Differentiation of young and replicatively aged mouse C2C12 muscle cells

Age-related impairments in myoblast differentiation may contribute to reductions in muscle function in older adults but the underlying proteostasis processes are not well understood. We investigated young (P6-10) and replicatively aged (P48-50) C2C12 myoblast cultures during early (0h-24h) and late (72h-96h) stages of differentiation using deuterium oxide (D2O) labelling and mass spectrometry. The absolute dynamic profiling technique for proteomics (Proteo-ADPT) was used to quantify the absolute rates of abundance change, synthesis and degradation of individual proteins. Proteo-ADPT encompassed 116 proteins and 74 proteins exhibited significantly (P<0.05, FDR <5 %) different changes in abundance between young and aged cells at early and later periods of differentiation. Young cells exhibited a steady pattern of growth, protein accretion and fusion, whereas aged cells failed to gain protein mass or undergo fusion during later differentiation. Maturation of the proteome was retarded in aged myoblasts at the onset of differentiation, but the proteome appeared to ‘catch up’ with the young cells during the early differentiation period. However, this ‘catch up’ process in aged cells was not accomplished by higher levels of protein synthesis. Instead, a lower level of protein degradation in aged cells was responsible for the elevated gains in protein abundance. Our novel data point to a loss of proteome quality as a precursor to the lack of fusion of aged myoblasts and highlights dysregulation of protein degradation, particularly of ribosomal and chaperone proteins, as a key mechanism that may contribute to age-related declines in the capacity of myoblasts to undergo differentiation.

成肌细胞分化的年龄相关性损伤可能会导致老年人肌肉功能下降，但目前对其背后的蛋白稳态（proteostasis）机制尚不清楚。本研究针对分化早期（0h-24h）与晚期（72h-96h）阶段的年轻（P6-10）及复制性衰老（P48-50）C2C12成肌细胞培养物展开研究，采用氘代水（deuterium oxide, D₂O）标记与质谱技术进行分析。本研究使用蛋白组学绝对动态谱分析技术（Proteo-ADPT），对单个蛋白的丰度变化、合成与降解的绝对速率进行定量分析。通过Proteo-ADPT技术共鉴定到116种蛋白，其中74种蛋白在年轻与衰老细胞的分化早期及后期阶段的丰度变化存在显著差异（P<0.05，错误发现率（False Discovery Rate, FDR）<5%）。年轻组成肌细胞呈现出稳定的生长、蛋白积累与融合模式，而衰老组成肌细胞在分化后期无法实现蛋白质量积累或细胞融合。在分化起始阶段，衰老成肌细胞的蛋白质组（proteome）成熟过程受到阻滞，但在分化早期阶段，其蛋白质组似乎能够追赶至年轻细胞的水平。然而，衰老细胞的这种“追赶”过程并非通过提升蛋白合成水平实现，反而得益于其更低的蛋白降解速率，使得蛋白丰度得以显著升高。本研究的新颖数据表明，蛋白质组质量丧失是衰老成肌细胞无法发生融合的前兆，并凸显了蛋白降解调控异常——尤其是核糖体蛋白与分子伴侣蛋白的降解调控异常——作为可能导致成肌细胞分化能力随年龄下降的关键机制。