Cytokine and Chemokine Release from Immortalised Human Myotubes in Culture: Effect of Contractions and Comparison with Isolated Intact Mature Adult Murine Muscle Fibres

Abstract

Skeletal muscle releases a number of signalling molecules, including immunomodulatory cytokines and chemokines which can act in an autocrine, paracrine and endocrine fashion. For example, neuromuscular integrity is dependent on efficient and appropriate signalling from muscle to nerve and vice versa and when such signalling fails then tissue deterioration occurs. The aim of this study was to characterise the pattern and time course of release of pro‐ and anti‐ inflammatory cytokines and chemokines from cultured immortalised human myotubes at rest and following contractions compared with release from isolated viable intact mouse muscle fibres. Immortalised human myoblasts were fused to mature into myotubes for 7 days (Donna et al, 2003). Alternatively, single muscle fibres were isolated from the flexor digitorum brevis (FDB) muscle of adult C57Bl/6 mice (Pye et al, 2007). Cells and isolated fibres were subjected to electrical field stimulation with trains of biphasic square wave pulses of 2 ms duration for 0.5 s repeated every 5 s at 50 Hz and 30 V per well of fibres for a total stimulation time of 15 min. Media from all samples was analysed for cytokine content at 15 minutes and 24 hours following contractions using a multiplexed cytokine assay (Luminex) and data were standardised to protein content of the well. At 15 minutes following contractions of isolated muscle fibres, a significant increase in a number of cytokines and chemokines were seen in the media, including IL‐5, IL‐6, IL‐10 and G‐CSF although this was not fully replicated in 2D cultured myotubes. At 24 hours following contractions, IL‐6 content was significantly increased in the media of both isolated fibres and 2D cultured myotubes. Thus, data demonstrate some commonality of cytokines/chemokines released but future studies will examine release from more physiological 3D muscle constructs as well as the effects of such release on neuronal cell structure and function.Pye et al, J Physiol. 2007 May 15; 581(Pt 1): 309–318.Donna et al, Mol Cancer Res. 2003 (1) (9) 643‐653.