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Mutated p.4894 RyR1 Function Related to Malignant Hyperthermia and Congenital Neuromuscular Disease with Uniform Type 1 Fiber (CNMDU1)

Haraki, Toshiaki MD*; Yasuda, Toshimichi MD, PhD*; Mukaida, Keiko MD, PhD; Migita, Takako MD, PhD*; Hamada, Hiroshi MD, PhD*; Kawamoto, Masashi MD, PhD*

doi: 10.1213/ANE.0b013e318232053e
Pediatric Anesthesiology: Research Reports
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BACKGROUND: Ryanodine receptor 1 (RyR1) is a Ca2+ release channel located in the sarcoplasmic reticulum membrane of skeletal muscle. More than 200 variants in RyR1 have been identified in DNA from patients with malignant hyperthermia (MH) and congenital myopathies; only 30 have been sufficiently studied so as to be identified as MH-causative mutations. The Ala4894Thr RyR1 variant was found in a Japanese patient with susceptibility to MH, and the Ala4894Pro variant in a rare case of myopathy: congenital neuromuscular disease with uniform type 1 fiber (CNMDU1). We hypothesized that different Ala4894 variants of RyR1 cause different pathophysiological changes that are identifiable by having differing pharmacological sensitivities to RYR1 agonists.

METHODS: Expression vector with a mutation in RYR1 corresponding to the Ala4894Thr, Ala4894Pro, Ala4894Ser, or Ala 4894Gly variant of human RyR1 was transfected into human embryonic kidney 293 cells. At 72 hours after transfection, we determined the intracellular Ca2+ changes induced by caffeine and 4-chloro-m-cresol (4CmC), in the presence or absence of dantrolene.

RESULTS: Ala4894Thr-transfected cells and Ala4894Ser-transfected cells were more sensitive to caffeine than the wild type, and Ala4894Thr-transfected cells were also more sensitive to 4CmC than the wild type, whereas Ala4894Pro-transfected cells had no response to caffeine or 4CmC. Ala4894Gly-transfected cells were significantly less sensitive to caffeine than the wild type. In addition, the responses of Ala4894Thr-transfected cells and Ala4894Ser-transfected cells to caffeine were suppressed by dantrolene.

CONCLUSION: We concluded that different Ala4894 variants of RyR1 lead to different agonist/antagonist sensitivities, which may predict differing RYR1 functionality during excitation-contraction coupling and sensitivity to MH. The hypersensitive Ala4894Thr-RyR1 is associated with MH and the poorly functional Ala4894Pro-RyR1 with CNMDU1.

Published ahead of print September 16, 2011 Supplemental Digital Content is available in the text.

From the *Department of Anesthesiology and Critical Care, Hiroshima University, Hiroshima; and Division of Anesthesia, Hiroshima Prefectural Rehabilitation Center, Hiroshima, Japan.

Supported in part by the Tsuchiya Foundation.

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Toshiaki Haraki, MD, Department of Anesthesiology and Critical Care, Division of Clinical Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. Address e-mail to tsakharaki@gmail.com.

Accepted July 26, 2011

Published ahead of print September 16, 2011

Malignant hyperthermia (MH) is an autosomal dominant, pharmacogenetic disorder triggered by volatile anesthetics and/or succinylcholine. Major MH symptoms are manifested as abnormal hyperthermia, muscle rigidity, acidosis, and hyperkalemia. If not treated adequately, a patient with MH can develop a life-threatening condition. Since the Ala614Cys ryanodine receptor 1 (RyR1) mutation was first reported in 1991,1 a large number of RyR1 mutations associated with MH have been reported. For example, the Ala4894Thr RyR1 variant in the C-terminal domain, which has been reported to be responsible for MH, was found in a Japanese MH patient,2 but the function of the mutated RyR1 was not evaluated.

Ryanodine receptors are large homotetrameric channel proteins, with each subunit consisting of approximately 5000 amino acids. These receptors are located in the membrane of the sarcoplasmic reticulum (SR) and regulate intracellular Ca2+ concentrations by releasing Ca2+ from the SR into the cytoplasm.3 RyR1 is predominantly expressed in skeletal muscle and has an important role in excitation-contraction (EC) coupling. More than 200 RyR1 variants have been reported.47 It has been reported that most of the variants are located within 3 hotspots (N-terminal, central, and C-terminal domains).8 However, a number of mutations have been found outside the hotspots2,9,10; therefore, the possibility exists that the hotspots result from screening bias.4,5,11 Some RyR1 variants have been implicated in MH and congenital myopathies, including central core disease (CCD), multi-minicore disease, and congenital neuromuscular disease with uniform type 1 fiber (CNMDU1).1214

CNMDU1, a rare congenital myopathy first reported in 1983,15 is pathologically characterized by the exclusive presence of type 1 muscle fibers (>99%) with no muscle structure abnormalities. It is clinically characterized by delayed motor milestones, mild proximal weakness, hypo- or nonreflexes, and a normal level of serum creatine kinase. These symptoms appear within the first few years of life.16,17 The Ala4894Pro RyR1 mutation was found in a CNMDU1 patient16; however, its function was not evaluated and the pathogenesis of CNMDU1 remains uncertain.

We have investigated the functions of RyR1 mutants associated with MH and congenital myopathy,18 and hypothesized that each of the different Ala4894 variants in RyR1 causes different pathophysiological conditions. The purpose of this study was to evaluate the function of mutated RyR1 channels. Expression of the RyR1 gene in human embryonic kidney (HEK)-293 cells and measurement of intracellular Ca2+ changes induced by caffeine and 4-chloro-m-cresol (4CmC) are methods frequently used to evaluate RyR1 functions.19,20 We transfected the Ala4894Thr and Ala4894Pro RyR1 genes into HEK-293 cells, and evaluated the function of RyR1. In addition, we transfected new artificial variants (Ala4894Ser and Ala4894Gly RyR1 genes) and examined their functions.

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METHODS

Rabbit-RyR1/pcDNA was a generous gift from Professor David H. MacLennan (University of Toronto). The HEK-293 Tet-off® Advanced Cell Line (HEK-293) was purchased from Clontech Laboratories (Shiga, Japan). Restriction enzymes were purchased from Takara Bio Inc. (Shiga, Japan). Caffeine, 4CmC, thapsigargin, and other chemicals were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

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Preparation of RyR1/pTRE-Tight-BI-AcGFP

To coexpress the RyR1 and Aequorea coerulescens green fluorescent protein (AcGFP) genes, rabbit-RyR1 cDNA was removed from rabbit-RyR1/pcDNA using the XbaI/HindIII restriction enzymes. Rabbit-RyR1 cDNA was then ligated into the NheI-HindIII site of pTRE-Tight-BI-AcGFP (pBI; Clontech Laboratories) (RyR1/pBI). Ligation was performed using a Takara DNA ligation kit LONG (Takara Bio Inc.).

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Mutagenesis

C-terminal RyR1 was removed from RyR1/pBI using the restriction enzymes. The fragment was inserted into a pBluescript II KS(+) (Stratagene, La Jolla, CA) vector and used as a template for mutagenesis. Mutagenesis was performed using a QuickChange®II XL Site-Directed Mutagenesis Kit (Stratagene). Primers for the Ala4894Thr and Ala4894Pro mutations, obtained from MH and CNMDU1 patients, respectively, were custom designed, as were primers for the Ala4894Ser and Ala4894Gly variants. Serine is classified as a polar amino acid because of the hydroxyl group and its characteristics are similar to threonine. Glycine has the simplest structure among amino acids, and its molecular weight and polar characteristics are similar to alanine. The primers used for mutagenesis were 5′-cgtgggcgtccggactggcggaggc for Ala4894Thr, 5′-cgtgggcgtccggcctggcggaggc for Ala4894Pro, 5′-cgtgggcgtccggtctggcggaggc for Ala4894Ser, and 5′-cgtgggcgtccgggctggcggaggc for Ala4894Gly. Mutagenesis was performed according to the methods described by the manufacturer. After mutagenesis, we confirmed that mutated pBluescript II KS(+) plasmid contained the desired mutation by sequencing. Analyses of the sequences were performed using an ABI 3100 DNA sequencer and a BigDye® Terminator v3.1 Cycle Sequence Kit (Applied Biosystems, Tokyo, Japan). The primers used for sequencing were T7, T3, and 5′-aagttcggggtcatcttcacg. The mutated fragment was removed from the pBluescript II KS(+) plasmid using the restriction enzymes ClaI/HindIII and ligated into RyR1/pBI to make the expression vectors inserted into the mutated RyR1 gene.

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Transfection

HEK-293 cells were maintained in Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, CA) supplemented with 10% Tet System–Approved fetal bovine serum (Clontech Laboratories), 100 U/mL penicillin (Sigma, St. Louis, MO), and 100 mg/mL streptomycin (Sigma) at 37°C under 5% CO2. Next, 1 × 105 HEK-293 cells were subcultured on 35-mm poly-L-lysine–coated glass-bottomed dishes (Matsunami, Osaka, Japan) for 24 hours. DNA transfection was performed using FuGENE HD Transfection Reagent (Roche Applied Science, Indianapolis, IN). An expression vector (pBI) was transfected as the control.

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Ca2+ Fluorescence Measurements

Seventy-two hours after transfection, the HEK-293 cells were washed twice with HEPES-buffered salt solution (HBSS) containing 130 mM NaCl, 5.4 mM KCl, 20 mM HEPES, 2.5 mM CaCl2, 1 mM MgCl2, and 5.5 mM glucose at pH 7.4. The cells were then loaded with 5.0 μM Fura-2 AM (Dojindo, Tokyo, Japan) in HBSS for 1 hour at 37°C. Measurements were performed after perfusion with HBSS for 15 minutes at a rate of 1.2 mL/min at 37°C.

Next, the cells were excited at 490 nm and fluorescence emissions of AcGFP were observed at 510 nm to identify AcGFP-positive cells, which were regarded as successfully transfected cells. The AcGFP-positive cells were excited alternately at 340 and 380 nm, and the fluorescence emissions of Fura-2 AM were observed at 510 nm using a fluorescence microscope (Nikon, Tokyo, Japan) at 5-second intervals to evaluate the intracellular Ca2+ changes. Images were acquired using a cooled high-speed digital video camera (ORCA-AG; Hamamatsu Photonics, Hamamatsu, Japan). The 340/380-nm signal ratio was calculated using a Ca2+ imaging system (Aquacosmos 2.5; Hamamatsu Photonics), and Fura-2 photobleaching was minimized by Aquacosmos 2.5 software.

HBSS containing incremental levels of caffeine or 4CmC was added to 1 side of the culture dish and aspirated on the opposite side for 2 minutes at a rate of 1.2 mL/min at 37°C. Each solution was washed out a few minutes before addition of the next incremental levels of caffeine or 4CmC. At the end of experiments, the cells were perfused with HBSS containing 500 nM thapsigargin, an inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase, to confirm that Ca2+ stores in the endoplasmic reticulum (ER) were replete.

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Data Analysis

To trace dose-response curves for caffeine and 4CmC, the data were normalized to the maximal response of each cell. Concentrations of caffeine and 4CmC required to reach half-maximal activation were calculated from the acquired dose-response curves. All data analyses were performed using PRISM 4.0 software (GraphPad Software, San Diego, CA). A P value of <0.05 was considered to be statistically significant.

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RESULTS

The proportions of AcGFP-positive cells were 50% to 70%. There was no obvious difference in transfection efficiency between wild-type (WT)- or mutated-RyR1–transfected cells, and 2 to 5 cells followed per experiment.

Figure 1 shows representative data for WT- or mutated-RyR1 cDNA transfected into HEK-293 cells with caffeine. WT-RyR1–transfected cells and Ala4894Thr-RyR1–transfected cells were sensitive to caffeine, whereas Ala4894Pro-RyR1 and vector alone (control)–transfected cells showed no reaction. Likewise, WT-RyR1–transfected cells and Ala4894Thr-RyR1–transfected cells were sensitive to 4CmC, whereas Ala4894Pro-RyR1–transfected cells were not (data not shown). We plotted the dose-response curves of WT-RyR1–transfected cells and Ala4894Thr-RyR1–transfected cells to caffeine (Fig. 2A). The dose-response curves of Ala4894Thr-RyR1–transfected cells were shifted to the left as compared with WT-transfected cells, with the 50% effective concentration of WT-RyR1–transfected cells and Ala4894Thr-RyR1–transfected cells for caffeine 1.38 ± 0.10 and 0.52 ± 0.04 mM, respectively (Table 1). We also plotted the dose-response curves of WT-RyR1–transfected cells and Ala4894Thr-RyR1–transfected cells for 4CmC (Fig. 2B). The dose-response curves of the Ala4894Thr-RyR1–transfected cells were shifted to the left as compared with the WT-RyR1–transfected cells, and the 50% effective concentration values of WT-RyR1–transfected cells and Ala4894Thr-RyR1–transfected cells for 4CmC were 176.6 ± 9.0 and 77.9 ± 8.6 μM, respectively (Table 2). The Ala4894Ser-RyR1–transfected cells and Ala4894Gly-RyR1–transfected cells were exposed to increasing concentration of caffeine, and we plotted the dose-response curves (Fig. 2, C and D), respectively. The 50% effective concentration value for Ala4894Ser-RyR1–transfected cells was 0.79 ± 0.05 mM, which was significantly lower than that for WT-RyR1–transfected cells, whereas that for Ala4894Gly-RyR1–transfected cells was significantly greater at 11.57 ± 1.34 mM (Table 1).

Figure 1

Figure 1

Figure 2

Figure 2

Table 1

Table 1

Table 2

Table 2

Dantrolene is a skeletal muscle relaxant and the only clinically available drug for treatment of MH.21 We examined the effects of dantrolene on WT-RyR1–transfected cells, Ala4894Thr-RyR1–transfected cells, and Ala4894Ser-RyR1–transfected cells. Cells were perfused with 10 μM dantrolene in HBSS for 15 minutes, then loaded with increasing concentrations of caffeine solutions containing 10 μM dantrolene for 2 minutes. We plotted the dose-response curves of WT-RyR1–transfected cells, Ala4894Thr-RyR1–transfected cells, and Ala4894Ser-RyR1–transfected cells to caffeine with dantrolene and found that the curves were shifted to the right as compared with those without dantrolene (Fig. 3). The 50% effective concentration values of WT-RyR1–transfected cells, Ala4894Thr-RyR1–transfected cells, and Ala4894Ser-RyR1–transfected cells for caffeine with dantrolene were 3.18 ± 0.31, 2.03 ± 0.54, and 1.21 ± 0.03 mM, respectively (Table 1).

Figure 3

Figure 3

Thapsigargin inhibits the uptake of Ca2+ into the SR and induces leakage from the SR into cytoplasm.22 Changes in the 340/380-nm signal ratio induced by thapsigargin allow us to estimate the amount of Ca2+ in the SR.23 We calculated the 340/380-nm signal ratios in a resting condition and maximal response induced by 500 nM thapsigargin in HBSS. The 340/380-nm signal ratios in the resting condition for WT-RyR1–transfected cells, Ala4894Thr-RyR1–transfected cells, and Ala4894Pro–transfected cells were 0.99 ± 0.10, 0.95 ± 0.08, and 0.94 ± 0.07, respectively, which are not statistically different (Table 3). The WT-RyR1–transfected cells, Ala4894Thr-RyR1–transfected cells, and Ala4894Pro-RyR1–transfected cells responded to 500 nM thapsigargin, with maximal 340/380-nm signal ratios of 2.71 ± 0.72, 2.79 ± 0.83, and 2.98 ± 0.68, respectively, which are not statistically different (Table 3).

Table 3

Table 3

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DISCUSSION

The present study revealed that MH-associated, Ala4894Thr-RyR1–transfected cells are more sensitive to caffeine and 4CmC than WT-RyR1 cells, whereas those responses were inhibited by dantrolene. In addition, CNMDU1-associated, Ala4894Pro-RyR1–transfected cells were found to be insensitive to caffeine and 4CmC, whereas cells transfected with Ala4894Ser, a new artificial variant, were also more sensitive to caffeine than WT cells. Our findings also showed that cells transfected with Ala4894Gly, another new artificial variant, were less sensitive to caffeine than WT cells. Together, these results indicate that different RyR1 mutations at Ala4894 lead to different physiological responses, which may represent clinically relevant findings.

Most mutations reported in studies of MH patients are located in the N-terminal or central domains. Ala4894Thr, found in a Japanese MH patient, is a mutation located in the C-terminal domain. That patient was clinically diagnosed with MH and examined with the calcium-induced calcium release (CICR) rate test.2 In Japan, a CICR rate test using skinned muscle fibers was performed to diagnose susceptibility to MH2426 and the patient had a significantly accelerated CICR rate (Table 4). The present results indicate that the Ala4894Thr-RyR1 mutation is consistent with known responses of the MH-causal RyR1 mutation.

Table 4

Table 4

We found that dantrolene suppressed the response of Ala4894Thr-transfected cells to caffeine. Dantrolene shows clinical efficacy in many MH cases; however, the scientific evidence that dantrolene is effective for any mutations of RyR1 that cause MH has not been obtained, and it is difficult to solve the problem. Therefore, it is important to show the effect of dantrolene for each variant reported from MH. Our results indicate that dantrolene may be effective for MH associated with Ala4894Thr.

We also found that Ala4894Pro-transfected cells, as a homozygous model, were completely insensitive to caffeine and 4CmC. Because humans are heterozygous for nearly all RyR1 mutations, and because such patients survive, albeit with modified skeletal muscle function, these patients are presumably heterozygous as well. We therefore hypothesize that this mutation would bestow a relative insensitivity of RyR1 to normal agonist stimuli during normal physiological function in these individuals, resulting in a decrement of excitation-coupled Ca2+ release and decreased contractile function. This is in line with the recent classification of mutated RyR1 into 3 types: leaky channels, EC uncoupling, and overactive channels. Leaky channels, which remain in an open state and leak Ca2+ from the SR, cause depletion of Ca2+ in the SR and increase of intracellular Ca2+ in a resting state. As a result, there is a decrease in Ca2+ release in an excited state, which causes muscle weakness.27 The Tyr523Ser CCD mutant was reported as leaky channels.28 EC uncoupling is a dysfunction of the timing of the open states of 2 receptors, dihydropyridine receptor (DHPR) and RYR1, disabling their coupling. DHPR is an L-type Ca2+ channel and known as a voltage sensor of skeletal muscle. The interaction of DHPR with RYR1 regulates the release of Ca2+ from SR. Consequently, EC uncoupling leads to lack of Ca2+ release from the SR.29 The Ile4897Thr CCD mutant was reported as EC uncoupling.30 Overactive channels are hypersensitive channels associated with MH. In our experiments, there was no difference in intracellular Ca2+ between WT- and Ala4894Pro-transfected cells in a resting state and maximal response. These results indicate that the amount of Ca2+ in the ER of Ala4894Pro-transfected cells is not different from that of WT-transfected cells, which leads us to conclude that the Ala4894Pro mutant is not the leaky channels type. EC uncoupling is insensitive to caffeine loading.27 Therefore, our results may indicate that Ala4894Pro-RyR1 is the EC uncoupling type. However, it is necessary to evaluate the coupling functions of DHPR and RyR1 before making a final conclusion. There are no data yet to link this RyR1 mutation and perturbations in Ca2+ release fluxes to the lack of development of type 2 fibers in CNMDU1, which also must contribute to the myopathy seen in these patients.

We also noted that Ala4894Ser-RyR1–transfected cells were more sensitive to caffeine. Because both serine and threonine contain a hydroxyl group, the hydroxyl group of amino acids at Ala4894 may enhance RyR1 function. Amino acids 4895 to 4901 correspond to pore-forming regions,3133 and Ala4894 is the amino acid adjacent to the beginning of this region. Mutations in the pore-forming region have been reported to change channel conductance and ion selectivity.31 Similarly, replacement of Ala4894 with an amino acid that contains a hydroxyl group may change channel gating and/or Ca2+ conductance in such a manner as to enhance RyR1 function and make it more sensitive to trigger MH.

Ala4894Gly-transfected cells were less sensitive to caffeine. The characteristics of glycine are similar to alanine. However, the function of Ala4894Gly was found to be different from that of the WT. Most mutations in the C-terminal domain have been reported in patients with congenital myopathy,3437 and various artificial variations in the pore-forming region are reported to lead to RyR1 dysfunction.30 Additional studies of variants in the pore-forming region may provide useful knowledge regarding RyR1 function.

The raw Ca2+ release traces of both WT and Ala4894Thr cells (Fig. 1, A and B) had 2 phases: the first phase was the peak of the response and the second phase was the decay of the Ca2+ transient. Our results may correspond to not only pure release of Ca2+ but also to store-operated Ca2+ entry (SOCE), a mechanism of an external Ca2+ entry into cells to replenish the ER/SR store.3840 Our experiments were not able to exclude the influence of SOCE on the development of the cellular Ca2+ transient, but SOCE in these cells is likely coupled to the degree of RyR1 functioning.40 In any case, because the only difference between these cells is the nature of the RyR1 expressed, the results lead us to believe that the differences in 50% effective concentration to RyR1 agonists reflect mutation-induced changes in RyR1 function as it relates to those aspects of cellular Ca2+ homeostasis that are under its control.

The present study was a cell culture–based, functional evaluation of transfected transgene-RyR1, both WT and specifically mutated. Although it is preferable to use tissue-specific biological models, such as murine models, with a specific knock-in transgene in appropriate tissue, i.e., skeletal muscle, our results in cell culture correspond well with patient clinical episodes. We suggest, therefore, that Ala4894Thr and Ala4894Pro are associated with MH and CNMDU1, respectively.

In conclusion, we evaluated RyR1 function after transfection of Ala4894-mutated RyR1 into HEK-293 cells. The Ala4894Thr-transfected cells were more sensitive to caffeine and 4CmC than the WT. In contrast, Ala4894Pro-transfected cells had no response to caffeine or 4CmC. Our results indicate that hypersensitive Ala4894Thr-RyR1 is associated with MH, whereas the poorly functional Ala4894Pro-RyR1 is associated with CNMDU1.

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DISCLOSURES

Name: Toshiaki Haraki, MD.

Attestation: Conduct of study, data analysis, manuscript preparation.

Name: Toshimichi Yasuda, MD, PhD.

Attestation: Study design, conduct of study, manuscript preparation.

Name: Keiko Mukaida, MD, PhD.

Attestation: Study design, manuscript preparation.

Name: Takako Migita, MD, PhD.

Attestation: Manuscript preparation.

Name: Hiroshi Hamada, MD, PhD.

Attestation: Manuscript preparation.

Name: Masashi Kawamoto, MD, PhD.

Attestation: Manuscript preparation.

This manuscript was handled by: Peter J. Davis, MD.

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ACKNOWLEDGMENTS

We thank Professor David H. MacLennan for the generous gift of Rabbit-RyR1/pcDNA. We also thank Professor Ichizo Nishino (Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Japan) for his kind advice. This study was performed at the Analysis Center of Life Science, Hiroshima University, and was supported in part by the Tsuchiya Foundation.

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