Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC327

Poster Communications

Artificially Aged Skeletal Myoblasts Display Reduced Regeneration in Bio-engineered Skeletal Muscle

A. P. Sharples1, D. J. Player1, N. R. Martin1, S. Passey1, C. E. Stewart3, M. P. Lewis1,2

1. Department of Sport and Exercise Sciences, Institute for Sport and Physical Activity Research (ISPAR), Muscle Cellular and Molecular Physiology Research Group (MCMPRG), University of Bedfordshire, Bedford, United Kingdom. 2. School of Life and Medical Sciences, UCL, London, United Kingdom. 3. Institute for Biomedical Research into Human Movement and Health (IRM), Manchester Metropolitan University, Manchester, United Kingdom.

The degeneration of skeletal muscle (SkM) with age (sarcopenia) is a major consideration for life long health and wellbeing. The extrinsic environment (the skM ‘niche’) has attracted much attention (Conboy et al. 2005) versus the contribution of intrinsic ageing in muscle “stem” cells and their progeny (myoblasts). However, it has become apparent that reduced myotube formation in parental C2 vs. daughter C2C12 myoblasts (Sharples et al. 2010) are associated with key molecular changes during growth and differentiation, which are also altered in artificially aged, serially passaged, myoblasts (unpublished observations). Furthermore, proliferation/differentiation in aged human myoblasts are comparable when cultured in serum from young vs. older humans (George et al. 2010) supporting the notion of intrinsic ageing. To date, studies have been conducted in monolayer cultures, however, to substantiate and extend these findings the current studies utilised an established 3-dimensional (3D) in-vitro model (Mudera et al. 2010) to assess artificially aged vs. un-passaged myoblasts capacity to create in-vivo-like SkM constructs. Serially passaged C2C12 skeletal myoblasts (63 doublings; passage 20) were compared with un-passaged cells. Cells were seeded at 4×106 cells/ml in type-1 rat tail collagen (3ml) and plated in chamber slides. Floatation bars at either end provided attachment points, enabling the polymerised collagen/myoblast constructs to be suspended in growth medium (20% FBS) for 24hrs prior to transfer into low serum medium (2% HS) to enable differentiation over 3, 7 and 14 days(d). Morphology, immuno-histological (desmin) analyses and preliminary transcript changes in matrix metalloproteinases (MMP2, MMP9), myogenic regulatory factors (MyoD, Myogenin), insulin-like growth factor members (IGF-I, IGF-IEa, MGF, IGF-IR, IGFBP2, IGFBP5) and myostatin were investigated (qRT-PCR). Morphology suggested aged muscle constructs showed reduced ability to attach to the collagen matrix with a 3.3 fold reduction in MMP2 at 7d and a 2 fold decrease in MMP9 at 14d vs. un-aged. This was associated with diminished myotube formation and a 2.6 fold reduction in myogenin at 3d. IGF-I mRNA was reduced 2.1 and 6.3 fold at 3 and 14d respectively together with a 5.1 and 3.8 fold decrease in IGF-IEa and MGF respectively at 14d. The differences were observed in parallel with 3.5 and 6.6 fold increase in aged cells production of IGFBP5 and 8 and 6.5 fold larger myostatin mRNA levels at 7 and 14d respectively vs. un-aged. This study provides an important 3-D model for studying ageing muscle in-vitro. It consolidates key findings in monolayer cultures of rodent and human muscle cell ageing and further provides important insight into the impact of multiple divisions (artificial ageing), independent of stem cell niche, on the cellular/molecular mechanisms underpinning degeneration.

Where applicable, experiments conform with Society ethical requirements