Cystathionine ß-synthase-derived hydrogen sulfide is involved in human malignant hyperthermia.

Hydrogen sulfide is an endogenous gasotransmitter and its mechanism of action involves activation of ATP-sensitive K(+) channels and phosphodiesterase inhibition. As both mechanisms are potentially involved in malignant hyperthermia (MH), in the present study we addressed the involvement of the L-cysteine/hydrogen sulfide pathway in MH. Skeletal muscle biopsies obtained from 25 MH-susceptible (MHS) and 56 MH-negative (MHN) individuals have been used to perform the in vitro contracture test (IVCT). Quantitative real-time PCR (qPCR) and Western blotting studies have also been performed. Hydrogen sulfide levels are measured in both tissue samples and plasma. In MHS biopsies an increase in cystathionine ß-synthase (CBS) occurs, as both mRNA and protein expression compared with MHN biopsies. Hydrogen sulfide biosynthesis is increased in MHS biopsies (0.128±0.12 compared with 0.943±0.13 nmol/mg of protein per min for MHN and MHS biopsies, respectively; P<0.01). Addition of sodium hydrosulfide (NaHS) to MHS samples evokes a response similar, in the IVCT, to that elicited by either caffeine or halothane. Incubation of MHN biopsies with NaHS, before caffeine or halothane challenge, switches an MHN to an MHS response. In conclusion we demonstrate the involvement of the L-cysteine/hydrogen sulfide pathway in MH, giving new insight into MH molecular mechanisms. This finding has potential implications for clinical care and could help to define less invasive diagnostic procedures.

Elevación crónica de creatincinasa / Chronic creatinine elevation

Las miopatías son entidades en las que se produce afectación musculo esquelética con elevación de creatincinasa en suero. La Enfermedad de Addison y la Hipertermia Maligna constituyen causas poco comunes de miopatía. La Enfermedad de Addison corresponde a una insuficiencia suprarrenal primaria producida por un déficit de glucocorticoides, mineral corticoides y andrógenos por destrucción de la corteza suprarrenal, cuyos síntomas son inespecíficos y el tratamiento es con hidrocortisona. La Hipertermia Maligna se produce por una alteración genética en la que los anestésicos inhalados y los relajantes musculares desencadenan una hipertermia progresiva, con rigidez muscular, taquicardia, hipotensión, rabdomiolisis, y coagulación intravascular diseminada (AU)

Myopathy is a disease that affects the musculoskeletal system with elevated serum creatinine. Addison’s Disease and Malignant Hyperthermia, are rare causes of myopathy. Addison’s disease, also known as primary adrenal insufficiency, occurs when the adrenal gland does not produce enough glucocorticoids, mineralocorticoids and androgens because of the destruction of the adrenal cortex. Symptoms are non-specific and it is treated with hydrocortisone. Malignant Hyperthermia is a genetic disorder, in which inhaled anaesthetics and muscle relaxants, trigger progressive hyperthermia with muscle rigidity, tachycardia, hypotension, rhabdomyolysis, and disseminated intravascular coagulation (AU)

Genetic variation in RYR1 and malignant hyperthermia phenotypes.

BACKGROUND: Malignant hyperthermia (MH) is associated, in the majority of cases, with mutations in RYR1, the gene encoding the skeletal muscle ryanodine receptor. Our primary aim was to assess whether different RYR1 variants are associated with quantitative differences in MH phenotype. METHODS: The degree of in vitro pharmacological muscle contracture response and the baseline serum creatine kinase (CK) concentration were used to generate a series of quantitative phenotypes for MH. We then undertook the most extensive RYR1 genotype-phenotype correlation in MH to date using 504 individuals from 204 MH families and 23 RYR1 variants. We also determined the association between a clinical phenotype and both the laboratory phenotype and RYR1 genotype. RESULTS: We report a novel correlation between the degree of in vitro pharmacological muscle contracture responses and the onset time of the clinical MH response in index cases (P<0.05). There was also a significant correlation between baseline CK concentration and clinical onset time (P=0.039). The specific RYR1 variant was a significant determinant of the severity of each laboratory phenotype (P<0.0001). CONCLUSIONS: The MH phenotype differs significantly with different RYR1 variants. Variants leading to more severe MH phenotype are distributed throughout the gene and tend to lie at relatively conserved sites in the protein. Differences in phenotype severity between RYR1 variants may explain the variability in clinical penetrance of MH during anaesthesia and why some variants have been associated with exercise-induced rhabdomyolysis and heat stroke. They may also inform a mutation screening strategy in cases of idiopathic hyperCKaemia.

Clinical significance and neuropathology of primary MADD in C34-T and G468-T mutations of the AMPD1 gene.

OBJECTIVE: Primary myoadenylate deaminase deficiency (MADD) is probably the most frequent inborn metabolic myopathy with a prevalence of up to 2%. It is the result of mutations in the AMPDI gene, the most common of which is a C34-T transition in exon 2. The importance of the more rare mutation G468-T in exon 5 is uncertain. Primary objective was to elucidate the clinical significance of the enzyme disorder, which remains unclear since its first description in 1978. We further examined the existence of an association of MADD with other muscle disorders, such as malignant hyperthermia and rhabdomyolysis, as was suspected in earlier studies. MATERIAL AND METHODS: In a large collection of 1673 muscle biopsies that had been stored deep frozen we identified 33 cases of primary MADD, 12 of which without any other coinciding muscle diseases, by histochemical, biochemical and molecular genetic examinations. Clinical and laboratory data was collected. By additional examination of randomly chosen blood samples we identified one person carrying the rare compound heterozygosity C34-T/ G468-T, who was examined in clinical respects and a muscle biopsy was taken. RESULTS: As underlying mutation, the most common transition C34-T/C 143-T was detected in 33 cases. One patient carried the compound heterozygosity C34-T/G468-T. The overall frequency of MADD in the contingent was 1.8%. Only three patients out of 12 with isolated primary MADD suffered from muscle complaints, one of whom did not experience the typical symptoms of exercise related myalgia, muscle cramps and weakness as described by Fishbein. The patient carrying C34-T/G468-T was a fully healthy female. She had never experienced any muscle complaints. Any association with other neuromuscular disorders, if not completely ruled out, was found to be very unlikely. CONCLUSION: The results suggest that MADD itself is unlikely to be solely responsible for the manifestation of muscular symptoms. It is probable that either the loss of a compensation mechanism or coexistent disturbances in muscle metabolism which are unidentified so far are required for the emergence of complaints.

In vitro and in vivo effects of the phosphodiesterase-III inhibitor enoximone on malignant hyperthermia-susceptible swine.

BACKGROUND: In human skeletal muscles, the phosphodiesterase-III inhibitor enoximone induces in vitro contracture development, and it has been suggested that enoximone could trigger malignant hyperthermia (MH). In this study, the in vitro and in vivo effects of enoximone in MH-normal (MHN) and MH-susceptible (MHS) swine were investigated. METHODS: Malignant hyperthermia trigger-free general anesthesia was performed in MHS and MHN swine. Skeletal muscle specimens were excised for an in vitro contracture test with 0.6 mm enoximone. Thereafter, MHS and MHN swine were exposed to cumulative administration of 0.5, 1, 2, 4, 8, 16, and 32 mg/kg enoximone intravenously. Clinical occurrence of MH was defined by a Pco(2) greater than 70 mmHg, a pH less than 7.20, and an increase in body temperature of more than 2.0 degrees C. RESULTS: Enoximone induced marked contractures in all MHS muscle specimens in vitro. In contrast, only small or no contracture development was observed in MHN muscle specimens, without an overlap in contractures between MHS and MHN muscles. However, in vivo, no clinical differences were found between MHS and MHN swine following cumulative enoximone doses. According to the defined criteria, none of the swine developed MH during the experiment. Furthermore, high enoximone doses induced progressive circulatory insufficiency, and after receiving 32 mg/kg enoximone, all animals died of cardiovascular failure. CONCLUSIONS: The cumulative enoximone doses used in this study were 30- to 50-fold higher than therapeutic doses in humans. Enoximone does not trigger MH in genetically determined swine. However, enoximone might be useful for in vitro diagnosis of MH.

Altered expression of cardiac myosin isozymes associated with the malignant hyperthermia genotype in swine.

BACKGROUND: Anesthetic-induced malignant hyperthermia (MH) in humans and pigs is associated with dramatic alterations in cardiac function. However, it remains controversial as to whether MH-associated cardiac symptoms represent a primary difference of myocardium or a secondary alteration consequent to increases in the hyperthermic stress. Here the authors describe changes in myosin isoform expression in the hearts of MH-susceptible pigs with and without prior exposure to halothane. METHODS: One group of pigs was diagnosed as MH susceptible by halothane challenge and Hal-1843 nucleotide examination. To determine if there is an effect of halothane exposure, another group of pigs was diagnosed by simple MH genotyping without exposure to halothane. After diagnosis and genotyping, animals with and without exposure to halothane were killed to study cardiac myosin isozyme distributions, cardiac myofibrillar adenosine triphosphatase (ATPase) activity, and the steepness of the Ca2+-ATPase activity relation in the hearts of normal and susceptible pigs. The altered myosin isozyme expression was analyzed by pyrophosphate gel electrophoresis. RESULTS: Malignant hyperthermia-susceptible animals with the prior halothane challenge showed an increased V1 myosin (-44%) expression, increased myofibrillar ATPase activity (-25%) and increased steepness of the Ca2+-ATPase activity relation. Without exposure to halothane, no change of myofibrillar ATPase activity was found in the hearts of different genotyped pigs, but there was a small increase in expression of V1 myosin (-5%) in the mutant (TT). CONCLUSIONS: The potential modulation of V1 myosin expression occurs in the hearts of MH-susceptible pigs. The added stress by halothane challenge would further cause a V3 --> V1 shift, which may be attributed to the long-term effects of hyperthermic stress.

[Are morphologic changes in skeletal muscles of patients with malignant hyperthermia diagnostically useful?]. / Sind morphologische Veränderungen im Skelettmuskel von Patienten mit Maligner Hyperthermie diagnostisch verwertbar?

Malignant Hyperthermia (MH) represents a functional myopathy triggered by volatile anesthetics and depolarizing muscle relaxants, and leading to metabolic disturbances of intracellular Calcium homeostasis. Central-Core-like-structures (CCLS) were recently described as central defects in enzyme-histochemical stains and well correlated to the autosomal-dominant MH-predisposition. We studied the correlation of a MH-predisposition with specific myopathological signs. Skeletal muscles of suspected MH-individuals were histochemically stained by SDH-, NADH-, COX-, Gomori-Trichrome-, ATPase-, Acid Phosphatase-, Oil-red O- und PAS-stain und evaluated without knowing MH-diagnosis by the in-vitro-contracture test. Out of 118 patients (30% MHS ["susceptible"], 63% MHN [normal], 7% MHE ["equivocal"]) 19% revealed pathological findings corresponding to CCLS. 45% of these findings were associated with MHS/MHE. With HE-staining internal nuclei were not specific, but increased with the probability of MHS/MHE from 24% to 80%. Central Cores were correlated in 100% with MHS/MHE (4 out of 118 patients). CCLS were found with about similar frequency in skeletal muscle of MHS/MHE and MHN individuals. Internal nuclei were, however, not specifically, associated with MHS. In contrast, Central Cores correlated significantly with MHS/MHE diagnosis. In conclusion, histopathological findings in skeletal muscle seem to be a reliable marker for MH-predisposition only with Central Cores.

Characterization of mitochondria from pig muscle: higher activity of exo-NADH oxidase in animals suffering from malignant hyperthermia.

Mitochondria were isolated from biopsies of the biceps femoris muscle of Danish landrace pigs. Three groups of animals were compared: (1) normal pigs; (2) pigs that were homozygous with respect to the gene Hal(n)/Hal(n) coding for the porcine malignant hyperthermia syndrome; and (3) heterozygote animals. A newly developed micro-method for preparation and assaying of small quantities of intact mitochondria was employed. With this technique mitochondria from biopsies weighing less than 100 mg were examined with respect to cytochrome content as well as phosphorylating and respiratory activities, including the nonphosphorylating exo-NADH oxidase activity. The mitochondria, prepared in a yield of 48%, showed high respiratory activities with tricarboxylic acid-cycle intermediates and pyruvate, and somewhat lower activity with palmitoyl-carnitine as substrate. The ATP synthase activity was about 1000 micromol ATP/min per g of protein and the maximal respiratory activity approx. 700 micromol of O2/min per g of protein. No differences among the three groups of animals were detected, except for the exo-NADH oxidase activities, which were 43, 78 and 107 micromol of O2/min per g of protein in the groups of normal, heterozygous and homozygous animals respectively. It is concluded that the exo-NADH oxidase activity may be a genetic manifestation of malignant hyperthermia and may play a significant role in the heat production characteristic of the syndrome.

Creatine kinase alterations after acute malignant hyperthermia episodes and common surgical procedures.

Skeletal muscle may release creatine kinase (CK) during a malignant hyperthermia (MH) episode; however, muscle damaged during surgery may also release CK. This study examined the overlap between peak plasma CK levels in patients suspected of having had a MH episode (data obtained from North American MH Registry) and previously published CK changes occurring after common surgeries. For patients who were subsequently proven to be MH positive by muscle biopsy, there was considerable overlap. This was most significant with surgeries having substantial tissue damage, such as major vascular surgery and abdominal surgery. Overlap was much less with minimally invasive surgery, such as cystoscopy. Approximately 30% of MH positive patients treated with dantrolene had peak CK in the range of most surgical procedures, and approximately 50% of MH positive patients not given succinylcholine had peak CK similar to those of most surgical procedures. Dantrolene did not significantly alter peak CK in MH positive patients; however, succinylcholine was associated with significantly higher peak CK. These data suggest that patients who have had an acute MH episode during a surgical procedure may have peak CK values within the range of CK values expected from the procedure itself.

Masseter muscle rigidity and malignant hyperthermia susceptibility in pediatric patients. An update on management and diagnosis.

BACKGROUND: Controversy exists regarding the definition of masseter muscle rigidity (MMR) and anesthetic management after MMR. This study reports current anesthetic management after MMR, estimates the incidence of clinical malignant hyperthermia (MH) in patients with MMR, and is the first to evaluate the coincidence of MMR with malignant hyperthermia susceptibility (MHS) according to the 1987 North American Malignant Hyperthermia Group protocol. METHODS: Practicing anesthesiologists referred pediatric patients for biopsy between 1986 and 1991 based on evidence of MMR after succinylcholine (1975-1991). The clinical scenario was described as MMR alone or MMR followed by signs of MH, including arterial CO2 tension > 50 mmHg, arterial pH < or = 7.25, and base deficit > 8. Patients had caffeine-halothane muscle contracture testing to determine MHS. RESULTS: Seventy patients (50 boys and 20 girls) were evaluated. Eighty-three percent (58 of 70) of anesthetics were halothane-succinylcholine. In 68% (48 of 70) of cases, the anesthetic was discontinued, whereas anesthesia was continued with nontriggering agents in 11% (8 of 70) and with triggering agents in 13% (9 of 70). Fifty-nine percent (41 of 70) of patients were diagnosed as MHS by muscle biopsy. In 7% (5 of 70) of patients, clinical MH developed within 10 min of MMR. CONCLUSIONS: This study, by using the current North American Malignant Hyperthermia Group protocol, reaffirms the high incidence (59%, 41 of 70) of MHS associated with MMR as confirmed by muscle biopsy. Of the MHS patients, 5 developed signs of clinical MH. Most anesthesiologists in this study, when confronted with MMR, discontinued anesthesia. Because of the potential lethality of MH and the > 50% concordance between MMR and MHS, the most conservative course of action after MMR is to discontinue the anesthetic and observe the patient for clinical evidence of MH. An acceptable alternative, depending on the urgency of the surgery, would be to continue anesthesia with nontriggering agents for MH, with appropriate monitoring.

Carnitine palmitoyl transferase deficiency in malignant hyperthermia.

The activity of carnitine palmitoyl transferase, an enzyme that catalyzes the transport of long-chain acylcarnitines into mitochondria, was quantitated in EB-virus-transformed lymphoblasts from 7 patients with susceptibility for malignant hyperthermia. Immunoreactive enzyme protein was also measured using an enzyme-linked immunosorbent assay. Cell lines derived from patients with carnitine palmitoyl transferase deficiency of muscle and from normal individuals were used as positive and negative controls, respectively. One patient with malignant hyperthermia had a deficiency in the enzyme activity which was comparable with that of the known carnitine palmitoyl transferase deficient patients. This individual's lymphoblasts were also deficient in immunoreactive enzyme protein. All of the remaining patients with malignant hyperthermia were deficient only when the backward assay for carnitine palmitoyl transferase was used for quantitation. It is likely that a subset of individuals with a malignant hyperthermia phenotype have a primary deficiency of carnitine palmitoyl transferase and that others have a milder enzyme deficiency secondary to the primary defect in malignant hyperthermia.

Ca(2+)-ATPase and Na(+)-K(+)-ATPase content in skeletal muscle from malignant hyperthermia patients.

The purpose of this study was to determine the concentration of Ca(2+)-ATPase and Na(+)-K(+)-ATPase in biopsies from vastus lateralis muscle of 24 patients, who underwent a diagnostic contracture test for susceptibility to malignant hyperthermia (MH). Ca(2+)-ATPase was quantified as the Ca(2+)-dependent 32P incorporation in whole muscle homogenates. Na(+)-K(+)-ATPase was quantified as the [3H]ouabain-binding capacity in intact muscle samples. These methods avoid isolation of membranes, a procedure that may influence the results due to interindividual variation in recovery. The results show that both enzymes can be determined in (frozen) muscle biopsies weighing 50 mg. Neither the concentration of Ca(2+)-ATPase nor that of Na(+)-K(+)-ATPase differed in biopsies from subjects diagnosed as susceptible (MHS) or nonsusceptible (MHN) to MH. Our data support the view that changes in the concentration of Ca(2+)-ATPase and/or Na(+)-K(+)-ATPase do not play a primary role in the pathogenesis of MH.

Pitfalls in the histochemical demonstration of alpha-glucan phosphorylase activity in glycogen-depleted skeletal muscle fibres.

In the present communication, an investigation is described into the reliability of histochemical methods for the demonstration of alpha-glucan phosphorylase activity in glycogen-depleted skeletal muscle fibres. Human skeletal muscles with glycogen-depleted fibres from patients with diseases of the neuromuscular system and from subjects who had suffered from malignant hyperthermia were used for the study. The location of phosphorylase activity and glycogen was demonstrated with histochemical techniques. Biochemical techniques were used to assay the activity of phosphorylase and the content of glycogen. Biochemical determinations of phosphorylase activity did frequently not reveal significant differences between glycogen-depleted and non-depleted skeletal muscle fibres. In contrast, all histochemical methods investigated, showed little or no phosphorylase activity in the glycogen depleted fibres, indicating that none of the existing histochemical methods revealed reliable staining results in these fibres. Owing to the invalid staining results of the histochemical methods for glycogen-depleted muscle fibres, it is necessary that for metabolic studies a biochemical assay for phosphorylase activity is also to be performed.

Calcium ATPase in the sarcoplasmic reticulum of muscle from normal and malignant hyperthermia susceptible pigs.

The density of Mg(2+)-dependent Ca2+ ATPase in the terminal cisternae of pig skeletal muscle fibers was investigated to discover whether a reduction in Ca2+ ATPase content impairs Ca2+ sequestration and contributes to the elevated myoplasmic Ca2+ concentration in malignant hyperthermia. Unexpectedly, immunogold electron microscopy showed an increase in Ca2+ ATPase, while densitometry of SDS-polyacrylamide gels suggested that the Ca2+ ATPase content of terminal cisternae vesicles did not change. The affinity of Ca2+ ATPase in vesicles for our monoclonal antibody was not altered. We suggest that the availability of antigenic sites in malignant hyperthermia increases after processing for electron microscopy, perhaps as a consequence of altered sarcoplasmic reticulum membrane properties.

The effect of azumolene on hypercontractility and sarcoplasmic reticulum Ca(2+)-dependent ATPase activity of malignant hyperpyrexia-susceptible porcine skeletal muscle.

1. Azumolene sodium is a new water-soluble derivative of dantrolene sodium that also acts as a skeletal-muscle relaxant. 2. Azumolene (6 mumol/L) inhibited the hypercontractility induced separately by 3% halothane, 2 mmol/L caffeine and 80 mmol/L potassium chloride in isolated malignant hyperpyrexia (MH)-susceptible muscle. Azumolene was equipotent with dantrolene in inhibiting the abnormal responses. 3. Like dantrolene, azumolene (6 mumol/L) not only prevented but reversed the abnormal contractures induced by halothane and caffeine. Contracture responses to caffeine were also modified by azumolene in control preparations. 4. In the presence of maximal effective concentrations of dantrolene, azumolene failed to further relax caffeine-induced contractures, and the converse was also true. This was observed in both MH-susceptible and control preparations. 5. Sarcoplasmic reticulum Ca(2+)-dependent ATPase activity from MH-susceptible and control muscle was not affected by azumolene. 6. Like dantrolene, azumolene may inhibit Ca2+ release directly from the sarcoplasmic reticulum and be of therapeutic value for the treatment of MH.

Serum creatine kinase activity as a selection criterion for stress susceptibility after standardised stress in pigs.

Estimation of serum creatine kinase isoenzyme activity was used as a selection criterion for stress sensitivity in pigs 8-12 weeks of age, classified for stress susceptibility as positive to halothane anaesthesia. Standardized stress was provoked by administration of adrenocorticotropic hormone or synstigmine. The experiments were performed under experimental conditions in permanently catheterized animals and in a field trial. It was shown that the stress inductors can be used as a selection criterion for stress sensitivity in catheterized animals by estimation of the CK-MM isoenzyme activity 20-24 h after administration of the inductor. In cases where the individual reaction to halothane anaesthesia is unclear and non classifiable, CK-MM isoenzyme assay performed before and 24 h after the halothane test, may also be useful under practical conditions as an additional indicator of stress sensitivity.

Erythrocyte osmotic fragility, serum creatine kinase and its isoenzymes related to body weight in porcine malignant hyperthermia.

Erythrocyte osmotic fragility, serum creatine kinase (CK) and its isoenzymes (CK-MM, CK-MB and CK-BB) were determined in pigs susceptible (SMH) and resistant (RMH) to malignant hyperthermia at three different body weights (Group 1, 10 to 25 kg; Group 2, 26 to 55 kg; Group 3, 56 to 95 kg). The halothane challenge test was used for determining susceptibility to malignant hyperthermia (MH). Significantly (P less than 0.05) greater erythrocyte haemolysis at saline concentrations of 80 to 100 mM NaCl occurred only between SMH and RMH pigs for body weight group 2. The determination of blood enzyme activities revealed also a significant (P less than 0.001) increase for total CK, CK-MM and CK-BB in SMH and RMH pigs of body weight Group 2. Furthermore in SMH pigs, total CK, CK-MM and CK-MB activities were significantly (P less than 0.05) greater in the animals of body weight Group 2. It was concluded that, in SMH pigs, weighing between 26 and 55 kg, there is a relationship between erythrocyte osmotic fragility, plasma CK, CK isoenzyme activities and muscle development.