Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC333
A Putative Model of Endurance Exercise Using Bio-Engineered Skeletal Muscle
D. Player1, N. R. Martin1, P. C. Castle1, A. P. Sharples1, S. Passey1, V. Mudera3, M. P. Lewis1,2
1. Cellular and Molecular Physiology Group (MCMPRG), Institute for Sport and Physical Activity Research (ISPAR), University of Bedfordshire, Bedford, United Kingdom. 2. UCL School of Life and Medical Sciences, UCL, London, United Kingdom. 3. UCL Institute of Orthopaedics and Musculoskeletal Science, UCL, Stanmore, United Kingdom.
The metabolic flux with in vivo exercise, including increases in Lactate production and Glucose uptake, have been used in exercise physiology to characterise bouts of exercise with respect to the intensity and energy systems involved. The use of in vitro skeletal muscle models for physiological testing can allow for greater amounts of experimentation and control compared to in vivo investigations. Models of skeletal muscle in vitro which respond physiologically will allow for insight into the cellular and molecular mechanisms involved in the adaptation of skeletal muscle following exercise. This investigation aimed to characterise a 3D in vitro skeletal muscle model (Mudera et al., 2010) with respect to endurance exercise. 4x106/ml C2C12 mouse muscle cells were seeded in 3ml of type-1 rat tail collagen and plated into chamber slides (n=3). Each chamber held an “A-frame” at either end to provide attachment points. Once set the collagen construct was cut away from the sides of the chamber and suspended in growth medium (20% FBS). The “A-frames” provided tension to allow for the development of myotubes. The constructs were left in culture for 14 days for optimal myotube development. The construct was then tethered to the tensioning culture force monitor (t-CFM) for mechanical stimulation. The protocol was as follows: 7.5% strain, cyclic continuous stretch at 0.4Hz for 60 minutes. Conditioned media was sampled every 10 minutes throughout for analysis. Control samples were tethered to the t-CFM without stretch. An Analox Analyser was used to analyse both [Lactate] and [Glucose] within the conditioned media. Following stimulation the construct was immediately sampled. qPCR was performed using primers for β-Globin (Nuclear encoding) and COXII (Mitochondria encoding). mtDNA copy number was represented per nuclear diploid genome. 60 minutes of cyclic stretch increased [Lactate] at every time point compared to 0 minutes from 1.83±0.23mmol.L to 4.52±0.21mmol.L (p<0.05). There were no significant differences between each time point after 0 minutes (p>0.05) indicative of a ‘steady state’ in Lactate production. The index of uptake ([Glucose] at 0 minutes - [Glucose] at experimental time point) of Glucose was significant after 30 minutes of the stretch protocol from 0mmol.L to 1.5±0.12mmol.L (p<0.05). There were no changes in Lactate or Glucose for controls. mtDNA copy number per nuclear diploid genome increased compared to control immediately post stretch (p<0.05). This model of mechanical stretch (‘exercise’) has shown in vivo-like responses with respect to both Lactate and Glucose metabolism. This is the first investigation to show an increase in mtDNA copy number following an acute bout of mechanical stimulation in vitro. This model will allow for future investigations to understand the cellular and molecular adaptation to acute exercise.
Where applicable, experiments conform with Society ethical requirements