Stretched Fibroblasts Regulate Acetylcholine Receptor (AChR) Expression and Clustering on Skeletal Muscle

Abstract

Repetitive tasks reduce fine motor control and manipulative therapy improves physical performance. Stretched muscle fascia may influence the contraction of the underlying skeletal muscle by secreting cytokines that regulate AChRs. A co‐culture was setup by seeding fibroblasts on deformable membranes and C2C12 myoblasts on non‐deformable coverslips for 24hrs before transferring the C2C12s two mm above the fibroblasts, within diffusible range of secreted cytokines. Fibroblasts were then subjected to repetitive stretch and/or manipulative therapy. Agrin‐induced myotubes in 2% horse serum and non‐strained C2C12s in co‐culture served as positive and negative controls. After 96hrs, C2C12s were analyzed for AChR expression by Western Blot (N=7) and for clustering by fluorescent imaging (130 images/treatment). Data were analyzed via ANOVA, p<0.05. AChR clusters numbered 196±21 for agrin and 65±16 for non‐strain co‐culture. Manipulative therapy did not affect cluster number 56±12, but repetitive stretch attenuated clusters 38±6. Inversely we found repetitive strain increased AChR expression by >;2‐fold vs. non‐strain, while manipulative therapy did not increase expression. Stretched fibroblasts differentially regulate AChRs on skeletal muscle cells through paracrine mediators. Opposite AChR expression and clustering results suggests that unique extrinsic factors regulate each outcome.AOA