Secretion of Interleukin 6 in Human Skeletal Muscle Cultures Depends on Ca(2+) Signalling

SIMPLE SUMMARY: Skeletal muscle releases numerous hormones into circulation that interact with other organs, such as the liver, bone or the brain. These hormones, termed myokines, mediate the effects of physical activity in health, aging and disease. Interleukin 6 was the first discovered myokine and is actively investigated due to its participation in inflammation, immunity and metabolism. However, there is little information regarding the mechanisms that induce its release from muscle cells, especially in humans. Our aim was to investigate whether changes in the concentration of calcium ions participate in the stimulated release of interleukin 6 in human muscle cells. Using muscle cultures, we have found that several proteins responsible for the calcium increase during stimulation induce the release of interleukin 6 from the muscle cells. This could help to unveil how interleukin 6 and other myokines are released in pathological conditions such as trauma, infections or cancer. ABSTRACT: The systemic effects of physical activity are mediated by the release of IL-6 and other myokines from contracting muscle. Although the release of IL-6 from muscle has been extensively studied, the information on the cellular mechanisms is fragmentary and scarce, especially regarding the role of Ca(2+) signals. The aim of this study was to characterize the role of the main components of Ca(2+) signals in human skeletal muscle cells during IL-6 secretion stimulated by the Ca(2+) mobilizing agonist ATP. Primary cultures were prepared from surgical samples, fluorescence microscopy was used to evaluate the Ca(2+) signals and the stimulated release of IL-6 into the medium was determined using ELISA. Intracellular calcium chelator Bapta, low extracellular calcium and the Ca(2+) channels blocker La(3+) reduced the ATP-stimulated, but not the basal secretion. Secretion was inhibited by blockers of L-type (nifedipine, verapamil), T-type (NNC55-0396) and Orai1 (Synta66) Ca(2+) channels and by silencing Orai1 expression. The same effect was achieved with inhibitors of ryanodine receptors (ryanodine, dantrolene) and IP3 receptors (xestospongin C, 2-APB, caffeine). Inhibitors of calmodulin (calmidazolium) and calcineurin (FK506) also decreased secretion. IL-6 transcription in response to ATP was not affected by Bapta or by the T channel blocker. Our results prove that ATP-stimulated IL-6 secretion is mediated at the post-transcriptional level by Ca(2+) signals, including the mobilization of calcium stores, the activation of store-operated Ca(2+) entry, and the subsequent activation of voltage-operated Ca(2+) channels and calmodulin/calcineurin pathways.