Mouse masseter muscle activity induces IL1β and IL6 expression mediated by extracellular ATP signaling

Abstract

Masticatory apparatus is a complex musculoskeletal structure, highly adapted to environmental demands. Increased levels of cytokines such as interleukin‐6 (IL6) and interleukin‐1β (IL1β) has been reported in the synovial fluid of patients with temporomandobular disorders, and it is usually thought coming from inflammatory cells. However, in trunk and limb muscles it is known that IL6 and IL1β are “myokines”, released from muscle fibers during activity. We demonstrated that IL6 synthesis and release is mediated by the “excitation‐transcription coupling”: depolarization sensing by dihydropyridine receptor (Cav1.1), ATP release through pannexin‐1 channel, activation of P2Y/P2X receptors, and rise in Ca2+ leading to gene expression. Masticatory muscles, such as masseter, are embryological and biochemically different than trunk and limbs ones. The aim of this study was to assess the role of mouse masseter muscle activity (in vitro and in vivo) over IL1β and IL6 expression and their dependence on the extracellular ATP (eATP) signaling pathway.Masseter muscles from 6–8 w/o mice were isolated. ATP release evoked by ES (270 pulses, 0.3 msec each, 20–90 Hz) was quantitated by a luciferin‐luciferase assay. After ES or exogenous ATP (0.001–100μM), mRNA and protein levels of IL1β and IL6 were assessed by qPCR and immunoblot. Pharmacological blockers of eATP signaling were used. For masseter remodeling in vivo, changes in diet hardness were addressed. Mice fed for 14 days with a control diet (normal pellet) were compared with those fed with extra‐hard diet (EHD, normal pellet + inert gnawing devices). Masseter muscles isolated from both groups were measured for ATP release and myokine expression, at rest or after ES.In isolated masseter muscles, ES at 20–60 Hz evoked up to 5‐fold increase in ATP release. In contrast, ES at 90 Hz did not evoke ATP release. ES‐20 Hz increased‐‐‐ IL1β and IL6 mRNA levels (25 and 90‐fold, respectively) and protein levels of active IL1β (15‐fold increase) and IL6 (5 fold increase). This effect was inhibited by Nifedipine (Cav1.1 blocker), Carbenoxolone (connexins/pannexins blocker), Apyrase (eATP metabolizing enzyme) and Suramin (P2X/P2YR antagonist). Exogenous ATP increased IL1β and IL6 mRNA levels in a concentration‐dependent manner, up to 25 and 10‐fold, respectively. Either Suramin or Brilliant Blue G (BBG, P2X7R blocker) blockade the effect of eATP over IL1β expression. However, IL6 mRNA expression evoked by eATP was just blocked by Suramin, without any effect of BBG.Masseter muscles derived from EHD‐fed animals had no changes in basal eATP, or in mRNA levels of IL1β or IL6. However, ATP release evoked by ES was higher than in masseters from control mice (7‐ and 4‐folds, respectively). IL1β and IL6 expression evoked by ES was also significantly higher in EHD‐fed animals than in control.Our results demonstrate for the first time that masseter muscle activity in vitro promotes IL1β/IL6 expression, mediated by eATP. When masseter activity is increased in vivo by changes in diet hardness, this pathway becomes more responsive. Molecular changes accounting for that are now being sought.Support or Funding InformationFondecyt 1151353 (SB/MC), CONICYT‐PCHA 21150059(CB), FONDEF ID16/10101 (MC/SB).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.