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.

In Duchenne muscular dystrophy (DMD), a lack of functional dystrophin leads to myofiber instability and progressive muscle damage that results in fibrosis. While fibrosis is primarily characterized by an accumulation of extracellular matrix (ECM) components, there are changes in ECM architecture during fibrosis that relate more closely to functional muscle stiffness. One of these architectural changes in dystrophic muscle is collagen cross-linking, which has been shown to increase the passive muscle stiffness in models of fibrosis including the mdx mouse, a model of DMD. We tested whether the intraperitoneal injections of beta-aminopropionitrile (BAPN), an inhibitor of the cross-linking enzyme lysyl oxidase, would reduce collagen cross-linking and passive stiffness in young and adult mdx mice compared to saline-injected controls. We found no significant differences between BAPN treated and saline treated mice in collagen cross-linking and stiffness parameters. However, we observed that while collagen cross-linking and passive stiffness scaled positively in dystrophic muscles, collagen fiber alignment scaled with passive stiffness distinctly between muscles. We also observed that the dystrophic diaphragm showed the most dramatic fibrosis in terms of collagen content, cross-linking, and stiffness. Overall, we show that while BAPN was not effective at reducing collagen cross-linking, the positive association between collagen cross-linking and stiffness in dystrophic muscles still show cross-linking as a viable target for reducing passive muscle stiffness in DMD or other fibrotic muscle conditions.