Håkan Westerblad

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.

EFFECT OF HYDROGEN PEROXIDE AND DITHIOTHREITOL ON CONTRACTILE FUNCTION OF SINGLE SKELETAL MUSCLE FIBRES FROM THE MOUSE

1 We used intact single fibres from a mouse foot muscle to study the role of oxidation‐reduction in the modulation of contractile function. 2 The oxidant hydrogen peroxide (H2O2, 100‐300 μM) for brief periods did not change myoplasmic Ca2+ concentrations ([Ca2+]i) during submaximal tetani. However, force increased by 27 % during the same contractions. 3 The effects of H2O2 were time dependent. Prolonged exposures resulted in increased resting and tetanic [Ca2+]i, while force was significantly diminished. The force decline was mainly due to reduced myofibrillar Ca2+ sensitivity. There was also evidence of altered sarcoplasmic reticulum (SR) function: passive Ca2+ leak was increased and Ca2+ uptake was decreased. 4 The reductant dithiothreitol (DTT, 0.5‐1 mM) did not change tetanic [Ca2+]i, but decreased force by over 40 %. This was completely reversed by subsequent incubations with H2O2. The force decline induced by prolonged exposure to H2O2 was reversed by subsequent exposure to DTT. 5 These results show that the elements of the contractile machinery are differentially responsive to changes in the oxidation‐reduction balance of the muscle fibres. Myofibrillar Ca2+ sensitivity appears to be especially susceptible, while the SR functions (Ca2+ leak and uptake) are less so.

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.

Role of phosphate and calcium stores in muscle fatigue

Intensive activity of muscles causes a decline in performance, known as fatigue, that is thought to be caused by the effects of metabolic changes on either the contractile machinery or the activation processes. The concentration of inorganic phosphate (Pi) in the myoplasm ([Pi]myo) increases substantially during fatigue and affects both the myofibrillar proteins and the activation processes. It is known that a failure of sarcoplasmic reticulum (SR) Ca2+ release contributes to fatigue and in this review we consider how raised [Pi]myo contributes to this process. Initial evidence came from the observation that increasing [Pi]myo causes reduced SR Ca2+ release in both skinned and intact fibres. In fatigued muscles the store of releasable Ca2+ in the SR declines mirroring the decline in SR Ca2+ release. In muscle fibres with inoperative creatine kinase the rise of [Pi]myo is absent during fatigue and the failure of SR Ca2+ release is delayed. These results can all be explained if inorganic phosphate can move from the myoplasm into the SR during fatigue and cause precipitation of CaPi within the SR. The relevance of this mechanism in different types of fatigue in humans is considered.

Increased mitochondrial mass in mitochondrial myopathy mice

We have generated an animal model for mitochondrial myopathy by disrupting the gene for mitochondrial transcription factor A (Tfam) in skeletal muscle of the mouse. The knockout animals developed a myopathy with ragged-red muscle fibers, accumulation of abnormally appearing mitochondria, and progressively deteriorating respiratory chain function in skeletal muscle. Enzyme histochemistry, electron micrographs, and citrate synthase activity revealed a substantial increase in mitochondrial mass in skeletal muscle of the myopathy mice. Biochemical assays demonstrated that the increased mitochondrial mass partly compensated for the reduced function of the respiratory chain by maintaining overall ATP production in skeletal muscle. The increased mitochondrial mass thus was induced by the respiratory chain deficiency and may be beneficial by improving the energy homeostasis in the affected tissue. Surprisingly, in vitro experiments to assess muscle function demonstrated that fatigue development did not occur more rapidly in myopathy mice, suggesting that overall ATP production is sufficient. However, there were lower absolute muscle forces in the myopathy mice, especially at low stimulation frequencies. This reduction in muscle force is likely caused by deficient formation of force-generating actin–myosin cross bridges and/or disregulation of Ca2+ homeostasis. Thus, both biochemical measurements of ATP-production rate and in vitro physiological studies suggest that reduced mitochondrial ATP production might not be as critical for the pathophysiology of mitochondrial myopathy as thought previously.

Role of reactive oxygen species in contraction-mediated glucose transport in mouse skeletal muscle

Exercise increases glucose transport into skeletal muscle via a pathway that is poorly understood. We investigated the role of endogenously produced reactive oxygen species (ROS) in contraction‐mediated glucose transport. Repeated contractions increased 2‐deoxyglucose (2‐DG) uptake roughly threefold in isolated, mouse extensor digitorum longus (fast‐twitch) muscle. N‐Acetylcysteine (NAC), a non‐specific antioxidant, inhibited contraction‐mediated 2‐DG uptake by ∼50% (P < 0.05 versus control values), but did not significantly affect basal 2‐DG uptake or the uptake induced by insulin, hypoxia or 5‐aminoimidazole‐4‐carboxamide‐1‐β‐d‐ribofuranoside (AICAR, which mimics AMP‐mediated activation of AMP‐activated protein kinase, AMPK). Ebselen, a glutathione peroxidase mimetic, also inhibited contraction‐mediated 2‐DG uptake (by almost 60%, P < 0.001 versus control values). Muscles from mice overexpressing Mn2+‐dependent superoxide dismutase, which catalyses H2O2 production from superoxide anions, exhibited a ∼25% higher rate of contraction‐mediated 2‐DG uptake versus muscles from wild‐type control mice (P < 0.05). Exogenous H2O2 induced oxidative stress, as judged by an increase in the [GSSG]/[GSH + GSSG] (reduced glutathione + oxidized glutathione) ratio to 2.5 times control values, and this increase was substantially blocked by NAC. Similarly, NAC significantly attenuated contraction‐mediated oxidative stress as judged by measurements of glutathione status and the intracellular ROS level with the fluorescent indicator 5‐(and‐6)‐chloromethyl‐2′,7′‐dichlorodihydrofluorescein (P < 0.05). Finally, contraction increased AMPK activity and phosphorylation ∼10‐fold, and NAC blocked ∼50% of these changes. These data indicate that endogenously produced ROS, possibly H2O2 or its derivatives, play an important role in contraction‐mediated activation of glucose transport in fast‐twitch muscle.

Properly formed but improperly localized synaptic specializations in the absence of laminin alpha4.

Precise apposition of pre- to postsynaptic specializations is required for optimal function of chemical synapses, but little is known about how it is achieved. At the skeletal neuromuscular junction, active zones (transmitter release sites) in the nerve terminal lie directly opposite junctional folds in the postsynaptic membrane. Few active zones or junctional folds form in mice lacking the laminin β2 chain, which is normally concentrated in the synaptic cleft. β2 and the broadly expressed γ1 chain form heterotrimers with α chains, three of which, α2, α4 and α5, are present in the synaptic cleft. Thus, α2β2γ1, α4β2γ1 and α5β2γ1 heterotrimers are all lost in β2 mutants. In mice lacking laminin α4, active zones and junctional folds form in normal numbers, but are not precisely apposed to each other. Thus, formation and localization of synaptic specializations are regulated separately, and α4β2γ1 (called laminin-9) is critical in the latter process.

Contractile response of skeletal muscle to low peroxide concentrations: myofibrillar calcium sensitivity as a likely target for redox-modulation

Endogenous peroxides and related reactive oxygen species may influence various steps in the contractile process. Single mouse skeletal muscle fibers were used to study the effects of hydrogen peroxide (H2O2) and t‐butyl hydroperoxide (f‐BOOH) on force and myoplasmic Ca2+ concentration ([Ca2+]i). Both peroxides (10−1 to 10−5 M) decreased tetanic [Ca2+]i and increased force during submaximal tetani. Catalase (1 kU/ml) blocked the effect of H2O2, but not of t‐BOOH. The decrease in tetanic [Ca2+]i was constant, while the effect on force was biphasic: A transitory increase was followed by a steady decline to the initial level. Myofibrillar Ca2+ sensitivity remained increased during incubation with either peroxide. Only the highest peroxide concentration (10 μM) increased resting [Ca2+]i and slowed the return of [Ca2+]i to its resting level after a contraction, evidence of impaired sarcoplasmic reticulum Ca2+ re‐uptake. The peroxides increased maximal force production and the rate of force redevelopment, and decreased maximum shortening velocity. N‐ethylmaleimide (25 μM, thiol‐alkylating agent) prevented the response to 1 μM H2O2. These results show that myofibrillar Ca2+ sensitivity and cross‐bridge kinetics are influenced by H2O2 and t‐BOOH concentrations that approach those found physiologically, and these findings indicate a role for endogenous oxidants in the regulation of skeletal muscle function.

Skeletal muscle: energy metabolism, fiber types, fatigue and adaptability.

Skeletal muscles cope with a large range of activities, from being able to support the body weight during long periods of upright standing to perform explosive movements in response to an unexpected threat. This requires systems for energy metabolism that can provide energy during long periods of moderately increased energy consumption as well as being able to rapidly increasing the rate of energy production more than 100-fold in response to explosive contractions. In this short review we discuss how muscles can deal with these divergent demands. We first outline the major energy metabolism pathways in skeletal muscle. Next we describe metabolic differences between different muscle fiber types. Contractile performance declines during intense activation, i.e. fatigue develops, and we discuss likely underlying mechanisms. Finally, we discuss the ability of muscle fibers to adapt to altered demands, and mechanisms behind these adaptations. The accumulated experimental evidence forces us to conclude that most aspects of energy metabolism involve multiple and overlapping signaling pathways, which indicates that the control of energy metabolism is too important to depend on one single molecule or mechanism.