Effects of propofol on calcium homeostasis in human skeletal muscle.
Individuals who are malignant hyperthermia susceptible (MHS) have been identified in Europe (the European Malignant Hyperthermia group) and North America (the North American Malignant Hyperthermia group) using an in vitro contracture test (2,7). In Japan, however, predisposition to MH is mainly diagnosed using the [Ca.sup.2+]-induced [Ca.sup.2+] release (CICR) rate test (8-10). This test shows the increase in CICR rate in individuals with predisposition to MH.
Numerous studies have shown that volatile anaesthetics activate RYR1 to evoke MH (11,12). The intravenous anaesthetic agent propofol has been suggested to be safe for MHS individuals in clinical settings (13,14). In vitro, propofol has been examined in animals (15,16) or by measuring human muscle contracture (14). However, its safety has not been confirmed by studies at the human cellular level on [Ca.sup.2+] homeostasis. Recently, we examined the effects of propofol on [Ca.sup.2+] homeostasis in isolated human myotubes from two patients carrying RYR1 mutations linked to MH (17). With the addition of more cases, the aim of this study was to investigate the safety of propofol and its mode of action upon skeletal muscle cells.
PATIENTS AND METHODS
Including the two patients previously reported17, 20 individuals underwent muscle biopsy from their quadriceps or biceps brachii muscle to determine their susceptibility to MH. They were classified into an accelerated group and a non-accelerated group using the CICR rate test according to the protocol developed by Endo et al (18). Their characteristics and results from the CICR rate test are summarised in Table 1. The accelerated group included two individuals carrying an RYR1 mutation and two patients who had experienced an MH episode (CGS 5-6). Case no. 1 had a C>G point mutation in RYR1 exon 101 at position 14512. Case no. 2 had a C>T point mutation in RYR1 exon 47 at position 7522 and the pathologic diagnosis was myopathic changes with some fibres exhibiting cores or core-like structures, type 1 fibre predominance and type 2b fibredeficiency (10,17). The non-accelerated group did not include individuals with muscle disease or anyone who reported a previous MH episode.
This study was investigated by using surplus muscle after the CICR rate test. Prior to the study, written informed consent was obtained from the individuals and their families and this study was approved by the ethics committee of Hiroshima University.
Effects of propofol on CICR
By using skinned fibres, we measured the effects of propofol on [Ca.sup.2+] release. Namely, purified 2, 6-diisopropylphenol (propofol, Biomedical, France) was prepared as a 1.0 M stock solution in dimethyl sulfoxide (DMSO, Sigma) and diluted to 10, 100 and 1000 [micro]M with 1.0 [micro]M [Ca.sup.2+] for testing.
Thin bundles of intact fibres were isolated from the biopsied specimens. To destroy the semipermeability of the surface membrane, but not that of the sarcoplasmic reticulum (SR), fibres were treated with 50 [micro]g x [ml.sup.-1] saponin for 30 minutes. Two or three fibres were tied together with a single silk thread and connected to a strain gauge transducer (N-3193, Capto, Norway) and an amplifier (DSA-601B, Minebea, Japan). Solutions used for the measurements (solution A in Table 2) were placed in 0.5 ml wells in an aluminum plate. The temperature of the solutions was maintained at 20[degrees]C by using circulating water underneath the plate. The SR was loaded with a fixed amount of [Ca.sup.2+] through the [Ca.sup.2+] pumps in the presence of Mg-ATP and ATP was removed to prevent re-uptake. The muscle specimens were then treated with various concentrations of propofol (0, 10, 100 and 1000 [micro]M) with 1.0 [micro]M [Ca.sup.2+]. In order to assay the remaining [Ca.sup.2+] in the SR, high concentrations of caffeine caused emptying of the SR [Ca.sup.2+] store (Figure 1, A-1). The CICR rate was estimated by comparing the amount of [Ca.sup.2+] remaining in the SR to the fixed amount of [Ca.sup.2+].
To measure baseline [Ca.sup.2+] release from the SR independent of CICR, [Ca.sup.2+] release was measured using [Ca.sup.2+]-free solution, and after the addition of 1000 [micro]M propofol to the [Ca.sup.2+]-free solution, [Ca.sup.2+] release was again measured (Figure 1, A-2).
In skinned fibre experiments, we measured the effects of propofol on [Ca.sup.2+] uptake in the accelerated group. A 1.0 M propofol stock solution was diluted to 100 and 1000 [micro]M with loading solution for testing (solution B in Table 2). The SR was loaded with [Ca.sup.2+] though the [Ca.sup.2+] pumps with various concentrations of propofol (0, 100 and 1000 [micro]M) for various periods of time (0.25, 0.5, 1.0 and 2.0 minutes). The [Ca.sup.2+] content in the SR was assayed after inducing its release with a high concentration of caffeine. Uptake of [Ca.sup.2+] into the SR was estimated from the [Ca.sup.2+] content of the SR (Figure 1B). Values were calculated as the percentage of the control, which was taken with 0 [micro]M propofol for two minutes.
The skeletal muscle cells isolated from the patients were maintained in dulbecco's modified Eagle medium (Invitrogen, USA) supplemented with 10% heat-inactivated bovine calf serum (FBS, Sigma-Aldrich, USA) containing 1% ampicillin sodium salt, kanamycin sulphate (Sigma, USA) and amphotericin B (Invitrogen, USA), in 25 [cm.sup.2] cell culture flasks (Corning, USA) and a 5% C[O.sub.2] atmosphere at 37[degrees]C. The medium was changed every three days. After two or three weeks in culture, the cells were plated on 35 mm glass-bottom culture dishes with 10 mm microwells (MatTek, USA), and allowed to grow for 10 to 14 days (20) in dulbecco's modified Eagle medium with 2% FBS until the myoblasts fused to form myotubes. We used the myotubes formed by fusion of satellite cells with a fibre-like shape and multiple nuclei.
[FIGURE 1 OMITTED]
[Ca.sup.2+] imaging of myotubes
Cells were washed in HEPES-buffered salt solution (HBSS) containing 130 mM NaCl, 5.4 mM KCl, 20 mM HEPES, 2.5 mM Ca[Cl.sub.2], 1 mM Mg[Cl.sub.2] and 5.5 mM glucose at pH 7.4. The cells were loaded with 5.0 [micro]M Fura-2 AM (Dojindo, Japan) in HBSS for one hour at room temperature (24 to 26[degrees]C) and washed with HBSS. The cells were then stimulated alternately at 340 nm and 380 nm. Fluorescence emission at 510 nm was measured using a fluorescence microscope (Nikon, Japan). Images were acquired using a cooled, high-speed digital video camera (ORCA-AG, Hamamatsu, Japan). HBSS was perfused into the sample dishes at a rate of 1.2 ml per minute at 37[degrees]C. The 1.0 M propofol stock solution in DMSo was diluted with HBSS to make the following test concentrations: 1, 3, 10, 30, 100, 300, 1000, 3000 and 5000 [micro]M. only the cells that racted to 20 mM caffeine (Wako, Japan) with an increase in the intracellular [Ca.sup.2+] concentration were used for experiments. Propofol-induced changes in Fura-2 aM fluorescence were measured and the 340/380 nm signal ratio was calculated using a [Ca.sup.2+] imaging system (Aquacosmos 2.5, Hamamatsu Photonics, Japan) within 90 minutes after washing away the excess Fura-2 AM. The dish was rinsed with HBSS for three minutes before the next dose was given. Similarly, caffeine-induced changes in Fura-2 AM fluorescence were measured using various caffeine concentrations: 0.25, 0.5, 1.0, 2.5, 5.0, 10.0 and 20.0 mM. The two fluorescence ratios were converted into [Ca.sup.2+] concentrations using a calibration curve constructed with a calibration kit (Fura-2 Calcium Imaging Calibration Kit, Invitrogen, USA).
L-type [Ca.sup.2+] channel blocker
Nifedipine dissolved in DMSO was diluted with HBSS to 50 [micro]M. Myotubes were treated with 1000 [micro]M propofol for three minutes, which was used as the control and washed with HBSS. Subsequently, after pretreatment with 50 [micro]M nifedipine, 1000 [micro]M propofol was added to the solution for three minutes. Fura-2 AM fluorescence was measured and the 340/380 nm ratio was calculated using a [Ca.sup.2+] imaging system. The same procedure was performed with 10 mM caffeine.
Procaine hydrochloride was diluted with HBSS to 10 mM. Myotubes were treated in 1000 [micro]M propofol for three minutes, which was used as the control and washed with HBSS. Subsequently, after pretreatment with 10 mM procaine, 1000 [micro]M propofol was added to the solution for three minutes. Fura-2 AM fluorescence was measured and the 340/380 nm ratio was calculated using a [Ca.sup.2+] imaging system. The same procedure was performed with 10 mM caffeine.
[FIGURE 2 OMITTED]
The changes in the ratios were calculated from the difference between the maximal response and the preceding baseline. To obtain dose-response curves, data for propofol and caffeine were normalised to the maximum response observed with 20 mM and 10 mM caffeine, respectively. Data analysis was performed using PRISM software (GraphPad Software, USA) with Excel-based templates (Microsoft, USA).
Unpaired t-tests were used to generate statistical comparisons between the accelerated group and the non-accelerated group. Values are shown as the means [+ or -] SEM. P values less than 0.05 were considered to be significant.
Effects of propofol on CICR
In 1 [micro]M [Ca.sup.2+], an increase in the [Ca.sup.2+] release rate was observed only with 1000 [micro]M propofol (Figure 2); compared with the propofol-free condition, the rates increased by 210 [+ or -] 5% in the accelerated group and 194 [+ or -] 15% in the non-accelerated group. There was no significant difference between the groups.
In [Ca.sup.2+]-free solution, 1000 [micro]M propofol increased the [Ca.sup.2+] release rate by 143 [+ or -] 3% in the accelerated group and 127 [+ or -] 20% in the non-accelerated group, as compared with the propofol-free condition. The values were not significantly different between the groups. There was, however, a significant difference between the groups in the results obtained with 1.0 [micro]M [Ca.sup.2+] and the [Ca.sup.2+]-free solution. DMSO at the same concentrations had no effect on the CICR rate.
Uptake of [Ca.sup.2+]
Uptake of [Ca.sup.2+] into the SR did not change with 100 [micro]M propofol. However, inhibition of uptake was observed with 1000 [micro]M propofol, which was significantly different from the propofol-free condition (Figure 3).
[FIGURE 3 OMITTED]
[FIGURE 4 OMITTED]
[EC.sub.50] for propofol
The 340/380 nm ratio did not increase when the concentration of propofol remained within 1 to 30 [micro]M, whereas it increased significantly when the concentration was greater than 100 [micro]M. A dose-figure response curve was computed by normalising the rise of the myoplasmic [Ca.sup.2+] concentration observed with each dose to the maximum response using 20 mM caffeine (Figure 4). The curve of the accelerated group shifted to the left of the non-accelerated group. The calculated half-maximal activation concentrations ([EC.sub.50]) for propofol were 274.7 [+ or -] 33.9 [micro]M in the accelerated group and 543.6 [+ or -] 79.5 [micro]M in the non-accelerated group (Table 3). The [EC.sub.50] values for caffeine were 3.04 [+ or -] 0.27 mM in the accelerated group and 4.99 [+ or -] 0.30 mM in the non-accelerated group. DMSO at the same concentrations had no effect on the 340/380 nm ratio.
[FIGURE 5 OMITTED]
L-type [Ca.sup.2+] channel blocker
The propofol-induced [Ca.sup.2+] response was inhibited with 50 [micro]M nifedipine by 18.0 [+ or -] 9.0% of the control response (taken with the same concentration of propofol in nifedipine-free conditions) in the accelerated group and by 21.3 [+ or -] 6.0% in the non-accelerated group (Figure 5). There was no statistical significance between the groups. However, nifedipine significantly inhibited the propofol-induced [Ca.sup.2+] response compared with the caffeine-induced [Ca.sup.2+] response.
[FIGURE 6 OMITTED]
The propofol-induced [Ca.sup.2+] response was inhibited with 10 mM procaine by 4.2 [+ or -] 0.9% of the control response (taken with the same concentration of propofol in procaine-free conditions) in the accelerated group and by 7.6 [+ or -] 4.1% of the control response in the non-accelerated group (Figure 6). There was no statistical significance between the groups. Compared with the results obtained with caffeine however, procaine inhibited the propofol-induced [Ca.sup.2+] response compared with caffeine. Procaine attenuated caffeine-induced [Ca.sup.2+] response by approximately 30% of the control response.
By investigating intracellular [Ca.sup.2+] homeostasis in human skeletal muscle, we have demonstrated that the [EC.sub.50] of propofol is much higher than the concentrations used clinically, i.e. low concentrations of propofol did not promote CICR and inhibit [Ca.sup.2+] uptake into the SR and the mode of the action of propofol differed from that of caffeine in the presence of a [Ca.sup.2+] channel blocker or CICR inhibitor. These findings provide a possible physiologic basis for the clinical observation that propofol is safe in individuals with predisposition to MH. In addition, propofol and caffeine may interact differently with the RYR1 [Ca.sup.2+] release channel.
CICR from the SR
In skinned fibre experiments, intracellular [Ca.sup.2+] levels become equal to extracellular [Ca.sup.2+] levels by influx of extracellular fluid, namely treated solution, into the cytoplasm due to destruction of the plasma membrane function with saponin. We measured the effects of propofol on the [Ca.sup.2+] release rate in 1.0 [micro]M [Ca.sup.2+]. Propofol at concentrations of 10 and 100 [micro]M did not enhance [Ca.sup.2+] release, whereas 1000 [micro]M doubled the [Ca.sup.2+] release rate compared with the propofol-free condition. Only high concentrations of propofol affected RYR1-mediated [Ca.sup.2+] efflux from SR vesicles. A study of the effects of propofol on [Ca.sup.2+] regulation in swine with MHS showed that propofol at concentrations ranging from 10 to 500 [micro]M had no effect on ryanodine receptors, whereas concentrations higher than 500 [micro]M propofol produced a response. In addition, propofol at concentrations greater than 300 [micro]M competed with [[sup.3]H]ryanodine for binding to ryanodine receptors (21). Our results involving human muscle experiments are consistent with those of the previous animal study. Thus, it appears that only high concentrations of propofol affect RYR1 as an agonist.
At the same concentration of propofol (1000 [micro]M), we also measured [Ca.sup.2+] release from the SR in [Ca.sup.2+]-free solution to determine the degree of [Ca.sup.2+] leak from the SR that was independent of cytosolic [Ca.sup.2+]. There was a significant difference between the results observed with 1.0 [micro]M [Ca.sup.2+] (CICR) and the [Ca.sup.2+]-free solution ([Ca.sup.2+] leak). In the presence of cytosolic [Ca.sup.2+], [Ca.sup.2+] release rate was increased. Therefore, the increase in [Ca.sup.2+] release induced by propofol was thought to result from augmented CICR.
Uptake of [Ca.sup.2+]
[Ca.sup.2+] released from the SR is taken back up into the SR via [Ca.sup.2+] pumps. We investigated [Ca.sup.2+] uptake into the SR in skinned fibres. Using loading solution that included 100 [micro]M or 1000 [micro]M propofol, we measured changes in the amount of [Ca.sup.2+] taken up into the SR. Due to the reduced [Ca.sup.2+] influx into the SR by propofol, the amount of [Ca.sup.2+] in the SR fell. Propofol at a concentration of 1000 [micro]M, but not 100 [micro]M, inhibited [Ca.sup.2+] uptake. In addition, it has previously been reported that [Ca.sup.2+]-ATPase activity in the SR is inhibited by concentrations of propofol greater than 200 [micro]M21, which was consistent with our results.
Muscle contraction is driven by an increase in the intracellular [Ca.sup.2+] concentration, which occurs as [Ca.sup.2+] is released from the SR through activation of ryanodine receptors. The intracellular [Ca.sup.2+] level is regulated by [Ca.sup.2+] release from and uptake into the SR. In skeletal muscle, extrusion of [Ca.sup.2+] from the cytosol occurs mainly by uptake through [Ca.sup.2+]-ATPase pumps into the SR, although some [Ca.sup.2+] are pumped out of cells via [Na.sup.+]/[Ca.sup.2+] exchange.
In muscles with predisposition to MH, although the [Ca.sup.2+] release rate is accelerated compared with muscles without predisposition, the [Ca.sup.2+] release rate is slower than the [Ca.sup.2+] uptake rate. Triggering by agents that cause the [Ca.sup.2+] release rate to overtake the [Ca.sup.2+] uptake rate result in an uncontrolled rise in the cytoplasmic [Ca.sup.2+] level and a clinical MH crisis. Halothane did not affect the [Ca.sup.2+] uptake rate, and the [Ca.sup.2+] uptake rate was unchanged in both MH susceptible and normal pigs (22).
High concentrations of propofol affected [Ca.sup.2+] homeostasis, whereas propofol in low concentrations did not inhibit uptake of [Ca.sup.2+] into the SR or increase of [Ca.sup.2+] release from the SR. Thus, clinically relevant concentrations of propofol have no marked effects on CICR and [Ca.sup.2+]-ATPases, indicating that propofol should not trigger MH.
[EC.sub.50] of propofol
Propofol is used for general anaesthesia and sedation in intensive care units at clinical concentrations ranging from 2 to 6 [micro]g/ml, equivalent to 11 to 34 [micro]M. Because the serum-protein binding rate ranges from 97 to 98%, free propofol concentrations under these conditions are assumed to be approximately 1 [micro]M (23). The 340/380 nm ratio did not increase with a propofol concentration range of 1 to 30 [micro]M. The [EC.sub.50] values for propofol in the previous two cases, which mutations were located in C-terminal or central region,
were 118.1 [micro]M and 420.5 [micro]M respectively (17), and those from 10 individuals with predisposition to MH (the accelerated group) in the present study ranged from 118.1 [micro]M to 420.5 [micro]M. The average value was 274.7 [micro]M, which is 100-fold greater than the clinically used concentrations. The [EC.sub.50] value in the non-accelerated group was 543.6 [micro]M. Though we could not investigate individuals with other mutations, we investigated 10 MH predisposed individuals, including two patients who had experienced a MH episode (CGS 5-6) (24). Our findings, therefore, confirm that propofol has no significant clinical effect on intracellular [Ca.sup.2+] homeostasis. Consequently, we conclude that propofol does not trigger MH.
Comparison of the accelerated and the non-accelerated groups
We found the [EC.sub.50] value of propofol in the accelerated group was approximately half of that in the non-accelerated group. Similarly, the [EC.sub.50] value of caffeine in the accelerated group was approximately half of that in the non-accelerated group. The [EC.sub.50] value of halothane and 4-chloro-m-cresol in MHS individuals were reported to be half of those in MH-negative individuals (25-27). MH mutations increase the extent of ryanodine receptors in the closed [Ca.sup.2+]-sensitive state and reduce the voltage dependence of activation (28-30). MH mutations may contribute to the abnormal sensitivity to channel activation induced by propofol due to destabilisation of the closed-state of the channel. This probably contributes to the differences between the [EC.sub.50] values from the two groups.
Comparison with caffeine
Caffeine is known to be a specific activator of ryanodine receptors and is used to investigate potential interactions on the SR via [Ca.sup.2+] channels. According to previous reports (31-33), binding of caffeine to ryanodine receptors induces conformational changes in these channels that result in increased channel sensitivity to [Ca.sup.2+]. In the presence of nifedipine, transient increases in [Ca.sup.2+] induced by propofol were remarkably attenuated, whereas those mediated by caffeine were slightly attenuated. As for skeletal muscle, dihydropyridine receptor (DHPR, L-type [Ca.sup.2+] channel) acts as a voltage sensor, and stimulates RYR1 without significant [Ca.sup.2+] influx by direct mechanical coupling of DHPR [[alpha].sub.1] subunit with RYR1. And the [Ca.sup.2+] blocker binds to the [[alpha].sub.1] subunit, which is involved in channel function. A previous report showed that nifedipine increases the intracellular [Ca.sup.2+] concentration by inducing the release of [Ca.sup.2+] from the SR, which involves a membrane potential-dependent mechanism (34). In other words, nifedipine functions as a RYR1 channel opener. In the present study, transient [Ca.sup.2+] increases induced by propofol were significantly attenuated in the presence of nifedipine. Therefore, the RYR1 channels were probably opened by the nifedipine before propofol was added. our results suggest that propofol may compete for binding of ryanodine receptors with nifedipine.
Procaine inhibits CiCR activity and acts on E-C coupling subsequent to inhibition of the T-tubule voltage sensor (35). In the presence of procaine, the effects of propofol were almost completely inhibited due to competition with procaine both on CICR and/or DHPR. In addition, a previous report showed that propofol is more sensitive to DHPR than RYR1 (21). Also, caffeine reacts on only RYR1. Thus, the mechanism by which propofol acts on RYR1 (CICR function) is likely to be different from that of caffeine. Based on the specific mechanism involved in skeletal muscle E-C coupling, our results suggest that propofol reacts on the connecting region between DHPR and RYR1.
We conclude that propofol is clinically safe in individuals with predisposition to MH, as long as it is used within the recommended dosage range, and also that the mode of the action of propofol upon ryanodine receptors is likely to be different from that of caffeine.
This study was supported in part by a Grant-in-aid (No. 17390428) for Scientific Research from the Japan Society for the Promotion of Science, Tokyo, Japan.
Accepted for publication on December 18, 2008.
(1.) Monnier N, Kozak-Ribbens G, Krivosic-Horber R, Nivoche Y, Qi D, Kraev N et al. Correlations between genotype and pharmacological, histological, functional, and clinical phenotypes in malignant hyperthermia susceptibility. Hum Mutat 2005; 26:413-425.
(2.) Rosenberg H, Davis M, James D, Pollock N, Stowell K. Malignant hyperthermia. Orphanet Journal of Rare Diseases 2007; 2:21-34.
(3.) Migita T, Mukaida K, Kawamoto M, Kobayashi M, Yuge O. Fulminant-type malignant hyperthermia in Japan: cumulative analysis of 383 cases. J Anesth 2007; 21:285-288.
(4.) Stowell KM. Malignant hyperthermia: a pharmacogenetic disorder. Pharmacogenomics 2008; 9:1657-1672.
(5.) D'arcy CE, Bjorksten A, Yiu EM, Bankier A, Gillies R, McLean CA et al. King-Denborough syndrome caused by a novel mutation in the ryanodine receptor gene. Neurology 2008; 71:776-777.
(6.) Robinson R, Carpenter D, Shaw MA, Halsall J, Hopkins P. Mutations in RYR1 in malignant hyperthermia and central core disease. Hum Mutat 2006; 27:977-989.
(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.) Wu S, Ibarra MCA, Malicdan MCV, Murayama K, Ichihara Y, Kikuchi H et al. Central core disease is due to RYR1 mutations in more than 90% of patients. Brain 2006; 129:1470-1480.
(9.) Ibarra MCA, Wu S, Murayama K, Minami N, Ichihara Y, Kikuchi H et al. Malignant hyperthermia in Japan. Anesthesiology 2006; 104:1146-1154.
(10.) 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.
(11.) Matsui K, Fujioka Y, Kikuchi H, Yuge O, Fujii K, Morio M et al. Effects of several volatile anesthetics on the [Ca.sup.2+]-related functions of skinned skeletal muscle fibers from the guinea pig. Hiroshima J Med Sci 1991; 40:9-13.
(12.) Kunst G, Grsaf BM, Schreiner R, Martin E, Fink RHA. Differential effects of sevoflurane, isoflurane, and halothane on [Ca.sup.2+] release from the sarcoplasmic reticulum of skeletal muscle. Anesthesiology 1999; 91:179-186.
(13.) Gallen JS. Propofol dose not trigger malignant hyperthermia. Anesth Analg 1991; 72:413-414.
(14.) McKenzie AJ, Couchman KG, Pollock N. Propofol is a 'safe' anaesthetic agent in malignant hyperthermia susceptible patients. Anaesth Intensive Care 1992; 20:165-168.
(15.) Krivosic-Hrber R, Reyfort H, Becq MC, Adnet P. Effect of propofol on the malignant hyperthermia susceptible pig model. Br J Anaesth 1989; 62:691-693.
(16.) Raff M, Harrison GG. The screening of propofol in MHS swine. Anesth Analg 1989; 68:750-751.
(17.) Migita T, Mukaida K, Kawamoto M, Kobayashi M, Nishino I, Yuge 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.) Endo M, Iino M. Measurement of Ca release in skinned fibers from skeletal muscle. Methods in Enzymology 1988; 157:12-26.
(19.) 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.
(20.) 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.
(21.) Fruen BR, Mickelson JR, Roghair TJ, Litterer LA, Louis CF. Effects of propofol on [Ca.sup.2+] regulation by malignant hyperthermia-susceptible muscle membranes. Anesthesiology 1995; 82:1274-1282.
(22.) Ohta T, Endo M, Nakano T, Morohoshi Y, Wanikawa K, Ohga A. Ca-induced Ca release in malignant hyperthermia-susceptible pig skeletal muscle. Am J Physiol 1989; 256:C358-367.
(23.) Franks NP, Lieb WR. Molecular and cellular mechanisms of general anesthesia. Nature 1994; 367:607-614.
(24.) 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.
(25.) Weigl LG, Ludwig-Papst C, Kress HG. 4-chloro-m-cresol can not detect malignant hyperthermia equivocal cells in an alternative minimally invasive diagnostic test of malignant hyperthermia susceptibility. Anesth Analg 2004; 99:103-107.
(26.) Wehner M, Rueffert H, Koening F, Meinecke CD, Olthoff D. The Ile2453Thr mutation in the ryanodine receptor gene 1 is associated with facilitated calcium release from sarcoplasmic reticulum by 4-chloro-m-cresol in human myotubes. Cell Calcium 2003; 34:163-168.
(27.) 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.
(28.) Balog EM, Fruen BR, Shomer NH, Louis CF. Divergent effects of the malignant hyperthermia-susceptible Arg(615)--Cys mutation on the [Ca.sup.2+] and [Mg.sup.2+] dependence of the RYR1. Biophys J 2001; 81:2050-2058.
(29.) Louis CF, Balog EM, Fruen BR. Malignant hyperthermia: an inherited disorder of skeletal muscle [Ca.sup.2+] regulation. Bioscience Reports 2001; 21:155-168.
(30.) Tong J, McCarthy TV, MacLennan DH. Measurement of resting cytosolic [Ca.sup.2+] concentrations and [Ca.sup.2+] store size in HEK-293 cells transfected with malignant hyperthermia or central core disease mutant [Ca.sup.2+] release channels. J Biol Chem 1999; 274:693-702.
(31.) Du GG, MacLennan DH. [Ca.sup.2+] inactivation sites are located in the COOH-terminal quarter of recombinant rabbit skeletal muscle [Ca.sup.2+] release channels (ryanodine receptors). J Biol Chem 1999; 274:26120-26126.
(32.) gallant EM, Hart J, Eager K, Curtis S, Dulhunty AF. Caffeine sensitivity of native RyR channels from normal and malignant hyperthermic pigs: effects of a DHPR II-III loop peptide. Am J Physiol Cell Physiol 2004; 286:C821-830.
(33.) Treves S, Pouliquin P, Moccagatta L, Zorzato F. Functional properties of EGFP-tagged skeletal muscle calcium-release channel (ryanodine receptor) expressed in COS-7 cells: sensitivity to caffeine and 4-chloro-m-cresol. Cell Calcium 2002; 31:1-12.
(34.) Weigl LG, Hohenegger M, Kress HG. Dihydropyridine-induced [Ca.sup.2+] release from ryanodine-sensitive [Ca.sup.2+] pools in human skeletal muscle cells. J Physiol 2000; 525:461-469.
(35.) Klein Mg, Simon BJ, Schneider MF. Effects of procaine and caffeine on calcium release from the sarcoplasmic reticulum in frog skeletal muscle. J Physiol 1992; 453:341-366.
T. MIGITA *, K. MUKAIDA ([dagger]), H. HAMADA ([double dagger]), M. KOBAYASHI ([section]), I. NISHINO **, O. YUGE ([dagger])([dagger]), M. KAWAMOTO ([double dagger])([double dagger])
Department of Anesthesiology and Critical Care, Hiroshima University, Hiroshima, Japan
* M.D., Assistant Professor.
([dagger]) M.D., Ph.D., Staff Anesthesiologist, Division of Anesthesia, Hiroshima Prefectural Rehabilitation Center.
([double dagger]) M.D., Ph.D., Associate Professor.
([section]) M.D., Staff Anesthesiologist, Department of Anesthesia, Hiroshima City Funairi Hospital.
** M.D., Ph.D., Director, Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira.
([dagger])([dagger]) M.D., Ph.D., ex-Trustee, Hiroshima University.
([double dagger])([double dagger]) M.D., Ph.D., Professor and Chair.
Address for reprints: Dr T. Migita, Department of Anesthesiology and Critical Care, Hiroshima University Hospital, 1-2-3 Kasumi, Minami-ku, Hiroshima, Japan.
TABLE 1 Patient characteristics and results from the [Ca.sup.2+]-induced [Ca.sup.2+] release (CICR) rate test No. Age (y) Gender Reason for testing 1 58 M MH family 2 13 F MH family/myopathy * 3 14 M MH family 4 27 M MH family 5 31 M MH (CGS 63 rank 6) 6 31 M MH (CGS 48 rank 5) 7 45 M MH family 8 11 M MH family 9 46 F MH family 10 56 F MH family 11 10 M MH family 12 75 M shock and unknown fever 13 28 F unknown fever 14 47 M MH family 15 35 M unknown fever 16 2 M MH family 17 6 M MH family 18 56 F MH family 19 32 M MH family 20 14 M high serum creatine kinase Standard mean + 2 SD [Ca.sup.2+] concentration ([micro]M) in the CICR rate test No. RYR1 0.3 1 mutation 1 p.L4838V 0.415 1.294 2 p.R2508C 0.151 0.262 3 0.274 0.738 4 0.423 1.567 5 0.120 0.297 6 0.098 0.257 7 0.084 0.292 8 0.080 0.263 9 0.082 0.305 10 0.142 0.472 11 0.057 0.129 12 0.053 0.095 13 0.052 0.091 14 0.046 0.095 15 0.052 0.099 16 0.053 0.136 17 0.101 0.100 18 0.040 0.064 19 0.047 0.100 20 0.061 0.076 Standard 0.081 0.108 mean + 2 SD [Ca.sup.2+] concentration ([micro]M) in the CICR rate test No. 3 10 Result of test 1 4.497 6.988 accelerated 2 0.970 2.669 accelerated 3 2.627 3.926 accelerated 4 5.163 7.354 accelerated 5 0.845 3.308 accelerated 6 0.916 2.920 accelerated 7 1.273 2.727 accelerated 8 1.488 2.408 accelerated 9 1.425 1.900 accelerated 10 1.247 2.978 accelerated 11 0.413 1.299 not accelerated 12 0.386 1.327 not accelerated 13 0.602 2.089 not accelerated 14 0.434 1.301 not accelerated 15 0.408 1.162 not accelerated 16 0.493 1.012 not accelerated 17 0.247 1.304 not accelerated 18 0.308 1.477 not accelerated 19 0.402 1.402 not accelerated 20 0.278 1.211 not accelerated Standard 0.594 2.511 mean + 2 SD RYR1=ryanodine receptor type 1, MH=malignant hyperthermia, CGS=clinical grading scale. * Congenital myopathy with cores. Italicised values indicate the data more than two standard deviations greater than the standard mean, which are calculated from 12 individuals showed negative in vitro contracture test results (European Malignant Hyperthermia group and North American Malignant Hyperthermia group protocol) (19). TABLE 2 Constituents of the solutions used to measure the [Ca.sup.2+]-induced [Ca.sup.2+] release (CICR) rate induced by propofol Constituents [Mg.sup.2+] [MgATP.sup.2-] (mM) (mM) Solution A G2 relaxing solution 1.5 3.5 Loading solution 1.5 3.5 G10 relaxing solution 1.5 3.5 G2 Rigor solution 1.5 0 Prereleasing solution 0 0 Testing solution (A-1) 0 0 Stopping solution 10 0 Preassay solution 1.5 3.5 Assay solution 0.1 1 [Ca.sup.2+]-free 1.5 0 solution (A-2) Solution B 1.5 3.5 Constituents EGTA [Ca.sup.2+] (mM) (M) Solution A G2 relaxing solution 2 0 Loading solution 10 2 x [10.sup.-6.7] G10 relaxing solution 10 0 G2 Rigor solution 2 0 Prereleasing solution 2 0 Testing solution (A-1) 10 1 x [10.sup.-6] Stopping solution 10 0 Preassay solution 0 0 Assay solution 0 0 [Ca.sup.2+]-free 10 0 solution (A-2) Solution B 10 2 x [10.sup.-6.7] Constituents PiPES Caffeine (mM) (mM) Solution A G2 relaxing solution 20 0 Loading solution 20 0 G10 relaxing solution 20 0 G2 Rigor solution 20 0 Prereleasing solution 20 0 Testing solution (A-1) 20 0 Stopping solution 20 0 Preassay solution 20 0 Assay solution 20 50 [Ca.sup.2+]-free 20 0 solution (A-2) Solution B 20 0 Constituents Procaine Propofol (mM) ([micro]M) Solution A G2 relaxing solution 0 Loading solution 0 G10 relaxing solution 0 G2 Rigor solution 0 Prereleasing solution 0 Testing solution (A-1) 0 variable Stopping solution 10 Preassay solution 5 Assay solution 0 [Ca.sup.2+]-free 0 1000 solution (A-2) Solution B 0 variable The pH was adjusted to 7.0 with KOH. The concentration of ATP was 5 mM. EGTA=ethylene glycol-bis ([beta]-amino ethyl ether)-N,N,N',N'-tetraacetic acid, PIPES=piperazine-N,N'-bis (2-ethanesulfonic acid), G=EGTA, Rigor=ATP-free. Variable for the testing solution a refers to 0, 10, 100 or 1000 [micro]M, and solution B refers 0, 100 or 1000 [micro]M. TABLE 3 The [EC.sub.50] values for propofol and caffeine Group [EC.sub.50] for propofol ([micro]M) n Accelerated 274.7 [+ or -] 33.9 * 10 (46 myotubes) Non-accelerated 543.6 [+ or -] 79.5 10 (61 myotubes) Group [EC.sub.50] for caffeine (mM) n Accelerated 3.04 [+ or -] 0.27 * 10 (66 myotubes) Non-accelerated 4.99 [+ or -] 0.30 10 (76 myotubes) N shows number of tested individuals. Values are expressed as the means [+ or -] SEM. * P <0.05 vs the non-accelerated group.
|Printer friendly Cite/link Email Feedback|
|Author:||Migita, T.; Mukaida, K.; Hamada, H.; Kobayashi, M.; Nishino, I.; Yuge, O.; Kawamoto, M.|
|Publication:||Anaesthesia and Intensive Care|
|Date:||May 1, 2009|
|Previous Article:||Laboratory validation of the M-COVX metabolic module in measurement of oxygen uptake.|
|Next Article:||Anaesthesia and right ventricular failure.|