Myotubes differentiate optimally on substrates with tissue-like stiffness

TLDR

Contractile myocytes are used to test whether cells sense mechanical and molecular cues, with adhesion strength increasing monotonically with substrate stiffness and peaking on glass. Myoblasts were cultured on collagen strips mounted on glass or polymer gels spanning a range of elasticities. Fusion into myotubes occurs regardless of substrate flexibility, but myosin/actin striations form only on gels with muscle‑like stiffness (~12 kPa), whereas glass, softer, or stiffer gels, including dystrophic‑muscle mimics, lack striations, and compliant glass‑attached myotube layers promote striation while the bottom cells form stress fibers, indicating that mechanical tuning is essential for stem‑cell‑based muscle repair.

Abstract

Contractile myocytes provide a test of the hypothesis that cells sense their mechanical as well as molecular microenvironment, altering expression, organization, and/or morphology accordingly. Here, myoblasts were cultured on collagen strips attached to glass or polymer gels of varied elasticity. Subsequent fusion into myotubes occurs independent of substrate flexibility. However, myosin/actin striations emerge later only on gels with stiffness typical of normal muscle (passive Young's modulus, E ∼12 kPa). On glass and much softer or stiffer gels, including gels emulating stiff dystrophic muscle, cells do not striate. In addition, myotubes grown on top of a compliant bottom layer of glass-attached myotubes (but not softer fibroblasts) will striate, whereas the bottom cells will only assemble stress fibers and vinculin-rich adhesions. Unlike sarcomere formation, adhesion strength increases monotonically versus substrate stiffness with strongest adhesion on glass. These findings have major implications for in vivo introduction of stem cells into diseased or damaged striated muscle of altered mechanical composition.