title: Combination of agrin and laminin increase acetylcholine receptor clustering and enhance functional neuromuscular junction formation In vitro.
abstract: Clustering of acetylcholine receptors (AChR) at the postsynaptic membrane is a crucial step in the development of neuromuscular junctions (NMJ). During development and after denervation, aneural AChR clusters form on the sarcolemma. Recent studies suggest that these receptors are critical for guiding and initiating synaptogenesis. The aim of this study is to investigate the effect of agrin and laminin-1; agents with known AChR clustering activity; on NMJ formation and muscle maturation. Primary myoblasts were differentiated in vitro on collagen, laminin or collagen and laminin-coated surfaces in the presence or absence of agrin and laminin. The pretreated cells were then subject to innervation by PC12 cells. The number of neuromuscular junctions was assessed by immunocytochemical co-localization of AChR clusters and the presynaptic marker synaptophysin. Functional neuromuscular junctions were quantitated by analysis of the level of spontaneous as well as neuromuscular blocker responsive contractile activity and muscle maturation was assessed by the degree of myotube striation. Agrin alone did not prime muscle for innervation while a combination of agrin and laminin pretreatment increased the number of neuromuscular junctions formed and enhanced acetylcholine based neurotransmission and myotube striation. This study has direct clinical relevance for treatment of denervation injuries and creating functional neuromuscular constructs for muscle tissue repair.
authors.affiliation: Department of Orthopaedics, St. Vincent's Hospital Melbourne and the University of Melbourne, Fitzroy, VIC, 3065, Australia.|Department of Surgery, St. Vincent's Hospital Melbourne and the University of Melbourne, Fitzroy, VIC, 3065, Australia.
authors.forename: Bill G X
authors.initials: BG
authors.lastname: Zhang
authors.affiliation: Department of Medicine, the University of Melbourne, St Vincent's Hospital Melbourne, Fitzroy, VIC, 3065, Australia.|ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, University of Wollongong, Wollongong, NSW, 2522, Australia.
authors.forename: Anita F
authors.initials: AF
authors.lastname: Quigley
authors.affiliation: Department of Medicine, the University of Melbourne, St Vincent's Hospital Melbourne, Fitzroy, VIC, 3065, Australia.
authors.forename: Justin L
authors.initials: JL
authors.lastname: Bourke
authors.affiliation: Walter and Eliza Hall Institute, Parkville, VIC, 3052, Australia.
authors.forename: Cameron J
authors.initials: CJ
authors.lastname: Nowell
authors.affiliation: Department of Orthopaedics, St. Vincent's Hospital Melbourne and the University of Melbourne, Fitzroy, VIC, 3065, Australia.|Department of Surgery, St. Vincent's Hospital Melbourne and the University of Melbourne, Fitzroy, VIC, 3065, Australia.
authors.forename: Damian E
authors.initials: DE
authors.lastname: Myers
authors.affiliation: Department of Orthopaedics, St. Vincent's Hospital Melbourne and the University of Melbourne, Fitzroy, VIC, 3065, Australia.|Department of Surgery, St. Vincent's Hospital Melbourne and the University of Melbourne, Fitzroy, VIC, 3065, Australia.
authors.forename: Peter F M
authors.initials: PF
authors.lastname: Choong
authors.affiliation: ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, University of Wollongong, Wollongong, NSW, 2522, Australia.
authors.forename: Robert M I
authors.initials: RM
authors.lastname: Kapsa
mesh_terms: D018171:Agrin / Q000008:administration & dosage / Q000378:metabolism*; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques; D003470:Culture Media; D007797:Laminin / Q000008:administration & dosage / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D009119:Muscle Contraction / Q000502:physiology; D018485:Muscle Fibers, Skeletal / Q000378:metabolism / Q000648:ultrastructure; D032446:Myoblasts / Q000378:metabolism / Q000648:ultrastructure; D009469:Neuromuscular Junction / Q000254:growth & development* / Q000378:metabolism* / Q000648:ultrastructure; D016716:PC12 Cells; D051381:Rats; D011950:Receptors, Cholinergic / Q000378:metabolism*
chemical_list: D018171:Agrin; D003470:Culture Media; D007797:Laminin; D011950:Receptors, Cholinergic; C121346:laminin 1
keywords: acetylcholine receptors; agrin; laminin; neuromuscular junction; prepatterning