Alakhdar Amira A, Sivakumar Sruthi, Kopchak Rylee M, Hunter Allison N, Ambrosio Fabrisia, Washburn Newell R
Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, 15213, USA.
Retro Biosciences Inc., Redwood City, CA, USA.
Adv Biol (Weinh). 2024 Dec;8(12):e2400334. doi: 10.1002/adbi.202400334. Epub 2024 Nov 27.
The stiffening of the extracellular matrix (ECM) with age hinders muscle regeneration by causing intrinsic muscle stem cell (MuSC) dysfunction through a poorly understood mechanism. Here, the study aims to study those age-related molecular changes in the differentiation of MuSCs due to age and/or stiffness. Hence, young and aged MuSCs are seeded onto substrates engineered to mimic a soft and stiff ECM microenvironment to study those molecular changes using single-cell RNA sequencing (scRNA). The trajectory of scRNA data of the MuSCs under four different conditions undergoing differentiation is analyzed as well as the active molecular pathways and transcription factors driving those differentiation fates. Data revealed the presence of a branching point within the trajectory leading to the emergence of an age-related fibroblastic population characterized by activation of the TNF-related apoptosis-inducing ligand (TRAIL) pathway, which is significantly activated in aged cells cultured on stiff substrates. Next, using the collagen cross-linking inhibitor β-aminopropionitrile (BAPN) in vivo, the study elucidates stiffness changes on TRAIL downstream apoptotic targets (caspase 8 and caspase 3) using immunostaining. TRAIL activity is significantly inhibited by BAPN in aged animals, indicating a complex mechanism of age-related declines in muscle function through inflammatory and apoptotic mediators.
细胞外基质（ECM）随年龄增长而硬化，通过一种尚不清楚的机制导致内在肌肉干细胞（MuSC）功能障碍，从而阻碍肌肉再生。在此，该研究旨在探究由于年龄和/或硬度导致的MuSCs分化过程中与年龄相关的分子变化。因此，将年轻和老年的MuSCs接种到经工程改造以模拟柔软和坚硬ECM微环境的底物上，使用单细胞RNA测序（scRNA）来研究这些分子变化。分析了处于四种不同分化条件下的MuSCs的scRNA数据轨迹，以及驱动这些分化命运的活跃分子途径和转录因子。数据显示，在轨迹中存在一个分支点，导致出现一个与年龄相关的成纤维细胞群体，其特征是肿瘤坏死因子相关凋亡诱导配体（TRAIL）途径的激活，在坚硬底物上培养的老年细胞中该途径被显著激活。接下来，在体内使用胶原蛋白交联抑制剂β-氨基丙腈（BAPN），该研究通过免疫染色阐明了硬度变化对TRAIL下游凋亡靶点（半胱天冬酶8和半胱天冬酶3）的影响。在老年动物中，BAPN显著抑制了TRAIL活性，表明通过炎症和凋亡介质，与年龄相关的肌肉功能下降存在复杂机制。
