Interleukin 6 in Masticatory Muscles: Dual Role Related to Muscle Activity and Muscle‐Bone Loss After Unloading

Abstract

The masticatory system is a coordinated machine, highly adaptable to environmental demands. Increased levels of interleukin 6 (IL6) in the synovial fluid have been associated with temporomandibular disorders. Although they are mostly suggested released from inflammatory cells, it has been reported that IL6 is also a “myokine”, released by muscle cells during activity or in pathological processes. A pivotal role of IL6 in skeletal muscle and bone has been suggested, being related to both anabolic and catabolic processes. We here propose that IL6 is released by masseter muscle during activity and that a deregulated increased level of muscle-derived IL6 correlates to the musculoskeletal impairment in animal models of masticatory muscles unloading. We induced masseter muscle hypofunction in male BalbC mice (8 weeks old) by either soft diet (SD) consumption (pellet:water=1:3) or unilateral paralysis of masseter muscles injected with Botulinum Toxin Type A (BoNTA; 0.2U/10 µL). Both masseter muscles and mandibles were dissected after 2-14 days for morphology, histology, or molecular measurements. Masseter muscles were electrically stimulated (ES) in vitro for demonstrating activity-evoked changes in IL6 expression and secretion. All procedures were approved by the Institutional Committee for the Care and Use of Animals of the Universidad de Chile (# 17011-OD-UCH). We demonstrated that IL6 is expressed and released from masseter muscle after ES, in a frequency-dependent manner. IL6 expression after ES requires the release of ATP from muscle, and activation of P2Y/P2X receptors. In the animal models of SD or BoNTA, atrophy of masseter muscle is observed (reduction of muscle volume, mass, and fibers diameter, as well as increased expression of Atrogin-1/MURF-1/Myogenin) after 7-14 days. Increased resting levels of extracellular ATP and IL6 expression were detected in masseter muscles of SD-fed mice and BoNTA-injected muscles. In the later, an increased expression of ATP releaser molecules (pannexin 1, connexin 43-45) was reported, as well as increased levels of ATP receptors P2Y2 and P2X7. Interestingly, the metabolization of extracellular ATP using Apyrase restored the resting levels of IL6 expression in masseter muscles derived from SD-fed mice. Also, changes in morphology and bone loss were observed in mandibular condyles associated with BoNTA-injected masseter muscles. We here report that IL6 is expressed and released by masseter muscles, either at rest and after muscle activity. Moreover, masseter muscle hypofunction induces an overactivation of the extracellular ATP-IL6 axis in muscles. We are currently addressing if this deregulated signaling is involved in the muscle atrophy and bone loss evoked by masticatory muscle unloading. Understanding the molecular pathways involved in muscle-bone crosstalk at the masticatory system will contribute to propose strategies to avoid non-desirable effects in pathologies or clinical interventions presenting muscle unloading.