Co-segregation of the malignant hyperthermia and the Arg615-Cys615 mutation in the skeletal muscle calcium release channel protein in five European Landrace and Pietrain pig breeds

A total of 392 pigs of European Landrace and Pietrain origin segregating for malignant hyperthermia (MH) were genotyped using a polymerase chain reaction (PCR)/restriction endonuclease test for the C—T mutation at nucleotide (nt) 1843 in the skeletal muscle ryanodine receptor (RYR1) gene, earlier identified as the causal mutation for MH. All pigs had been halothane tested and genotyped at linked polymorphic marker loci. There was complete correlation between MH status of the 392 animals, as diagnosed by a combination of the halothane challenge test with S, GPI, H, A1BG, PGD haplotyping, and the DNA-based test. DNA-based detection of the MH status in 238 MH-susceptible heterozygous (N/n) and homozygous (n/n) pigs was shown to be accurate, eliminating the 2% diagnostic error that is associated with the halothane challenge test. The mutation was also associated with an allele of a polymorphic microsatellite (ETH5 001) at the RYR1 locus.     

Discordance, in a Malignant Hyperthermia Pedigree, between in Vitro Contracture-Test Phenotypes and Haplotypes for the MHS1 Region on Chromosome 19q12–13.2, Comprising the C1840T Transition in the RYR1 Gene

A point mutation in the gene encoding the skeletal muscle calcium release channel (RYR1) has been proposed as the probable cause of malignant hyperthermia (MH) in swine, where it segregates with the disease in all MH–prone strains investigated. The same C-to-T exchange in nucleotide position 1840 of the human RYR1 cDNA sequence was found in a few human MH pedigrees. We report a German MH pedigree where in vitro contracture test (IVCT) results and haplotypes of markers for the MHS1/RYR1 region including this base transition have yielded several discrepancies. The MH-susceptible phenotype was defined by IVCT performed according to the European standard protocol. Haplotypes were constructed for markers for the MHS1/RYR1 region on chromosome 19 and include the C1840T base exchange. Discussing the probabilities for a number of hypotheses to explain these data, we suggest that our results may challenge the causative role of this mutation—and possibly the role of the RYR1 gene itself—in human MH susceptibility, at least in some cases.     

Mice expressing T4826I-RYR1 are viable but exhibit sex- and genotype-dependent susceptibility to malignant hyperthermia and muscle damage

Mutation T4825I in the type 1 ryanodine receptor (RYR1(T4825I/+)) confers human malignant hyperthermia susceptibility (MHS). We report a knock-in mouse line that expresses the isogenetic mutation T4826I. Heterozygous RYR1(T4826I/+) (Het) or homozygous RYR1(T4826I/T4826I) (Hom) mice are fully viable under typical rearing conditions but exhibit genotype- and sex-dependent susceptibility to environmental conditions that trigger MH. Hom mice maintain higher core temperatures than WT in the home cage, have chronically elevated myoplasmic[Ca(2+)](rest), and present muscle damage in soleus with a strong sex bias. Mice subjected to heat stress in an enclosed 37°C chamber fail to trigger MH regardless of genotype, whereas heat stress at 41°C invariably triggers fulminant MH in Hom, but not Het, mice within 20 min. WT and Het female mice fail to maintain euthermic body temperature when placed atop a bed whose surface is 37°C during halothane anesthesia (1.75%) and have no hyperthermic response, whereas 100% Hom mice of either sex and 17% of the Het males develop fulminant MH. WT mice placed on a 41°C bed maintain body temperature while being administered halothane, and 40% of the Het females and 100% of the Het males develop fulminant MH within 40 min. Myopathic alterations in soleus were apparent by 12 mo, including abnormally distributed and enlarged mitochondria, deeply infolded sarcolemma, and frequent Z-line streaming regions, which were more severe in males. These data demonstrate that an MHS mutation within the S4-S5 cytoplasmic linker of RYR1 confers genotype- and sex-dependent susceptibility to pharmacological and environmental stressors that trigger fulminant MH and promote myopathy.     

Voltage-dependent calcium release in human malignant hyperthermia muscle fibers.

Malignant hyperthermia (MH) results from a defect of calcium release control in skeletal muscle that is often caused by point mutations in the ryanodine receptor gene (RYR1). In malignant hyperthermia-susceptible (MHS) muscle, calcium release responds more sensitively to drugs such as halothane and caffeine. In addition, experiments on the porcine homolog of malignant hyperthermia (mutation Arg615Cys in RYR1) indicated a higher sensitivity to membrane depolarization. Here, we investigated depolarization-dependent calcium release under voltage clamp conditions in human MHS muscle. Segments of muscle fibers dissected from biopsies of the vastus lateralis muscle of MHN (malignant hyperthermia negative) and MHS subjects were voltage-clamped in a double vaseline gap system. Free calcium was determined with the fluorescent indicator fura-2 and converted to an estimate of the rate of SR calcium release. Both MHN and MHS fibers showed an initial peak of the release rate, a subsequent decline, and rapid turn-off after repolarization. Neither the kinetics nor the voltage dependence of calcium release showed significant deviations from controls, but the average maximal peak rate of release was about threefold larger in MHS fibers.     

The Ile2453Thr mutation in the ryanodine receptor gene 1 is associated with facilitated calcium release from sarcoplasmic reticulum by 4-chloro-m-cresol in human myotubes

Central core disease (CCD) is a congenital disorder of skeletal muscle that is characterised histologically by typical central cores in type 1 skeletal muscle fibres. This disease is associated with malignant hyperthermia susceptibility and has been linked to the gene of skeletal muscle ryanodine receptor RYR1. In this study, we present a family with the spontaneous occurrence of the RYR1 Ile2453Thr mutation. Affected individuals were diagnosed as susceptible to malignant hyperthermia in the in vitro contracture test (IVCT) and showed histological signs of CCD. Myotubes were derived from the index patient. The calcium homeostasis in response to the ryanodine receptor agonist 4-chloro-m-cresol (4CmC) was investigated by calcium imaging using the Ca(2+)-sensitive fluorescent probe FURA 2. In the myotubes derived from the mutation carrier, the EC(50) of 4CmC was reduced to 94 micro as compared to 201 microM in a control group of 16 individuals non-susceptible to malignant hyperthermia. In the myotubes of the non-affected family members, the EC(50) was found within the same range as that of the control group. The reduction of EC(50) indicates a facilitated calcium release from sarcoplasmic reticulum in the myotubes of the index patient suggesting that the RYR1 Ile2453Thr mutation is pathogenic for the malignant hyperthermia susceptibility and CCD of the two affected individuals.     

Cosegregation of porcine malignant hyperthermia and a probable causal mutation in the skeletal muscle ryanodine receptor gene in backcross families.

A study of the inheritance of malignant hyperthermia (MH) in the British Landrace breed revealed the same substitution of T for C at nucleotide 1843 in the ryanodine receptor (RYR1) gene that was previously shown to be correlated with MG in five Canadian swine breeds. Cosegregation of the mutation with MH in 338 informative meioses led to a lod score of 101.75 for linkage at Omax = 0.0. The substitution was also associated with a HinPI- BanII+ RsaI- haplotype in this breed, as in the five breeds tested earlier, suggesting its origin in a common founder animal. DNA-based detection of the MH status in 376 MH-susceptible heterozygous (N/n) and homozygous (n/n) pigs was shown to be accurate, eliminating the 5% diagnostic error that is associated with the halothane challenge test and flanking marker haplotyping procedures in current diagnostic use. These results strongly support the view that the substitution of T for C at nucleotide 1843 is the causative mutation in porcine MH and demonstrate the feasibility of rapid, accurate, noninvasive, large-scale testing for porcine MH status using DNA-based tests for the mutation.     

Evidence for genetic heterogeneity of malignant hyperthermia susceptibility

A locus for malignant hyperthermia susceptibility (MHS) has been localized on chromosome 19q12-13.2, while at the same time the gene encoding the skeletal muscle ryanodine receptor (RYR1) also has been mapped to this region and has been found to be tightly linked to MHS. RYR1 was consequently postulated as the candidate for the molecular defect causing MHS, and a point mutation in the gene has now been identified and is thought to be the cause of MH in at least some MHS patients. Here we report the results of a linkage study done with 19q12-13.2 markers, including the RYR1 cDNA, in two Bavarian families with MHS. In one of the families, three unambiguous recombination events between MHS and the RYR1 locus were found. In the second family only one informative meiosis was seen with RYR1. However, segregation analysis with markers for D19S75, D19S28, D19S47, CYP2A, BCL3, and APOC2 shows that the crossovers in the first family involve the entire haplotype defined by these markers flanking RYR1 and, furthermore, reveals multiple crossovers between these haplotypes and MHS in the second family. In these families, pairwise and multipoint lod scores below -2 exclude MHS from an interval spanning more than 26 cM and comprising the RYR1 and the previously described MHS locus. Our findings thus strongly suggest genetic heterogeneity of the MHS trait and prompt the search for another MHS locus.     

In Vivo 31P NMR Spectroscopy Studies of Halothane Induced Porcine Stress Syndrome. No Effect of C-Phenyl N-Tertbutyl Nitrone (PBN)

Porcine stress syndrome (PSS) which is an example of malignant hyperthermia (MH) in swine has previously been attributed to oxidative stress primarily due to an inherited antioxidant abnormality in MH susceptible (MHS) animals. C-phenyl-N-tert-butyl nitrone (PBN), a free radical spin trap, was selected to investigate whether free radicals are involved in MH. If free radicals cause the MH stress attack, then PBN should alter the time required for the onset of the stress attack, or perhaps protect the animal from experiencing the stress attack. In vivo phosphorus-31 (³¹P) magnetic resonance spectroscopy (MRS) was used to monitor metabolism in three to four week old normal and MHS piglets administered halothane as the stress challenge. Malignant hyperthermia was not reproducibly induced by halothane anesthesia. For those animals which did develop MH a dramatic fall in the level of PCr and a rise in the level of Pi was detected by ³¹P MRS. Intravenous administration of PBN prior to halothane exposure had no effect on the number of animals experiencing the stress attack. PBN does not appear to prevent, delay or reverse the onset of halothane-induced MH in three to four week old MHS piglets. The primary events leading to the MH syndrome do not appear to be influenced by the intervention of the type of free radicals normally trapped by PBN.     

B-lymphocytes from malignant hyperthermia-susceptible patients have an increased sensitivity to skeletal muscle ryanodine receptor activators.

Malignant hyperthermia (MH) is a pharmacogenetic disease triggered by volatile anesthetics and succinylcholine in genetically predisposed individuals. The underlying feature of MH is a hypersensitivity of the calcium release machinery of the sarcoplasmic reticulum, and in many cases this is a result of point mutations in the skeletal muscle ryanodine receptor calcium release channel (RYR1). RYR1 is mainly expressed in skeletal muscle, but a recent report demonstrated the existence of this isoform in human B-lymphocytes. As B-cells can produce a number of cytokines, including endogenous pyrogens, we investigated whether some of the symptoms seen during MH could be related to the involvement of the immune system. Our results show that (i) Epstein-Barr virus-immortalized B-cells from MH-susceptible individuals carrying the V2168M RYR1 gene mutation were more sensitive to the RYR activator 4-chloro-m-cresol and (ii) their peripheral blood leukocytes produce more interleukin (IL)-1beta after treatment with the RYR activators caffeine and 4-chloro-m-cresol, compared with cells from healthy controls. Our result demonstrate that RYR1-mediated calcium signaling is involved in release of IL-1beta from B-lymphocytes and suggest that some of the symptoms seen during an MH episode may be due to IL-1beta production.     

Identification of novel mutations in the ryanodine-receptor gene (RYR1) in malignant hyperthermia: genotype-phenotype correlation.

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle that is triggered in genetically predisposed individuals by common anesthetics and muscle relaxants. The ryanodine receptor (RYR1) is mutated in a number of MH pedigrees, some members of which also have central core disease (CCD), an inherited myopathy closely associated with MH. Mutation screening of 6 kb of the RYR1 gene has identified four adjacent novel mutations, C6487T, G6488A, G6502A, and C6617T, which result in the amino acid alterations Arg2163Cys, Arg2163His, Val2168Met, and Thr2206Met, respectively. Collectively, these mutations account for 11% of MH cases and identify the gene segment 6400-6700 as a mutation hot spot. Correlation analysis of the in vitro contracture-test data available for pedigrees bearing these and other RYR1 mutations showed an exceptionally good correlation between caffeine threshold and tension values, whereas no correlation was observed between halothane threshold and tension values. This finding has important ramifications for assignment of the MH-susceptible phenotype, in genotyping studies, and indicates that assessment of recombinant individuals on the basis of caffeine response is justified, whereas assessment on the basis of halothane response may be problematic. Interestingly, the data suggest a link between the caffeine threshold and tension values and the MH/CCD phenotype.     

Polymorphisms and deduced amino acid substitutions in the coding sequence of the ryanodine receptor (RYR1) gene in individuals with malignant hyperthermia.

Twenty-one polymorphic sequence variants of the RYR1 gene, including 13 restriction fragment length polymorphisms (RFLPs), were identified by sequence analysis of human ryanodine receptor (RYR1) cDNAs from three individuals predisposed to malignant hyperthermia (MH). All RFLPs were detectable in PCR-amplified products, and their segregation was consistent with our initial finding of linkage to MH in the nine families previously informative for one or more intragenic markers (MacLennan et al., 1990, Nature 343:559-561). Four amino acid substitutions were identified in the study: Arg for Gly248, Cys for Arg470, Leu for Pro1785, and Cys for Gly2059. Of 45 families tested, a single family presented the Arg for Gly248 substitution where it segregated with malignant hyperthermia, making it a candidate mutation for predisposition to MH in man. The other three polymorphic substitutions failed to segregate with malignant hyperthermia in those families in which they occurred, implying that they represent polymorphisms with little or no effect on the function of the RYR1 gene.     

Does the A3333G mutation in the CACNL1A3 gene, detected in malignant hyperthermia, also occur in central core disease?

Malignant hyperthermia (MH) and central core disease (CCD) are two conditions associated with susceptibility to volatile anesthetics and depolarizing muscle relaxants. The gene RYR1, encoding the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum, is responsible for about 50% of the cases of MH and some cases of CCD. However, genetic heterogeneity occurs in MH and a mutation in a second gene (CACLN1A3), encoding the alpha1-subunit of the dihydropyridine (DHP) channel, has recently been found in a large MH French family. The presence of this mutation in patients with CCD has not yet been reported. In this study, we analyzed the A3333G mutation in 5 unrelated patients affected by CCD and 31 MH-susceptible relatives (from 19 MH families) and did not find this mutation in any of them. Nevertheless, the report of data on newly described mutations in different populations is important to estimate the contributions of each gene mutation to the phenotype of MH and CCD.     

Prediction of susceptibility to the porcine stress syndrome

An experiment designed to compare different predictors of porcine stress syndrome (PSS) was conducted. Animals were exposed to the anesthetic gas, halothane, and their reactions monitored to determine susceptibility or resistance to PSS. Two blood creatine phosphokinase (CPK) traits plus their logs10 were examined as predictors of PSS susceptibility. These were Sigma CPK, Antonik CPK, log Sigma CPK, and log Antonik CPK. The accuracy of these predictors varied from 87 percent to 91 percent in agreement with halothane-determined stress susceptibility. In addition, the relationship of PSS and blood types systems (AO,H) were studied. Two blood types, (+,-/-) and (-,+/+), were consistently stress susceptible while three blood types, (+,a/a), (+,a/c), and (+,c/-), were consistently stress resistant. However, one blood type (+,a/-) contained both stress-susceptible and stress-resistant individuals.     

Effect of ryanodine receptor mutations on interleukin-6 release and intracellular calcium homeostasis in human myotubes from malignant hyperthermia-susceptible individuals and patients affected by central core disease

In this study we report for the first time the functional properties of human myotubes isolated from patients harboring the native RYR1 I4898T and R4893W mutations linked to central core disease. We examined two aspects of myotube physiology, namely excitation-contraction and excitation-secretion coupling. Our results show that upon activation of the ryanodine receptor (RYR), myotubes release interleukin-6 (IL-6); this was dependent on de novo protein synthesis and could be blocked by dantrolene and cyclosporine. Myotubes from the two patients affected by central core disease showed a 4-fold increase in the release of the inflammatory cytokine IL-6, compared with cells derived from control or malignant hyperthermia susceptible individuals. All tested myotubes released calcium from intracellular stores upon stimulation via surface membrane depolarization or direct RYR activation by 4-chloro-m-cresol. The functional impact on calcium release of RYR1 mutations linked to central core disease or malignant hyperthermia is different: human myotubes carrying the malignant hyperthermia-linked RYR1 mutation V2168M had a shift in their sensitivity to the RYR agonist 4-chloro-m-cresol to lower concentrations, whereas human myotubes harboring C-terminal mutations linked to central core disease exhibited reduced [Ca2+]i increase in response to 4-chloro-m-cresol, caffeine, and KCl. Taken together, these results suggest that abnormal release of calcium via mutated RYR enhances the production of the inflammatory cytokine IL-6, which may in turn affect signaling pathways responsible for the trophic status of muscle fibers.     

Influence of Nutrition on Malignant Hyperthermia in Pigs

In 2 experiments malignant hyperthermia susceptible Danish Landrace pigs were fed, for 2 or 4 weeks, synthetic diets containing casein as protein source or no protein. Minerals and vitamins were supplied to both groups. The animals were anaesthetized weekly for a maximum of 20 min with a halothane-oxygen mixture. In the first experiment malignant hyperthermia was equally delayed in both groups. If malignant hyperthermia developed, the appearance was at the end of the anaesthetic period. In the second experiment a deeper anaesthesia was employed. Malignant hyperthermia was delayed in both groups, but most markedly in the protein-deficient animals. Malignant hyperthermia developed faster after return to the original feed. These results provide evidence for a nutritional influence on the penetrance of malignant hyperthermia susceptibility during halothane anaesthesia in pigs.     

Dantrolene inhibition of ryanodine receptor Ca2+ release channels. Molecular mechanism and isoform selectivity

As an inhibitor of Ca(2+) release through ryanodine receptor (RYR) channels, the skeletal muscle relaxant dantrolene has proven to be both a valuable experimental probe of intracellular Ca(2+) signaling and a lifesaving treatment for the pharmacogenetic disorder malignant hyperthermia. However, the molecular basis and specificity of the actions of dantrolene on RYR channels have remained in question. Here we utilize [(3)H]ryanodine binding to further investigate the actions of dantrolene on the three mammalian RYR isoforms. The inhibition of the pig skeletal muscle RYR1 by dantrolene (10 microm) was associated with a 3-fold increase in the K(d) of [(3)H]ryanodine binding to sarcoplasmic reticulum (SR) vesicles such that dantrolene effectively reversed the 3-fold decrease in the K(d) for [(3)H]ryanodine binding resulting from the malignant hyperthermia RYR1 Arg(615) --> Cys mutation. Dantrolene inhibition of the RYR1 was dependent on the presence of the adenine nucleotide and calmodulin and reflected a selective decrease in the apparent affinity of RYR1 activation sites for Ca(2+) relative to Mg(2+). In contrast to the RYR1 isoform, the cardiac RYR2 isoform was unaffected by dantrolene, both in native cardiac SR vesicles and when heterologously expressed in HEK-293 cells. By comparison, the RYR3 isoform expressed in HEK-293 cells was significantly inhibited by dantrolene, and the extent of RYR3 inhibition was similar to that displayed by the RYR1 in native SR vesicles. Our results thus indicate that both the RYR1 and the RYR3, but not the RYR2, may be targets for dantrolene inhibition in vivo.     

Fatty acids modulate calcium-induced calcium release from skeletal muscle heavy sarcoplasmic reticulum fractions: implications for malignant hyperthermia

Based on studies in swine, the malignant hyperthermia syndrome has been postulated to result from an enhanced sensitivity (low threshold) of the Ca2(+)-induced Ca2(+)-release process. However, fatty acid production is elevated in homogenates of skeletal muscle from pigs and humans susceptible to malignant hyperthermia. In the present study, we demonstrate that the threshold of Ca2(+)-induced Ca2+ release is normal in susceptible humans and in susceptible swine depleted of triglycerides. Exogenously added unsaturated fatty acids decreased the threshold of Ca2(+)-induced Ca2+ release to a much greater extent in porcine and equine muscle than in human muscle. When triglyceride and free fatty acid values were reduced to about 40 and 60%, respectively, of control values, malignant hyperthermia-susceptible swine did not exhibit muscle rigidity when challenged in vivo with halothane and succinylcholine and the threshold of the Ca2(+)-induced Ca2(+)-release process in heavy sarcoplasmic reticulum fractions was normal. Despite the reduced triglyceride and fatty acid levels, these swine had a positive in vitro contracture test for malignant hyperthermia. A low Ca2(+)-induced Ca2(+)-release threshold is not essential for malignant hyperthermia susceptibility, but appears to be the result of excessive free fatty acids produced during organelle isolation.     

Single-amino-acid deletion in the RYR1 gene, associated with malignant hyperthermia susceptibility and unusual contraction phenotype

Malignant hyperthermia (MH) is an anesthetic-drug-induced, life-threatening hypermetabolic syndrome caused by abnormal calcium regulation in skeletal muscle. Often inherited as an autosomal dominant trait, MH has linkage to 30 different mutations in the RYR1 gene, which encodes a calcium-release-channel protein found in the sarcoplasmic reticulum membrane in skeletal muscle. All published RYR1 mutations exclusively represent single-nucleotide changes. The present report documents, in exon 44 of RYR1 in two unrelated, MH-susceptible families, a 3-bp deletion that results in deletion of a conserved glutamic acid at position 2347. This is the first deletion, in RYR1, found to be associated with MH susceptibility. MH susceptibility was confirmed among some family members by in vitro diagnostic pharmacological contracture testing of biopsied skeletal muscle. Although a single-amino-acid deletion appears to be a subtle change in the protein, the deletion of Glu2347 from RYR1 produces an unusually large electrically evoked contraction tension in MH-positive individuals, suggesting that this deletion produces an alteration in skeletal-muscle calcium regulation, even in the absence of pharmacological agents.     

Thermochemiluminescent assay of porcine, rat, and human erythrocytes for antioxidative deficiencies

The thermal induction of chemiluminescence of luminol-horseradish peroxidase-labeled erythrocytes from pigs, rats, and man was studied. The luminescent responses of rat, porcine, and human erythrocytes to heating were linear in respect to logs of counts per minute versus temperature. Landrace-Duroc crossbred pigs with a history of malignant hyperthermia (porcine stress syndrome) and Poland-China-miniature pigs inbred for malignant hyperthermia (MH) yielded erythrocytes with high-level thermochemiluminescence (TCL). Sprague-Dawley rat erythrocytes were intermediate in their production of TCL. Normal human and MH-resistant miniature swine erythrocytes produced low-level TCL. However, pretreatment of human erythrocytes with 1-chloro-2,4-dinitrobenzene (CDNB) resulted in high-level TCL. Furthermore, halothane enhanced the TCL of CDNB-treated human erythrocytes and Landrace-Duroc porcine erythrocytes that were not treated with CDNB. Red blood cells from pigs susceptible to the porcine stress syndrome demonstrated a TCL response very similar to CDNB-treated erythrocytes.     

High-resolution physical mapping of four microsatellite repeat markers near the RYR1 locus on chromosome 19q13.1 and apparent exclusion of the MHS locus from this region in two malignant hyperthermia susceptible families.

Malignant hyperthermia susceptibility (MHS) is a potentially lethal, hereditary disorder of skeletal muscle that may be triggered by inhalation anesthetics and depolarizing muscle relaxants. Defects in the gene encoding the ryanodine receptor (RYR1) localized on human chromosome 19q13.1 have been proposed to be responsible for MHS. Using a chromosome 19-specific human/hamster somatic cell hybrid mapping panel, we were able to determine that four closely linked microsatellite repeat markers bracket RYR1 with the order 19cen-D19S75-D19S191-RYR1-(D19S47, D19S190)-19ter. Application of the four markers to genetic studies of MHS showed recombination between the markers and MHS in two families, with linkage analysis apparently excluding the MHS locus from the RYR1 region of 19q13.1. These results therefore support the recent observations of genetic heterogeneity in MHS.