Limited oxygen diffusion accelerates fatigue development in mouse skeletal muscle

Isolated whole skeletal muscles fatigue more rapidly than isolated single muscle fibres. We have now employed this difference to study mechanisms of skeletal muscle fatigue. Isolated whole soleus and extensor digitorum longus (EDL) muscles were fatigued by repeated tetanic stimulation while measuring force production. Neither application of 10 m m lactic acid nor increasing the [K⁺] of the bath solution from 5 to 10 m m had any significant effect on the rate of force decline during fatigue induced by repeated brief tetani. Soleus muscles fatigued slightly faster during continuous tetanic stimulation in 10 m m [K⁺]. Inhibition of mitochondrial respiration with cyanide resulted in a faster fatigue development in both soleus and EDL muscles. Single soleus muscle fibres were fatigued by repeated tetani while measuring force and myoplasmic free [Ca²⁺] ([Ca²⁺]_i). Under control conditions, the single fibres were substantially more fatigue resistant than the whole soleus muscles; tetanic force at the end of a series of 100 tetani was reduced by about 10% and 50%, respectively. However, in the presence of cyanide, fatigue developed at a similar rate in whole muscles and single fibres, and tetanic force at the end of fatiguing stimulation was reduced by ∼80%. The force decrease in the presence of cyanide was associated with a ∼50% decrease in tetanic [Ca²⁺]_i, compared with an increase of ∼20% without cyanide. In conclusion, lactic acid or [K⁺] has little impact on fatigue induced by repeated tetani, whereas hypoxia speeds up fatigue development and this is mainly due to an impaired Ca²⁺ release from the sarcoplasmic reticulum.     

Calmodulin kinase modulates Ca2+ release in mouse skeletal muscle

Activation of the contractile machinery in skeletal muscle is initiated by the action-potential-induced release of Ca²⁺ from the sarcoplasmic reticulum (SR). Several proteins involved in SR Ca²⁺ release are affected by calmodulin kinase II (CaMKII)-induced phosphorylation in vitro , but the effect in the intact cell remains uncertain and is the focus of the present study. CaMKII inhibitory peptide or inactive control peptide was injected into single isolated fast-twitch fibres of mouse flexor digitorum brevis muscles, and the effect on free myoplasmic [Ca²⁺] ([Ca²⁺]_i) and force during different patterns of stimulation was measured. Injection of the inactive control peptide had no effect on any of the parameters measured. Conversely, injection of CaMKII inhibitory peptide decreased tetanic [Ca²⁺]_i by ≈25 %, but had no significant effect on the rate of SR Ca²⁺ uptake or the force-[Ca²⁺]_i relationship. Repeated tetanic stimulation resulted in increased tetanic [Ca²⁺]_i, and this increase was smaller after CaMKII inhibition. In conclusion, CaMKII-induced phosphorylation facilitates SR Ca²⁺ release in the basal state and during repeated contractions, providing a positive feedback between [Ca²⁺]_i and SR Ca²⁺ release.     

Interpolated twitches in fatiguing single mouse muscle fibres: implications for the assessment of central fatigue

An electrically evoked twitch during a maximal voluntary contraction (twitch interpolation) is frequently used to assess central fatigue. In this study we used intact single muscle fibres to determine if intramuscular mechanisms could affect the force increase with the twitch interpolation technique. Intact single fibres from flexor digitorum brevis of NMRI mice were dissected and mounted in a chamber equipped with a force transducer. Free myoplasmic [Ca²⁺] ([Ca²⁺]_i) was measured with the fluorescent Ca²⁺ indicator indo-1. Seven fibres were fatigued with repeated 70 Hz tetani until 40% initial force with an interpolated pulse evoked every fifth tetanus. Results showed that the force generated by the interpolated twitch increased throughout fatigue, being 9 ± 1% of tetanic force at the start and 19 ± 1% at the end ( P < 0.001). This was not due to a larger increase in [Ca²⁺]_i induced by the interpolated twitch during fatigue but rather to the fact that the force–[Ca²⁺]_i relationship is sigmoidal and fibres entered a steeper part of the relationship during fatigue. In another set of experiments, we observed that repeated tetani evoked at 150 Hz resulted in more rapid fatigue development than at 70 Hz and there was a decrease in force ('sag') during contractions, which was not observed at 70 Hz. In conclusion, the extent of central fatigue is difficult to assess and it may be overestimated when using the twitch interpolation technique.     

Sensitivity limits for voltage control of P2Y receptor-evoked Ca2+ mobilization in the rat megakaryocyte

G-protein-coupled receptor signalling has been suggested to be voltage dependent in a number of cell types; however, the limits of sensitivity of this potentially important phenomenon are unknown. Using the non-excitable rat megakaryocyte as a model system, we now show that P2Y receptor-evoked Ca²⁺ mobilization is controlled by membrane voltage in a graded and bipolar manner without evidence for a discrete threshold potential. Throughout the range of potentials studied, the peak increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in response to depolarization was always larger than the maximal reduction in [Ca²⁺]_i following an equivalent amplitude hyperpolarization. Significant [Ca²⁺]_i increases were observed in response to small amplitude (<5 mV, 5 s duration) or short duration (25 ms, 135 mV) depolarizations. Individual cardiac action potential waveforms were also able to repeatedly potentiate P2Y receptor-evoked Ca²⁺ release and the response to trains of normally paced stimuli fused to generate prolonged [Ca²⁺]_i increases. Furthermore, elevation of the temperature to physiological levels (36°C) resulted in a more sustained depolarization-evoked Ca²⁺ increase compared with more transient or oscillatory responses at 20–24°C. The ability of signalling via a G-protein-coupled receptor to be potentiated by action potential waveforms and small amplitude depolarizations has broad implications in excitable and non-excitable tissues.     

Clathrin-mediated endocytosis in snake motor terminals is directly facilitated by intracellular Ca2+

At the snake neuromuscular junction, low temperature (LT, 5–7°C) blocks clathrin-mediated endocytosis (CME) while exocytosis is largely unaffected. Thus compensatory endocytosis that normally follows transmitter release is inhibited, or 'delayed' until the preparation is warmed to room temperature (RT). This delay was exploited to observe how changes in bulk [Ca²⁺]_i directly affect CME. Motor terminals were loaded with fura-2 to monitor [Ca²⁺]_i. With brief stimulation at LT, [Ca²⁺]_i transiently increased but returned to baseline (∼63 n m ) in < 8 min. After 15 min at LT, [Ca²⁺]_i was altered by incubating preparations in the Ca²⁺ ionophore ionomyocin. Preparations were then warmed to RT to initiate delayed endocytosis, which was quantified as uptake of the fluorescent optical probe sulforhodamine 101. Endocytosis was more rapid when [Ca²⁺]_i increased; the rate at 300 n m Ca²⁺ was ∼double that under basal conditions. Thus the rate of CME – isolated from stimulation, transmitter release, and other forms of endocytosis – is directly influenced by intraterminal Ca²⁺.     

Propagation in the transverse tubular system and voltage dependence of calcium release in normal and mdx mouse muscle fibres

Using a two-microelectrode voltage clamp technique, we investigated possible mechanisms underlying the impaired excitation–contraction coupling in skeletal muscle fibres of the mdx mouse, a model of the human disease Duchenne muscular dystrophy. We evaluated the role of the transverse tubular system (T-system) by using the potentiometric indicator di-8 ANEPPS, and that of the sarcoplasmic reticulum (SR) Ca²⁺ release by measuring Ca²⁺ transients with a low affinity indicator in the presence of high EGTA concentrations under voltage clamp conditions. We observed minimal differences in the T-system structure and the T-system electrical propagation was not different between normal and mdx mice. Whereas the maximum Ca²⁺ release elicited by voltage pulses was reduced by ∼67% in mdx fibres, in agreement with previous results obtained using AP stimulation, the voltage dependence of SR Ca²⁺ release was identical to that seen in normal fibres. Taken together, our data suggest that the intrinsic ability of the sarcoplasmic reticulum to release Ca²⁺ may be altered in the mdx mouse.     

Local recovery of Ca2+ release in rat ventricular myocytes

Excitation–contraction coupling in the heart depends on the positive feedback process of Ca²⁺-induced Ca²⁺ release (CICR). While CICR provides for robust triggering of Ca²⁺ sparks, the mechanisms underlying their termination remain unknown. At present, it is unclear how a cluster of Ca²⁺ release channels (ryanodine receptors or RyRs) can be made to turn off when their activity is sustained by the Ca²⁺ release itself. We use a novel experimental approach to investigate indirectly this issue by exploring restitution of Ca²⁺ sparks. We exploit the fact that ryanodine can bind, nearly irreversibly, to an RyR subunit (monomer) and increase the open probability of the homotetrameric channel. By applying low concentrations of ryanodine to rat ventricular myocytes, we observe repeated activations of individual Ca²⁺ spark sites. Examination of these repetitive Ca²⁺ sparks reveals that spark amplitude recovers with a time constant of 91 ms whereas the sigmoidal recovery of triggering probability lags behind amplitude recovery by ∼80 ms. We conclude that restitution of Ca²⁺ sparks depends on local refilling of SR stores after depletion and may also depend on another time-dependent process such as recovery from inactivation or a slow conformational change after rebinding of Ca²⁺ to SR regulatory proteins.     

Presynaptic N-type and P/Q-type Ca2+ channels mediating synaptic transmission at the calyx of Held of mice

At the nerve terminal, both N- and P/Q-type Ca²⁺ channels mediate synaptic transmission, with their relative contribution varying between synapses and with postnatal age. To clarify functional significance of different presynaptic Ca²⁺ channel subtypes, we recorded N-type and P/Q-type Ca²⁺ currents directly from calyces of Held nerve terminals in α_1A-subunit-deficient mice and wild-type (WT) mice, respectively. The most prominent feature of P/Q-type Ca²⁺ currents was activity-dependent facilitation, which was absent for N-type Ca²⁺ currents. EPSCs mediated by P/Q-type Ca²⁺ currents showed less depression during high-frequency stimulation compared with those mediated by N-type Ca²⁺ currents. In addition, the maximal inhibition by the GABA_B receptor agonist baclofen was greater for EPSCs mediated by N-type channels than for those mediated by P/Q-type channels. These results suggest that the developmental switch of presynaptic Ca²⁺ channels from N- to P/Q-type may serve to increase synaptic efficacy at high frequencies of activity, securing high-fidelity synaptic transmission.     

External Ca2+-dependent excitation–contraction coupling in a population of ageing mouse skeletal muscle fibres

In the present work, we investigate whether changes in excitation–contraction (EC) coupling mode occur in skeletal muscles from ageing mammals by examining the dependence of EC coupling on extracellular Ca²⁺. Single intact muscle fibres from flexor digitorum brevis muscles from young (2–6 months) and old (23–30 months) mice were subjected to tetanic contractile protocols in the presence and absence of external Ca²⁺. Contractile experiments in the absence of external Ca²⁺ show that about half of muscle fibres from old mice are dependent upon external Ca²⁺ for maintaining maximal tetanic force output, while young fibres are not. Decreased force in the absence of external Ca²⁺ was not due to changes in charge movement as revealed by whole-cell patch-clamp experiments. Ca²⁺ transients, measured by fluo-4 fluorescence, declined in voltage-clamped fibres from old mice in the absence of external Ca²⁺. Similarly, Ca²⁺ transients declined in parallel with tetanic contractile force in single intact fibres. Examination of inward Ca²⁺ current and of mRNA and protein assays suggest that these changes in EC coupling mode are not due to shifts in dihydropyridine receptor (DHPR) and/or ryanodine receptor (RyR) isoforms. These results indicate that a change in EC coupling mode occurs in a population of fibres in ageing skeletal muscle, and is responsible for the age-related dependence on extracellular Ca²⁺.     

Calcium oscillations in interstitial cells of the rabbit urethra

Measurements were made (using fast confocal microscopy) of intracellular Ca²⁺ levels in fluo-4 loaded interstitial cells isolated from the rabbit urethra. These cells exhibited regular Ca²⁺ oscillations which were associated with spontaneous transient inward currents recorded under voltage clamp. Interference with d - myo -inositol 1,4,5-trisphosphate (IP₃) induced Ca²⁺ release using 100 μ m 2-aminoethoxydiphenyl borate, and the phospholipase C (PLC) inhibitors 2-nitro-4-carboxyphenyl N , N -diphenylcarbamate and U73122 decreased the amplitude of spontaneous oscillations but did not abolish them. However, oscillations were abolished when ryanodine receptors were blocked with tetracaine or ryanodine. Oscillations ceased in the absence of external Ca²⁺, and frequency was directly proportional to the external Ca²⁺ concentration. Frequency of Ca²⁺ oscillation was reduced by SKF-96365, but not by nifedipine. Lanthanum and cadmium completely blocked oscillations. These results suggest that Ca²⁺ oscillations in isolated rabbit urethral interstitial cells are initiated by Ca²⁺ release from ryanodine-sensitive intracellular stores, that oscillation frequency is very sensitive to the external Ca²⁺ concentration and that conversion of the primary oscillation to a propagated Ca²⁺ wave depends upon IP₃-induced Ca²⁺ release.     

Hypoxic pulmonary vasoconstriction: mechanisms and controversies

The pulmonary circulation differs from the systemic in several important aspects, the most important being that pulmonary arteries constrict to moderate physiological (∼20–60 mmHg P _O2) hypoxia, whereas systemic arteries vasodilate. This phenomenon is called hypoxic pulmonary vasoconstriction (HPV), and is responsible for maintaining the ventilation–perfusion ratio during localized alveolar hypoxia. In disease, however, global hypoxia results in a detrimental increase in total pulmonary vascular resistance, and increased load on the right heart. Despite many years of study, the precise mechanisms underlying HPV remain unresolved. However, as we argue below, there is now overwhelming evidence that hypoxia can stimulate several pathways leading to a rise in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in pulmonary artery smooth muscle cells (PASMC). This rise in [Ca²⁺]_i is consistently found to be relatively small, and HPV seems also to require rho kinase-mediated Ca²⁺ sensitization. There is good evidence that HPV also has an as yet unexplained endothelium dependency. In this brief review, we highlight selected recent findings and ongoing controversies which continue to animate the study of this remarkable and unique response of the pulmonary vasculature to hypoxia.     

Autocrine activation of nicotinic acetylcholine receptors contributes to Ca2+ spikes in mouse myotubes during myogenesis

It is widely accepted that nicotinic acetylcholine receptor (nAChR) channel activity controls myoblast fusion into myotubes during myogenesis. In this study we explored the possible role of nAChR channels after cell fusion in a murine cell model. Using videoimaging techniques we showed that embryonic muscle nAChR channel openings contribute to the spontaneous transients of intracellular concentration of Ca²⁺ ([Ca²⁺]_i) and to twitches characteristic of developing myotubes before innervation. Moreover, we observed a choline acetyltransferase immunoreactivity in the myotubes and we detected an acetylcholine-like compound in the extracellular solution. Therefore, we suggest that the autocrine activation of nAChR channels gives rise to [Ca²⁺]_i spikes and contractions. Spontaneous openings of the nAChR channels may be an alternative, although less efficient, mechanism. We report also that blocking the nAChRs causes a significant reduction in cell survival, detectable as a decreased number of myotubes in culture. This led us to hypothesize a possible functional role for the autocrine activation of the nAChRs. By triggering mechanical activity, such activation could represent a strategy to ensure the trophism of myotubes in the absence of nerves.     

Role of phosphate and calcium stores in muscle fatigue

Intensive activity of muscles causes a decline in performance, known as fatigue, that is thought to be caused by the effects of metabolic changes on either the contractile machinery or the activation processes. The concentration of inorganic phosphate (P_i) in the myoplasm ([P_i]_myo) increases substantially during fatigue and affects both the myofibrillar proteins and the activation processes. It is known that a failure of sarcoplasmic reticulum (SR) Ca²⁺ release contributes to fatigue and in this review we consider how raised [P_i]_myo contributes to this process. Initial evidence came from the observation that increasing [P_i]_myo causes reduced SR Ca²⁺ release in both skinned and intact fibres. In fatigued muscles the store of releasable Ca²⁺ in the SR declines mirroring the decline in SR Ca²⁺ release. In muscle fibres with inoperative creatine kinase the rise of [P_i]_myo is absent during fatigue and the failure of SR Ca²⁺ release is delayed. These results can all be explained if inorganic phosphate can move from the myoplasm into the SR during fatigue and cause precipitation of CaP_i within the SR. The relevance of this mechanism in different types of fatigue in humans is considered.     

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.     

Lipid rafts, the sarcoplasmic reticulum and uterine calcium signalling: an integrated approach

The pathways involved in Ca²⁺ signalling in the uterus remain incompletely understood, impairing our ability to prevent preterm and difficult labours. In this review we focus on two elements in the pathway of Ca²⁺ signalling that have recently emerged as playing important roles: membrane lipid rafts and the sarcoplasmic reticulum. We examine the evidence for lipid rafts in the uterus and discuss their functional role. We suggest that the increases in cytosolic [Ca²⁺] and contractility that occur with raft disruption are due, at least in part, to effects on large conductance Ca²⁺-activated K⁺ (BK) channels that are localized to rafts. The role of the SR in contributing to subsarcolemmal cytosolic microdomains in uterus is evaluated, along with its interactions with ion channels on the plasma membrane. Thus, signalling microdomains play an important, but incompletely understood, role in the uterus, and integrating them into other Ca²⁺ signalling pathways is a challenge for further research. We suggest that the role of the SR changes in pregnancy, from promoting quiescence via BK channels or SR Ca²⁺ uptake, to promoting Ca²⁺ entry and contractility at term, and relate data on lipid rafts to clinical outcome in obese pregnant women.     

Regenerative component of slow waves in the guinea-pig gastric antrum involves a delayed increase in [Ca2+]i and Cl− channels

Regenerative potentials were initiated by depolarizing short segments of single bundles of circular muscle isolated from the gastric antrum of guinea-pigs. When changes in [Ca²⁺]_i and membrane potential were recorded simultaneously, regenerative potentials were found to be associated with an increase in [Ca²⁺]_i, with the increase starting after a minimum latency of about 1 s. Although the increase in [Ca²⁺]_i was reduced by nifedipine, the amplitudes of the regenerative responses were little changed. Regenerative responses and associated changes in [Ca²⁺]_i were abolished by loading the preparations with the Ca²⁺ chelator MAPTA-AM. Regenerative potentials were abolished by 2-aminoethoxydiphenyl borate (2APB), an inhibitor of IP₃ induced Ca²⁺ release, by N -ethylamaleimide (NEM), an alkylating agent which blocks activation of G-proteins and were reduced in amplitude by two agents which block chloride (Cl⁻)-selective channels in many tissues. The observations suggest that membrane depolarization triggers IP₃ formation. This causes Ca²⁺ release from intracellular stores which activates Ca²⁺-dependent Cl⁻ channels.     

Calcium events in smooth muscles and their interstitial cells; physiological roles of sparks

The observation of spontaneous sporadic releases of packets of stored calcium made 20 years ago has opened up a number of new concepts in smooth muscle physiology: (1) the calcium release sites are ryanodine and inositol 1,4,5-trisphosphate (IP₃) receptor channels which contribute to cell-wide increases in [Ca²⁺]_i in response to cell depolarization, activation of IP₃-generating receptors, or other stimuli; (2) changes in [Ca²⁺]_i act back on the cell membrane to activate or modulate K⁺, Cl⁻ and cation channel activity so affecting contraction, in arterial smooth muscle for example affecting blood pressure; (3) IP₃ production is voltage dependent and is believed to contribute to pacemaker potentials and to refractory periods which control the rhythmical motility of many hollow organs. Most smooth muscle tissues contain interstitial cells (ICs) in addition to contractile smooth muscle cells (SMCs). The interactions of these internal mechanisms, and in turn the interactions of SMCs and ICs in various smooth muscle tissues, are major factors in determining the unique physiological profiles of individual smooth muscles.     

Dual Ca2+ modulation of glycinergic synaptic currents in rodent hypoglossal motoneurones

Glycinergic synapses are implicated in the coordination of reflex responses, sensory signal processing and pain sensation. Their activity is pre- and postsynaptically regulated, although mechanisms are poorly understood. Using patch-clamp recording and Ca²⁺ imaging in hypoglossal motoneurones from rat and mouse brainstem slices, we address here the role of cytoplasmic Ca²⁺ (Ca_i) in glycinergic synapse modulation. Ca²⁺ influx through voltage-gated or NMDA receptor channels caused powerful transient inhibition of glycinergic IPSCs. This effect was accompanied by an increase in both the failure rate and paired-pulse ratio, as well as a decrease in the frequency of mIPSCs, suggesting a presynaptic mechanism of depression. Inhibition was reduced by the cannabinoid receptor antagonist SR141716A and occluded by the agonist WIN55,212-2, indicating involvement of endocannabinoid retrograde signalling. Conversely, in the presence of SR141716A, glycinergic IPSCs were potentiated postsynaptically by glutamate or NMDA, displaying a Ca²⁺-dependent increase in amplitude and decay prolongation. Both presynaptic inhibition and postsynaptic potentiation were completely prevented by strong Ca_i buffering (20 m m BAPTA). Our findings demonstrate two independent mechanisms by which Ca²⁺ modulates glycinergic synaptic transmission: (i) presynaptic inhibition of glycine release and (ii) postsynaptic potentiation of GlyR-mediated responses. This dual Ca²⁺-induced regulation might be important for feedback control of neurotransmission in a variety of glycinergic networks in mammalian nervous systems.