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Abstract

Dantrolene is currently the only drug known to specifically treat malignant hyperthermia (MH) crises. Although dantrolene attenuates Ca²⁺ disorders by acting mainly on the ryanodine receptor type 1 (RYR1), some patients who manifest MH without RYR1 variants have also been successfully treated with dantrolene. Thus, dantrolene appears to have an inhibitory effect on patients with and without RYR1 variants. This study aimed to investigate whether the effects of dantrolene differed depending on the presence or absence of RYR1 variants using muscle cells from MH-predisposed individuals. The study participants were individuals diagnosed with MH predisposition by the Ca²⁺-induced Ca²⁺ release rate test. They were genetically tested and divided into two groups: with and without RYR1 variants. We investigated whether these two groups showed differences in the changes in the half-maximal effective concentration (EC₅₀) for caffeine and the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) before and after dantrolene administration. Dantrolene administration significantly increased the EC₅₀ (P < 0.0001) and decreased the resting [Ca²⁺]_i (P < 0.0001). The inhibitory effects of dantrolene and the presence of RYR1 variants showed no statistically significant interactions related to the EC₅₀ (P = 0.59) and resting [Ca²⁺]_i (P = 0.21). In conclusion, the presence or absence of RYR1 variants does not appear to influence the effect of dantrolene.

Keywords

Hyperthermia anaesthesia malignant hyperthermia pharmacology ryanodine receptor calcium release channel dantrolene

Introduction

Malignant hyperthermia (MH) is a pharmacogenetic disorder of the skeletal muscles triggered by volatile anaesthetics or the depolarising muscle relaxant succinylcholine. The pathophysiology of MH is related to malfunction of intracellular Ca²⁺ homeostasis, and MH symptoms manifest as a lethal hypermetabolic crisis associated with a rapid and uncontrolled increase in intracellular Ca²⁺ levels in skeletal muscle cells. Abnormal Ca²⁺ metabolism is mainly caused by functional, structural and morphological abnormalities of ryanodine receptor type 1 (RYR1), which have a significant involvement in susceptibility to MH. The gene variants of RYR1 contribute to MH susceptibility, and 48 RYR1 variants have been identified as diagnostic genetic variants by the European Malignant Hyperthermia Group (EMHG).¹

Many studies have confirmed that the muscles of MH-susceptible individuals exhibit increased sensitivity to direct RYR1 agonists, such as caffeine, halothane and 4-chloro-m-cresol (4CmC), and this increased sensitivity is also likely to be the primary underlying mechanism for triggering the clinical MH syndrome.² Moreover, defective or disordered RYR1 results in uncontrolled release of intracellular Ca²⁺ from the skeletal muscle sarcoplasmic reticulum (SR). In fact, a high resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) has been found in MH-susceptible patients.³

Dantrolene is the only clinically available agent for the speciﬁc treatment of an MH crisis, and its discovery was followed by a substantial reduction in the mortality and morbidity of MH.⁴ Dantrolene is a direct-acting skeletal muscle relaxant that blocks Ca²⁺ release from intracellular storage in the SR. Previous studies have shown that dantrolene acts mainly on RYR1 to inhibit the extent of channel activation. It also decreases Ca²⁺ sensitivity of channel activation and interacts with RYR1 to suppress the channel dysfunction that occurs with MH variants.⁵

Despite the fact that dantrolene shows inhibitory effects on abnormal Ca²⁺ metabolism by acting mainly on RYR1, some individuals showing MH susceptibility without RYR1 variants have also been successfully treated with dantrolene administration.⁶ Thus, dantrolene appears to have an inhibitory effect on both patients with and without RYR1 variants. If the effect of dantrolene is mainly attributable to its effect on RYR1 dysfunction, its effects may differ depending on the presence or absence of RYR1 variants. To date, no reports have examined whether the presence or absence of RYR1 variants causes differences in the potency of dantrolene in human muscle cells. In this study, we investigated whether the effects of dantrolene differed depending on the presence or absence of RYR1 variants using muscle cells from MH-predisposed individuals.

Methods

Protocol

This study was approved by the ethics committee of Hiroshima University Hospital (ヒ-151-9), and written consent was obtained from all participants before the Ca²⁺-induced Ca²⁺ release (CICR) rate test for the use of residual skeletal muscle for the experiments.

Eleven individuals diagnosed with MH predisposition by the CICR rate test were included in this study. They were genetically tested and divided into two groups: those with RYR1 variants (with RYR1 group) and those without RYR1 variants (without RYR1 group). We investigated whether these two groups showed differences in the changes in the half-maximal effective concentration (EC₅₀) for caffeine and resting [Ca²⁺]_i before and after dantrolene administration. Five individuals, diagnosed as MH-negative (MHN) because their CICR was not accelerated, were excluded from the dantrolene study. Thus, no genetic testing was performed on them.

CICR rate test

The CICR rate test was performed as described previously.⁷ Accelerated CICR was defined as a CICR rate more than two standard deviations above the mean of the control CICR values, which were derived from 12 patients with negative results in in vitro contracture tests (IVCT; EMHG protocol) and caffeine–halothane contracture tests (CHCT; North American MH Group protocol) at two or more Ca²⁺ concentrations in accordance with a previous study.⁸

Genetic testing

After obtaining written consent from the study participants, we asked the National Centre of Neurology and Psychiatry to conduct genetic tests to examine the gene variants in the RYR1 region. In one case (case no. 4), genetic testing was performed at Keio Hospital.

Myotube cultures

As described previously,⁹ myoblasts were obtained from the surplus muscle pieces after CICR examination and cultured in Dulbecco’s modified Eagle’s medium and F12 medium (DMEM/F12; Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma–Aldrich, St Louis, MO, USA). The myoblasts were then frozen and stored at –70°C. The frozen myoblasts were defrosted and plated on 35 mm glass-bottom culture dishes with DMEM/F12 medium supplemented with 10% FBS at 37°C under 5% CO₂. At 80% confluence, the medium was changed to DMEM/F12 supplemented with 2% FBS to induce differentiation into myotubes. Seven to 14 days after the medium change, measurements were performed on cells that had morphologically changed into multinucleated and spindle-shaped cells. The following experiments were conducted multiple times using multiple myotubes cultured from the surplus muscle pieces of each case.

Ca²⁺ imaging

Ca²⁺ imaging from a single myotube was performed using the Ca²⁺ fluorescence indicator Fura-2/AM (Dojindo Molecular Technologies, Tokyo, Japan), as previously described.^10,11 Cells were loaded on Fura2/AM at 37°C for one hour and then washed in Hank’s balanced salt solution (HBSS). The ionic composition of HBSS was as follows: 130 mM NaCl, 5.4 mM KCl, 20 mM 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 2.5 mM CaCl₂, 1 mM MgCl₂ and 5.5 mM glucose (pH 7.4). The HBSS used in the experiment was prepared by us. The myotubes on the glass-bottom dishes were maintained for 30 minutes. The myotubes were perfused with HBSS for 30 minutes at a rate of 1.2 ml/min at 37°C prior to the experiments. The myotubes were excited at 340 and 380 nm at 37°C, and the measurements to evaluate intracellular Ca²⁺ changes were obtained at 510 nm using a fluorescence microscope (Nikon, Tokyo, Japan). Perfusion with HBSS was performed, and a Ca²⁺ image analyser (Aquacosmos 2.5; Hamamatsu Photonics, Hamamatsu City, Japan) was used to calculate the 340/380 nm wavelength ratio.

A calibration kit (Invitrogen™, ThermoFisher Scientific, Tokyo, Japan) was used to convert the 340/380 nm wavelength ratio to [Ca²⁺]_i values. Of the myotubes that were successfully cultured, only those in which the 340/380 nm wavelength ratio increased in response to 10 mM caffeine were determined to have successfully expressed RYR1, and were included in the measurement.

Measurement of the sensitivity for 4-chloro-m-cresol and caffeine

Myotubes were perfused with HBSS containing 10 mM caffeine before measurements were obtained. The 340/380 nm wavelength ratio was measured when HBSS containing incremental levels of 4 chloro-m-cresol (4CmC) was perfused into the culture dish. Each solution was washed out for two minutes prior to adding the next incremental level of 4CmC; 4CmC was evaluated at 3, 10, 30, 100, 300, 500 and 1000 μM. The sensitivity of caffeine was measured in the same manner. Various concentrations of caffeine (0.25, 0.5, 1.0, 2.5, 5.0, 10.0 and 20.0 mM) were added to HBSS, and caffeine-induced changes in the 340/380 nm wavelength ratio were measured.

Measurement of the caffeine sensitivity following dantrolene administration

The sensitivity of dantrolene to caffeine was measured by replacing HBSS with HBSS supplemented with 5 or 15 μM dantrolene. Caffeine was added to the HBSS at increasing concentrations in the same way and at the same concentrations as described above, and the caffeine-induced changes in the 340/380 nm wavelength ratio with the addition of dantrolene were measured. After the measurements, myotubes were perfused with HBSS without dantrolene, and the 340/380 nm wavelength ratio was measured with the addition of 10 mM caffeine. When the 340/380 nm wavelength ratio reduced to < 80% of the 340/380 nm wavelength ratio measured before dantrolene addition, the values were discarded and removed from this study (Figure 1).

Figure 1.

Representative recording of Ca²⁺ fluorescence measurement for caffeine in the myotube. The horizontal axis shows the time, and the vertical axis shows the 340/380 nm wavelength ratio of Fura-2 AM fluorescence intensity. The upper bar and the concentration below it indicate the duration of caffeine addition and its concentration. The lower bar indicates perfusion of HBSS with dantrolene. HBSS: Hank’s balanced salt solution.

Measurement of resting [Ca²⁺]_i before and after dantrolene addition

The resting [Ca²⁺]_i was calculated from the 340/380 nm wavelength ratio before stimulation with 10 mM caffeine. After measurement of resting [Ca²⁺]_i, the resting [Ca²⁺]_i with the addition of dantrolene was measured by replacing HBSS with HBSS supplemented with 50 μM dantrolene. The resting [Ca²⁺]_i with the addition of dantrolene was calculated from the 340/380 nm wavelength ratio two minutes after replacing HBSS.

Data analysis

For both the EC₅₀ value and the resting [Ca²⁺]_i value, the median value of each case was used to eliminate outliers. The EC₅₀ of 4CmC and caffeine were calculated from the dose–response curves. To trace these curves, the data were normalised to the maximal response of each myotube. With regard to changes in EC₅₀ and resting [Ca²⁺]_i, two-way repeated-measures analysis of variance was used to evaluate differences between the groups with and without RYR1 and between the conditions before and after dantrolene addition. For the examination of the effect of dantrolene on EC₅₀, Tukey’s multiple comparison test was used for the post hoc test. Regression analysis was used to examine the correlation between resting [Ca²⁺]_i and the EC₅₀ values of caffeine. The difference was considered statistically significant when the P-value was < 0.05. The data were analysed using PRISM v8.0 (GraphPad Software, San Diego, CA, USA).

Results

Gene testing

Of the 11 cases, six showed RYR1 variants. Table 1 shows the results of gene testing and the RYR1 variants found in each case.

Table 1.

The RYR1 variants that were identified.

Case no.	RYR1 variants identified
1	T84M
2	Q155K
3	G341R
4	R614L
5	R2508C
6	L4838V
7	None
8	None
9	None
10	None
11	None

RYR1: ryanodine receptor type 1.

The median EC₅₀ values of 4CmC and caffeine

EC₅₀ values of 4CmC and caffeine and their statistical significance for each group and for the five individuals with MHN whose CICR rate was not accelerated are presented in Tables 2 and 3, respectively. There was no significant difference between the with RYR1 and without RYR1 groups in the EC₅₀ for either 4CmC or caffeine. The median EC₅₀ values of 4CmC and caffeine in the groups with and without RYR1 variants were significantly lower than those in the MHN group.

Table 2.

The median EC₅₀ value of 4CmC and caffeine in each case.

Case no.	EC₅₀ of4CmC (μM)	EC₅₀ ofcaffeine (mM)
With RYR1 group
1	64.025 (n = 6)	2.85 (n = 13)
2	123.20 (n = 9)	2.59 (n = 11)
3	74.41 (n = 5)	2.28 (n = 18)
4	81.83 (n = 4)	2.32 (n = 16)
5	127.90 (n = 4)	2.31 (n = 14)
6	115.60 (n = 5)	1.90 (n = 4)
Mean (standard deviation)	97.83 (27.61)	2.37 (0.32)
Without RYR1 group
7	141.90 (n = 14)	2.64 (n = 26)
8	115.90 (n = 10)	2.75 (n = 7)
9	117.30 (n = 8)	2.62 (n = 11)
10	141.10 (n = 9)	2.39 (n = 16)
11	135.55 (n = 6)	3.25 (n = 15)
Mean (standard deviation)	130.35 (12.80)	2.73 (0.32)
MHN (CICR–) group
12	321.55 (n = 6)	5.73 (n = 11)
13	240.50 (n = 9)	5.38 (n = 7)
14	214.45 (n = 8)	5.05 (n = 23)
15	313.40 (n = 5)	3.90 (n = 13)
16	190.10 (n = 10)	4.85 (n = 9)
Mean (standard deviation)	256.00 (58.96)	4.98 (0.69)

The median EC50 values of 4CmC and caffeine in each individual, and the mean (standard deviation) of those values for each group (with RYRI, without RYR1, and MHN).

EC₅₀: half-maximal effective concentration; 4CmC: 4-chloro-m-cresol; MHN: malignant hyperthermia negative; n: number of myotubes.

Table 3.

Results of one-way analysis of variance for EC₅₀ values of 4CmC and caffeine between the with RYR1 group, without RYR1 group and MHN group.

	P-value (4CmC)	P-value (caffeine)
With RYR1 group vs. without RYR1 group	0.3555	0.443
With RYR1 group vs. MHN group	<0.0001	<0.0001
Without RYR1 group vs. MHN group	0.0004	<0.0001

There was no significant difference between the with RYR1 group and without RYR1 group in the EC₅₀ for either 4CmC or caffeine. There were significant differences between the with RYR1 group and MHN group and between the without RYR1 group and MHN group in the EC₅₀ for both 4CmC and caffeine.

Note that the MHN group was used only as a negative control and was not the subject of this study.EC₅₀: half-maximal effective concentration; 4CmC: 4-chloro-m-cresol; RYRI: ryanodine receptor type 1; MHN: malignant hyperthermia negative.

Median EC₅₀ values of caffeine and the changes in the EC₅₀ of caffeine with dantrolene

The median EC₅₀ values of caffeine for the two groups before dantrolene addition and with the addition of 5 and 15 μm dantrolene are shown in Figure 2(a). The P-value for the median EC₅₀ between the with and without RYR1 groups was 0.04 (Figure 2(b)). The EC₅₀ of caffeine was significantly increased by the addition of dantrolene (P < 0.0001; Figure 2(c)). However, the changes in the EC₅₀ values of caffeine did not show a significant interaction between dantrolene addition and the presence of RYR1 variants (P = 0.59). Results indicated that dantrolene had a significant effect on the changes in the EC₅₀ values.

Figure 2.

The median EC₅₀ values for caffeine before and after dantrolene addition in individuals with and without RYR1 variants. (a) Scatter plot showing the median EC₅₀ for each individual in both groups at each dantrolene concentration. In the two-way ANOVA, there was statistically no influence of interaction between dantrolene and RYR1 variants on the EC₅₀ (dantrolene×RYR1 variant: P=0.59). (b) The graph shows the median EC₅₀ of each group (with or without RYR1 variation) in the present study. In the two-way ANOVA, the factor RYR1 variant was significant (RYR1 variant: P=0.04). (c) In the two-way ANOVA, the factor dantrolene was significant (dantrolene: P < 0.0001). Tukey’s multiple comparison test showed that EC₅₀ changed significantly depending on the dantrolene dose. Data represent mean (standard deviation). The circles and open circles show the median EC₅₀ for each individual; n indicates the number of myotubes used. ANOVA: analysis of variance; EC₅₀: the half-maximal effective concentration; RYR1: ryanodine receptor type 1. *P < 0.05; **P < 0.0001.

Changes in the resting [Ca²⁺]_i with dantrolene

The median resting [Ca²⁺]_i varied among the RYR1 variants, with the T84M and R614L variants presenting the lowest (77.04 nM) and the highest (123.75 nM) values, respectively. Similarly, the median resting [Ca²⁺]_i varied from 57.35 to 123.33 nM in the group without RYR1 variants (Figure 3(a)). The mean (standard deviation (SD)) values of the median resting [Ca²⁺]_i in the two groups were 104.08 (16.50) and 81.76 (26.82) nM in the with RYR1 group and without RYR1 group, respectively. The mean (SD) values of the median resting [Ca²⁺]_i with dantrolene in the two groups were 74.05 (14.00) and 60.53 (19.49) nM in the with RYR1 group and without RYR1 group, respectively. Although there was no significant difference in the median resting [Ca²⁺]_i values between the two groups (P = 0.15; Figure 3(b)), the addition of dantrolene significantly decreased the median resting [Ca²⁺]_i values (P < 0.0001; Figure 3(c)). There was no significant interaction between the reduction of resting [Ca²⁺]_i by dantrolene addition and the presence of RYR1 variants (P = 0.21). The scatter plot data (Figure 3(a)) shows comparisons between the two groups for the relative contribution of dantrolene to the resting [Ca²⁺]_i.

Figure 3.

The median resting [Ca²⁺]_i values before and after dantrolene addition in cases with and without RYR1 variants. (a) The scatter plot data showing comparisons between the groups with and without RYR1 variant for the relative contribution of dantrolene to the resting [Ca²⁺]_i. In the two-way ANOVA, there was no significant interaction between the reduction of resting [Ca²⁺]_i values by dantrolene administration and the presence of RYR1 variant (dantrolene×RYR1 variant: P = 0.21). (b) The graph shows the resting [Ca²⁺]_i values of each group (with or without RYR1 variation) in this study. In the two-way ANOVA, the factor RYR1 variant was not significant (RYR1 variant: P = 0.15). (c) The graph shows the resting [Ca²⁺]_i values before and after dantrolene administration for all participants in this study. In the two-way ANOVA analysis, the resting [Ca²⁺]_i values were significantly decreased by dantrolene administration (P < 0.0001). Data represent mean (standard deviation). The circles and open circles show the median EC₅₀ for each individual; n indicates the number of myotubes used. **P < 0.0001.RYR1: ryanodine receptor type 1; ANOVA: analysis of variance.

Correlation between the resting [Ca²⁺]_i and caffeine EC₅₀ values

Our results showed no correlation between resting [Ca²⁺]_i and the EC₅₀ values of caffeine (r² = 0.08, P = 0.40).

Discussion

We have shown that the inhibitory effect of dantrolene did not differ in the presence or absence of RYR1 variants in human muscle cells with an MH predisposition. This confirms that dantrolene is an effective treatment for patients with MH, regardless of whether they have RYR1 variants.

The experimental data showed that RYR1 variants did not influence the alleviating effect of dantrolene on sensitivity to caffeine. The EC₅₀ values for 4CmC and caffeine were significantly lower in both the with and without RYR1 groups than in the MHN group.

The median resting [Ca²⁺]_i value varied in myotubes with or without RYR1 variants, which is consistent with the findings from a previous report in which the resting [Ca²⁺]_i differed depending on the site of the variant.¹²

According to a previous report,¹³ spontaneous Ca²⁺ leakage through MH mutant channels can lead to changes in the resting [Ca²⁺]_i, but compensatory mechanisms exist, leading to the rapid re-establishment of Ca²⁺ homeostasis. The compensatory control of resting [Ca²⁺]_i in skeletal muscle is thought to involve the expression of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase, Ca²⁺ pumps that take up Ca²⁺ from the cytoplasm into the SR, and other channels, pumps and exchangers involved in Ca²⁺ metabolism in both the sarcolemma and SR. The decrease in resting [Ca²⁺]_i in all myotubes suggests that dantrolene suppresses Ca²⁺ leakage from the SR to the cytoplasm independent of RYR1 variants. Dantrolene has other sites of action in addition to RYR1, and previous studies using animal models have reported that dantrolene acts on Ca²⁺ regulatory mechanisms other than RYR1. For example, dantrolene has been previously shown to block non-specific sarcolemmal cation entry channels and it has been suggested that the action at these channels may be important for the therapeutic effects of dantrolene in MH. This result is consistent with the results of our study using human muscle cells.¹⁴

Caffeine increases the open probability of RYR1 and facilitates Ca²⁺ release.¹⁵ A reduction in the EC₅₀ for caffeine is a sign of accelerated Ca²⁺ release via RYR1 variants. Tong et al. reported that the resting [Ca²⁺]_i showed no linear correlation with caffeine sensitivity.¹³ Our results also showed no correlation between the resting [Ca²⁺]_i and the caffeine EC₅₀ values. Thus, in assessments regarding abnormalities of the Ca²⁺ metabolism in MH, EC₅₀ can be considered as an independent factor of resting [Ca²⁺]_i.

A limitation of this study is that the presence of MH predisposition was determined by the CICR rate test rather than the more widely accepted CHCT/IVCT. However, the CICR rate test can reveal abnormalities associated with Ca²⁺ release from the SR, a process mainly controlled by RYR1 in skeletal muscle.⁹ The EC₅₀ values of 4CmC and caffeine in participants with accelerated CICR rates in this study were lower than the previously reported cut-off values to diagnose MH,⁹ which confirmed that the participants in this study had MH predisposition. The RYR1 variants identified in this study are shown in Table 1. Of these, G341R, R614L, R2508C and L4838V have been recognised as diagnostic MH variants by EMHG.¹ T84M and Q155K were functionally analysed in a previous study and have been suggested to contribute to MH crisis.^16,17 Using a modified version of the American College of Medical Genetics and Genomics guidelines,¹⁸ the T84M and Q155K variants can be classified as uncertain significance and likely pathogenic, respectively.

Another major limitation of this study was the limited number of samples. Therefore, care should be taken in interpreting the results of the statistical tests in this study.

Another limitation is that RYR1 expression was not consistent in all the cultured and differentiated myotubes. Since RYR1 expression itself significantly increases the resting [Ca²⁺]_i,¹⁹ differences in the degree of differentiation of the sample myotubes may have resulted in differential expression of RYR1 and affected the assessment of the resting [Ca²⁺]_i. Because of the limited number of myotubes that could be cultured from muscle biopsies, it was difficult to evaluate the RYR1 expression by western blotting in this study.

In the present study, the resting [Ca²⁺]_i was calculated by converting the 340/380 nm wavelength ratio to [Ca²⁺]_i. The calibration curve was constructed by adding Fura-2 to the Ca²⁺ buffer, of known Ca²⁺ concentration, measuring the fluorescence intensity ratio of the buffer and then plotting the Ca²⁺ concentration against the ratio value. However, it has been reported that fluorescence indicators in cells, such as Fura-2, interact with high concentrations of intracellular proteins and cell membranes, altering their fluorescence properties and chelation constants and changing their fluorescence spectra with Ca²⁺ buffer.^20,21 Reportedly, when the protein concentration increases, [Ca²⁺]_i produced by the calibration curve using Ca²⁺ buffer is measured to be lower than the actual [Ca²⁺]_i concentration. Furthermore, it is difficult to make a buffer with the exact intracellular composition, and therefore an accurate calibration curve cannot be plotted. Thus, it should be noted that the resting [Ca²⁺]_i in this study may differ from the actual values.

In skeletal muscle, the resting [Ca²⁺]_i is determined by Ca²⁺ movement between the cytosol, SR and the extracellular matrix. Both RYR1 and dihydropyridine receptor (DHPR) are involved in Ca²⁺ release from SR, and mutations in calcium channel, voltage-dependent, L type, α1S subunit (CACNA1S), which encodes DHPR, have been implicated in MH. The influx of extracellular Ca²⁺ occurs through store-operated Ca²⁺ entry (SOCE), transient receptor potential and excitation-coupled Ca²⁺ entry. Orai1 and stromal interaction molecule 1 are Ca²⁺ proteins mainly responsible for SOCE in skeletal muscle. Therefore other factors aside from RYR1 variants may influence resting [Ca²⁺]_i in myotubes from individuals predisposed to MH.

In addition to RYR1, CACNA1S and STAC3 have also been associated with MH susceptibility and severe dysregulation of skeletal muscle Ca²⁺ homeostasis, which results in the clinical features of an MH reaction under anaesthesia. The relationship between genetic variants in these genes and the effects of dantrolene should be investigated in the future.

In conclusion, as there was no significant interaction between the inhibitory effect of dantrolene and the presence or absence of the RYR1 variants in either EC₅₀ for caffeine or resting [Ca²⁺]_i, these inhibitory effects of dantrolene did not differ in relation to the presence or absence of RYR1 variants.

Footnotes

Author Contribution(s)

Yuko Noda: Conceptualization; Data curation; Funding acquisition; Investigation; Methodology; Project administration; Writing – original draft; Writing – review & editing.

Keiko Mukaida: Investigation; Methodology; Writing – original draft; Writing – review & editing

Hirotsugu Miyoshi: Investigation; Writing – original draft; Writing – review & editing

Ryuji Nakamura: Investigation; Writing – original draft; Writing – review & editing

Toshimichi Yasuda: Conceptualization; Investigation; Writing – review & editing.

Noboru Saeki: Conceptualization; Supervision; Writing – review & editing.

Ichizo Nishino: Investigation; Resources; Writing – review & editing.

Yasuo M Tsutsumi: Conceptualization; Funding acquisition; Supervision; Writing – original draft; Writing – review & editing.

Declaration of conflicting interests

The authors have no conflicts of interest to declare.

Funding

The authors received no financial support for the research, authorship and/or publication of this article.

ORCID iDs

Yuko Noda

Noboru Saeki

Yasuo M Tsutsumi

References

European Malignant Hyperthermia Group. Genetics in malignant hyperthermia, https://www.emhg.org/diagnostic-mutations (accessed 20 March 2021).

Riazi

Kraeva

Hopkins

PM.

Malignant hyperthermia in the post-genomics era: new perspectives on an old concept. Anesthesiology 2018; 128: 168–180.

Lopez

Gerardi

Lopez

, et al. Effects of dantrolene on myoplasmic free [Ca2+] measured in vivo in patients susceptible to malignant hyperthermia. Anesthesiology 1992; 76: 711–719.

Krause

Gerbershagen

Fiege

, et al. Dantrolene – a review of its pharmacology, therapeutic use and new developments. Anaesthesia 2004; 59: 364–373.

Paul-Pletzer

Yamamoto

Bhat

, et al. Identification of a dantrolene-binding sequence on the skeletal muscle ryanodine receptor. J Biol Chem 2002; 277: 34918–34923.

Toppin

Chandy

Ghanekar

, et al. A report of fulminant malignant hyperthermia in a patient with a novel mutation of the CACNA1S gene. Can J Anesth 2010; 57: 689–693.

Endo

Calcium-induced calcium release in skeletal muscle. Physiol Rev 2009; 89: 1153–1176.

Oku

Mukaida

Nosaka

, et al. 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.

Kobayashi

Mukaida

Migita

, et al. Analysis of human cultured myotubes responses mediated by ryanodine receptor 1. Anaesth Intensive Care 2011; 39: 252–261.

10.

Watanabe

Miyoshi

Noda

, et al. Effects of remimazolam and propofol on Ca2+ regulation by ryanodine receptor 1 with malignant hyperthermia mutation. Biomed Res Int 2021; 2021: 8845129.

11.

Miyoshi

Yasuda

Otsuki

, et al. Several ryanodine receptor type 1 gene mutations of p.Arg2508 are potential sources of malignant hyperthermia. Anesth Analg 2015; 121: 994–1000.

12.

Yang

Esteve

Pessah

, et al. 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–C1598.

13.

Tong

McCarthy

MacLennan

DH.

Measurement of resting cytosolic Ca2+ concentrations and Ca2+ store size in HEK-293 cells transfected with malignant hyperthermia or central core disease mutant Ca2+ release channels. J Biol Chem 1999; 274: 693–702.

14.

Eltit

Ding

Pessah

, et al. Nonspecific sarcolemmal cation channels are critical for the pathogenesis of malignant hyperthermia. FASEB J 2013; 27: 991–1000.

15.

Zucchi

Ronca-Testoni

The sarcoplasmic reticulum Ca2+ channel/ryanodine receptor: modulation by endogenous effectors, drugs and disease states.

Pharmacol Rev 1997; 49: 1–51.

16.

Kondo

Yasuda

Mukaida

, et al. Genetic and functional analysis of the RYR1 mutation p.Thr84Met revealed a susceptibility to malignant hyperthermia. J Anesth 2018; 32: 174–181.

17.

Nakano

Oyamada

Yamazawa

, et al. Construction and expression of ryanodine receptor mutants relevant to malignant hyperthermia patients in Japan. Showa Univ J Med Sci 2014; 26: 27–38.

18.

Johnston

Dirksen

Girard

, et al. Variant curation expert panel recommendations for RYR1 pathogenicity classifications in malignant hyperthermia susceptibility. Genet Med 2021; 23: 1288–1295.

19.

Eltit

Yang

, et al. RyR1-mediated Ca2+ leak and Ca2+ entry determine resting intracellular Ca2+ in skeletal myotubes. J Biol Chem 2010; 285: 13781–13787.

20.

Konishi

Olson

Hollingworth

, et al. Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements. Biophys J 1988; 54: 1089–1104.

21.

Baylor

Hollingworth

Measurements and interpretation of cytoplasmic [Ca²⁺]_i signals from calcium-indicator dyes. News Physiol Sci 2000; 15: 19–26.

The effects of dantrolene in the presence or absence of ryanodine receptor type 1 variants in individuals predisposed to malignant hyperthermia

Abstract

Abstract

Keywords

Introduction

Methods

Protocol

CICR rate test

Genetic testing

Myotube cultures

Ca2+ imaging

Measurement of the sensitivity for 4-chloro-m-cresol and caffeine

Measurement of the caffeine sensitivity following dantrolene administration

Measurement of resting [Ca2+]i before and after dantrolene addition

Data analysis

Results

Gene testing

The median EC50 values of 4CmC and caffeine

Median EC50 values of caffeine and the changes in the EC50 of caffeine with dantrolene

Changes in the resting [Ca2+]i with dantrolene

Correlation between the resting [Ca2+]i and caffeine EC50 values

Discussion

Footnotes

Author Contribution(s)

Declaration of conflicting interests

Funding

ORCID iDs

References

Ca²⁺ imaging

Measurement of resting [Ca²⁺]_i before and after dantrolene addition

The median EC₅₀ values of 4CmC and caffeine

Median EC₅₀ values of caffeine and the changes in the EC₅₀ of caffeine with dantrolene

Changes in the resting [Ca²⁺]_i with dantrolene

Correlation between the resting [Ca²⁺]_i and caffeine EC₅₀ values