A. Köchling

In vitro diagnosis of malignant hyperthermia susceptibility with ryanodine-induced contractures in human skeletal muscles

The in vitro contracture test with ryanodine is a new method to distinguish malignant hyperthermia (MH) susceptible (MHS) from normal (MHN) patients.The purpose of our investigation was to determine whether smaller concentrations of ryanodine than those used previously may result in a better differentiation. We performed a ryanodine contracture test (RCT) using concentrations of 1 and 2 micro Meter in muscle specimens of 41 MHS, 58 MHN, and 19 MH-equivocal (MHE) patients. Nine patients were excluded from the study due to neuromuscular diseases. All contracture levels (i.e., start of contractures, contractures of 0.2 g and 1.0 g) were attained significantly earlier in MHS than in MHN muscles at both concentrations of ryanodine. Using a ryanodine concentration of 2 micro Meter, all contracture levels were reached significantly faster than with 1 micro Meter. There was no overlap in the range of times between groups at all contracture levels with ryanodine 1 and 2 micro Meter. The median threshold times for all MHE patients were always between those of MHS and MHN. Defining arbitrarily threshold times for MHS and MHN, an assignment of MHE patients to either MHS or MHN using 1 or 2 micro Meter ryanodine was possible in most cases. Ryanodine administration at a concentration of 1 micro Meter led to a better distinction of MHS from MHN patients than 2 micro Meter. The RCT with ryanodine 1 micro Meter should therefore be added to the current diagnostic methods. (Anesth Analg 1996;82:1230-6)

Fulminant malignant hyperthermia associated with ketoacidotic diabetic coma

Malignant hyperthermia (MH) in humans is usually triggered by volatile anaesthetics and depolarizing muscle relaxants. However, other factors or drugs (e.g. cresol) are thought to induce MH. We report a case of fulminant MH associated with a ketoacidotic diabetic coma. After therapy for diabetic coma with insulin (containing the preservative cresol) and electrolyte solutions was started, the patient complained of increasing myalgia, developed a high fever and respiratory and metabolic acidosis and lost consciousness. MH was treated immediately with dantrolene; the patient recovered within 14 days. Five months later the patient was diagnosed as MH-susceptible by the in vitro caffeine and halothane contracture test. This case supports the assessment that MH and diabetes are associated diseases and that cresol could possibly trigger MH. Furthermore, therapy with dantrolene has been demonstrated to the beneficial in the treatment of MH associated with diabetic coma.

Effects of the serotonin2 receptor agonist DOI on skeletal muscle specimens from malignant hyperthermia-susceptible patients

Background 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. Methods 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. Results Muscle specimens from 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. Conclusions A functional or structural altered serotonin system might be involved in the development of MH in humans.

C1840-T mutation in the human skeletal muscle ryanodine receptor gene: frequency in northern German families susceptible to malignant hyperthermia and the relationship to in vitro contracture response

In swine, a point mutation in the ryanodine receptor gene can account for all cases of malignant hyperthermia (MH). The frequency of a corresponding mutation in humans (C1840-T) and its relationship to the in vitro contracture profile is unknown. We screened 192 patients from 28 unrelated northern German families for the C1840-T mutation in the human ryanodine receptor gene and tested for MH susceptibility using the in vitro contracture test (IVCT) according to the European MH Protocol. In our patients 106 revealed MH susceptible (MHS), 56 MH nonsusceptible and 30 MH equivocal status following IVCT. In each family one or two individuals had developed clinical signs of MH or a MH crisis. All of these patients were classified MHS. The C1840-T mutation was found in 2 of 28 families (7.1%). All eight individuals of the two families characterized by this mutation revealed MHS status following IVCT. The thresholds for halothaneand caffeine-induced contractures as well as the contracture profiles following cumulative (0.4–10.0 μmol/l every 3 min) and bolus (10 μmol/l) administration of ryanodine were found to be similar in MHS patients with and without the C1840-T mutation. In conclusion, the C1840-T mutation in the human ryanodine receptor gene is a rare abnormality in MHS families. Similar contracture profiles in the presence and absence of this mutation might imply no major functional role with respect to the contracture response. At present, molecular genetic analysis cannot replace IVCT to discover MH susceptibility in humans.

Attenuation of serotonin–induced contractures in skeletal muscle from malignant hyperthermia–susceptible patients with dantrolene

Background: Porcine malignant hyperthermia (MH) can be triggered by administration of certain serotonin2 receptor agonists. Pretreatment with dantrolene completely abolished serotonininduced MH. The purpose of this study was to investigate the effects of the serotonin2 receptor agonist l–(2,5–dimethoxy–4–iodophenyl)–2–aminopropane (DOI) in skeletal muscle specimens from MH–susceptible (MHS) and MH–nonsusceptible (MHN) patients following pretreatment with dantrolene.

Effects of serotonin 2 receptor agonists on skeletal muscle preparations in patients with a disposition toward malignant hyperthermia

ZusammenfassungIm Tierexperiment löste die Applikation von Agonisten für Serotonin2-Rezeptoren eine maligne Hyperthermie (MH) aus, Antagonisten konnten hingegen die Entwicklung einer MH beim Schwein verhindern. In einer weiteren Studie wurde ein Anstieg des Serotonins im Plasma während einer Halothan-induzierten MH beim Schwein nachgewiesen. Ziel dieser Untersuchung war die Registrierung der in vitro-Effekte des Serotonin2-Rezeptoragonisten 1-(2,5-Dimethoxy-4-Iodophenyl)-2-Aminopropan (DOI) auf menschliche Skelettmuskelpräparate von MH-Anlageträgern (MHS) und Nichtanlageträgern (MHN). Nach Gabe von 0,02 mmol/l DOI entwickelten die Muskelpräparate aller 37 Patienten Kontrakturen. Allerdings begann in der MHS-Gruppe (n= 17) die Kontrakturentwicklung mit 17,0±1,8 min signifikant früher als in der MHN-Gruppe (64,7± 5,9 min; n=15). Die Kontrakturen der MHS-Muskeln waren signifikant stärker als in der Vergleichsgruppe. Am Ende des Beobachtungszeitraums von 120 min wurde ein Kontrakturmaximum von 12,5±0,9 mN bei MHS- und von 5,1±0,7 mN bei MHN-Muskeln registriert, die Muskelkontraktionskraft zeigte sich in beiden Gruppen signifikant reduziert. Die Ergebnisse von vier als MHE klassifizierten Muskeln waren vergleichbar mit denen der MHS-Muskeln. Die vorgelegte Studie unterstützt die Vermutung, daß bei der MH ein verändertes Serotoninsystem ursächlich beteiligt sein könnte. In weiteren Studien sollte untersucht werden, ob Serotonin2-Rezeptoren der Skelettmuskulatur von MHS Individuen in ihrer Funktion oder Struktur alteriert sind. Serotonin2-Rezeptoragonisten sollten als Triggersubstanzen der MH gelten.AbstractIn pigs genetically susceptible to malignant hyperthermia (MH), it has been shown that serotonin (5-HT2) receptor agonists can induce MH and “psychotic” behaviour. Both can be prevented by 5-HT2 receptor antagonists. Furthermore, free levels of serotonin in plasma increased concomitantly with clinical and laboratory parameters during halothane-induced MH in pigs. In this study the in vitro-effects of the 5-HT2 receptor agonist1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) were investigated in muscle specimens of MH-susceptible (MHS) and normal (MHN) patients. Methods. Muscle biopsies were obtained from 37 patients aged 5–69 years (23.6±5.3 years) with clinical suspicion for MH. The patients were first classified as MHS, MHN or MHE (MH equivocal) by the in vitro contracture test (IVCT) according to the European MH protocol. After MH classification, surplus muscle specimens were subjected to the DOI study. DOI was added to the organ bath in a concentration of 0.02 mmol/l. The in vitro effects on contracture development and muscle twitch were observed for 120 min. Results. Muscle specimens of all patients developed contractures after administration of DOI. However, DOI produced an earlier development of contracture in MHS (17.0±1.8 min; n=17) than in MHN (64.7±5.9 min; n=15) muscles. In MHS muscles, contractures were more distinct than in MHN muscles; at the end of the experiment, contractures had reached a maximum of 12.5±0.9 mN in MHS and 5.1±0.7 mN in MHN muscles. Muscle twitch following DOI administration was reduced significantly in both MHS and MHN muscles. The results of four MHE muscles were comparable with MHS. Conclusion. The present study supports the assumption that an altered serotonin system might be involved in the development of MH. In further studies it should investigated whether 5-HT2 receptors of skeletal muscles from MHS subjects are disordered in function or structure. 5-HT2 receptor agonists should be considered as MH-triggering agents.

Modulation of ryanodine‐induced contractures in human skeletal muscle pretreated with dantrolene

Dantrolene seems to be the causal therapy in malignant hyperthermia (MH) crisis but the complex mechanisms of MH and dantrolene therapy are still not fully understood. The influence of dantrolene on ryanodine‐induced contractures has been reported in animal studies only. In the present study 20 patients from] 7 families were tested for MH using the protocol of the European Malignant Hyperthermia Group. In addition ryanodine‐induced contractures were evaluated following bolus application of 10.0 μmol · 1‐1 ryanodine. After pretreatment with 1 μimol · 1‐1 dantrolene ryanodine‐provoked contractures developed significantly later in MHS (15.8±1.8 min) and MHN (46.0±4.2 min) muscle specimens than after ryanodine alone (MHS 4.8±0.7 min), (MHN 13.7±0.9 min). They were no longer observed in either group after pretreatment with 5 μimol · 1‐1 dantrolene. We conclude that dantrolene is able to attenuate ryanodine‐induced contractures dose‐dependendy, and therefore it is speculated that dantrolene could specifically act at the ryanodine receptor binding site.