Decreased total nitric oxide production in patients with duchenne muscular dystrophy

Plasma nitric oxide (NO) levels in Duchenne muscular dystrophy (DMD) patients were significantly lower than those observed in both healthy controls and in patients with other neuromuscular disorders. The correlation between NO level and ejection fraction was significant (r=–0.384, p=0.0391) in the DMD group. Disruption of NO systems may contribute to the development of muscular dystrophy and have implications for therapeutic strategies.     

Proteomic profiling of giant skeletal muscle proteins

Introduction: Distinct subtypes of contractile fibres are highly diverse in their proteomic profile and greatly adaptable to physiological or pathological challenges. A striking biochemical feature of heterogeneous skeletal muscle tissues is the presence of a considerable number of extremely large protein species, which often present a bioanalytical challenge for the systematic separation and identification of muscle proteomes during large-scale screening surveys.

Areas covered: This review outlines the proteomic characterization of skeletal muscles with a special focus on giant proteins of the sarcomere, the cytoskeleton and the sarcoplasmic reticulum. This includes an overview of the involvement of large muscle proteins, such as titin, nebulin, obscurin, plectin, dystrophin and the ryanodine receptor calcium release channel, during normal muscle functioning, swift adaptations to changed physiological demands and changes in relation to pathobiochemical insults.

Expert commentary: The proteomic screening and characterization of total muscle extracts and various subcellular fractions has confirmed the critical role of large skeletal muscle proteins in the regulation of ion homeostasis, the maintenance of contraction-relaxation cycles and fibre elasticity, and the stabilisation of supramolecular complexes of the muscle periphery and cytoskeletal networks of contractile fibres. These findings will be helpful for the future functional systems analysis of giant muscle proteins.     

Proteomic study of calpain interacting proteins during skeletal muscle aging

Aging is associated with a progressive and involuntary loss of muscle mass also known as sarcopenia. This condition represents a major public health concern. Although sarcopenia is well documented, the molecular mechanisms of this condition still remain unclear. The calcium-dependent proteolytic system is composed of calcium-dependent cysteine proteases named calpains. Calpains are involved in a large number of physiological processes such as muscle growth and differentiation, and pathological conditions such as muscular dystrophies. The aim of this study was to determine the involvement of this proteolytic system in the phenotype associated with sarcopenia by identifying key proteins (substrates or regulators) interacting with calpains during muscle aging.     

Cyclic ADP-ribose induced Ca release in rabbit skeletal muscle sarcoplasmic reticulum

The Ca²⁺-mobilizing metabolite cyclic ADP-ribose (cADPR) has been shown to release Ca²⁺ from ryanodine-sensitive stores in many cells. We show that this metabolite at a concentration of 17μM, but not its precursor β-NAD⁺ nor non-cyclic ADPR at the same concentration, is active in releasing Ca²⁺ from rabbit skeletal muscle sarcoplasmic reticulum. The release was not sensitive to Ruthenium red (1μM) nor to the ryanodine receptor-specific scorpion toxin Buthotus₁-1 (10 μM). In planar bilayer single channel recordings, concentrations up to 50μM cADPR did not increase the open probability of Ruthenium red and toxin-sensitive Ca²⁺ release channels. Thus Ca²⁺ release induced by cADPR in skeletal muscle sarcoplasmic reticulum may not involve opening of ryanodine receptors.     

Inactivation of Brain Tryptophan Hydroxylase by Nitric Oxide

Abstract: Tryptophan hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is inactivated by nitric oxide (NO) and by the NO generators sodium nitroprusside, diethylamine/NO, S -nitroso- N -acetylpenicillamine, and S -nitrosocysteine. The inactivation occurs in an oxygen-free environment and is enhanced by dithiothreitol and ascorbic acid. Protection against the effect of NO on tryptophan hydroxylase is afforded by oxyhemoglobin, reduced glutathione, and exogenous Fe(II). Catalase partially protects the enzyme from NO-induced inactivation, whereas both superoxide dismutase and uric acid are without effect. These findings indicate that tryptophan hydroxylase is a target for NO and suggest that critical iron-sulfur groups in this enzyme serve as the substrate for NO-induced nitrosylation of the protein, resulting in enzyme inactivation.     

On the footsteps of Triadin and its role in skeletal muscle

Calcium is a crucial element for striated muscle function. As such, myoplasmic free Ca2+ concentration is delicately regulated through the concerted action of multiple Ca2+ pathways that relay excitation of the plasma membrane to the intracellular contractile machinery. In skeletal muscle, one of these major Ca2+ pathways is Ca2+ release from intracellular Ca2+ stores through type-1 ryanodine receptor/Ca2+ release channels (RyR1), which positions RyR1 in a strategic cross point to regulate Ca2+ homeostasis. This major Ca2+ traff ic point appears to be highly sensitive to the intracellular environment, which senses through a plethora of chemical and protein-protein interactions. Among these modulators, perhaps one of the most elusive is Triadin, a musclespecif ic protein that is involved in many crucial aspect of muscle function. This family of proteins mediates complex interactions with various Ca2+ modulators and seems poised to be a relevant modulator of Ca2+ signaling in cardiac and skeletal muscles. The purpose of this review is to examine the most recent evidence and current understanding of the role of Triadin in muscle function, in general, with particular emphasis on its contribution to Ca2+ homeostasis.     

S-nitrosylation of proteins at the leading edge of migrating trophoblasts by inducible nitric oxide synthase promotes trophoblast invasion

Nitric oxide regulates many important cellular processes including motility and invasion. Many of its effects are mediated through the modification of specific cysteine residues in target proteins, a process called S-nitrosylation. Here we show that S-nitrosylation of proteins occurs at the leading edge of migrating trophoblasts and can be attributed to the specific enrichment of inducible nitric oxide synthase (iNOS/NOS2) in this region. Localisation of iNOS to the leading edge is co-incidental with a site of extensive actin polymerisation and is only observed in actively migrating cells. In contrast endothelial nitric oxide synthase (eNOS/NOS3) shows distribution that is distinct and non-colocalised with iNOS, suggesting that the protein S-nitrosylation observed at the leading edge is caused only by iNOS and not eNOS. We have identified MMP-9 as a potential target for S-nitrosylation in these cells and demonstrate that it co-localises with iNOS at the leading edge of migrating cells. We further demonstrate that iNOS plays an important role in promoting trophoblast invasion, which is an essential process in the establishment of a successful pregnancy.     

Calsequestrin and the calcium release channel of skeletal and cardiac muscle

Calsequestrin is by far the most abundant Ca(2+)-binding protein in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle. It allows the Ca2+ required for contraction to be stored at total concentrations of up to 20mM, while the free Ca2+ concentration remains at approximately 1mM. This storage capacity confers upon muscle the ability to contract frequently with minimal run-down in tension. Calsequestrin is highly acidic, containing up to 50 Ca(2+)-binding sites, which are formed simply by clustering of two or more acidic residues. The Kd for Ca2+ binding is between 1 and 100 microM, depending on the isoform, species and the presence of other cations. Calsequestrin monomers have a molecular mass of approximately 40 kDa and contain approximately 400 residues. The monomer contains three domains each with a compact alpha-helical/beta-sheet thioredoxin fold which is stable in the presence of Ca2+. The protein polymerises when Ca2+ concentrations approach 1mM. The polymer is anchored at one end to ryanodine receptor (RyR) Ca2+ release channels either via the intrinsic membrane proteins triadin and junctin or by binding directly to the RyR. It is becoming clear that calsequestrin has several functions in the lumen of the SR in addition to its well-recognised role as a Ca2+ buffer. Firstly, it is a luminal regulator of RyR activity. When triadin and junctin are present, calsequestrin maximally inhibits the Ca2+ release channel when the free Ca2+ concentration in the SR lumen is 1mM. The inhibition is relieved when the Ca2+ concentration alters, either because of small changes in the conformation of calsequestrin or its dissociation from the junctional face membrane. These changes in calsequestrin's association with the RyR amplify the direct effects of luminal Ca2+ concentration on RyR activity. In addition, calsequestrin activates purified RyRs lacking triadin and junctin. Further roles for calsequestrin are indicated by the kinase activity of the protein, its thioredoxin-like structure and its influence over store operated Ca2+ entry. Clearly, calsequestrin plays a major role in calcium homeostasis that extends well beyond its ability to buffer Ca2+ ions.     

Effect of respiratory muscle fatigue on subsequent exercise performance.

The purpose of this study was to determine whether induction of inspiratory muscle fatigue might impair subsequent exercise performance. Ten healthy subjects cycled to volitional exhaustion at 90% of their maximal capacity. Oxygen consumption, breathing pattern, and a visual analogue scale for respiratory effort were measured. Exercise was performed on three separate occasions, once immediately after induction of fatigue, whereas the other two episodes served as controls. Fatigue was achieved by having the subjects breathe against an inspiratory threshold load while generating 80% of their predetermined maximal mouth pressure until they could no longer reach the target pressure. After induction of fatigue, exercise time was reduced compared with control, 238 +/- 69 vs. 311 +/- 96 (SD) s (P less than 0.001). During the last minute of exercise, oxygen consumption and heart rate were lower after induction of fatigue than during control, 2,234 +/- 472 vs. 2,533 +/- 548 ml/min (P less than 0.002) and 167 +/- 15 vs. 177 +/- 12 beats/min (P less than 0.002). At exercise isotime, minutes ventilation and the visual analogue scale for respiratory effort were larger after induction of fatigue than during control. In addition, at exercise isotime, relative tachypnea was observed after induction of fatigue. We conclude that induction of inspiratory muscle fatigue can impair subsequent performance of high-intensity exercise and alter the pattern of breathing during such exercise.     

Coexistence of potentiation and low-frequency fatigue during voluntary exercise in human skeletal muscle

The role of muscle potentiation in overcoming low-frequency fatigue (LFF) as it developed during submaximal voluntary exercise was investigated in eight males (age 26.4 +/- 0.7 years, mean +/- SE) performing isometric leg extension at approximately 30% of maximal voluntary contraction for 60 min using a 0.5-duty cycle (1 s contraction, 1 s rest). At 5, 20, 40, and 60 min, exercise was interrupted for 3 min, and the maximum positive rate of force development (+dF/dtmax) and maximal twitch force (Pt) were measured in maximal twitch contractions at 0, 1, 2, and 3 min of rest (R0, R1, R2, R3); they were also measured at 15 min of recovery following the entire 60-min exercise period. These measures were compared with pre-exercise (PRE) as an indicator of potentiation. Force at low frequency (10 Hz) was also measured at R0, R1, R2, and R3, and at 15 min of recovery, while force at high frequency (100 Hz) was measured only at R0 and R3 and in recovery. Voluntary exercise increased twitch +dF/dtmax at R0 following 5, 20, 40, and 60 min of exercise, from 2553 +/- 150 N/s at PRE to 39%, 41%, 42%, and 36% above PRE, respectively (P<0.005). Twitch +dF/dtmax decayed at brief rest (R3) following 20, 40, and 60 min of exercise (P<0.05). Pt at R0 following 5 and 20 min of exercise was above that at PRE (P<0.05), indicating that during the early phase of moderate-intensity repetitive exercise, potentiation occurs in the relative absence of LFF. At 40 and 60 min of exercise, Pt at R0 was unchanged from PRE. The LFF (10 Hz) induced by the protocol was evident at 40 and 60 min (R0-R3; P<0.05) and at 15 min following exercise (P<0.05). High-frequency force was not significantly compromised by the protocol. Since twitch force was maintained, these results suggest that as exercise progresses, LFF develops, which can be compensated for by potentiation.     

Regulation of skeletal muscle myofibrillar protein degradation: relationships to fatigue and exercise

1. Exercise results in large alterations in cellular metabolic homeostasis and protein turnovers. Exhaustive exercise (as well as starvation, dystrophy, motor nerve disease) results in myofibrillar degradation and has been associated with the decreased force generating capabilities of muscle at fatigue. 2. Complete protein degradation is accomplished by the combined actions of non-lysosomal and lysosomal proteases and the initial breakdown of myofibrillar protein appears to be non-lysosomal mediated. 3. Current evidence suggests that covalent modification (mixed-function oxidation, formation of mixed disulfides, oxidation of methionine residues and phosphorylation) of proteins may mark them for degradation by rendering them more susceptible to proteolytic attack. 4. The rate of covalent modification can be controlled by the level of stabilizing and destabilizing ligands and by factors affecting the activity of the marking reaction. 5. The activities of individual proteases may be controlled by activators and inhibitors. 6. It is suggested that the large alterations in metabolism (hormonal profiles, energy status, redox status and Ca2+ levels) which accompany exercise serve to activate specific proteases and/or induce covalent modifications which mark specific myofibrillar proteins for subsequent proteolytic attack.     

The swimming performance of brown trout and whitefish: the effects of exercise on Ca2 + handling and oxidative capacity of swimming muscles

The swimming performance of two fish species, the brown trout and whitefish, having initially different swimming strategies, was measured after nine different training programs in order to relate the effects of exercise on Ca²⁺ handling and oxidative capacity of swimming muscles. The time to 50% fatigue was measured during the training period, and compared with the density of dihydropyridine (DHP) and ryanodine (Ry) receptors and succinate dehydrogenase (SDH) and phosphorylase activity determined by histochemical analysis of the swimming muscles. Overall, both trained brown trout and whitefish had superior swimming performance as compared to control ones. Interestingly, the training programs had different effect on the two species studied since brown trout achieved the highest swimming performance, swimming against the water flow velocity of 2 BL s⁻¹ while among whitefish the best efficiency was seen after training with lower swimming velocities. Training also induced a significant increase in DHP and Ry receptor density in both species. Generally, in brown trout the most notable increase in the receptor densities was observed in red muscle sections from the fish swimming for 6 weeks against water currents of 1 BL s⁻¹ (DHPR 176.5 ± 7.7% and RyR 231.4 ± 11.8%) and white muscle sections against 2 BL s⁻¹ (DHPR 129.6 ± 12.4% and RyR 161.9 ± 15.5%). In whitefish the most prominent alterations were noted in samples from both muscle types after 6 weeks of training against water current of 1.5 BL s⁻¹ (DHPR 167.1 ± 16.9% and RyR 190.4 ± 19.4%). Finally, after all the training regimens the activity of SDH increased but the phosphorylase activity decreased significantly in both the species. To conclude, our findings demonstrate an improved swimming performance and enhanced Ca²⁺ regulation and oxidative capacity after training. Moreover, there seems to be a connection between the swimming performance and receptor levels, especially in white swimming muscles of different fish species, regardless of their initially deviant swimming behaviours. However, depending on the training regimen the divergent swimming behaviours do cause a different response, resulting in the most prominent adaptational changes in the receptor levels of red muscle samples with lower swimming velocities in brown trout and with higher ones in whitefish.     

Expression and functional activity of ryanodine receptors (RyRs) during skeletal muscle development

Two isoforms of ryanodine receptors are expressed in skeletal muscles, RyR1 and RyR3. We investigated the relative level of expression of RyRs in developing murine skeletal muscles using [3H]ryanodine binding and immunoprecipitation experiments. In the diaphragm RyR3 accounted for 11% of total RyRs in 5-day-old mice and for 3% of total RyRs in 60-day-old mice. In hindlimb muscles, RyR3 accounted for 3% and 1% of total RyRs in 5-day-old and adult mice, respectively. The activity of RyR1 channels in native microsomal vesicles from murine muscles was found to be as low as 35% of that measured after CHAPS exposure, while no inhibition was observed for RyR3. CHAPS sensitivity of recombinant RyR1 and RyR3 expressed in HEK293 cells was also investigated. The activity of recombinant RyR1 but not RyR3 channels was found to be inhibited in native conditions, suggesting that this property may not be dependent on a muscle environment.     

Effects of contraction duration on low-frequency fatigue in voluntary and electrically induced exercise of quadriceps muscle in humans

The aims of this study were to investigate if low-frequency fatigue (LFF) dependent on the duration of repeated muscle contractions and to compare LFF in voluntary and electrically induced exercise. Male subjects performed three 9-min periods of repeated isometric knee extensions at 40% maximal voluntary contraction with contraction plus relaxation periods of 30 plus 60 s, 15 plus 30 s and 5 plus 10 s in protocols 1, 2 and 3, respectively. The same exercise protocols were repeated using feedback-controlled electrical stimulation at 40% maximal tetanic torque. Before and 15 min after each exercise period, knee extension torque at 1, 7, 10, 15, 20, 50 and 100 Hz was assessed. During voluntary exercise, electromyogram root mean square (EMG_rms) of the vastus lateralis muscle was evaluated. The 20-Hz torque:100-Hz torque (20:100 Hz torque) ratio was reduced more after electrically induced than after voluntary exercise (P < 0.05). During electrically induced exercise, the decrease in 20:100 Hz torque ratio was gradually (P < 0.05) reduced as the individual contractions shortened. During voluntary exercise, the decrease in 20:100 Hz torque ratio and the increase in EMG_rms were greater in protocol 1 (P < 0.01) than in protocols 2 and 3, which did not differ from each other. In conclusion, our results showed that LFF is dependent on the duration of individual muscle contractions during repetitive isometric exercise and that the electrically induced exercise produced a more pronounced LFF compared to voluntary exercise of submaximal intensity. It is suggested that compensatory recruitment of faster-contracting motor units is an additional factor affecting the severity of LFF during voluntary exercise. Accepted: 5 November 1997     

Relationship between performance at different exercise intensities and skeletal muscle characteristics

The hypothesis investigated whether exercise performance over a broad range of intensities is determined by specific skeletal muscle characteristics. Seven subjects performed 8-10 exhaustive cycle trials at different workloads, ranging from 150 to 700 W (150 min to 20 s). No relationships between the performance times at high and low workloads were observed. A relationship (P < 0.05) was noticed between the percentage of fast-twitch x fibers and the exercise time at 579 ± 21 W (～30 s; r(2) = 0.88). Capillary-to-fiber-ratio (r(2): 0.58-0.85) was related (P < 0.05) to exercise time at work intensities ranging from 395 to 270 W (2.5-21 min). Capillary density was correlated (r(2) = 0.68; P < 0.05) with the net rate of plasma K(+) accumulation during an ～3-min bout and was estimated to explain 50-80% (P < 0.05) of the total variance observed in exercise performances lasting ～30 s to 3 min. The Na(+)-K(+) pump β(1)-subunit expression was found to account for 13-34% (P < 0.05) during exhaustive exercise of ～1-4 min. In conclusion, exercise performance at different intensities is related to specific physiological variables. A large distribution of fast-twitch x fibers may play a role during very intense efforts, i.e., ～30 s. Muscle capillaries and the Na(+)-K(+) pump β(1)-subunit seem to be important determinants for performance during exhaustive high-intensity exercises lasting between 30 s and 4 min.     

Akt signaling in skeletal muscle: regulation by exercise and passive stretch

Akt/protein kinase B is a serine/threonine kinase that has emerged as a critical signaling component for mediating numerous cellular responses. Contractile activity has recently been demonstrated to stimulate Akt signaling in skeletal muscle. Whether physiological exercise in vivo activates Akt is controversial, and the initiating factors that result in the stimulation of Akt during contractile activity are unknown. In the current study, we demonstrate that treadmill running exercise of rats using two different protocols (intermediate high or high-intensity exhaustive exercise) significantly increases Akt activity and phosphorylation in skeletal muscle composed of various fiber types. To determine if Akt activation during contractile activity is triggered by mechanical forces applied to the skeletal muscle, isolated skeletal muscles were incubated and passively stretched. Passive stretch for 10 min significantly increased Akt activity (2-fold) in the fast-twitch extensor digitorum longus (EDL) muscle. However, stretch had no effect on Akt in the slow-twitch soleus muscle, although there was a robust phosphorylation of the stress-activated protein kinase p38. Similar to contraction, stretch-induced Akt activation in the EDL was fully inhibited in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin, whereas glycogen synthase kinase-3 (GSK3) phosphorylation was only partially inhibited. Stretch did not cause dephosphorylation of glycogen synthase on GSK3-targeted sites in the absence or presence of wortmannin. We conclude that physiological exercise in vivo activates Akt in multiple skeletal muscle fiber types and that mechanical tension may be a part of the mechanism by which contraction activates Akt in fast-twitch muscles.     

ADP-ribose stimulates the calcium release channel RyR1 in skeletal muscle of rat

The Ca(2+) mobilizing metabolite cyclic ADP-ribose has been shown to release Ca(2+) from intracellular ryanodine sensitive stores in many cells. However, the activation of the ryanodine receptor of skeletal muscle by cADP-ribose (cADPr) and its precursor and metabolite (beta-NAD(+) and ADPr) remains to be discussed. We studied the effect of ADPr on the Ca(2+) release channel of skeletal muscle RyR1 after incorporation of microsomes isolated from fast muscles of rat in planar lipid bilayers. We observed an increase in the electrophysiological activity of the channel after addition of ADPr (10 microM) at micromolar Ca(2+) concentrations, characterized by a time-lag. The increase in P(o) is mainly due to an increase in the open frequency. The long time course observed for the development of the ADPr effect may indicate that this activation induces a change in the conformation of the RyR1 channel, which increases its sensitivity to calcium.     

A method for assessing muscle fatigue during sprint exercise in humans using a friction-loaded cycle ergometer

This study investigated the mechanical changes induced by muscle fatigue caused by repeated sprints and determined whether a friction-loaded cycle ergometer has any advantages for assessing muscle fatigue. Nine subjects performed 15 sprints, each of 5 s with a 25-s rest, on a friction-loaded cycle ergometer. The averaged force, power and velocity of each push-off were calculated. Maximal power decreased by 17.9%, with a concomittent slowing of muscle contraction, but without any change in the maximal force. These results demonstrated that repeated sprints slow down muscle contraction, leading to a fall in maximal power without any loss of force. This would suggest that fast twitch fibres are selectively fatigued by repeated sprints. However, the ergometer used in the present study made it difficult to evaluate the relative influences of contraction velocity and sprinting time. This was certainly the most important limitation. On the other hand, it showed the advantage of measuring instantaneous power and total work dissipated in the environment simultaneously. It also permitted a force-velocity relationship to be obtained from a single sprint and this relationship is known to be closely related to the muscle fibre composition. Accepted: 5 March 1998