Administration of serotonin2 (5-HT2) receptor agonists in pigs triggers malignant hyperthermia (MH) and psychotic-like behavior. Both can be reduced by 5-HT2 receptor antagonists. Furthermore, an increase in the plasma concentration of 5-HT has been found during onset of halothane-induced MH in pigs. Therefore, in this study, the in vitro effects of the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) were investigated in muscle specimens from MH-susceptible (MHS) and -negative (MHN) patients.
After MH classification using the caffeine-halothane contracture test (CHCT), surplus muscle specimens from 23 MHS and 17 MHN patients were used to examine the effects of DOI. In the first study, DOI was added to the bath in a concentration of 0.02 mM. In a second experiment, muscles were preincubated for 60 min with 0.02 mM DOI, and subsequently, halothane was added incrementally to the organ bath (0.11-0.22-0.44 mM) for 15 min according to the CHCT protocol. The in vitro effects of DOI on contracture development and muscle twitch were measured for 120 min in both investigations.
Muscle specimens form all patients developed contractures after administration of DOI, characterized by a significantly earlier development of contracture in MHS (16.8 +/- 1.7 min) than in MHN (66.3 +/- 5.8 min) muscles (P < 0.05). There was no overlap between the groups in the range of times. The onset of contracture development after DOI was prolonged by halothane in specimens from MHN patients (89.7 +/- 5.6 min) but not MHS patients. Preincubation with DOI increased the halothane-induced contractures in specimens from MHS patients compared to the results of the CHCT. The contracture development in specimens from MHS patients was larger than from MHN patients. At the end of the experiment, contractures had reached a maximum of 12.9 +/- 1.1 mN in specimens from MHS and 5.3 +/- 0.6 mN in MHN patients (P < 0.05). The additional administration of halothane led to significantly increased contractures in specimens from MHS individuals (15.9 +/- 0.9 mN) at 120 min. However, the contracture development decreased significantly to 3.1 +/- 0.4 mN in MHN muscles. Muscle twitch after DOI administration was reduced significantly in specimens from MHS and MHN patients.
A functional or structural altered serotonin system might be involved in the development of MH in humans.
MALIGNANT hyperthermia (MH) is a clinically well characterized heterogeneous inherited disorder of skeletal muscle. [1,2]It is widely believed that susceptibility to MH is caused by abnormal calcium metabolism within the skeletal muscle fiber. [3,4]An enhanced calcium release from intracellular stores produces an overload of the muscle cell with calcium, which accounts for sustained muscle contraction as well as accentuated glycolytic and aerobic metabolism. The site of these defects is presumably a calcium release channel of the skeletal muscle sarcoplasmic reticulum (SR), the ryanodine receptor, the footplate protein between the dihydropyridine receptor and the SR. [6,7]Several in vitro studies have demonstrated pharmacologic and biochemical abnormalities in the ryanodine receptor of MHS individuals. [8–11]Linkage studies in MHS pedigrees support the existence of genetic heterogeneity, and it has been estimated that mutations in the ryanodine receptor (RYR1) gene account for approximately 50% of MH cases. [12,13]Sequence analysis of the porcine Ry1receptor gene revealed a single-point mutation in all MHS pigs. However, the corresponding mutation in humans has been found in only approximately 5% of MHS families. [15,16].
It has been reported that serotonin2(5-HT2) antagonists prevent the onset of halothane-induced MH in susceptible pigs and also muscle damage in pigs submitted to physical stress, indicating that serotonin is involved in MH induction. In line with this assumption, the stimulation of 5-HT2receptors with specific ligands induces MH and a psychotic-like behavior, characterized by grimacing, backward locomotion, and blank stares in conscious pigs. Both, MH and this abnormal behavior could be weakened or prevented by 5-HT2receptor antagonists, although this is a matter of debate. [19,20]Furthermore, the free plasma concentration of physiologically active serotonin increased concomitantly with body temperature, plasma lactate, venous carbon dioxide, and muscle tone during a halothane-induced MH crisis in pigs. .
The detection of serotonergic receptors in the membranes of skeletal muscles and the increased sensitivity of MHS pigs in response to environmental stress, which is associated with release of serotonin, support the theory that the serotonin system might be involved in mechanisms triggering the MH syndrome or tends to potentiate the effects of any triggering agents. Moreover, 5-HT2receptors mediate their effects via the second messenger inositol-1,4,5-trisphosphate, which is suspected to play a role in the pathophysiology of MH. [24,25]The current study investigated the effects of the serotonin2(5-HT2) receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) on skeletal muscle specimens from MHS and MHN patients.
Materials and Methods
After approval by the local ethics committee, written informed consent for the different investigations was obtained from the patients or their parents, as appropriate.
We investigated 40 patients from 26 families, 29 adults (age 18–72 yr) and 11 children (age 5–17 yr), with clinical suspicion for MH. Before starting the investigation, we obtained a complete personal and family history, electrocardiogram, and laboratory parameters, including creatine kinase concentration.
Adult muscle biopsies were taken after performing regional anesthesia (3-in-1 nerve block) with 40 ml 1% prilocaine. Biopsies in children were obtained during triggerfree general anesthesia. Patients received 0.1 mg/kg midazolam orally 2 h before anesthetic induction. Anesthesia was induced with an intravenous bolus of 50 micro gram/kg alfentanil followed by 2–2.5 mg/kg propofol. Before laryngoscopy and tracheal intubation, 0.1 mg/kg vecuronium was administered intravenously. A continuous infusion of propofol (less or equal to 150 micro gram *symbol* kg sup -1 *symbol* min sup -1) was used for maintenance of anesthesia. Nitrous oxide (66%) in oxygen was administered to all patients throughout surgery. Two or three muscle bundles were excised carefully from the vastus lateralis, avoiding any trauma. The fresh specimens were placed in a container filled with Krebs-Ringer solution (in mM: NaCl 118.1, KCl 3.4, CaCl22.5, MgSO sub 4 0.8, KH2PO41.2, NaHCO325.0, glucose 11.1) equilibrated with carbogen (95% O2, 5% CO2) and transported without delay to our laboratory. Apart from these specimens, an additional small muscle sample (weight approximately 250 mg) was taken for evaluation of histomorphologic changes.
In Vitro Contracture Test
The muscle bundles were divided carefully into six to ten strips (length 15–25 mm, width 2–3 mm, weight 120–250 mg). Each muscle specimen was secured with silk sutures to a fixed point and connected with a Fleck TF 3V force displacement transducer (Mainz, Germany). The whole system was suspended in a 80 ml organ bath perfused with Krebs-Ringer solution bubbled with carbogen continuously, temperature was constant at 37 degrees Celsius, and pH was 7.4. Isometric tension was amplified by a Gould THE (Cleveland, OH) and recorded with a Gould polygraph TA 2000. The muscles were stimulated electrically with square waves to achieve a supramaximal response by a Grass Stimulator SD 9 (Quincy, MA) with a duration of 1 ms and a frequency of 0.2 Hz. The initial baseline tension was set at 20 mN (corresponding to 2 g).
The patients were first classified as MHS and MHN by the in vitro contracture test with caffeine and halothane (CHCT) according to the procedure of the European Malignant Hyperthermia Group (EMHG). The CHCT gave halothane and caffeine thresholds for each patient as follows: MHS = muscle contractures greater or equal to 2 mN (corresponding to greater or equal to 0.2 g in the original version of the protocol) at a caffeine concentration of 2.0 mM or less and a halothane threshold concentration at 0.44 mM or less; MHN = muscle contractures greater or equal to 2 mN at a caffeine concentration of 3.0 mM or more and a halothane threshold concentration greater than 0.44 mM.
Patients tested as MH-equivocal according to the European protocol were excluded from the investigation.
After investigation of MH susceptibility, surplus muscle specimens from patients tested as MHS and MHN were subjected to the DOI study. After achieving at least 10 min stable baseline tension, DOI was added as a bolus to the organ bath to obtain a concentration of 0.02 mM. In the second investigation, muscle specimens were preincubated with 0.02 mM DOI for 60 min. Subsequently, halothane was added incrementally to the organ bath according to the European protocol. The halothane concentrations used were 0.5%, 1.0%, and 2.0% in the gas phase. Muscles were exposed to halothane for 15 min. The in vitro effects on contracture development and muscle twitch were observed for 120 min in both studies. Furthermore, the effects of halothane on skeletal muscle specimens after 60 min preincubation of 0.02 mM DOI were compared to the results of the contracture test with halothane from the CHCT.
The following chemicals were used: caffeine (Sigma, Deisenhofen, Germany), halothane (Hoechst, Frankfurt, Germany), 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI; as hydrochloride; Research Biochemical, Wayland, MA). All other substances used were pro analysi, or highest purity available. Solutions were prepared fresh before each investigation in carboxygenated Krebs-Ringer solution at 37 degrees Celsius and administered directly to the organ bath. Halothane was added to the carbogen from a Drager vaporizer (Lubeck, Germany), the concentration of halothane was measured with an anesthetic gas monitor (Normac, Datex, Helsinki, Finland). Bath concentration of halothane was determined by gas chromatography. .
Statistical evaluation of our data was performed using a computer-based program (StatView 4.0, Abacus Concepts, Berkeley, CA). Unless otherwise indicated, data are presented as mean+/-SEM. The effects of DOI and DOI plus halothane on contracture development and muscle twitch variables were assessed with repeated-measures analysis of variance. When appropriate, subsequent comparisons were performed using Scheffe's method. Statistical analysis of demographic data and the start of contractures were calculated using the Mann-Whitney test. Results were considered significant if P values were less than 0.05.
Forty patients were included in the study. Twenty-three patients were characterized as MHS and 17 as MHN according to the criteria of the European MH Group. In one patient, a Duchenne's muscular dystrophy was diagnosed; the data of this patient were presented separately. Patients' characteristics did not differ significantly in age, weight, height, and gender (Table 1). However, creatine kinase concentration values at rest were significantly greater in MHS (102+/-26 U/l) than in MHN (54 +/-8 U/l) patients. In 8 of 23 MHS patients (35%), neuromuscular diseases were diagnosed by histologic examination. Seven neuromuscular diseases were nonspecific (i.e., atrophic or hypertrophic fibers, variation in fiber size, signs of necrosis or regeneration). In the MHN group four nonspecific neuromuscular diseases (24%) were found.
After administration of 0.02 mM DOI, muscle specimens of all patients developed contractures. The results of the MHS and MHN muscles are presented in Table 2. The start of contracture development was attained significantly earlier in MHS (16.8+/-1.7 min) than in MHN muscles (66.3+/-5.8 min). There was no overlap between both groups in the range of times. The additional administration of halothane to the organ bath led to a significantly delayed onset of contracture in MHN; the contractures started after 89.7+/-5.6 min. There was no difference between the results of DOI alone and DOI plus halothane in the MHS group.
A 60-min preincubation with 0.02 mM DOI led to a significantly increased contracture development after administration of halothane in MHS muscles compared to the contracture course of the CHCT (Figure 1). Significant contractures in muscle specimens of MHN patients were not observed (data not shown).
In Figure 2, muscle twitch of specimens from both MHS and MHN patients is shown after addition of halothane. In this figure, the effects of halothane after preincubation of 0.02 mM DOI were compared to the results of the CHCT. In both the MHS and MHN groups, a significant difference in muscle twitch was observed before starting administration of halothane. The incremental application of halothane in the CHCT led to an increase of muscle twitch in both groups. After preincubation with 0.02 mM DOI halothane also led to an increased twitch in specimens from MHS and MHN patients. However, this increase was smaller than with halothane alone.
The contractures achieved 120 min after administration of 0.02 mM DOI are shown in Table 2. The contracture development was significantly larger in specimens from MHS than MHN individuals. In the MHS group contractures had reached 12.9+/-1.1 mN and, in the MHN group, 5.3+/-0.6 mN. The additional administration of halothane increased the contracture development in specimens from MHS patients up to 15.9+/-0.9 mN. However, contractures at 120 min decreased significantly to 3.1+/-0.4 mN in specimens from MHN patients.
At the beginning of the investigation, twitch was 32.4 +/-6.2 mN in the specimens from MHS patients and 29.5+/- 4.5 mN in the specimens from MHN individuals (Table 2). Muscle twitch after DOI administration was reduced significantly in specimens from both MHS and MHN patients. After 120 min, twitch decreased in the MHS muscles to 13.5+/-4.4 mN and in MHN muscles to 11.4+/-4.9 mN. In the second investigation with 0.02 mM DOI plus halothane, muscle twitch also decreased in specimens from both groups. Twitch was significantly reduced from 43.9+/-7.2 mN to 15.8+/-4.2 mN in specimens from MHS patients and from 35.8+/-3.9 mN to 13.7+/-3.0 mN in specimens from MHN individuals.
The results of the MHS patient with Duchenne's muscular dystrophy were quite similar to the results of the other MHS individuals. The contracture development after administration of 0.02 mM DOI started after 23.2 min, and the maximum contracture was 12.1 mN. In the second experiment, contracture started after 21.7 min and reached a maximum of 14.2 mN. Muscle twitch was reduced after 0.02 mM DOI plus halothane as well as with 0.02 mM DOI alone.
These data demonstrate that the 5-HT2receptor agonist DOI induces a significant contracture in skeletal muscle specimens of MHS individuals and, in contrast, only small contractures in MHN muscle. Furthermore, DOI decreases muscle twitch in both MHS and MHN muscles. To evaluate whether 5-HT2receptors are involved in halothane-induced MH, halothane was added to skeletal muscles pretreated with DOI in a second experiment. Pretreatment with DOI led to accelerated and increased contracture development after halothane administration only in specimens from MHS patients.
Serotonin is widely distributed in body tissues and occurs in the enterochromaffin cells of gastrointestinal mucosa, the central nervous system, and blood platelets. Radioligand binding techniques revealed different 5-HT receptor subtypes (5-HT1-4) with different properties, e.g., modulation of neuronal activity, regulation of smooth muscle tone, platelet aggregation, mediation of membrane depolarization, behavioral changes, and gastrokinetic action. [23,28].
It has been shown that 5-HT receptors also occur in skeletal muscles. [22,29]In skeletal muscles of the rat, increasing concentrations of 5-HT produced a decrease in the rate of alanine and glutamine release from muscle, indicating a direct effect of 5-HT on skeletal muscle metabolism. These effects seem to require the intermediary participation of muscle cyclic AMP. These findings were supported by the determination of serotonergic receptors in the plasma membranes in skeletal muscles of the rat, and it was hypothesized that 5-HT regulates skeletal muscle metabolism under basal circumstances. .
In swine, MH can be triggered by environmental stress, such as exercise, heat, and excitement. The sympathetic nervous system enhances skeletal muscle responses during stress, but its role in MH appears to be secondary. On the other hand, stress significantly enhances 5-HT release in the brain and circulating 5-HT levels in the blood, [31,32]which may lead to an activation of skeletal muscle by direct effects on 5-HT receptors. Thus, it has been suggested that an activation of 5-HT receptors could trigger MH. .
Previous investigations showed the efficacy of the 5-HT2receptor antagonist ketanserin in preventing or treating halothane- and stress-induced MH in pigs and smaller mammals. Furthermore, ketanserin counteracts the halothane-induced increase of calcium levels in MHS muscle. In contrast, in subsequent in vivo experiments, pretreatment with ketanserin did not prevent development of MH induced by halothane and succinylcholine in susceptible pigs. Furthermore, it was reported that 5-HT2receptor antagonists, such as ketanserin or ritanserin, could not prevent or treat halothane-induced MH in pigs. However, the designs of these investigations were different, e.g., exposure time to halothane, concentration of halothane, and the additional administration of succinylcholine. [17,19]In the treatment of stress-induced hyperthermia in elks, ketanserin produced significant decreases in temperature, heart, and respiratory rates. However, ketanserin showed only incomplete protection; three of nine elks died, but none of the control animals.
It was shown that the stimulation of 5-HT2receptors with specific ligands triggers MH and psychotic-like behavior in conscious MHS pigs. These pigs died within 60 min. In MHN pigs, stimulation of 5-HT2receptors induced transient muscle rigidity, an increase in body temperature, and psychotic-like behavior. In contrast to MHS pigs, the MHN pigs recovered from this syndrome after about 3 h. The 5-HT2receptor antagonists ritanserin and ketanserin were capable of reducing the induction of MH. The authors concluded that induction of MH is due to direct serotonergic effects on skeletal muscle, because 5-HT, which does not cross the blood-brain barrier, also induced skeletal muscle activation. The 5-HT2receptor was suggested as the site of action, because of the MH induction by 5-HT2agonists and the finding that selective or partial agonists at other 5-HT receptor subtypes did not induce MH in susceptible pigs. .
In the current study, DOI induced marked contractures in skeletal muscle specimens from MHS but only small contractures in muscle specimens from MHN patients. 5-HT2receptors mediate their effects by the second messenger inositol-l,4,5-trisphosphate (IP3), which has a key role in controlling both the mobilization of calcium from internal stores and the entry of external calcium. Studies have suggested that an altered inositol phosphate system might be involved in MH. [24,25]Higher basal contents of IP3were measured in skeletal and cardiac muscles of MHS swine and humans than in MHN. Furthermore, in rat aortic myocytes, serotoninstimulated inositol phosphate accumulation was mediated by 5-HT2receptors, whereas selective 5-HT sub 2 receptor antagonists inhibited the serotonin-induced response. In calf aortic smooth muscle cells, 5-HT increases inositol phosphate accumulation. It has been reported that the activation of phospholipase C produces IP3and diacylglycerol in MHS skeletal muscle. Because diacylglycerol is an activator of protein kinase C, it has been discussed that this pathway could play a modulating role in MH, perhaps via an elevation of free fatty acids, which has been suggested as an cause of lowered threshold for calcium-induced calcium release in skeletal muscles. .
Basal concentrations of physiologically active 5-HT are comparable in humans (3.4 ng/ml) and pigs (3.5 ng/ml). Recently, an increase of 5-HT in plasma on an average of 4.9 ng/ml during onset of halothane-induced MH in pigs was measured. The concentrations of 5-HT increased concomitantly with the increases of muscle tone, body temperature, venous PCO2, and plasma lactate. However, it remains unclear whether these changes were induced by halothane or were secondary to the development of MH. On the other hand, the increased levels of serotonin may represent part of a positive feedback loop that tends to potentiate the effects of any triggering agent. Volatile anesthetics were shown to reduce pulmonary 5-HT removal and inhibit 5-HT uptake by blood platelets [21,40]; both could lead to an elevation of 5-HT in blood.
In humans, administration of 5-HT precursors in patients treated for depression is suggested to be dangerous, particularly in combination with monoamine oxidase (MAO) inhibitors and lithium. In two case reports, patients suffered a syndrome similar to MH with fatal outcome. [41,42]The authors concluded that an increase of 5-HT could trigger the syndrome. Another possible explanation in these cases could be a neuroleptic malignant syndrome (NMS). The clinical picture of NMS is similar to MH, but in contrast to MH, the NMS is due to central effects of chronic administration of psychoactive drugs. In this respect, it is interesting to note that the interaction of MAO inhibitors and analgesics such as meperidine triggers a central serotonergic overactivity, presenting as an excitatory form with hyperpyrexia, rigidity, hypertension, unmanageable behavior, and coma. This response could be caused by an increase in the cerebral 5-HT concentration, potentiated by meperidine, which blocks the neuronal uptake of 5-HT. However, it remains uncertain whether a drug-induced increase of cerebral 5-HT concentration could lead to MH and/or NMS.
An increasing number of specific agonists and antagonists at 5-HT receptors were revealed in the past years. The 5-HT1Dreceptor agonist sumatriptan has been introduced for the palliation of migraine headache. The 5-HT precursors tryptophan and 5-hydroxytryptophan are able, in contrast to 5-HT, to pass the blood-brain barrier and stimulate the central 5-HT synthesis. This led to the therapeutic application in an effort to remedy 5-HT deficiency syndromes and the treatment of depressions. The alpha1-receptor agonist urapidil is an antihypertensive drug that also affects central 5-HT1A-receptors. However, there is no evidence that these drugs could trigger MH.
The development of DOI-induced contractures in vitro indicates that the 5-HT2receptor agonist DOI exerts effects not only on the brain 5-HT receptors but also on peripheral receptors at human skeletal muscles. The differences between specimens of MHS and MHN patients in contracture development after administration of DOI support the speculation that an altered serotonin system might be involved in MH. Further pharmacologic and biochemical studies must determine whether 5-HT sub 2 receptors of skeletal muscles from MHS subjects are disordered in function or structure. Receptor binding studies are required to investigate which 5-HT receptor subtypes are possibly involved in the development of MH.
The authors thank Andrea Oldag and Monika Weber, for their excellent technical assistance.