Store-operated Ca2+ entry in malignant hyperthermia-susceptible human skeletal muscle.

In malignant hyperthermia (MH), mutations in RyR1 underlie direct activation of the channel by volatile anesthetics, leading to muscle contracture and a life-threatening increase in core body temperature. The aim of the present study was to establish whether the associated depletion of sarcoplasmic reticulum (SR) Ca(2+) triggers sarcolemmal Ca(2+) influx via store-operated Ca(2+) entry (SOCE). Samples of vastus medialis muscle were obtained from patients undergoing assessment for MH susceptibility using the in vitro contracture test. Single fibers were mechanically skinned, and confocal microscopy was used to detect changes in [Ca(2+)] either within the resealed t-system ([Ca(2+)](t-sys)) or within the cytosol. In normal fibers, halothane (0.5 mM) failed to initiate SR Ca(2+) release or Ca(2+)(t-sys) depletion. However, in MH-susceptible (MHS) fibers, halothane induced both SR Ca(2+) release and Ca(2+)(t-sys) depletion, consistent with SOCE. In some MHS fibers, halothane-induced SR Ca(2+) release took the form of a propagated wave, which was temporally coupled to a wave of Ca(2+)(t-sys) depletion. SOCE was potently inhibited by "extracellular" application of a STIM1 antibody trapped within the t-system but not when the antibody was denatured by heating. In conclusion, (i) in human MHS muscle, SR Ca(2+) depletion induced by a level of volatile anesthetic within the clinical range is sufficient to induce SOCE, which is tightly coupled to SR Ca(2+) release; (ii) sarcolemmal STIM1 has an important role in regulating SOCE; and (iii) sustained SOCE from an effectively infinite extracellular Ca(2+) pool may contribute to the maintained rise in cytosolic [Ca(2+)] that underlies MH.

DHPR activation underlies SR Ca2+ release induced by osmotic stress in isolated rat skeletal muscle fibers.

Changes in skeletal muscle volume induce localized sarcoplasmic reticulum (SR) Ca(2+) release (LCR) events, which are sustained for many minutes, suggesting a possible signaling role in plasticity or pathology. However, the mechanism by which cell volume influences SR Ca(2+) release is uncertain. In the present study, rat flexor digitorum brevis fibers were superfused with isoosmotic Tyrode's solution before exposure to either hyperosmotic (404 mOsm) or hypoosmotic (254 mOsm) solutions, and the effects on cell volume, membrane potential (E(m)), and intracellular Ca(2+) ([Ca(2+)](i)) were determined. To allow comparison with previous studies, solutions were made hyperosmotic by the addition of sugars or divalent cations, or they were made hypoosmotic by reducing [NaCl](o). All hyperosmotic solutions induced a sustained decrease in cell volume, which was accompanied by membrane depolarization (by 14-18 mV; n = 40) and SR Ca(2+) release. However, sugar solutions caused a global increase in [Ca(2+)](i), whereas solutions made hyperosmotic by the addition of divalent cations only induced LCR. Decreasing osmolarity induced an increase in cell volume and a negative shift in E(m) (by 15.04 +/- 1.85 mV; n = 8), whereas [Ca(2+)](i) was unaffected. However, on return to the isoosmotic solution, restoration of cell volume and E(m) was associated with LCR. Both global and localized SR Ca(2+) release were abolished by the dihydropyridine receptor inhibitor nifedipine by sustained depolarization of the sarcolemmal or by the addition of the ryanodine receptor 1 inhibitor tetracaine. Inhibitors of the Na-K-2Cl (NKCC) cotransporter markedly inhibited the depolarization associated with hyperosmotic shrinkage and the associated SR Ca(2+) release. These findings suggest (1) that the depolarization that accompanies a decrease in cell volume is the primary event leading to SR Ca(2+) release, and (2) that volume-dependent regulation of the NKCC cotransporter contributes to the observed changes in E(m). The differing effects of the osmotic agents can be explained by the screening of fixed charges by divalent ions.

The presence of a functional t-tubule network increases the sensitivity of RyR1 to agonists in skinned rat skeletal muscle fibres.

Single mechanically skinned extensor digitorum Longus (EDL) rat fibres were used as a model to study the influence of functional t-tubules on the properties of RyR1 in adult skeletal muscle. Fibres were superfused with solutions approximating to the intracellular milieu. Following skinning, the t-tubules re-seal and repolarise, allowing the sarcoplasmic reticulum (SR) Ca2+ release to be activated by field stimulation. However, in the present study, some fibres exhibited localised regions where depolarisation-induced SR Ca2+ release was absent, due to failure of the t-tubules to re-seal. When these fibres were exposed to caffeine to directly activate RyR1, regions with re-sealed t-tubules exhibited greater sensitivity to submaximal (2-5 mM) levels of caffeine (n = 8), while the response to a supramaximal SR Ca2+ release stimulus was uniform (n = 8, p < 0.05). This difference in RyR1 sensitivity was unaffected by sustained depolarisation of the t-tubule network. However, after saponin permeabilization of the t-tubules or withdrawal of Ca2+ from the t-tubules before skinning, the difference in agonist sensitivity was abolished. These results suggest that in adult skeletal muscle fibres, the presence of a functional t-tubule network increases the sensitivity of RyR1 to agonists via a mechanism that involves binding of Ca2+ to an extracellular regulatory site.

Defective Mg2+ regulation of RyR1 as a causal factor in malignant hyperthermia.

In skeletal muscle, Mg(2+) exerts a dual inhibitory effect on RyR1, by competing with Ca(2+) at the activation site and binding to a low affinity Ca(2+)/Mg(2+) inhibitory site. Pharmacological activators of RyR1 must overcome the inhibitory action of Mg(2+) before Ca(2+) efflux can occur. In normal muscle, where the free [Mg(2+)](i) is approximately 1mM, even prolonged exposure to millimolar levels of volatile anesthetics does not initiate SR Ca(2+) release. However, when the cytosolic [Mg(2+)] is reduced below the physiological range, low levels of volatile anesthetic within the clinically relevant range (1mM) can initiate SR Ca(2+) release, in the form of a propagating Ca(2+) wave. In human muscle fibers from malignant hyperthermia susceptible patients, such Ca(2+) waves occur when 1mM halothane is applied at physiological [Mg(2+)](i). There is increasing evidence to suggest that defective Mg(2+) regulation of RyR1 confers susceptibility to malignant hyperthermia. At the molecular level, interactions between critical RyR1 subdomains may explain the clustering of RyR1 mutations and associated effects on Mg(2+) regulation.

Mg2+ dependence of halothane-induced Ca2+ release from the sarcoplasmic reticulum in skeletal muscle from humans susceptible to malignant hyperthermia.

BACKGROUND: Recent work suggests that impaired Mg(2+) regulation of the ryanodine receptor is a common feature of both pig and human malignant hyperthermia. Therefore, the influence of [Mg(2+)] on halothane-induced Ca(2+) release from the sarcoplasmic reticulum was studied in malignant hyperthermia-susceptible (MHS) or -nonsusceptible (MHN) muscle. METHODS: Vastus medialis fibers were mechanically skinned and perfused with solutions containing physiologic (1 mm) or reduced concentrations of free [Mg(2+)]. Sarcoplasmic reticulum Ca(2+) release was detected using fura-2 or fluo-3. RESULTS: In MHN fibers, 1 mm halothane consistently did not induce sarcoplasmic reticulum Ca(2+) release in the presence of 1 mm Mg(2+). It was necessary to increase the halothane concentration to 20 mm or greater before Ca release occurred. However, when [Mg(2+)] was reduced below 1 mm, halothane became an increasingly effective stimulus for Ca(2+) release; e.g., at 0.4 mm Mg(2+), 58% of MHN fibers responded to halothane. In MHS fibers, 1 mm halothane induced Ca(2+) release in 57% of MHS fibers at 1 mm Mg(2+). Reducing [Mg(2+)] increased the proportion of MHS fibers that responded to 1 mm halothane. Further experiments revealed differences in the characteristics of halothane-induced Ca(2+) release in MHS and MHN fibers: In MHN fibers, at 1 mm Mg(2+), halothane induced a diffuse increase in [Ca(2+)], which began at the periphery of the fiber and spread slowly inward. In MHS fibers, halothane induced a localized C(2+)a release, which then propagated along the fiber. However, propagated Ca(2+) release was observed in MHN fibers when halothane was applied at an Mg(2+) concentration of 0.4 mm or less. CONCLUSIONS: When Mg(2+) inhibition of the ryanodine receptor is reduced, the halothane sensitivity of MHN fibers and the characteristics of the Ca release process approach that of the MHS phenotype. In MHS fibers, reduced Mg(2+) inhibition of the ryanodine receptor would be expected to have a major influence on halothane sensitivity. The Mg dependence of the halothane response in MHN and MHS may have important clinical implications in circumstances where intracellular [Mg(2+)] deviates from normal physiologic concentrations.

Mg2+ dependence of halothane-induced Ca2+ release from the sarcoplasmic reticulum in rat skeletal muscle.

The effect of cytosolic Mg2+ on halothane-induced Ca2+ release from the sarcoplasmic reticulum (SR) was investigated in mechanically skinned fibres from the rat extensor digitorum longus (EDL) muscle. Preparations were perfused with solutions mimicking the intracellular milieu and changes in [Ca2+] were detected using Fura-2 fluorescence. In the presence of 1 mM Mg2+, brief (500 ms) applications of 40 mM halothane failed to induce Ca2+ release from the SR. However, Ca2+ release became detectable when [Mg2+] was reduced to 0.4 mM, and the amplitude of the response increased progressively as [Mg2+] was further reduced to 0.2 and 0.1 mM. Lower halothane concentrations within the range found during anaesthesia or induction (0.1-1.2 mM) failed to induce SR Ca2+ release at 0.2 or 0.4 mM Mg2+. However, in further experiments, preparations were exposed to 1 mM halothane for 2-3 min under conditions where the volume of solution surrounding the preparation was restricted by stopping the flow. In the absence of perfusion, 1 mM halothane induced Ca2+ release from the SR at 0.4 mM Mg2+ in two out of six preparations, and release was observed consistently at 0.2 and 0.1 mM Mg2+. Responses to 1 mM halothane induced in the presence of 0.4 and 0.2 mM Mg2+ were typically delayed in onset and involved a localised release of Ca2+ that propagated along the fibre. These results suggest that halothane-induced Ca2+ release is strongly inhibited at normal physiological levels of Mg2+. However, when Mg2+-induced inhibition of the ryanodine receptor (RYR) is reduced, levels of halothane within the range found during anaesthesia can induce a marked efflux of Ca2+ from the SR. This may be of relevance to the condition of malignant hyperthermia, where the inhibition of RYRs by Mg2+ is reportedly reduced.

Effects of Mg(2+) and SR luminal Ca(2+) on caffeine-induced Ca(2+) release in skeletal muscle from humans susceptible to malignant hyperthermia.

Regulation of the ryanodine receptor (RYR) by Mg(2+) and SR luminal Ca(2+) was studied in mechanically skinned malignant hyperthermia susceptible (MHS) and non-susceptible (MHN) fibres from human vastus medialis. Preparations were perfused with solutions mimicking the intracellular milieu and changes in [Ca(2+)] were detected using fura-2 fluorescence. At 1 mM cytosolic Mg(2+), MHS fibres had a higher sensitivity to caffeine (2-40 mM) than MHN fibres. The inhibitory effect of Mg(2+) on caffeine-induced Ca(2+) release was studied by increasing [Mg(2+)] of the solution containing 40 mM caffeine. Increasing [Mg(2+)] from 1 to 3 mM reduced the amplitude of the caffeine-induced Ca(2+) transient by 77 +/- 7.4 % (n = 8) in MHN fibres. However, the caffeine-induced Ca(2+) transient decreased by only 24 +/- 8.1 % (n = 9) in MHS fibres. In MHN fibres, reducing the Ca(2+) loading period from 4 to 1 min (at 1 mM Mg(2+)) decreased the fraction of the total sarcoplasmic reticulum (SR) Ca(2+) content released in response to 40 mM caffeine by 90.4 +/- 6.2 % (n = 6). However, in MHS fibres the response was reduced by only 31.2 +/- 17.4 % (n = 6) under similar conditions. These results suggest that human malignant hyperthermia (MH) is associated with reduced inhibition of the RYR by (i) cytosolic Mg(2+) and (ii) SR Ca(2+) depletion. Both of these effects may contribute to increased sensitivity of the RYR to caffeine and volatile anaesthetics.