Contracting C₂C₁₂ myotubes release CCL2 in an NF-κB-dependent manner to induce monocyte chemoattraction

Abstract

Miyatake S, Bilan PJ, Pillon NJ, Klip A. Contracting C2C12 myotubes release CCL2 in an NF-B-dependent manner to induce monocyte chemoattraction. Am J Physiol Endocrinol Metab 310: E160 -E170, 2016. First published November 10, 2015; doi:10.1152/ajpendo.00325.2015.-Muscle inflammation following exercise is characterized by expression of inflammatory cytokines and chemokines. Exercise also increases muscle macrophages derived from circulating monocytes. However, it is unknown whether muscle cells themselves attract circulating monocytes, or what is the underlying mechanism. We used an in vitro system of electrical stimulation (ES) causing C 2C12 myotube contraction to explore whether monocyte chemoattraction ensues and investigated the mediating chemoattractants. Conditioned medium from ES-contracted myotubes caused robust chemoattraction of THP-1 monocytes across Boyden chambers. Following ES, expression of several known monocyte chemokines [C-C motif ligand 2 (CCL2) and C-X-C motif ligand (CXCL)1, -2, and -5] was elevated, but of these, only recombinant CCL2 effectively reproduced monocyte migration. Electrically stimulated myotubes secreted CCL2, and neutralization of CCL2 in conditioned medium or antagonizing the CCL2 receptor (CCR2) in THP-1 monocytes inhibited ES-induced monocyte migration. N-benzyl-p-toluene sulfonamide (BTS), a myosin II-ATPase inhibitor, prevented ES-induced myotube contraction but not CCL2 gene expression and secretion. The membrane-permeant calcium chelator BAPTA-AM reduced ESinduced CCL2 secretion. Hence, electrical depolarization, rather than mechanical contraction, drives the rise in CCL2, with partial calcium input. ES activated the NF-B pathway; NF-B inhibitors reduced ES-induced CCL2 gene expression and secretion and repressed ESinduced THP-1

Activating transcription factor 3 regulates chemokine expression in contracting C2C12 myotubes and in mouse skeletal muscle after eccentric exercise

“…NFkB activation was necessary for the up-regulation of ccl2 expression upon EPS [15]. Here, we demonstrate that ATF3 is also necessary.…”

“…We have previously shown in mice that endurance exercise increases ATF3 protein levels 3 h post-exercise [7]. Here, we electrically stimulated C 2 C 12 myotubes with a protocol that mimics endurance exercise in vitro [15], and analyzed the responses 3 h after. EPS increased atf3 mRNA and protein levels in C 2 C 12 myotubes, suggesting part of the ATF3 up-regulation in contracting skeletal muscle occurs in myofibers.…”

“…After additional 72 h in differentiation medium, EPS was applied on C 2 C 12 myotubes using the C-PACE EP Cell Culture Stimulator (Ion Optix, Milton, MA, USA). We used an EPS protocol demonstrated to mimic endurance exercise without damaging cells even after 8 h of stimulation [15]. Since this protocol was specifically tested in C 2 C 12 myotubes [15], we selected these cells as our in vitro model.…”

“…We used an EPS protocol demonstrated to mimic endurance exercise without damaging cells even after 8 h of stimulation [15]. Since this protocol was specifically tested in C 2 C 12 myotubes [15], we selected these cells as our in vitro model. Briefly, myotubes were incubated in differentiation medium containing 5.5 mM glucose, and then 2-ms pulses of 20 V at 1 Hz were applied for 4 h. Three hours post-EPS, myotubes were washed with PBS and recovered in lysis buffer for protein extraction, or in Trizol ® for RNA extraction.…”

Anti-inflammatory drugs for Duchenne muscular dystrophy: focus on skeletal muscle-releasing factors

“…Besides immune cells, the main source of chemokines in skeletal muscle, muscle fibers can also chemoattract myeloid cells secreting chemokines. We recently found that NF-κB is activated in C2C12 myotubes upon muscle contraction and upregulate C-C motif chemokine ligand (CCL) 2 secretion, inducing THP-1 monocyte chemoattraction 61. In addition, several CC chemokines including CCL2, which are the components of the largest family of chemokines, and chemoattractive for diverse inflammatory cells, showed increased expression in mdx muscle 62.…”