Jan Lännergren

Muscle cell function during prolonged activity: cellular mechanisms of fatigue

Muscle performance declines during prolonged and intense activity; important components are a reduction in force production and shortening velocity and a prolongation of relaxation. In this review we consider how the changes in metabolites (particularly H+, inorganic phosphate (Pi), ATP and ADP) and changes in sarcoplasmic reticulum Ca2+ release lead to the observed changes in force, shortening velocity and relaxation. The reduced force is caused by a combination of reduced maximum force‐generating capacity, reduced myofibrillar Ca2+ sensitivity and reduced Ca2+ release. The reduced maximum force and Ca2+ sensitivity are largely explained by the effects of H+ and Pi that have been observed in skinned fibres. At least three different forms of reduced Ca2+ release can be recognized but the mechanisms involved are incompletely understood. The reduced shortening velocity can be partly explained by the effects of H+ that have been observed in skinned fibres. In addition it is proposed that ADP, which depresses shortening velocity, increases during contractions to a level that is considerably higher than existing measurements suggest. Changes in Ca2+ release are probably unimportant for the reduced shortening velocity. The prolongation of relaxation can arise both from slowing of the rate of decline of myoplasmic calcium concentration and from slowing of cross‐bridge detachment rates. A method of analysis which separates these components is described. The increase in H+ and the other metabolite changes during fatigue can independently affect both components. Finally we show that reduced force, shortening velocity and slowed relaxation all contribute to the decline in muscle performance during a working cycle in which the muscle first shortens actively and then is stretched passively by an antagonist muscle.

The effect of intracellular pH on contractile function of intact, single fibres of mouse muscle declines with increasing temperature.

1. The effect of altered intracellular pH (pHi) on isometric contractions and shortening velocity at 12, 22 and 32 degrees C was studied in intact, single fibres of mouse skeletal muscle. Changes in pHi were obtained by exposing fibres to solutions with different CO2 concentrations. 2. Under control conditions (5% CO2), pHi (measured with carboxy SNARF‐1) was about 0.3 pH units more alkaline than neutral water at each temperature. An acidification of about 0.5 pH units was produced by 30% CO2 and an alkalinization of similar size by 0% CO2. 3. In acidified fibres tetanic force was reduced by 28% at 12 degrees C but only by 10% at 32 degrees C. The force increase with alkalinization showed a similar reduction with increasing temperature. Acidification caused a marked slowing of relaxation and this slowing became less with increasing temperature. 4. Acidification reduced the maximum shortening velocity (V0) by almost 20% at 12 degrees C, but had no significant effect at 32 degrees C. Alkalinization had no significant effect on V0 at any temperature. 5. In conclusion, the effect of pHi on contraction of mammalian muscle declines markedly with increasing temperature. Thus, the direct inhibition of force production by acidification is not a major factor in muscle fatigue at physiological temperatures.

Functional significance of Ca2+ in long-lasting fatigue of skeletal muscle

Abstract Repeated activation of skeletal muscle causes fatigue, which involves a reduced ability to produce force and slowed contraction regarding both the speed of shortening and relaxation. One important component in skeletal muscle fatigue is a reduced sarcoplasmic reticulum (SR) Ca2+ release. In the present review we will describe different types of fatigue-induced inhibition of SR Ca2+ release. We will focus on a type of long-lasting failure of SR Ca2+ release which is called low-frequency fatigue, because this type of fatigue may be involved in the muscle dysfunction and chronic pain experienced by computer workers. Paradoxically it appears that the Ca2+ released from the SR, which is required for contraction, may actually be responsible for the failure of SR Ca2+ release during low-frequency fatigue. We will also discuss the relationship between gross morphological changes in muscle fibres and long-lasting failure of SR Ca2+ release. Finally, a model linking muscle cell dysfunction and muscle pain is proposed.

The temperature dependence of isometric contractions of single, intact fibres dissected from a mouse foot muscle.

1. Isometric tension responses to electrical stimulation have been studied at 7.5 37.5 degrees C in single, intact fibres of the flexor digitorum brevis muscle of the mouse. A large number of reproducible tetani could be obtained at temperatures less than or equal to 35 degrees C. 2. The tetanic force per cross‐sectional area generated at 25.0 degrees C was 375 +/‐ 56 kPa (mean +/‐ S.D., n = 16). 3. The curve relating maximum tetanic tension to temperature exhibited a transition between a level of almost unaltered force (25.0‐32.5 degrees C) and a marked force decline (less than or equal to 20.0 degrees C). At temperatures higher than 35.0 degrees C force production was markedly depressed and this reduction was in some cases irreversible. 4. Twitch tension showed less regular dependence on temperature; it was reduced less than tetanic tension at low temperatures. Thus, the twitch/tetanus tension ratio was higher at low temperatures. 5. The times for twitch contraction and for twitch half‐relaxation (i) ranged from 7 to 14 ms and from 6 to 15 ms at 35.0 degrees C and (ii) exhibited Q10 values of 3.2 +/‐ 0.4 and 4.0 +/‐ 0.6, respectively. 6. It is concluded that it is possible to use intact, single fibres dissected from mammalian skeletal muscle in physiological studies. Our results are close to previous results obtained from mammalian muscles except that the tetanic tension per cross‐sectional area was found to be higher than commonly reported.

Effects of concentric and eccentric contractions on phosphorylation of MAPKerk1/2 and MAPKp38 in isolated rat skeletal muscle

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 (MAPKerk1/2) and stress‐activated protein kinase p38 (MAPKp38). 3 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. 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 MAPKerk1/2, respectively, whereas phosphorylation of MAPKp38 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 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.

Force decline due to fatigue and intracellular acidification in isolated fibres from mouse skeletal muscle.

1. Single, intact muscle fibres from the flexor brevis foot muscle of the mouse have been fatigued at 25 degrees C by 350 ms, 70 Hz stimulation trains, initially delivered every 3.8 s and then at stepwise decreasing intervals until tension was down to about 30% of the original (Po). Rested fibres generated a specific force of 372 +/‐ 8.4 kPa (mean +/‐ S.E.M., n = 25). 2. Endurance, defined as time to attain 0.5 Po, varied from 2.5 to 24 min, with the majority of fibres falling in the range 4‐8 min, corresponding to 70‐160 tetani. In all fibres where it was followed, tension recovery after cessation of stimulation was 90% or better. 3. Tetanic force declined in a characteristic way during fatiguing stimulation: initially tension fell to about 0.85 Po during eight to fourteen tetani (phase 1), then followed a long period of nearly steady tension generation (phase 2) and finally there was a rapid force decline (phase 3). 4. Caffeine (15 or 25 mM) caused a slight potentiation of tetanic force in the rested state (4.7 +/‐ 0.9%, n = 21) and slowed relaxation. No change in resting tension was seen with caffeine at concentrations up to 25 mM. 5. Caffeine (15‐25 mM) caused a rapid and dramatic increase in tetanic force when applied to severely fatigued fibres: force output rose from 29.8 +/‐ 1.5 to 82.5 +/‐ 1.2% (n = 13) of Po. During phase 2 force potentiation with caffeine was much smaller. 6. A 10 s pause resulted in a large, transient force increase when imposed during phase 3 but had little effect on force production during phase 2. 7. Intracellular acidosis, induced by superfusion with Tyrode solution gassed with 30% CO2 instead of the normal 5% (extracellular pH 6.5 vs. 7.3), resulted in a fall in tetanic tension to about 0.85 Po (n = 7). This depression could to some extent be counteracted by 15 mM‐caffeine, which brought tension back to about 0.90 Po. 8. It is concluded that there are at least two mechanisms for force decline during fatiguing stimulation: one which manifests itself early and is likely to be related to cross‐bridge function and another representing deficient Ca2+ handling which becomes prominent at a later stage. For severe fatigue (0.3 Po) the latter mechanism is dominant.

The force—velocity relation of isolated twitch and slow muscle fibres of Xenopus laevis

1. A study has been made of the relation between force and speed of shortening, or lengthening, in isolated twitch and slow muscle fibres, dissected from the iliofibularis muscle of Xenopus laevis. Both after‐loaded and quick‐release contractions were studied. Twitch fibres were stimulated electrically to give tetanic contractions (5‐20 °C); slow fibres were activated by a rapid change to solutions with high K concentration (30‐75 m M; experiments at 21‐24 °C).

Contractile properties and myosin isoenzymes of various kinds ofXenopus twitch muscle fibres

SummarySingle twitch muscle fibres have been isolated from various parts of the iliofibularis muscle ofXenopus laevis. After measurements of their isotonic contractile properties, myosin extraction was performed on individual fibres and the extract analysed by various forms of gel electrophoresis. In agreement with previous results three major fibre types, types 1, 2, and 3 could be discerned. Both mechanical data and native isomyosin patterns indicated a further subdivision of types 1 and 2 into subtypes (1n, 1s and 2f, 2n, respectively). Transitional forms between 2n and type 3 were also identified. Types 1 and 2 had the same kinds of light chains (LC1f, LC2, LC3f), but different heavy chains (HC) as observed on 7% SDS gels. Type 3 lacked LC3 and had a more slowly migrating LC1 (LC1s); their HC migration velocity was indistinguishable from that of type 2 HC. A comparison was made between LC1/LC3 ratio and contractile parameters for nine type 1n fibres and six type 2n fibres. No clear correlation was observed between light chain proportions on the one hand and force per cross-sectional area or shortening velocity on the other. It is concluded that myosin heavy chain composition is the major determinant for contractile performance inXenopus skeletal muscle fibres.

Mitochondrial and myoplasmic [Ca2+] in single fibres from mouse limb muscles during repeated tetanic contractions

Previous studies on single fast‐twitch fibres from mouse toe muscles have shown marked fatigue‐induced changes in the free myoplasmic [Ca2+] ([Ca2+]i), while mitochondrial [Ca2+] remained unchanged. We have now investigated whether muscle fibres from the legs of mice respond in a similar way. Intact, single fibres were dissected from the soleus and extensor digitorum longus (EDL) muscles of adult mice. To measure [Ca2+]i, indo‐1 was injected into the isolated fibres. Mitochondrial [Ca2+] was measured using Rhod‐2 and confocal laser microscopy. Fatigue was induced by up to 1000 tetanic contractions (70 Hz) given at 2 s intervals. In soleus fibres, there was no significant decrease in tetanic [Ca2+]i at the end of the fatiguing stimulation, whereas tetanic force was significantly reduced by about 30 %. In 10 out of 14 soleus fibres loaded with Rhod‐2 and subjected to fatigue, mitochondrial [Ca2+] increased to a maximum after about 50 tetani; this increase was fully reversed within 20 min after the end of stimulation. The force‐frequency curve of the non‐responding soleus fibres was shifted to higher frequencies compared to that of the responding fibres. In addition, eight out of nine Rhod‐2‐loaded EDL fibres showed similar changes in mitochondrial [Ca2+] during and after a period of fatiguing stimulation. The stimulation‐induced increase in mitochondrial [Ca2+] was reduced when mitochondria were depolarised by application of carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone, whereas it was increased by application of an inhibitor of the mitochondrial Na+/Ca2+ exchange (CGP‐37157). In conclusion, isolated slow‐twitch muscle fibres show only modest changes in tetanic force and [Ca2+]i during repeated contractions. The increase in mitochondrial Ca2+ does not appear to be essential for activation of mitochondrial ATP production, nor does it cause muscle damage.

Role of Excitation-Contraction Coupling in Muscle Fatigue*

SummaryThe force produced by muscles declines during prolonged activity and this decline arises largely from processes within the muscle. At a cellular level the reduced force could be caused by: (a) reduced intracellular calcium release during activity; (b) reduced sensitivity of the myofilaments to calcium; or (c) reduced maximal force development. Experiments involving intracellular calcium measurements in isolated single fibres show that all 3 of the above contribute to the decline of force during fatigue. Metabolic changes associated with fatigue are probably involved in each of the 3 factors. Thus the accumulation of phosphate and protons which occur during fatigue cause a reduction in calcium sensitivity and a decline in maximal force. The cause of the reduced intracellular calcium during contractions in fatigue is less clear. During prolonged tetani the conduction of the action potential in the T-tubules appears to fail leading to reduced intracellular calcium in the central part of the muscle fibre. However, during repeated tetani there is a uniform decline of intracellular calcium across the fibre and this remains one of the least understood processes which contribute to fatigue.