Detection of Proton Release from Cultured Human Myotubes to Identify Malignant Hyperthermia Susceptibility

2002 ◽  
Vol 97 (5) ◽  
pp. 1059-1066 ◽  
Author(s):  
Werner Klingler ◽  
Christoph Baur ◽  
Michael Georgieff ◽  
Frank Lehmann-Horn ◽  
Werner Melzer
Keyword(s):  
Skeletal Muscle ◽  
Malignant Hyperthermia ◽  
Metabolic Activation ◽  
Malignant Hyperthermia Susceptibility ◽  
Proton Release ◽  
Release Channel ◽  
Proton Secretion ◽  
In Vitro Contracture Test ◽  
Human Myotubes

Background Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle. During general anesthesia, a life-threatening hypermetabolic state may occur resulting from increased release of Ca2+ from the sarcoplasmic reticulum in skeletal muscle. Diagnosis of MH susceptibility requires surgical muscle biopsies to measure force in response to chemical stimulation (in vitro contracture test, IVCT). Here, the authors investigated an alternative way of discriminating MH-susceptible (MHS) from normal (MHN) subjects by using cultured human myotubes and measuring proton release as an indicator of cellular metabolism. Methods Myotubes were stimulated with the Ca2+ release channel agonist 4-chloro-m-cresol (4-CmC), leading to metabolic activation and proton secretion. The rate of extracellular acidification was recorded with a silicon sensor chip. Results A stepwise increase in 4-CmC concentration led to a phasic-tonic increase in the acidification rate. The response, measured at different concentrations of 4-CmC, was considerably larger in cultures from MHS compared with MHN subjects and correlated well with the force response in the IVCT. Conclusions The enhanced metabolism of cultured skeletal myotubes, likely originating from an increased myoplasmic Ca2+ concentration, can be monitored by studying the proton secretion rate. Because the method seems to be able to distinguish normal from pathologic phenotypes, it is a promising technique for possible future use in less invasive MH testing.

Suspected Malignant Hyperthermia Reactions in New Zealand

2002 ◽  
Vol 30 (4) ◽  
pp. 453-461 ◽  
Author(s):  
A. N. Pollock ◽  
E. E. Langton ◽  
K. Couchman ◽  
K. M. Stowell ◽  
M. Waddington
Keyword(s):  
New Zealand ◽  
Malignant Hyperthermia ◽  
Dna Analysis ◽  
Anaesthetic Agent ◽  
Clinical Signs ◽  
Malignant Hyperthermia Susceptibility ◽  
In Vitro Contracture Test ◽  
Grading Scale ◽  
Clinical Grading

Early clinical signs, triggering agents, time to onset of reaction, mortality and methods of treatment were identified in 123 suspected malignant hyperthermia reactions. In vitro contracture test results were compared with clinical signs and the Malignant Hyperthermia Clinical Grading Scale. Increased end-tidal carbon dioxide is the earliest sign when not preceded by masseter spasm. Earlier diagnosis reduces the incidence of rigidity and severe metabolic acidosis. The combination of suxamethonium and a potent volatile anaesthetic agent triggers an earlier reaction compared with a volatile agent alone. There has been zero mortality since 1981, essentially due to a combination of advanced monitoring capability, increased anaesthetist awareness of malignant hyperthermia, and dantrolene availability. DNA analysis has identified nine New Zealand families with ryanodine receptor gene mutations. A positive DNA test indicates malignant hyperthermia susceptibility with “causative” mutations but discordance requires that negative DNA tests are confirmed with in vitro contracture test. This test also demonstrated the shortcomings of the Malignant Hyperthermia Clinical Grading Scale.

scholarly journals Muscle Biopsy and In Vitro  Contracture Test in Subjects with Idiopathic HyperCKemia

2008 ◽  
Vol 109 (4) ◽  
pp. 625-628 ◽  
Author(s):  
Alessandro Malandrini ◽  
Alfredo Orrico ◽  
Carmen Gaudiano ◽  
Simona Gambelli ◽  
Lucia Galli ◽  
...  
Keyword(s):  
Malignant Hyperthermia ◽  
Muscle Biopsy ◽  
Malignant Hyperthermia Susceptibility ◽  
Manual Muscle Testing ◽  
In Vitro Contracture Test ◽  
Muscle Testing ◽  
Malignant Hyperthermia Susceptible ◽  
Fiber Size ◽  
Precise Diagnosis

Background Persistent high creatine kinase (CK) levels may reflect underlying subclinical myopathies. In most cases, pathogenesis is unknown and clinical management is unclear. Though clinically asymptomatic, these subjects are potentially susceptible to malignant hyperthermia. Methods The authors analyzed 37 subjects with persistent elevation of CK without significant weakness or other neurologic symptoms. Neurologic examination was performed according to manual muscle testing. Muscle biopsy and the in vitro contracture test were performed in all subjects. Results Twenty-three subjects (51.1%) were completely asymptomatic. The others had minor symptoms such as occasional cramps (11 subjects, 24.4%), fatigue (5 subjects, 11.1%), a combination of cramps and fatigue (5 subjects, 11.1%), and muscle pain (1 case, 2.2%). Muscle biopsy enabled precise diagnosis in 3 cases and was normal in 3 cases. The more frequent changes were variation in fiber size (31.1%), a combination of nuclear internalization and variation in fiber size (26.6%), nuclear internalization (6.6%), minor mitochondrial changes (4.4%), and neurogenic atrophy (4.4%). Immunocytochemical analysis was normal in all patients. In vitro contracture testing detected one malignant hyperthermia-susceptible and one malignant hyperthermia-equivocal subject. Conclusions The evidence of malignant hyperthermia susceptibility by in vitro contracture test seems to be relatively infrequent among subjects with idiopathic hyperCKemia, but the incidence of true malignant hyperthermia in idiopathic hyperCKemia is unknown. Muscle biopsy should be considered a useful, though not very sensitive, diagnostic tool in idiopathic hyperCKemia, because it enables potentially treatable disorders, such as inflammatory myopathies, to be discovered. No uniform morphologic finding typical of idiopathic hyperCKemia or malignant hyperthermia susceptibility was identified by muscle biopsy.

scholarly journals Scanning for Mutations of the Ryanodine Receptor (RYR1) Gene by Denaturing HPLC: Detection of Three Novel Malignant Hyperthermia Alleles

2003 ◽  
Vol 49 (5) ◽  
pp. 761-768 ◽  
Author(s):  
Angela Tammaro ◽  
Adele Bracco ◽  
Santolo Cozzolino ◽  
Maria Esposito ◽  
Antonietta Di Martino ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Sudden Increase ◽  
Gene Encoding ◽  
In Vitro Contracture Test ◽  
Ryr1 Gene ◽  
Molecular Tests ◽  
Anesthetic Agents

Abstract Background: Malignant hyperthermia (MH) is a fatal autosomal dominant pharmacogenetic disorder characterized by skeletal muscle hypertonicity that causes a sudden increase in body temperature after exposure to common anesthetic agents. The disease is genetically heterogeneous, with mutations in the gene encoding the skeletal muscle ryanodine receptor (RYR1) at 19q13.1 accounting for up to 80% of the cases. To date, at least 42 RYR1 mutations have been described that cause MH and/or central core disease. Because the RYR1 gene is huge, containing 106 exons, molecular tests have focused on the regions that are more frequently mutated. Thus the causative defect has been identified in only a fraction of families as linked to chromosome 19q, whereas in others it remains undetected. Methods: We used denaturing HPLC (DHPLC) to analyze the RYR1 gene. We set up conditions to scan the 27 exons to identify both known and unknown mutations in critical regions of the protein. For each exon, we analyzed members from 52 families with positive in vitro contracture test results, but without preliminary selection by linkage analysis. Results: We identified seven different mutations in 11 MH families. Among them, three were novel MH alleles: Arg44Cys, Arg533Cys, and Val2117Leu. Conclusion: Because of its sensitivity and speed, DHPLC could be the method of choice for the detection of unknown mutations in the RYR1 gene.

Malignant hyperthermia: excitation-contraction coupling, Ca2+ release channel, and cell Ca2+ regulation defects

1996 ◽  
Vol 76 (2) ◽  
pp. 537-592 ◽  
Author(s):  
J. R. Mickelson ◽  
C. F. Louis
Keyword(s):  
Skeletal Muscle ◽  
Malignant Hyperthermia ◽  
Contractile Activity ◽  
Underlying Mechanism ◽  
Clinical Test ◽  
Release Channel ◽  
Excitation Contraction Coupling ◽  
Contraction Coupling ◽  
Anesthetic Agents

Malignant hyperthermia (MH) is a disorder of skeletal muscle in which certain anesthetic agents trigger a sustained elevation in myoplasmic Ca2+ concentration that activates metabolic and contractile activity. This review focuses on the biochemical and physiological alterations in the skeletal muscle of MH-susceptible (MHS) pigs and humans that appear responsible for this inherited disorder. In porcine MH, these studies identified the skeletal muscle sarcoplasmic reticulum Ca2+ release channel gene (RYR1) as the site of the defect. A mutation in this protein results in altered excitation-contraction coupling and secondary changes in porcine muscle structure and function. Although RYR1 mutations have been reported in many MHS human families, there is also significant genetic heterogeneity, and much less is known as to the underlying mechanism responsible for altered human myoplasmic Ca2+ regulation. The effects of caffeine and anesthetic agents on MHS and normal muscle are also discussed to better understand the basis for the in vitro clinical test for this disorder and mechanisms responsible for the initiation and maintenance of MH episodes in susceptible individuals. Finally, we examine the possiblity of a defect in Ca2+ regulation in tissues other than skeletal muscle. Current understanding of the molecular basis of MH elegantly illustrates the successful integration of knowledge obtained from all fields of biological and clinical science.

Autosomal Dominant Canine Malignant Hyperthermia Is Caused by a Mutation in the Gene Encoding the Skeletal Muscle Calcium Release Channel (RYR1 )

2001 ◽  
Vol 95 (3) ◽  
pp. 716-725 ◽  
Author(s):  
Monica C. Roberts ◽  
James R. Mickelson ◽  
Edward E. Patterson ◽  
Thomas E. Nelson ◽  
P. Jane Armstrong ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Calcium Release ◽  
Autosomal Dominant ◽  
Calcium Release Channel ◽  
Gene Encoding ◽  
Release Channel ◽  
In Vitro Contracture Test ◽  
Mixed Breed ◽  
Muscle Calcium

Background Malignant hyperthermia (MH) is an inherited disorder of skeletal muscle characterized by hypercarbia, rhabdomyolysis, generalized skeletal muscle contracture, cardiac dysrhythmia, and renal failure, that develops on exposure to succinylcholine or volatile anesthetic agents. All swine and up to 50% of human MH events are thought to be associated with mutations in the calcium release channel of the sarcoplasmic reticulum, also known as the ryanodine receptor (RYR1). Events resembling MH have been reported in other species, but none have undergone genetic investigation to date. Methods To determine the molecular basis of canine MH, a breeding colony was established with a male, mixed-breed, MH-susceptible (MHS) dog that survived an in vivo halothane-succinylcholine challenge. He was mated to three unaffected females to produce four litters and back-crossed to an affected daughter to produce one litter. One of his MHS sons was mated to an unaffected female to produce an additional litter. Forty-seven dogs were phenotyped with an in vitro contracture test and diagnosed as MHS or MH normal based on the North American in vitro contracture test protocol. Nine microsatellite markers in the vicinity of RYR1 on canine chromosome 1 (CFA01) were tested for linkage to the MHS phenotype. Mutational analysis in two MHS and two MH-normal dogs was performed with direct sequencing of polymerase chain reaction products and of cloned fragments that represent frequently mutated human RYR1 regions. A restriction fragment length polymorphism was chosen to detect the candidate mutation in the pedigree at large. Results Pedigree inspection revealed that MHS in this colony is transmitted as an autosomal dominant trait. FH2294, the marker closest to RYR1, is linked to MHS at a theta = 0.03 with a LOD score of 9.24. A T1640C mutation gives rise to an alanine for valine substitution of amino acid 547 in the RYR1 protein, generating a maximum LOD score of 12.29 at theta = 0.00. All dogs diagnosed as MHS by in vitro contracture test were heterozygous for the mutation, and all MH-normal dogs were homozygous for the T1640 allele. Conclusions These results indicate that autosomal dominant canine MH is caused by a mutation in the gene encoding the skeletal muscle calcium release channel and that the MHS trait in this pedigree of mixed-breed dogs is in perfect cosegregation with the RYR1 V547A mutation.

scholarly journals Masseter muscle rigidity and the role of DNA analysis to confirm malignant hyperthermia susceptibility

2019 ◽  
Vol 47 (1) ◽  
pp. 60-68 ◽  
Author(s):  
Kate Hudig ◽  
Neil Pollock ◽  
Terasa Bulger ◽  
Roslyn G Machon ◽  
Andrew Woodhead ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Malignant Hyperthermia ◽  
Masseter Muscle ◽  
Dna Analysis ◽  
Malignant Hyperthermia Susceptibility ◽  
Muscle Rigidity ◽  
Autosomal Dominant Disorder ◽  
In Vitro Contracture Test ◽  
Normal Individuals

Malignant hyperthermia (MH) is an uncommon, autosomal dominant disorder of skeletal muscle, triggered by inhalational anaesthetics or depolarizing muscle relaxants. Masseter muscle rigidity (MMR) can be regarded as potentially a preceding sign for an MH reaction. Susceptibility to MH can be determined by the in vitro contracture test (IVCT) or DNA analysis where a familial variant is known. Our aims were to review patients with MMR, where IVCT and DNA analysis had been undertaken, to determine if DNA analysis could be used as an initial screening tool for MH susceptibility, and, by reviewing standard monitored variables (SMVs), to determine if any clinical characteristics could be used to differentiate between MMR patients who are MH susceptible (MHS) and those who are not. Patients with MMR were identified from the Palmerston North Hospital MH Reactions Database. IVCT and DNA analysis results were documented. DNA testing was performed retrospectively in the majority of patients as many patients had presented before DNA analysis was available. Forty-one patients were analysed. Fourteen were DNA positive/IVCT positive and six DNA positive only (48% in total), seven were IVCT positive/DNA negative and 14 were IVCT normal. Increased creatine kinase (>18,000 units/L) was consistent with MH susceptibility. Severity of MMR was not linked to MH susceptibility. This study confirmed that DNA analysis can be used as a first-line test for MH susceptibility in patients presenting with MMR (consistent with European MH Group recommendations). Creatine kinase was the only SMV that was significantly different between MHS and MH normal individuals.

Histomorphologic Examination of Skeletal Muscle Preparations Does Not Differentiate between Malignant Hyperthermia–Susceptible and –Normal Patients

2004 ◽  
Vol 100 (4) ◽  
pp. 789-794 ◽  
Author(s):  
Franziska von Breunig ◽  
Frank Wappler ◽  
Christian Hagel ◽  
Verena von Richthofen ◽  
Marko Fiege ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Malignant Hyperthermia ◽  
Fiber Type ◽  
Type I ◽  
Muscle Sample ◽  
In Vitro Contracture Test ◽  
Pathologic Findings ◽  
Specific Alteration ◽  
Small Muscle

Background It has been suggested that malignant hyperthermia (MH) can be diagnosed by specific myopathologic alterations. The purpose of this study was to investigate whether there are characteristic myopathologic changes in skeletal muscles of MH-susceptible (MHS) compared with MH-normal (MHN) patients. Methods Four hundred forty patients with clinical suspicion of MH were classified as MHN, MH equivocal (MHE), or MHS by the in vitro contracture test with halothane and caffeine. In addition, a small muscle sample excised from each patient was analyzed by histologic, histochemical, immunohistochemical, and computer-aided morphometric methods. Results MHN was diagnosed in 243 patients, MHE was diagnosed in 65, and MHS was diagnosed in 132. No myopathologic abnormalities were found in 53.5% of the MHN, 53.9% of the MHE, and 56.1% of the MHS patients. Thirty-five percent of all patients showed one, 9.8% showed two, and only 0.9% showed three different pathologic findings within skeletal muscle preparations. The frequency of pathologic findings did not differ between the MHN and the MHS patients; only fiber type I predominance was observed more often in MHN. MHE patients could not be assigned to a diagnostic group by detection of myopathologic alterations. In six clinically unaffected patients, a former unrecognized myopathy, such as central core disease, was diagnosed. This disease is characterized by a specific alteration (cores). Conclusions Histologic differences between MHS and MHN statuses could not be demonstrated in this study. Histopathologic examinations can neither improve the diagnosis of MH nor contribute to a better definition of the MH status. However, histopathologic examinations might be useful to detect formerly unrecognized specific myopathies.

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