Age‐Related ECM Stiffness Mediates TRAIL Activation in Muscle Stem Cell Differentiation

Abstract 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.