Cytokine Response of Cultured Skeletal Muscle Cells Stimulated with Proinflammatory Factors Depends on Differentiation Stage

Abstract

Myoblast proliferation and myotube formation are critical early events in skeletal muscle regeneration. The attending inflammation and cytokine signaling are involved in regulation of skeletal muscle cell proliferation and differentiation. Secretion of muscle-derived cytokines upon exposure to inflammatory factors may depend on the differentiation stage of regenerating muscle cells. Cultured human myoblasts and myotubes were exposed to 24-hour treatment with tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS). Secretion of interleukin 6 (IL-6), a major muscle-derived cytokine, and interleukin 1 (IL-1), an important regulator of inflammatory response, was measured 24 hours after termination of TNF-α or LPS treatment. Myoblasts pretreated with TNF-α or LPS displayed robustly increased IL-6 secretion during the 24-hour period after removal of treatments, while IL-1 secretion remained unaltered. IL-6 secretion was also increased in myotubes, but the response was less pronounced compared with myoblasts. In contrast to myoblasts, IL-1 secretion was markedly stimulated in LPS-pretreated myotubes. We demonstrate that preceding exposure to inflammatory factors stimulates a prolonged upregulation of muscle-derived IL-6 and/or IL-1 in cultured skeletal muscle cells. Our findings also indicate that cytokine response to inflammatory factors in regenerating skeletal muscle partially depends on the differentiation stage of myogenic cells.

PGC‐1α4 gene expression is suppressed by the IL‐6—MEK—ERK 1/2 MAPK signalling axis and altered by resistance exercise, obesity and muscle injury

“…Concentrations of recombinant proteins IL-6 and TNFa were determined by a combination of examining plasma IL-6 levels in pathological conditions and previous use of the recombinant proteins in vivo and in vitro. Doses of IL-6 and TNF-a were selected from previous literature and optimized in our laboratory for these experiments (Yuen et al 2009, Bhatnagar et al 2010, Bonetto et al 2012, De Larichaudy et al 2012, Podbregar et al 2013, White et al 2013, Meyer et al 2015. Experimental concentrations of IL-6 frequently range from 5 to 100 ng/mL in vitro for studying IL-6 signalling (Yuen et al 2009, Bonetto et al 2012, White et al 2013).…”

“…Experimental concentrations of IL-6 frequently range from 5 to 100 ng/mL in vitro for studying IL-6 signalling (Yuen et al 2009, Bonetto et al 2012, White et al 2013). Furthermore, experimental concentrations of TNFa in vitro range from 5 to 100 ng/mL (Bhatnagar et al 2010, De Larichaudy et al 2012, Podbregar et al 2013, Meyer et al 2015. To verify efficacy of inhibitors on protein phosphorylation, treatments were performed for 30 min.…”

“…Notably, the PGC-1a gene contains a pair of promoter regions, with both a canonical promoter regulating expression of PGC-1a1 and an alternative promoter from which the splice variants PGC-1a2, PGC-1a3 and PGC-1a4 are regulated (Ruas et al 2012). Recent evidence suggests that PGC-1a4 expression may be promoted through b2 adrenergic receptor signalling to the alternative promoter region (Popov et al 2015). Pgc1a4 alternate splicing likely operates through small nuclear proteins assembling upon the pre-mRNA substrate to excise intron sequences (House & Lynch 2008).…”

Effect of ionizing radiation on human skeletal muscle precursor cells

“…Experiments were performed on cultures of human myoblasts prepared as previously described 5,15–16. Human myoblasts were derived from satellite cells obtained from muscle tissue routinely discarded during orthopaedic operations on donor patients aged 5, 11 and 20 years, without diagnosed muscular disease.…”

“…Myokine interleukin 6 (IL-6) is released from activated myoblasts in response to injury and IL-6 is an important factor promoting myoblast proliferation and differentiation during muscle regeneration 2–5. Several studies have investigated radiation effects on skeletal muscle, demonstrating that skeletal muscle damage after irradiation remains for many years.…”