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 invitro 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 8days 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-0.20mM, 0.40mM, 0.6mM, 0.8mM and above 1.0mM, 1.5mM, 2.0mM 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 invitro 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.
Highlights
Skeletal muscle is the largest organ in the human body comprising of approximately 50 % of our total body mass [1]
Muscle mass is regulated by a balance of muscle protein synthesis (MPS) and muscle protein breakdown (MPB), the rate of which can be altered through nutrition, age, exercise and sexual dimorphism [3]
Myoblasts were cultured using E-16 VIEW plates in a 10 % FBS media, and monitored until they reached 70 % confluence. These plates have rows of microelectrode sensors removed within the centre of the base of the plate to facilitate clearer imaging of cell performance while still enabling impedance measurement from the xCELLigenceTM platform. 70 % confluency was achieved after 30 h when cells were seeded at 5000 cells per well (Figure 1B (iii))
Summary
Skeletal muscle is the largest organ in the human body comprising of approximately 50 % of our total body mass [1]. The function of the skeletal muscle is to maintain the skeleton, moving, breathing and thermoregulation and is an important determinant in over-all body health [2]. Maintaining healthy muscle mass is a critical factor in metabolic health, bone strength, body weight control and resilience to stress and disease. Defects in skeletal muscle growth and metabolism can lead to diseases such as diabetes, obesity, heart disease and rheumatoid arthritis [4,5,6]. Healthy muscle mass promotes overall general good health, preventing many pathologic conditions and chronic diseases [2]
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