Myofibers cultured in viscoelastic hydrogels reveal the effects of integrin-binding and mechanosensing on muscle satellite cells.

Quiescent skeletal muscle satellite cells (SCs) located on myofibers activate in response to muscle injury to regenerate muscle; however, identifying the role of specific matrix signals on SC behavior in vivo is difficult. Therefore, we developed a viscoelastic hydrogel with tunable properties to encapsulate myofibers while maintaining stem cell niche polarity and SC-myofiber interactions to investigate how matrix signals, including viscoelasticity and the integrin-binding ligand arginyl-glycyl-aspartic acid (RGD), influence SC behavior during muscle regeneration. Viscoelastic hydrogels support myofiber culture while preserving SC stemness for up to 72 hours post-encapsulation, minimizing myofiber hypercontraction and SC hyperproliferation compared to Matrigel. Pax7 is continuously expressed in SCs on myofibers embedded in hydrogels with higher stress relaxation while SCs differentiate when embedded in elastic hydrogels. Increasing RGD concentrations activates SCs and translocates YAP/TAZ to the nucleus as revealed by photo-expansion microscopy. Deleting YAP/TAZ abrogates RGD-mediated activation of SCs, and thus, YAP/TAZ mediates RGD ligand-induced SC activation and subsequent proliferation. STATEMENT OF SIGNIFICANCE: Satellite cells (SCs) are responsible for muscle maintenance and regeneration, but how the extracellular matrix regulates SC function is less understood and would benefit from new biomaterial models that can recapitulate the complexity of SC niche in vitro. Upon isolation of myofibers, SCs exit quiescence, becoming activated. To circumvent this issue, we developed a viscoelastic hydrogel for encapsulating myofibers, which maintains SC quiescence and limits differentiation, allowing the study of RGD effects. We showed that increasing RGD concentration promotes activation and suppresses differentiation. Finally, to allow high resolution imaging for resolving the subcellular localization of YAP/TAZ transcriptional co-activators, we applied photo-expansion microscopy and gel-to-gel transfer techniques to quantify YAP/TAZ nuclear-cytoplasmic ratio, revealing that RGD-mediated activation relies on YAP/TAZ nuclear translocation.