Research Institute for Sport & Exercise Sciences, Liverpool John Moores University, Liverpool, UK.
J Cachexia Sarcopenia Muscle. 2022 Oct;13(5):2562-2575. doi: 10.1002/jcsm.13034. Epub 2022 Jul 12.
Cell assays are important for investigating the mechanisms of ageing, including losses in protein homeostasis and 'proteostasis collapse'. We used novel isotopic labelling and proteomic methods to investigate protein turnover in replicatively aged (>140 population doublings) murine C2C12 myoblasts that exhibit impaired differentiation and serve as a model for age-related declines in muscle homeostasis.
The Absolute Dynamic Profiling Technique for Proteomics (Proteo-ADPT) was used to investigate proteostasis in young (passage 6-10) and replicatively aged (passage 48-50) C2C12 myoblast cultures supplemented with deuterium oxide (D O) during early (0-24 h) or late (72-96 h) periods of differentiation. Peptide mass spectrometry was used to quantify the absolute rates of abundance change, synthesis and degradation of individual proteins.
Young cells exhibited a consistent ~25% rise in protein accretion over the 96-h experimental period. In aged cells, protein accretion increased by 32% (P < 0.05) during early differentiation, but then fell back to baseline levels by 96-h. Proteo-ADPT encompassed 116 proteins and 74 proteins exhibited significantly (P < 0.05, FDR < 5% interaction between age × differentiation stage) different changes in abundance between young and aged cells at early and later periods of differentiation, including proteins associated with translation, glycolysis, cell-cell adhesion, ribosomal biogenesis, and the regulation of cell shape. During early differentiation, heat shock and ribosomal protein abundances increased in aged cells due to suppressed degradation rather than heightened synthesis. For instance, HS90A increased at a rate of 10.62 ± 1.60 ng/well/h in aged which was significantly greater than the rate of accretion (1.86 ± 0.49 ng/well/h) in young cells. HS90A synthesis was similar in young (21.23 ± 3.40 ng/well/h) and aged (23.69 ± 1.13 ng/well/h), but HS90A degradation was significantly (P = 0.05) greater in young (19.37 ± 2.93 ng/well/h) versus aged (13.06 ± 0.76 ng/well/h) cells. During later differentiation the HS90A degradation (8.94 ± 0.38 ng/well/h) and synthesis (7.89 ± 1.28 ng/well/h) declined and were significantly less than the positive net balance between synthesis and degradation (synthesis = 28.14 ± 3.70 ng/well/h vs. degradation = 21.49 ± 3.13 ng/well/h) in young cells.
Our results suggest a loss of proteome quality as a precursor to the lack of fusion of aged myoblasts. The quality of key chaperone proteins, including HS90A, HS90B and HSP7C was reduced in aged cells and may account for the disruption to cell signalling required for the later stages of differentiation and fusion.
细胞分析对于研究衰老机制非常重要，包括蛋白质内稳态的丧失和“蛋白质稳态崩溃”。我们使用新的同位素标记和蛋白质组学方法来研究复制性衰老（超过 140 个群体倍增）的鼠 C2C12 成肌细胞中的蛋白质周转，这些细胞表现出分化受损，并可作为与年龄相关的肌肉内稳态下降的模型。
使用绝对动态蛋白质组学分析技术（Proteo-ADPT）来研究早期（0-24 小时）或晚期（72-96 小时）分化时用重水（D O）补充的年轻（第 6-10 代）和复制性衰老（第 48-50 代）C2C12 成肌细胞培养物中的蛋白质稳态。肽质谱用于定量个体蛋白质丰度变化、合成和降解的绝对速率。
年轻细胞在 96 小时的实验期间表现出一致的约 25%的蛋白质积累增加。在衰老细胞中，早期分化时蛋白质积累增加了 32%（P < 0.05），但到 96 小时时又回落到基线水平。Proteo-ADPT 涵盖了 116 种蛋白质，74 种蛋白质在年轻和衰老细胞的早期和晚期分化阶段之间表现出明显（P < 0.05，FDR < 5%，年龄×分化阶段之间的相互作用）的丰度变化，包括与翻译、糖酵解、细胞-细胞粘附、核糖体生物发生和细胞形状调节相关的蛋白质。在早期分化过程中，由于降解受到抑制而不是合成增加，热休克和核糖体蛋白的丰度在衰老细胞中增加。例如，HS90A 在衰老细胞中的增加速度为 10.62±1.60ng/孔/小时，明显高于年轻细胞中的积累速度（1.86±0.49ng/孔/小时）。HS90A 的合成在年轻（21.23±3.40ng/孔/小时）和衰老（23.69±1.13ng/孔/小时）细胞中相似，但 HS90A 的降解在年轻（19.37±2.93ng/孔/小时）细胞中明显大于衰老（13.06±0.76ng/孔/小时）细胞（P = 0.05）。在后期分化过程中，HS90A 的降解（8.94±0.38ng/孔/小时）和合成（7.89±1.28ng/孔/小时）下降，并且明显小于年轻细胞中合成和降解之间的正净平衡（合成=28.14±3.70ng/孔/小时与降解=21.49±3.13ng/孔/小时）。
我们的结果表明，随着衰老成肌细胞融合的缺失，蛋白质组质量的丧失可能是其前兆。关键伴侣蛋白（包括 HS90A、HS90B 和 HSP7C）的质量在衰老细胞中降低，这可能导致分化和融合后期所需的细胞信号转导中断。
