Exercise and contractions of isolated skeletal muscle induce phosphorylation of mitogen‐activated protein kinases (MAPKs) by undefined mechanisms. The aim of the present study was to determine exercise‐related triggering factors for the increased phosphorylation of MAPKs in isolated rat extensor digitorum longus (EDL) muscle. Concentric or eccentric contractions, or mild or severe passive stretches were used to discriminate between effects of metabolic/ionic and mechanical alterations on phosphorylation of two MAPKs: extracellular signal‐regulated kinase 1 and 2 (MAPKerk1/2) and stress‐activated protein kinase p38 (MAPKp38). Concentric contractions induced a 5‐fold increase in MAPKerk1/2 phosphorylation. Application of the antioxidants N‐acetylcysteine (20 mM) or dithiothreitol (5 mM) suppressed concentric contraction‐induced increase in MAPKerk1/2 phosphorylation. Mild passive stretches of the muscle increased MAPKerk1/2 phosphorylation by 1.8‐fold, whereas the combination of acidosis and passive stretches resulted in a 2.8‐fold increase. Neither concentric contractions, nor mild stretches nor acidosis significantly affected phosphorylation of MAPKp38. High force applied upon muscle by means of either eccentric contractions or severe passive stretches resulted in 5.7‐ and 9.5‐fold increases of phosphorylated MAPKerk1/2, respectively, whereas phosphorylation of MAPKp38 increased by 7.6‐ and 1.9‐fold (not significant), respectively. We conclude that in isolated rat skeletal muscle an increase in phosphorylation of both MAPKerk1/2 and MAPKp38 is induced by mechanical alterations, whereas contraction‐related metabolic/ionic changes (reactive oxygen species and acidosis) cause increased phosphorylation of MAPKerk1/2 only. Thus, contraction‐induced phosphorylation can be explained by the combined action of increased production of reactive oxygen species, acidification and mechanical perturbations for MAPKerk1/2 and by high mechanical stress for MAPKp38.

The mitogen-activated protein (MAP) kinase pathways have been highlighted as a possible link between exercise and adaptive changes in skeletal muscle. In this study, the effect of exercise intensity on the activation of the ERK/MAP kinase pathway was investigated in human skeletal muscle. One-leg exercise at low (40% maximal oxygen consumption, VO2max for 30 min) and high (75% VO2max for 30 min) intensity resulted in 11.5+8. I-fold and 39.7+/-6.3-fold (mean +/-SEM) increases in ERK1/2 phosphorylation (P<0.001), respectively. The phosphorylation of MEK1/2, the upstream kinase of ERK1/2, increased with exercise intensity (P<0.05) to 2.5+/-0.9 and 4.8+/-1.1 times the basal level at the low and high intensity, respectively. The statistical analysis revealed a systematic difference between basal, low and high intensity exercise levels for both kinases. There was no change in the phosphorylation of either kinase in the non-exercised leg. The phosphorylation of the transcription factor cyclic AMP response element binding protein (CREB), a possible downstream target of the ERK/MAP kinase signalling pathway, was unaffected by exercise. The phosphorylation of ERK1/2 was significantly higher in purified freeze-dried compared to crude wet muscle after exercise, whereas the opposite pattern was observed for CREB. In conclusion, phosphorylation of ERK1/2 and MEK1/2 increases in an exercise intensity-dependent manner in human skeletal muscle and this seems to originate in the muscle fibres themselves.

The involvement of Ca2+ in insulin‐mediated glucose uptake in skeletal muscle is uncertain. Here we study the possible role of Ca2+ influx via canonical transient receptor potential 3 (TRPC3) channels in insulin‐mediated glucose uptake. Experiments were performed on adult skeletal mouse muscle fibers. Ca2+ influx and glucose uptake were measured with fluorescent indicators and confocal microscopy. TRPC3 protein expression was knocked down using a novel technique where functionalized carbon nanotubes were used to transfect cells with small interfering RNA. The interaction between TRPC3 and the glucose transporter 4 (GLUT4) was studied with immunoprecipitation and immunofluorescence staining. Knock down of TRPC3 resulted in ∼ 80% decrease in insulin‐mediated glucose uptake. TRPC3 can be activated by diacylglycerol (DAG) and knock down of TRPC3 inhibited the DAG‐induced Ca2+ influx. TRPC3 and GLUT4 co‐immunoprecipitated and showed co‐localization in the proximity of the t‐tubular system, which is the major site of insulin‐mediated glucose transport. In conclusion, TRPC3 interacts functionally and physically with GLUT4 and Ca2+ influx through TRPC3 has a large impact on insulin‐mediated glucose uptake.

Activation of mitogen-activated protein (MAP) kinases has been implicated in the signal transduction pathways linking exercise to adaptive changes of muscle protein expression. In the present study, we investigated whether contractions of isolated muscles induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38 MAPK in a fibre-type dependent manner. Slow-twitch (soleus) and fast-twitch (epitrochlearis, extensor digitorum longus) rat skeletal muscles were exposed to intermittent tetanic stimulation. Compared with the contralateral non-stimulated muscle, contractions increased ERK1/2 phosphorylation to the same extent in fast- and slow-twitch muscles. Significant increase in phosphorylation of p38 MAPK was observed in the fast-twitch muscles only. The total amount of ERK1/2 and p38 MAPK proteins was higher in the slow-twitch soleus muscle. In conclusion, MAP kinase signalling pathways are differentially activated and expressed in slow- and fast-twitch muscles. In addition, this activation is owing to muscle contraction per se and do not demand additional external influence.

Muscle performance is improved after a brief period of exercise (warm-up). One factor that is known to strongly affect force production is the myoplasmic concentration of inorganic phosphate ([P(i)]). Improved performance after warm-up may therefore be due to a reduction of [P(i)]. Herein, we show that after a warm-up protocol (15 tetani at 2-s intervals), tetanic force is increased by approximately 6% (P < 0.05) and [P(i)] is almost halved (P < 0.05) in isolated mouse soleus muscle. A warm-up protocol with longer intervals (15 tetani at 5-s intervals) reduced tetanic force and did not alter [P(i)]. We conclude that a reduction of [P(i)] contributes to the force-potentiating effect of warm-up.