Aged Muscle Stem Cell Sensitivity to Matrix Stiffening Disrupts Differentiation Kinetics through Dysregulation of SIRT3

Aging is typically associated with decreased functional mobility, which can be caused by declines in skeletal muscle regenerative capacity and functional recovery after injury. For efficient muscle regeneration, resident muscle stem cells (MuSCs) are essential. However, over time, MuSCs display a progressively diminished myogenic lineage specification. While aged MuSCs display cell-autonomous deficits that drive impaired regeneration, a young microenvironment restores a youthful cellular phenotype. Although most studies to date have focused on the effects of circulating factors on age-related MuSC dysfunction, little is known about the impact of biophysical niche alterations on MuSC behavior over time. In this dissertation work, we evaluated whether aged MuSC dysfunction is mediated by increased muscle stiffness. Further, given the role of mitochondria in dictating stem cell fate, we investigated whether mitochondria-associated gene expression is perturbed in response to a stiff microenvironment. First, the impact of substrate stiffness on MuSC characteristics was assessed at the level of nuclear morphology, single-cell transcripts, and protein expression. In vitro data revealed that aged MuSC nuclear morphology recapitulates that of young MuSCs when cells were exposed to a substrate engineered to mimic the stiffness of young muscle. As nuclear morphological changes influence gene expression and, thus, stem cell fate, we next examined whether changes in nuclear morphology were associated with improved myogenicity. Single cell RNA-seq and imaging flow cytometry revealed that exposure to a soft substrate increased aged MuSC activation as evidenced by Pax7 and MyoD1 expression at both mRNA and protein levels, respectively, suggesting rejuvenation of aged MuSCs. Notably, young MuSCs were resistant to stiffness alterations, and a stiff substrate did not significantly affect lineage progression. Further investigation implicated SIRT3, a master regulator of mitochondria, as a novel mechano-sensitive factor regulating MuSC fate. Finally, we tested whether reduction of aged muscle stiffness enhances regenerative capacity in vivo. We found that modulation of aged muscle elasticity led to enhanced myofiber cross-sectional area and force recovery. Consistent with in vitro findings, SIRT3 expression at the injury site was also enhanced with reduced stiffness in aged muscle. Our findings highlight a previously unrecognized role of SIRT3 in MuSC activation and muscle regeneration in response to microenvironmental stiffness