Graham D. Lamb

Skeletal Muscle Fatigue: Cellular Mechanisms

Repeated, intense use of muscles leads to a decline in performance known as muscle fatigue. Many muscle properties change during fatigue including the action potential, extracellular and intracellular ions, and many intracellular metabolites. A range of mechanisms have been identified that contribute to the decline of performance. The traditional explanation, accumulation of intracellular lactate and hydrogen ions causing impaired function of the contractile proteins, is probably of limited importance in mammals. Alternative explanations that will be considered are the effects of ionic changes on the action potential, failure of SR Ca2+ release by various mechanisms, and the effects of reactive oxygen species. Many different activities lead to fatigue, and an important challenge is to identify the various mechanisms that contribute under different circumstances. Most of the mechanistic studies of fatigue are on isolated animal tissues, and another major challenge is to use the knowledge generated in these studies to identify the mechanisms of fatigue in intact animals and particularly in human diseases.

Effects of creatine phosphate and P(i) on Ca2+ movements and tension development in rat skinned skeletal muscle fibres.

1. Mechanically skinned fast‐twitch (FT) and slow‐twitch (ST) muscle fibres of the rat were used to investigate the effects of fatigue‐like changes in creatine phosphate (CP) and inorganic phosphate (P(i)) concentration on Ca(2+)‐activation properties of the myofilaments as well as Ca2+ movements into and out of the sarcoplasmic reticulum (SR). 2. Decreasing CP from 50 mM to zero in FT fibres increased maximum Ca(2+)‐activated tension (Tmax) by 16 +/‐ 2% and shifted the mid‐point of the tension‐pCa relation (pCa50) to the left by 0.28 +/‐ 0.03 pCa units. In ST fibres, a decrease of CP from 25 mM to zero increased Tmax by 9 +/‐ 1% and increased the pCa50 by 0.16 +/‐ 0.01 pCa units. The effect of CP on Tmax was suppressed in both fibre types by prior treatment with 0.3 mM FDNB (1‐fluoro‐2,4‐dinitrobenzene), suggesting that these effects may occur via changes in creatine kinase activity. 3. Increases of P(i) in the range 0‐50 mM reduced the pCa50 and Tmax in both fibre types. These effects were more pronounced in ST fibres than in FT fibres in absolute terms. However, normalization of the results to resting P(i) levels appropriate to both fibre types (1 mM for FT and 5 mM for ST fibres) revealed similar decreases in Tmax (approximately 39% at 25 mM P(i) and approximately 48% at 50 mM P(i)) and pCa50 (0.25 pCa units at 25‐50 mM P(i)). The depressant action of P(i) on both parameters was considerably reduced when the rise in P(i) was accompanied by an equivalent reduction in [CP]. 4. Tension development in the presence of complex, fatigue‐like milieu changes (40 mM P(i) for FT; 20 mM P(i) for ST) was decreased by 35‐40% at a constant myoplasmic [Ca2+] of 6 microM in both fibre types. 5. SR Ca2+ loading at a myoplasmic [Ca2+] of 100 nM was found to increase abruptly when the [P(i)] during loading was increased to near 9 mM. At a myoplasmic [Ca2+] of 300 nM, the threshold P(i) for this effect dropped to approximately 3 mM. 6. Tension responses evoked by caffeine in the absence of P(i) were smaller and slower to peak if fibres were exposed to P(i) in a restricted myoplasmic Ca2+ pool after SR Ca2+ loading. This indicated that myoplasmic P(i) can decrease and prolong the rate of Ca2+ release from the SR.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of intracellular pH and [Mg2+] on excitation‐contraction coupling in skeletal muscle fibres of the rat.

1. The effects on normal excitation‐contraction (E‐C) coupling of two important intracellular ions, H+ and Mg2+, were examined in skinned fibres from the extensor digitorum longus muscle of rat. 2. A single depolarization (2‐3 s duration) in the presence of 1 mM Mg2+ (pH 7.1, 23 degrees C) released most of the available Ca2+ in the sarcoplasmic reticulum (SR), but a similar depolarization in the presence of 10 mM Mg2+ was unable to release almost any Ca2+. Thus, raised [Mg2+] potently inhibits depolarization‐induced Ca2+ release in mammalian muscle. 3. Depolarization at pH 6.2 (1 mM Mg2+, 23 degrees C) induced a large force response, which was on average 78 +/‐ 2%, n = 6, of the depolarization‐induced response at pH 7.1; this reduction resulted from a corresponding reduction in maximum Ca(2+)‐activated force at pH 6.2. Similar results were obtained at 37 degrees C. Also, a single depolarization at pH 6.2 caused almost complete depletion of the releasable Ca2+ in the SR. Thus, low pH does not prevent depolarization‐induced Ca2+ release in mammalian muscle. 4. Lowering the free [Mg2+] from 1 mM to 15 microM caused massive release of Ca2+, and depletion of the SR, at both pH 7.1 and 6.2, indicating that over this pH range, H+ did not readily substitute for Mg2+ at its inhibitory site on the Ca2+ release channel.(ABSTRACT TRUNCATED AT 250 WORDS)

RAISED INTRACELLULAR CA2+ ABOLISHES EXCITATION-CONTRACTION COUPLING IN SKELETAL MUSCLE FIBRES OF RAT AND TOAD

1. Raising the intracellular [Ca2+] for 10 s at 23 degrees C abolished depolarization‐induced force responses in mechanically skinned muscle fibres of toad and rat (half‐maximal effect at 10 and 23 microM, respectively), without affecting the ability of caffeine or low [Mg2+] to open the ryanodine receptor (RyR)/Ca2+ release channels. Thus, excitation‐contraction coupling was lost, even though the Ca2+ release channels were still functional. Coupling could not be restored in the duration of an experiment (up to 1 h). 2. The Ca(2+)‐dependent uncoupling had a Q10 > 3.5, and was three times slower at pH 5.8 than at pH 7.1. Sr2+ caused similar uncoupling at twenty times higher concentration, but Mg2+, even at 10 mM, was ineffective. Uncoupling was not noticeably affected by removal of ATP or application of protein kinase or phosphatase inhibitors. 3. Confocal laser scanning microscopy showed that the transverse tubular system was sealed in its entirety in mechanically skinned fibres and that its integrity was maintained in uncoupled fibres. Electron microscopy revealed distorted or severed triad junctions and Z‐line aberrations in uncoupled fibres. 4. Only when uncoupling was induced at a relatively slow rate (e.g. over 60 s with 2.5 microM Ca2+) could it be prevented by the protease inhibitor leupeptin (1 mM). Immunostaining of Western blots showed no evidence of proteolysis of the RyR, the alpha 1‐subunit of dihydropyridine receptor (DHPR) or triadin in uncoupled fibres. 5. Fibres which, whilst intact, were stimulated repeatedly by potassium depolarization with simultaneous application of 30 mM caffeine showed reduced responsiveness after skinning to depolarization but not to caffeine. Rapid release of endogenous Ca2+, or raised [Ca2+] under conditions which minimized the loss of endogenous diffusible myoplasmic molecules from the skinned fibre, caused complete uncoupling. Taken together, these results suggest that Ca(2+)‐dependent uncoupling can also occur in intact fibres. 6. This Ca(2+)‐dependent loss of depolarization‐induced Ca2+ release may play an important feedback role in muscle by stopping Ca2+ release in localized areas where it is excessive and may be responsible for long‐lasting muscle fatigue after severe exercise, as well as contributing to muscle weakness in various dystrophies.

Excitation–Contraction Coupling In Skeletal Muscle: Comparisons With Cardiac Muscle

1. The present review describes the mechanisms involved in controlling Ca2+ release from the sarcoplasmic reticulum (SR) of skeletal muscle, which ultimately regulates contraction.

Effect of Mg2+ on the control of Ca2+ release in skeletal muscle fibres of the toad.

1. The effect of myoplasmic Mg2+ on Ca2+ release was examined in mechanically skinned skeletal muscle fibres, in which the normal voltage‐sensor control of Ca2+ release is preserved. The voltage sensors could be activated by depolarizing the transverse tubular (T‐) system by lowering the [K+] in the bathing solution. 2. Fibres spontaneously contracted when the free [Mg2+] was decreased from 1 to 0.05 mM, with no depolarization or change of total ATP, [Ca2+] or pH (pCa 6.7, 50 microM‐EGTA). After such a ‘low‐Mg2+ response’ the sarcoplasmic reticulum (SR) was depleted of Ca2+ and neither depolarization nor caffeine (2 mM) could induce a response, unless the [Mg2+] was raised and the SR reloaded with Ca2+. Exposure to 0.05 mM‐Mg2+ at low [Ca2+] (2 mM‐free EGTA, pCa greater than 8.7) also induced Ca2+ release and depleted the SR. 3. The response to low [Mg2+] was unaffected by inactivation of the voltage sensors, but was completely blocked by 2 microM‐Ruthenium Red indicating that it involved Ca2+ efflux through the normal Ca2+ release channels. 4. In the absence of ATP (and creatine phosphate), complete removal of Mg2+ (i.e. no added Mg2+ with 1 mM‐EDTA) did not induce Ca2+ release. Depolarization in the absence of Mg2+ and ATP also did not induce Ca2+ release. 5. Depolarization in 10 mM‐Mg2+ (pCa 6.7, 50 microM‐EGTA, 8 mM‐total ATP) did not produce any response. In the presence of 1 mM‐EGTA to chelate most of the released Ca2+, depolarizations in 10 mM‐Mg2+ did not noticeably deplete the SR of Ca2+, whereas a single depolarization in 1 mM‐Mg2+ (and 1 mM‐EGTA) resulted in marked depletion. Depolarization in the presence of D600 and 10 mM‐Mg2+ produced use‐dependent ‘paralysis’, indicating that depolarization in 10 mM‐Mg2+ did indeed activate the voltage sensors. 6. Depolarization in the presence of 10 mM‐Mg2+ and 25 microM‐ryanodine neither interfered with the normal voltage control of Ca2+ release nor caused depletion of the Ca2+ in the SR even after returning to 1 mM‐Mg2+ for 1 min, indicating that few if any of the release channels had been opened by the depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium release in skinned muscle fibres of the toad by transverse tubule depolarization or by direct stimulation.

1. Skeletal muscle fibres from the toad were mechanically skinned under paraffin oil and then bathed in a potassium HDTA solution (HDTA: hexamethylenediamine‐tetraacetate) which mimicked the ionic composition of the myoplasm. 2. Rapid transient contractions could be triggered by substitution of K+ with Na+ (with no change of anion), which should have virtually no direct effect on the electrical polarization of the sarcoplasmic reticulum (SR) membrane. Up to thirty or more contractions could be evoked by repeated substitutions if there was sufficient ‘repriming’ time (about 30 s) between them; these rapid contractions were analagous to potassium contractures in intact fibres. 3. When the SR was not heavily loaded, substitution of potassium HDTA with choline chloride also produced a rapid, brief contraction. 4. All treatments designed to ‘inactivate’ the voltage sensor in the T‐system invariably abolished the rapid contractions. Thus, rapid contractions were absent if (i) the T‐system was permanently depolarized by pre‐soaking the muscle in a high potassium solution with ouabain before skinning, (ii) a fibre was split rather than skinned, (iii) the T‐system was temporarily depolarized by Na+ substitution immediately before choline chloride substitution, or vice versa, (iv) a skinned fibre was briefly exposed to saponin (50 micrograms/ml) to selectively disrupt the T‐system membrane or (v) the muscle was pre‐soaked in a solution with 1 mM‐EGTA and no Ca2+ or Mg2+ before skinning. In contrast to (v), if 10 mM‐Mg2+ was present in the EGTA solution before skinning, rapid contractions could be elicited, presumably because the presence of Mg2+ prevented the inactivation of the T‐system voltage sensor in low [Ca2+]. 5. These results unequivocally demonstrate that (a) the T‐system reseals and repolarizes after mechanical skinning under oil and (b) the fast contractions are produced by activation of the voltage sensor in the T‐system. 6. When the SR had been heavily loaded, choline chloride substitution (but not Na+ substitution) could also induce an unphysiological, slow contraction (second component’). In total contrast to the fast contraction, this slow component was unaffected by any of the treatments (i‐v) above, indicating that it did not depend on activation of the voltage sensor in the T‐system but resulted from a direct action of choline chloride on the SR.(ABSTRACT TRUNCATED AT 400 WORDS)

Calsequestrin content and SERCA determine normal and maximal Ca2+ storage levels in sarcoplasmic reticulum of fast- and slow-twitch fibres of rat

Whilst calsequestrin (CSQ) is widely recognized as the primary Ca2+ buffer in the sarcoplasmic reticulum (SR) in skeletal muscle fibres, its total buffering capacity and importance have come into question. This study quantified the absolute amount of CSQ isoform 1 (CSQ1, the primary isoform) present in rat extensor digitorum longus (EDL) and soleus fibres, and related this to their endogenous and maximal SR Ca2+ content. Using Western blotting, the entire constituents of minute samples of muscle homogenates or segments of individual muscle fibres were compared with known amounts of purified CSQ1. The fidelity of the analysis was proven by examining the relative signal intensity when mixing muscle samples and purified CSQ1. The CSQ1 contents of EDL fibres, almost exclusively type II fibres, and soleus type I fibres [SOL (I)] were, respectively, 36 ± 2 and 10 ± 1 μmol (l fibre volume)−1, quantitatively accounting for the maximal SR Ca2+ content of each. Soleus type II [SOL (II)] fibres (∼20% of soleus fibres) had an intermediate amount of CSQ1. Every SOL (I) fibre examined also contained some CSQ isoform 2 (CSQ2), which was absent in every EDL and other type II fibre except for trace amounts in one case. Every EDL and other type II fibre had a high density of SERCA1, the fast‐twitch muscle sarco(endo)plasmic reticulum Ca2+‐ATPase isoform, whereas there was virtually no SERCA1 in any SOL (I) fibre. Maximal SR Ca2+ content measured in skinned fibres increased with CSQ1 content, and the ratio of endogenous to maximal Ca2+ content was inversely correlated with CSQ1 content. The relative SR Ca2+ content that could be maintained in resting cytoplasmic conditions was found to be much lower in EDL fibres than in SOL (I) fibres (∼20 versus >60%). Leakage of Ca2+ from the SR in EDL fibres could be substantially reduced with a SR Ca2+ pump blocker and increased by adding creatine to buffer cytoplasmic [ADP] at a higher level, both results indicating that at least part of the Ca2+ leakage occurred through SERCA. It is concluded that CSQ1 plays an important role in EDL muscle fibres by providing a large total pool of releasable Ca2+ in the SR whilst maintaining free [Ca2+] in the SR at sufficiently low levels that Ca2+ leakage through the high density of SERCA1 pumps does not metabolically compromise muscle function.

Acute effects of reactive oxygen and nitrogen species on the contractile function of skeletal muscle

Reactive oxygen and nitrogen species (ROS/RNS) are important for skeletal muscle function under both physiological and pathological conditions. ROS/RNS induce long‐term and acute effects and the latter are the focus of the present review. Upon repeated muscle activation both oxygen and nitrogen free radicals likely increase and acutely affect contractile function. Although fluorescent indicators often detect only modest increases in ROS during repeated activation, there are numerous studies showing that manipulations of ROS can affect muscle fatigue development and recovery. Exposure of intact muscle fibres to the oxidant hydrogen peroxide (H2O2) affects mainly the myofibrillar function, where an initial increase in Ca2+ sensitivity is followed by a decrease. Experiments on skinned fibres show that these effects can be attributed to H2O2 interacting with glutathione and myoglobin, respectively. The primary RNS, nitric oxide (NO•), may also acutely affect myofibrillar function and decrease the Ca2+ sensitivity. H2O2 can oxidize the sarcoplasmic reticulum Ca2+ release channels. This oxidation has a large stimulatory effect on Ca2+‐induced Ca2+ release of isolated channels, whereas it has little or no effect on the physiological, action potential‐induced Ca2+ release in skinned and intact muscle fibres. Thus, acute effects of ROS/RNS on muscle function are likely to be mediated by changes in myofibrillar Ca2+ sensitivity, which can contribute to the development of muscle fatigue or alternatively help counter it.

Calcium currents, charge movement and dihydropyridine binding in fast- and slow-twitch muscles of rat and rabbit.

1. The Vaseline‐gap technique was used to record slow calcium currents and asymmetric charge movement in single fibres of fast‐twitch muscles (extensor digitorum longus (e.d.l.) and sternomastoid) and slow‐twitch muscles (soleus) from rat and rabbit, at a holding potential of ‐90 mV. 2. The slow calcium current in soleus fibres was about one‐third of the size of the current in e.d.l. fibres, but was very similar otherwise. In both e.d.l. and soleus fibres, the dihydropyridine (DHP), nifedipine, suppressed the calcium current entirely. 3. In these normally polarized fibres, nifedipine suppressed only part (qns) of the asymmetric charge movement. The proportion of qns suppressed by various concentrations of nifedipine was linearly related to the associated reduction of the calcium current. Half‐maximal suppression of both parameters was obtained with about 0.5 microM‐nifedipine. The calcium current and the qns component of the charge movement also were suppressed over the same time course by nifedipine. Another DHP calcium antagonist, (+)PN200/110, was indistinguishable from nifedipine in its effects of suppressing calcium currents and qns. 4. In all muscle types, the total amount of qns in each fibre was linearly related to the size of the calcium current (in the absence of DHP). On average, qns was 3.3 times larger in e.d.l. fibres than in soleus fibres. 5. In contrast to the other dihydropyridines, (‐)bay K8644, a calcium channel agonist, did not suppress any asymmetric charge movement. 6. The potential dependence of the slow calcium current implied a minimum gating charge of about five or six electronic charges. The movement of qns occurred over a more negative potential range than the change in calcium conductance. 7. Experiments on the binding of (+)PN200/110 indicated that e.d.l. muscles had between about 2 and 3 times more specific DHP binding sites than did soleus muscle. 8. These results point to a close relationship between slow calcium channels, the qns component of the charge movement and DHP binding sites, in both fast‐ and slow‐twitch mammalian muscle. qns appears to be part of the gating current of the T‐system calcium channels.