Halothane-induced Calcium Release in Cultured Human Skeletal Muscle Cells from a Family Susceptible to Malignant Hyperthermia with an Unidentified Mutation in Chromosome 19

Muscle biopsies were obtained from four individuals without any known muscular disorder and from six MHS patients. All patients gave written informed consent, formulated by the Committee on Medical Ethics of the University of Nijmegen (Nijmegen, The Netherlands). The MHS patients were members of one single family in which a man in 1972 had died from MH during appendectomy. The MHS phenotypes were recognized by IVCT 2 yr before this study (table 1). Genetic analysis in this family showed linkage to a candidate locus on chromosome 19 (lod score greater than +3.0).

Samples of the quadriceps femoris muscle were obtained by percutaneous needle biopsies (25–30 mg). Fragments were attached on the bottom of a 35-mm culture dish containing 1 ml proliferation medium (Dulbecco's modified Eagle medium [DMEM], 20% fetal calf serum [FCS], 4.5 mg/ml glucose, and 4 mm glutamine) and cultured in a humidified CO²incubator (5% CO²) at 37°C. The next day, this medium was substituted by proliferation medium containing 4% Ultroser G and 10% rat brain extract instead of FCS. After 7–10 days, the explants were removed, and the myoblasts were plated out on glass coverslips (10 × 30 mm) in 35-mm dishes. Further proliferation took place in 20% FCS containing medium until confluency was reached. Differentiation to polynucleated myotubes was achieved in DMEM containing 10% horse serum for 7 days. For cryopreservation, about 10⁶myoblasts/ml DMEM containing 20% FCS and 10% dimethyl sulfoxide were stored in liquid nitrogen.

The free cytosolic Ca²⁺concentration ([Ca²⁺]ⁱ) was determined in skeletal muscle cells using Fura-2. 11Myotubes were loaded with 5 μm Fura-2/acetoxymethyl ester (Fura-2/AM) and 10 μm Pluronic acid for 90 min at 37°C in physiologic salt solution (PSS; containing 10 mm HEPES, 125 mm NaCl, 10 mm NaHCO³, 1 mm NaH²PO⁴, 5 mm KCl, 2 mm MgSO⁴, 1.8 mm CaCl², and 10 mm glucose, pH 7.4). On-line ratio measurements were recorded with a Shimadzu RF-5301 spectrofluorophotometer. Fura-2 fluorescence was measured at an emission wavelength of 492 nm (bandwidth, 5 nm) and alternating excitation wavelengths of 340 and 380 nm (bandwidth, 3 nm). During the measurements, the cells in the cuvette were superfused with PSS (4.0 ml/min; 37°C) without or with different concentrations of halothane (0.11, 0.22, 0.44, 1.0, 2.0, 4.0, 8.0, 12.0 mm). Halothane was solved in dimethyl sulfoxide (DMSO) and added to PSS in airtight, dark bottles. All fluorescence signals were corrected for autofluorescence.

The 340/380 ratios (Fura-2) were calibrated using PSS containing 4 μm ionomycin and 10 mm Ca²⁺(pH 7.7; R^max) or 4 μm ionomycin and 20 mm EGTA without external Ca²⁺(pH 8.5; R^min). [Ca²⁺]ⁱwas calculated using the equation:

12 

Halothane concentrations in PSS were measured using a head space gas chromatographic technique (Chrompack CP-9001, equipped with a flame ionization detector). 13 

Dulbecco's modified Eagle medium and Ultroser G were from Gibco BRL Life Technologies, Paisly, United Kingdom; FCS and horse serum were from Flow Laboratories, Irvine, United Kingdom. Brain extract was prepared from brains of 10-day-old Wistar albino rats as a 10% (w/v) homogenate. Fura-2/AM, ionomycine, and Pluronic acid were purchased from Molecular Probes, Eugene, Oregon. Halothane was from Tempus b.v., Oegstgeest, The Netherlands.

Data are represented as mean (SD). Statistical analysis is performed using the unpaired Student t  test. Significance was set at P < 0.05. Curve fittings were obtained by linear regression analysis. Individual areas under the curves were determined from the curves by interpolation.

Halothane concentrations in PSS were measured in the cuvette on completion of the calcium measurements. We found a substantial difference between the intended concentrations and the measured concentrations (intended → measured concentrations, respectively: 0.11 → 0.095 (0.012) mm, 0.22 → 0.187 (0.029) mm, 0.44 → 0.33 (0.039) mm, 1.0 → 0.69 (0.16) mm, 2.0 → 1.4 (0.20) mm, 4.0 → 2.75 (0.42) mm, 8.0 → 5.2 (0.99) mm, 12.0 →7.5 (1.3) mm).

The 340/380 ratios of the Fura-2 calibration resulted in an R^maxof 11.88 (1.03) and an R^minof 1.59 (0.15), mean (SD) of eight experiments. The constant β, i.e. , the ratio of the fluorescence emission of the free dye and the Ca²⁺-saturated dye measured at 380 nm, is 3.09 (0.33) (n = 7). We did not find a statistical difference between the mean resting [Ca²⁺]ⁱin cultured muscle cells of MHS or control individuals, which were respectively 65 (20) nm (n = 72) and 58 (17) nm (n = 50).

Halothane produced a dose-dependent increase of [Ca²⁺]ⁱ(fig. 1). In the MHS group, the Ca²⁺response is observed in the clinically used halothane concentrations (< 1 mm). The Ca²⁺response in cultured muscle cells of MHS individuals is significantly different from that of control individuals, and there is no overlap beyond 0.44 mm halothane (equivalent to 2% v/v).

Cultured human skeletal muscle cells have been very useful for studying ion homeostasis in relation to MH. 14–17Interpretation of the results is complicated by the existence of incomplete maturation and differences related to the heterogeneous population of cells at the myotube stage. By analyzing the fluorescence signal composed of the whole monolayer of myoblasts/myotubes on glass coverslips, we were able to compare the common behavior of thousands of cells instead of a small selection of cells, which was done in previous studies with comparable aims. 14,15,17This could explain why we could discriminate MHS from control muscle tissue using halothane in clinically used concentrations, whereas the other investigators needed much higher halothane concentrations. 15,17Further standardization of cell culturing could improve reproducibility of test results even more. In the IVCT, a vaporizer is used to add halothane to carboxygen to solve halothane in Krebs-Ringer's solution. 8To reach supraclinical halothane concentrations, halothane was solved in DMSO before it was added to the physical salt solution. This procedure requires assay of halothane concentration in the test bath at completion of each experiment because the variations are common.

In this study, we confirm that there is no difference in the resting intracellular calcium concentrations between cultured muscle cells from MHS and control individuals. 2,14,15 

A test based on cultured muscle cells obtained by needle biopsy is less invasive and is easily reproducible. The time between biopsy and performance of the test is not critical because cell cultures can be expanded, frozen, and thawed. In contrast, the IVCT requires large muscle samples (surgically collected) and must be performed within 5 h. 8 

In conclusion, cultured human muscle cells obtained from needle biopsies may well be applied in diagnostic tests for MH susceptibility. More studies comparing data from the IVCT and cultured cells have to be undertaken to determine sensitivity (and specificity).