Kynurenine aminotransferase isoforms display fiber-type specific expression in young and old human skeletal muscle.

Conversion of kynurenine (KYN) to kynurenic acid (KYNA) is the main pathway for free tryptophan degradation in skeletal muscle and has emerged as an important mechanism of how exercise is linked to promotion of mental health. Metabolism of KYN to KYNA mainly depends on the expression of kynurenine aminotransferases (KATs) that is under control of the mitochondria biogenesis regulator PGC-1α. We therefore hypothesized that expression of KATs would vary between muscle fibers that differ in mitochondrial content, i.e. oxidative type I vs more glycolytic type II muscle fibers. Moreover, we tested the hypothesis that KAT expression differs with age. Single muscle fibers were isolated from biopsies taken from the vastus lateralis muscle in young and old healthy subjects. In young and old subjects the abundance of KAT I, KAT III and KAT IV was greater in Type I than Type II fibers without age-dependent difference in the KAT isoform expressions. The link to mitochondrial content was further seen as the expression of KAT IV correlated to mitochondrial cytochrome c oxidase IV (COX IV) abundance in both fiber types. In conclusion, we describe for the first time the expression pattern of KAT isoforms with respect to specific fiber types and age in human skeletal muscle.

The decrease in electrically evoked force production is delayed by a previous bout of stretch-shortening cycle exercise.

AIM: Unaccustomed physical exercise with a large eccentric component is accompanied by muscle damage and impaired contractile function, especially at low stimulation frequencies. A repeated bout of eccentric exercise results in less damage and improved recovery of contractile function. Here we test the hypotheses that (1) a prior stretch-shortening cycle (SSC) exercise protects against impaired muscle function during a subsequent bout of SSC exercise and (2) the protection during exercise is transient and becomes less effective as the exercise progresses. METHODS: Healthy untrained men (n = 7) performed SSC exercise consisting of 100 maximal drop jumps at 30 s intervals. The same exercise was repeated 4 weeks later. Peak quadriceps muscle force evoked by electrical stimulation at 15 (P15) and 50 (P50) Hz was measured before exercise, after 10, 25, 50 and 100 jumps as well as 1 and 24 h after exercise. RESULTS: P15 and P50 were higher during the initial phase of the repeated bout compared with the first exercise bout, but there was no difference between the bouts at the end of the exercise periods. P15 and P50 were again larger 24 h after the repeated bout. The P15/P50 ratio during exercise was not different between the two bouts, but it was higher after the repeated bout. CONCLUSION: A prior bout of SSC exercise temporarily protects against impaired contractile function during a repeated exercise bout. The protection can again be seen after exercise, but the underlying mechanism then seems to be different.

Thyroid hormone receptor alpha can control action potential duration in mouse ventricular myocytes through the KCNE1 ion channel subunit.

AIMS: The reduced heart rate and prolonged QT(end) duration in mice deficient in thyroid hormone receptor (TR) alpha1 may involve aberrant expression of the K(+) channel alpha-subunit KCNQ1 and its regulatory beta-subunit KCNE1. Here we focus on KCNE1 and study whether increased KCNE1 expression can explain changes in cardiac function observed in TRalpha1-deficient mice. METHODS: TR-deficient, KCNE1-overexpressing and their respective wildtype (wt) mice were used. mRNA and protein expression were assessed with Northern and Western blot respectively. Telemetry was used to record electrocardiogram and temperature in freely moving mice. Patch-clamp was used to measure action potentials (APs) in isolated cardiomyocytes and ion currents in Chinese hamster ovary (CHO) cells. RESULTS: KCNE1 was four to 10-fold overexpressed in mice deficient in TRalpha1. Overexpression of KCNE1 with a heart-specific promoter in transgenic mice resulted in a cardiac phenotype similar to that in TRalpha1-deficient mice, including a lower heart rate and prolonged QT(end) time. Cardiomyocytes from KCNE1-overexpressing mice displayed increased AP duration. CHO cells transfected with expression plasmids for KCNQ1 and KCNE1 showed an outward rectifying current that was maximal at equimolar plasmids for KCNQ1-KCNE1 and decreased at higher KCNE1 levels. CONCLUSION: The bradycardia and prolonged QT(end) time in hypothyroid states can be explained by altered K(+) channel function due to decreased TRalpha1-dependent repression of KCNE1 expression.

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.

Activation of glucose transport and AMP-activated protein kinase during muscle contraction in adenylate kinase-1 knockout mice.

AIM: Recently it was reported that adenylate kinase-1 knockout mice (AK(-/-)) exhibit elevated rates of glucose uptake following repeated contractions and hypoxia, but the mechanism was not investigated. The purpose of the present study was to measure the changes in glucose transport and AMP-activated protein kinase (AMPK) phosphorylation/activity following repeated contractions in isolated muscles from AK(-/-) mice. METHODS: Extensor digitorum longus muscles underwent an intense stimulation protocol that decreased force to less than 10% of initial by the end of 10 min. Glucose uptake was measured with 2-deoxy-D-[1,2-(3)H]glucose. RESULTS: Muscle glucose uptake in the basal state was identical between control and AK(-/-) mice and increased twofold in both groups during contraction. The general antioxidant: N-acetylcysteine, decreased contraction-mediated glucose uptake by 30% in both groups. AMPK activity and phosphorylation were similar in the two groups in the basal state and, surprisingly, after contraction as well (approximately threefold increase). Both groups exhibited marked decreases in adenosine triphosphate following contraction (60-70% depletion), which coincided with stoichiometric increases in the content of inosine monophosphate, an indirect marker of AMP production. Adenylate kinase activity averaged 2081 +/- 106 micromol min(-1) (g dry wt)(-1) for control and 37 +/- 10 for AK(-/-) muscles; the activity in the AK(-/-) muscle is likely accounted for by isoforms other than AK1. CONCLUSION: In conclusion, AK(-/-) mice have a normal capacity for contraction-mediated glucose uptake. This appears to occur via increases in AMP and reactive oxygen species that result in the activation of AMPK.

Impaired calcium release during fatigue.

Impaired calcium release from the sarcoplasmic reticulum (SR) has been identified as a contributor to fatigue in isolated skeletal muscle fibers. The functional importance of this phenomenon can be quantified by the use of agents, such as caffeine, which can increase SR Ca(2+) release during fatigue. A number of possible mechanisms for impaired calcium release have been proposed. These include reduction in the amplitude of the action potential, potentially caused by extracellular K(+) accumulation, which may reduce voltage sensor activation but is counteracted by a number of mechanisms in intact animals. Reduced effectiveness of SR Ca(2+) channel opening is caused by the fall in intracellular ATP and the rise in Mg(2+) concentrations that occur during fatigue. Reduced Ca(2+) available for release within the SR can occur if inorganic phosphate enters the SR and precipitates with Ca(2+). Further progress requires the development of methods that can identify impaired SR Ca(2+) release in intact, blood-perfused muscles and that can distinguish between the various mechanisms proposed.

Activation of Ca(2+)-dependent protein kinase II during repeated contractions in single muscle fibres from mouse is dependent on the frequency of sarcoplasmic reticulum Ca(2+) release.

AIM: To investigate the importance and contribution of calmodulin-dependent protein kinase II (CaMKII) activity on sarcoplasmic reticulum (SR) Ca(2+)-release in response to different work intensities in single, intact muscle fibres. METHODS: CaMKII activity was blocked in single muscle fibres using either the inhibitory peptide AC3-I or the pharmacological inhibitor KN-93. The effect on tetanic force production and [Ca(2+)](i) was determined during work of different intensities. The activity of CaMKII was assessed by mathematical modelling. RESULTS: Using a standard protocol to induce fatigue (50x 70 Hz, 350 ms duration, every 2 s) the number of stimuli needed to induce fatigue was decreased from 47 +/- 3 contractions in control to 33 +/- 3 with AC3-I. KN-93 was a more potent inhibitor, decreasing the number of contractions needed to induce fatigue to 15 +/- 3. Tetanic [Ca(2+)](i) was 100 +/- 11%, 97 +/- 11% and 67 +/- 11% at the end of stimulation in control, AC3-I and KN-93 respectively. A similar inhibition was obtained using a high intensity protocol (20x 70 Hz, 200 ms duration, every 300 ms). However, using a long interval protocol (25x 70 Hz, 350 ms duration, every 5 s) no change was observed in either tetanic [Ca(2+)](i) or force when inhibiting CaMKII. A mathematical model used to investigate the activation pattern of CaMKII suggests that there is a threshold of active CaMKII that has to be surpassed in order for CaMKII to affect SR Ca(2+) release. CONCLUSION: Our results show that CaMKII is crucial for maintaining proper SR Ca(2+) release and that this is regulated in a work intensity manner.

The effects of the myosin-II inhibitor N-benzyl-p-toluene sulphonamide on fatigue in mouse single intact toe muscle fibres.

AIM: This study determined whether fatigue in skeletal muscle is primarily due to the repeated elevations of myoplasmic free calcium concentration ([Ca(2+)](i)) or to metabolite accumulation. METHODS: We examined the effects of N-benzyl-p-toluene sulphonamide (BTS) which is a potent and specific inhibitor of fast muscle myosin-II on the development of fatigue in mouse flexor digitorum brevis (FDB) muscle fibres. Single intact FDB fibres were micro-injected with indo-1 to monitor changes in [Ca(2+)](i) and stimulated repeatedly for a maximum of 150 tetani or until force declined to 40%. RESULTS: BTS markedly reduced tetanic force but had no effect on the tetanic [Ca(2+)](i) transients. When fatigue was induced in the presence of BTS, the reduction in [Ca(2+)](i) and force transients occurred much more slowly than in the absence of BTS. The extent of force depression was similar after induction of fatigue in fibres exposed to Tyrode only or to BTS and force recovered to the same extent. CONCLUSION: The results suggest that the decrease in tetanic [Ca(2+)](i) and force caused during fatigue are due mainly to accumulated metabolic changes.

Effects of a myosin-II inhibitor (N-benzyl-p-toluene sulphonamide, BTS) on contractile characteristics of intact fast-twitch mammalian muscle fibres.

We have examined the effects of N-benzyl-p-toluene sulphonamide (BTS), a potent and specific inhibitor of fast muscle myosin-II, using small bundles of intact fibres or single fibres from rat foot muscle. BTS decreased tetanic tension reversibly in a concentration-dependent manner with half-maximal inhibition at approximately approximately 2 microM at 20 degrees C. The inhibition of tension with 10 microM BTS was marked at the three temperatures examined (10, 20 and 30 degrees C), but greatest at 10 degrees C. BTS decreased active muscle stiffness to a lesser extent than tetanic tension indicating that not all of the tension inhibition was due to a reduced number of attached cross-bridges. BTS-induced inhibition of active tension was not accompanied by any change in the free myoplasmic Ca2+ transients. The potency and specificity of BTS make it a very suitable myosin inhibitor for intact mammalian fast muscle and should be a useful tool for the examination of outstanding questions in muscle contraction.

Generation and phenotypic characterization of a galanin overexpressing mouse.

In most parts of the peripheral nervous system galanin is expressed at very low levels. To further understand the functional role of galanin, a mouse overexpressing galanin under the platelet-derived growth factor-B was generated, and high levels of galanin expression were observed in several peripheral tissues and spinal cord. Thus, a large proportion of neurons in autonomic and sensory ganglia were galanin-positive, as were most spinal motor neurons. Strong galanin-like immunoreactivity was also seen in nerve terminals in the corresponding target tissues, including skin, blood vessels, sweat and salivary glands, motor end-plates and the gray matter of the spinal cord. In transgenic superior cervical ganglia around half of all neuron profiles expressed galanin mRNA but axotomy did not cause a further increase, even if mRNA levels were increased in individual neurons. In transgenic dorsal root ganglia galanin mRNA was detected in around two thirds of all neuron profiles, including large ones, and after axotomy the percentage of galanin neuron profiles was similar in overexpressing and wild type mice. Axotomy reduced the total number of DRG neurons less in overexpressing than in wild type mice, indicating a modest rescue effect. Aging by itself increased galanin expression in the superior cervical ganglion in wild type and transgenic mice, and in the latter also in preganglionic cholinergic neurons projecting to the superior cervical ganglion. Galanin overexpressing mice showed an attenuated plasma extravasation, an increased pain response in the formalin test, and changes in muscle physiology, but did not differ from wild type mice in sudomotor function. These findings suggest that overexpressed galanin in some tissues of these mice can be released and via a receptor-mediated action influence pathophysiological processes.

Prolonged force increase following a high-frequency burst is not due to a sustained elevation of [Ca2+]i.

A brief high-frequency burst of action potentials results in a sustained force increase in skeletal muscle. The present study investigates whether this force potentiation is the result of a sustained increase of the free myoplasmic [Ca2+] ([Ca2+]i). Single fibers from mouse flexor brevis muscles were stimulated with three impulses at 150 Hz (triplet) at the start of a 350-ms tetanus or in the middle of a 700-ms tetanus; the stimulation frequency of the rest of the tetanus ranged from 20 to 60 Hz. After the triplet, force was significantly (P < 0.05) increased between 17 and 20% when the triplet was given at the start of the tetanus and between 5 and 18% when the triplet was given in the middle (n = 7). However, during this potentiation, [Ca2+]i was not consistently increased. Hence, the increased force following a high-frequency burst is likely due to changes in the myofibrillar properties.

Muscle fatigue: the role of intracellular calcium stores.

Force declines when muscles are used repeatedly and intensively and a variety of intracellular mechanisms appear to contribute to this muscle fatigue. Intracellular calcium release declines during fatigue and has been shown to contribute to the reduction in force. Three new approaches have helped to define the role of calcium stores to this decline in calcium release. Skinned fibre experiments show that when intracellular phosphate is increased the amount of Ca2+ released from the sarcoplasmic reticulum (SR) declines. Intact fibre experiments show that the size of the calcium store declines during fatigue and recovers on rest. Intact muscles which lack the enzyme creatine kinase, do not exhibit the usual rise of phosphate during fatigue and, under these conditions, the decline of Ca2+ release is absent or delayed. These results can be explained by the "calcium phosphate precipitation" hypothesis. This proposes that if phosphate in the myoplasm rises, it enters the SR and binds to Ca2+ as Ca2+ phosphate. The resultant reduction in free Ca2+ within the SR contributes to the reduced Ca2+ release during fatigue.

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 (P(i)) in the myoplasm ([P(i)](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) Ca(2+) release contributes to fatigue and in this review we consider how raised [P(i)](myo) contributes to this process. Initial evidence came from the observation that increasing [P(i)](myo) causes reduced SR Ca(2+) release in both skinned and intact fibres. In fatigued muscles the store of releasable Ca(2+) in the SR declines mirroring the decline in SR Ca(2+) release. In muscle fibres with inoperative creatine kinase the rise of [P(i)](myo) is absent during fatigue and the failure of SR Ca(2+) 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 CaP(i) within the SR. The relevance of this mechanism in different types of fatigue in humans is considered.

Effects of concentric and eccentric contractions on phosphorylation of MAPK(erk1/2) and MAPK(p38) 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 (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).

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.

Role of myoplasmic phosphate in contractile function of skeletal muscle: studies on creatine kinase-deficient mice.

1. Increased myoplasmic inorganic phosphate (P(i)) has been suggested to have an important role in skeletal muscle fatigue, especially in the early phase. In the present study we used intact fast-twitch muscle cells from mice completely deficient in creatine kinase (CK(-/-)) to test this suggestion. These CK(-/-) muscle cells provide a good model since they display a higher P(i) concentration in the unfatigued state and fatigue without significant increase of P(i). 2. Tetanic contractions (350 ms duration) were produced in intact single muscle fibres. The free myoplasmic [Ca(2+)] ([Ca(2+)](i)) was measured with the fluorescent indicator indo-1. The force-[Ca(2+)](i) relationship was constructed from tetani at different frequencies. 3. Compared with wild-type fibres, CK(-/-) fibres displayed lower force in 100 Hz tetani and at saturating [Ca(2+)](i) (i.e. 100 Hz stimulation during caffeine exposure), higher tetanic [Ca(2+)](i) during the first 100 ms of tetanic stimulation, reduced myofibrillar Ca(2+) sensitivity when measurements were performed 100-200 ms into tetani, and slowed force relaxation that was due to altered cross-bridge kinetics rather than delayed Ca(2+) removal from the myoplasm. 4. In wild-type fibres, a series of 10 tetani resulted in reduced tetanic force, slowed force relaxation, and increased amplitude of [Ca(2+)](i) tails after tetani. None of these changes were observed in CK(-/-) fibres. 5. Complementary experiments on isolated fast-twitch extensor digitorum longus muscles showed a reduction of tetanic force and relaxation speed in CK(-/-) muscles similar to those observed in single fibres. 6. In conclusion, increased P(i) concentration can explain changes observed in the early phase of skeletal muscle fatigue. Increased P(i) appears to be involved in both fatigue-induced changes of cross-bridge function and SR Ca(2+) handling.

Inhibition of creatine kinase reduces the rate of fatigue-induced decrease in tetanic [Ca(2+)](i) in mouse skeletal muscle.

1. Ca(2+)-phosphate (P(i)) precipitation in the sarcoplasmic reticulum (SR) may cause reduced SR Ca(2+) release in skeletal muscle fatigue. To study this, we inhibited the creatine kinase (CK) reaction with 2,4-dinitro-1-fluorobenzene (DNFB). The hypothesis was that with inhibition of CK, phosphocreatine would not break down to creatine and P(i). Therefore P(i) transport into the SR would be limited and Ca(2+)-P(i) precipitation would not occur. 2. Intact single fibres from a mouse foot muscle were fatigued by repeated short tetani under control conditions or after exposure to DNFB (10 microM). The free myoplasmic concentrations of Ca(2+) ([Ca(2+)](i)) and Mg(2+) ([Mg(2+)](i)) were measured with indo-1 and mag-indo-1, respectively. Changes in [Mg(2+)](i) were assumed to reflect alterations in myoplasmic ATP concentration. 3. During the first 10 fatiguing tetani, tetanic [Ca(2+)](i) increased both in control and after DNFB exposure. Thereafter tetanic [Ca(2+)](i) fell and the rate of fall was about fourfold lower after DNFB exposure compared with control. 4. Under control conditions, there was a good relationship between declining tetanic [Ca(2+)](i) and increasing [Mg(2+)](i) during the final part of fatiguing stimulation. This correlation was lost after DNFB exposure. 5. In conclusion, the present data fit with a model where Ca(2+)-P(i) precipitation inhibits SR Ca(2+) release in fatigue produced by repeated short tetani. Furthermore, the results suggest that the rate of P(i) transport into the SR critically depends on the myoplasmic Mg(2+)/ATP concentration.

The role of Ca2+ and calmodulin in insulin signalling in mammalian skeletal muscle.

The role of Ca2+ in mediating effects of insulin on skeletal muscle has been widely debated. It is believed that in skeletal muscle Ca2+ has a permissive role, necessary but not of prime importance in mediating the stimulatory actions of insulin. In this review, we present evidence that insulin causes a localized increase in the concentration of Ca2+. Specifically, insulin induces a rise in near-membrane Ca2+ but not the bulk Ca2+ in the myoplasm. The rise in near-membrane Ca2+ is because of an influx through channels that can be blocked by L-type Ca2+ channel inhibitors. Calcium appears to exert some of its subsequent effects via calmodulin-dependent processes as calmodulin inhibitors block the translocation of glucose transporters and other enzymes as well as the insulin-stimulated increase in glucose transport.

Contraction and intracellular Ca(2+) handling in isolated skeletal muscle of rats with congestive heart failure.

A decreased exercise tolerance is a common symptom in patients with congestive heart failure (CHF). This decrease has been suggested to be partly due to altered skeletal muscle function. Therefore, we have studied contractile function and cytoplasmic free Ca(2+) concentration ([Ca(2+)](i), measured with the fluorescent dye indo 1) in isolated muscles from rats in which CHF was induced by ligation of the left coronary artery. The results show no major changes of the contractile function and [Ca(2+)](i) handling in unfatigued intact fast-twitch fibers isolated from flexor digitorum brevis muscles of CHF rats, but these fibers were markedly more susceptible to damage during microdissection. Furthermore, CHF fibers displayed a marked increase of baseline [Ca(2+)](i) during fatigue. Isolated slow-twitch soleus muscles of CHF rats displayed slower twitch contraction and tetanic relaxation than did muscles from sham-operated rats; the slowing of relaxation became more pronounced during fatigue in CHF muscles. Immunoblot analyses of sarcoplasmic reticulum proteins and sarcolemma Na(+),K(+)-ATPase showed no difference in flexor digitorum brevis muscles of sham-operated versus CHF rats. In conclusion, functional impairments can be observed in limb muscle isolated from rats with CHF. These impairments seem to mainly involve structures surrounding the muscle cells and sarcoplasmic reticulum Ca(2+) pumps, the dysfunction of which becomes obvious during fatigue.