1 Departments of Anaesthesiology and Research, Kantonsspital/University of Basel, CH-4031 Basel, Switzerland. 2 Institut fuer Humangenetik, Biozentrum, University of Wuerzburg, D-97074 Wuerzburg, Germany. 3 Department of Experimental and Diagnostic Medicine, General Pathology Section, University of Ferrara, Via Borsari 46, I-44100 Ferrara, Italy.*Corresponding author
Accepted for publication: June 11, 2002
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods. Primary cultures of human skeletal muscle cells were established from 54 individuals diagnosed by the IVCT according to the protocol of the European MH Group as: MH susceptible (n=22), MH negative (n=18) or MH equivocal (n=14). All individuals were screened for the presence of the most common mutations in the RYR1 gene. [Ca2+]i was measured by fluorescent digital microscopy using fura-2/AM in 10 cells from each patient at five different halothane concentrations.
Results. The halothane-induced increase in [Ca2+]i differed significantly between the three diagnostic groups. Different mutations of the RYR1 gene did not have a specific impact on halothane-induced increases in [Ca2+]i.
Conclusions. Measurements of [Ca2+]i in human skeletal muscle cells can be used to phenotype MH susceptibility; however, we did not observe a specific effect of any mutation in the RYR1 gene on the halothane-induced increase in [Ca2+]i.
Br J Anaesth 2002; 89: 5719
Keywords: anaesthesia; anaesthetics volatile, halothane; ions, calcium; muscle, skeletal, myoblasts
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In their daily lives MH-predisposition does not pose a threat to MH-susceptible (MHS) individuals, so the major goal of MH diagnostics is to identify susceptible individuals before the administration of trigger agents.7 8 To date the gold standard of MH diagnosis is the in vitro contracture test (IVCT). This invasive procedure involves an open muscle biopsy and in vitro challenge of muscle strips with halothane or caffeine. According to the guidelines of the European MH Group, patients are then diagnosed as MHS or MH negative (MHN) on the basis of contractile threshold and sensitivity of the muscle bundles to halothane and caffeine. If contracture is achieved only with either caffeine or halothane, the patient is diagnosed as MH equivocal (MHE).6 9
The underlying cause of MH is an abnormality in skeletal muscle calcium metabolism.10 11 Therefore, alterations in proteins involved in the regulation of the intracellular calcium concentration ([Ca2+]i), such as the calcium pump, the calcium release channel or other proteins implicated in excitationcontraction coupling, could potentially cause MH. Studies into the molecular mechanisms underlying this disease have demonstrated that the ryanodine receptor (RYR1) gene on human chromosome 19q is the primary locus of MH.2 7 12Mutation screening has identified more than 30 mutations in the RYR1 gene so far. Although approximately 50% of MH families have mutations in the RYR1 gene, linkage studies have revealed that this is a heterogenetic disease.7 1216
The aim of the present study was to determine if halothane-induced increases in [Ca2+]i in human skeletal muscle cells can be used to phenotype MH susceptibility and if different mutations in the RYR1 gene have a distinct effect on the halothane-induced increases in [Ca2+]i.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In order to determine if different mutations have a different effect on halothane-induced increases in [Ca2+]i, all patients were screened for nine of the most common mutations in the RYR1 gene (described below) and the increases in [Ca2+]i in subjects carrying different mutations, as well as in subjects carrying the same mutation in the RYR1 gene, were analysed and compared.
Patient selection
We selected 54 patients from 26 families with a positive history of MH without clinical signs of neuromuscular disease. The age range of the patients was 859 yr. Twenty-two patients were diagnosed as MHS, 18 as MHN and 14 as MHE by IVCT. Patients were classified as MHS if a contracture force 0.2 g was elicited by at least 2% halothane and 2 mM caffeine, as MHE if a contracture force
0.2 g was elicited only by either caffeine or halothane, and MHN if contractures
0.2 g were not reached with either trigger agent. The characteristics of the individuals involved in this study, including contractures obtained from the muscle strips during the IVCT, family of origin and mutation found are given in Table 1.
|
Materials
Dulbecco modified Eagle (DME) medium containing 4.5 mg ml1 glucose, fetal calf serum (FCS), horse serum, penicillin G and streptomycin was purchased from Life Technologies Ltd, Paisley, UK. Insulin was purchased from Eli Lilly and Co., Indianapolis, IN, USA. Cell culture material was from Becton Dickinson GmbH, Heidelberg, Germany. Halothane was from Halocarbon Labs, Inc., Hackensack, NJ, USA. The mammalian blood DNA isolation kit and Taq polymerase and DNA restriction enzymes were from Roche Molecular Biochemicals (Basel, Switzerland). The kit for DNA isolation from tissue was from Machery-Nagel GmbH, Düren, Germany. The polymerase chain reaction (PCR) purification kit was from Qiagen GmbH, Hilden, Germany. Fura-2/AM and ionomycin were from Sigma Chemical Co., St Louis, MO, USA. Primers were from Microsynth GmbH, Balgach, Switzerland. All other chemicals were reagent grade or of highest available grade.
Human skeletal muscle cell cultures
Primary human muscle cell cultures were established from surplus fragments taken from biopsies of patients undergoing diagnostic IVCT as described previously.17 Cells were grown in DME, 10% horse serum, insulin 5 ng ml1, 2 mM glutamine, antibiotics and 7 mM HEPES, pH 7.4 (proliferative medium) under standard cell culture conditions. For cryopreservation, about 106 cells were resuspended in DME containing 40% FCS and 10% DMSO and were stored in liquid nitrogen.
[Ca2+]i measurements
For measurements of [Ca2+]i, cells were trypsinized and transferred from tissue culture flasks to glass coverslips and allowed to grow in proliferative medium until groups of cells were visible. We have previously demonstrated that under these culture conditions the cells acquire skeletal-muscle-specific proteins such as sarcomeric -actinin and type-1 RYR,17 although they do not fully differentiate into multinuclear myotubes. Such primary cultures exhibit a degree of variability in the maturity of individual cells.
Cells to be tested were loaded with the fluorescent calcium indicator fura-2/AM. Single-cell calcium measurements using fluorescence microscopy were performed on 10 cells before and after the addition of halothane. A new coverslip containing fura-2/AM-loaded cells was used for each halothane concentration. For each experiment a total of 72 digital images were recorded at excitations of 340 and 380 nm and the ratio calculated, as per our previous study.17 The values obtained during the first 10 cells (i.e. before the application of halothane) were used to calculate the resting [Ca2+]i. Values were then taken during halothane perfusion as well as after addition of EGTA/ionomycin and calcium ions to obtain Rmin (the value obtained in the presence of EGTA) and Rmax (the value obtained in the presence of high calcium after the addition of ionomycin). Each run lasted approximately 6 min, including the time taken to run the calibration at the end of the experiment. Halothane in DMSO was administered at the indicated concentrations from a gas-tight vial by means of a roller pump. Its actual concentration was verified by gas chromatography in several test runs using fixed flow rates, temperature and tubing. Calibration was performed using the EGTA/ionomycin/manganese chloride method.18 The changes in fluorescence were converted into [Ca2+]i using the formula [Ca2+]i=[kDx(RRmin)/(RmaxR)] xSf2/Sb2, where kD (dissociation constant) of fura-2/AM was assumed to be 225 nM and Sf2 and Sb2 are the fluorescent values for Ca2+ free (f) and bound (b) of the indicator. These values are a constant.
The ratios were analysed and converted into [Ca2+]i values (nM) using a programmable database application (Omnis 7/3 from Blyth Holding Inc., Suffolk, UK). The increase in [Ca2+]i was determined by the difference between resting [Ca2+]i and peak [Ca2+]i after the addition of halothane. The whole transient elicited by a given halothane concentration was used to calculate the integral calcium, which reflects the total amount of calcium released.
Mutation screening
Screening for the presence of nine of the most frequent MH-linked mutations (Arg163Cys, Gly341Arg, Arg614Cys, Arg614Leu, Arg2163Cys, Val2168Met, Gly2434Arg, Arg2458Cys, Arg2458His) was performed by genomic DNA PCR amplification followed by restriction enzyme digestion and polyacrylamide gel electrophoresis. Total genomic DNA was isolated from either peripheral blood or muscle fragments not used for IVCT. PCR conditions and primer sequences were as described previously.19
Statistical analysis
The [Ca2+]i measurements from the three diagnostic groups were compared using repeated measurements ANOVA. Within each halothane concentration, the results from the three diagnostic groups were compared using Fisherss protected least significant difference (PLSD) post-hoc test.
To determine normal [Ca2+]i values, all measurements of the MHN cells were pooled and median values, as well as 75th, 90th, 95th, 97th and 99th percentiles, were calculated for each halothane concentration. These percentile values were used to define different cut-off values. Median values were calculated for each individual at the five halothane concentrations and compared with the cut-off values defined by the MHN population. If the cut-off value was exceeded at any concentration of halothane, the patient was classified as MHS.
StatView (SAS Institute Inc., Cary, NC, USA) was used for statistical analysis.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In seven of 22 individuals the IVCT diagnosis of MHS could not be confirmed by halothane-induced increases in [Ca2+]i. This could be due to either a reduced specificity of the IVCT (not every MHS diagnosis can be confirmed by molecular genetic analysis), or to an overestimation of the halothane-induced increases in [Ca2+]i of the true normal populationin the present study normal [Ca2+]i values were obtained from individuals from MHS families, not from non-MH-linked individuals.
In this study we confirm and extend our previous results. In fact, our earlier work was based on observations carried out on two MHS subjects and three MHN subjects while in this study we also included 14 MHE individuals, and the number of patients involved was 10 times larger. In the study by Censier and colleagues,17 we observed that the maximum difference between MHS and MHN, as far as halothane-induced increases in [Ca2+]i are concerned, occurred at halothane concentrations of 5.79.5 mM. In addition, we confirm that the mean increase in [Ca2+]i of cells from MHN individuals occurring at 8.6 mM halothane is similar to that of cells from MHS individuals at 5.7 mM halothane (203 nM vs 167 nM, respectively; P=0.269, students t-test for unpaired samples).
The response of cells presented in our previous report17 seem to be different from those found in this study. In the present study we examined a larger sample than previously and we believe that the values obtained are more likely to represent the MHN population. We would like to point out that it is very important to perform [Ca2+]i measurements at several halothane concentrations and we suggest concentrations ranging around 5.7 mM since this is the concentration where maximal differences can be observed.
In this study we used small fragments of tissue (24 mm3) left over from muscle biopsies. We have already assessed the feasibility of obtaining primary cultures from needle biopsies (unpublished observations). This approach offers obvious physical advantages for the patients, as well as being cheaper. Obviously there are some limitations in the use of this novel approach: (i) the need for cell culture and fluorescent calcium imaging facilities, (ii) the fact that cells cultured from the biopsies give rise to heterogeneous cell populations that do not respond to halothane in a uniform way, (iii) not all individuals diagnosed as MHS by IVCT had an halothane-induced increase in [Ca2+]i beyond that of the MHN population.
As to the future of MH diagnostics, one can envisage that individuals from families with defined mutations could first undergo genetic testing.22 If no mutation is found, they could undergo a skeletal muscle needle biopsy followed by measurements of halothane-induced increases in [Ca2+]i. If measurements of [Ca2+]i do not reveal MH susceptibility, then an IVCT could be performed. With this less invasive and stepwise approach, the high sensitivity of the IVCT and thus the high safety level of MH diagnostics is maintained. In fact if these criteria were used, only three of the 22 MHS individuals and seven of the 14 MHE individuals would still require the invasive IVCT. For the time being, however, all the MHN individuals would still require IVCT testing.
The common final pathway for MH is the loss of the fine regulation of calcium homeostasis in muscle cells. The present investigation is not only potentially important from a clinical point of view, but also from a biological one. In fact, it indicates that although a variety of factors such as metabolic processes, muscle training, protein composition and enzyme activation may influence the IVCT (at least as far as contracture force is concerned), there is an intrinsic defect in the machinery involved in [Ca2+]i homeostasis in MHS individuals. Each individual may compensate for the molecular defect in a variety of ways, which may be influenced by the specific mutation present as well as other genetic and environmental factors. This hypothesis is supported by the fact that the halothane-induced changes in [Ca2+]i in cells from MHS individuals bearing the same point mutation but with a different genetic background do not coincide, and because MHS individuals with RYR1 mutations do not undergo MHS reactions at every contact with trigger agents.4 5 Our results also show that the penetrance of a given mutation varies between individuals. This observation would be expected in view of the fact that the RYR must assemble as a tetramer in order to function as a calcium channel. The results of this study, as well as data from IVCT, tend to favour the hypothesis that mutated channels are probably randomly distributed, giving rise to heterogeneous responses, but the presence of a single mutated channel is sufficient to confer the MHS phenotype. This hypothesis is supported by our previous work,17 in which the normal phenotype was not reconstituted by transfecting MHS cells with wild-type channels.
In conclusion, measurements of [Ca2+]i may be useful in increasing the accuracy of MH phenotyping and may therefore be used as a complementary method for the diagnosis of MH susceptibility.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 Denborough M. Malignant hyperthermia. Lancet 1998; 352: 11316[ISI][Medline]
3 Gronert G, Antonigni J, Pessah I. Malignant hyperthermia. In: Miller R, ed. Anesthesia. New York: Churchill Livingstone, 2000; 103352
4 Bendixen D, Skovgaard LT, Ording H. Analysis of anaesthesia in patients suspected to be susceptible to malignant hyperthermia before diagnostic in vitro contracture test. Acta Anaesthesiol Scand 1997; 41: 4804[ISI][Medline]
5 Puschel K, Schubert-Thiele I, Hirth L, Benkmann HG, Brinkmann B. Malignant hyperthermia during the 13th general anaesthesia. Anaesthesist 1978; 27: 48891[ISI][Medline]
6 Larach MG, Localio AR, Allen GC, et al. A clinical grading scale to predict malignant hyperthermia susceptibility. Anesthesiology 1994; 80: 7719[ISI][Medline]
7 MacLennan DH, Phillips MS. Malignant hyperthermia. Science 1992; 256: 78994[ISI][Medline]
8 Ording H. Diagnosis of susceptibility to malignant hyperthermia in man. Br J Anaesth 1988; 60: 287302[ISI][Medline]
9 European Malignant Hyperpyrexia Group. A protocol for the investigation of malignant hyperpyrexia (MH) susceptibility. Br J Anaesth 1984; 56: 12679[Abstract]
10 Iaizzo PA, Klein W, Lehmann-Horn F. Fura-2 detected myoplasmic calcium and its correlation with contracture force in skeletal muscle from normal and malignant hyperthermia susceptible pigs. Pflugers Arch 1988; 411: 64853[ISI][Medline]
11 Mickelson JR, Gallant EM, Litterer LA, et al. Abnormal sarcoplasmic reticulum ryanodine receptor in malignant hyperthermia. J Biol Chem 1988; 263: 93105
12 McCarthy TV, Healy JM, Heffron JJ, et al. Localization of the malignant hyperthermia susceptibility locus to human chromosome 19q1213.2. Nature 1990; 343: 5624[ISI][Medline]
13 Fujii J, Otsu K, Zorzato F, et al. Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia. Science 1991; 253: 44851[ISI][Medline]
14 Manning BM, Quane KA, Ording H, et al. Identification of novel mutations in the ryanodine-receptor gene (ryr1) in malignant hyperthermia: Genotype-phenotype correlation. Am J Hum Genet 1998; 62: 599609[ISI][Medline]
15 Robinson RL, Monnier N, Wolz W, et al. A genome wide search for susceptibility loci in three european malignant hyperthermia pedigrees. Hum Mol Genet 1997; 6: 95361
16 Jurkat-Rott K, McCarthy T, Lehmann-Horn F. Genetics and pathogenesis of malignant hyperthermia. Muscle Nerve 2000; 23: 417[ISI][Medline]
17 Censier K, Urwyler A, Zorzato F, Treves S. Intracellular calcium homeostasis in human primary muscle cells from malignant hyperthermia-susceptible and normal individuals. Effect of overexpression of recombinant wild-type and arg163cys mutated ryanodine receptors. J Clin Invest 1998; 101: 123342
18 Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 344050[Abstract]
19 Girard T, Urwyler A, Censier K, et al. Genotype-phenotype comparison of the swiss malignant hyperhermia population. Hum Mutat 2001; 18: 3578
20 Ording H, Brancadoro V, Cozzolino S, et al. In vitro contracture test for diagnosis of malignant hyperthermia following the protocol of the European MH Group: Results of testing patients surviving fulminant mh and unrelated low-risk subjects. The european malignant hyperthermia group. Acta Anaesthesiol Scand 1997; 41: 95566[ISI][Medline]
21 Tong J, Oyamada H, Demaurex N, et al. Caffeine and halothane sensitivity of intracellular Ca2+ release is altered by 15 calcium release channel (ryanodine receptor) mutations associated with malignant hyperthermia and/or central core disease. J Biol Chem 1997; 272: 263329
22 Urwyler A, Deufel T, McCarthy T, West S. Guidelines for molecular genetic detection of susceptibility to malignant hyperthermia. Br J Anaesth 2001; 86: 2837