Joseph D. Bruton

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 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.

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.

Dietary nitrate increases tetanic [Ca2+]i and contractile force in mouse fast-twitch muscle.

•  Dietary supplementation with inorganic nitrate has beneficial effects on skeletal muscle responses to exercise. •  Both mitochondrial and extra‐mitochondrial explanations have been proposed. •  Contractile force of fast‐twitch muscles was enhanced in mice supplemented with 1 mm NaNO3 in drinking water for 7 days. •  Myoplasmic free [Ca2+] during tetanic stimulation was increased in fast‐twitch muscles of nitrate‐supplemented mice and this was accompanied by increased expression of calsequestrin 1 and the dihydropyridine receptor. •  These results provide a new mechanism by which nitrate exerts beneficial effects on muscle function with applications to sports performance and a potential therapeutic role in conditions with muscle weakness.

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.

Effects of Palmitate on Ca2+ Handling in Adult Control and ob/ob Cardiomyocytes : Impact of Mitochondrial Reactive Oxygen Species

Obesity and insulin resistance are associated with enhanced fatty acid utilization, which may play a central role in diabetic cardiomyopathy. We now assess the effect of the saturated fatty acid palmitate (1.2 mmol/l) on Ca2+ handling, cell shortening, and mitochondrial production of reactive oxygen species (ROS) in freshly isolated ventricular cardiomyocytes from normal (wild-type) and obese, insulin-resistant ob/ob mice. Cardiomyocytes were electrically stimulated at 1 Hz, and the signal of fluorescent indicators was measured with confocal microscopy. Palmitate decreased the amplitude of cytosolic Ca2+ transients (measured with fluo-3), the sarcoplasmic reticulum Ca2+ load, and cell shortening by ∼20% in wild-type cardiomyocytes; these decreases were prevented by the general antioxidant N-acetylcysteine. In contrast, palmitate accelerated Ca2+ transients and increased cell shortening in ob/ob cardiomyocytes. Application of palmitate rapidly dissipated the mitochondrial membrane potential (measured with tetra-methyl rhodamine-ethyl ester) and increased the mitochondrial ROS production (measured with MitoSOX Red) in wild-type but not in ob/ob cardiomyocytes. In conclusion, increased saturated fatty acid levels impair cellular Ca2+ handling and contraction in a ROS-dependent manner in normal cardiomyocytes. Conversely, high fatty acid levels may be vital to sustain cardiac Ca2+ handling and contraction in obesity and insulin-resistant conditions.

Muscle fatigue: from observations in humans to underlying mechanisms studied in intact single muscle fibres

Prolonged dynamic exercise and sustained isometric contractions induce muscle fatigue, as manifested by decreased performance and a reduction in the maximum voluntary contraction force. Studies with non-invasive measurements in exercising humans show that mechanisms located beyond the sarcolemma are important in the fatigue process. In this review, we describe probable cellular mechanisms underlying fatigue-induced changes in excitation–contraction (E–C) coupling occurring in human muscle fibres during strenuous exercise. We use fatigue-induced changes observed in intact single muscle fibres, where force and cellular Ca2+ handling can be directly measured, to explain changes in E–C coupling observed in human muscle during exercise.

Reactive oxygen species and fatigue-induced prolonged low-frequency force depression in skeletal muscle fibres of rats, mice and SOD2 overexpressing mice

Skeletal muscle often shows a delayed force recovery after fatiguing stimulation, especially at low stimulation frequencies. In this study we focus on the role of reactive oxygen species (ROS) in this fatigue‐induced prolonged low‐frequency force depression. Intact, single muscle fibres were dissected from flexor digitorum brevis (FDB) muscles of rats and wild‐type and superoxide dismutase 2 (SOD2) overexpressing mice. Force and myoplasmic free [Ca2+] ([Ca2+]i) were measured. Fibres were stimulated at different frequencies before and 30 min after fatigue induced by repeated tetani. The results show a marked force decrease at low stimulation frequencies 30 min after fatiguing stimulation in all fibres. This decrease was associated with reduced tetanic [Ca2+]i in wild‐type mouse fibres, whereas rat fibres and mouse SOD2 overexpressing fibres instead displayed a decreased myofibrillar Ca2+ sensitivity. The SOD activity was ∼50% lower in wild‐type mouse than in rat FDB muscles. Myoplasmic ROS increased during repeated tetanic stimulation in rat fibres but not in wild‐type mouse fibres. The decreased Ca2+ sensitivity in rat fibres could be partially reversed by application of the reducing agent dithiothreitol, whereas the decrease in tetanic [Ca2+]i in wild‐type mouse fibres was not affected by dithiothreitol or the antioxidant N‐acetylcysteine. In conclusion, we describe two different causes of fatigue‐induced prolonged low‐frequency force depression, which correlate to differences in SOD activity and ROS metabolism. These findings may have clinical implications since ROS‐mediated impairments in myofibrillar function can be counteracted by reductants and antioxidants, whereas changes in SR Ca2+ handling appear more resistant to interventions.

Limited oxygen diffusion accelerates fatigue development in mouse skeletal muscle.

Isolated whole skeletal muscles fatigue more rapidly than isolated single muscle fibres. We have now employed this difference to study mechanisms of skeletal muscle fatigue. Isolated whole soleus and extensor digitorum longus (EDL) muscles were fatigued by repeated tetanic stimulation while measuring force production. Neither application of 10 mm lactic acid nor increasing the [K+] of the bath solution from 5 to 10 mm had any significant effect on the rate of force decline during fatigue induced by repeated brief tetani. Soleus muscles fatigued slightly faster during continuous tetanic stimulation in 10 mm[K+]. Inhibition of mitochondrial respiration with cyanide resulted in a faster fatigue development in both soleus and EDL muscles. Single soleus muscle fibres were fatigued by repeated tetani while measuring force and myoplasmic free [Ca2+] ([Ca2+]i). Under control conditions, the single fibres were substantially more fatigue resistant than the whole soleus muscles; tetanic force at the end of a series of 100 tetani was reduced by about 10% and 50%, respectively. However, in the presence of cyanide, fatigue developed at a similar rate in whole muscles and single fibres, and tetanic force at the end of fatiguing stimulation was reduced by ∼80%. The force decrease in the presence of cyanide was associated with a ∼50% decrease in tetanic [Ca2+]i, compared with an increase of ∼20% without cyanide. In conclusion, lactic acid or [K+] has little impact on fatigue induced by repeated tetani, whereas hypoxia speeds up fatigue development and this is mainly due to an impaired Ca2+ release from the sarcoplasmic reticulum.

Neuromuscular junction disassembly and muscle fatigue in mice lacking neurotrophin-4.

Neurotrophin-4 (NT-4) is produced by slow muscle fibers in an activity-dependent manner and promotes growth and remodeling of adult motorneuron innervation. However, both muscle fibers and motor neurons express NT-4 receptors, suggesting bidirectional NT-4 signaling at the neuromuscular junction. Mice lacking NT-4 displayed enlarged and fragmented neuromuscular junctions with disassembled postsynaptic acetylcholine receptor (AChR) clusters, reduced AChR binding, and acetylcholinesterase activity. Electromyographic responses, posttetanic potentiation, and action potential amplitude were also significantly reduced in muscle fibers from NT-4 knock-out mice. Slow-twitch soleus muscles from these mice fatigued twice as rapidly as those from wild-type mice during repeated tetanic stimulation. Thus, muscle-derived NT-4 is required for maintenance of postsynaptic AChR regions, normal muscular electrophysiological responses, and resistance to muscle fatigue. This neurotrophin may therefore be a key component of an activity-dependent feedback mechanism regulating maintenance of neuromuscular connections and muscular performance.