Effects of CX3CR1 and CXCR2 antagonists on running-dependent intramuscular neutrophil recruitments and myokine upregulation.

Muscle contractile activity stimulates intramuscular recruitment of immune cells including neutrophils emerging to serve as a prerequisite for exerting proper muscular performance, although the underlying mechanisms and their contributions to myokine upregulation remain ill-defined. We previously reported that pharmacological inhibition of CX3CR1, a fractalkine receptor, dampens gnawing-dependent neutrophil recruitment into masseter muscles along with compromising their masticatory activity. By using a running exercise model, we herein demonstrated that hindlimb muscles require collaborative actions of both CX3CR1- and CXCR2-mediated signals for achieving neutrophil recruitment, upregulation of myokines including interleukin (IL)-6, enhanced GLUT4 translocation, and adequate endurance capability. Mechanistically, we revealed that a combination of CX3CR1 and CXCR2 antagonists, i.e., AZD8797 and SB2205002, inhibits exercise-inducible ICAM-1 and fractalkine upregulations in the area of the endothelium and muscle-derived CXCL1 upregulation, both of which apparently contribute to the intramuscular neutrophil accumulation in working muscles. Intriguingly, we also observed that 2 h of running results in intramuscular augmentation of innate lymphoid type 2 cells (ILC2s) markers, i.e., Bcl11b mRNA levels and anti-GATA-3-antibody-positive signals, and that these effects are completely abolished by administration of the combination of CX3CR1 and CXCR2 antagonists. Taken together, our findings strongly suggest that the exercise-evoked regional interplay among working myofibers, the adjacent endothelium, and recruited immune cells including neutrophils and possibly ILC2s, mediated through these local factors, plays a key role in the organization of the intramuscular microenvironment supporting the performance of hindlimb muscles during running.NEW & NOTEWORTHY This study provides compelling evidence that running-dependent intramuscular neutrophil recruitment requires both CX3CR1- and CXCR2-mediated signals that prime not only myofiber-derived myokine upregulations but also endothelium ICAM-1 and fractalkine expressions. The results revealed the importance of the exercise-evoked regional interplay among working myofibers, the adjacent endothelium, and recruited immune cells, including neutrophils and possibly ILC2s, which plays a key role in the organization of the intramuscular microenvironment supporting the performance of hindlimb muscles during running.

RSPO3 is a novel contraction-inducible factor identified in an "in vitro exercise model" using primary human myotubes.

The physiological significance of skeletal muscle as a secretory organ is now well known but we can only speculate as to the existence of as-yet-unidentified myokines, especially those upregulated in response to muscle contractile activity. We first attempted to establish an "insert-chamber based in vitro exercise model" allowing the miniature but high cell-density culture state enabling highly developed contractile human myotubes to be readily obtained by applying electric pulse stimulation (EPS). By employing this in vitro exercise model, we identified R-spondin 3 (RSPO3) as a novel contraction-inducible myokine produced by cultured human myotubes. Contraction-dependent muscular RSPO3 mRNA upregulation was confirmed in skeletal muscles of mice subjected to sciatic nerve mediated in situ contraction as well as those of mice after 2 h of running. Pharmacological in vitro experiments demonstrated a relatively high concentration of metformin (millimolar range) to suppress the contraction-inducible mRNA upregulation of human myokines including RSPO3, interleukin (IL)-6, IL-8 and CXCL1. Our data also suggest human RSPO3 to be a paracrine factor that may positively participate in the myogenesis processes of myoblasts and satellite cells. Thus, the "insert chamber-based in vitro exercise model" is a potentially valuable research tool for investigating contraction-inducible biological responses of human myotubes usually exhibiting poorer contractility development even in the setting of EPS treatment.

TRPA1 and TRPV1 channels participate in atmospheric-pressure plasma-induced [Ca2+]i response.

Despite successful clinical application of non-equilibrium atmospheric pressure plasma (APP), the details of the molecular mechanisms underlying APP-inducible biological responses remain ill-defined. We previously reported that exposure of 3T3L1 cells to APP-irradiated buffer raised the cytoplasmic free Ca2+ ([Ca2+]i) concentration by eliciting Ca2+ influx in a manner sensitive to transient receptor potential (TRP) channel inhibitors. However, the precise identity of the APP-responsive channel molecule(s) remains unclear. In the present study, we aimed to clarify channel molecule(s) responsible for indirect APP-responsive [Ca2+]i rises. siRNA-mediated silencing experiments revealed that TRPA1 and TRPV1 serve as the major APP-responsive Ca2+ channels in 3T3L1 cells. Conversely, ectopic expression of either TRPA1 or TRPV1 in APP-unresponsive C2C12 cells actually triggered [Ca2+]i elevation in response to indirect APP exposure. Desensitization experiments using 3T3L1 cells revealed APP responsiveness to be markedly suppressed after pretreatment with allyl isothiocyanate or capsaicin, TRPA1 and TRPV1 agonists, respectively. APP exposure also desensitized the cells to these chemical agonists, indicating the existence of a bi-directional heterologous desensitization property of APP-responsive [Ca2+]i transients mediated through these TRP channels. Mutational analyses of key cysteine residues in TRPA1 (Cys421, Cys621, Cys641, and Cys665) and in TRPV1 (Cys258, Cys363, and Cys742) have suggested that multiple reactive oxygen and nitrogen species are intricately involved in activation of the channels via a broad range of modifications involving these cysteine residues. Taken together, these observations allow us to conclude that both TRPA1 and TRPV1 channels play a pivotal role in evoking indirect APP-dependent [Ca2+]i responses.

In vitro exercise model using contractile human and mouse hybrid myotubes.

Contraction of cultured myotubes with application of electric pulse stimulation (EPS) has been utilized for investigating cellular responses associated with actual contractile activity. However, cultured myotubes derived from human subjects often exhibit relatively poor EPS-evoked contractile activity, resulting in minimal contraction-inducible responses (i.e. myokine secretion). We herein describe an "in vitro exercise model", using hybrid myotubes comprised of human myoblasts and murine C2C12 myoblasts, exhibiting vigorous contractile activity in response to EPS. Species-specific analyses including RT-PCR and the BioPlex assay allowed us to separately evaluate contraction-inducible gene expressions and myokine secretions from human and mouse constituents of hybrid myotubes. The hybrid myotubes, half of which had arisen from primary human satellite cells obtained from biopsy samples, exhibited remarkable increases in the secretions of human cytokines (myokines) including interleukins (IL-6, IL-8, IL-10, and IL16), CXC chemokines (CXCL1, CXCL2, CXCL5, CXCL6, CXCL10), CC chemokines (CCL1, CCL2, CCL7, CCL8, CCL11, CCL13, CCL16, CCL17, CCL19, CCL20, CCL21, CCL22, CCL25, CCL27), and IFN-γ in response to EPS-evoked contractile activity. Together, these results indicate that inadequacies arising from human muscle cells are effectively overcome by fusing them with murine C2C12 cells, thereby supporting the development of contractility and the resulting cellular responses of human-origin muscle cells. Our approach, using hybrid myotubes, further expands the usefulness of the "in vitro exercise model".