title: Biomechanical strain vehicles for fibroblast-directed skeletal myoblast differentiation and myotube functionality in a novel coculture.
abstract: Skeletal muscle functionality is governed by multiple stimuli, including cytokines and biomechanical strain. Fibroblasts embedded within muscle connective tissue respond to biomechanical strain by secreting cytokines that induce myoblast differentiation and, we hypothesize, regulate myotube function. A coculture was established to allow cross talk between fibroblasts in Bioflex wells and myoblasts on nondeformable coverslips situated above Bioflex wells. Cyclic short-duration strain (CSDS) modeling repetitive stress/injury, acyclic long-duration strain (ALDS) modeling manipulative therapy, and combined strain paradigms (CSDS + ALDS) were applied to fibroblasts. Nonstrained myoblasts in uniculture and coculture served as controls. After fibroblasts had induced myoblast differentiation, myotube contraction was assessed by perfusion of ACh (10(-11)-10(-3) M). CSDS-treated fibroblasts increased myotube contractile sensitivity vs. uniculture (P < 0.05). As contraction is dependent on ACh binding, expression and clustering of nicotinic ACh receptors (nAChRs) were measured. CSDS-treated fibroblasts increased nAChR expression (P < 0.05), which correlated with myotube contraction. ALDS-treated fibroblasts did not significantly affect contraction or nAChR expression. Agrin-treated myotubes were then used to design a computer algorithm to identify α-bungarotoxin-stained nAChR clusters. ALDS-treated fibroblasts increased nAChR clustering (P < 0.05), while CSDS-treated fibroblasts disrupted cluster formation. CSDS-treated fibroblasts produced nAChRs preferentially located in nonclustered regions (P < 0.05). Strain-activated fibroblasts mediate myotube differentiation with multiple functional phenotypes. Similar to muscle injury, CSDS-treated fibroblasts disrupted nAChR clusters and hypersensitized myotube contraction, while ALDS-treated fibroblasts aggregated nAChRs in large clusters, which may have important clinical implications. Cellular strategies aimed at improving muscle functionality, such as through biomechanical strain vehicles that activate fibroblasts to stabilize postsynaptic nAChRs on nearby skeletal muscle, may serve as novel targets in neuromuscular disorders.
authors.affiliation: The University of Arizona College of Medicine-Phoenix, Phoenix, Arizona; and School of Life Sciences, Arizona State University, Tempe, Arizona Michael.Hicks@asu.edu.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Hicks
authors.affiliation: The University of Arizona College of Medicine-Phoenix, Phoenix, Arizona; and.
authors.forename: Thanh V
authors.initials: TV
authors.lastname: Cao
authors.affiliation: The University of Arizona College of Medicine-Phoenix, Phoenix, Arizona; and.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Standley
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001696:Biomechanical Phenomena; D002454:Cell Differentiation; D002460:Cell Line; D048429:Cell Size; D018920:Coculture Techniques; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000502:physiology*; D051379:Mice; D009119:Muscle Contraction; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000502:physiology; D032448:Myoblasts, Skeletal / Q000502:physiology*; D011978:Receptors, Nicotinic / Q000378:metabolism; D012038:Regeneration; D013312:Stress, Physiological
chemical_list: D011978:Receptors, Nicotinic; D000109:Acetylcholine
keywords: biomechanical strain; contraction; cytokines; fibroblasts; nicotinic acetylcholine receptors; skeletal muscle