Adrian M. Duke

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) Ca2+ triggers sarcolemmal Ca2+ influx via store-operated Ca2+ 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 [Ca2+] either within the resealed t-system ([Ca2+]t-sys) or within the cytosol. In normal fibers, halothane (0.5 mm) failed to initiate SR Ca2+ release or Ca2+t-sys depletion. However, in MH-susceptible (MHS) fibers, halothane induced both SR Ca2+ release and Ca2+t-sys depletion, consistent with SOCE. In some MHS fibers, halothane-induced SR Ca2+ release took the form of a propagated wave, which was temporally coupled to a wave of Ca2+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 Ca2+ depletion induced by a level of volatile anesthetic within the clinical range is sufficient to induce SOCE, which is tightly coupled to SR Ca2+ release; (ii) sarcolemmal STIM1 has an important role in regulating SOCE; and (iii) sustained SOCE from an effectively infinite extracellular Ca2+ pool may contribute to the maintained rise in cytosolic [Ca2+] that underlies MH.

Effects of creatine phosphate on Ca2+ regulation by the sarcoplasmic reticulum in mechanically skinned rat skeletal muscle fibres

1 The effect of creatine phosphate (PCr) on sarcoplasmic reticulum (SR) Ca2+ regulation was studied in mechanically skinned skeletal muscle fibres from rat extensor digitorium longus (EDL). Preparations were perfused with solutions mimicking the intracellular milieu and the [Ca2+] within the muscle was monitored continuously using fura‐2. 2 Brief application of 40 mM caffeine caused a transient increase in [Ca2+] due to SR Ca2+ release, and an associated tension response. Withdrawal of PCr resulted in (i) a slow transient release of Ca2+ from the SR (ii) a marked prolongation of the descending phase of the caffeine‐induced fluorescence ratio transient and (iii) a decrease in the Ca2+ transient amplitude to 69.2 ± 2.7% (n= 16) of control responses. 3 Prolongation of the caffeine‐induced Ca2+ transient also occurred following application of the SR Ca2+ pump inhibitor cyclopiazonic acid (CPA). This suggests that (i) the descending phase of the caffeine‐induced Ca2+ transient is dependent on the rate of Ca2+ uptake by the SR and (ii) prolongation associated with PCr withdrawal may also reflect a decrease in the net Ca2+ uptake rate. 4 The effects of PCr withdrawal were mimicked by addition of the creatine kinase (CK) inhibitor 2,4‐dinitro‐1‐fluorobenzene (DNFB). Hence, reducing the [PCr] may influence SR Ca2+ regulation by limiting local ATP regeneration by endogenous CK. After treatment with DNFB, PCr withdrawal had no effect on the Ca2+ transient, confirming that PCr does not have an additional direct effect on the SR. 5 The Ca2+ efflux associated with PCr withdrawal was insensitive to ryanodine or Ruthenium Red, but was effectively abolished by pretreatment with the SR Ca2+ pump inhibitor cyclopiazonic acid (CPA). This suggests that the Ca2+ efflux associated with PCr withdrawal is independent of the SR Ca2+ channel, but may involve reversal or inhibition of the Ca2+ ATPase. 6 These data suggest that Ca2+ regulation by the SR is strongly dependent on the supply of ATP via endogenous CK. Depletion of PCr may contribute to impaired SR Ca2+ regulation known to occur in intact skeletal muscle under conditions of fatigue.

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) Ca2+ 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 Ca2+ release is uncertain. In the present study, rat flexor digitorum brevis fibers were superfused with isoosmotic Tyrodes solution before exposure to either hyperosmotic (404 mOsm) or hypoosmotic (254 mOsm) solutions, and the effects on cell volume, membrane potential (Em), and intracellular Ca2+ ([Ca2+]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 Ca2+ release. However, sugar solutions caused a global increase in [Ca2+]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 Em (by 15.04 ± 1.85 mV; n = 8), whereas [Ca2+]i was unaffected. However, on return to the isoosmotic solution, restoration of cell volume and Em was associated with LCR. Both global and localized SR Ca2+ 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 Ca2+ release. These findings suggest (1) that the depolarization that accompanies a decrease in cell volume is the primary event leading to SR Ca2+ release, and (2) that volume-dependent regulation of the NKCC cotransporter contributes to the observed changes in Em. The differing effects of the osmotic agents can be explained by the screening of fixed charges by divalent ions.

Effects of caffeine and adenine nucleotides on Ca2+ release by the sarcoplasmic reticulum in saponin-permeabilized frog skeletal muscle fibres

1 The effect of caffeine and adenine nucleotides on the sarcoplasmic reticulum (SR) Ca2+ release mechanism was investigated in permeabilized frog skeletal muscle fibres. Caffeine was rapidly applied and the resulting release of Ca2+ from the SR detected using fura‐2 fluorescence. Decreasing the [ATP] from 5 to 0.1 mm reduced the caffeine‐induced Ca2+ transient by 89 ± 1.4 % (mean ± s.e.m., n= 16), while SR Ca2+ uptake was unaffected. 2 The dependence of caffeine‐induced Ca2+ release on cytosolic [ATP] was used to study the relative ability of other structurally related compounds to substitute for, or compete with, ATP at the adenine nucleotide binding site. It was found that AMP, ADP and the non‐hydrolysable analogue adenylyl imidodiphosphate (AMP‐PNP) partially substituted for ATP, although none was as potent in facilitating the Ca2+‐releasing action of caffeine. 3 Adenosine reversibly inhibited caffeine‐induced Ca2+ release, without affecting SR Ca2+ uptake. Five millimolar adenosine markedly reduced the amplitude of the caffeine‐induced Ca2+ transient by 64 ± 4 % (mean ± s.e.m., n= 11). The degree of inhibition was dependent upon the cytosolic [ATP], suggesting that adenosine may act as a competitive antagonist at the adenine nucleotide binding site. 4 These data show that (i) the sensitivity of the in situ SR Ca2+ channel to caffeine activation is strongly dependent upon the cytosolic [ATP], (ii) the number of phosphates attached to the 5′ carbon of the ribose ring influences the efficacy of the ligand, and (iii) removal of a single phosphate group transforms AMP from a partial agonist, to adenosine, which acts as a competitive antagonist under these conditions.

Interdependent effects of inorganic phosphate and creatine phosphate on sarcoplasmic reticulum Ca2+ regulation in mechanically skinned rat skeletal muscle

1 The effects of creatine phosphate (CP) and inorganic phosphate (Pi) on sarcoplasmic reticulum (SR) Ca2+ regulation were investigated in mechanically skinned muscle fibres from rat extensor digitorum longus (EDL) muscles. Changes in [Ca2+] were detected using fura‐2 fluorescence, during continuous perfusion or when the solution surrounding the preparation was restricted to approximately 6 μl by stopping perfusion. 2 In solutions with 5 mm ATP and 10 mm CP, stopping the flow for 2‐3 min had no effect on [Ca2+] within the bath. This suggests that SR Ca2+ uptake is balanced by an efflux under these conditions. 3 In solutions with CP, the introduction of Pi induced a small transient rise in [Ca2+], due to Ca2+ loss from the SR. Following equilibration with solutions containing Pi (≥ 5 mm), a maintained decrease in [Ca2+] occurred when the flow was stopped. This is consistent with calcium phosphate (Ca‐Pi) precipitation within the SR, resulting in maintained Ca2+ uptake. 4 In the absence of CP, the [Ca2+] within the bath increased progressively when the flow was stopped. This rise in [Ca2+] was inhibited by an alternative ATP regenerating system comprising phosphoenolpyruvate (PEP) and pyruvate kinase (PK). Therefore, the loss of Ca2+ from the SR may result from local ADP accumulation and the consequent reversal of the SR Ca2+ pump. 5 In the absence of CP, the initial Ca2+ release associated with the introduction of Pi increased markedly. Following prolonged equilibration with solutions containing Pi, a rise in [Ca2+] occurred within the bath when the flow was stopped. Maintained Ca2+ uptake associated with Ca‐Pi precipitation was not apparent at any level of Pi tested (1–60 mm), when CP was absent. 6 These results suggest that withdrawal of CP is associated with activation of a SR Ca2+ efflux pathway. This may involve reversal of the SR Ca2+ pump, due to local ADP accumulation. In the absence of CP, the dominant influence of Pi appears to involve further Ca2+ efflux via the SR Ca2+ pump. The possible relevance of these effects to skeletal muscle fatigue is considered.

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 Mg2+ regulation of the ryanodine receptor is a common feature of both pig and human malignant hyperthermia. Therefore, the influence of [Mg2+] on halothane-induced Ca2+ 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 [Mg2+]. Sarcoplasmic reticulum Ca2+ release was detected using fura-2 or fluo-3. Results:In MHN fibers, 1 mm halothane consistently did not induce sarcoplasmic reticulum Ca2+ release in the presence of 1 mm Mg2+. It was necessary to increase the halothane concentration to 20 mm or greater before Ca2+ release occurred. However, when [Mg2+] was reduced below 1 mm, halothane became an increasingly effective stimulus for Ca2+ release; e.g., at 0.4 mm Mg2+, 58% of MHN fibers responded to halothane. In MHS fibers, 1 mm halothane induced Ca2+ release in 57% of MHS fibers at 1 mm Mg2+. Reducing [Mg2+] increased the proportion of MHS fibers that responded to 1 mm halothane. Further experiments revealed differences in the characteristics of halothane-induced Ca2+ release in MHS and MHN fibers: In MHN fibers, at 1 mm Mg2+, halothane induced a diffuse increase in [Ca2+], which began at the periphery of the fiber and spread slowly inward. In MHS fibers, halothane induced a localized Ca2+ release, which then propagated along the fiber. However, propagated Ca2+ release was observed in MHN fibers when halothane was applied at an Mg2+ concentration of 0.4 mm or less. Conclusions:When Mg2+ inhibition of the ryanodine receptor is reduced, the halothane sensitivity of MHN fibers and the characteristics of the Ca2+ release process approach that of the MHS phenotype. In MHS fibers, reduced Mg2+ inhibition of the ryanodine receptor would be expected to have a major influence on halothane sensitivity. The Mg2+ dependence of the halothane response in MHN and MHS may have important clinical implications in circumstances where intracellular [Mg2+] deviates from normal physiologic concentrations.

Effects of Mg2+ and SR luminal Ca2+ on caffeine-induced Ca2+ release in skeletal muscle from humans susceptible to malignant hyperthermia

Regulation of the ryanodine receptor (RYR) by Mg2+ and SR luminal Ca2+ 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 [Ca2+] were detected using fura‐2 fluorescence. At 1 mm cytosolic Mg2+, MHS fibres had a higher sensitivity to caffeine (2‐40 mm) than MHN fibres. The inhibitory effect of Mg2+ on caffeine‐induced Ca2+ release was studied by increasing [Mg2+] of the solution containing 40 mm caffeine. Increasing [Mg2+] from 1 to 3 mm reduced the amplitude of the caffeine‐induced Ca2+ transient by 77 ± 7.4 % (n= 8) in MHN fibres. However, the caffeine‐induced Ca2+ transient decreased by only 24 ± 8.1 % (n= 9) in MHS fibres. In MHN fibres, reducing the Ca2+ loading period from 4 to 1 min (at 1 mm Mg2+) decreased the fraction of the total sarcoplasmic reticulum (SR) Ca2+ 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 Mg2+ and (ii) SR Ca2+ depletion. Both of these effects may contribute to increased sensitivity of the RYR to caffeine and volatile anaesthetics.

Inhibition of the Cardiac Na+ Channel Nav1.5 by Carbon Monoxide

Background: CO poisoning causes cardiac arrhythmias, in part via modulation of the cardiac Na+ channel, Nav1.5. Results: CO inhibition of peak recombinant Nav1.5 current occurs via nitric oxide formation and is also dependent on channel redox state. Conclusion: CO inhibits peak recombinant Nav1.5 current via a mechanism distinct from activation of the late Na+ current. Significance: CO may induce Brugada-like arrhythmias via inhibition of peak Na+ current. Sublethal carbon monoxide (CO) exposure is frequently associated with myocardial arrhythmias, and our recent studies have demonstrated that these may be attributable to modulation of cardiac Na+ channels, causing an increase in the late current and an inhibition of the peak current. Using a recombinant expression system, we demonstrate that CO inhibits peak human Nav1.5 current amplitude without activation of the late Na+ current observed in native tissue. Inhibition was associated with a hyperpolarizing shift in the steady-state inactivation properties of the channels and was unaffected by modification of channel gating induced by anemone toxin (rATX-II). Systematic pharmacological assessment indicated that no recognized CO-sensitive intracellular signaling pathways appeared to mediate CO inhibition of Nav1.5. Inhibition was, however, markedly suppressed by inhibition of NO formation, but NO donors did not mimic or occlude channel inhibition by CO, indicating that NO alone did not account for the actions of CO. Exposure of cells to DTT immediately before CO exposure also dramatically reduced the magnitude of current inhibition. Similarly, l-cysteine and N-ethylmaleimide significantly attenuated the inhibition caused by CO. In the presence of DTT and the NO inhibitor Nω-nitro-l-arginine methyl ester hydrochloride, the ability of CO to inhibit Nav1.5 was almost fully prevented. Our data indicate that inhibition of peak Na+ current (which can lead to Brugada syndrome-like arrhythmias) occurs via a mechanism distinct from induction of the late current, requires NO formation, and is dependent on channel redox state.

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.

Properties of Store Operated Ca2+ Entry in Malignant Hyperthermia Susceptible Human Skeletal Muscle Fibres

Experiments were carried out to investigate the possibility that store operated Ca2+ entry (SOCE) may be triggered by volatile anaesthetics in malignant hyperthermia susceptible (MHS) human skeletal muscle. Samples of vastus medialis muscle were obtained from patients undergoing assessment for malignant hyperthermia (MH) susceptibility using the standardised in vitro contracture test. All experiments were performed with institutional Research Ethics Committee approval and informed patient consent, according to the Declaration of Helsinki. Single fibres were mechanically skinned and confocal microscopy used to detect changes [Ca2+] within the re-sealed t-tubules (with fluo-5N) or within the cytosol (with fluo-3). In normal fibres (MHN), exposure to 0.5 mM halothane failed to trigger SR Ca2+ release, or to induce depletion of t-tubule Ca2+ (n=8). However, in MHS fibres, 0.5 mM halothane induced both SR Ca2+ release and a rapid depletion of t-tubule Ca2+, consistent with SOCE (n=8). In ∼20% of MHS fibres, SR Ca2+ release took the form of a propagating Ca2+ wave and this was associated with a corresponding SOCE wave of t-tubule Ca2+ depletion. In MHN fibres, both SR Ca2+ release and SOCE could be induced by 0.5 mM halothane when the cytosolic [Mg2+] was decreased to 0.2 mM (n=6). In MHS fibres, SOCE was potently inhibited by inclusion of a STIM1 blocking antibody within the re-sealed t-tubules (n=6). These data suggest (i) that in MHS fibres the degree of SR Ca2+ depletion induced by a clinically relevant level of volatile anaesthetic is sufficient to induce SOCE and (ii) that STIM1 located within the sarcolemma modulates SOCE.