Muscle stem cells (MuSCs) are essential for the robust regenerative capacity of skeletal muscle. However, in fibrotic environments marked by abundant collagen and altered collagen organization, the regenerative capability of MuSCs is diminished. MuSCs are sensitive to their extracellular matrix environment, but their response to collagen architecture is largely unknown. The present study aimed to systematically test the effect of underlying collagen structures on MuSC functions. Collagen hydrogels were engineered with varied architectures: collagen concentration, crosslinking, fibril size, and fibril alignment, and the changes were validated with second harmonic generation imaging and rheology. Proliferation and differentiation responses of primary mouse MuSCs and immortal myoblasts (C2C12s) were assessed using EdU assays and immunolabeling skeletal muscle myosin expression, respectively. Changing collagen concentration and the corresponding hydrogel stiffness did not have a significant influence on MuSC proliferation or differentiation. However, MuSC differentiation on atelocollagen gels, which do not form mature pyridinoline crosslinks, was increased compared to the crosslinked control. In addition, MuSCs and C2C12 myoblasts showed greater differentiation on gels with smaller collagen fibrils. Proliferation rates of C2C12 myoblasts were also higher on gels with smaller collagen fibrils, while MuSCs did not show a significant difference. Surprisingly, collagen alignment did not have significant effects on muscle progenitor function. This study demonstrates that MuSCs are capable of sensing their underlying ECM structures and enhancing differentiation on substrates with less collagen crosslinking or smaller collagen fibrils. Thus, in fibrotic muscle, targeting crosslinking and fibril size rather than collagen expression may more effectively support MuSC-based regeneration.

Fibro-adipogenic progenitors (FAPs) are essential in supporting regeneration in skeletal muscle, but in muscle pathologies FAPs the are main source of excess extracellular matrix (ECM) resulting in fibrosis. Fibrotic ECM has altered mechanical and architectural properties, but the feedback onto FAPs of stiffness or ECM properties is largely unknown. In this study, FAPs’ sensitivity to their ECM substrate was assessed using collagen coated polyacrylamide to control substrate stiffness and collagen hydrogels to engineer concentration, crosslinking, fibril size, and alignment. FAPs on substrates of fibrotic stiffnesses had increased myofibroblast activation, depicted by αSMA expression, compared to substrates mimicking healthy muscle, which correlated strongly YAP nuclear localization. Surprisingly, fibrosis associated collagen crosslinking and larger fibril size inhibited myofibroblast activation, which was independent of YAP localization. Additionally, collagen crosslinking and larger fibril diameters were associated with decreased remodeling of the collagenous substrate as measured by second harmonic generation imaging. Inhibition of YAP activity through verteporfin reduced myofibroblast activation on stiff substrates but not substrates with altered architecture. This study is the first to demonstrate that fibrotic muscle stiffness can elicit FAP activation to myofibroblasts through YAP signaling. However, fibrotic collagen architecture actually inhibits myofibroblast activation through a YAP independent mechanism. These data expand knowledge of FAPs sensitivity to ECM and illuminate targets to block FAP’s from driving progression of muscle fibrosis.

BackgroundFibro-adipogenic progenitors (FAPs) are essential in supporting regeneration in skeletal muscle but pathologically become the main source of excess extracellular matrix (ECM) deposition in fibrosis. Fibrosis can create a progressively degenerative feedback loop with fibrotic tissue leading to an increase in the fibrotic FAP phenotype. Fibrotic ECM has altered mechanical and architectural properties, but the interactions between FAPs and the ECM are not well understood.MethodsIn this study, we aimed to examine FAPs’ sensitivity to their environment and its effect on cell fate. Polyacrylamide and collagen hydrogels were utilized to investigate substrate stiffness and ECM architecture. Collagen hydrogels were manipulated to mimic aspects of healthy and fibrotic ECM including density, crosslinking, fibril size, and alignment. Collagen-coated polyacrylamide gels were utilized to analyze substrate stiffnesses that are physiologically relevant. YAP nuclear localization, proliferation, and myofibroblast activation were measured using immunofluorescence and custom FIJI: ImageJ scripts. Results Substrates of fibrotic stiffnesses had increased myofibroblast activation compared to substrates mimicking healthy muscle. The increase in myofibroblast activation correlated strongly with in Yes-associated protein (YAP) nuclear localization. Collagen crosslinking and increased fibril size inhibited myofibroblast activation, independent of YAP localization. Alignment of collagen did not affect FAP behavior.ConclusionsThis study shows that FAPs possess the ability to sense their ECM environment and that influences cell fate. Therefore, ECM mechanics and architecture are key targets to reduce the progression of fibrosis.

Fibro-adipogenic progenitors (FAPs) are essential in supporting regeneration in skeletal muscle but in muscle pathologies FAPs the are main source of excess extracellular matrix (ECM) resulting in fibrosis. Fibrotic ECM has altered mechanical and architectural properties, but the feedback onto FAPs of stiffness or ECM properties is largely unknown. In this study, FAPs’ sensitivity to their ECM substrate was assessed using collagen coated polyacrylamide to control substrate stiffness and collagen hydrogels to engineer concentration, crosslinking, fibril size, and alignment. FAPs on substrates of fibrotic stiffnesses had increased myofibroblast activation, depicted by αSMA expression, compared to substrates mimicking healthy muscle which correlated strongly YAP nuclear localization. Surprisingly, fibrosis associated collagen crosslinking and larger fibril size inhibited myofibroblast activation, which was independent of YAP localization. Additionally, collagen crosslinking and larger fibril diameters were associated with decreased remodeling of the collagenous substrate as measured by second harmonic generation imaging. This study is the first to demonstrate that fibrotic muscle stiffness can elicit FAP activation to myofibroblasts through YAP signaling. However, fibrotic collagen architecture actually inhibits myofibroblast activation through a YAP independent mechanism. These data expand knowledge of FAPs sensitivity to ECM and illuminate targets to block FAP’s from driving progression of muscle fibrosis.