Investigations of back muscle fatigue by simultaneous 31P MRS and surface EMG measurements.

Investigations of back muscle fatigue are important for understanding the role of muscle strain in the development of low back pain. The aim of this contribution is to review the two main techniques used for in vivo investigations of metabolic and electrophysiological changes, namely magnetic resonance phosphorous spectroscopy ((31)P MRS) and surface electromyography (SEMG), and to report some of our recent results on simultaneous measurements using these techniques during isometric back-muscle contraction in volunteers. Since it appears that electrophysiological and metabolic factors are simultaneously involved in the processes of fatigue and muscle recovery during load application, simultaneous acquisition of complete information is quite promising for obtaining new insights into the metabolic origin of electrophysiological changes or vice versa. Performing these measurements simultaneously, however, is more intricate owing to the occurrence of signal artifacts caused by mutual signal interferences of both techniques. Besides these mutual disturbances, further experimental difficulties are related to spatial limitations within the bore of clinical whole-body high-field magnetic resonance (MR) systems (1.5 T) and the sensitivity of MR measurements to motion-induced artifacts. Our own experimental results are presented, and problems that occur using both techniques simultaneously, as well as possibilities to resolve them, are discussed. The results shed light on the interrelation of electrophysiological and metabolic changes during fatigue of the back muscle while performing an exercise.     

Evaluation of exercise muscle energetics by NMR

Magnetic resonance imaging (MRI) is superior to ultrasonography and X-CT especially in density resolution in soft tissue. 31P NMR provides information on metabolism, which has not been obtained in vivo by conventional methods, such as phosphocreatine (PCr), inorganic phosphate (Pi), ATP, and intracellular pH. We used MRI and 31P NMR spectroscopy to study skeletal muscle metabolism of human and rat. These NMR results suggested that 1) estimation of muscle fiber composition, 2) evaluation of muscle ATP turnover and 3) imaging of local muscle fatigue are possible.     

Effect of N-acetylcysteine on diaphragm fatigue

It has recently been postulated that diaphragm fatigue may be due, at least in part, to a form of low-grade injury to subcellular organelles. Moreover, several studies have shown that thiol-containing compounds can protect cardiac and striated skeletal muscle organelles from the deleterious effects of a number of physiological stresses. The purpose of the present study was to determine whether pretreatment with N-acetylcysteine (NAC), a thiol-containing compound, would attenuate the rate of development of diaphragmatic fatigue. Studies were performed with the use of an in situ rabbit diaphragm strip preparation that permitted direct and continuous measurement of diaphragm tension development. Diaphragm fatigue was induced by rhythmically stimulating strips to contract at 30/min (20-Hz trains) for 20 min. The diaphragm force-frequency relationship (10-, 20-, 50-, and 100-Hz stimuli) was assessed immediately before and after fatigue trials and then again 20 min into the period of recovery. Half the animals were treated with intravenous NAC before fatigue, whereas the remaining animals were given intravenous saline. The rate of development of fatigue was markedly greater in saline-treated control than in NAC-treated animals, with reductions in tension of 55 +/- 3 and 34 +/- 3%, respectively, in these two groups of animals over 20 min (P less than 0.001). Although rhythmic stimulation resulted in a downward shift in the force-frequency relationship in both NAC- and saline-treated animals, the magnitude of this shift was substantially greater in saline-treated animals (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphocreatine and pH recovery without restoration of mechanical function during prolonged activity of rat gastrocnemius muscle: an in vivo 31P NMR study

Metabolic impairment in skeletal muscle was suggested to be involved in the development of local mechanical fatigue but until now results have dealt with short activity periods whereas little data on exhaustive and prolonged exercises are available. Stimulations of rat leg muscle lasting 45 min were induced by tetanic trains delivered via sciatic nerve at five different rhythms. Energy metabolism of the stimulated gastrocnemius muscle was followed by 31P NMR spectroscopy using surface coil while mechanical function was recorded. Our data showed a decrease in the force level to very low values a few minutes after exercise onset. This mechanical impairment only induced a transient metabolic failure followed by rapid restoration of high phosphocreatine (PCr) values and intracellular pH, without mechanical recovery. In addition, at the end of exercise, the PCr content was proportional to the fatigue level. As these experiments could not have impaired neuromuscular junction, the data would indicate that fatigue was maintained by a mechanism which does not appear to depend directly on muscle cell energy stores.     

Free radical induced respiratory muscle dysfunction

It is now recognized that respiratory muscle fatigue contributes to the development of respiratory failure in some patients with lung disease. This observation has prompted an examination into the mechanisms of development of muscle fatigue, with the understanding that an elucidation of these processes may lead to new therapeutic approaches to the treatment of these patients. A series of recent studies examining this issue have, moreover, discovered that oxygen-derived free radicals generated during strenuous contraction may modulate respiratory muscle contractile function and contribute to the development of muscle fatigue. The data supporting this concept include: (a) direct (e.g. EPR, ESR studies) and indirect (evidence of lipid peroxidation, protein carbonyl formation, glutathione oxidation) evidence that there is heightened free radical production in contracting muscle, (b) evidence that pharmacologic depletion of muscle antioxidant stores increases degree of muscle fatigue present after a period of exercise, and (c) evidence that administration of agents that act as free radical scavengers retard the development muscle fatigue. Free radicals may produce these changes in muscle force generating capacity by interacting with and altering the function of a number of intracellular-biophysical processes (i.e. sarcolemmal action potential propagation, sarcoplasmic reticulum calcium handling, mitochondrial function, contractile protein interactions).     

Muscle fatigue and contraction intensity modulates the complexity of surface electromyography

Nonlinear dynamical techniques offer a powerful approach for the investigation of physiological time series. Multiscale entropy analyses have shown that pathological and aging systems are less complex than healthy systems and this finding has been attributed to degraded physiological control processes. A similar phenomenon may arise during fatiguing muscle contractions where surface electromyography signals undergo temporal and spectral changes that arise from the impaired regulation of muscle force production. Here we examine the affect of fatigue and contraction intensity on the short and long-term complexity of biceps brachii surface electromyography. To investigate, we used an isometric muscle fatigue protocol (parsed into three windows) and three contraction intensities (% of maximal elbow joint moment: 40%, 70% and 100%). We found that fatigue reduced the short-term complexity of biceps brachii activity during the last third of the fatiguing contraction. We also found that the complexity of surface electromyography is dependent on contraction intensity. Our results show that multiscale entropy is sensitive to muscle fatigue and contraction intensity and we argue it is imperative that both factors be considered when evaluating the complexity of surface electromyography signals. Our data contribute to a converging body of evidence showing that multiscale entropy can quantify subtle information content in physiological time series.     

New insights into the role of sphingosine 1-phosphate and lysophosphatidic acid in the regulation of skeletal muscle cell biology

Lysophospholipids are bioactive molecules that are implicated in the control of fundamental biological processes such as proliferation, differentiation, survival and motility in different cell types. Here we review the role of sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) in the regulation of skeletal muscle biology. Indeed, a wealth of experimental data indicate that these molecules are crucial players in the skeletal muscle regeneration process, acting by controllers of activation, proliferation and differentiation not only of muscle-resident satellite cells but also of mesenchymal progenitors that originate outside the skeletal muscle. Moreover, S1P and LPA are clearly involved in the regulation of skeletal muscle metabolism, muscle adaptation to different physiological needs and resistance to muscle fatigue. Notably, studies accomplished so far, have highlighted the complexity of S1P and LPA signaling in skeletal muscle cells that appears to be further complicated by their close dependence on functional cross-talks with growth factors, hormones and cytokines. Our increasing understanding of bioactive lipid signaling can individuate novel molecular targets aimed at enhancing skeletal muscle regeneration and reducing the fibrotic process that impairs full functional recovery of the tissue during aging, after a trauma or skeletal muscle diseases. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Fatigue in isometric contraction in a single muscle fibre: a compartmental calcium ion flow model

Fatigue in muscle is a complex biological phenomenon which has so far eluded a definite explanation. Many biochemical and physiological models have been suggested in the literature to account for the decrement in the ability of muscle to sustain a given level of force for a long time. Some of these models have been critically analysed in this paper and are shown to be not able to explain all the experimental observations. A new compartmental model based on the intracellular calcium ion movement in muscle is proposed to study the mechanical responses of a muscle fibre. Computer simulation is performed to obtain model responses in isometric contraction to an impulse and a train of stimuli of long duration. The simulated curves have been compared with experimentally observed mechanical responses of the semitendinosus muscle fibre of Rana pipiens. The comparison of computed and observed responses indicates that the proposed calcium ion model indeed accounts very well for the muscle fatigue.     

Activation of human quadriceps femoris muscle during dynamic contractions: effects of load on fatigue.

Muscle fatigue is both multifactorial and task dependent. Electrical stimulation may assist individuals with paralysis to perform functional activities [functional electrical stimulation (FES), e.g., standing or walking], but muscle fatigue is a limiting factor. One method of optimizing force is to use stimulation patterns that exploit the catchlike property of skeletal muscle [catchlike-inducing trains (CITs)]. Although nonisometric (dynamic) contractions are important parts of both normal physiological activation of skeletal muscles and FES, no previous studies have attempted to identify the effect that the load being lifted by a muscle has on the fatigue produced. This study examined the effects of load on fatigue during dynamic contractions and the augmentation produced by CITs as a function of load. Knee extension in healthy subjects was electrically elicited against three different loads. The highest load produced the least excursion, work, and average power, but it produced the greatest fatigue. CIT augmentation was greatest at the highest load and increased with fatigue. Because CITs were effective during shortening contractions for a variety of loads, they may be of benefit during FES applications.     

Locomotor muscle fatigue increases cardiorespiratory responses and reduces performance during intense cycling exercise independently from metabolic stress

Locomotor muscle fatigue, defined as an exercise-induced reduction in maximal voluntary force, occurs during prolonged exercise, but its effects on cardiorespiratory responses and exercise performance are unknown. In this investigation, a significant reduction in locomotor muscle force (-18%, P < 0.05) was isolated from the metabolic stress usually associated with fatiguing exercise using a 100-drop-jumps protocol consisting of one jump every 20 s from a 40-cm-high platform. The effect of this treatment on time to exhaustion during high-intensity constant-power cycling was measured in study 1 (n = 10). In study 2 (n = 14), test duration (871 +/- 280 s) was matched between fatigue and control condition (rest). In study 1, locomotor muscle fatigue caused a significant curtailment in time to exhaustion (636 +/- 278 s) compared with control (750 +/- 281 s) (P = 0.003) and increased cardiac output. Breathing frequency was significantly higher in the fatigue condition in both studies despite similar oxygen consumption and blood lactate accumulation. In study 2, high-intensity cycling did not induce further fatigue to eccentrically-fatigued locomotor muscles. In both studies, there was a significant increase in heart rate in the fatigue condition, and perceived exertion was significantly increased in study 2 compared with control. These results suggest that locomotor muscle fatigue has a significant influence on cardiorespiratory responses and exercise performance during high-intensity cycling independently from metabolic stress. These effects seem to be mediated by the increased central motor command and perception of effort required to exercise with weaker locomotor muscles.     

RELATIONS BETWEEN STRUCTURE AND FUNCTION IN RAT SKELETAL MUSCLE FIBERS

The fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus muscle of the rat consist of heterogeneous fiber populations. EDL muscle fibers differ in size, mitochondrial content, myoglobin concentration, and thickness of the Z line. The sarcoplasmic reticulum, on the other hand, is richly developed in all fibers, with only small variation. Myofibrils are clearly circumscribed at both the A and I band level. The soleus muscle is composed primarily of fibers with moderate mitochondrial content and myoglobin concentration. In most fibers the sarcoplasmic reticulum is poorly developed, with the exception of the portion of reticulum in phase with the Z line. As a consequence the myofibrillar fields are amply fused together. Contacts between sarcoplasmic reticulum and T system are discontinuous and may occur in the form of "dyads" instead of the typical triad structure. In a small proportion of soleus muscle fibers the organization and development of the sarcoplasmic reticulum is similar to that of EDL muscle fibers, with prominent fenestrated collars at the H band level. In these fibers mitochondria are larger and more abundant. The results are correlated with physiological studies on motor units in the same and in similar rat muscles. It is suggested that the variable structural pattern of rat muscle fibers is related to two distinct physiological parameters, speed of contraction and resistance to fatigue.     

Energy metabolism in relation to oxygen supply in contracting rat skeletal muscle

The regulation of the energy metabolism in contracting skeletal muscle is under close control, and several regulating factors have been reported. The aim of this study was to investigate the importance of the oxygen supply as a limiting factor for muscle performance during contractions and recovery from contractions. To perform well-controlled standardized experiments on contracting skeletal muscle, the perfused rat hind limb model was developed. The 31P NMR technique was adapted to the rat hind limb model. This enabled continuous nondestructive monitoring of the energy state at various levels of muscular activity. Significant correlations were found between oxygen delivery and oxygen consumption, lactate release, and glucose uptake, respectively. An increased degree of fatigue was observed at lower oxygen deliveries. In both soleus and gastrocnemius muscles, oxygen delivery correlated with the intramuscular concentrations of phosphocreatine (PCr), lactate, and glycogen. The 31P NMR experiments showed a correlation between oxygen delivery and the steady-state level of PCr/inorganic phosphate (Pi) during the contraction period. The rate of recovery in PCr/Pi after the contraction was also dependent on oxygen delivery. The results demonstrate a causal relationship between oxygen supply and energy state in contracting as well as recovering skeletal muscles.     

Interactions between intracellular calcium and phosphate in intact mouse muscle during fatigue

Fatigue was studied in intact tibialis anterior muscle of anesthetized mice. The distal tendon was detached and connected to a force transducer while blood flow continued normally. The muscle was stimulated with electrodes applied directly to the muscle surface and fatigued by repeated (1 per 4 s), brief (0.4 s), maximal (100-Hz stimulation frequency) tetani. Force declined monotonically to 49 ± 5% of the initial value with a half time of 36 ± 5 s and recovered to 86 ± 4% after 4 min. Intracellular phosphate concentration ([P(i)]) was measured by (31)P-NMR on perchloric acid extracts of muscles. [P(i)] increased during fatigue from 7.6 ± 1.7 to 16.0 ± 1.6 mmol/kg muscle wet wt and returned to control during recovery. Intracellular Ca(2+) was measured with cameleons whose plasmids had been transfected in the muscle 2 wk before the experiment. Yellow cameleon 2 was used to measure myoplasmic Ca(2+), and D1ER was used to measure sarcoplasmic reticulum (SR) Ca(2+). The myoplasmic Ca(2+) during tetani declined steadily during the period of fatigue and showed complete recovery over 4 min. The SR Ca(2+) also declined monotonically during fatigue and showed a partial recovery with rest. These results show that the initial phase of force decline is accompanied by a rise in [P(i)] and a reduction in the tetanic myoplasmic Ca(2+). We suggest that both changes contribute to the fatigue. A likely cause of the decline in tetanic myoplasmic Ca(2+) is precipitation of CaP(i) in the SR.     

Biochemical adaptations to training: implications for resisting muscle fatigue

Skeletal muscle activity is invariably associated with a decline in force-generating capacity (fatigue). The build-up of metabolic by-products such as intracellular H+ and inorganic phosphate (Pi) has been shown to be one of the potential mechanisms of muscle fatigue. The use of phosphorus magnetic resonance spectroscopy is a repeatable and useful tool to study the effect of pH and Pi on force development. When maximal exercise is preceded by submaximal exercise to reduce the starting muscle pH and increase Pi, the degree of muscle fatigue correlates more strongly with H2PO4- than pH or Pi alone. However, other studies in humans have found that H2PO4- does not always correlate well with fatigue. The use of ramp exercise protocols allow repeatable and sensitive measurement of changes in muscle metabolism in response to endurance training. Chronic electrical stimulation in dogs and endurance training in humans results in reduced pH and Pi changes at the same exercise intensities. This means that the effect of pH and Pi in depressing force development is reduced, which could partially explain the increased fatigue resistance seen following endurance training.     

Inositol trisphosphate, calcium and muscle contraction

The identity of organelles storing intracellular calcium and the role of Ins(1,4,5)P3 in muscle have been explored with, respectively, electron probe X-ray microanalysis (EPMA) and laser photolysis of 'caged' compounds. The participation of G-protein(s) in the release of intracellular Ca2+ was determined in saponin-permeabilized smooth muscle. The sarcoplasmic reticulum (SR) is identified as the major source of activator Ca2+ in both smooth and striated muscle; similar (EPMA) studies suggest that the endoplasmic reticulum is the major Ca2+ storage site in non-muscle cells. In none of the cell types did mitochondria play a significant, physiological role in the regulation of cytoplasmic Ca2+. The latency of guinea pig portal vein smooth muscle contraction following photolytic release of phenylephrine, an alpha 1-agonist, is 1.5 +/- 0.26 s at 20 degrees C and 0.6 +/- 0.18 s at 30 degrees C; the latency of contraction after photolytic release of Ins(1,4,5)P3 from caged Ins(1,4,5)P3 is 0.5 +/- 0.12 s at 20 degrees C. The long latency of alpha 1-adrenergic Ca2+ release and its temperature dependence are consistent with a process mediated by G-protein-coupled activation of phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P2) hydrolysis. GTP gamma S, a non-hydrolysable analogue of GTP, causes Ca2+ release and contraction in permeabilized smooth muscle. Ins(1,4,5)P3 has an additive effect during the late, but not the early, phase of GTP gamma S action, and GTP gamma S can cause Ca2+ release and contraction of permeabilized smooth muscles refractory to Ins(1,4,5)P3. These results suggest that activation of G protein(s) can release Ca2+ by, at least, two G-protein-regulated mechanisms: one mediated by Ins(1,4,5)P3 and the other Ins(1,4,5)P3-independent. The low Ins(1,4,5)P3 5-phosphatase activity and the slow time-course (seconds) of the contractile response to Ins(1,4,5)P3 released with laser flash photolysis from caged Ins(1,4,5)P3 in frog skeletal muscle suggest that Ins(1,4,5)P3 is unlikely to be the physiological messenger of excitation-contraction coupling of striated muscle. In contrast, in smooth muscle the high Ins(1,4,5)P3-5-phosphatase activity and the rate of force development after photolytic release of Ins(1,4,5)P3 are compatible with a physiological role of Ins(1,4,5)P3 as a messenger of pharmacomechanical coupling.     

Cellular responses to H(2)O(2) and bleomycin-induced oxidative stress in L6C5 rat myoblasts

In muscle cells, reactive oxygen species (ROS) are continually generated. It is believed that these molecules have a well-established role as physiological modulators of skeletal muscle functions, ranging from development to metabolism and from blood flow to contractile functions. Moreover, ROS may contribute to the development of muscle fatigue, inflammation, and degeneration, and may be implicated in many muscle diseases. The aim of the present study was to verify the role of short or prolonged exposure to oxidative stress, generated by different concentrations of H(2)O(2), on growth, chromosomal aberrations, and apoptosis induced in cultured L6C5 rat muscle cells used as model for myoblasts. Our results indicate that, in L6C5 cells, reactive oxygen intermediates (ROI) can activate distinct cell pathways leading to cell growth induction and development of resistant phenotype, or to chromosomal aberrations, cell cycle arrest, or cell death. The positive vs. negative effects of H(2)O(2)-altered redox potential in myoblasts are strictly related to the intensity of oxidative stress, likely depending on the types and number of cellular targets involved. Among these, DNA molecules appear to be very sensitive to breakage by H(2)O(2), although DNA damage is not directly responsible for ROI-induced apoptosis in L6C5 rat myoblasts.     

The role of sphingosine-1-phosphate in skeletal muscle: Physiology,mechanisms, and clinical perspectives

Sphingolipids were discovered more than a century ago and were simply considered as a class of cell membrane lipids for a long time. However, after the discovery of several intracellular functions and their role in the control of many physiological and pathophysiological conditions, these molecules have gained much attention. For instance, the sphingosine-1-phosphate (S1P) is a circulating bioactive sphingolipid capable of triggering strong intracellular reactions through the family of S1P receptors (S1PRs) spread in several cell types and tissues. Recently, the role of S1P in the control of skeletal muscle metabolism, atrophy, regeneration, and metabolic disorders has been widely investigated. In this review, we summarized the knowledge of S1P and its effects in skeletal muscle metabolism, highlighting the role of S1P/S1PRs axis in skeletal muscle regeneration, fatigue, ceramide accumulation, and insulin resistance. Finally, we discussed the physical exercise role in S1P/S1PRs signaling in skeletal muscle cells, and how this nonpharmacological strategy may be prospective for future investigations due to its ability to increase S1P levels.     

Dystrophic mdx mouse myoblasts exhibit elevated ATP/UTP-evoked metabotropic purinergic responses and alterations in calcium signalling

Pathophysiology of Duchenne Muscular Dystrophy (DMD) is still elusive. Although progressive wasting of muscle fibres is a cause of muscle deterioration, there is a growing body of evidence that the triggering effects of DMD mutation are present at the earlier stage of muscle development and affect myogenic cells. Among these abnormalities, elevated activity of P2X7 receptors and increased store-operated calcium entry myoblasts have been identified in mdx mouse. Here, the metabotropic extracellular ATP/UTP-evoked response has been investigated. Sensitivity to antagonist, effect of gene silencing and cellular localization studies linked these elevated purinergic responses to the increased expression of P2Y2 but not P2Y4 receptors. These alterations have physiological implications as shown by reduced motility of mdx myoblasts upon treatment with P2Y2 agonist. However, the ultimate increase in intracellular calcium in dystrophic cells reflected complex alterations of calcium homeostasis identified in the RNA seq data and with significant modulation confirmed at the protein level, including a decrease of Gq11 subunit α, plasma membrane calcium ATP-ase, inositol-2,4,5-trisphosphate-receptor proteins and elevation of phospholipase Cβ, sarco-endoplamatic reticulum calcium ATP-ase and sodium‑calcium exchanger. In conclusion, whereas specificity of dystrophic myoblast excitation by extracellular nucleotides is determined by particular receptor overexpression, the intensity of such altered response depends on relative activities of downstream calcium regulators that are also affected by Dmd mutations. Furthermore, these phenotypic effects of DMD emerge as early as in undifferentiated muscle. Therefore, the pathogenesis of DMD and the relevance of current therapeutic approaches may need re-evaluation.     

Proteins from rat liver cytosol which stimulate mRNA transport. Purification and interactions with the nuclear envelope mRNA translocation system

Two polysome-associated proteins with particular affinities for poly(A) have been purified from rat liver. These proteins stimulate the efflux of mRNA from isolated nuclei in conditions under which such efflux closely stimulates mRNA transport in vivo, and they are therefore considered as mRNA-transport-stimulatory proteins. Their interaction with the mRNA-translocation system in isolated nuclear envelopes has been studied. The results are generally consistent with the most recently proposed kinetic model of mRNA translocation. One protein, P58, has not been described previously. It inhibits the protein kinase that down-regulates the NTPase, it enhances the NTPase activity in both the presence and the absence of poly(A) and it seems to increase poly(A) binding in unphosphorylated, but not in phosphorylated, envelopes. The other protein, P31, which probably corresponds to the 35,000-Mr factor described by Webb and his colleagues, enhances the binding of poly(A) to the mRNA-binding site in the envelope, thus stimulating the phosphoprotein phosphatase and, in consequence, the NTPase. The possible physiological significance of these two proteins is discussed.     

Constraints on muscular performance: trade-offs between power output and fatigue resistance

An important functional and evolutionary constraint on the physical performance of vertebrates is believed to be the trade-off between speed and endurance capacity. However, despite the pervasiveness of physiological arguments, most studies have found no evidence of the trade-off when tested at the whole-animal level. We investigated the existence of this trade-off at the whole-muscle level, the presumed site of this physiological conflict, by examining inter-individual variation in both maximum power output and fatigue resistance for mouse extensor digitorum longus (EDL) muscle using the work-loop technique. We found negative correlations between several measures of in vitro maximum power output and force production with fatigue resistance for individual mouse EDL muscles, indicating functional trade-offs between these performance parameters. We suggest that this trade-off detected at the whole-muscle level has imposed an important constraint on the evolution of vertebrate physical performance.