Culture substrate stiffness impacts human myoblast contractility-dependent proliferation and nuclear envelope wrinkling

Abstract

The remarkable self-repair ability of skeletal muscle tissues is driven by muscle stem cells, whose activities are orchestrated by a variety of transient cues present in their microenvironment. Understanding how the cross-interactions of these biophysical and biochemical microenvironmental cues influences muscle stem cells and their progeny is crucial in strategizing remedies for pathological dysregulation of these cues in aging and disease. In this study, we investigated the cell-level influences of extracellular matrix ligands in the context of mechanically tuned culture substrates on primary human myoblast contractility and proliferation within 16 hours of plating. We found that fibronectin led to stronger stiffness-dependent responses in myoblasts compared to laminin and collagen as revealed by cell spreading area, focal adhesion morphology, traction stresses, and proliferation. While we did not observe stiffness-dependent changes in cell fate signatures at this early time-point, an analysis of the stiffness-sensitive myoblast proteome uncovered an enrichment for differentiation associated signatures on 17 kPa culture substrates, in addition to distinct profiles with relation to metabolism, cytoskeletal regulation, ECM interactions, and nuclear component enrichment. Interestingly, we found that softer substrates increased the incidence of myoblasts with a wrinkled nucleus, and that the extent of wrinkling could predict Ki67 expression. While nuclear wrinkling and Ki67 expression could be controlled by pharmacological manipulation of cellular contractility, cellular traction stresses did not correlate with nuclear envelope wrinkling level. These results provide new insights into the regulation of human myoblast stiffness-dependent contractility response by ECM ligands and also highlight a link between myoblast contractility and proliferation.