Anesth Analg 2002;94:1028-1033
© 2002 International Anesthesia Research Society
GENERAL ARTICLES
The Differential Effect of Halothane and 1,2-Dichlorohexafluorocyclobutane on In VitroMuscle Contractures of Patients Susceptible to Malignant Hyperthermia
Christoph H. Kindler, MD*,
Thierry Girard, MD*,
Diane Gong, BS , and
Albert Urwyler, MD*
*Departments of Anesthesia and Research, University of Basel, Kantonsspital, Basel, Switzerland; and Department of Anesthesia and Perioperative Care, University of California, San Francisco, California
Address correspondence and reprint requests to Christoph H. Kindler, MD, Department of Anesthesia, University of Basel, Kantonsspital, CH-4031 Basel, Switzerland. Address e-mail to ckindler{at}uhbs.ch
 |
Abstract
|
|---|
Malignant hyperthermia (MH) is an autosomal dominant, potentially fatal pharmacogenetic disorder of skeletal muscle. Approximately half of all known MH families show a linkage to the ryanodine receptor type 1 (RY1) gene. Although our knowledge of the diagnosis, genetics, and therapy of MH has improved, the exact pathogenesis and the role of volatile anesthetics as trigger substances for an MH crisis remain unknown. Compounds that do not obey the Meyer-Overton hypothesis (i.e., nonimmobilizers) are today an important part of research on anesthetic mechanisms. We designed this study to test the hypothesis that the nonimmobilizer 1,2-dichlorohexafluorocyclobutane (2N) compared with halothane has different effects on in vitro muscle contractures of muscle bundles from MH-susceptible (MHS) individuals. In vitro muscle contracture tests were performed with either halothane ( 660 µM, equivalent to 4 minimum alveolar anesthetic concentration [MAC]) or 2N (100 µM, equivalent to 5 times predicted MAC). MAC is defined as the anesthetic concentration that prevents nocifensive movements after a surgical stimulus in 50% of subjects. In contrast to halothane, 2N caused only minimal muscle contractures in muscle bundles from six MHS patients (0.13 g [0.04–0.31 g] vs 1.95 g [1.60–4.70 g], median values and ranges; P = 0.004). Halothane and 2N differ in their effects on muscle contractures of MHS individuals, possibly because of a differing action on MH RY1.
IMPLICATIONS: Using in vitro contracture tests, we showed that halothane and the nonimmobilizer 1,2-dichlorohexafluorocyclobutane differ in their effects on contractures of muscle bundles from individuals susceptible to malignant hyperthermia (MH) as a result of their differing action on MH ryanodine receptors. These findings render this receptor a possible molecular target for volatile anesthetic action.
|
Introduction
|
|---|
Malignant hyperthermia (MH), an autosomal dominant disorder of skeletal muscle, is a potentially life-threatening event in response to volatile anesthetics. In genetically predisposed individuals, volatile anesthetics and depolarizing neuromuscular blocking drugs lead to a hypermetabolic state characterized by an abnormally large release of calcium from the sarcoplasmic reticulum (1). Advances in molecular genetic techniques and heterologous expression systems have increased our understanding of the molecular etiology of MH. Early genetic linkage studies of MH families pointed to a locus on chromosome 19 (19q12.1–13.2) coding for the skeletal muscle sarcoplasmic reticulum calcium release channel, also named the ryanodine receptor type 1 (RY1) (2). Approximately half of all known MH families show linkage to this RY1 gene, and more than 23 mutations have been described (3). The complexity and heterogeneity of the MH disorder make a simple noninvasive DNA test for susceptibility to MH unrealistic, although recently published guidelines allow for molecular DNA genetic detection of MH susceptibility in a limited subset of individuals (4). However, for most patients the in vitro contracture test (IVCT) used for the last 30 yr remains the primary diagnostic tool to assess MH status. Both the North American MH Group (NAMHG) (5) and the European MH Group (EMHG) (6) have developed standardized protocols for the IVCT to measure muscle contractures upon exposure to given concentrations of halothane and caffeine. As a result of our increased knowledge of MH, increased presymptomatic diagnosis, and the availability of a specific treatment with dantrolene, the fatality rate from MH has decreased from 70% in the 1970s to 5% today (7).
However, the exact pathogenesis of MH other than that of a malfunction of RY1 is unknown. Particularly, the role of volatile anesthetics, which are the main trigger substances of an MH crisis, remains elusive. Early work showed no effect of halothane on [3H]ryanodine binding to the skeletal RY1 protein (8). Other reports suggested that halothane changes the process of activation/inactivation and increases conductance of RY1 (9) or that halothane triggers abnormal calcium homeostasis in MH via oligomerization of MH RY1 (10).
Over the past few years there has been increasing evidence that volatile anesthetics exert their effects by binding specifically to particular protein targets rather than by disrupting lipid membranes. Accumulated evidence for a direct interaction of volatile anesthetics with the skeletal RY1 protein (11) suggests that elucidation of the exact pathogenesis of MH might also contribute to our understanding of molecular mechanisms of inhaled anesthetics in addition to increasing our knowledge about MH.
Compounds that do not obey the Meyer-Overton hypothesis (i.e., that hydrophobicity strongly correlates with the clinical potency of volatile anesthetics) are today an integral part of research on anesthetic mechanisms. These new compounds are called nonimmobilizers because they do not cause immobility in response to noxious stimuli, even at partial pressures several-fold higher than their predicted (according to the Meyer-Overton hypothesis) minimum alveolar anesthetic concentration (MAC), which is defined as the anesthetic concentration that prevents nocifensive movements after a surgical stimulus in 50% of subjects (12). There are no data available on the effect of nonimmobilizers on the contractile behavior of muscles from MH-susceptible (MHS) patients. This study was therefore designed to test the hypothesis that the nonimmobilizer 1,2-dichlorohexafluorocyclobutane (2N) differs from halothane in its effects on in vitro muscle contractures of MHS patients. To establish the receptor protein possibly responsible for a difference in muscle contractures, all individuals were screened for published MH mutations in the RY1 gene by molecular genetic testing.
|
Methods
|
|---|
This study was conducted with the approval of the institutional committee for human investigation at the University of Basel, and informed consent was obtained from all tested individuals. MH screening with IVCT according to the protocols of the NAMHG (5) and EMHG (6) has been performed in the laboratory of the Departments of Anesthesia and Research in Basel, Switzerland, since 1986 and has previously been described in detail elsewhere (13). Briefly, muscle strips obtained from open biopsies of quadriceps muscle with a length of approximately 2–3 cm and a diameter of approximately 3 mm are mounted in a test bath. The test bath is continuously perfused with Krebs-Ringer solution and bubbled with 5% CO2 in oxygen. All experiments are performed at 37°C. According to the test protocols, muscle specimens are separately challenged with incremental concentrations of halothane (110, 220, 440, and 660 µM; EMHG) or exposed to a given concentration of halothane (660 µM; NAMHG), and challenged with increasing concentrations of caffeine (up to 32 mM for both EMHG and NAMHG). For the EMHG protocol, an increase in contracture of 0.2 g at concentrations of up to 440 µM halothane or 2 mM caffeine is considered a positive, i.e., pathological, test result. Three categories can result from the IVCT of a given patient: 1) pathological contractures under the thresholds of both test substances, halothane and caffeine, are considered MHS; 2) normal reactions to both drugs are considered MH negative (MHN); and 3) one pathological and one normal result are classified as MH equivocal. For clinical purposes, MH-equivocal patients are considered as MHS.
After the scheduled IVCT of an individual, muscle strips not used for clinical testing were exposed to the nonimmobilizer 2N (>97% purity; Lancaster Synthesis GmbH, Mühlheim, Germany). For the 2N experiments, an identical setup as for the clinical IVCT was used, with the exception of using two enfluratec vaporizers (Cyprane LTD, Keighley, England) in tandem position filled with 2N, both set at the maximal output of 5% to ensure adequate 2N concentrations in the test bath (2N has a low solubility, with a saline/gas partition coefficient of 0.0119). Also because of the low solubility we chose a protocol for the 2N experiments similar to the NAMHG IVCT protocol for halothane, with exposing the muscle strips to a given 2N concentration for 10 min. Serial solution samples from the recording chamber were drawn into gas-tight glass syringes (B-D Yale; Becton Dickinson, Franklin Lakes, NJ) and analyzed by gas chromatography (Gow-Mac Instruments Co., Bridgewater, NJ) by use of standard methods. Reported concentrations are the averages (mean ± SEM) of all analyzed samples at a given time point. Duplicate measurements were made for each experiment.
First the EMHG IVCT protocol was used to determine the MH status of the subject. To compare the effects of halothane and 2N on muscle bundles from MHS and MHN individuals, muscle strips from the same individual were exposed for 10 min to 660 µM (4 MAC) halothane (according to the NAMHG protocol) and to 100 µM (5 times the predicted MAC) 2N. For all experiments, new and viable muscle bundles were used, and they were exposed to only one experimental treatment. The maximal muscle contractures were compared between groups by use of the nonparametric two-tailed Mann-Whitney U-test. Statistical significance was defined as P < 0.05.
In addition, all individuals were screened for known MH mutations by use of polymerase chain reaction amplification of genomic DNA and restriction enzyme digestion following published conditions (14). DNA was extracted from whole blood and quantified by spectrophotometric measurements. Approximately 100 ng of DNA was used per polymerase chain reaction amplification with a PerkinElmer GeneAmp 2400 thermocycler (PerkinElmer Inc., Norwalk, CT). After an initial denaturation for 4 min at 95°C, 35 cycles with 30 s annealing at the specific temperature, 45 s extension at 72°C, and 30 s denaturing at 92°C, followed by a final extension of 3 min at 72°C, were performed.
|
Results
|
|---|
We exposed muscle strips from six MHS and four MHN individuals to halothane and the nonimmobilizer 2N. In clear contrast to halothane, 2N caused only minimal muscle contracture of muscle bundles from MHS patients (Fig. 1). For halothane, a maximal contracture amplitude of 1.95 g (1.60–4.70 g) was measured, compared with 0.13 g (0.04–0.31 g) for 2N (median values and ranges; P = 0.004). Similar to halothane, 2N showed only little effect on the baseline muscle tension of muscle strips from MHN patients (Fig. 2, Table 1). As expected, halothane produced significantly greater muscle contractures in muscle bundles from MHS patients compared with MHN patients (P = 0.01). It is interesting to note that the muscle contractures evoked by 2N were also greater in MHS patients (although much smaller compared with those produced by halothane) than in MHN patients (P = 0.01) (Table 1). Muscle lengths, muscle wet weights, and preexposure twitch heights are summarized in Table 1. Two individuals who were MH mutation carriers were identified in the MHS group: one individual was heterozygous for the R614C and one for the V2168M mutation. Figure 3 shows an electrophoresis polyacrylamide gel with the identification of these two mutations by restriction enzyme digestion.
View larger version (110K):
[in this window]
[in a new window]
|
Figure 1. In vitro muscle contractures of muscle bundles from a malignant hyperthermia-susceptible individual with the R614C mutation in the ryanodine receptor type 1 gene exposed to 10 min of halothane or 1,2-dichlorohexafluorocyclobutane (horizontal arrows). Note that traces run from right to left.
|
|
View larger version (82K):
[in this window]
[in a new window]
|
Figure 2. In vitro muscle contractures of muscle bundles from a malignant hyperthermia-negative individual exposed to 10 min of halothane or 1,2-dichlorohexafluorocyclobutane (horizontal arrows). Note that traces run from right to left.
|
|
View larger version (14K):
[in this window]
[in a new window]
|
Figure 3. Polymerase chain reaction (PCR) amplification of genomic DNA and detection of mutations R614C and V2168M in two malignant hyperthermia-susceptible (MHS) individuals: 6% polyacrylamide gel of PCR-amplified genomic DNA encompassing the R614C mutation (A) and the V2168M mutation (B). (A) PCR amplification of a 922-bp fragment (Lane 1). The presence of the R614C mutation abolishes a restriction site for the enzyme RsaI, resulting in an extra band of 729 bp (Lane 3; *) in the MHS individual which is not present in a control individual (Lane 2). (B) PCR amplification of a 345-bp fragment (Lane 4). The presence of the V2168M mutation creates an additional restriction site for the enzyme MslI, resulting in two extra bands of 201 and 85 bp (Lane 6; *) in the MHS individual which are not present in a control individual (Lane 5). The polyacrylamide gel shown in Panel A was left to run longer to increase the resolution for larger PCR fragments.
|
|
Halothane concentrations are routinely determined for clinical MH testing. Serial measurements of 2N concentration by gas chromatography showed a >50% decrease in concentration in the test bath during the experiments. Within the first 3 min, we were able to achieve 96.5 ± 10.1 µM 2N (mean ± SEM), corresponding to predicted MAC values for 2N of 4.9 ± 0.5, followed by an increasing loss of concentration over time. However, at the end of the 10-min exposure, we still measured 37.8 ± 2.8 µM 2N, corresponding to predicted MAC values for 2N of 1.9 ± 0.1.
|
Discussion
|
|---|
The results of this study show that halothane and the nonimmobilizer 2N differ in their effects on the contractile behavior of muscle bundles from MHS patients, possibly because of a differing action on MH RY1. Nonimmobilizers administered at partial pressures several-fold higher than those predicted to produce anesthesia by the Meyer-Overton hypothesis are important compounds in differentiating molecular anesthetic target sites (12). Physiological or biophysical changes produced by conventional anesthetics in in vitro systems relevant for anesthetic mechanisms should not be produced by nonimmobilizers. This prediction is valid for  -aminobutyric acid (15), glycine (16), glutamate (17), and neuronal nicotinic acetylcholine receptors (18) and for the volatile anesthetic-induced increase of intracellular calcium in neurons (19), neuronal voltage-gated sodium channels (20), and thermoregulation in rats (21).
Conventional volatile anesthetics are MH-triggering substances leading to an increase in the concentration of free myoplasmic calcium that is released from the sarcoplasmic reticulum calcium stores by MH RY1. However, the exact molecular interaction between anesthetics and the RY1 protein remains to be investigated, and, particularly, the cause for the increased calcium efflux through MH RY1 is unknown. Using IVCT we have demonstrated a clear difference between the effects of the anesthetic halothane and the nonimmobilizer 2N with respect to their action on muscle bundles from MHS individuals. The MH mutated RY1 might be sensitive to conventional anesthetics but not to the nonimmobilizer 2N.
In addition to increasing our understanding of RY1 function, the results of this study also have another potential implication. Volatile anesthetics increase intracellular calcium concentration not only in skeletal muscle cells (22), but also in cortical neurons (19). Because nonimmobilizers can pass the blood-brain barrier (23) and all three ryanodine receptor isoforms, including RY1, are expressed in the central nervous system (24), it is intriguing to speculate that the differential effect of conventional anesthetics and 2N on neuronal intracellular calcium concentration may be the result of their differing interaction with ryanodine receptor isoforms in the brain (19). This finding could lead to a more focused exploration of the physiological and pathological function of the ryanodine receptor genes and their implication for anesthetic mechanisms.
In interpreting the results of this study, some limitations should be considered. First, the number of subjects included is relatively small. However, ethical considerations prohibit receiving unnecessarily large invasive muscle biopsies. Because of the guidelines used in our laboratory, clinical testing of the MH status requires both the EMHG protocol for halothane and caffeine in duplicates followed by the NAMHG protocol for data comparison. Therefore, often there is not enough functional muscle left for further experimental testing with 2N. But despite the small number of tests, we are confident in the results, because all MHS muscle bundles, which clearly contracted to halothane, showed only minimal contracture to 2N. This is further reflected by the statistical significance of P = 0.004 with a conservative statistical test (i.e., the unpaired nonparametric two-tailed Mann-Whitney U-test). The variability in preexposure twitch heights (Table 1) represents normal skeletal muscle physiology and does not influence the results, because muscle contractures are independent of twitch height. The preexposure twitch heights are given in Table 1 to show viability of the muscle strips (5,6). Second, we observed a substantial loss of 2N concentration over time because we used an identical open chamber for the IVCT with halothane and 2N. The maximal muscle contracture in 660 µM halothane (4 MAC) was observed two to four minutes after the start of the anesthetic (Fig. 1). Time-series experiments showed that at these time points we were able to achieve 2N con-centrations approximately 5 times larger than the predicted MAC values, assuming sufficient concentrations to achieve muscle contracture. Although effective concentrations of halothane and 2N were not measured at the site of action within the muscle preparation (which is unknown), measured partial pressures of nonimmobilizers are the same as that within the putative site of action. Therefore, pharmacokinetics do not explain the failure of nonimmobilizers to have an effect (23). And third, we demonstrated a known MH mutation in the RY1 gene in only two of six subjects, leaving the theoretical possibility open that the four other tested MHS individuals in fact are linked to other MH loci on different chromosomes, such as the gene encoding the  1 subunit of the dihydropyridine receptor, an L-type calcium channel (25). However, at our institution we have implemented the recently published guidelines for molecular genetic detection of MH susceptibility (4). Relatives of an individual who is a known causative MH mutation carrier now undergo molecular DNA testing, and when they are found to carry the same causative mutation, they are considered MHS without a further IVCT. The implementation of these guidelines disproportionally increases the number of MHS individuals with no known RY1 mutation who will undergo IVCT. However, a recent linkage analysis addressed the issue of discordance in MH and suggested that recombinant families previously excluded from linkage to the RY1 gene may actually demonstrate linkage as the number of members tested within the pedigrees increases (26).
In summary, we observe that halothane and 2N have distinct effects on in vitro contractures of muscle bundles from MHS patients, possibly because of a differing action on MH RY1, which parallel those previously shown for the -aminobutyric acid, glycine, glutamate, and neuronal nicotinic acetylcholine receptors. These findings render RY1 an interesting molecular target for volatile anesthetic action. However, the complexity of the RY1 protein, with more than 5000 amino acids, will make the identification of the anesthetic binding sites or pockets difficult and time consuming.
|
Acknowledgments
|
|---|
Supported by a grant from the Swiss Society of Anesthesia and Resuscitation (CHK), National Institutes of Health Grant 1P01GM47818-07 (DG), and Swiss National Science Foundation Grant 32-63959.00 (AU).
The authors thank Dr. Edmond I Eger II for his generous help with the gas chromatographic analysis of 2N and for reviewing the manuscript. We also thank Dr. P. Reinhard and P. Hummel, Abbott AG, Baar, Switzerland, for providing the anesthesia circuit used for delivering 2N, and Joan Etlinger, for editorial assistance.
|
Footnotes
|
|---|
Presented in part at the Sixth International Conference on Molecular and Basic Mechanisms of Anesthesia MAC, Bonn, Germany, June 28–30, 2001.
|
References
|
|---|
-
Gronert GA, Antonigni JF, Pessah I. Malignant hyperthermia. In: Miller RD, ed. Anesthesia. New York: Churchill Livingstone, 2000: 1033–52.
-
McCarthy TV, Healy JM, Heffron JJ, et al. Localization of the malignant hyperthermia susceptibility locus to human chromosome 19q12–13.2. Nature 1990; 343: 562–4.[Medline]
-
McCarthy TV, Quane KA, Lynch PJ. Ryanodine receptor mutations in malignant hyperthermia and central core disease. Hum Mutat 2000; 15: 410–7.[Web of Science][Medline]
-
Urwyler A, Deufel T, McCarthy T, West S. Guidelines for molecular genetic detection of susceptibility to malignant hyperthermia. Br J Anaesth 2001; 86: 283–7.[Abstract/Free Full Text]
-
Larach MG. Standardization of the caffeine halothane muscle contracture test: North American Malignant Hyperthermia Group. Anesth Analg 1989; 69: 511–5.[Free Full Text]
-
A protocol for the investigation of malignant hyperpyrexia (MH) susceptibility: the European Malignant Hyperpyrexia Group. Br J Anaesth 1984; 56: 1267–9.[Abstract/Free Full Text]
-
Denborough M. Malignant hyperthermia. Lancet 1998; 352: 1131–6.[Web of Science][Medline]
-
Connelly TJ, Hayek RE, Rusy BF, Coronado R. Volatile anesthetics selectively alter [3H]ryanodine binding to skeletal and cardiac ryanodine receptors. Biochem Biophys Res Commun 1992; 186: 595–600.[Web of Science][Medline]
-
Nelson TE. Halothane effects on human malignant hyperthermia skeletal muscle single calcium-release channels in planar lipid bilayers. Anesthesiology 1992; 76: 588–95.[Web of Science][Medline]
-
Froemming GR, Dillane DJ, Ohlendieck K. Complex formation of skeletal muscle Ca2+-regulatory membrane proteins by halothane. Eur J Pharmacol 1999; 365: 91–102.[Web of Science][Medline]
-
O’Sullivan GH, McIntosh JM, Heffron JJ. Abnormal uptake and release of Ca2+ ions from human malignant hyperthermia-susceptible sarcoplasmic reticulum. Biochem Pharmacol 2001; 61: 1479–85.[Web of Science][Medline]
-
Koblin DD, Chortkoff BS, Laster MJ, et al. Polyhalogenated and perfluorinated compounds that disobey the Meyer-Overton hypothesis. Anesth Analg 1994; 79: 1043–8.[Abstract/Free Full Text]
-
Urwyler A, Censier K, Seeberger MD, et al. Diagnosis of susceptibility for malignant hyperthermia using in-vitro muscle contraction testing in Switzerland. Schweiz Med Wochenschr 1991; 121: 566–71.[Web of Science][Medline]
-
Girard T, Urwyler A, Censier K, et al. Genotype-phenotype comparison of the Swiss malignant hyperthermia population. Hum Mutat 2001; 18: 357–8.
-
Mihic SJ, McQuilkin SJ, Eger EI, et al. Potentiation of gamma-aminobutyric acid type A receptor-mediated chloride currents by novel halogenated compounds correlates with their abilities to induce general anesthesia. Mol Pharmacol 1994; 46: 851–7.[Abstract]
-
Mascia MP, Machu TK, Harris RA. Enhancement of homomeric glycine receptor function by long-chain alcohols and anesthetics. Br J Pharmacol 1996; 119: 1331–6.[Web of Science][Medline]
-
Liachenko S, Tang P, Somogyi GT, Xu Y. Comparison of anesthetic and non-anesthetic effects on depolarization-evoked glutamate and GABA release from mouse cerebrocortical slices. Br J Pharmacol 1998; 123: 1274–80.[Web of Science][Medline]
-
Cardoso RA, Yamakura T, Brozowski SJ, et al. Human neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes predict efficacy of halogenated compounds that disobey the Meyer-Overton rule. Anesthesiology 1999; 91: 1370–7.[Web of Science][Medline]
-
Kindler CH, Eilers H, Donohoe P, et al. Volatile anesthetics increase intracellular calcium in cerebrocortical and hippocampal neurons. Anesthesiology 1999; 90: 1137–45.[Web of Science][Medline]
-
Ratnakumari L, Vysotskaya TN, Duch DS, Hemmings HC. Differential effects of anesthetic and nonanesthetic cyclobutanes on neuronal voltage-gated sodium channels. Anesthesiology 2000; 92: 529–41.[Web of Science][Medline]
-
Maurer AJ, Sessler DI, Eger EI, Sonner JM. The nonimmobilizer 1,2-dichlorohexafluorocyclobutane does not affect thermoregulation in the rat. Anesth Analg 2000; 91: 1013–6.[Abstract/Free Full Text]
-
Klip A, Hill M, Ramlal T. Halothane increases cytosolic Ca2+ and inhibits Na+/H+ exchange in L6 muscle cells. J Pharmacol Exp Ther 1990; 254: 552–9.[Abstract/Free Full Text]
-
Chortkoff BS, Laster MJ, Koblin DD, et al. Pharmacokinetics do not explain the absence of an anesthetic effect of perfluoropropane or perfluoropentane. Anesth Analg 1994; 79: 234–7.[Abstract/Free Full Text]
-
Martin C, Chapman KE, Seckl JR, Ashley RH. Partial cloning and differential expression of ryanodine receptor/calcium-release channel genes in human tissues including the hippocampus and cerebellum. Neuroscience 1998; 85: 205–16.[Web of Science][Medline]
-
Jurkat-Rott K, McCarthy T, Lehmann-Horn F. Genetics and pathogenesis of malignant hyperthermia. Muscle Nerve 2000; 23: 4–17.[Web of Science][Medline]
-
Brown RL, Pollock AN, Couchman KG, et al. A novel ryanodine receptor mutation and genotype-phenotype correlation in a large malignant hyperthermia New Zealand Maori pedigree. Hum Mol Genet 2000; 9: 1515–24.[Abstract/Free Full Text]
Accepted for publication November 16, 2001.
|
Top
Abstract
Introduction
