Abstract
For successful growth and maintenance of primary myogenic cells in vitro, culture medium and addition of sera are the most important factors. At present it is not established as to what extent sera of different origin and composition, supplemented in media or serum-free media conditions influence myoblast function and responses to different stimuli. By assessing markers of proliferation, differentiation/fusion, quiescence, apoptosis and protein synthesis the aim of the current study was to elucidate how primary human myoblasts and myotubes are modulated by different commonly used serum using FCS (foetal calf serum), (CS-FCS charcoal-stripped FCS, a manufacturing process to remove hormones and growth factors from sera), HS (horse serum) as well as in serum free conditions (DMEM). To characterise the biological impact of the different serum, myoblasts were stimulated with Insulin (100 nM) and Vitamin D (100 nM; 1α,25(OH)2D3, 1α,25-Dihydroxycholecalciferol, Calcitriol), two factors with characterised effects on promoting fusion and protein synthesis or quiescence, respectively in human myoblasts/myotubes. We demonstrate that sera of different origin/formulation differentially affect myoblast proliferation and myotube protein synthesis. Importantly, we showed that quantifying the extent to which Insulin effects myoblasts in vitro is highly dependent upon serum addition and which type is present in the media. Upregulation of mRNA markers for myogenic fusion, Myogenin, with Insulin stimulation, relative to DMEM, appeared dampened at varying degrees with serum addition and effects on p70S6K phosphorylation as a marker of protein synthesis could not be identified unless serum was removed from media. We propose that these asymmetric molecular and biochemical responses in human myoblasts reflect the variable composition of mitogenic and anabolic factors in each of the sera. The results have implications for both the reproducibility and interpretation of results from experimental models in myoblast cells/myotubes.
Highlights
Age or injury-induced muscle weakness leading to frailty is a major public health problem
The different serum conditions were associated with either increasing levels of BrdU and Ki67 (HS) or increasing levels of MyoD and Myogenin (CS-foetal calf serum (FCS) and Dulbecco’s Modified Eagle Medium/Ham’s Nutrient Mixture F-12 (DMEM) only)
Insulin and Vitamin D treatments differed mainly on PC2 where Vitamin D was associated with lowered expression of MyoD and Myogenin and higher levels of Foxo3A and hairy and enhancer of split 1 (HES1)
Summary
Age or injury-induced muscle weakness leading to frailty is a major public health problem. An important characteristic of muscle is its capability for regeneration upon stimulation such as injury This involves the activation of normally quiescent satellite cells in adult muscle. Useful rodent cell lines such as mouse C2C12 or rat L6 myoblasts are available [4,5]. These cell lines are used extensively to explore the molecular mechanisms of muscle differentiation, fusion and function [6,7,8]. In some instances, they have been used in drug discovery screenings [9,10]. A potentially more predictive screening strategy would be to use primary human muscle cells
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