Reactive oxygen species contribute to Ca2+ signals produced by osmotic stress in mouse skeletal muscle fibres.

Ca(2+) sparks, localized elevations in cytosolic [Ca(2+)], are rarely detected in intact adult mammalian skeletal muscle under physiological conditions. However, they have been observed in permeabilized cells and in intact fibres subjected to stresses, such as osmotic shock and strenuous exercise. Our previous studies indicated that an excess in cellular reactive oxygen species (ROS) generation over the ROS scavenging capabilities could be one of the up-stream causes of Ca(2+) spark appearance in permeabilized muscle fibres. Here we tested whether the cytosolic ROS balance is compromised in intact skeletal muscle fibres that underwent osmotic shock and whether this misbalance contributes to unmasking Ca(2+) sparks. Spontaneous Ca(2+) sparks and the rate of ROS generation were assessed with single photon confocal microscopy and fluorescent indicators fluo-4, CM-H(2)DCFDA and MitoSOX Red. Osmotic shock produced spontaneous Ca(2+) sparks and a concomitant significant increase in ROS production. Preincubation of muscle cells with ROS scavengers (e.g. MnTBAP, Mn-cpx 3, TIRON) nearly eliminated Ca(2+) sparks. In addition, inhibitors of NAD(P)H oxidase (DPI and apocynin) significantly reduced ROS production and suppressed the appearance of Ca(2+) sparks. Taken together, the data suggest that ROS contribute to the abnormal Ca(2+) spark activity in mammalian skeletal muscle subjected to osmotic stress and also indicate that NAD(P)H oxidase is a possible source of ROS. We propose that ROS-dependent Ca(2+) sparks are an important component of adaptive/maladaptive muscle responses under various pathological conditions such as eccentric stretch, osmotic changes during ischaemia and reperfusion, and some muscle diseases.     

Reactive oxygen species contribute to Ca2+ signals produced by osmotic stress in mouse skeletal muscle fibres

Ca²⁺ sparks, localized elevations in cytosolic [Ca²⁺], are rarely detected in intact adult mammalian skeletal muscle under physiological conditions. However, they have been observed in permeabilized cells and in intact fibres subjected to stresses, such as osmotic shock and strenuous exercise. Our previous studies indicated that an excess in cellular reactive oxygen species (ROS) generation over the ROS scavenging capabilities could be one of the up-stream causes of Ca²⁺ spark appearance in permeabilized muscle fibres. Here we tested whether the cytosolic ROS balance is compromised in intact skeletal muscle fibres that underwent osmotic shock and whether this misbalance contributes to unmasking Ca²⁺ sparks. Spontaneous Ca²⁺ sparks and the rate of ROS generation were assessed with single photon confocal microscopy and fluorescent indicators fluo-4, CM-H₂DCFDA and MitoSOX Red. Osmotic shock produced spontaneous Ca²⁺ sparks and a concomitant significant increase in ROS production. Preincubation of muscle cells with ROS scavengers (e.g. MnTBAP, Mn-cpx 3, TIRON) nearly eliminated Ca²⁺ sparks. In addition, inhibitors of NAD(P)H oxidase (DPI and apocynin) significantly reduced ROS production and suppressed the appearance of Ca²⁺ sparks. Taken together, the data suggest that ROS contribute to the abnormal Ca²⁺ spark activity in mammalian skeletal muscle subjected to osmotic stress and also indicate that NAD(P)H oxidase is a possible source of ROS. We propose that ROS-dependent Ca²⁺ sparks are an important component of adaptive/maladaptive muscle responses under various pathological conditions such as eccentric stretch, osmotic changes during ischaemia and reperfusion, and some muscle diseases.     

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.     

Reactive oxygen species and inflammatory mediators enhance muscle spindles mechanosensitivity in rats

We tested the hypothesis that reactive oxygen species (ROS) and inflammatory mediators affect transduction properties of muscle spindles. In rats, muscle spindles response to high-frequency vibration (HFV) was recorded before and after (1) injection of hydrogen peroxide (H2O2) in control rats and animals pre-treated with diclofenac (anti-inflammatory substance), (2) injection of bradykinin and (3) fatigue induced by muscle stimulation (MS) in control rats and rats receiving diclofenac, superoxide dismutase (SOD) or H2O2. Muscular oxidative stress and inflammation induced by H2O2 or MS were assessed by measurements of isoprostanes and IL-6 levels. In control rats, H2O2, bradykinin and MS significantly enhanced the HFV response. Pre-treatment with SOD abolished the post-MS-enhanced HFV response whereas diclofenac lowered the peak HFV response to MS and H2O2. H2O2 injection and MS elicited significant and similar increases in isoprostanes and IL-6. We report a direct modulation of muscle spindles mechanosensitivity by ROS and inflammatory mediators.     

Reactive oxygen species activate the group IV muscle afferents in resting and exercising muscle in rats

We tested the hypothesis that the reactive oxygen species (ROS) produced at rest and mostly during muscle contraction may stimulate the group IV muscle afferents. In rats, afferent activity was recorded in the peroneal nerve innervating the tibialis anterior muscle. Group IV afferents were identified from measurements of their conduction velocity and response to lactic acid. Comparing the group IV response to an intramuscular injection of buffered isotonic NaCl solution, we searched for the effects of a ROS donor (H₂O₂) or a ROS inhibitor (superoxide dismutase, SOD) on the baseline afferent activity in resting muscles. We also explored the consequences of a pre-treatment with SOD on the afferent nerve response to H₂O₂ injection or electrical muscle stimulation (MS). In other animals, we measured the changes in intramuscular level of a marker of oxidative stress (isoprostanes) after each test agent. H₂O₂ injection markedly activated all recorded group IV afferents. SOD injection lowered the baseline activity of 50 out of 70 afferent units, suppressed the afferent response to H₂O₂ injection, and delayed and reduced the MS-induced activation of all recorded units. Intramuscular isoprostanes level significantly increased after H₂O₂ injection or MS, the oxidative stress being absent in muscles pre-treated with SOD. We concluded that ROS influence both the spontaneous and contraction-induced activities of the group IV muscle afferents and are a potent stimulus of muscle metaboreceptors.     

Endogenous MLC2 phosphorylation and Ca2+-activated force in mechanically skinned skeletal muscle fibres of the rat

A method has been developed for measuring the level of phosphorylation of myosin regulatory light chains (MLC2) by the endogenous myosin light chain kinase in mechanically skinned skeletal muscle fibres. The method was used to characterize the endogeous MLC2 phosphorylation capacity of single fast-twitch fibres from the rat and to investigate the relationship between the endogenous MLC2 phosphorylation and the Ca²⁺-activated force. The results show that (1) about 50% of MLC2 were ³²P-phosphorylated after activation of the skinned fibre preparation by 30 M [Ca²⁺] for longer than 30 s, but that there was variability between fibres; (2) most of the endogenous phosphorylating system diffused out of the skinned fibre preparation after 5 min exposure to an aqueous solution; (3) the MLC2 phosphorylation by the endogenous phosphorylating system followed with a delay of the order of 1–2 s after the sudden rise in [Ca²⁺] from below 10 nM to 30 M; and (4) the sensitivity of the contractile apparatus to Ca²⁺ was markedly increased when the MLC2 were phosphorylated by the endogenous phosphorylating system following a rise in [Ca²⁺]. The K _d for MgATP of the endogenous MLC2 phosphorylating system was estimated to be less than 300 M. These results unequivocally demonstrate that prolonged activation of the fast-twitch muscle fibre leads to increased Ca²⁺ sensitivity of the contractile apparatus and that mechanically skinned fibres can be successfully used to study the regulation of the endogenous MLC2 phosphorylation capacity at single muscle fibre level.     

Force and force transients in skeletal muscle fibres of the frog skinned by freeze-drying

A freeze-drying method is described by which single skinned skeletal muscle fibres or fibre bundles can readily be obtained. Skinned fibre segments of the ileofibularis and semitendinous muscles of the frog — activated by means of a rapid increase in the Ca-concentration — showed very stable and reproducible contractions. Complete activation occurred at a Ca-concentration of 1.6·10^–6 M and the mid-point of the pCa-tension curve occurred at 6.3·10^–7 M. Addition of phosphate (10^–2 M) had a depressing effect on the speed of the Ca-activated tension development as well as on the maximum tension reached.Addition of caffeine (10^–2 M) had no effect on the tension generation, indicating that the sarcoplasmic reticulum, if present, was not active. The force responses due to rapid length changes applied to the Ca-activated fibre preparations were found to be qualitatively similar to the force responses on intact tissue. This skinning technique might be employed on human biopsies, enabling the measurement of physiological parameters such as for example force and shortening velocity.     

Effects of amrinone on shortening velocity and force development in skinned skeletal muscle fibres

Summary The effects of amrinone were studied on single skinned fibres isolated from rat hindlimb muscles. In each fibre a force-velocity relation was determined during maximal calcium activation (pCa=4.45) in control conditions and in the presence of amrinone. The MgATP concentration was 3.93 mm, close to the physiological value. After the experiment the fibre was classified as fast or slow on the basis of its reactivity with anti-myosin monoclonal antibodies. In fast fibres amrinone (3 mm) potentiated isometric tension (P ₀) by 13.8±2.9% (n=13), reduced maximum shortening velocity (V _max ) by 32.6±3.2% and the curvature of the force-velocity relation (a/P ₀) was increased by 98.9±46.0%. All these effects were less pronounced in slow fibres, where V _max was reduced only by 11.4±3.6 (n=16). The effects of amrinone (0.3–6 mm) on the ATPase activity of myofibrils and myosin prepared from fast (tibialis anterior) and slow (soleus) rat skeletal muscles were studied. Amrinone was found to depress Ca–Mg dependent ATPase activity of myofibrillar preparations of the tibialis anterior (up to 16.6±2%) and, to a lesser extent, of the soleus (up to 7.2±1.2%). On the contrary, Ca-stimulated myosin ATPase activity was significantly increased by amrinone in myosin preparations from the tibialis anterior. Experiments were carried out to test whether amrinone (3 mm) might affect the sensitivity of the contractile system to MgATP concentration ([MgATP]). The results obtained showed that (1) the [MgATP] value at which isometric tension reached its maximum was shifted by amrinone from 0.1 mm to 0.3 mm, (2) the slope of the negative relation between [MgATP] and a/P ₀ was made more steep by amrinone, and (3) the Km of the hyperbolic relation between [MgATP] and V _max was increased from 0.39 to 1.71 mm by amrinone, thus indicating a reduced affinity of myosin for MgATP. These results are in accordance with the hypothesis that amrinone exerts a direct effect on the contractile mechanism.     

Control of reactive oxygen species production in contracting skeletal muscle

SIGNIFICANCE: The increased activities of free radicals or reactive oxygen species in tissues of exercising humans and animals were first reported ～30 years ago. A great deal has been learned about the processes that can generate these molecules, but there is little agreement on which are important, how they are controlled, and there are virtually no quantitative data. Superoxide and nitric oxide are generated by skeletal muscle and their reactions lead to formation of secondary species. A considerable amount is known about control of superoxide generation by xanthine oxidase activity, but similar information for other generation systems is lacking. RECENT ADVANCES: Re-evaluation of published data indicates potential approaches to quantification of the hydrogen peroxide concentration in resting and contracting muscle cells. Such calculations reveal that, during contractions, intracellular hydrogen peroxide concentrations in skeletal muscle may only increase by ～100 nM. The primary effects of this modest increase appear to be in "redox" signaling processes that mediate some of the responses and adaptations of muscle to exercise. These act, in part, to increase the expression of cytoprotective proteins (e.g., heat shock proteins and antioxidant enzymes) that help maintain cell viability. During aging, these redox-mediated adaptations fail and this contributes to age-related loss of skeletal muscle. CRITICAL ISSUES AND FUTURE DIRECTIONS: Understanding the control of ROS generation in muscle and the effect of aging and some disease states will aid design of interventions to maintain muscle mass and function, but is dependent upon development of new analytical approaches. The final part of this review indicates areas where such developments are occurring.     

Mitochondrial function in intact skeletal muscle fibres of creatine kinase deficient mice

Creatine kinase (CK) has a central role in skeletal muscle, acting as a fast energy buffer and shuttle between sites of energy production (mitochondria) and consumption (cross-bridges and ion pumps). Unexpectedly, isolated fast-twitch skeletal muscle cells of mice deficient in both cytosolic and mitochondrial CK (CK-/-) are highly fatigue resistant during stimulation protocols that stress aerobic metabolism. We have now studied different aspects of mitochondrial function in CK-/- skeletal muscle. Intact, single fibres of flexor digitorum brevis (FDB) muscles were fatigued by repeated tetanic stimulation (70 Hz, 350 ms duration, duty cycle 0.14). Under control conditions, CK-/- FDB fibres were more fatigue resistant than wild-type fibres. However, after mitochondrial inhibition with cyanide, force declined markedly faster in CK-/- fibres than in wild-type fibres. The rapid force decline in CK-/- fibres was not due to decreased myoplasmic [Ca²⁺] during tetani (measured with indo-1), which in these fibres remained virtually constant during fatigue in the presence of cyanide. Intact, single fibres of highly oxidative soleus muscles were fatigued by repeated tetani (50 Hz, 500 ms duration, duty cycle 0.5). All CK-/- soleus fibres tested ( n = 9) produced > 40% force at the end of the fatiguing stimulation period (500 tetani), whereas force fell to < 40% before 500 tetani in two of three wild-type fibres. Mitochondrial [Ca²⁺] (measured with rhod-2 and confocal microscopy) increased during repeated tetanic stimulation in CK-/- but not in wild-type FDB fibres. In conclusion, mitochondria and energy shuttling operate effectively in CK-/- fibres and this is associated with an increase in mitochondrial [Ca²⁺].     

Glycosylation pattern and enzyme activities in atrophic,angulated skeletal muscle fibres from ageing rats

In this study enzyme activities and lectin binding patterns in skeletal muscle from very old rats were investigated in order to evaluate changes in enzyme activity or carbohydrate expression in senile muscle. Activities for adenosine triphosphatase (ATPase), succinic dehydrogenase, non-specific esterase and the binding pattern for 31 lectins were investigated in the soleus muscles from very old (36 months) and young (3 months) rats. In ageing muscles atrophic, angulated muscle fibres are frequent. In cryostat sections these fibres were mostly but not always type II defined by the myosin ATPase reaction; few showed a strong esterase activity. Some showed strong activity for succinic dehydrogenase while others were weakly reacting. A number of lectins strongly bound to the sarcoplasm in angulated fibres while the binding to normal fibres in both old and young rat muscle was much weaker or even absent. Preferential binding to the ageing, angulated fibres was seen with Aleuria aurentia, Galantus nivalis, Caragana abborecens, Triticum vulgaris, Maackia amurensis, Sambucus nigra, Phaseolus vulgaris erythroagglutinin, and Phaseolus coccineus. Samples of homogenized and centrifuged muscles were run by electrophoresis and the gels blotted to nitrocellulose paper. Subsequent lectin staining of the blots detected that two glycoproteins with molecular weights around 25000 and 21000 daltons were present in old muscle, but not in young. Aberrant or elevated expression of sarcoplasmic glycoconjugates is involved in ageing muscle atrophy.     

Reversible depression of action potentials and force production in frog single muscle fibres by calmodulin-inhibitors

The effects of the calmodulin-inhibitors trifluoperazine, thioridazine and zaldaride maleate on the responses to electrical stimulation in isolated frog skeletal muscle fibres were investigated. All three drugs initially reduced the amplitude of the action potentials but potentiated twitch force. This was followed by a total loss of action potentials and force production. However, the resting membrane potential was not changed. The effects were completely reversible upon removal of the drugs. These results suggest that an intact calmodulin system is required for normal function of the sarcolemmal sodium channels of frog skeletal muscle.     

Exogenous reactive oxygen and nitric oxide alter intracellular oxidant status of skeletal muscle fibres

To test whether exogenous oxidants alter intracellular oxidant levels in skeletal muscle fibres, we exposed rat diaphragm to donors of nitric oxide (NO_x), reactive oxygen species (ROS) or hyperoxia, and monitored intracellular oxidant levels using a fluorescent probe. Fibre bundles were dissected from the diaphragm and loaded with 2’,7’-dichlorodihydrofluorescein (DCFH); emissions were monitored using a fluorescence microscope. DCFH-loaded muscles were exposed to either a NO_x donor (1 m M S-nitroso-N-acetyl penicillamine, SNAP; 1 m M sodium nitroprusside, SNP; 400 μM 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazen, NOC-7), an ROS donor (100 μM hydrogen peroxide, H₂O₂; 100 μM tert-butyl hydroperoxide; 1 m M hypoxanthine plus 0.01 U mL^–1 xanthine oxidase, HXXO) or a range of PO ₂s (25, 60 or 95% O₂ oxygenating Krebs–Ringer solution) for 40 min; time-matched control bundles remained in Krebs–Ringer solution. Control muscles oxidized DCFH at a rate of 0.32 ± 0.1 greyscale units min^–1. SNAP (766%), SNP (1244%), NOC-7 (851%), H₂O₂ (543%), and HXXO (541%) increased DCFH oxidation from control levels. The increase in emissions caused by NOC-7 and SNP were blunted by the NO_x scavenger haemoglobin (1 μM ). DCFH oxidation by HXXO was unaffected by 1000 U mL^–1 superoxide dismutase but was significantly decreased by 1000 U mL^–1 catalase and 1 m M salicylate. PO ₂ had no effect on intracellular oxidant levels. Therefore, extracellular NO_x and ROS can alter intracellular oxidant status in skeletal muscle fibres. These observations suggest that intrafibre oxidant levels could be the result of both intracellular and extracellular oxidant production.     

Intracellular sodium and potassium activities of skeletal muscle fibres of hypothyroid rats.

In soleus muscle fibres of hypothyroid rats, the membrane potential (Vm) and the intracellular K+ activity (aKi) were significantly lower than in control muscles. These results are consistent with the well-documented decrease in the number of Na(+)-K+ pumps which occurs in hypothyroid muscles. aNai was unchanged in the hypothyroid muscles but this may reflect a change in passive Na+ fluxes which has been reported to occur in association with changes in the number of pump units.     

Role of extracellular metal cations in the potential dependence of force inactivation in skeletal muscle fibres

Summary The present experiments were designed to further characterize a metal ion binding site at the voltage sensor in the T-tubular (TT) membrane which controls the release of Ca²⁺ from the sarcoplasmic reticulum. For this purpose the potential dependence of force inactivation was measured under voltage clamp control in short toe muscle fibres of the frog. External solutions contained in each case one species of metal ion (Ca²⁺, Ba²⁺, Na⁺ and Li⁺, respectively). Assuming that the metal ion binds with different affinities to the resting and active state of the sensor and that the metal ion free sensor is inactivated, we estimated the dissociation constants by using the inactivation midpoint voltages (V) at different concentrations of one species of metal ion. For Ca²⁺ the analysis resulted in a low apparent dissociation constant K _D1 (binding to the resting state) of 5×10^-8 M and a high apparent dissociation constant K _D2>23 mM (binding to the active state). The corresponding values for Ba²⁺ were: K _D1=5×10^-5 M and K _D2>125 mM. For different reasons, the data for Na⁺ and Li⁺ proved to be inconclusive.