Slow recovery of force in single skeletal muscle fibres

After a bout of intense exercise, especially in untrained persons, recovery of muscle force is often slow. Force depression is much more marked at low frequencies of stimulation than at high frequencies (recovery is also seen in single muscle fibres from frog and mouse after fatigue induced by repeated, brief contractions. Evidence from our own and other laboratories indicates that the impairment is unlikely to result from metabolic changes and points to a defect in excitation–contraction coupling. We demonstrate that the likely site of failure is in the coupling between t-tubule depolarization and release of Ca²⁺ from the SR. The causative agent appears to be a localized increase in cytoplasmic Ca²⁺ which initiates some disruptive process, which can, however, be fully reversed, albeit slowly. Our experimental evidence does not support the involvement of Ca²⁺-activated proteases. Attempts to clarify the possible role of Ca²⁺-activated lipases (phospholipase A₂) and Ca²⁺/calmodulin have been hampered by side-effects of available inhibitors. Efforts to clarify how Ca²⁺ exerts its effects are continuing.     

Skeletal muscle training in chronic heart failure

Patients with heart failure are limited in their ability to tolerate exercise. Recent research has suggested that this limitation cannot be entirely attributed to cardiac or lung impairment but rather that changes in peripheral muscles may play an important role. There are objective similarities between heart failure and muscular deconditioning. Deficiencies in peripheral blood flow and skeletal muscle function, morphology, metabolism and function are present in both conditions. Moreover, an exaggerated activity of the receptors sensitive to exercise‐derived metabolic signals (muscle ergoreceptors and peripheral and central chemoreceptors) leads to early and profound exercise‐induced fatigue and dyspnoea. These muscle afferents contribute to the ventilatory, haemodynamic and autonomic responses to exercise both in physiological and pathological conditions, including chronic heart failure. Against this background, a skeletal muscle origin of symptoms in heart failure has been proposed. The protective effects of physical training have been described in many recent studies: training improves ventilatory control, skeletal muscle metabolism and autonomic nervous system activity. The exercise training appears to induce its beneficial effects on skeletal muscle both directly (on muscle function, histological and biochemical features) and indirectly (by reducing the activation of the muscle afferents). The metabolic mediators of these muscle afferents may become a potential target in the future therapy of heart failure symptoms.     

Events of the excitation–contraction–relaxation (E–C–R) cycle in fast- and slow-twitch mammalian muscle fibres relevant to muscle fatigue

The excitation–contraction–relaxation cycle (E–C–R) in the mammalian twitch muscle comprises the following major events: (1) initiation and propagation of an action potential along the sarcolemma and transverse (T)-tubular system; (2) detection of the T-system depolarization signal and signal transmission from the T-tubule to the sarcoplasmic reticulum (SR) membrane; (3) Ca²⁺ release from the SR; (4) transient rise of myoplasmic [Ca²⁺]; (5) transient activation of the Ca²⁺-regulatory system and of the contractile apparatus; (6) Ca²⁺ reuptake by the SR Ca²⁺ pump and Ca²⁺ binding to myoplasmic sites. There are many steps in the E–C–R cycle which can be seen as potential sites for muscle fatigue and this review explores how structural and functional differences between the fast- and slow-twitch fibres with respect to the E–C–R cycle events can explain to a great extent differences in their fatiguability profiles.     

Effect of intracellular and extracellular ion changes on E–C coupling and skeletal muscle fatigue

The causative factors in muscle fatigue are multiple, and vary depending on the intensity and duration of the exercise, the fibre type composition of the muscle, and the individual's degree of fitness. Regardless of the aetiology, fatigue is characterized by the inability to maintain the required power output, and the decline in power can be attributed to a reduced force and velocity. Following high-intensity exercise, peak force has been shown to recover biphasically with an initial rapid (2 min) recovery followed by a slower (50 min) return to the pre-fatigued condition. The resting membrane potential depolarizes by 10–15 mV, while the action potential overshoot declines by a similar magnitude. Following high-frequency stimulation of the frog semitendinous muscle, we observed intracellular potassium [K⁺]_i decrease from 142±5 to 97±8 mm , while sodium [Na⁺]_i rose from 16±1 to 49±6 mm . The [K⁺]_i loss was similar to that observed in fatigued mouse and human skeletal muscle, which suggests that there may be a limit to which [K⁺]_i can decrease before the associated depolarization begins to limit the action potential frequency. Fibre depolarization to -60 mV (a value observed in some cells) caused a significant reduction in the t-tubular charge movement, and the extent of the decline was inversely related to the concentration of extracellular Ca²⁺. A decrease in intracellular pH (pH_i) to 6.0 was observed, and it has been suggested by some that low pH may disrupt E–C coupling by directly inhibiting the SR Ca²⁺ release channel. However, Lamb et al. (1992) observed that low pH had no effect on Ca²⁺ release, and we found low pH_i to have no effect on t-tubular charge movement (Q) or the Q vs. V_m relationship. The Ca²⁺ released from the SR plays three important roles in the regulation of E–C coupling. As Ca²⁺ rises, it binds to the inner surface of the t-tubular charge sensor to increase charge (Q_γ) and thus Ca²⁺ release, it opens SR Ca²⁺ channels that are not voltage-regulated, and as [Ca²⁺]_i increases further it feeds back to close the same channels. The late stages of fatigue have been shown to be in part caused by a reduced SR Ca²⁺ release. The exact cause of the reduced release is unknown, but the mechanism appears to involve a direct inhibition of the SR Ca²⁺ channel.     

Skeletal muscle fatigue in normal subjects and heart failure patients. Is there a common mechanism?

Skeletal muscle fatigue develops gradually during all forms of exercise, and develops more rapidly in heart failure patients. The fatigue mechanism is still not known, but is most likely localized to the muscle cells themselves. During high intensity exercise the perturbations of the Na⁺ and K⁺ balance in the exercising muscle favour depolarization, smaller action potentials and inexcitability. The Na⁺, K⁺ pump becomes strongly activated and limits, but does not prevent the rise in extracellular Na⁺, K⁺ pump concentration and intracellular Na⁺ concentration. However, by virtue of its electrogenic property the pump may contribute in maintaining excitability and contractility by keeping the cells more polarized than the ion gradients predict. With prolonged exercise perturbations of Na⁺ and K⁺ are smaller and fatigue may be associated with altered cellular handling of Ca²⁺ and Mg²⁺. Release of Ca²⁺ from the sarcoplasmic reticulum (SR) is reduced in the absence of changes of the cellular content of Ca²⁺ and Mg²⁺. In heart failure several clinical reports indicate severe electrolyte perturbations in skeletal muscle. However, in well controlled studies small or insignificant changes are found. We conclude that with high intensity exercise perturbations of Na⁺ and K⁺ in muscle cells may contribute to fatigue, whereas with endurance type of exercise and in heart failure patients the skeletal muscle fatigue is more likely to reside in the intracellular control of Ca²⁺ release and reuptake.     

Role of tyrosine phosphorylation in excitation–contraction coupling in vascular smooth muscle

Increasingly it is recognized that tyrosine phosphorylation plays an important part in the regulation of function in differentiated contractile vascular smooth muscle. Tyrosine kinases and phosphatases are present in large amounts in vascular smooth muscle and have been reported to influence a number of processes crucial to contraction, including ion channel gating, calcium homeostasis and sensitization of the contractile process to [Ca²⁺]_i. This review summarizes current understanding regarding the role of tyrosine phosphorylation in excitation–contraction coupling in blood vessels.     

Rat skeletal muscle metabolism in experimental heart failure: effects of physical training

Skeletal muscle metabolic abnormalities exist in chronic heart failure. The influence of physical training on muscle metabolism after myocardial infarction was studied in a rat model. ³¹P magnetic resonance spectroscopy and enzyme assays were performed in Wistar rats 12 weeks after coronary artery ligation. Infarcted rats were allocated randomly to either 6 weeks of training or non-training. Spectra were collected from the calf muscles during sciatic nerve stimulation at 2 Hz. Fibre typing and enzymatic assays were performed on the muscles of the contralateral non stimulated leg. Post-mortem rats were also divided into severe and moderate heart failure according to the lung weight per body weight. At 200 g twitch tension, phosphocreatine and pH were found to be significantly lower in the non-trained severe heart failure group compared with the other groups. Phosphocreatine recovery half-time was significantly longer in the non-trained group with severe heart failure and correlated with the citrate synthase activity in the muscle. The training did not induce a change in the enzyme activities in the infarcted animals with moderate heart failure but did correct the lower citrate synthase activity in the non-trained severe heart failure animals. This normalization of muscle metabolism was achieved by training without any change in calf muscle mass, making atrophy unlikely to be the sole cause of the metabolic changes in heart failure. Training in rats with severe heart failure can reverse the abnormalities of skeletal muscle metabolism, implicating decreased physical activity in the aetiology of these changes.     

Malignant hyperthermia and excitation–contraction coupling

Malignant hyperthermia (MH) is a state of elevated skeletal muscle metabolism that may occur during general anaesthesia in genetically pre‐disposed individuals. Malignant hyperthermia results from altered control of sarcoplasmic reticulum (SR) Ca²⁺ release. Mutations have been identified in MH‐susceptible (MHS) individuals in two key proteins of excitation–contraction (EC) coupling, the Ca²⁺ release channel of the SR, ryanodine receptor type 1 (RyR1) and the α1‐subunit of the dihydropyridine receptor (DHPR, L‐type Ca²⁺ channel). During EC coupling, the DHPR senses the plasma membrane depolarization and transmits the information to the ryanodine receptor (RyR). As a consequence, Ca²⁺ is released from the terminal cisternae of the SR. One of the human MH‐mutations of RyR1 (Arg614Cys) is also found at the homologous location in the RyR of swine (Arg615Cys). This animal model permits the investigation of physiological consequences of the homozygously expressed mutant release channel. Of particular interest is the question of whether voltage‐controlled release of Ca²⁺ is altered by MH‐mutations in the absence of MH‐triggering substances. This question has recently been addressed in this laboratory by studying Ca²⁺ release under voltage clamp conditions in both isolated human skeletal muscle fibres and porcine myotubes.     

Role of nitric oxide in skeletal muscle: synthesis,distribution and functional importance

Over the last two decades, nitric oxide (NO) has been established as a novel mediator of biological processes, ranging from vascular control to long-term memory, from tissue inflammation to penile erection. This paper reviews recent research which shows that NO and its derivatives also are synthesized within skeletal muscle and that NO derivatives influence various aspects of muscle function. Individual muscle fibres express one or both of the constitutive NO synthase (NOS) isoforms. Type I (neuronal) NOS is localized to the sarcolemma of fast fibres; type III (endothelial) NOS is associated with mitochondria. Isolated skeletal muscle produces NO at low rates under resting conditions and at higher rates during repetitive contraction. NO appears to mediate cell–cell interactions in muscle, including vasodilation and inhibition of leucocyte adhesion. NO also acts directly on muscle fibres to alter cell function. Muscle metabolism appears to be NO-sensitive at several sites, including glucose uptake, glycolysis, mitochondrial oxygen consumption and creatine kinase activity. NO also modulates muscle contraction, inhibiting force output by altering excitation–contraction coupling. The mechanisms of NO action are likely to include direct effects on redox-sensitive regulatory proteins, interaction with endogenous reactive oxygen species, and activation of second messengers such as cyclic guanosine monophosphate (cGMP). In conclusion, research published over the past few years makes it clear that skeletal muscle produces NO and that endogenous NO modulates muscle function. Much remains to be learned, however, about the physiological importance of NO actions and about their underlying mechanisms.     

Effects of perchlorate on myofibrillar calcium sensitivity in rat skinned skeletal muscles

The effects of perchlorate (1–20 mm ) on myofibrillar calcium responsiveness have been tested in Triton X-100-skinned fibre bundles from rat soleus (slow-twitch) and extensor digitorum longus (fast-twitch) skeletal muscles. In extensor digitorum longus and soleus, perchlorate dose-dependently shifted the pCa (-log[Ca²⁺])/tension relationship towards lower free calcium concentration (sensitizing effect) and maximal tension was unchanged. The degree of sensitization was greater in extensor digitorum longus than in soleus bundles. Reversibility after exposure to 12 mm perchlorate was complete in soleus but not in extensor digitorum longus muscles. In fact, the ‘return’ pCa/tension relationship in extensor digitorum longus was shifted to higher free calcium concentration (desensitizing effect) compared with control. Perchlorate (12 mm ) also enhanced myofibrillar calcium responsiveness of frog semitendinosus skinned skeletal fibres. Assuming a passive distribution of perchlorate across the sarcolemma, this sensitizing effect is probably not involved in perchlorate-induced potentiation of contractile responses of intact muscles and thereby supports the specificity of perchlorate as an agonist of the excitation/calcium release sequence in skeletal muscle fibres.     

Nandrolone decanoate treatment induces changes in contractile responses of rat untrained fast‐twitch skeletal muscle

This investigation was designed to examine whether short‐term administration of anabolic–androgenic steroids (AAS) (nandrolone decanoate) could produce changes in contractile responses of untrained rat fast‐ (edl) and slow‐ (soleus) twitch skeletal muscle. Twenty male rats were divided into two groups, one group received weekly (for 6 weeks) an intramuscular injection of AAS, nandrolone decanoate (15 mg kg^–1) and the second group received weekly the similar doses of vehicle (sterile peanut oil). In edl intact isolated small bundles (two to four cells), it was found that nandrolone decanoate treatment increases the K⁺ contracture tension (146 mM ) relative to maximum tension by 56%, whereas no change was observed in the time to peak tension and in the time constant of relaxation. By contrast, in treated soleus muscle, compared with control, no significant modification was found in the K⁺ contracture characteristics. The change in edl contractile responses was associated with a shift to more negative potential of the voltage‐dependence activation and the steady‐state inactivation curves which also shifted leftward in treated soleus fibres. Furthermore, in edl skinned Triton X‐100 fibres, the Ca²⁺ sensitivity of contractile proteins (pCa₅₀) was increased, while electrophoresis analysis indicates no significant effect of nandrolone decanoate treatment on myosin heavy chain (MHC) isoforms. The present results show that nandrolone decanoate treatment produces more pronounced changes in untrained fast muscle function rather than soleus by acting at different levels of the excitation–contraction coupling mechanism without changes in the MHC isoforms and that contractile responses became similar to those found in soleus muscle.     

Defective excitation‐contraction coupling in hearts of rats with congestive heart failure

Aim: We examined the cellular basis for depressed cardiac contractility in rats with congestive heart failure (CHF) secondary to myocardial infarction. Methods: Six weeks after ligation of the left coronary artery, CHF was confirmed by haemodynamic measures and echocardiographic demonstration of reduced myocardial contractility in vivo. Papillary muscles from CHF animals developed less force than those from sham operated (SHAM) animals. Cell shortening was measured in isolated ventricular myocytes voltage-clamped with high resistance electrodes. Ca²⁺ transients were measured in fluo-4 loaded myocytes. Results: Contractions triggered by depolarizing test steps from a post conditioning potential of −70 mV were significantly smaller and had significantly reduced velocity of shortening in CHF compared with SHAM myocytes. However, contractions initiated from −40 mV, were similar in amplitude and velocity of shortening in CHF and SHAM cells. L-type Ca²⁺ current was not significantly different between CHF and SHAM cells, whether activated from −70 or −40 mV. Therefore, in SHAM cells, excitation-contraction coupling exhibited higher gain when contractions were initiated from negative (−70 mV), as compared with depolarized potentials (−40 mV). However, in CHF myocytes, excitation-contraction coupling gain was selectively depressed with steps from −70 mV. This depression of gain in CHF was not accompanied by a significant reduction in sarcoplasmic reticulum Ca²⁺ content. Isoproterenol increased Ca²⁺ transients less in CHF than SHAM myocytes. Conclusion: In this post-infarction model of CHF, the contractile deficit was voltage dependent and the gain of excitation-contraction coupling was selectively depressed for contractions initiated negative to −40 mV.     

Confocal imaging of calcium release events in single smooth muscle cells

Localized [Ca²⁺]_i transients (‘sparks’) first directly detected in cardiac myocytes were considered to represent ‘elementary’ Ca²⁺-release events playing a key role during excitation–contraction coupling ( Cheng et al. 1993 ). In this study we employed confocal [Ca²⁺]_i imaging to characterize subcellular calcium signalling in fluo-3 loaded visceral and vascular smooth muscle cells. In some experiments membrane potential of the myocyte was controlled using whole-cell patch clamp technique and changes in membrane current were recorded simultaneously with [Ca²⁺]_i imaging. Some local [Ca²⁺]_i transients were very similar to ‘Ca²⁺ sparks’ observed in heart, i.e. lasting ≈200 ms with a peak fluorescence ratio of 1.75 ± 0.23 (mean ± SD, n = 33). Ca²⁺ sparks were found to occur in certain preferred locations in the cell, termed frequent discharge sites. Other events were faster and smaller, lasting only ≈40 ms with a peak normalized fluorescence of 1.36 ± 0.09 (mean ± SD, n = 28). A high correlation between spontaneous transient outward currents and spark occurrence was observed. Proliferating waves of elevated [Ca²⁺]_i initiated during membrane depolarization seem to arise from spatio-temporal recruitment of local Ca²⁺-release events. The spatial non-uniformity of sarcoplasmic reticulum and ryanodine receptor distribution within the cell may account for the existence of ‘frequent discharge sites’ and the wide variation in the Ca²⁺ wave propagation velocities observed.     

Skeletal muscle disorders in heart failure.

Heart failure is associated with reduction of exercise capacity that cannot be solely ascribed to reduced maximal oxygen uptake (VdotO2max). Therefore, research has focused on changes in skeletal muscle morphology, metabolism and function. Factors that can cause such changes in skeletal muscle comprise inactivity, malnutrition, constant or repeated episodes of inadequate oxygen delivery and prolonged exposure to altered neurohumoural stimuli. Most of these factors are not specific for the heart failure condition. On the other hand, heart failure is more than one clinical condition. Congestive heart failure (CHF) develops gradually as a result of deteriorating contractility of the viable myocardium, myocardial failure. Is it possible that development of this contractile deficit in the myocardium is paralleled by a corresponding contractile deficit of the skeletal muscles? This question cannot be answered today. Both patient studies and experimental studies support that there is a switch to a faster muscle phenotype and energy metabolism balance is more anaerobic. The muscle atrophy seen in many patients is not so evident in experimental studies. Few investigators have studied contractile function. Both fast twitch and slow twitch muscles seem to become slower, not faster as might be expected, and this is possibly linked to slower intracellular Ca2+ cycling. The neurohumoural stimuli that can cause this change are not known, but recently it has been reported that several cytokines are increased in CHF patients. Thus, the changes seen in skeletal muscles during CHF are partly secondary to inactivity, but the possibility remains that the contractility is altered because of intracellular changes of Ca2+ metabolism that are also seen in the myocardium.     

Effects of salbutamol on the contractile properties of human skeletal muscle before and after fatigue

Aim: The study examined the effects of an oral acute administration of the β2‐agonist salbutamol (Sal) (6 mg) vs. placebo on muscle strength and fatigability in 12 non‐asthmatic recreational male athletes in a randomized double‐blind protocol. Methods: Contractile properties of the right quadriceps muscle were measured during electrical stimulations, i.e. twitch, 1‐s pulse trains at 20 (P₂₀) and 80 Hz (P₈₀) and during maximal voluntary isometric contraction (MVIC) before (PRE) and after (POST) a fatigue‐producing protocol set by an electromyostimulation (30 contractions, frequency: 75 Hz, on–off ratio: 6.25–20 s). In addition, the level of muscle voluntary activation was measured. Results: In PRE and POST conditions, the peak torque (PT) of twitch, P₈₀ and MVIC were not modified by the treatment. The PT in POST P₂₀ was slightly, although not significantly, less affected by fatigue in Sal compared with placebo condition. Moreover, twitch half‐relaxation time at PRE was smaller under Sal than under placebo (P < 0.05). No significant changes in the degree of voluntary activation were observed with Sal treatment in PRE or POST condition. Conclusion: Although these findings did not exclude completely an effect of Sal on peripheral factors of human skeletal muscle, oral acute administration of the β2‐agonist Sal seems to be without any relevant ergogenic effect on muscle contractility and fatigability in non‐asthmatic recreational male athletes.     

Apoptosis and changes in contractile protein pattern in the skeletal muscle in heart failure

Chronic heart failure is characterized as a clinical disorder by exercise intolerance. There are two factors that are independently responsible for the reduced exercise capacity: (a) a shift from myosin heavy chain 1 (MHC1) to MHC2a and MHC2b and (b) muscle atrophy. We have demonstrated, both in experimental models of heart failure and in man, that the more severe the heart failure, the greater the magnitude of skeletal muscle apoptosis. In the monocrotaline treated rat, that develops a severe right‐sided heart failure, the increased number of apoptotic nuclei was paralleled by increasing levels of circulating TNFα. In agreement with some recent observations showing that sphingolipids can mediate programmed cell death, we found that in animals with heart failure and high number of apoptotic nuclei, circulating levels of sphingosine were significantly increased. In a study conducted in patients with heart failure we found a correlation between exercise capacity limitation and skeletal myocytes apoptosis. There was also a correlation between degree of muscle atrophy and magnitude of apoptosis. The shift in MHCs, although with a different mechanism, is also responsible for the reduced exercise capacity in these patients. In fact there is a strong correlation between indices of severity of CHF and MHC composition. Muscle fatigue, appears earlier in patients that have a greater skeletal muscle expression of ‘fast’ MHCs. We have also demonstrated that MHCs shift and apoptosis can be prevented by using angiotensin II converting enzyme inhibitors and angiotensin II receptor blockers.     

Heart failure alters matrix metalloproteinase gene expression and activity in rat skeletal muscle

Heart failure is associated with a skeletal muscle myopathy with cellular and extracellular alterations. The hypothesis of this investigation is that extracellular changes may be associated with enhanced mRNA expression and activity of matrix metalloproteinases (MMP). We examined MMP mRNA expression and MMP activity in Soleus (SOL), extensor digitorum longus (EDL), and diaphragm (DIA) muscles of young Wistar rat with monocrotaline-induced heart failure. Rats injected with saline served as age-matched controls. MMP2 and MMP9 mRNA contents were determined by RT-PCR and MMP activity by electrophoresis in gelatin-containing polyacrylamide gels in the presence of SDS under non-reducing conditions. Heart failure increased MMP9 mRNA expression and activity in SOL, EDL and DIA and MMP2 mRNA expression in DIA. These results suggest that MMP changes may contribute to the skeletal muscle myopathy during heart failure.     

Effect of LKB1 deficiency on mitochondrial content, fibre type and muscle performance in the mouse diaphragm

Aim: The liver kinase B1 (LKB1)/AMP‐activated protein kinase (AMPK) signalling pathway is a major regulator of skeletal muscle metabolic processes. During exercise, LKB1‐mediated phosphorylation of AMPK leads to its activation, promoting mitochondrial biogenesis and glucose transport, among other effects. The roles of LKB1 and AMPK have not been fully characterized in the diaphragm. Methods: Two methods of AMPK activation were used to characterize LKB1/AMPK signalling in diaphragms from muscle‐specific LKB1 knockout (KO) and littermate control mice: (1) acute injection of 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR) and (2) 5‐min direct electrical stimulation of the diaphragm. Diaphragms were excised 60 min post‐AICAR injection and immediately after electrical stimulation. Results: AMPK phosphorylation increased with AICAR and electrical stimulation in control but not KO mice. Acetyl CoA carboxylase phosphorylation increased with AICAR in control but not KO mice, but increased in both genotypes with electrical stimulation. While the majority of mitochondrial protein levels were lower in KO diaphragms, uncoupling protein 3, complex I and cytochrome oxidase IV protein levels were not different between genotypes. KO diaphragms have a lower percentage of IIx fibres and an elevated percentage of IIb fibres when compared with control diaphragms. While in vitro peak force generation was similar between genotypes, KO diaphragms fatigued more quickly and had an impaired ability to recover. Conclusion: LKB1 regulates AMPK phosphorylation, mitochondrial protein expression, fibre type distribution, as well as recovery of the diaphragm from fatigue.