Malignant Hyperthermia Susceptibility Research Articles

Mice expressing T4826I‐RYR1 are viable but exhibit sex‐ and genotype‐dependent susceptibility to malignant hyperthermia and muscle damage

Mutation T4825I in the type 1 ryanodine receptor (RYR1(T4825I/+)) confers human malignant hyperthermia susceptibility (MHS). We report a knock-in mouse line that expresses the isogenetic mutation T4826I. Heterozygous RYR1(T4826I/+) (Het) or homozygous RYR1(T4826I/T4826I) (Hom) mice are fully viable under typical rearing conditions but exhibit genotype- and sex-dependent susceptibility to environmental conditions that trigger MH. Hom mice maintain higher core temperatures than WT in the home cage, have chronically elevated myoplasmic[Ca(2+)](rest), and present muscle damage in soleus with a strong sex bias. Mice subjected to heat stress in an enclosed 37°C chamber fail to trigger MH regardless of genotype, whereas heat stress at 41°C invariably triggers fulminant MH in Hom, but not Het, mice within 20 min. WT and Het female mice fail to maintain euthermic body temperature when placed atop a bed whose surface is 37°C during halothane anesthesia (1.75%) and have no hyperthermic response, whereas 100% Hom mice of either sex and 17% of the Het males develop fulminant MH. WT mice placed on a 41°C bed maintain body temperature while being administered halothane, and 40% of the Het females and 100% of the Het males develop fulminant MH within 40 min. Myopathic alterations in soleus were apparent by 12 mo, including abnormally distributed and enlarged mitochondria, deeply infolded sarcolemma, and frequent Z-line streaming regions, which were more severe in males. These data demonstrate that an MHS mutation within the S4-S5 cytoplasmic linker of RYR1 confers genotype- and sex-dependent susceptibility to pharmacological and environmental stressors that trigger fulminant MH and promote myopathy.

Nonanesthetic Malignant Hyperthermia

Nonanesthetic Malignant Hyperthermia

Defects in Ca2+ release associated with local expression of pathological ryanodine receptors in mouse muscle fibres

Mutations of the gene encoding the type 1 ryanodine receptor (RyR1) are associated with skeletal muscle disorders including malignant hyperthermia susceptibility (MHS) and central core disease (CCD). We used in vivo expression of EGFP-RyR1 constructs in fully differentiated mouse muscle fibres to characterize the function of several RyR1 mutants. Wild-type and Y523S, R615C, R2163H and I4897T mutants of RyR1 were separately expressed and found to be present within restricted regions of fibres with a pattern consistent with triadic localization. Confocal measurements of voltage-clamp-activated myoplasmic Ca(2+) transients demonstrated alterations of sarcoplasmic reticulum (SR) Ca(2+) release spatially correlated with the presence of exogenous RyR1s. The Y523S, R615C and R2163H RyR1 MHS-related mutants were associated with enhanced peak Ca(2+) release for low and moderate levels of depolarization, whereas the I4897T CCD mutant produced a chronic reduction of peak SR Ca(2+) release. For example, peak Ca(2+) release in response to a depolarization to -20 mV in regions of fibres expressing Y523S and I4897T was 2.0 ± 0.3 (n = 9) and 0.46 ± 0.1 (n = 5) times the corresponding value in adjacent, non-expressing regions of the same fibre, respectively. Interestingly no significant change in the estimated total amount of Ca(2+) released at the end of large depolarizing pulses was observed for any of the mutant RyR1 channels. Overall, results are consistent with an 'inherent' increase in RyR1 sensitivity to activation by the voltage sensor for the MHS-related RyR1 mutants and a partial failure of voltage-gated release for the CCD-related I4897T mutant, that occur with no sign of change in SR Ca(2+) content. Furthermore, the results indicate that RyR1 channel density is tightly regulated even under the present conditions of forced exogenous expression.

A study of a family with the skeletal muscle RYR1 mutation (c.7354C>T) associated with central core myopathy and malignant hyperthermia susceptibility

Congenital myopathies are early onset hereditary muscle disorders. A sub-group of these is associated with malignant hyperthermia susceptibility. Mutations in the skeletal muscle ryanodine receptor ( RYR1) gene have been associated with various congenital myopathy phenotypes and may also cause malignant hyperthermia susceptibility. We describe nine affected members of an extended family presenting with a myopathy typically manifesting as upper eye lid ptosis, quadriceps atrophy and patellar dislocation. Three affected members underwent extensive genetic testing and have a RYR1 exon 46 c.7354C>T gene mutation; two of whom had muscle biopsies – both demonstrated central core myopathy. The only affected family member who underwent testing for malignant hyperthermia susceptibility was shown to be positive. The clinical phenotypes seen among affected family members varies widely in severity, and have features in common with those congenital myopathies associated with malignant hyperthermia susceptibility, raising the possibility that these conditions represent a spectrum of disease.

Excess comorbidities associated with malignant hyperthermia diagnosis in pediatric hospital discharge records

Case reports have linked malignant hyperthermia (MH) to several genetic diseases. The objective of this study was to quantitatively assess excess comorbidities associated with MH diagnosis in pediatric hospital discharge records. Data for this study came from the Kids' Inpatient Database (KID) for the years 2000, 2003, and 2006. The KID contains an 80% random sample of patients under the age of 21 discharged from short-term, non-Federal hospitals in the United States, with up to 19 diagnoses recorded for each patient. Using all pediatric inpatients as the reference, we calculated the standardized morbidity ratios (SMRs) and 95% confidence intervals (CIs) for children with MH diagnosis according to major disease groups and specific medical conditions. Of the 5,916,989 nonbirth-related hospital discharges studied, 175 had a recorded diagnosis of MH. Compared with the general pediatric inpatient population, children with MH diagnosis were significantly more likely to be diagnosed with diseases of the musculoskeletal system and connective tissue (SMR 5.7; 95% CI: 3.9-7.9), diseases of the circulatory system (SMR 3.3; 95% CI: 2.1-4.8), and congenital anomalies (SMR 3.2; 95% CI: 2.3-4.4). The specific diagnosis that was most strongly associated with MH was muscular dystrophies (SMR 31.3; 95% CI 12.6-64.6). Diseases of the musculoskeletal system and connective tissue are significantly associated with MH diagnosis in children. Further research is warranted to determine the clinical utility of these comorbidities in assessing MH susceptibility in children.

Fluoroquinolones influence the intracellular calcium handling in individuals susceptible to malignant hyperthermia

The mechanisms of fluoroquinolone-induced myotoxicity are unknown but an involvement of intracellular calcium handling is suspected. An in vitro contracture test used to investigate cellular processes in malignant hyperthermia (MH) can be applied to study the effects of fluoroquinolones. With approval of the local ethics committee, muscle biopsies of 18 MH susceptible (MHS) and 12 MHS non-susceptible (MHN) pigs were performed. Individual bundles were mounted on an isometric force transducer, preloaded, and electrically stimulated. After equilibration they were exposed to ciprofloxacin or levofloxacin. The measured baseline tension was analyzed (Wilcoxon test: P < 0.05). There were no differences in weight, length, or predrug tension between the groups. Both levofloxacin an ciprofloxacin induced significant contractures in MHS muscle bundles but not in MHN. Fluoroquinolones appear to have a pathological influence on intracellular calcium handling. A pre-existing impairment of the calcium homeostasis, however, seems to be necessary for this behavior.

Succinylcholine in malignant hyperthermia: Evaluation of a novel in vivo model

Malignant hyperthermia (MH) is a potentially lethal anesthetic complication characterized by muscle hypermetabolism and generalized rigor. The exact mechanism of succinylcholine as an MH trigger cannot be examined in existing in vitro models. Therefore, a novel in vivo model was used to examine the metabolic response to succinylcholine. With institutional review board approval, 6 MH susceptible (MHS) and 6 MH non-susceptible (MHN) pigs were anesthetized with hemodynamic and systemic metabolic monitoring. Microdialysis catheters were placed intramuscularly. After equilibration, succinylcholine, halothane, and Ringer solution were injected. Lactate was measured in the dialysate and statistically analyzed by Mann-Whitney U-test (significance level P < 0.05). Hemodynamic and systemic metabolic parameters were not different between the groups throughout the experiment. In the MHS pigs, halothane induced a significant increase of lactate. In MHN pigs, no substance induced a reaction. Halothane, but not succinylcholine, induced a hypermetabolic reaction in this model. Therefore, the role of succinylcholine as an MH trigger remains questionable.

King–Denborough syndrome with and without mutations in the skeletal muscle ryanodine receptor ( RYR1) gene

King–Denborough syndrome (KDS), first described in 1973, is a rare condition characterised by the triad of dysmorphic features, myopathy, and malignant hyperthermia susceptibility (MHS). Autosomal dominant inheritance with variable expressivity has been reported in several cases. Mutations in the skeletal muscle ryanodine receptor ( RYR1) gene have been implicated in a wide range of myopathies such as central core disease (CCD), the malignant hyperthermia (MH) susceptibility trait and one isolated patient with KDS. Here we report clinical, pathologic and genetic features of four unrelated patients with KDS. Patients had a relatively uniform clinical presentation but muscle biopsy findings were highly variable. Heterozygous missense mutations in RYR1 were uncovered in three out of four families, of which one mutation was novel and two have previously been reported in MH. Further RyR1 protein expression studies performed in two families showed marked reduction of the RyR1 protein, indicating the presence of allelic RYR1 mutations not detectable on routine sequencing and potentially explaining marked intrafamilial variability. Our findings support the hypothesis that RYR1 mutations are associated with King–Denborough syndrome but that further genetic heterogeneity is likely.

Novel missense mutations and unexpected multiple changes of RYR1 gene in 75 malignant hyperthermia families

Malignant hyperthermia (MH) is an autosomal dominant pharmacogenetic disorder of skeletal muscle characterized by disturbance of intracellular calcium homeostasis in the sarcoplasmic reticulum. Mutations of the ryanodine receptor 1 (RYR1) gene account for most cases, with some studies claiming up to 86% of mutations in this locus. However, RYR1 gene is large and variants are common even in the normal population. We examined 54 families with MH susceptibility and 21 diagnosed with equivocal MH. Thirty-five were selected for an anesthetic reaction, whereas the remainder for hyperCKemia. In these, we studied all 106 exons of the RYR1 gene. When no mutation was found, we also screened: sodium channel voltage-gated, type IV alpha subunit (SCN4A), calcium channel voltage-dependent, L type, alpha 1S subunit (CACNA1S), and L-type voltage-gated calcium channel alpha 2/delta-subunit (CACNL2A). Twenty-nine different RYR1 mutations were discovered in 40 families. Three other MH genes were tested in negative cases. Fourteen RYR1 amino acid changes were novel, of which 12 were located outside the mutational 'hot spots'. In two families, the known mutation p.R3903Q was also observed in malignant hyperthermia-nonsusceptible (MHN) individuals. Unexpectedly, four changes were also found in the same family and two in another. Our study confirms that MH is genetically heterogeneous and that a consistent number of cases are not due to RYR1 mutations. The discordance between in vitro contracture test status and the presence of a proven causative RYR1 mutation suggests that the penetrance may vary due to as yet unknown factors.

Continued progress in understanding the molecular genetics of malignant hyperthermia

A little over 50 years ago, Denborough and Lovell described the syndrome known today as malignant hyperthermia (MH). Little was known about this disorder at the time other than it carried a high fatality rate and was characterized by explosive hyperthermia in an otherwise apparently healthy individual undergoing general anesthesia. The unravelling of the principal features of the syndrome involved a combination of astute clinical observations, a fortuitous association with a similar syndrome in pigs, and assiduous laboratory investigations involving contributions from many countries. Seminal contributions to the understanding of MH were made by individuals associated with the University of Toronto, particularly Drs. Beverly Britt, Werner Kalow, Roderick Gordon, David MacLennan, and their many coworkers. In this issue of the Journal, Dr. MacLennan et al. continue to expand on earlier work in MH, particularly the molecular genetics of MH and the allied myopathy, Central Core Disease. Although the inheritance pattern of MH in humans was known by the 1980s, a turning point in the MH story occurred in 1990 when Dr. MacLennan’s team identified the mutation in the ryanodine receptor gene causal for MH in the swine model of MH. However, the molecular genetics of MH in humans has proven to be far more complex and obscure. This is due, in part, to the inherent complexity of the MH syndrome; to a variety of presentations that may be mimicked by several clinical situations; to a lack of typical phenotypic signs in the absence of anesthesia; to an association with several genes; and potentially, to scores of DNA variants that may lead to the pathophysiologic abnormality in MH, namely, calcium dysregulation in the skeletal muscle cell. Since MH may become manifest without warning and may progress rapidly to life-threatening hypercarbia, muscle breakdown, acidosis, and hyperthermia, much effort has been expended in attempting to identify those at risk for the syndrome. The mainstay diagnostic test for MH is the muscle biopsy contracture test first described by Drs. Britt and Kalow in the early 1970s. The test entails exposure of biopsied skeletal muscle to calcium-releasing agents, such as halothane, caffeine, ryanodine, and /or chloro-m-cresol. Since that time, the contracture test has served to clarify the inheritance of MH, the association with Central Core Disease, and multiple phenotypic presentations of the syndrome. Clearly, it is not applicable either to screening or to rapid identification of susceptibility. Furthermore, as with most laboratory tests, sensitivity and specificity are not 100%. Nevertheless, the test has served as the standard against which other tests are judged. One of the main current goals of research related to MH is the development of a minimally invasive, highly accurate diagnostic test for MH. Molecular genetics holds the promise of meeting that goal. In fact, clinical genetic testing for MH susceptibility is already available in many countries at the present time, but it requires careful patient selection because of the limited sensitivity of genetic testing. The road to the goal line of a sensitive and specific genetic test has proven to be replete with obstacles, just as it has been for many other inherited disorders. For instance, although the ryanodine receptor gene (RYR-1) and the ryanodine receptor or calcium release channel in the sarcoplasmic reticulum are most often associated with the syndrome, other calcium regulatory proteins, such as the dihydropyridine receptor and, more recently, calsequestrin, lead to intracellular calcium dysregulation and clinical MH H. Rosenberg, MD (&) Department of Medical Education and Clinical Research, Saint Barnabas Medical Center, Livingston, NJ, USA e-mail: hrosenberg@sbhcs.com

Ryanodine receptor type 1 gene mutations found in the Canadian malignant hyperthermia population

Malignant hyperthermia (MH) is an autosomal dominant pharmacogenetic disorder that is manifested on exposure of susceptible individuals to halogenated anesthetics or succinylcholine. Since MH is associated primarily with mutations in the ryanodine receptor type 1 (RYR1) gene, the purpose of this study was to determine the distribution and frequency of MH causative RyR1 mutations in the Canadian MH susceptible (MHS) population. In this study, we screened a representative cohort of 36 unrelated Canadian MHS individuals for RYR1 mutations by sequencing complete RYR1 transcripts and selected regions of CACNA1S transcripts. We then analyzed the correlation between caffeine-halothane contracture test (CHCT) results and RYR1 genotypes within MH families. Eighty-six percent of patients had at least one RyR1 mutation (31 out of 36), five of which were unrelated individuals who were double-variant carriers. Fifteen of the 27 mutations identified in RYR1 were novel. Eight novel mutations, involving highly conserved amino acid residues, were predicted to be causal. Two of the mutations co-segregated with the MHS phenotype within two large independent families (a total of 79 individuals). Fourteen percent of MHS individuals (five out of 36) carried neither RYR1 nor known CACNA1S mutations. The distribution and frequency of MH causative RyR1 mutations in the Canadian MHS population are close to those of European MHS populations. Novel mutations described in this study will contribute to the worldwide pool of MH-associated mutations in the RYR1 gene, ultimately increasing the value of MH genetic diagnostic testing.

Cardiopulmonary bypass in malignant hyperthermia susceptible patients: A systematic review of published cases

Malignant hyperthermia susceptibility is an important risk factor during general anesthesia. Affected patients have an asymptomatic but potentially lethal hypermetabolic reaction after contact with volatile anesthetics or succinylcholine. Classic symptoms include hemodynamic instability, combined with acidosis, rigor, and hyperthermia. During cardiopulmonary bypass, these signs may be obscured, delaying correct diagnosis and lifesaving treatment. Malignant hyperthermia-susceptible individuals are more sensitive to heat and stress, so rewarming and catecholamine administration may trigger an episode, necessitating prophylactic measures. This systematic review identified typical malignant hyperthermia symptoms during cardiopulmonary bypass and investigated other factors in cardiac surgery that might trigger an episode in susceptible individuals. Approaches used to treat and prevent malignant hyperthermia during cardiopulmonary bypass were systematically analyzed. We conducted a systematic search for reports about malignant hyperthermia and cardiopulmonary bypass. Search terms included malignant hyperthermia and cardiopulmonary bypass, extracorporeal circulation, or cardiac surgery. We found 24 case reports and case series including details of 26 patients. In 14 cases, malignant hyperthermia crises during or shortly after cardiopulmonary bypass were described. Fourteen reports discussed prevention of an episode. Early symptoms of a malignant hyperthermia episode include excessive carbon dioxide production and metabolic acidosis. Massively increased creatine kinase levels are a strong indicator of a malignant hyperthermia reaction. Rewarming is associated with development of clinical signs of malignant hyperthermia. In potentially susceptible patients, apart from avoiding classic trigger substances, aggressive rewarming should not be applied. Hemodynamic instability in conjunction with the described symptoms should result in a diagnostic algorithm.

Recessive RYR1 mutations cause unusual congenital myopathy with prominent nuclear internalization and large areas of myofibrillar disorganization

To report the clinical, pathological and genetic findings in a group of patients with a previously not described phenotype of congenital myopathy due to recessive mutations in the gene encoding the type 1 muscle ryanodine receptor channel (RYR1). Seven unrelated patients shared a predominant axial and proximal weakness of varying severity, with onset during the neonatal period, associated with bilateral ptosis and ophthalmoparesis, and unusual muscle biopsy features at light and electron microscopic levels. Muscle biopsy histochemistry revealed a peculiar morphological pattern characterized by numerous internalized myonuclei in up to 51% of fibres and large areas of myofibrillar disorganization with undefined borders. Ultrastructurally, such areas frequently occupied the whole myofibre cross section and extended to a moderate number of sarcomeres in length. Molecular genetic investigations identified recessive mutations in the ryanodine receptor (RYR1) gene in six compound heterozygous patients and one homozygous patient. Nine mutations are novel and four have already been reported either as pathogenic recessive mutations or as changes affecting a residue associated with dominant malignant hyperthermia susceptibility. Only two mutations were located in the C-terminal transmembrane domain whereas the others were distributed throughout the cytoplasmic region of RyR1. Our data enlarge the spectrum of RYR1 mutations and highlight their clinical and morphological heterogeneity. A congenital myopathy featuring ptosis and external ophthalmoplegia, concomitant with the novel histopathological phenotype showing fibres with large, poorly delimited areas of myofibrillar disorganization and internal nuclei, is highly suggestive of an RYR1-related congenital myopathy.

Sarcoplasmic Reticulum Ca 2+ Release in Mouse Muscle Fibers Expressing Pathological Mutant Forms of the Type 1 Ryanodine Receptor

Mutations of the gene encoding type 1 ryanodine receptor (RyR1) are associated with susceptibility to malignant hyperthermia (SMH) and/or central core disease (CCD). We used in vivo expression of EGFP-RyR1 DNA constructs to study the function of pathological forms of RyR1 in fully differentiated muscle fibers. The Y522S, R615C, R2163H and I4897T mutant RyR1s were separately expressed in mouse flexor digitorum brevis muscles, using plasmid injection and electroporation. All EGFP-RyR1s were present within restricted regions of the transfected fibers with a pattern consistent with triadic localization. Confocal measurements of voltage-activated rhod-2 Ca2+ transients demonstrated spatially confined alterations of SR Ca2+ release closely correlated with the presence of the mutant channels; in fiber regions expressing Y522S-RyR1 channels, Ca2+ release was specifically enhanced at low and moderate levels of depolarization: for instance peak Ca2+ release in response to pulses from −80 to −30 mV and −10 mV was 3.0 ± 0.6 and 1.5 ± 0.3 (n=9) times larger in the Y522S-positive fiber regions than in adjacent negative ones, respectively. When expressing wild-type EGFP-RyR1s, the corresponding ratio value was 0.86 ± 0.1 (n=9) at the two same voltages. Results similar to the ones with Y522S were observed with R615C and R2163H RyR1s. Conversely, expression of I4897T-RyR1s tended to be associated with a reduced SR Ca2+ release: for instance peak value at −10 mV was depressed by 45 ± 10 % (n=6). Results are thus consistent with an inherent increased sensitivity to activation by the voltage sensor of SMH-associated RyR1 mutant channels and an inherent functional failure of the CCD-associated I4897T ones. Overall, the present strategy proves to be highly effective to reveal the dysfunction of SR Ca2+ release due to mutant-RyR1s in differentiated muscle fibers.

Effects of Theophylline on Anesthetized Malignant Hyperthermia‐Susceptible Pigs

Background. Theophylline was shown to induce contracture development in porcine malignant hyperthermia (MH) susceptible (MHS) skeletal muscles in vitro. The purpose of the current study was to investigate the in vivo effects of theophylline in MHS and MH normal (MHN) swine. Methods. MH-trigger-free general anesthesia was performed in MHS and MHN swine. Theophylline was administered intravenously in cumulative doses up to 93.5 mg·kg−1. The clinical occurrence of MH was defined by changes of central-venous pCO2, central-venous pH, and body core temperature. Results. Theophylline induced comparable clinical alterations in the anesthetized MHS and MHN swine, especially in regard to hemodynamic data. No pig developed hypermetabolism and/or MH according to defined criteria. All animals died with tachycardia followed by ventricular fibrillation. Conclusions. The cumulative theophylline doses used in this study were much higher than doses used therapeutically in humans, as demonstrated by measured blood concentrations. Theophylline is thus not a trigger of MH in genetically determined swine.

In-Vitro and In-Vivo Effects of the Local Anaesthetic Dibucaine on Malignant Hyperthermia–Susceptible and Normal Swine

Background: Ten case reports have described clinical signs and symptoms of malignant hyperthermia (MH) that developed following local anaesthetics. In this study, the In-Vitro effects of dibucaine, an amide local anaesthetic, were compared in skeletal-muscle samples from MH-susceptible (MHS) and normal (MHN) swine. In addition, we investigated the In-Vivo MH trigger potency of dibucaine in MHS swine. Methods: Trigger-free general anaesthesia was induced in six MHS and six MHN swine. Muscle biopsies were obtained. In the In-Vivo experiment, cumulative doses of dibucaine (1, 2, 4, 8, 16, and 32 mg/kg) were administered i.v. In the In-Vitro contracture test (IVCT), dibucaine was added cumulatively to muscle specimens from eight MHS and eight MHN muscles (bath concentrations: 0.1, 0.2, 0.4, 0.6, and 1.0 mmol/l). Results: In the In-Vivo test, no signs or symptoms of MH were observed. In-Vitro contractures developed in all muscle specimens, but they developed significantly faster and were more intense in MHS than in MHN muscles. Conclusion: Dibucaine is not a MH trigger in swine. The IVCT with dibucaine demonstrated overlaps between the diagnostic groups, so that the possibility of 100 % accurate discrimination between MHS and MHN individuals by this means does not seem likely.

Functional and Biochemical Properties of Ryanodine Receptor Type 1 Channels from Heterozygous R163C Malignant Hyperthermia-Susceptible Mice

Mutations in ryanodine receptor type 1 (RyR1) confer malignant hyperthermia susceptibility. How inherent impairments in Ca(2+) channel regulation affect skeletal muscle function in myotubes and adult fibers under basal (nontriggering) conditions are not understood. Myotubes, adult flexor digitorum brevis (FDB) fibers, and sarcoplasmic reticulum skeletal membranes were isolated from heterozygous knockin R163C and wild-type (WT) mice. Compared with WT myotubules, R163C myotubes have reduced Ca(2+) transient amplitudes in response to electrical field pulses; however, R163C FDB fibers do not differ in their responses to electrical stimuli, despite heightened cellular cytoplasmic resting Ca(2+) ([Ca(2+)](rest)) and sensitivity to halothane. Immunoblotting of membranes from each genotype shows similar expression of RyR1, FK506 binding protein 12 kDa, and Ca(2+)-ATPase, but RyR1 (2844)Ser phosphorylation in R163C muscle is 31% higher than that of WT muscle (p < 0.001). RyR1 channels reconstituted in planar lipid bilayers reveal ∼65% of R163C channels exhibit ≥2-fold greater open probability (P(o)) than WT, with prolonged mean open dwell times and shortened closed dwell times. [(3)H]Ryanodine (Ry) binding and single-channel analyses show that R163C-RyR1 has altered regulation compared with WT: 1) 3-fold higher sensitivity to Ca(2+) activation; 2) 2-fold greater [(3)H]Ry receptor occupancy; 3) comparatively higher channel activity, even in reducing glutathione buffer; 4) enhanced RyR1 activity both at 25 and 37°C; and 5) elevated cytoplasmic [Ca(2+)](rest). R163C channels are inherently more active than WT channels, a functional impairment that cannot be reversed by dephosphorylation with protein phosphatase. Dysregulated R163C channels produce a more overt phenotype in myotubes than in adult fibers in the absence of triggering agents, suggesting tighter negative regulation of R163C-RyR1 within the Ca(2+) release unit of adult fibers.

Statins Alter Intracellular Calcium Homeostasis in Malignant Hyperthermia Susceptible Individuals

Statines, HMG-CoA reductase inhibitors, are widely used to treat hypercholesterinemia. These substances are well tolerated, but myotoxic effects have been reported. The exact mechanisms of the induced myotoxicity are unknown but an involvement of intracellular calcium handling is suspected. Individuals susceptible to malignant hyperthermia (MH) have an impaired calcium homeostasis. An in vitro test measuring contracture responses of isolated muscle bundles is used to investigate cellular processes of MH. Aim of this study was to investigate if statins modify the contracture response of isolated muscle bundles from MH susceptible (MHS) and nonsusceptible (MHN) pigs. With approval of the local ethics committee muscle biopsies of 18 MH susceptible and 12 nonsusceptible pigs were performed. Muscle bundles were mounted on an isometric force transducer, preloaded, and electrically stimulated. After establishment of a stable baseline, muscle bundles were exposed to simvastatin, atorvastatin, gemfibrocil, and the pure solvent. Baseline tension was measured and analyzed for changes with P < 0.05 considered to be significant. There were no differences in weight, length, and predrug baseline tension between the groups. Both simvastatin and atorvastatin induced significant contractures in muscle bundles from MHS pigs. Gemfibrocil and the solvent methanol showed no effect. In MHN muscle bundles, none of the tested substances induced a contracture. Statines induce contractures only in MHS muscle bundles. We therefore conclude that the underlying mechanism may be a pathologic influence on intracellular calcium handling that is absent in MHN. A preexisting impairment of the calcium homeostasis seems to be necessary for this behavior because muscle bundles of MHN pigs showed no pathologic reaction. A higher muscle cell vulnerability toward statins is assumed in MHS patients. Statins ought to be used with caution in these individuals. Analogous a diagnostic workup for MH should be considered for patients with statin-induced rhabdomyolyis.

Malignant hyperthermia in India

Malignant hyperthermia in India

Identification and functional characterization of malignant hyperthermia mutation T1354S in the outer pore of the Cavalpha1S-subunit.

To identify the genetic locus responsible for malignant hyperthermia susceptibility (MHS) in an Italian family, we performed linkage analysis to recognized MHS loci. All MHS individuals showed cosegregation of informative markers close to the voltage-dependent Ca(2+) channel (Ca(V)) α(1S)-subunit gene (CACNA1S) with logarithm of odds (LOD)-score values that matched or approached the maximal possible value for this family. This is particularly interesting, because so far MHS was mapped to >178 different positions on the ryanodine receptor (RYR1) gene but only to two on CACNA1S. Sequence analysis of CACNA1S revealed a c.4060A>T transversion resulting in amino acid exchange T1354S in the IVS5-S6 extracellular pore-loop region of Ca(V)α(1S) in all MHS subjects of the family but not in 268 control subjects. To investigate the impact of mutation T1354S on the assembly and function of the excitation-contraction coupling apparatus, we expressed GFP-tagged α(1S)T1354S in dysgenic (α(1S)-null) myotubes. Whole cell patch-clamp analysis revealed that α(1S)T1354S produced significantly faster activation of L-type Ca(2+) currents upon 200-ms depolarizing test pulses compared with wild-type GFP-α(1S) (α(1S)WT). In addition, α(1S)T1354S-expressing myotubes showed a tendency to increased sensitivity for caffeine-induced Ca(2+) release and to larger action-potential-induced intracellular Ca(2+) transients under low (≤ 2 mM) caffeine concentrations compared with α(1S)WT. Thus our data suggest that an additional influx of Ca(2+) due to faster activation of the α(1S)T1354S L-type Ca(2+) current, in concert with higher caffeine sensitivity of Ca(2+) release, leads to elevated muscle contraction under pharmacological trigger, which might be sufficient to explain the MHS phenotype.