Human Malignant Hyperthermia Research Articles

Understanding The Molecular Defects Of Human MH And CCD

Malignant Hyperthermia (MH) and Central Core Disease (CCD) are skeletal muscle disorders linked to mutations in the skeletal muscle ryanodine receptor (RyR1). Based on their phenotypes, disease-causing RyR1 mutations can be separated into three major groups: MH-only, both MH and CCD (MH/CCD), and CCD-only. The molecular basis for these different genotype-phenotype relationships is largely undefined. We have recently demonstrated that the porcine MH mutation, R615C, increases the sensitivity of the RyR1 channel to luminal Ca2+ activation and reduces the threshold for spontaneous Ca2+ release during store Ca2+ overload, also known as store-overload-induced Ca2+ release (SOICR). To investigate whether human MH and CCD mutations also alter the luminal Ca2+ activation of RyR1 and SOICR, we have generated a number of MH-only, MH/CCD, and CCD-only mutations located in the NH2 terminal, central, and COOH-terminal regions, and established stable, inducible HEK293 cell lines expressing these mutants. Using single cell Ca2+ imaging, we found that MH-only and MH/CCD mutations enhance the propensity for SOICR. On the other hand, some CCD-only mutations suppress or abolish SOICR, but retain caffeine-induced Ca2+ release, while other CCD-only mutants display little or no ryanodine- or caffeine-sensitive channel activity. Single channel studies reveal that, like the R615C MH mutation, MH/CCD mutations markedly sensitize the RyR1 channel to activation by luminal Ca2+. To assess their impact in the context of muscle cells, we are currently establishing stable, inducible mouse skeletal muscle C2C12 cell lines expressing MH and CCD RyR1 mutants. Further studies will address the question of whether altered luminal Ca2+ activation of RyR1 underlies a common defect of human MH and CCD mutations.

Clues to the Formation of Cores in a Mouse Model of Malignant Hyperthermia

Malignant hyperthermia (MH) and central core disease (CCD) are closely related diseases of skeletal muscle linked to mutations in the ryanodine receptor (RyR1) gene. Heterozygous mice harbouring a human MH mutation (Y522S), which is linked to MH susceptibility (MHS) with cores, were shown to display MH susceptibility that is also associated with heat-induced sudden death and mitochondrial damage (Durham et al., 2008; Cell: 133, 53). Here we show that RyR1Y522S/wt fibers develop amorphous cores which mimic, at least in later stages, the structural alterations observed in muscle biopsies from CCD patients. By examining mice at various ages (2 m - 1 y), we identified early steps in the formation of cores, a feat that has not been possible in human CCD muscle. The earliest and most obvious event in “core” formation is the swelling and partial disruption of mitochondria within discrete regions of the cell. This defect quickly leads to disarray of closely associated sarcoplasmic reticulum (SR) and calcium release units (CRUs), ultimately leading to the eventual complete disorganization of both organelles. The core regions also exhibit clearly delimited myofibril contractures, probably due increased Ca2+ leak from the SR diffusing from adjacent regions that cannot be sequestered. In later stages, amorphous cores, lacking all structural components become more frequent. We suggest that the initial triggering event in core formation is a local imbalance in Ca2+ release due to increased RyR1 leak that initially alters mitochondrial activity and then eventually disrupts both mitochondria and SR structure/function. Loss of mitochondrial ATP production and SR Ca2+ sequestration in these regions leads to local contractures and sarcomeric disruption within the core regions.

Reduced Threshold for Luminal Ca2+ Activation of RyR1 Underlies a Causal Mechanism of Porcine Malignant Hyperthermia

Naturally occurring mutations in the skeletal muscle Ca(2+) release channel/ryanodine receptor RyR1 are linked to malignant hyperthermia (MH), a life-threatening complication of general anesthesia. Although it has long been recognized that MH results from uncontrolled or spontaneous Ca(2+) release from the sarcoplasmic reticulum, how MH RyR1 mutations render the sarcoplasmic reticulum susceptible to volatile anesthetic-induced spontaneous Ca(2+) release is unclear. Here we investigated the impact of the porcine MH mutation, R615C, the human equivalent of which also causes MH, on the intrinsic properties of the RyR1 channel and the propensity for spontaneous Ca(2+) release during store Ca(2+) overload, a process we refer to as store overload-induced Ca(2+) release (SOICR). Single channel analyses revealed that the R615C mutation markedly enhanced the luminal Ca(2+) activation of RyR1. Moreover, HEK293 cells expressing the R615C mutant displayed a reduced threshold for SOICR compared with cells expressing wild type RyR1. Furthermore, the MH-triggering agent, halothane, potentiated the response of RyR1 to luminal Ca(2+) and SOICR. Conversely, dantrolene, an effective treatment for MH, suppressed SOICR in HEK293 cells expressing the R615C mutant, but not in cells expressing an RyR2 mutant. These data suggest that the R615C mutation confers MH susceptibility by reducing the threshold for luminal Ca(2+) activation and SOICR, whereas volatile anesthetics trigger MH by further reducing the threshold, and dantrolene suppresses MH by increasing the SOICR threshold. Together, our data support a view in which altered luminal Ca(2+) regulation of RyR1 represents a primary causal mechanism of MH.

Effects of Azumolene on Normal and Malignant Hyperthermia‐Susceptible Skeletal Muscle

Malignant hyperthermia is a pharmacogenetic disease of skeletal muscle in which a life-threatening, hypermetabolic syndrome is induced by exposure of susceptible patients to halogenated general anaesthetics and/or succinylcholine. Dantrolene sodium, the only drug effective for treatment of malignant hyperthermia, has low water solubility that makes its clinical use difficult. The aim of this study was to investigate the potency of azumolene, a 30-fold more water-soluble analogue, in comparison to the prototype dantrolene sodium, on mammalian and human skeletal muscles. The twitches of extensor digitorum longus and soleus muscles from mice were inhibited by azumolene with IC(50) of 2.8 +/- 0.8 and 2.4 +/- 0.6 microM, respectively. The IC(50) of dantrolene sodium in these muscles was 1.6 +/- 0.4 and 3.5 +/- 1.2 microM, respectively, with no difference in comparison to azumolene. Previous in vitro exposure of mouse soleus muscle to azumolene and dantrolene sodium (10 microM) significantly inhibited 8 mM caffeine-induced contractures. Azumolene was just effective as dantrolene sodium in relaxing caffeine-induced contractures of mouse soleus muscle. Intravenous injection caused dose-dependent decreases in twitches of guinea pig gastrocnemius muscle with IC(50) of 1.2 +/- 0.1 and 1.5 +/- 0.2 mg/kg for azumolene and dantrolene sodium, respectively. Azumolene, 10 microM, was effective in blocking and reversing caffeine-induced contracture of human malignant hyperthermia susceptible skeletal muscle in vitro. These studies provide evidence that azumolene is equipotent to dantrolene sodium in blocking pharmacologic-induced muscle contractures and that azumolene should be efficacious for treatment/prevention of malignant hyperthermia.

Intramuscular Injection of Sevoflurane Detects Malignant Hyperthermia Predisposition in Susceptible Pigs

The authors hypothesized that intramuscular sevoflurane injection allows diagnostic differentiation between malignant hyperthermia-susceptible (MHS) and -nonsusceptible (MHN) pigs by measurement of intramuscular lactate and carbon dioxide partial pressure (PCO2), and that dantrolene reduces the sevoflurane-induced PCO2 increase. With approval of the local animal care committee, microdialysis probes with attached microtubing for sevoflurane injection were placed in the adductor muscles of nine MHS and six MHN pigs, and PCO2 probes with microtubing were positioned in the triceps muscle of eight MHS and six MHN pigs. After equilibration, sevoflurane boluses at different concentrations and a sevoflurane-dantrolene bolus were injected synchronously. Lactate, pyruvate, and glucose as well as PCO2 were measured spectrophotometrically, and the rate of PCO2 increase was calculated. Intramuscular sevoflurane injection increased local lactate and PCO2 dose dependently, and significantly higher in MHS than in MHN pigs. Measurement of the rate of PCO2 increase allowed a distinct differentiation between single MHS and MHN pigs. No significant increase in PCO2 was found with sevoflurane and dantrolene. Local sevoflurane induces a hypermetabolic reaction measured by PCO2 and lactate increases. The reduced PCO2 increase in MHS after sevoflurane and dantrolene injection is likely to be a result of the sevoflurane-mediated calcium release and its antagonism by dantrolene. Sevoflurane may be useful for a less invasive diagnostic test for malignant hyperthermia in humans.

Basic Skeletal Muscle Phenotype of a Knock-in Mouse Model of Malignant Hyperthermia

Malignant hyperthermia (MH) is an inheritable disorder that can result in skeletal muscle hypermetabolism, limb rigidity, body temperature elevation, and death after exposure to halogenated anesthetics. Gene mutations of the skeletal muscle ryanodine receptor (i.e., SR Ca2+ release channel, or RYR1) have been associated with MH in humans. Recently, the first mouse model of MH was created in which tyrosine 522 was replaced with serine (Y522S) in RYR1. The heterozygous expression of the Y522S mutation mimics an autosomal dominant condition in humans. PURPOSE: To describe basic skeletal muscle morphology and contractile characteristics in Y522S and wild-type (WT) control mice. METHODS: Isometric torque as a function of stimulation frequency (20–400Hz) was measured in vivo from the anterior crural muscles of anesthetized Y522S (n = 15) and WT (n = 4) mice. In vivo twitch potentiation was measured at 20 Hz. The sensitivity of the EDL muscle to caffeine (0-50 mM) was assessed in vitro at 30 degrees C. RESULTS: There were no differences between Y522S and WT mice for body weight (24.9+ 1.0 vs. 22.5+ 1.4 g), wet weight of the tibialis anterior (42.8 + 1.5 vs. 41.4 + 3.4 g) and extensor digitorum longus (9.4 + 0.5 vs. 8.7 + 0.8 g) muscles, or isometric torque as a function of stimulation frequency. However, anterior crural muscles from Y522S mice did exhibit 25.1 + 1% potentiation of twitches at 20 Hz stimulation, whereas WT mice displayed none (−8.0 + 2.3%). EDL muscles from Y522S mice were more sensitive to caffeine than WT mice, with peak baseline isometric forces being greater for Y522S mice at caffeine concentrations greater than 4 mM. CONCLUSION: Anterior crural skeletal muscles from Y522S mice appear similar to WT control mice in size and isometric strength. However, anterior crural skeletal muscles of Y522S mice exhibit twitch potentiation and have a greater sensitivity to caffeine than WT controls.

Pharmacologic and Functional Characterization of Malignant Hyperthermia in the R163C RyR1 Knock-in Mouse

Malignant hyperthermia is a pharmacogenetic disorder affecting humans, dogs, pigs, and horses. In the majority of human cases and all cases in animals, malignant hyperthermia has been associated with missense mutations in the skeletal ryanodine receptor (RyR1). The authors used a "knock-in" targeting vector to create mice carrying the RyR1 R163C malignant hyperthermia mutation. Validation of this new mouse model of human malignant hyperthermia susceptibility includes (1) proof of transcription of the R163C allele and expression of ryanodine receptor protein in R163C heterozygous and R163C homozygous animals; (2) fulminant malignant hyperthermia episodes in R163C heterozygous mice after exposure to 1.25-1.75% halothane or an ambient temperature of 42 degrees C characterized by increased rectal temperature, respiratory rate, and inspiratory effort, with significant blood biochemical changes indicating metabolic acidosis, ending in death and hyperacute rigor mortis; (3) intraperitoneal pretreatment with dantrolene provided 100% protection from the halothane-triggered fulminant malignant hyperthermia episode; (4) significantly increased sensitivity (decreased effective concentration causing 50% of the maximal response) of R163C heterozygous and homozygous myotubes to caffeine, 4-chloro-m-cresol, and K-induced depolarization; (5) R163C heterozygous and homozygous myotubes have a significantly increased resting intracellular Ca concentration compared with wild type; (6) R163C heterozygous sarcoplasmic reticulum membranes have a twofold higher affinity (Kd = 35.4 nm) for [H]ryanodine binding compared with wild type (Kd = 80.1 nm) and a diminished inhibitory regulation by Mg. Heterozygous R163C mice represent a valid model for studying the mechanisms that cause the human malignant hyperthermia syndrome.

In Hot Pursuit

In Hot Pursuit

Heat‐ and anesthesia‐induced malignant hyperthermia in an RyR1 knock‐in mouse

Malignant hyperthermia (MH) is a life-threatening disorder characterized by skeletal muscle rigidity and elevated body temperature in response to halogenated anesthetics such as isoflurane or halothane. Mutation of tyrosine 522 of RyR1 (the predominant skeletal muscle calcium release channel) to serine has been associated with human malignant hyperthermia. In the present study, mice created harboring this mutation were found to represent the first murine model of human malignant hyperthermia. Mice homozygous for the Y522S mutation exhibit skeletal defects and die during embryonic development or soon after birth. Heterozygous mice, which correspond to the human occurrence of this mutation, are MH susceptible, experiencing whole body contractions and elevated core temperatures in response to isoflurane exposure or heat stress. Skeletal muscles from heterozygous mice exhibit increased susceptibility to caffeine- and heat-induced contractures in vitro. In addition, the heterozygous expression of the mutation results in enhanced RyR1 sensitivity to activation by temperature, caffeine, and voltage but not uncompensated sarcoplasmic reticulum calcium leak or store depletion. We conclude that the heterozygous expression of the Y522S mutation confers susceptibility to both heat- and anesthetic-induced MH responses.

Mg2+Dependence of Halothane-induced Ca2+Release from the Sarcoplasmic Reticulum in Skeletal Muscle from Humans Susceptible to Malignant Hyperthermia

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. 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. 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. 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.

Dantrolene Use in 3,4-Methylenedioxymethamphetamine (“Ecstasy”)–Mediated Hyperthermia

We thank Dr. Rusyniak et al. for their critical comments on our study about the induction of malignant hyperthermia (MH) in susceptible swine by 3,4-methylenedioxymethamphetamine (MDMA) (“ecstasy”).1However, some of the criticisms of our study must be relativized.First, to our knowledge, this is the first controlled study investigating the association between MDMA-induced hypermetabolic syndrome and MH. MH crisis is an acute clinical complication; therefore, the experimental setting for this study was following the clinical situation, and diagnosis of MH in our experiment could only be based on clinical parameters. The definition of the clinical cutoff parameters for MH crisis in our study was following the recommendations for clinical diagnosis of human MH crisis and previous animal studies. Increasing doses of MDMA induced a hypermetabolic state in MH-susceptible (MHS) as well as MH-normal (MHN) swine. However, the changes in the MHN swine after receiving a higher dose of MDMA (12 mg/kg) were moderate compared with the changes in MHS swine after 8 mg/kg MDMA, and all MHS swine fulfilled the defined criteria for MH.The only known differentiation between MHS and MHN swine is the presence of the Arg615-Cys point mutation on chromosome 6 leading to a functional impairment of the skeletal muscle ryanodine receptor (RyR1). We share the opinion of Dr. Rusyniak et al. that MDMA-induced hypermetabolism is not solely mediated through RyR1 complexes. However, the different reactions of MHS and MHN swine in our study are an indirect hint for activation of RyR1 after in vivo MDMA administration. The current study was aimed to prove whether MDMA is capable of inducing an MH syndrome, not to clarify the exact pathomechanism, i.e. , a possible mediation via the RyR1. Whether the RyR1 activation could be attributed to a direct effect of MDMA at the skeletal muscle or to a secondary effect of central stimulation, hyperthermia, or an MDMA-metabolite must therefore be clarified in future studies.The definition of an MH “trigger” is not as clear as mentioned in the letter of Dr. Rusyniak et al. From a clinical point of view, an MH trigger is a substance that is able to induce an MH crisis in a genetically determined individual in a clinically relevant dosage without any relevant cofactors. Following this definition, MDMA triggered MH in MHS swine in our study. We agree that cumulative intravenous administration of MDMA is not the common method of MDMA abuse. However, this course of action and measurement of corresponding MDMA plasma concentrations allowed us to determine a dose response and to underline the clinical relevance.The therapeutic regimen of MDMA-induced MH in our study was based on the standard clinical therapy of MH. Standardized therapy of MH in the MHS swine performed with dantrolene, sodium bicarbonate, and hyperventilation partly removed the clinical signs of MH immediately. The body temperature of the swine remained unchanged 15 min after therapy induction. We agree that the short observation time without the possibility to detect changes in body temperature was a weakness in our study design.Whether administration of dantrolene is useful in all patients with MDMA-induced hyperthermia could not be answered by our study. However, in a life-threatening clinical situation, “simple hyperthermia” could not be distinguished from “true malignant hyperthermia.” Therefore, in our opinion, dantrolene might be a lifesaving therapy option, and consequently, administration of dantrolene should be considered with respect to patient safety in cases of MDMA-mediated hyperthermic syndrome.* University Hospital Hamburg-Eppendorf, Hamburg, Germany. fiege@uke.uni-hamburg.de

Dantrolene – A review of its pharmacology, therapeutic use and new developments

Human malignant hyperthermia is a life-threatening genetic sensitivity of skeletal muscles to volatile anaesthetics and depolarizing neuromuscular blocking drugs occurring during or after anaesthesia. The skeletal muscle relaxant dantrolene is the only currently available drug for specific and effective therapy of this syndrome in man. After its introduction, the mortality of malignant hyperthermia decreased from 80% in the 1960s to < 10% today. It was soon discovered that dantrolene depresses the intrinsic mechanisms of excitation-contraction coupling in skeletal muscle. However, its precise mechanism of action and its molecular targets are still incompletely known. Recent studies have identified the ryanodine receptor as a dantrolene-binding site. A direct or indirect inhibition of the ryanodine receptor, the major calcium release channel of the skeletal muscle sarcoplasmic reticulum, is thought to be fundamental in the molecular action of dantrolene in decreasing intracellular calcium concentration. Dantrolene is not only used for the treatment of malignant hyperthermia, but also in the management of neuroleptic malignant syndrome, spasticity and Ecstasy intoxication. The main disadvantage of dantrolene is its poor water solubility, and hence difficulties are experienced in rapidly preparing intravenous solutions in emergency situations. Due to economic considerations, no other similar drugs have been introduced into routine clinical practice.

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.

Testing for malignant hyperthermia.

Testing for malignant hyperthermia.

Autosomal dominant canine malignant hyperthermia is caused by a mutation in the gene encoding the skeletal muscle calcium release channel (RYR1).

Malignant hyperthermia (MH) is an inherited disorder of skeletal muscle characterized by hypercarbia, rhabdomyolysis, generalized skeletal muscle contracture, cardiac dysrhythmia, and renal failure, that develops on exposure to succinylcholine or volatile anesthetic agents. All swine and up to 50% of human MH events are thought to be associated with mutations in the calcium release channel of the sarcoplasmic reticulum, also known as the ryanodine receptor (RYR1). Events resembling MH have been reported in other species, but none have undergone genetic investigation to date. To determine the molecular basis of canine MH, a breeding colony was established with a male, mixed-breed, MH-susceptible (MHS) dog that survived an in vivo halothane-succinylcholine challenge. He was mated to three unaffected females to produce four litters and back-crossed to an affected daughter to produce one litter. One of his MHS sons was mated to an unaffected female to produce an additional litter. Forty-seven dogs were phenotyped with an in vitro contracture test and diagnosed as MHS or MH normal based on the North American in vitro contracture test protocol. Nine microsatellite markers in the vicinity of RYR1 on canine chromosome 1 (CFA01) were tested for linkage to the MHS phenotype. Mutational analysis in two MHS and two MH-normal dogs was performed with direct sequencing of polymerase chain reaction products and of cloned fragments that represent frequently mutated human RYR1 regions. A restriction fragment length polymorphism was chosen to detect the candidate mutation in the pedigree at large. Pedigree inspection revealed that MHS in this colony is transmitted as an autosomal dominant trait. FH2294, the marker closest to RYR1, is linked to MHS at a theta = 0.03 with a LOD score of 9.24. A T1640C mutation gives rise to an alanine for valine substitution of amino acid 547 in the RYR1 protein, generating a maximum LOD score of 12.29 at theta = 0.00. All dogs diagnosed as MHS by in vitro contracture test were heterozygous for the mutation, and all MH-normal dogs were homozygous for the T1640 allele. These results indicate that autosomal dominant canine MH is caused by a mutation in the gene encoding the skeletal muscle calcium release channel and that the MHS trait in this pedigree of mixed-breed dogs is in perfect cosegregation with the RYR1 V547A mutation.

Xenon does not induce contracture in human malignant hyperthermia muscle

Xenon has many characteristics of an ideal anaesthetic agent. It is not known whether xenon is a safe alternative to the potent inhalational anaesthetics in patients susceptible to malignant hyperthermia (MH). We investigated the effect of xenon, halothane and caffeine on muscle specimens of 31 individuals, referred to the MH Unit of the University of Ulm, and performed genetic epidemiology. Thirteen individuals were classified as MH susceptible and 18 as MH negative. Xenon 70% did not cause an increase in baseline tension of any MH-susceptible muscle specimen in contrast to halothane and caffeine. The evoked twitch response increased transiently in MH-susceptible and normal specimens indicating a mechanism independent of MH susceptibility. These results suggest that xenon, in concentrations up to 70% may be a safe anaesthetic for MH-susceptible patients.

The effects of mutated skeletal ryanodine receptors on calreticulin and calsequestrin expression in the brain and pituitary gland of boars

Mutations in skeletal ryanodine receptors (sRyR) result in malignant hyperthermia in humans and Porcine Stress Syndrome (PSS) in pigs. Whether the sRyR is expressed in neuronal tissue and what impact it has on neuronal function is relatively unexplored. We have hypothesized that the presence of mutated sRyR may be accompanied by compensatory changes in Ca 2+-binding protein expression. We were interested in whether pigs heterozygous for mutated sRyR would show changes in the expression of Ca 2+-binding proteins, in specific regions of the brain, and whether changes in this expression would be accompanied by the presence of sRyR within that region. The objectives of the current experiments were to determine (1) whether calreticulin (CR) and calsequestrin (CS) are expressed in the pituitary gland and brain of the pig, (2) if boars heterozygous for mutated sRyR differed from wild-type boars in the expression of CR or CS, and (3) if altered Ca 2+-binding protein expression would be accompanied by the presence of sRyR mRNA. Boars either heterozygous or wild-type ( n=6) for the mutation in sRyR known to cause PSS, were euthanized and the pituitary gland and brains were collected for western blotting for CR and CS. An additional four wild-type boars were sacrificed and brains were collected for in situ hybridization for sRyR mRNA. Immunoreactive CR was expressed in porcine tissues with highest ( p<0.0001) expression in the pituitary gland and lower but equivalent expression in the hypothalamus, frontal cortex, and hippocampus. Immunoreactive CS was not detectable in the pituitary gland while low levels were observed in the hypothalamus and frontal cortex. Dramatically higher ( p<0.0001) levels of CS were found in the hippocampus. Genotype did not affect CR expression in the pituitary gland or any brain region examined. Immunoreactive CS levels were lower ( p<0.002) in the hippocampus of heterozygous compared to wild-type boars. In situ hybridization experiments revealed the presence of sRyR mRNA in the hippocampus equally distributed across all cell subfields. In conclusion, both CR and CS were expressed in the porcine brain with specific patterns of expression across the brain regions examined. Boars heterozygous for mutated sRyR had lower CS in the hippocampus, which was accompanied by the expression of mRNA for sRyR.

No evidence that xenon triggers malignant hyperthermia in humans

No evidence that xenon triggers malignant hyperthermia in humans

5-HT2 receptor antagonist-mediated inhibition of halothane-induced contractures in skeletal muscle specimens from malignant hyperthermia susceptible patients.

Administration of 5-HT2 receptor agonists induced malignant hyperthermia (MH) in susceptible pigs. Furthermore, the 5-HT2 receptor antagonist ritanserin prevented 5-HT-induced porcine MH. It has been shown that 5-HT2 receptor agonists induce marked contractures in skeletal muscle specimens from MH susceptible (MHS) but not in specimens from normal patients. The purpose of this study was to investigate the effects of ritanserin on halothane-induced contractures in muscle specimens from MHS patients. Twenty-five patients aged 8-56 years (29.5+/-13.6) classified as MHS by the in vitro contracture test (IVCT) with halothane and caffeine according to the protocol of the European MH Group participated in this study. Muscle specimens were pretreated with ritanserin 10 micromol/l (n= 14), 20 micromol/l (n=14) and 100 micromol/l (n=12) for 10 min and subsequently halothane was added incrementally (0.11-0.22-0.44 mmol/l) to the tissue bath as described in the European MH protocol. The results of the halothane contracture test were used as control. Following administration of halothane, muscle contractures reached a maximum of 16.9+/-4.2 mN. Ritanserin led to a significant inhibition of halothane-induced contractures in MHS muscles. Following pretreatment with ritanserin, halothane-induced contracture maximum was significantly smaller with 7.5+/-3.1 mN after 10 micromol/l ritanserin, 4.9+/-1.5 mN after 20 micromol/l ritanserin and 0.5+/- 0.2 mN after 100 micromol/l ritanserin than without pretreatment. Administration of ritanserin induced at all concentrations a decrease in muscle twitch height. Increase in muscle twitch following halothane was reduced in a concentration-dependent manner by ritanserin. The presented findings indicate that 5-HT might be involved in the mechanisms of halothane-induced MH in humans. Further studies have to determine the pathophysiological role of the 5-HT system in MH, and whether ritanserin could be an alternative for treatment or prevention of halothane-induced MH.

The anesthetic myopathies and malignant hyperthermias.

Fatal destruction of skeletal muscle coincident with exposure to specific drugs used during anethesia has been recognized as a potentially heritable disorder for more than 30 years. Variable expressivity and incomplete penetrance of the clinical malignant hyperthermia phenotype, together with inherent drawbacks of the in-vitro contracture test confounded efforts to discover the underlying pathogenesis until the application of molecular genetic techniques. On the basis of linkage analysis and mapping of positional candidate genes, mutant alleles at loci on chromosomes 1q, (dihydropyridine-sensitive L-type calcium channel-A1S); 3q, 5p, 7q (dihydropyridine-sensitive L-type calcium channel-LA2), and 19q (ryanodine receptor) are now believed to account for up to 50% of human malignant hyperthermia susceptibility. Although inconsistent genotype-phenotype correlations and doubts regarding the causality of each mutant allele persist, the definition of malignant hyperthermia and relevance of molecular genetic data to the problems of family counseling, population screening, and improved resolution of the malignant hyperthermia phenotype must now be appraised in view of significant locus and allelic genetic heterogeneity.