Effects of concentric and eccentric contractions on phosphorylation of MAPK(erk1/2) and MAPK(p38) in isolated rat skeletal muscle

Abstract

1. 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. 2. 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 (MAPK(erk1/2)) and stress-activated protein kinase p38 (MAPK(p38)). 3. Concentric contractions induced a 5-fold increase in MAPK(erk1/2) phosphorylation. Application of the antioxidants N-acetylcysteine (20 mM) or dithiothreitol (5 mM) suppressed concentric contraction-induced increase in MAPK(erk1/2) phosphorylation. Mild passive stretches of the muscle increased MAPK(erk1/2) 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 MAPK(p38). 4. 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 MAPK(erk1/2), respectively, whereas phosphorylation of MAPK(p38) increased by 7.6- and 1.9-fold (not significant), respectively. 5. We conclude that in isolated rat skeletal muscle an increase in phosphorylation of both MAPK(erk1/2) and MAPK(p38) is induced by mechanical alterations, whereas contraction-related metabolic/ionic changes (reactive oxygen species and acidosis) cause increased phosphorylation of MAPK(erk1/2) only. Thus, contraction-induced phosphorylation can be explained by the combined action of increased production of reactive oxygen species, acidification and mechanical perturbations for MAPK(erk1/2) and by high mechanical stress for MAPK(p38).

Figure 8. Original records of muscle force (top) and length (bottom) from an experiment with severe stretches

An isometric contraction was induced before (A) and at the end (D) of a series of severe stretches (B and C, the first and the last stretch, respectively). In order to induce high passive force, a resting muscle was prestretched to a passive tension close to half of isometric force. Then the muscle was intermittently stretched by 23 % of its optimal length at a speed ≈2.6 l_o s⁻¹. Horizontal bars represent 170 ms trains of stimuli at 100 Hz.

Figure 6. Original records of muscle force (top) and length (bottom) from an experiment with eccentric contractions

An isometric contraction was induced before (A) and at the end (D) of a series of eccentric contractions (B and C, the first and the last eccentric contraction, respectively). The muscle was initially stretched by 10 % of its optimal length at a speed 1 l_o s⁻¹. Horizontal bars represent 170 ms trains of stimuli at 100 Hz.

Figure 5. Original records of muscle force (top) and length (bottom) from an experiment with mild stretches

An isometric contraction was induced before (A) and at the end (D) of a series of intermittent passive stretches (B and C, the first and the last stretch, respectively). The muscle was stretched by 30 % of its optimal length at a speed 3 l_o s⁻¹. Horizontal bars represent 170 ms trains of stimuli at 100 Hz.

Figure 1. Original records of muscle force (top) and length (bottom) in a concentric contraction experiment

An isometric contraction was induced before (A) and at the end (D) of a series of concentric contractions (B and C). In concentric contractions, a rapid ramp release allowed the muscle to shorten at near maximal speed, thereby minimising force generation in the beginning of the series of contractions (B). By the end of the series, concentric force was undetectable (C). Horizontal bar represents 170 ms trains of stimuli at 100 Hz.

Figure 2. Concentric contractions of isolated muscle induce MAPK^erk1/2 but not MAPK^p38 phosphorylation

EDL muscles were subjected to concentric contractions (▪) and contralateral muscles served as resting controls (□). Representative immunoblots of phosphorylated and total proteins are presented above corresponding bars. Data are normalised to the corresponding average of resting controls. Results are presented as means ± s.e.m. for 6 muscles per bar. † P < 0.01 versus resting control.

Figure 3. Effect of antioxidants on concentric contraction-induced phosphorylation of MAPK^erk1/2

EDL muscles were incubated in 20 mm of N-acetylcysteine (NAC) or 5 mm of dithiothreitol (DTT) and subjected to concentric contractions (+conc) or an equally long rest period. Muscles treated with NAC or DTT (▪) are compared with contralateral muscles serving as resting controls (□). Representative immunoblots of phosphorylated and total proteins are presented above corresponding bars. Concentric contractions only (conc) from Fig. 2 are added for comparison. Data are normalised to the corresponding average of resting controls. Results are presented as means ± s.e.m. for 6 muscles per bar in all groups except DTT (5 muscles per bar). ‡ P < 0.05, ‡‡ P < 0.01 versus concentric contractions only.

Figure 4. Effect of acidosis and mild passive stretches on phosphorylation of MAPK^erk1/2

EDL muscles were acidified by exposure to 30 % CO₂ (acidosis) and/or subjected to intermittent passive stretches (stretch) (▪); contralateral muscles served as resting controls (□). Representative immunoblots of phosphorylated and total proteins are presented above corresponding bars. Data are normalised to the corresponding average of resting controls. Results are presented as means ± s.e.m. for 6 muscles per bar. † P < 0.01, *P < 0.05 versus resting control.

Figure 7. Effect of eccentric contractions and severe stretch on phosphorylation of MAPK^erk1/2 and MAPK^p38

EDL muscles were subjected (▪) to eccentric contractions (Ecc) or severe stretch. Contralateral muscles served as resting controls (□). Representative immunoblots of phosphorylated and total proteins are presented above corresponding bars. Data are normalised to the corresponding average of resting controls. Results are presented as means ± s.e.m. for 6 muscles per bar. * P < 0.05, † P < 0.01 versus resting control.