCs，根据肌生成标志物CD56（N-CAM）的存在情况进行免疫磁珠分选，并培养为纯（100% CD56）或混合群体（MP：90% CD56）。使用结合超滤和尺寸排阻色谱（UF + SEC）的优化方案分离SM-EV，并使用拉曼光谱（RS）和液相色谱质谱（LC-MS）对其生物学内容进行广泛表征。在年轻和老年群体之间，基本EV参数（颗粒数量、大小、蛋白质标志物）的变化极小。然而，RS的生化指纹图谱显示，老年SM-EV的蛋白质（酰胺I）、脂质（磷脂和磷脂酰胆碱）和次黄嘌呤特征增加。通过LC-MS，我们鉴定出84种共享蛋白质，其功能主要与细胞体内平衡、肌肉维持和转录调控有关。值得注意的是，老年参与者的SM-EV中相对富含参与氧化应激和DNA/RNA诱变的蛋白质，如E3泛素蛋白连接酶TTC3（TTC3）、小延伸复合体亚基1（ICE1）和乙酰辅酶A羧化酶1（ACACA）。这些数据表明，SM-EV可能为SM衰老过程中的体内平衡和解毒提供一条替代途径。
