A defect in the ryanodine (Ry1) receptor Ca2+ channel has been implicated as one of the possible underlying causes of malignant hyperthermia (MH), a pharmacogenetic disorder characterized by sustained muscle contracture. The disease is triggered by common halogenated anesthetics and skeletal muscle relaxants, such as succinylcholine. This study tested whether the functional properties of the Ry1 receptor Ca2+ channel are affected by chlorocresol, a preservative added to a commercial preparation of succinylcholine (Midarine) and other parenteral compounds.
In vitro contracture testing was carried out on muscle biopsies from malignant hyperthermia-susceptible (MHS) and -negative (MHN) individual according to the protocol of the European MH group. Ca2+ flux studies on isolated rabbit sarcoplasmic reticulum fractions were measured spectrophotometrically by following the A710-790 of the Ca2+ indicator antipyrylazo III.
Chlorocresol causes muscle contracture in MHS muscles at a concentration of 25-50 microM and potentiates the caffeine contracture response in human MHS muscles. Sub-threshold (20 microM) concentrations of chlorocresol increase both the Kd and the Vmax of caffeine-induced Ca2+ release from isolated rabbit terminal cisternae.
These data suggest that, in muscle from MHS individuals, the enhanced Ca2+ released from the sarcoplasmic reticulum may not be due to the effect of succinylcholine alone but rather to the action of the preservative chlorocresol added to the drug.
MALIGNANT hyperthermia (MH) is a potentially lethal pharmacogenetic disease characterized by sustained muscle contracture that occurs during general anesthesia. In 75% of the cases, the disease is triggered by a combination of halogenated anesthetics and succinylcholine. Use of succinylcholine has been correlated with a variety of clinical forms of the disease, including malignant hyperthermia and masseter spasm. The former is a life-threatening condition particularly relevant in pediatric anesthetics; the latter has been estimated to occur in 1 of 100 anesthesia procedures and is often considered a warning sign of malignant hyperthermia. Massive myoglobinuria and increased concentrations of creatine phosphate kinase also have been reported as a consequence of succinylcholine administration. The exact mechanisms underlying the reaction to succinylcholine have not been fully understood, although succinylcholine-triggered muscle rigidity is thought to be a consequence of membrane depolarization, which activates massive Calcium2+ release from the sarcoplasmic reticulum. The sarcoplasmic reticulum is the organelle responsible for intracellular Calcium2+ homeostasis in skeletal muscle; it is endowed with a Calcium2+ pump and a Calcium sup 2+ channel (ryanodine (Ry1) receptor) for uptake and release of calcium, respectively. [4,5]The Ry1receptor Calcium2+ channel gene has been cloned, localized to human chromosome 19, and linked to a mutation causing MH susceptibility in some human families and in pigs, the latter being an animal model of the disease. [6–11]The Ry sub 1 receptor Calcium2+ channel is modulated by putative physiologic agonists, such as Calcium2+, ATP, Magnesium2+, and calmodulin. Electrophysiologic studies have shown an alteration of the calcium-dependent regulation of Calcium2+ channel activity in both human and pig MH-susceptible (MHS) muscles. [13,14]The Ry1receptor Calcium2+ channel also is activated in vitro by a variety of unrelated compounds (for review, see ). Thus, in vivo, the Ry sub 1 receptor could be a potential target of many chemicals added as preservatives to pharmacologic preparations of clinically relevant drugs. The effect of these preservatives may be enhanced in the presence of Ry sub 1 receptor Calcium2+ channels that are defective in the mechanisms controlling their activity. As part of a comprehensive study of MH-triggering drugs, we investigated the action of a commercial preparation of succinylcholine (Midarine, Wellcome, Pomezia, Italy) on muscle bundles from MH-negative (MHN) and MHS patients. We demonstrate that chlorocresol induces contracture in muscles from MHS patients; this drug also potentiates caffeine-induced Calcium2+ release from isolated sarcoplasmic reticulum terminal cisternae via the Ry1receptor Calcium2+ release channel.
Materials and Methods
Ruthenium red, 4-chloro-m-cresol, antipyrylazo III were from Fluka (Buchs, Switzerland); preservative-free succinylcholine and caffeine were from Sigma (St. Louis, MO); and halothane was from ICI Pharma (Macclesfield, UK). All chemicals were reagent grade.
Halothane and Caffeine Contracture Test
The muscle bundles used in these experiments were isolated from vastus lateralis muscles of 11 patients with suspected MH susceptibility who consented to the biopsy. The biopsy was performed under spinal anesthesia, and eight muscle bundles 15–25 mm in length and 2–3 mm thick were excised from each patient and bathed in a solution containing carboxygenated (95% O2, 5% CO2) Krebs-Ringer solution at 37 degrees C. Each bundle was attached by one end to a hook and by the other to an isometric force transducer via a thin steel wire. The muscle bundles were immersed in a 15-ml bath perfused at a flow rate of 2 ml/min with a Krebs-Ringer solution and stimulated by coaxial platinum electrodes that extended the entire length of the bundles. Before the experiments, muscle bundles were preloaded to 2–3 g and supramaximally stimulated ((280–350 mA)-(15–35 Volt)- 0.2 Hz), 1 ms stimulus duration). The basal line was stabilized within 20–30 min. Values of isometric tension were recorded as follows:
(1) Two bundles were sequentially exposed to increasing doses of halothane (0.5%, 1%, 2%, and 3%) changed every 5 min.
(2) Two bundles were sequentially exposed to increasing concentrations of caffeine (0.5, 1, 2, 3, 4, and 32 mM) changed every 3 min.
(3) One bundle was sequentially exposed to increasing doses of caffeine in the presence 10 micro liter Midarine/ml of Krebs-Ringer solution. The Midarine ampules contained 500 mg succinylcholine and 10 mg chlorocresol dissolved in 10 ml of distilled water; thus, the final concentrations of chlorocresol and succinylcholine in the Krebs-Ringer solution were 70 micro Meter and 1.25 mM, respectively.
(4) One bundle was sequentially exposed to increasing doses of caffeine in the presence of 1.25 mM succinylcholine.
(5) One bundle was sequentially exposed to increasing doses of chlorocresol (1, 10, 25, 50, 70, 100, 150, and 200 micro Meter) changed every 5 min.
(6) One bundle was kept on supply in case the test had to be repeated.
Following the European MH group protocol, the calculated isometric tension was the difference between the low point and the value chosen. In the current report, a positive in vitro contracture test was defined as that in which 2 mM caffeine or less and 2% halothane or less caused a force development of 0.2 g. Using this protocol, six patients were classified as MHS and five were MHN. MH-equivocal results were discarded from this study.
Calcium sup 2+ Flux Assays
Rabbit sarcoplasmic reticulum (SR) was isolated and fractionated into light and terminal cisternae fractions as previously described by Saito et al. SR fractionation was carried out in the presence of antiproteolytic agents. The SR fractions were resuspended in 0.3 M sucrose, 5 mM imidazole (pH 7.4), 100 micro Meter PMSF, and 1 micro gram/ml leupeptin and stored in liquid nitrogen until used. Protein concentration was measured according to Lowry using bovine serum albumin as standard. Calcium2+ release from isolated SR fractions was measured in a Beckman (Palo Alto, CA) DU 7,400 diode array spectrophotometer by monitoring the A710-A790of the Calcium sup 2+ indicator antipyrylazo III as described by Palade. Briefly, the assay was carried out at 37 degrees C in a medium containing, in a final volume of 1.5 ml, 7.5 mM K-pyrophosphate, 18.5 mM K-MOPS, pH 7.0, 100 mM KCl, 1.0 mM MgATP, 250 micro Meter antipyrylazo III, 5 mM creatine phosphate, 20 micro gram/ml creatine phosphate kinase, and 50 micro gram of SR fractions. Pulses of 25 nmol Calcium2+ were administered to load SR fractions with approximately 2.5 micro mol of Calcium2+/mg protein. When steady-state was reached, Calcium2+ release was triggered by the addition of different drugs.
Student's t test for paired samples was performed.
Effect of Midarine and Succinylcholine on the Contracture Response to Caffeine
(Figure 1) shows a representative caffeine contracture dose-response curve of muscle bundles isolated from patients defined as MHS and MHN according to the European MH group IVCT protocol. The threshold caffeine concentration for development of 0.2 g isometric force contracture in MHS was 1.5 mM (Figure 1(A); upper trace), whereas no contracture was obtained in MHN muscle bundles, up to a caffeine concentration of 4 mM (Figure 1(B); upper trace). We tested the effect of Midarine (final concentration 10 micro liter/ml Krebs solution) on isometric force contracture (Figure 1, middle traces). As can be seen, the commercial preparation of succinylcholine affected the threshold caffeine concentration for the development of significant force contracture in both MHS (Figure 1(A)) and MHN (Figure 1(B)) muscle bundles. However, the most striking effect was observed in MHS patients; their muscles developed significant contracture even in the absence of caffeine (Figure 1(A), middle trace), whereas in MHN, in the presence of Midarine, the threshold concentration for caffeine-induced force development of 1 g was 1.5 mM (Figure 1(B), middle trace).
The decreased caffeine threshold concentration for force contracture could be due to either the effect of succinylcholine on the electrical properties of the surface membrane or the effect of chlorocresol, the preservative added to the commercial preparation of succinylcholine, on the Calcium2+ release process, or both. To discriminate between these possibilities, we investigated the effect of succinylcholine alone (1.25 mM) on the caffeine contracture test. Under these conditions, the threshold caffeine concentration was similar to that of caffeine alone, i.e., 1.5 mM in MHS muscle bundles, whereas no force developed in MHN muscle bundles (Figure 1, bottom traces).
Dose-dependence of Chlorocresol-induced Muscle Contracture
The data of Figure 1(bottom traces) indicate that the effect on caffeine contracture of Midarine is likely due to the preservative. To address this issue, we determined the threshold concentration for chlorocresol-induced contracture in MHS and MHN muscles. Figure 2shows a dose-response curve for chlorocresol-induced isometric tension development in normal and MHS muscles. As shown, 25–50 micro Meter chlorocresol was capable of causing > 0.2 g contracture force in fibers isolated from the MHS patients, whereas in normal fibers, tension developed only at concentrations greater than 150 micro Meter.
Effect of Chlorocresol on Caffeine-induced Calcium sup 2+ Release
(Figure 1) indicates that chlorocresol is a potent activator of caffeine contracture in intact muscle fibers. In a previous study, we demonstrated that chlorocresol acts on the Calcium2+ release channel (Ry1receptor) of the skeletal muscle sarcoplasmic reticulum. In the following set of experiments, we wish to examine whether the effect of chlorocresol on caffeine-induced contracture could be due to the presence of a drug binding site(s) in the Calcium2+ release molecular complex that cooperates with caffeine to open the release channel (Ry1receptor). The Calcium2+ release assay was carried out with rabbit SR fractions. We chose this species for two reasons:(1) subcellular fractionation procedures have been well described for rabbit skeletal muscle, and (2) we are not limited by the amount of tissue. Figure 3shows a dose-response curve of caffeine-induced Calcium2+ release in the presence and absence of subthreshold concentrations (20 micro Meter of chlorocresol. Chlorocresol potentiated caffeine-induced Calcium2+ release by increasing both the Kd (6.0 vs. 10 mM) and the Vmax(2.2 + 0.21 vs. 1.55 + 0.21 micro mol Calcium2+/min/mg protein; n = 5).
In this study, we show that Midarine, a commercial preparation of succinylcholine containing chlorocresol as a preservative, potentiates caffeine-induced contracture in human MHS muscles. Preservative-free succinylcholine at a concentration of 1.25 mM had no effect on the threshold caffeine concentration for muscle contracture. On the contrary, exposure of MHS muscles to 10 micro liter Midarine/ml elicited contracture even in the absence of caffeine. The effect of Midarine is due to the preservative, because chlorocresol alone is capable of inducing contracture in isolated muscle bundles from MHS patients. This effect is evident at concentrations 30–40 times lower than that observed with caffeine. Because of its potency and short half-life, succinylcholine is a widely used drug, therefore the elucidation of whether it is an MH-triggering agent is crucial for its safe clinical application. The use of succinylcholine has been associated not only with the MH reaction in vivo but with alterations of the in vitro contracture response of MH muscles. [21–24]However, the exact mechanism by which succinylcholine affects in vitro contractures has been debated: In some cases, succinylcholine elicits muscle contracture, whereas in others, succinylcholine induces muscle contracture in MHS muscle bundles only in the presence of a second MH-triggering agent, such as halothane. These discrepancies might be linked to the heterogeneity of muscle preparations in which MH susceptibility may arise from a variety of causes, with only 50% of cases appearing to be caused by abnormalities in the Ry1receptor. In view of our results, differences in the composition of the succinylcholine preparation used for the experiments may contribute to divergent responses. Although we cannot exclude the possibility that an agent may trigger an MH episode and yet not elicit contracture of isolated muscle bundles, we think that chlorocresol is the active agent in Midarine that affects the response of MHS muscles to triggering compounds. The sensitivity of MHS muscles to chlorocresol is three- to fourfold greater than that observed in MHN muscles, and thus, it might be useful for the in vitro contracture test because it is potent and freely permeable across membranes.
Our results with human MHS muscles and Calcium2+ release from isolated sarcoplasmic reticulum confirm and extend previous observations by Galloway and Denborough regarding the effect of chlorocresol on MHS pig muscles. Several mechanisms may account for the mode of action by which chlorocresol affects the caffeine contracture response. The drug might alter the permeability to Calcium2+ of the surface membrane and/or sensitize the sarcoplasmic reticulum Calcium2+ release channel to the releasing action of caffeine and other MH-triggering agents. On the basis of the previous study, we believe that the effect of chlorocresol on caffeine contracture is mainly due to its action at a specific site on the sarcoplasmic reticulum Calcium sup 2+ release channel (Ry1receptor), as opposed to a perturbation of surface membranes. Activation of Calcium2+ efflux from terminal cisternae by chlorocresol is mediated by the Calcium2+-induced Calcium2+ release mechanism, because its effect appears to be prevented by ruthenium red, a known blocker of this type of Calcium2+ release. This conclusion is supported by the results showing that subthreshold concentrations of chlorocresol increased both the Kdand Vmaxof Calcium2+ release induced by caffeine, the latter being a well characterized modulator of the Calcium2+-induced Calcium2+ release mechanism.
The clinical dose of succinylcholine varies considerably; however, on average, the injection of 100 mg succinylcholine (Midarine) would be associated with the exposure of a patient to 2 mg chlorocresol. Because the exact volume of distribution of chlorocresol is not known, it is difficult to establish the clinical concentration of the drug after injection of an average dose of succinylcholine. Nevertheless, it should be mentioned that injection of solutions containing m-cresol have been reported to increase serum creatine kinase activity and to cause myalgia and pain at the site of subcutaneous injection. These results support the conclusions that chlorocresols are not safe preservatives and that their use should be considered with caution, not only in MHS individuals and/or in myopathic patients. Furthermore, these agents are used as preservatives in a variety of drugs, including insulin, sodium-heparin, and growth hormone, administered to millions of patients worldwide. As a consequence, the use of these preparations by MHS patients or those with certain myopathies may contribute to activation of MH, either directly or by decreasing the threshold for other triggering agents.