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Analysis of human cultured myotubes responses mediated by ryanodine receptor 1.

Malignant hyperthermia (MH) is a life-threatening pharmacogenetic disorder triggered by exposure to inhalation anaesthetics and/or depolarising muscle relaxants in genetically susceptible patients. MH is characterised by an increase in the intracellular calcium concentration ([[Ca.sup.2+]]) in skeletal muscle cells (1-4), and, in these cells, both the ryanodine receptor type 1 (RYR1) and the dihydropyridine receptor (DHPR) regulate [Ca.sup.2+] release from the sarcoplasmic reticulum (SR). RYR1 forms a [Ca.sup.2+] release channel in the SR membrane and DHPR is a voltage sensor and [Ca.sup.2+] channel in the plasmalemma. Mutations in RYR1 and the a1 subunit of DHPR (CACNA1S) are linked to MH, central core disease and multiminicore disease (1,2,5,6). RYR1 is among the largest genes in humans, containing 159,000 nucleotides including 106 exons. More than 200 sequence variants of RYR1 have been identified1. However, only 32 mutations have been functionally characterised with respect to affecting cellular [Ca.sup.2+] homeostasis1 and, accordingly, causative mutations in RYR1 have been identified in only 50% of patients with MH and/or central core disease, and their families (7-10). Thus, until further studies identify the functional significance of more RYR1 polymorphisms, functional tests of [Ca.sup.2+] homeostasis are needed to diagnose and/or identify patients susceptible to MH.

In Europe and North America, susceptibility to MH is traditionally identified using the in vitro contracture test (IVCT), in which viable muscle strips are exposed to halothane and caffeine (11-13), but in Japan, the Ca-induced Ca release (CICR) test is performed on a small number of chemically skinned fibres (14-15). The CICR test can reveal abnormalities associated with calcium release from the SR, a process primarily controlled by the RYR1 channel in skeletal muscle (16). A large multi-protein complex containing the voltage sensing DHPR in the plasmalemma and RYR1 in the SR membrane are involved in Ca release and influx1, but because the plasmalemma is functionally destroyed in skinned muscle fibres, the CICR test may not detect abnormalities of [Ca.sup.2+] homeostasis associated with the plasmalemma, including those of DHPR. In the present study, we used intact muscle cells to assess [Ca.sup.2+] homeostasis in muscle cells with an emphasis on a complement to CICR test.

We used cultured myotubes derived from surplus muscle pieces and examined changes in myoplasmic [[Ca.sup.2+]] at the single cell level following exposure to the RYR1 activators caffeine, halothane and 4-CmC in conjunction with traditional CICR testing. Based on CICR results, samples were placed in the accelerated CICR rate group (accelerated group) or non-accelerated CICR rate group (non-accelerated group), and the dose-response curves to three RYR1 activators were compared. Finally, we evaluated the validity of the calculated the half maximal effective concentrations ([EC.sub.50]) for these RYR1 activators and determined the cut-off point for the [EC.sub.50] for identifying patients susceptible to the development of MH.



This study was approved by the ethics committee of Hiroshima University. Muscle specimens (0.1 to 0.9 g) obtained from patients who underwent muscle biopsy to diagnose a predisposition to MH were cultured after obtaining written informed consent from patients and their next of kin to use waste material from their muscle specimens. Muscle biopsies from the quadriceps or biceps brachii were performed at different hospitals and sent to our institution within 24 hours, immersed in a relaxing solution at 4[degrees]C, by an express delivery service. The relaxing solution contained [Mg.sup.2+] 1.5 mM, MgATP 3.5 mM, [Ca.sup.2+] 0 M, EGTA 2 mM, PIPES 20 mM, EGTA; ethylene glycol-bis (P-amino ether)-N,N,N',N',-tetra acetic acid, PIPES; piperazine-N,N'-bis (2-ethanesulfonic acid). CICR testing was performed within 48 hours after muscle biopsy, and patients were classified into the accelerated and non-accelerated groups based on the pattern of CICR. Myotubes were cultured from the surplus muscle pieces remaining after CICR testing, and calcium imaging experiments were performed using these myotubes.


Thirty-four patients were enrolled in this study (Table 1), including 17 individuals described in a previous study (17,18). Four patients (numbers 5, 6, 15 and 16) experienced MH events diagnosed as rank 5 or 6 according to clinical grading scale19 during general anaesthesia. Six patients experienced an MH-like episode, but the presence of fever or rhabdomyolysis after anaesthesia was unknown. Twenty patients had a family history of MH and one patient had a family history of exertional heat stroke. This patient's son had died at age 16 while running. The father of patients numbers 6 and 28 had a possible episode of MH. Patients numbers 5 and 30 were brothers. Only three patients (numbers 1, 2 and 18) had undergone RYR1 gene analysis in a previous study20,21. The p.L4838V mutation was identified in patient number 1 and the p.R2508C mutation was identified in patient number 2. The son of patient number 1 had a suspected MH event, and the same mutation was identified in RYR1. His mother, patient number 18, had no identifiable mutations in RYR1 (20). The clinical presentation of patient number 2, suspected myopathy, had been reported previously (21).

Calcium-induced calcium release rate test

The CICR rate was measured according to Endo's method (15-18). In brief, bundles of skeletal muscle fibres 5 to 6 mm in width and 10 to 15 mm in length were dissected into smaller pieces, and these pieces were isolated in the relaxing solution under a stereomicroscope. Fibres were chemically skinned with saponin (50 [micro]g/ml) for 30 minutes in the relaxing solution, and the membrane of skinned skeletal muscle fibres was chemically destroyed. The CICR rates were assessed by measuring induced calcium release from the SR at five different calcium concentrations (16,18).

The CICR rate was defined as 'accelerated' if the CICR values were more than two standard deviations above the mean of the control patients at two or more calcium concentrations. Control CICR values were derived from 12 patients with negative IVCT according to both the protocols of the European MH Group and the North American MH Group (14,18).

Cell cultures

After measuring the CICR rate, surplus muscle pieces were stripped of visible connective tissue, dissected into smaller pieces (2 to 4 mm in diameter) and placed in uncoated 25 [cm.sup.2] culture flasks (Corning, USA) in Dulbecco's modified Eagle medium and F12 medium (DMEM/F12; Invitrogen, USA) supplemented with 10% heat-inactivated bovine calf serum (FBS; Sigma-Aldrich, USA), 1% amipicillin sodium salt, kanamycin sulphate (Sigma, USA) and 2.5 [micro]g/ml amphotericin B (Invitrogen, USA) in a 5% C[O.sub.2] atmosphere at 37[degrees]C for two to four weeks. The growth medium was changed every three days. When the cells were nearly confluent, cells were passaged and split using trypsinEDTA (Invitrogen, USA). Proliferating myoblasts were resuspended in growth medium and seeded onto 10 to 35 mm culture dishes with 10 mm glass-bottom micro-wells (MatTek, USA) for calcium imaging experiments. Differentiation of myoblasts was induced by replacing growth medium with DMEM/F12 containing 2% FBS, 1% amipicillin sodium salt, kanamycin sulphate and 2.5 [micro]g/ml amphotericin B, when 90% confluence was almost reached. Myoblasts fused and differentiated to form multinuclear myotubes during 10 to 14 days with incubation in DMEM/F12 supplemented with 2% FBS. We confirmed that cells cultured under these conditions acquire skeletal muscle-specific proteins such as sarcomeric [alpha]-actinin and RYR1 by indirect immunofluorescent staining (data not shown). Excess myoblasts were cryopreserved using FBS supplemented with 10% dimethyl sulphoxide (Sigma, USA) and stored in liquid nitrogen. If additional experiments were needed, cells were thawed and seeded onto 35 mm culture dishes.

Calcium imaging

Myotubes were identified morphologically, i.e. long and narrow multinuclear cells, and calcium imaging from a single myotube was performed using the [Ca.sup.2+]-sensitive fluorescent dye Fura-2 aceto-oxymethyl ester (Fura-2/AM, Dojindo, Japan) as previously described (17,18). Briefly, cells were loaded with 5 [micro]M Fura-2/AM in HEPES buffered saline (HBSS; 130.0 mM NaCl, 5.4 mM KCl, 20 mM HEPES, 2.5 mM Ca[Cl.sub.2], 1.0 mM Mg[Cl.sub.2] and 5.5 mM glucose at pH 7.4) for 60 minutes at 37[degrees]C. The myotubes were then washed with HBSS to eliminate excess Fura-2/AM and maintained at 37[degrees]C for 30 minutes. Cell culture dishes were continuously perfused with HBSS at a rate of 1.2 ml per minute in a perfusion chamber placed on the stage of an inverted microscope (TE2000U-EF-S, Nikon, Japan). All experiments were performed at 36 to 37[degrees]C with an in-line heating system monitoring the temperature of the solution on the coverslip. The field of interest contained three to 10 cells. Single cell fluorescence was imaged by alternate excitation at 340 and 380 nm, and measurement of emission at 510 nm. Image sequences (0.4 to 0.9 second exposure time, 2x2 binning) were acquired by an ORCAAG cooled digital charge coupled device camera (Hamamatsu Photonics, Hamamatsu, Japan) and imaged with AQUACOSMOS 2.0 (Hamamatsu Photonics). Emission ratios were sampled once every five seconds.

Relative changes in [[Ca.sup.2+]] were determined by calculating the ratio of the fluorescence intensity at 340 nm to that at 380 nm (340 nm/380 nm), and absolute [[Ca.sup.2+]] was calculated by comparing the measured 340 nm/380 nm ratio against a standard curve. The standard curve was generated using commercially available Fura-2 calcium imaging calibration kit (F-6774; Molecular Probes, Eugene, OR, USA). Resting [[Ca.sup.2+]] was defined as the average of six data points obtained over 30 seconds prior to the initiation of stimulus. Responses to stimuli were determined by comparing the difference between the peak 340 nm/380 nm ratio during exposure to drug and the resting [[Ca.sup.2+]].

Responses to caffeine, halothane and 4-CmC were assessed by monitoring for an increase in the 340/380 nm ratio at different doses of the indicated compounds. Caffeine was tested incrementally at 0.25, 0.5, 1.0, 2.5, 5.0, 10.0 and 20.0 mM, halothane was tested incrementally at 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 mM, and 4-CmC was tested incrementally at 3, 10, 30, 100, 300, 500 and 1000 [micro]M. Following each concentration of compound, cells were washed and the 340/380 nm ratios returned to baseline. Finally, the cell responses to 10.0 mM caffeine were measured to very consistent reactivity. The 340/380 nm ratios were normalised to the responses at 10 mM caffeine, 8 mM halothane and 1000 [micro]M 4-CmC. The dose response curves and the [EC.sub.50] for each RYR1 activator were obtained for each myotube by fitting a nonlinear regression with a variable slope sigmoid curve using Prism (GraphPad Software, La Jolla, USA). The [EC.sub.50] for the RYR1 activators were averaged in some myotubes.

Data analysis

Receiver operating characteristic (ROC) curve analyses were used to estimate cut-off points and the associated diagnostic sensitivities and specificities for the [EC.sub.50] of each myotube for the diagnosis of a predisposition to MH. The best statistical 'cut-off point' was determined by minimising the distance between the point with specificity=1 and sensitivity=1.

Statistical analysis was performed using t-test for unpaired values. Correlation among the [EC.sub.50] for the three RYR1 activators, and between CICR rate and [EC.sub.50] for RYR1 activators were assessed by linear regression analysis. P values of less than 0.05 were considered statistically significant. Statistical analyses, dose response curve and ROC curve analyses were conducted using Prism (GraphPad Software, La Jolla, USA). Values other than age are expressed as the mean [+ or -] SD. Age is presented as median, with range in brackets.


Patients and CICR rate

Samples from 34 patients (25 male, 9 female) were analysed for this study. Based on their CICR rate, 17 patients were classified into the accelerated and non-accelerated groups. The CICR values corresponding to five different [Ca.sup.2+] stimuli concentrations in the accelerated group were significantly higher compared to the values of the control and non-accelerated groups (Figure 1), but the non-accelerated and control groups did not significantly differ at any Ca2+ concentration (Figure 1). The biometric data, indications for CICR testing and results of CICR testing are presented in Table 1. The accelerated and nonaccelerated groups did not differ significantly with respect to age (34 [6 to 71] years versus 32 [2 to 74] years). The four patients with a documented episode of MH during anaesthesia were in the accelerated group, and no patients with an MH event were in the non-accelerated group.



Response to RYR1 activators

Two representative traces showing the 340/380 nm ratios in response to treatment with incremental doses of caffeine from patients in the accelerated and non-accelerated groups are shown in Figure 3A. The myotubes derived from a patient in the accelerated group had a lower threshold for responding to caffeine than that derived from a patient in the non-accelerated group. The dose-response curve to caffeine for a myotube derived from patient number 1 in the accelerated group was shifted to the left compared with that from patient number 20 in the non-accelerated group (Figure 2). Similar results were obtained for halothane and 4-CmC (Figure 2). When [EC.sub.50] values were calculated for caffeine, halothane and 4-CmC, the [EC.sub.50] values of the accelerated group was significantly less than that of the non-accelerated group (P <0.001). The [EC.sub.50] values for caffeine, halothane and 4-CmC were 2.52 [+ or -]0.77 mM, 1.71 [+ or -] 0.36 mM and 138.3 [+ or -] 42.7 //M in the accelerated group, and 5.13 [+ or -] 0.59 mM, 3.63 [+ or -] 0.64 mM and 275.4 [+ or -] 60.2 [micro]M in the nonaccelerated group, respectively.


There was greater variability in the resting [[Ca.sup.2+]] levels in the accelerated group (Table 1). The resting [[Ca.sup.2+]] of some patients in the accelerated group were higher than that in the non-accelerated group, but others were not different from the non-accelerated group.

Correlation among [EC.sub.50] for the three RYR1 activators, and between the CICR rate and [EC.sub.50] for the three RYR1 activators

There were strong positive correlations among the [EC.sub.50] values for the different RYR1 activators, which were highly statistically significant (caffeine [EC.sub.50] and halothane [EC.sub.50], caffeine [EC.sub.50] and 4-CmC [EC.sub.50], halothane [EC.sub.50] and 4-CmC [EC.sub.50]; r=0.80, P <0.001, r=0.74, P <0.001, r=0.73, P <0.001, respectively). Similar statistically significant positive correlations were found for the relationship between the [EC.sub.50] values for caffeine, halothane, 4-CmC and CICR rate at 0.3, 1.0, 3.0, 10.0 [micro]M of stimulating [Ca.sup.2+] concentration (Table 2).

Calculated cut-off points of [EC.sub.50] for three RYR1 activators

Myotubes from accelerated patients were considered true positives for statistical purposes and, to determine the best cut-off values to diagnose predisposition to MH, ROC curves were constructed and analysed (Figure 3). The area under the ROC curves were 0.901, 0.921 and 0.833 for caffeine, halothane and 4-CmC, respectively (Table 3), and the calculated cut-off values of [EC.sub.50] were 3.62 mM, 2.28 mM and 197.0 /M for caffeine, halothane and 4-CmC, respectively (Table 3). With these cut-off values, sensitivity was 0.85, 0.85 and 0.77 for caffeine, halothane and 4-CmC, respectively, and specificity was 0.83, 0.89 and 0.77 for caffeine, halothane and 4-CmC, respectively (Table 3). We considered the test positive, i.e. the patient is predisposed to develop MH, if three or more [EC.sub.50] values for caffeine obtained from five different myotubes were lower than the cut-off value. Sensitivity was calculated as greater than 0.97 using the following equation: sensitivity= [sub.5][C.sub.5] x [0.85.sup.5] + [sub.5][C.sub.4] x [0.85.sub.4] x 0.15 + [sub.5][C.sub.3] x [0.85.sub.3] x [0.15.sub.2.]

The sensitivities and specificities for caffeine, halothane and 4-CmC calculated using this equation are shown in Table 3.


MH is a potentially fatal complication of general anaesthesia, and we wished to develop new screening methods to identify patients at risk of developing MH. In this study, we demonstrated that changes in intracellular [Ca.sup.2+] concentration in myotubes derived from patients in the accelerated group were more pronounced at lower concentrations of all three RYR1 activators tested compared to the non-accelerated group (Figure 2). We observed increased [Ca.sup.2+] release in myotubes treated with caffeine, halothane or 4-CmC in a dose-dependent manner, and the [EC.sub.50] values for all three RYR1 activators significantly differed between the accelerated and non-accelerated groups. These characteristics suggest that some combination of RYR1 activators may be used for identifying predisposition to MH, and we conducted an ROC analysis to determine the optimal cut-off for each compound.

Several studies have confirmed that muscles from patients susceptible to MH are more sensitive to direct RYR1 activators such as caffeine, halothane and 4-CmC (22-25), but the criteria of [EC.sub.50] for MH susceptible have not been shown. In the present study, the [EC.sub.50] values for the three RYR1 activators tested in cultured myotubes from the accelerated group were two-fold lower than those of the nonaccelerated group. These results are similar to those previously obtained using the same experimental system (human cultured myotubes expressing MH mutations (22-25)) as well as other experimental models (human embryonic kidney cells (26) or dyspedic myotubes (4) transfected with RyR1 cDNAs possessing some MH mutations, and lymphoblastoid cells (27,28)).

Not all RYR1 mutations cause MH, and several studies have shown that some RYR1 mutations are actually hyposensitive to RYR1 activators (29,30). We did not perform a genetic analysis on all of the patients enrolled in this study, but three of the study subjects had undergone RYR1 analysis as part of earlier studies (20,21). Patient number 18, the parent of an individual with a documented episode of MH, carried no mutations of RYR1, but two causative mutations (p.L4838V and p.R2508C) were identified in patients number 1 and 2, respectively. The [EC.sub.50] values for the three RYR1 activators for patient number 18 were higher than those observed for patients number 1 and 2, and at least for this limited number of patients, the [EC.sub.50] values corresponded to RYR1 genotype.

Myotubes from some individuals in the accelerated group had a 2.0 times higher resting [[Ca.sup.2+]] than the non-accelerated group, but others did not differ from the non-accelerated group. Some studies examining human cultured myotubes observed elevated resting [[Ca.sup.2+]] for cells derived from MH susceptible patients (22,23), but others did not find similar changes (23-25). It was recently demonstrated that resting [[Ca.sup.2+]] in skeletal muscle is partly determined by [Ca.sup.2+] passively leaking through RYR1 and increased basal sarcolemmal [Ca.sup.2+] entry (31,32). The observed differences in resting [Ca.sup.2+] could be due to underlying differences in RYR1-leak as well as variations in the activity of other [Ca.sup.2+] transporters including the SR calcium ATPase (SERCA), plasma membrane [Ca.sup.2+] ATPase, and [Na.sup.+]/[Ca.sup.2+] exchange pump rather than heightened sensitivity to RYR1 activators or accelerated CICR.

Both IVCT and CICR are functional tests of muscle reaction following exposure to [Ca.sup.2+] or RYR1 activators, and our method directly measures intracellular [[Ca.sup.2+]] at the single-cell level. In this study, there was a good correlation between CICR rate and the [EC.sub.50] values of the RYR1 activators. Concordance between the CICR and IVCT tests has been evaluated previously, and there was agreement in only 13 of 21 cases (14). The CICR test is sensitive to abnormalities in [Ca.sup.2+] release from the SR, and this process is primarily controlled by the RYR1 channel in the SR. However, [Ca.sup.2+] release from SR and [Ca.sup.2+] influx requires a large complex containing many different proteins including the voltage sensor DHPR and others in the plasma membrane1. Because the plasma membrane is functionally destroyed in skinned muscle fibres, the CICR test cannot detect abnormalities in [Ca.sup.2+] homeostasis associated with plasma membrane proteins. The method we describe in this paper is theoretically able to detect abnormalities associated with plasma membrane proteins and it may be a complement to CICR test. More discordance between CICR results may become apparent as we test more patients.

IVCT is currently considered the most reliable diagnostic test for identifying patients susceptible to MH (1,11). It has a high sensitivity (99% for European MH group, 92 to 97% for North American MH group) and specificity (94% for European MH group, 53 to 78% for North American MH group) (12,13). In this study, the sensitivity and specificity calculated by ROC curves for the [EC.sub.50] values for each myotube was about 0.77 to 0.85 and lower compared to IVCT. However, the sensitivity and specificity of our method were increased by measuring five different myotubes. If three of the five [EC.sub.50] values were less than the cut-off value, a patient was considered predisposed to MH. Using this approach, sensitivity and specificity could approach 0.95, which makes this a potentially clinically useful test. Because more than five myotubes are present in most microscopy fields, increasing the number of tested myotubes is very practical. There was a problem that we defined true positives according to CICR test which was not more reliable than IVCT.

Caffeine, halothane and 4-CmC are well characterised RYR1 activators used in numerous studies, and a strong positive correlation among the [EC.sub.50] values obtained for the three RYR1 activators was found in this study. The site of action of 4-CmC on RYR1 is different from that of caffeine (33), and there was some discordance between the [EC.sub.50] values for caffeine and 4-CmC. Accordingly, it is essential to use at least two different RYR1 activators for assessing cellular [Ca.sup.2+] responses. Unlike caffeine, higher concentrations of halothane and 4-CmC were required to generate ideal dose-response curves, but high concentrations of halothane caused the myotubes to detach from the coverslip. The number of cells used to calculate the mean [EC.sub.50] for halothane was 143, the fewest of the three RYR1 activators. Concentrations of 4-CmC above 1000 [micro]M inhibit the SERCA pump, and increases in [Ca.sup.2+] caused by high concentrations of 4-CmC could be nonspecific rather than the result of RYR1 activation (34).

The relative maturity of cells contained in primary cultures can vary, and the expression of ion channels related to Ca2+ homeostasis depends on the duration of culture with differentiating media. Inositol 4,4,5-triophosphate ([InsP.sub.3]) receptors and DHPR are expressed by 0 to 6 days after exposure to differentiating media, but RYR1 was not expressed until after 10 days of culture in differentiating media in a human skeletal muscle cell line (35). Additionally, RYR3 is expressed at low levels by skeletal muscle cells (36), and it is much more sensitive to activation by caffeine compared with RYR1 (36). However, 500 [micro]M of 4-CmC is supposed to activated RYR1 fully, but not RYR3 (36). Additionally, InsP3 receptors are directly inhibited by caffeine (37). Taken together, these data indicate that if the muscle cells we selected for analysis had not yet gained expression of RYR1, then identification of MH susceptible patients would be seriously impaired. However, we previously determined that the culture conditions used in this study are compatible with the expression of skeletal muscle-specific proteins such as sarcomeric [alpha]-actinin and RYR1, and we have shown this with indirect immunofluorescent staining (data not shown). Finally, we selected myotubes for complete analysis with maximal increases in [Ca.sup.2+] elicited by both 10 mM caffeine and 500 [micro]M 4-CmC.

In previous genetic studies of MH, there were significant differences in the [EC.sub.50] values of caffeine, halothane and 4-CmC between MH susceptible subjects with RYR1 mutations and MH negative subjects (4,28,29). Recently, cells harbouring MH causative mutations were found to be more sensitive to RYR1 activators, but some mutations associated with MH completely inhibit RYR1 activity due to the site of mutation (3,4,10,29,38). We did not perform a genetic analysis in this study and we could not evaluate whether RYR1 variants were associated with significant differences in the [EC.sub.50] values for RYR1 activators. Sequencing of the entire RYR1 coding region was performed on only three individuals in our previous study (20,21) and, for these patients, the MH susceptibility estimated by [EC.sub.50] values corresponded to the genetic analysis.

In the satellite cells that lie between the plasma membrane and the basal lamina, mitosis is quiescent in adult muscle, but they become activated by stimuli such as myotrauma. Activated satellite cells proliferate and generate myoblasts, which express myogenic markers. These cells fuse together to form multinucleated cells, myotubes. The culture of satellite cells can be performed using small pieces of tissue (around 0.1 g) and/or damaged skeletal muscle. In contrast, larger amounts of muscle fibre are needed for the performance of IVCT and CICR testing. Additionally, the tension of children's muscles is not sufficient for performance of the CICR or other contraction tests, but it is relatively easy to culture myotubes from children's muscles once an appropriate specimen is obtained (39). In this study, we derived myotubes from specimens obtained from patients over 70 years old. Furthermore, contraction tests require the use of a fresh muscle bundle, but myotubes can be successfully grown from crushed muscle or muscle stored at 4[degrees]C for three days. However, myotube culture requires at least four to six weeks to go from muscle pieces to [EC.sub.50] analysis.

At the present time, a phenotypic screening method for the diagnosis of MH susceptibility is preferred to a genetic test because only 35 RYR1 mutations have been functionally characterised with respect to [Ca.sup.2+] homeostasis1. As more mutations are identified, correlation between functional studies such as ours and genotypes should be undertaken to better understand the pathophysiology of MH.

In conclusion, we used human cultured myotubes to examine the cellular response to RYR1 activators. Among samples from CICR-accelerated patients, there was an increased sensitivity to RYR1 activators compared to non-accelerated patients. The [EC.sub.50] values for these different compounds correlated with results of CICR testing. Using this approach may be a sensitive and specific method of identifying patients predispose to MH.


This study was supported in part by Grant-in-Aid number 21591973 for Scientific Research from the Japan Society for the Promotion of Sciences, Tokyo, Japan and Tsuchiya-foundation, Hiroshima, Japan.


(1.) Stowell KM. Malignant hyperthermia: a pharmacogenetic disorder. Pharmacogenomics 2008; 9:1657-1672.

(2.) Treves S, Anderson AA, Ducreux S, Divet A, Bleunven C, Grasso C et al. Ryanodine receptor 1 mutations, dysregulation of calcium homeostasis and neuromuscular disorders. Neuromuscul Disord 2005; 15:577-587.

(3.) Avila G. Intracellular Ca2+ dynamics in malignant hyperthermia and central core disease: established concepts, new cellular mechanisms involved. Cell Calcium 2005; 37:121-127.

(4.) Yang T, Ta TA, Pessah IN, Allen PD. Functional defects in six ryanodine receptor isoform-1 (RyR1) mutations associated with malignant hyperthermia and their impact on skeletal excitation-contraction coupling. J Biol Chem 2003; 278:25722-25730.

(5.) Robinson R, Carpenter D, Shaw M-A, Halsall J, Hopkins P. Mutations in RYR1 in malignant hyperthermia and central core disease. Hum Mutat 2006; 27:977-989.

(6.) Sambuughin N, Holley H, Muldoon S, Brandom BW, de Bantel AM, Tobin JR et al. Screening of the entire ryanodine receptor type 1 coding region for sequence variants associated with malignant hyperthermia susceptibility in the north american population. Anesthesiology 2005; 102:515-521.

(7.) Gillies RL, Bjorksten AR, Davis M, Du Sart D. Identification of genetic mutations in Australian malignant hyperthermia families using sequencing of RYR1 hotspots. Anaesth Intensive Care 2008; 36:391-403.

(8.) Ibarra MCA, Wu S, Murayama K, Minami N, Ichihara Y, Kikuchi H et al. Malignant hyperthermia in Japan: mutation screening of the entire ryanodine receptor type 1 gene coding region by direct sequencing. Anesthesiology 2006; 104:1146-1154.

(9.) Weiss RG, O'Connell KMS, Flucher BE, Allen PD, Grabner M, Dirksen RT. Functional analysis of the R1086H malignant hyperthermia mutation in the DHPR reveals an unexpected influence of the III-IV loop on skeletal muscle EC coupling. Am J Physiol Cell Physiol 2004; 287:C1094-1102.

(10.) Levano S, Vukcevic M, Singer M, Matter A, Treves S, Urwyler A et al. Increasing the number of diagnostic mutations in malignant hyperthermia. Hum Mutat 2009; 30:590-598.

(11.) Rosenberg H, Davis M, James D, Pollock N, Stowell K. Malignant hyperthermia. Orphanet J Rare Dis 2007; 2:21.

(12.) Ording H, Brancadoro V, Cozzolino S, Ellis FR, Glauber V, Gonano EF 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; 41:955-966.

(13.) Allen GC, Larach MG, Kunselman AR. The sensitivity and specificity of the caffeine-halothane contracture test: a report from the North American Malignant Hyperthermia Registry. The North American Malignant Hyperthermia Registry of MHAUS. Anesthesiology 1998; 88:579-588.

(14.) Oku S, Mukaida K, Nosaka S, Sai Y, Maehara Y, Yuge O. Comparison of the in vitro caffeine-halothane contracture test with the Ca-induced Ca release rate test in patients suspected of having malignant hyperthermia susceptibility. J Anesth 2000; 14:6-13.

(15.) Maehara Y, Mukaida K, Hiyama E, Morio M, Kawamoto M, Yuge O. Genetic analysis with calcium-induced calcium release test in Japanese malignant hyperthermia susceptible (MHS) families. Hiroshima J Med Sci 1999; 48:9-15.

(16.) Endo M. Calcium-induced calcium release in skeletal muscle. Physiol Rev 2009; 89:1153-1176.

(17.) Migita T, Mukaida K, Kawamoto M, Kobayashi M, Nishino I, Yuget O. Propofol-induced changes in myoplasmic calcium concentrations in cultured human skeletal muscles from RYR1 mutation carriers. Anaesth Intensive Care 2007; 35:894-898.

(18.) Migita T, Mukaida K, Hamada H, Kobayashi M, Nishino I, Yuge O et al. Effects of propofol on calcium homeostasis in human skeletal muscle. Anaesth Intensive Care 2009; 37:415-425.

(19.) Larach MG, Localio AR, Allen GC, Denborough MA, Ellis FR, Gronert GA et al. A clinical grading scale to predict malignant hyperthermia susceptibility. Anesthesiology 1994; 80:771-779.

(20.) Tanabe T, Fukusaki M, Terao Y, Yamashita K, Sumikawa K, Mukaida K et al. Malignant hyperthermia susceptibility diagnosed with a family-specific ryanodine receptor gene type 1 mutation. J Anesth 2008; 22:70-73.

(21.) Migita T, Mukaida K, Hamada H, Yasuda T, Haraki T, Nishino I et al. Functional analysis of ryanodine receptor type 1 p.R2508C mutation in exon 47. J Anesth 2009; 23:341-346.

(22.) Wehner M, Rueffert H, Koenig F, Neuhaus J, Olthoff D. Increased sensitivity to 4-chloro-m-cresol and caffeine in primary myotubes from malignant hyperthermia susceptible individuals carrying the ryanodine receptor 1 Thr2206Met (C6617T) mutation. Clin Genet 2002; 62:135-146.

(23.) Wehner M, Rueffert H, Koenig F, Olthoff D. Functional characterization of malignant hyperthermia-associated RyR1 mutations in exon 44, using the human myotube model. Neuromuscul Disord 2004; 14:429-437.

(24.) Weigl LG, Ludwig-Papst C, Kress HG. 4-chloro-m-cresol cannot detect malignant hyperthermia equivocal cells in an alternative minimally invasive diagnostic test of malignant hyperthermia susceptibility. Anesth Analg 2004; 99:103-107.

(25.) Kaufmann A, Kraft B, Michalek-Sauberer A, Weigl LG. Novel ryanodine receptor mutation that may cause malignant hyperthermia. Anesthesiology 2008; 109:457-64.

(26.) Tong J, Oyamada H, Demaurex N, Grinstein S, McCarthy TV, MacLennan DH. 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; 272:26332-26339.

(27.) Loke JCP, Kraev N, Sharma P, Du GG, Patel L, Kraev A et al. Detection of a novel ryanodine receptor subtype 1 mutation (R328W) in a malignant hyperthermia family by sequencing of a leukocyte transcript. Anesthesiology 2003; 99:297-302.

(28.) Anderson AA, Brown RL, Polster B, Pollock N, Stowell KM. Identification and biochemical characterization of a novel ryanodine receptor gene mutation associated with malignant hyperthermia. Anesthesiology 2008; 108:208-215.

(29.) Du GG, Oyamada H, Khanna VK, MacLennan DH. Mutations to Gly2370, Gly2373 or Gly2375 in malignant hyperthermia domain 2 decrease caffeine and cresol sensitivity of the rabbit skeletal-muscle Ca2+-release channel (ryanodine receptor isoform 1). Biochem J 2001; 360:97-105.

(30.) Ducreux S, Zorzato F, Ferreiro A, Jungbluth H, Muntoni F, Monnier N et al. Functional properties of ryanodine receptors carrying three amino acid substitutions identified in patients affected by multi-minicore disease and central core disease, expressed in immortalized lymphocytes. Biochem J 2006; 395:259-266.

(31.) Yang T, Esteve E, Pessah IN, Molinski TF, Allen PD, Lopez JR. Elevated resting [Ca(2+)](i) in myotubes expressing malignant hyperthermia RyR1 cDNAs is partially restored by modulation of passive calcium leak from the SR. Am J Physiol Cell Physiol 2007; 292:C1591-1598.

(32.) Eltit JM, Yang T, Li H, Molinski TF, Pessah IN, Allen PD et al. RyR1-mediated Ca2+ leak and Ca2+ entry determine resting intracellular Ca2+ in skeletal myotubes. J Biol Chem 2010; 285:13781-13787.

(33.) Fessenden JD, Feng W, Pessah IN, Allen PD. Amino acid residues Gln4020 and Lys4021 of the ryanodine receptor type 1 are required for activation by 4-chloro-m-cresol. J Biol Chem 2006; 281:21022-21031.

(34.) Al-Mousa F, Michelangeli F. Commonly used ryanodine receptor activator, 4-chloro-m-cresol (4CmC), is also an inhibitor of SERCA Ca2+ pumps. Pharmacol Rep 2009; 61:838-842.

(35.) Liberona JL, Caviedes P, Tascon S, Hidalgo J, Giglio JR, Sampaio SV et al. Expression of ion channels during differentiation of a human skeletal muscle cell line. J Muscle Res Cell Motil 1997; 18:587-598.

(36.) Conti A, Gorza L, Sorrentino V. Differential distribution of ryanodine receptor type 3 (RyR3) gene product in mammalian skeletal muscles. Biochem J 1996; 316:19-23.

(37.) Hume JR, McAllister CE, Wilson SM. Caffeine inhibits InsP3 responses and capacitative calcium entry in canine pulmonary arterial smooth muscle cells. Vascul Pharmacol 2009; 50:89-97.

(38.) Ghassemi F, Vukcevic M, Xu L, Zhou H, Meissner G, Muntoni F et al. A recessive ryanodine receptor 1 mutation in a CCD patient increases channel activity. Cell Calcium 2009; 45:192-197.

(39.) Lees SJ, Rathbone CR, Booth FW. Age-associated decrease in muscle precursor cell differentiation. Am J Physiol Cell Physiol 2006; 290:C609-615.

M. KOBAYASHI *, K. MUKAIDA ([dagger]), T. MIGITA ([double dagger]), H. HAMADA ([section]), M. KAWAMOTO **, O. YUGE ([dagger])([dagger])

Department of Anesthesiology and Critical Care, Hiroshima University, Hiroshima, Japan

* M.D., Staff Anesthesiologist, Division of Anesthesia, Hiroshima General Hospital, Hatsukaichi.

([dagger]) M.D., Ph.D., Staff Anesthesiologist, Department of Anesthesia, Hiroshima Prefectural Rehabilitation Center.

([double dagger]) M.D., Ph.D., Assistant Professor.

([section]) M.D., Ph.D., Associate Professor.

** M.D., Ph.D., Professor and Chair.

([dagger]) ([dagger]) M.D., Ph.D., Ex-Trustee.

Address for correspondence: Dr M. Kobayashi, Division of Anesthesia, Hiroshima General Hospital, 1-3-3 Jigozen Hatsukaichi-city, 738-8503, Japan. Email:

Accepted for publication on September 30, 2010.
Biometric data, indications for CICR testing, results of CICR testing
and [EC.sub.50] values for caffeine, halothane, 4-CmC and resting

Patient Indications for CICR
no. Gender Age, y testing

1 * M 58 MH family
2 * F 13 MH family/myopahty
3 * M 14 MH family
4 * M 27 MH family
5 * M 31 MH (CGS 63 rank 6)
6 * M 31 MH (CGS 48 rank 5)
7 * M 45 MH family
8 M 60 MH family
9 * F 46 MH family
10 * F 56 MH family
11 M 48 Heat stroke family
12 M 34 MH family
13 F 40 MH family
14 M 71 MH family
15 M 6 MH (CGS 68 rank 6)
16 M 18 MH (CGS 48 rank 5)
17 M 58 HyperCKaemia
18 F 59 MH family
19 * M 10 MH family
20 * M 75 Shock and unknown
21 * F 28 Postoperative
 unknown fever
22 * M 47 MH family
23 * M 35 Postoperative
 unknown fever
24 * M 2 MH family
25 * M 6 MH family
26 M 21 Postoperative
27 M 32 MH family
28 F 37 MH family
29 M 15 Postoperative
30 * M 32 MH family
31 F 32 HyperCKaemia family
32 M 74 Postoperative
 unknown fever
33 M 4 HyperCKaemia
34 F 48 MH family

Patient Results of CICR RYR1 mutation Caffeine
no. testing amino acid change (mM)

1 * accelerated p.L4838V 3.28
2 * accelerated p.R2508C 2.07
3 * accelerated 1.93
4 * accelerated 1.91
5 * accelerated 4.05
6 * accelerated 2.21
7 * accelerated 2.89
8 accelerated 3.92
9 * accelerated 2.71
10 * accelerated 1.82
11 accelerated 1.80
12 accelerated 1.77
13 accelerated 3.38
14 accelerated 2.52
15 accelerated 1.62
16 accelerated 2.10
17 accelerated 2.93
18 not accelerated -- ** 4.38
19 * not accelerated 5.13
20 * not accelerated 5.90
21 * not accelerated 4.35
22 * not accelerated 5.44
23 * not accelerated 5.43
24 * not accelerated 5.28
25 * not accelerated 5.61
26 not accelerated 5.72
27 not accelerated 6.23
28 not accelerated 4.77
29 not accelerated 5.12
30 * not accelerated 4.97
31 not accelerated 4.10
32 not accelerated 4.84
33 not accelerated 4.47
34 not accelerated 5.53


Patient Halothane 4-CmC [[[Ca.sup.2+]].sub.I]
no. (mM) ([micro]M) (nM) resting

1 * 1.60 88.1 83.2
2 * 1.71 130.9 125.8
3 * 1.39 123.4 100.5
4 * 1.95 131.4 74.5
5 * 2.21 93.3 87.2
6 * 2.24 144.7 84.9
7 * 2.03 163.0 74.4
8 1.20 97.5 61.9
9 * 2.06 175.2 69.9
10 * 2.02 170.1 54.0
11 1.79 206.0 59.5
12 1.80 164.8 69.4
13 1.73 179.0 73.9
14 1.39 186.7 83.9
15 1.11 68.1 107.1
16 1.13 160.7 58.9
17 1.66 68.6 87.4
18 3.72 248.6 60.3
19 * 5.09 257.0 57.1
20 * 4.55 379.2 58.5
21 * 4.52 229.9 46.3
22 * 3.24 215.6 61.2
23 * 3.19 314.4 60.5
24 * 3.21 308.6 53.1
25 * 2.86 255.6 61.4
26 2.92 296.4 73.6
27 4.20 212.3 66.1
28 2.86 302.0 63.1
29 3.15 410.3 55.8
30 * 3.61 204.7 62.7
31 3.56 225.3 78.1
32 3.52 339.3 64.1
33 3.65 228.6 66.9
34 3.90 253.8 62.9

CICR=Ca-induced Ca release, 4-CmC=4-chloro-m-creso, RYR1=ryanodine
receptor type 1, MH=malignant hyperthermia, CGS=Clinical Grading
Scale, HyperCKaemia=raised blood levels of creatine kinase. * Seventy
patients were used in our previous study, ** Minus sign indicates that
no mutation was detected within the entire RYR1 coding region from
genomic DNA.

Correlations between CICR rate and EC50 values for RYR1 activators

 Rate of CICR
[Ca.sup.2+] concentration
([micro]M) 0.3 1.0

Caffeine r=0.63 (P <0.001) r=0.64 (P <0.001)
Halothane r=0.69 (P <0.001) r=0.69 (P <0.001)
4-CmC r=0.61 (P <0.001) r=0.60 (P <0.002)

 Rate of CICR
 [Ca.sup.2+] concentration
([micro]M) 3.0 10

Caffeine r=0.66 (P <0.001) r=0.67 (P <0.001)
Halothane r=0.69 (P <0.001) r=0.71 (P <0.001)
4-CmC r=0.63 (P <0.001) r=0.65 (P <0.001)

CICR=Ca-induced Ca release, RYR1=ryanodine receptor type 1,

Diagnostic accuracy based on ROC curves and cut-off points of
[EC.sub.50] values


 Caffeine Halothane 4-CmC

Area under the ROC 0.901 0.921 0.833
SEM 0.027 0.025 0.031
95% CI 0.858-0.943 0.872-0.970 0.772-0.893
Cut off value 3.62 mM 2.28 mM 197 [micro]M
Accelerated (n) 103 67 82
Non-accelerated (n) 95 76 90
Sensitivity 0.85 0.85 0.77
Specificity 0.83 0.89 0.77
Sensitivity * 0.97 0.97 0.92
Specificity * 0.96 0.99 0.92

ROC=receiver operating characteristic, SEM=standard error
mean, CI=confidence interval. * If 3 or more values of five
[EC.sub.50] values obtained from five myotubes were lower than
its cut-off value, the individual was considered predisposed to MH.
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Article Details
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Author:Kobayashi, M.; Mukaida, K.; Migita, T.; Hamada, H.; Kawamoto, M.; Yuge, O.
Publication:Anaesthesia and Intensive Care
Article Type:Report
Geographic Code:9JAPA
Date:Mar 1, 2011
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