title: Optimization of an in vitro bioassay to monitor growth and formation of myotubes in real time.
abstract: The importance of growth and maintenance of skeletal muscle is vital for long term health and quality of life. Appropriate nutrition with specific bioactivities relevant to the functionalities of tissues such as skeletal muscle, can assist in maintaining and promoting adaptive responses to biological and environmental stresses which prevent muscle atrophy and promote hypertrophy. The aim of this investigation was to develop a novel in vitro cell-based electric impedance assay to study myoblast to myotube formation on the real time cell analysis (RTCA) platform (xCELLigence™, ACEA) and to validate the system by testing myotube responses to hypertrophic stimuli. C2C12 myoblasts were proliferated until 70% confluent in Dulbecco's Modified Eagles Medium (DMEM) (10% FBS) and subsequently differentiated to myotubes over 8 days in DMEM [2% horse serum (HS)]. Changes in cell behaviour and adhesion properties were monitored by measuring impedance via interdigitated microelectrodes in the base of E-16 cell culture dishes. To establish the suitability of this assay to monitor nutrient regulation of muscle hypertrophy, leucine, a known potent regulator of MPS was then supplemented to the fully formed myotubes in physiologically relevant conditions-0.20 mM, 0.40 mM, 0.6 mM, 0.8 mM and above 1.0 mM, 1.5 mM, 2.0 mM and impedance subsequently monitored. Parallel experiments highlighting alterations in myotube thickness, muscle protein synthesis (MPS) (mammalian target of rapamycin; mTOR) and differentiation (myogenin) were conducted to support RTCA bioassay findings. This in vitro bioassay can be used to monitor skeletal muscle behaviour and identify nutrient compounds with bioactivities promoting skeletal muscle hypertrophy, reducing muscle atrophy and thus inform the development of novel nutrient formulations for the maintenance of skeletal muscle.
authors.affiliation: Food for Health Ireland, University of Limerick, Limerick, Ireland Department of Life Sciences, Materials and Surface Sciences Institute and Stokes Institute, University of Limerick, Limerick, Ireland 4i Centre for Interventions in Infection, Inflammation and Immunity, Graduate Entry Medical School, University of Limerick, Limerick, Ireland Physical Education and Sport Sciences, University of Limerick, Limerick, Ireland.
authors.forename: Sylvia M
authors.initials: SM
authors.lastname: Murphy
authors.affiliation: Food for Health Ireland, University of Limerick, Limerick, Ireland Physical Education and Sport Sciences, University of Limerick, Limerick, Ireland.
authors.forename: Maeve
authors.initials: M
authors.lastname: Kiely
authors.affiliation: Food for Health Ireland, University of Limerick, Limerick, Ireland 4i Centre for Interventions in Infection, Inflammation and Immunity, Graduate Entry Medical School, University of Limerick, Limerick, Ireland.
authors.forename: Philip M
authors.initials: PM
authors.lastname: Jakeman
authors.affiliation: Food for Health Ireland, University of Limerick, Limerick, Ireland Department of Life Sciences, Materials and Surface Sciences Institute and Stokes Institute, University of Limerick, Limerick, Ireland 4i Centre for Interventions in Infection, Inflammation and Immunity, Graduate Entry Medical School, University of Limerick, Limerick, Ireland Health Research Institute, University of Limerick, Limerick, Ireland.
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Kiely
authors.affiliation: Food for Health Ireland, University of Limerick, Limerick, Ireland 4i Centre for Interventions in Infection, Inflammation and Immunity, Graduate Entry Medical School, University of Limerick, Limerick, Ireland Physical Education and Sport Sciences, University of Limerick, Limerick, Ireland Health Research Institute, University of Limerick, Limerick, Ireland brian.carson@ul.ie.
authors.forename: Brian P
authors.initials: BP
authors.lastname: Carson
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D049108:Cell Enlargement; D002460:Cell Line; D048429:Cell Size; D017097:Electric Impedance; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000166:cytology* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000254:growth & development / Q000378:metabolism; D009133:Muscular Atrophy / Q000378:metabolism; D032446:Myoblasts / Q000166:cytology* / Q000378:metabolism; D009997:Osmotic Pressure
keywords: C2C12; MPB; MPS; RTCA; hypertrophy; skeletal muscle