Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, PC67
Muscle precursor cell number as a determinant of skeletal muscle hypertrophic potential
N. R. Martin1, P. C. Castle1, V. Mudera2, M. P. Lewis1,3
1. Sport and Exercise Sciences, University of Bedfordshire, Bedford, United Kingdom. 2. Institute of Orthopaedics and Musculoskeletal Science, University College London, London, United Kingdom. 3. Eastman Dental Institute, University College London, London, United Kingdom.
Insights into the mechanisms involved in postnatal muscle growth are beginning to be elucidated utilising various in vivo animal and human models. Of particular interest is the observation that individuals who experience a high degree of hypertrophy appear to have more muscle stem (satellite) cells in an untrained state than individuals that undergo no, or moderate hypertrophy following a resistance training period (1). In vivo testing is limited by the difficulty of manipulating the muscle environment, and thus the use of a bioengineered in vitro 3D muscle construct can be of great use in further understanding how muscle responds to injury, disease states and exercise. This study uses a 3D muscle model to determine if the number of muscle precursor cells predicts the potential of skeletal muscle to undergo hypertrophy. Muscle derived cells (MDCs), obtained from patients undergoing orthopaedic surgery wth informed consent, were separated into myogenic (CD56+) and non-myogenic (CD56-) fractions using immunomagnetic techniques (MACS®). Cells were then seeded into neutralised type 1 rat tail collagen at a range of myogenic fractions using established techniques (2). Briefly, each collagen-cell mixture was cast into a custom built chamber between two floatation bars attached to wire ‘A-frames’ tethered to immovable points providing isometric tension within the culture. Connecting the A-frames to a culture force monitor (CFM) allows for real time analysis of force generation within the muscle construct. Use of a tensioning CFM (t-CFM) also allows programmable regimens of mechanical strain to be applied to the construct to mimic resistance or endurance training. Molecular and cellular changes were analysed by fluorescence immunohistochemistry using confocal microscopy and PCR. MDCs align in a single plane along the lines of principle longitudinal strain within the collagen gels, and differentiation of single myogenic cells into multinucleated myotubes capable of directed contraction is observed, analogous to skeletal muscle in vivo. Immunohistochemical analysis confirmed the formation of primary myotubes and preliminary PCR and CFM data suggests that the myotubes present in these 3D cultures are representative of in vivo muscle development and regeneration. The seeding of different proportions of myogenic fractions in the collagen gels followed by different mechanical stimulation regimes allows investigation of the relationship between the extent of hypertrophy and the proportion of satellite cells. This study addresses the relationship between satellite cell number and the hypertrophic response of skeletal muscle to resistance training. The ability to manipulate the satellite cell population in engineered muscle and measure the effects in vitro exercise training of the muscle constructs is of great benefit in further understanding the basis of muscle hypertrophy at a molecular level.
Where applicable, experiments conform with Society ethical requirements