A cultured C2C12 myotube contraction system was examined for application as a model for acute contraction-induced phenotypes of skeletal muscle. C2C12 myotubes seeded into 4-well rectangular plates were placed in a contraction system equipped with a carbon electrode at each end. The myotubes were stimulated with electric pulses of 50 V at 1 Hz for 3 ms at 997-ms intervals. Approximately 80% of the myotubes were observed to contract microscopically, and the contractions lasted for at least 3 h with electrical stimulation. Calcium ion (Ca2+) transient evoked by the electric pulses was detected fluorescently with Fluo-8. Phosphorylation of protein kinase B/Akt (Akt), 5′ AMP-activated protein kinase (AMPK), p38 mitogen-activated protein kinase (p38), and c-Jun NH2-terminal kinase (JNK)1/2, which are intracellular signaling proteins typically activated in exercised/contracted skeletal muscle, was observed in the electrically stimulated C2C12 myotubes. The contractions induced by the electric pulses increased glucose uptake and depleted glycogen in the C2C12 myotubes. C2C12 myotubes that differentiated after exogenous gene transfection by a lipofection or an electroporation method retained their normal contractile ability by electrical stimulation. These findings show that our C2C12 cell contraction system reproduces the muscle phenotypes that arise in vivo (exercise), in situ (hindlimb muscles in an anesthetized animal), and in vitro (dissected muscle tissues in incubation buffer) by acute muscle contraction, demonstrating that the system is applicable for the analysis of intracellular events evoked by acute muscle contraction.

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

Lipids in skeletal muscle play a fundamental role both in normal muscle metabolism and in disease states. Skeletal muscle lipid accumulation is associated with several chronic metabolic disorders, including obesity, insulin resistance, and type 2 diabetes. However, it is poorly understood whether the lipid composition of skeletal muscle changes by contraction, due to the complexity of lipid molecular species. In this study, we used matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) to investigate changes in skeletal muscle lipid composition induced by contraction. We successfully observed the reduction of diacylglycerol and triacylglycerol, which are generally associated with muscle contraction. Interestingly, we found the accumulation of some saturated and mono-unsaturated fatty acids and poly-unsaturated fatty acids containing phosphatidylcholine in contracted muscles. Moreover, the distributions of several types of lipid were changed by contraction. Our results show that changes in the lipid amount, lipid composition, and energy metabolic activity can be evaluated in each local spot of cells and tissues at the same time using MALDI-IMS. In conclusion, MALDI-IMS is a powerful tool for studying lipid changes associated with contractions.

Duchenne muscular dystrophy (DMD), an incurable and a progressive muscle wasting disease, is caused by the absence of dystrophin protein, leading to recurrent muscle fiber damage during contraction. The inflammatory response to fiber damage is a compelling candidate mechanism for disease exacerbation. The only established pharmacological treatment for DMD is corticosteroids to suppress muscle inflammation, however this treatment is limited by its insufficient therapeutic efficacy and considerable side effects. Recent reports show the therapeutic potential of inhibiting or enhancing pro- or anti-inflammatory factors released from DMD skeletal muscles, resulting in significant recovery from muscle atrophy and dysfunction. We discuss and review the recent findings of DMD inflammation and opportunities for drug development targeting specific releasing factors from skeletal muscles. It has been speculated that nonsteroidal anti-inflammatory drugs targeting specific inflammatory factors are more effective and have less side effects for DMD compared with steroidal drugs. For example, calcium channels, reactive oxygen species, and nuclear factor-κB signaling factors are the most promising targets as master regulators of inflammatory response in DMD skeletal muscles. If they are combined with an oligonucleotide-based exon skipping therapy to restore dystrophin expression, the anti-inflammatory drug therapies may address the present therapeutic limitation of low efficiency for DMD.

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