Malignant hyperthermia is a pharmacogenetic skeletal muscle disorder of intracellular calcium ..... macological, histological, functional, and clinical phenotypes.
Anaesth Intensive Care 2009; 37: 415-425
Effects of propofol on calcium homeostasis in human skeletal muscle T. Migita*, K. Mukaida†, H. Hamada‡, M. Kobayashi§, I. Nishino**, O. Yuge††, M. Kawamoto‡‡ Department of Anesthesiology and Critical Care, Hiroshima University, Hiroshima, Japan
Summary Malignant hyperthermia is a pharmacogenetic skeletal muscle disorder of intracellular calcium (Ca2+) homeostasis with an autosomal dominant inheritance. The objective of this study was to investigate the safety of propofol by investigating its effects on calcium homeostasis and its effect sites in human skeletal muscles. Muscle specimens were obtained from 10 individuals with predisposition to malignant hyperthermia. In skinned fibre experiments, we measured the effects of propofol on the Ca2+-induced Ca2+ release and the uptake of Ca2+ into the sarcoplasmic reticulum. Ca2+ imaging in primary myotubes was employed to analyse propofol-mediated alternations in the Ca2+ regulation and propofol-induced Ca2+ responses in the presence of Ca2+ channel blocker or Ca2+-induced Ca2+ release inhibitor. Increased Ca2+ release from the sarcoplasmic reticulum and inhibition of Ca2+ uptake into the sarcoplasmic reticulum were not observed with 100 μM propofol. A rise of Ca2+ was not seen under 100 μM propofol and the EC50 value for propofol was 274.7±33.9 µM, which is higher than the clinical levels for anaesthesia. Propofolinduced Ca2+ responses were remarkably attenuated in the presence of Ca2+ channel blocker or Ca2+-induced Ca2+ release inhibitor compared with the results obtained with caffeine. We conclude firstly that propofol is safe for individuals with predisposition to malignant hyperthermia when it is used within the recommended clinical dosage range, and secondly that its mode of action upon ryanodine receptors is likely to be different from that of caffeine. Key Words: malignant hyperthermia, propofol, calcium
Malignant hyperthermia (MH) is a pharmacogenetic disorder triggered by such agents as volatile anaesthetics and depolarising muscle relaxants, which induce a hypermetabolic response in skeletal muscle1-4. A number of myopathies, such as central core disease, multiminicore disease and King-Denborough syndrome5 predispose to MH. MH is autosomally dominant inherited and over 200 mutations have been identified in the ryanodine receptor 1 (RYR1) gene, which is expressed in skeletal muscle4,6. * M.D., Assistant Professor. † M.D., Ph.D., Staff Anesthesiologist, Division of Anesthesia, Hiroshima Prefectural Rehabilitation Center. ‡ M.D., Ph.D., Associate Professor. § 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. †† M.D., Ph.D., ex-Trustee, Hiroshima University. ‡‡ 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. Accepted for publication on December 18, 2008. Anaesthesia and Intensive Care, Vol. 37, No. 3, May 2009
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 test2,7. In Japan, however, predisposition to MH is mainly diagnosed using the Ca2+-induced Ca2+ release (CICR) rate test8-10. 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 MH11,12. The intravenous anaesthetic agent propofol has been suggested to be safe for MHS individuals in clinical settings13,14. In vitro, propofol has been examined in animals15,16 or by measuring human muscle contracture14. However, its safety has not been confirmed by studies at the human cellular level on Ca2+ homeostasis. Recently, we examined the effects of propofol on Ca2+ homeostasis in isolated human myotubes from two patients carrying RYR1 mutations linked to MH17. 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.
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PATIENTS AND METHODS Patients 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 al18. 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 fibredeficiency10,17. 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. Skinned fibres Effects of propofol on CICR By using skinned fibres, we measured the effects of propofol on Ca2+ release. Namely, purified
Table 1 Patient characteristics and results from the Ca2+-induced Ca2+ release (CICR) rate test Ca2+ concentration (µM) in the CICR rate test No.
Age (y)
Gender
Reason for testing
RYR1 mutation
0.3
1
3
10
Result of test
1
58
M
MH family
p.L4838V
0.415
1.294
4.497
6.988
accelerated
2
13
F
MH family/myopathy*
p.R2508C
0.151
0.262
0.970
2.669
accelerated
3
14
M
MH family
0.274
0.738
2.627
3.926
accelerated
4
27
M
MH family
0.423
1.567
5.163
7.354
accelerated
5
31
M
MH (CGS 63 rank 6)
0.120
0.297
0.845
3.308
accelerated
6
31
M
MH (CGS 48 rank 5)
0.098
0.257
0.916
2.920
accelerated
7
45
M
MH family
0.084
0.292
1.273
2.727
accelerated
8
11
M
MH family
0.080
0.263
1.488
2.408
accelerated
9
46
F
MH family
0.082
0.305
1.425
1.900
accelerated
10
56
F
MH family
0.142
0.472
1.247
2.978
accelerated
11
10
M
MH family
0.057
0.129
0.413
1.299
not accelerated
12
75
M
shock and unknown fever
0.053
0.095
0.386
1.327
not accelerated
13
28
F
unknown fever
0.052
0.091
0.602
2.089
not accelerated
14
47
M
MH family
0.046
0.095
0.434
1.301
not accelerated
15
35
M
unknown fever
0.052
0.099
0.408
1.162
not accelerated
16
2
M
MH family
0.053
0.136
0.493
1.012
not accelerated
17
6
M
MH family
0.101
0.100
0.247
1.304
not accelerated
18
56
F
MH family
0.040
0.064
0.308
1.477
not accelerated
19
32
M
MH family
0.047
0.100
0.402
1.402
not accelerated
20
14
M
high serum creatine kinase
0.061
0.076
0.278
1.211
not accelerated
0.081
0.108
0.594
2.511
Standard 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. Anaesthesia and Intensive Care, Vol. 37, No. 3, May 2009
Effects of propofol on human skeletal muscle 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 µM with 1.0 μM Ca2+ 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 µg.ml-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 (DSA601B, 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°C by using circulating water underneath the plate. The SR was loaded with a fixed amount of Ca2+ through the Ca2+ 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 μM) with 1.0 μM Ca2+. In order to assay the remaining Ca2+ in the SR, high concentrations of caffeine caused emptying of the SR Ca2+ store (Figure 1, A-1). The CICR rate was estimated by comparing the amount of Ca2+ remaining in the SR to the fixed amount of Ca2+. To measure baseline Ca2+ release from the SR independent of CICR, Ca2+ release was measured using Ca2+-free solution, and after the addition of
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1000 μM propofol to the Ca2+-free solution, Ca2+ release was again measured (Figure 1, A-2). Ca2+ uptake In skinned fibre experiments, we measured the effects of propofol on Ca2+ uptake in the accelerated group. A 1.0 M propofol stock solution was diluted to 100 and 1000 µM with loading solution for testing (solution B in Table 2). The SR was loaded with Ca2+ though the Ca2+ pumps with various concentrations of propofol (0, 100 and 1000 μM) for various periods of time (0.25, 0.5, 1.0 and 2.0 minutes). The Ca2+ content in the SR was assayed after inducing its release with a high concentration of caffeine. Uptake of Ca2+ into the SR was estimated from the Ca2+ content of the SR (Figure 1B). Values were calculated as the percentage of the control, which was taken with 0 μM propofol for two minutes. Cell cultures 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, SigmaAldrich, USA) containing 1% ampicillin sodium salt, kanamycin sulphate (Sigma, USA) and amphotericin B (Invitrogen, USA), in 25 cm2 cell culture flasks (Corning, USA) and a 5% CO2 atmosphere at 37°C. The medium was changed every three days. After two or three weeks in culture, the cells were plated
Table 2 Constituents of the solutions used to measure the Ca2+-induced Ca2+ release (CICR) rate induced by propofol Constituents
Mg2+ (mM)
MgATP2(mM)
EGTA (mM)
Ca2+ (M)
PIPES (mM)
Caffeine (mM)
Procaine (mM)
G2 relaxing solution
1.5
3.5
2
0
20
0
0
Loading solution
1.5
3.5
10
2×10-6.7
20
0
0
G10 relaxing solution
1.5
3.5
10
0
20
0
0
G2 Rigor solution
1.5
0
2
0
20
0
0
Prereleasing solution
0
0
2
0
20
0
0
Propofol (µM)
Solution A
Testing solution (A-1)
0
0
10
1×10
20
0
0
Stopping solution
10
0
10
0
20
0
10
Preassay solution
1.5
3.5
0.1
0
20
0
5
Assay solution
0.1
1
0.1
0
20
50
0
Ca -free solution (A-2)
1.5
0
10
0
20
0
0
1000
Solution B
1.5
3.5
10
2×10
20
0
0
variable
2+
-6
-6.7
variable
The pH was adjusted to 7.0 with KOH. The concentration of ATP was 5 mM. EGTA=ethylene glycol-bis (β-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 µM, and solution B refers 0, 100 or 1000 µM. Anaesthesia and Intensive Care, Vol. 37, No. 3, May 2009
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T. Migita, K. Mukaida et al
Figure 1: Experimental protocol used to measure the effects of propofol on Ca2+-induced Ca2+ release (CICR) (A) and Ca2+ uptake (B). Dashed lines represent the cell membrane and solid lines denote the sarcoplasmic reticulum (SR). A-1: Loading: the Ca2+ pumps were used to load a fixed amount of Ca2+ into the SR. Releasing: solutions with various concentration propofol and Ca2+ were applied to induce CICR. Assay: the remaining Ca2+ in the SR was released by addition of assay solution containing a high concentration of caffeine. A-2: Releasing: propofol and Ca2+-free solution were applied. B: Loading: the Ca2+ pumps were used to load various concentration propofol and a fixed amount of Ca2+ into the SR. Assay: the Ca2+ in the SR was released by addition of assay solution containing a high concentration of caffeine.
on 35 mm glass-bottom culture dishes with 10 mm microwells (MatTek, USA), and allowed to grow for 10 to 14 days20 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. Ca2+ imaging of myotubes Propofol Cells were washed in 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 loaded with 5.0 µM Fura-2 AM (Dojindo, Japan) in HBSS for one hour at room temperature (24 to 26°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°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 µM. Only the cells that racted to 20 mM caffeine (Wako, Japan) with an increase in the intracellular Ca2+ concentration were used for experiments. Propofolinduced changes in Fura-2 AM fluorescence were measured and the 340/380 nm signal ratio was Anaesthesia and Intensive Care, Vol. 37, No. 3, May 2009
Effects of propofol on human skeletal muscle calculated using a Ca2+ 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, caffeineinduced 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 Ca2+ concentrations using a calibration curve constructed with a calibration kit (Fura-2 Calcium Imaging Calibration Kit, Invitrogen, USA). L-type Ca2+ channel blocker Nifedipine dissolved in DMSO was diluted with HBSS to 50 μM. Myotubes were treated with 1000 μM propofol for three minutes, which was used as the control and washed with HBSS. Subsequently, after pretreatment with 50 μM nifedipine, 1000 μM 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 Ca2+ imaging system. The same procedure was performed with 10 mM caffeine. CICR inhibitor Procaine hydrochloride was diluted with HBSS to 10 mM. Myotubes were treated in 1000 μM propofol for three minutes, which was used as the control and washed with HBSS. Subsequently, after pretreatment with 10 mM procaine, 1000 μM 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 Ca2+
imaging system. The same procedure was performed with 10 mM caffeine. Data analysis 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 ± SEM. P values less than 0.05 were considered to be significant. RESULTS Effects of propofol on CICR In 1 μM Ca2+, an increase in the Ca2+ release rate was observed only with 1000 μM propofol (Figure 2); compared with the propofol-free condition, the rates increased by 210±5% in the accelerated group and 194±15% in the non-accelerated group. There was no significant difference between the groups. In Ca2+-free solution, 1000 μM propofol increased the Ca2+ release rate by 143±3% in the accelerated group and 127±20% in the nonaccelerated group, as compared with the propofolfree condition. The values were not significantly
Figure 2: The Ca2+-induced Ca2+ release (CICR) rate induced by propofol with 1.0 µM Ca2+. Data represent the means ± SEM. * P