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Propofol-induced changes in myoplasmic calcium concentrations in cultured human skeletal muscles from RYR1 mutation carriers.

SUMMARY

Malignant hyperthermia is a pharmacogenetic disorder caused by autosomal dominant mutations in the ryanodine receptor type 1 gene. Propofol has been reported as a safe anaesthetic for malignant hyperthermia susceptible patients but has not been tested on cultured cells from patients with the ryanodine receptor type 1 mutation. The aim of this study was to determine whether propofol could trigger abnormal calcium fluxes in human myotubes isolated from malignant hyperthermia susceptible patients harbouring the native ryanodine receptor type 1 mutation. Muscle specimens were obtained from the patients to diagnose malignant hyperthermia disposition and the calcium-induced calcium release test and molecular genetic analyses were performed. Using the calcium sensitive probe Fura 2, we determined the 340/380 nm wave-length ratios by measuring alterations in calcium homeostasis in isolated myotubes from cultured skeletal muscle specimens. Two patients, one with ryanodine receptor type 1 R2508C and one with the L4838V mutation had accelerated calcium-induced calcium release rates. The 340/380 nm ratios increased when the propofol concentration exceeded 100 [micro]M. The half-maximal activation concentrations ([EC.sub.50]) for propofol from patients 1 and 2 were 181.1 and 420.5 [micro]M, respectively. Increases in calcium concentrations in response to propofol dosage were limited to doses at least 100-fold greater than those used in clinical settings. These observations correlate well with clinical observations that propofol does not trigger malignant hyperthermia in susceptible humans.

Key Words: malignant hyperthermia, propofol, myotubes, calcium

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Malignant hyperthermia (MH) is a pharmaco-genetic disorder triggered by agents such as volatile anaesthetics and depolarising muscle relaxants that induce excessive calcium release from the sarcoplasmic reticulum (SR) in skeletal muscle (1). Central core disease (CCD) closely resembles MH, with a congenital myopathy and muscle pathology characterised by the presence of distinct cores, interstitial fibrosis and type 2 fibre deficiencies (2). CCD and MH both have autosomal dominant inheritance and more than 100 MH- and CCD-associated mutations have been reported in the ryanodine receptor 1 (RYR1) gene of skeletal muscle (3). MH susceptible patients (MHS) are diagnosed in North America by a caffeine-halothane contracture test and in Europe by an in vitro contracture test. In Japan, because of limitations in muscle biopsy size and examination times, MHS is diagnosed by detecting an accelerated calcium-induced calcium (CICR) rate from the sarcoplasmic reticulum (CICR rate test). The CICR test can be performed on smaller specimens up to 48 hours after biopsy, as opposed to the six hours required for caffeine contracture or halothane caffeine contracture tests used elsewhere.

Previous reports have shown that propofol is safe as a general anaesthetic for MHS patients (4-6). In these studies propofol was examined in MHS animals or humans by measuring general anaesthesia or by examining human muscle contraction. Propofol has not been tested on cultured cells from the patients with RYR1 mutations. Our present study measured calcium regulation in propofol-treated human cells and showed that propofol is safe for MHS patients. we investigated calcium homeostasis in human myotubes formed from cells isolated from patients harbouring the native RYR1 mutation linked to MH.

MATERIALS AND METHODS

Patients

Muscle specimens were obtained from two patients referred to Hiroshima University Hospital or the National Center of Neurology and Psychiatry for the diagnosis of MH disposition. Prior to the study, written informed consent was obtained from the patients and their families.

CICR rate test and molecular genetic methods

CICR rate tests were performed at Hiroshima University according to Endo's protocol (7-8). In brief, chemical skinned fibres were made from biopsied muscle tissues using saponin and then treated with different calcium concentrations (0, 0.3, 1.0, 3.0 and 10 [micro]M). The tension of each sample was measured with a force transducer and CICR rates were calculated. CICR values above 2 SD of the normal average were defined as "accelerated". The average normal values were obtained from 12 individuals with negative in vitro contracture tests and caffeine-halothane contracture tests (9).

Molecular genetic analyses were conducted at the National Center of Neurology and Psychiatry (10).

Cell cultures

Skeletal muscle cells isolated from the patients were maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen, U.S.A.) supplemented with 10% heat-inactivated bovine calf serum (FBS; Sigma-Aldrich, U.S.A.) containing 1% amikacin sulphate, kanamycin sulphate (Sigma) and amphotericin B (Invitrogen), in 25 [cm.sup.2] cell culture flasks (Corning, U.S.A.) in a 5% C[O.sub.2] atmosphere at 37[degrees]C. The medium was changed every three days. After two to three weeks, the cells were plated on 35 mm dishes with a 10 mm micro-well glass bottom (MatTek, U.S.A.) and grown for 10 to 14 days in DMEM with 2% FBS until the myoblasts fused to form myotubes.

Calcium imaging

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], 1.0 mM Mg[Cl.sub.2] and 5.5 mM glucose, with the pH adjusted to 7.4 with NaOH. The cells were loaded with 5 [micro]M Fura-2 AM (Dojindo, Japan) in HBSS for one hour, then excited alternately at 340 nm and 380 nm, after which fluorescence emission at 510 nm was measured using a fluorescent microscope (Nikon, Japan) equipped with a cooled high-speed digital video camera (ORCA-AG; Hamamatsu, Japan). HBSS was perfused at a rate of 1.2 ml per minute. Propofol (biomedical, France) was dissolved in dimethyl sulphoxide (DMSO; Sigma) and diluted with HBSS to make the following test concentrations: 1, 3, 10, 30, 100, 300, 1000, 3000 and 5000 M. Only cells reacting to 20 mM caffeine (Wako, Japan) with an increase in intracellular calcium concentration were used for experiments. Propofol-induced changes in Fura-2 AM fluorescence were measured and the 340/380 nm ratios were calculated using a calcium imaging system (Aquacosmos 2.5; Hamamatsu, Japan). Similarly, caffeine-induced changes in Fura-2 AM fluorescence were measured at 0.25, 0.5, 1, 2.5, 5, 10 and 20 mM. The two fluorescence ratios were converted into [Ca.sup.2+] concentrations, using a calibration curve drawn with a calibration kit (Fura-2 Calcium Imaging Calibration Kit; Invitrogen).

Data analysis

To obtain dose response curves, data for propofol and caffeine were normalised to the maximum response of 20 mM and 10 mM caffeine respectively. Data analysis was performed using PRISM software (graphPad Software Inc, USA). Values are shown as mean [+ or -] SD.

An unpaired t-test was used for statistical comparisons of mean values between samples and control. Statistical significance was set at P <0.05.

RESULTS

Patients, CICR test and molecular genetic methods

Patient 1

A 13-year-old female with congenital polyarthrogryposis and scoliosis underwent surgery for scoliosis safely with propofol. Her father had an MH episode with general anaesthesia and her sister presented with congenital myopathy. The patient's serum creatine kinase (CK) was 561 [U.l.sup-1] at rest. Prior to the surgery, a muscle biopsy was performed to diagnose MH disposition. The patient had an accelerated CICR rate (Figure 1). genetic analysis showed a point mutation in RYR1 7522 C [right arrow] T, resulting in the amino acid change R2508C in exon 47. The pathological diagnosis was myopathic changes with some fibres with cores or a core-like structure, type 1 fibre predominance and type 2B fibre deficiency. The patient was diagnosed MHS with CCD.

Patient 2

A 58-year-old male consulted with us to confirm MH, because his son had experienced an MH-related incident with general anaesthesia, although he had recently received general anaesthesia with propofol safely. The serum CK of the patient was 432 [U.l.sup.-1] at rest and CICR rate was accelerated (Figure 1). genetic analysis demonstrated a RYR1 mutation of 14642C [right arrow] G, resulting in the amino acid change L4838V in exon 101.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Controls

Ten individuals who were referred to our laboratory for MH deposition and had normal CICR rates were assigned to the control group (Figure 1).

Calcium imaging results

The average (range) for resting calcium concentrations in the myotubes of cases 1 and 2 were 107 (42-193) and 91 (56-138) nM respectively. After application of 20 mM caffeine, the average myoplasmic calcium concentrations rose to 227 and 237 nM respectively.

The 340/380 nm ratios did not increase when propofol was within 1 to 30 [micro]M, although it rose significantly when the concentration was greater than 100 [micro]M (Figure 2). A dose-response curve was obtained by normalising the increase in myoplasmic calcium concentrations for each dose to the maximum response with 20 mM caffeine (Figure 3). The half-maximal activation concentrations ([EC.sub.50]) for propofol for patients 1 and 2 were 181.1 and 420.5 [micro]M (T1) respectively. There were no changes with DMSO at the same concentrations.

[FIGURE 3 OMITTED]

DISCUSSION

In the present study, we found that differences in effects of propofol on in vitro calcium induced calcium release for MHS patients compared with MHN patients occurred only at concentrations well above the clinical range using cultured human cells obtained from skeletal muscle specimens. This fits well with propofol's clinical record as safe for MHS patients. For the experiments, we used myotubes formed by satellite cells with a fibre-like shape and multiple nuclei. The myotubes also shared functional properties with striated skeletal muscle cells, such as calcium release via RYR1. Using this system, the total calcium regulation of the whole cell was investigated (11-12).

Caffeine, a ryanodine receptor agonist, has been used as a tool to investigate ryanodine receptor stimulated calcium transients (11-12). In the present study, reactions to caffeine and propofol were shown only at high concentrations. Therefore no increase in the 340/380 nm ratios at lower propofol dose was regarded as non-reactivity to propofol.

Propofol is used for general anaesthesia and sedation in intensive care units at clinical concentrations ranging from 2 to 6 [micro]g/ml, which is equivalent to 11 to 34 [micro]M. Since the serum-protein combination rate ranges from 97 to 98%, free concentrations at these doses are assumed to be approximately 1 [micro]M. The 340/380 nm ratios did not increase when propofol ranged from 1 to 30 [micro]M. In the two MHS patients, the half-maximal activation concentrations ([EC.sub.50]) for propofol were 181.1 and 420.5 [micro]M, which are much higher than clinical concentrations. Our findings showed that intracellular calcium homeostasis did not significantly increase in response to propofol, even though the concentrations tested were more than 100-fold greater than those used in clinical settings.

In an investigation of the effects of propofol on calcium regulation in swine with MHS, it was reported that concentrations ranging from 10 to 500 [micro]M had no effect on ryanodine receptor-mediated [Ca.sup.++] efflux from sarcoplasmic reticulum vesicles. This was considered further evidence for propofol's lack of triggering of abnormal calcium fluxes in MHS ryanodine receptor channel activity, even at concentrations more than 100-fold greater than used clinically (13). In our study using myotubes, we investigated not only the function of RYR1 but also the total calcium regulation of whole cell and showed that calcium concentrations rose at propofol levels lower than 500 [micro]M. Accordingly, our results are consistent with the previous study (13). We conclude that measuring calcium homeostasis with Fura 2 was useful to test the effects of propofol.

It has previously been reported that the RYR1 mutation in patient 2 (L4838V) was responsible for MH incidents, based on its expression in Chinese hamster ovary cells in functional assay (14). Both the L4838L and R2508C mutations are in the distribution of sequence variations of the RYR1 gene which are pathogenic for MH (10). Using myotubes from MHS patients, especially carriers with the mutations, propofol showed no effect at clinical concentrations. These results correlate well with the observation that clinical doses of propofol do not trigger MH in MHS patients in vivo.

There was a significant difference in the [EC.sub.50] of propofol between patient 1 and controls but not between patient 2 and controls. We studied only two MHS individuals and therefore could not make general comparisons between MHS subjects and controls. Futhermore, it was unclear whether the distinctions between the two patients were caused by variations in the mutation region. Futher studies are needed to compare MHS subjects and genetic sub-types of MHS and controls.

In conclusion, we observed propofol-induced changes in myoplasmic calcium concentrations in cultured human skeletal muscle cells obtained from carriers of the RYR1 R2508C and L4838V mutations. An increase in response to propofol was demonstrated only at concentrations 100-fold greater than clinical doses. This supports clinical observations that propofol does not trigger MH in MHS individuals.

ACKNOWLEDGEMENT

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 July 10, 2007.

REFERENCES

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(2.) 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.

(3.) Robinson R, Carpenter D, Shaw MA, Halsall J, Hopkins P. Mutations in RYR1 in malignant hyperthermia and central core disease. Human Mutation 2006; 27:977-989.

(4.) Krivosic-Horber R, Reyfort H, Becq MC, Adnet P. Effect of propofol on the malignant hyperthermia susceptible pig model. Br J Anaesth 1989; 62:691-693.

(5.) Raff M, Harrison GG. The screening of propofol in MHS swine. Anesth Analg 1989; 68:750-751.

(6.) McKenzie AJ, Couchman Kg, Pollock N. Propofol is a 'safe' anaesthetic agent in malignant hyoerthermia susceptible patients. Anaesth Intensive Care 1992; 20:165-168.

(7.) Endo M, Iino M. Measurement of Ca release in skinned fibers from skeletal muscle. Methods Enzymol 1988; 157:12-26.

(8.) Matsui K, Fujioka Y, Kikuchi H, Yuge O, Fujii K, Morio M et al. Effects of several volatile anesthetics on the Ca(2+)-related functions of skinned skeletal muscle fibers from the guinea pig. Hiroshima J Med Sci 1991; 40:9-13.

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

(10.) Ibarra MCA, Wu S, Murayama K, Minami N, Ichihara Y, Kikuchi H et al. Malignant hyperthermia in Japan. Anesthesiology 2006; 104:1146-1154.

(11.) Wehner M, Rueffert H, Koenig F, Olthoff D. Functional characterization of malignant hyperthermia-associated RYR1 mutation in exon 44, using the human myotube model. Neuromuscul Disord 2004; 14:429-437.

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

(13.) 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.

(14.) Yamada H, Oguchi K, Saitoh N, Yamazawa T, Hirose K, Kawana Y et al. Novel mutations in C-terminal channel region of the ryanodine receptor in malignant hyperthermia patients. Jpn J Pharmacol 2002; 88:159-166.

T. MIGITA *, K. MUKAIDA [[dagger]], M. KAWAMOTO [[double dagger]], M. KOBAYASHI [[section]], I. NISHINO **, O. YUGE [[dagger]][[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., Professor and Chair.

[[section]] M.D., Staff Anesthesiologist, Department of Anesthesiology, 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., Trustee, Hiroshima University.

Address for reprints: Dr T. Migita, Department of Anesthesiology and Critical Care, Hiroshima University Hospital,1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
TABLE 1
E[C.sub.50] for propofol

Patient Mutation E[C.sub.50] for propofol n

1 R2508C 118.1 [+ or -] 87.9 * 11
2 L4838V 420.5 [+ or -] 125.1 4
Control 485.3 [+ or -] 249.2 10 (44 myotubes)

Patient E[C.sub.50] for caffeine n

1 2.08 [+ or -] 0.31 * 6
2 3.90 [+ or -] 1.50 * 4
Control 5.01 [+ or -] 0.96 10 (61 myotubes)

n = number of tested myotubes. Values are expressed as
mean [+ or -] SD. * P <0.05 vs. control
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Article Details
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Author:Migita, T.; Mukaida, K.; Kawamoto, M.; Kobayashi, M.; Nishino, I.; Yuge, O.
Publication:Anaesthesia and Intensive Care
Article Type:Clinical report
Geographic Code:9JAPA
Date:Dec 1, 2007
Words:2763
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