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.

Skeletal muscle is considered a secretory organ that produces bioactive proteins known as myokines, which are released in response to various stimuli. However, no experimental evidence exists regarding the mechanism by which acute muscle contraction regulates myokine secretion. Here, we present evidence that acute contractions induced myokine secretion from C2C12 myotubes. Changes in the cell culture medium unexpectedly triggered the release of large amounts of proteins from the myotubes, and these proteins obscured the contraction-induced myokine secretion. Once protein release was abolished, the secretion of interleukin-6 (IL-6), the best-known regulatory myokine, increased in response to a 1-hour contraction evoked by electrical stimulation. Using this experimental condition, intracellular calcium flux, rather than the contraction itself, triggered contraction-induced IL-6 secretion. This is the first report to show an evidence for acute contraction-induced myokine secretion by skeletal muscle cells.

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Myokines are skeletal muscle-derived hormones. In this study, using a C2C12 myotube contraction system, we sought to determine whether the skeletal muscle secreted thioredoxin (TRX) and related redox proteins. Redox proteins such as TRXs, peroxiredoxins, and glutaredoxins were detected in the C2C12 myotube culture medium in the absence of any stimulation. The amounts of TRXs, peroxiredoxins, and glutaredoxins secreted by the C2C12 myotubes were not affected by the contraction, unless the myotubes were injured. Because TRX-1 was known to be a secreted protein that lacks a signal peptide, we examined whether this protein was secreted via exosome vesicles. The results indicated that TRX-1 was not secreted via exosome vesicles. We concluded that TRX-1 and related redox proteins are myokines that are constitutively secreted by the skeletal muscle cells. Although the mechanism of TRX-1 secretion remains unclear, our findings suggest that the skeletal muscle is an endocrine organ and the redox proteins that are constitutively secreted from the skeletal muscle may exert antioxidant and systemic health-promoting effects.

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Case summaryA 10-month-old male domestic shorthair cat was brought to Kitanomori Animal Hospital for routine castration. Preoperative thoracic radiography revealed a mildly enlarged heart silhouette, and transthoracic echocardiography (ECHO) suggested a quadricuspid aortic valve associated with central aortic regurgitation (regurgitant fraction 31%). After sedation with intramuscular medetomidine and midazolam for castration, heart rate decreased from 193 to 76 beats per minute. ECHO under sedation revealed two equally small and two equally large aortic valve cusps, suggesting a type C quadricuspid aortic valve. The findings were confirmed by real-time three-dimensional ECHO.Relevance and novel informationThis case reveals the echocardiographic features of a feline quadricuspid aortic valve and shows that transthoracic ECHO is useful to examine aortic valve morphology in cats. It also suggests that echocardiographic screening may be beneficial for detecting congenital cardiac anomalies in apparently healthy cats.

Skeletal muscle has only recently been considered a secretory organ. Muscle-derived proteins are now termed myokines. Until date, about 20 proteins known as cytokines, growth factors, and adipokines have been reported as myokines. However, only a few studies have been able to demonstrate secretion from the skeletal muscle. Furthermore, many reports are still uncertain of whether proteins are secreted from skeletal muscle cells or from the surrounding tissue, because some studies have measured myokine concentration in blood taken from human and animal subjects, which also contains other organ-derived proteins. Secretion of some myokines is promoted by muscle contraction or insulin stimulation, whereas others seem to be constitutively secreted. The mechanisms of action and roles of myokines are also complicated. Some are believed to affect distant organs through endocrine and paracrine mechanisms, while others affect organs through an autocrine mechanism. In this article, we review updates of myokines, including their history. Furthermore, the article discusses the need to re-define myokines in order to avoid possible misunderstandings because of insufficient data.

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