Heat-hypersensitive mutants of ryanodine receptor type 1 revealed by microscopic heating.

Thermoregulation is an important aspect of human homeostasis, and high temperatures pose serious stresses for the body. Malignant hyperthermia (MH) is a life-threatening disorder in which body temperature can rise to a lethal level. Here we employ an optically controlled local heat-pulse method to manipulate the temperature in cells with a precision of less than 1 °C and find that the mutants of ryanodine receptor type 1 (RyR1), a key Ca2+ release channel underlying MH, are heat hypersensitive compared with the wild type (WT). We show that the local heat pulses induce an intracellular Ca2+ burst in human embryonic kidney 293 cells overexpressing WT RyR1 and some RyR1 mutants related to MH. Fluorescence Ca2+ imaging using the endoplasmic reticulum-targeted fluorescent probes demonstrates that the Ca2+ burst originates from heat-induced Ca2+ release (HICR) through RyR1-mutant channels because of the channels' heat hypersensitivity. Furthermore, the variation in the heat hypersensitivity of four RyR1 mutants highlights the complexity of MH. HICR likewise occurs in skeletal muscles of MH model mice. We propose that HICR contributes an additional positive feedback to accelerate thermogenesis in patients with MH.

Putative malignant hyperthermia mutation CaV1.1-R174W is insufficient to trigger a fulminant response to halothane or confer heat stress intolerance.

Malignant hyperthermia susceptibility (MHS) is an autosomal dominant pharmacogenetic disorder that manifests as a hypermetabolic state when carriers are exposed to halogenated volatile anesthetics or depolarizing muscle relaxants. In animals, heat stress intolerance is also observed. MHS is linked to over 40 variants in RYR1 that are classified as pathogenic for diagnostic purposes. More recently, a few rare variants linked to the MHS phenotype have been reported in CACNA1S, which encodes the voltage-activated Ca2+ channel CaV1.1 that conformationally couples to RyR1 in skeletal muscle. Here, we describe a knock-in mouse line that expresses one of these putative variants, CaV1.1-R174W. Heterozygous (HET) and homozygous (HOM) CaV1.1-R174W mice survive to adulthood without overt phenotype but fail to trigger with fulminant malignant hyperthermia when exposed to halothane or moderate heat stress. All three genotypes (WT, HET, and HOM) express similar levels of CaV1.1 by quantitative PCR, Western blot, [3H]PN200-110 receptor binding and immobilization-resistant charge movement densities in flexor digitorum brevis fibers. Although HOM fibers have negligible CaV1.1 current amplitudes, HET fibers have similar amplitudes to WT, suggesting a preferential accumulation of the CaV1.1-WT protein at triad junctions in HET animals. Never-the-less both HET and HOM have slightly elevated resting free Ca2+ and Na+ measured with double barreled microelectrode in vastus lateralis that is disproportional to upregulation of transient receptor potential canonical (TRPC) 3 and TRPC6 in skeletal muscle. CaV1.1-R174W and upregulation of TRPC3/6 alone are insufficient to trigger fulminant malignant hyperthermia response to halothane and/or heat stress in HET and HOM mice.

Evaluation of Malignant Hyperthermia Features in Patients with Pathogenic or Likely Pathogenic RYR1 Variants Disclosed through a Population Genomic Screening Program.

BACKGROUND: Malignant hyperthermia (MH) susceptibility is a heritable musculoskeletal disorder that can present as a potentially fatal hypermetabolic response to triggering anesthesia agents. Genomic screening for variants in MH-associated genes RYR1 and CACNA1S provides an opportunity to prevent morbidity and mortality. There are limited outcomes data from disclosing variants in RYR1, the most common MH susceptibility gene, in unselected populations. The authors sought to identify the rate of MH features or fulminant episodes after triggering agent exposure in an unselected population undergoing genomic screening including actionable RYR1 variants. METHODS: The MyCode Community Health Initiative by Geisinger (USA) is an electronic health record-linked biobank that discloses pathogenic and likely pathogenic variants in clinically actionable genes to patient-participants. Available electronic anesthesia and ambulatory records for participants with actionable RYR1 results returned through December 2020 were evaluated for pertinent findings via double-coded chart reviews and reconciliation. Descriptive statistics for observed phenotypes were calculated. RESULTS: One hundred fifty-two participants had an actionable RYR1 variant disclosed during the study period. None had previous documented genetic testing for MH susceptibility; one had previous contracture testing diagnosing MH susceptibility. Sixty-eight participants (44.7%) had anesthesia records documenting triggering agent exposure during at least one procedure. None received dantrolene treatment or had documented muscle rigidity, myoglobinuria, hyperkalemia, elevated creatine kinase, severe myalgia, or tea-colored urine. Of 120 possibly MH-related findings (postoperative intensive care unit admissions, hyperthermia, arterial blood gas evaluation, hypercapnia, or tachycardia), 112 (93.3%) were deemed unlikely to be MH events; 8 (6.7%) had insufficient records to determine etiology. CONCLUSIONS: Results demonstrate a low frequency of classic intraanesthetic hypermetabolic phenotypes in an unselected population with actionable RYR1 variants. Further research on the actionability of screening for MH susceptibility in unselected populations, including economic impact, predictors of MH episodes, and expanded clinical phenotypes, is necessary.

Ryanodine receptor 1 (RYR1) mutations in two patients with tubular aggregate myopathy.

Two likely causative mutations in the RYR1 gene were identified in two patients with myopathy with tubular aggregates, but no evidence of cores or core-like pathology on muscle biopsy. These patients were clinically evaluated and underwent routine laboratory investigations, electrophysiologic tests, muscle biopsy and muscle magnetic resonance imaging (MRI). They reported stiffness of the muscles following sustained activity or cold exposure and had serum creatine kinase elevation. The identified RYR1 mutations (p.Thr2206Met or p.Gly2434Arg, in patient 1 and patient 2, respectively) were previously identified in individuals with malignant hyperthermia susceptibility and are reported as causative according to the European Malignant Hyperthermia Group rules. To our knowledge, these data represent the first identification of causative mutations in the RYR1 gene in patients with tubular aggregate myopathy and extend the spectrum of histological alterations caused by mutation in the RYR1 gene.

Sex-specific alterations in whole body energetics and voluntary activity in heterozygous R163C malignant hyperthermia-susceptible mice.

Malignant hyperthermia (MH) is characterized by induction of skeletal muscle hyperthermia in response to a dysregulated increase in myoplasmic calcium. Although altered energetics play a central role in MH, MH-susceptible humans and mouse models are often described as having no phenotype until exposure to a triggering agent. The purpose of this study was to determine the influence of the R163C ryanodine receptor 1 mutation, a common MH mutation in humans, on energy expenditure, and voluntary wheel running in mice. Energy expenditure was measured by indirect respiration calorimetry in wild-type (WT) and heterozygous R163C (HET) mice over a range of ambient temperatures. Energy expenditure adjusted for body weight or lean mass was increased (P < .05) in male, but not female, HET mice housed at 22°C or when housed at 28°C with a running wheel. In female mice, voluntary wheel running was decreased (P < .05) in the HET vs WT animals when analyzed across ambient temperatures. The thermoneutral zone was also widened in both male and female HET mice. The results of the study show that the R163C mutations alters energetics even at temperatures that do not typically induce MH.

Junctional membrane Ca2+ dynamics in human muscle fibers are altered by malignant hyperthermia causative RyR mutation.

We used the nanometer-wide tubules of the transverse tubular (t)-system of human skeletal muscle fibers as sensitive sensors for the quantitative monitoring of the Ca2+-handling properties in the narrow junctional cytoplasmic space sandwiched between the tubular membrane and the sarcoplasmic reticulum cisternae in single muscle fibers. The t-system sealed with a Ca2+-sensitive dye trapped in it is sensitive to changes in ryanodine receptor (RyR) Ca2+ leak, the store operated calcium entry flux, plasma membrane Ca pump, and sodium-calcium exchanger activities, thus making the sealed t-system a nanodomain Ca2+ sensor of Ca2+ dynamics in the junctional space. The sensor was used to assess the basal Ca2+-handling properties of human muscle fibers obtained by needle biopsy from control subjects and from people with a malignant hyperthermia (MH) causative RyR variant. Using this approach we show that the muscle fibers from MH-susceptible individuals display leakier RyRs and a greater capacity to extrude Ca2+ across the t-system membrane compared with fibers from controls. This study provides a quantitative way to assess the effect of RyR variants on junctional membrane Ca2+ handling under defined ionic conditions.

A recurrent pathogenic variant in TPM2 reveals further phenotypic and genetic heterogeneity in multiple pterygium syndrome-related disorders.

Multiple pterygium syndrome (MPS) disorders are a phenotypically and genetically heterogeneous group of conditions characterized by multiple joint contractures (arthrogryposis), pterygia (joint webbing) and other developmental defects. MPS is most frequently inherited in an autosomal recessive fashion but X-linked and autosomal dominant forms also occur. Advances in genomic technologies have identified many genetic causes of MPS-related disorders and genetic diagnosis requires large targeted next generation sequencing gene panels or genome-wide sequencing approaches. Using the Illumina TruSightOne clinical exome assay, we identified a recurrent heterozygous missense substitution in TPM2 (encoding beta tropomyosin) in three unrelated individuals. This was confirmed to have arisen as a de novo event in the two patients with parental samples. TPM2 mutations have previously been described in association with a variety of dominantly inherited neuromuscular phenotypes including nemaline myopathy, congenital fibre-type disproportion, distal arthrogryposis and trismus pseudocamptodactyly, and in a patient with autosomal recessive Escobar syndrome and a nemaline myopathy. The three cases reported here had overlapping but variable features. Our findings expand the range of TMP2-related phenotypes and indicate that de novo TMP2 mutations should be considered in isolated cases of MPS-related conditions.

Transient Receptor Potential Cation Channels and Calcium Dyshomeostasis in a Mouse Model Relevant to Malignant Hyperthermia.

BACKGROUND: Until recently, the mechanism for the malignant hyperthermia crisis has been attributed solely to sustained massive Ca release from the sarcoplasmic reticulum on exposure to triggering agents. This study tested the hypothesis that transient receptor potential cation (TRPC) channels are important contributors to the Ca dyshomeostasis in a mouse model relevant to malignant hyperthermia. METHODS: This study examined the mechanisms responsible for Ca dyshomeostasis in RYR1-p.G2435R mouse muscles and muscle cells using calcium and sodium ion selective microelectrodes, manganese quench of Fura2 fluorescence, and Western blots. RESULTS: RYR1-p.G2435R mouse muscle cells have chronically elevated intracellular resting calcium and sodium and rate of manganese quench (homozygous greater than heterozygous) compared with wild-type muscles. After exposure to 1-oleoyl-2-acetyl-sn-glycerol, a TRPC3/6 activator, increases in intracellular resting calcium/sodium were significantly greater in RYR1-p.G2435R muscles (from 153 ± 11 nM/10 ± 0.5 mM to 304 ± 45 nM/14.2 ± 0.7 mM in heterozygotes P < 0.001] and from 251 ± 25 nM/13.9 ± 0.5 mM to 534 ± 64 nM/20.9 ± 1.5 mM in homozygotes [P < 0.001] compared with 123 ± 3 nM/8 ± 0.1 mM to 196 ± 27 nM/9.4 ± 0.7 mM in wild type). These increases were inhibited both by simply removing extracellular Ca and by exposure to either a nonspecific (gadolinium) or a newly available, more specific pharmacologic agent (SAR7334) to block TRPC6- and TRPC3-mediated cation influx into cells. Furthermore, local pretreatment with SAR7334 partially decreased the elevation of intracellular resting calcium that is seen in RYR1-p.G2435R muscles during exposure to halothane. Western blot analysis showed that expression of TRPC3 and TRPC6 were significantly increased in RYR1-p.G2435R muscles in a gene-dose-dependent manner, supporting their being a primary molecular basis for increased sarcolemmal cation influx. CONCLUSIONS: Muscle cells in knock-in mice expressing the RYR1-p.G2435R mutation are hypersensitive to TRPC3/6 activators. This hypersensitivity can be negated with pharmacologic agents that block TRPC3/6 activity. This reinforces the working hypothesis that transient receptor potential cation channels play a critical role in causing intracellular calcium and sodium overload in malignant hyperthermia-susceptible muscle, both at rest and during the malignant hyperthermia crisis.

Dantrolene requires Mg2+ to arrest malignant hyperthermia.

Malignant hyperthermia (MH) is a clinical syndrome of skeletal muscle that presents as a hypermetabolic response to volatile anesthetic gases, where susceptible persons may develop lethally high body temperatures. Genetic predisposition mainly arises from mutations on the skeletal muscle ryanodine receptor (RyR). Dantrolene is administered to alleviate MH symptoms, but its mechanism of action and its influence on the Ca2+ transients elicited by MH triggers are unknown. Here, we show that Ca2+ release in the absence of Mg2+ is unaffected by the presence of dantrolene but that dantrolene becomes increasingly effective as cytoplasmic-free [Mg2+] (free [Mg2+]cyto) passes mM levels. Furthermore, we found in human muscle susceptible to MH that dantrolene was ineffective at reducing halothane-induced repetitive Ca2+ waves in the presence of resting levels of free [Mg2+]cyto (1 mM). However, an increase of free [Mg2+]cyto to 1.5 mM could increase the period between Ca2+ waves. These results reconcile previous contradictory reports in muscle fibers and isolated RyRs, where Mg2+ is present or absent, respectively, and define the mechanism of action of dantrolene is to increase the Mg2+ affinity of the RyR (or "stabilize" the resting state of the channel) and suggest that the accumulation of the metabolite Mg2+ from MgATP hydrolysis is required to make dantrolene administration effective in arresting an MH episode.

Permeabilised skeletal muscle reveals mitochondrial deficiency in malignant hyperthermia-susceptible individuals.

BACKGROUND: Individuals genetically susceptible to malignant hyperthermia (MH) exhibit hypermetabolic reactions when exposed to volatile anaesthetics. Mitochondrial dysfunction has previously been associated with the MH-susceptible (MHS) phenotype in animal models, but evidence of this in human MH is limited. METHODS: We used high resolution respirometry to compare oxygen consumption rates (oxygen flux) between permeabilised human MHS and MH-negative (MHN) skeletal muscle fibres with or without prior exposure to halothane. A substrate-uncoupler-inhibitor titration protocol was used to measure the following components of the electron transport chain under conditions of oxidative phosphorylation (OXPHOS) or after uncoupling the electron transport system (ETS): complex I (CI), complex II (CII), CI+CII and, as a measure of mitochondrial mass, complex IV (CIV). RESULTS: Baseline comparisons without halothane exposure showed significantly increased mitochondrial mass (CIV, P=0.021) but lower flux control ratios in CI+CII(OXPHOS) and CII(ETS) of MHS mitochondria compared with MHN (P=0.033 and 0.005, respectively) showing that human MHS mitochondria have a functional deficiency. Exposure to halothane triggered a hypermetabolic response in MHS mitochondria, significantly increasing mass-specific oxygen flux in CI(OXPHOS), CI+CII(OXPHOS), CI+CII(ETS), and CII(ETS) (P=0.001-0.012), while the rates in MHN samples were unaltered by halothane exposure. CONCLUSIONS: We present evidence of mitochondrial dysfunction in human MHS skeletal muscle both at baseline and after halothane exposure.

Strenuous exercise triggers a life-threatening response in mice susceptible to malignant hyperthermia.

In humans, hyperthermic episodes can be triggered by halogenated anesthetics [malignant hyperthermia (MH) susceptibility] and by high temperature [environmental heat stroke (HS)]. Correlation between MH susceptibility and HS is supported by extensive work in mouse models that carry a mutation in ryanodine receptor type-1 (RYR1Y522S/WT) and calsequestrin-1 knockout (CASQ1-null), 2 proteins that control Ca2+ release in skeletal muscle. As overheating episodes in humans have also been described during exertion, here we subjected RYR1Y522S/WT and CASQ1-null mice to an exertional-stress protocol (incremental running on a treadmill at 34°C and 40% humidity). The mortality rate was 80 and 78.6% in RYR1Y522S/WT and CASQ1-null mice, respectively, vs. 0% in wild-type mice. Lethal crises were characterized by hyperthermia and rhabdomyolysis, classic features of MH episodes. Of importance, pretreatment with azumolene, an analog of the drug used in humans to treat MH crises, reduced mortality to 0 and 12.5% in RYR1Y522S/WT and CASQ1-null mice, respectively, thanks to a striking reduction of hyperthermia and rhabdomyolysis. At the molecular level, azumolene strongly prevented Ca2+-dependent activation of calpains and NF-κB by lowering myoplasmic Ca2+ concentration and nitro-oxidative stress, parameters that were elevated in RYR1Y522S/WT and CASQ1-null mice. These results suggest that common molecular mechanisms underlie MH crises and exertional HS in mice.-Michelucci, A., Paolini, C., Boncompagni, S., Canato, M., Reggiani, C., Protasi, F. Strenuous exercise triggers a life-threatening response in mice susceptible to malignant hyperthermia.

[Telephone enquiries on the topic of malignant hyperthermia: Evaluation of the content and subsequent diagnostic results at the MH Center Leipzig]. / Telefonische Anfragen zum Thema maligne Hyperthermie: Auswertung der Inhalte und der resultierenden Diagnostikergebnisse am MH-Zentrum Leipzig.

BACKGROUND: Malignant hyperthermia (MH) is an autosomal dominant metabolic myopathy. The in vitro contracture test (IVCT) is still considered to be the gold standard for diagnosing a disposition for MH. However, advances in genetic testing for MH disposition have supplemented or even replaced the invasive procedure of the IVCT. Information about MH can be obtained by either contacting the hotline for MH as a nationwide 24 h/7 days a week service or one of the regional MH centers. METHODS: The protocols of telephone conversations concerning MH at the MH Center University Leipzig were retrospectively analyzed. Data were collected from January 2011 to March 2015. Additionally, the results of the IVCT and genetic testing evolving from the counseling interviews were examined. RESULTS: A total of 205 telephone calls were documented during the period in question and an IVCT was performed as a consequence of 112 of the telephone calls. The IVCT resulted in 27 individuals being identified as MH susceptible which was subsequently diagnosed in 15 individuals with known familial MH disposition and 12 individuals were identified as new index patients. In 24 individuals a total of 13 different mutations were detected and of these 4 mutations were causative concerning MH. Of the 205 telephone calls 131 were private and 74 of medical professional origin. Among the private enquiries MH disposition within the family was a frequent reason for contacting the MH Center (61.8%). Conversations relating to MH-like symptoms during general anesthesia were carried out with 35.1% of medical doctors and with 22.9% of private callers. Advice about neuromuscular symptoms of unknown genesis was given to 15.3% of private individuals and to 24.3% of medical doctors. Overall MH topics were discussed with 23% (N = 17) of the medical profession and approximately half of these were anesthesiologists (N = 8). Not a single call was documented for the treatment of a suspected MH crisis. CONCLUSION: Private individuals and families affected by a MH disposition often showed good compliance with respect to counseling and diagnostics for MH and contacted the MH center more often than medical doctors. A more comprehensive cooperation with the medical profession is preferable and necessary to obtain a systematic and broad synopsis of characteristic and uncharacteristic signs and symptoms of MH. The telephone conversations analyzed as well as the diagnostic results (IVCT and genetic testing) underline that MH disposition is still a current and relevant topic.

Ca2+ influx via the Na+/Ca2+ exchanger is enhanced in malignant hyperthermia skeletal muscle.

Malignant hyperthermia (MH) is potentially fatal pharmacogenetic disorder of skeletal muscle caused by intracellular Ca(2+) dysregulation. NCX is a bidirectional transporter that effluxes (forward mode) or influxes (reverse mode) Ca(2+) depending on cellular activity. Resting intracellular calcium ([Ca(2+)]r) and sodium ([Na(+)]r) concentrations are elevated in MH susceptible (MHS) swine and murine muscles compared with their normal (MHN) counterparts, although the contribution of NCX is unclear. Lowering [Na(+)]e elevates [Ca(2+)]r in both MHN and MHS swine muscle fibers and it is prevented by removal of extracellular Ca(2+) or reduced by t-tubule disruption, in both genotypes. KB-R7943, a nonselective NCX3 blocker, reduced [Ca(2+)]r in both swine and murine MHN and MHS muscle fibers at rest and decreased the magnitude of the elevation of [Ca(2+)]r observed in MHS fibers after exposure to halothane. YM-244769, a high affinity reverse mode NCX3 blocker, reduces [Ca(2+)]r in MHS muscle fibers and decreases the amplitude of [Ca(2+)]r rise triggered by halothane, but had no effect on [Ca(2+)]r in MHN muscle. In addition, YM-244769 reduced the peak and area under the curve of the Ca(2+) transient elicited by high [K(+)]e and increased its rate of decay in MHS muscle fibers. siRNA knockdown of NCX3 in MHS myotubes reduced [Ca(2+)]r and the Ca(2+) transient area induced by high [K(+)]e. These results demonstrate a functional NCX3 in skeletal muscle whose activity is enhanced in MHS. Moreover reverse mode NCX3 contributes to the Ca(2+) transients associated with K(+)-induced depolarization and the halothane-triggered MH episode in MHS muscle fibers.

Effect of caffeine on intrinsic mechanical properties of normal and malignant hyperthermia-susceptible muscle.

INTRODUCTION: Malignant hyperthermia (MH) is a potentially lethal anesthesic complication. Pathological symptoms develop after exposure to triggering substances. It remains uncertain whether cellular alterations pre-exist. Mechanical properties of isolated muscle bundles were examined before and after exposure to a triggering substance. METHODS: With prior written consent, muscle bundles of 12 MH-susceptible (MHS) and 56 MH-nonsusceptible (MHN) individuals were examined before and after exposure to incremental doses of caffeine. Mechanical properties (baseline tension, peak tension, time to peak tension, and relaxation time) were measured. Contraction and relaxation derivatives and contraction-relaxation coupling were calculated and analyzed. RESULTS: Mechanical properties were not different between the groups before caffeine application. Caffeine increased peak tension in both groups and baseline tension only in MHS muscle bundles; relaxation time/derivative and contraction-relaxation coupling were prolonged. CONCLUSIONS: Cellular changes seen in MH are not pre-existing. Exposure to triggering substance impairs relaxation in MHS muscle.

Nonspecific sarcolemmal cation channels are critical for the pathogenesis of malignant hyperthermia.

Malignant hyperthermia (MH) susceptibility has been attributed to a leaky sarcoplasmic reticulum (SR) caused by missense mutations in RYR1 or CACNA1S, and the MH crisis has been attributed solely to massive self-sustaining release of Ca(2+) from SR stores elicited by triggering agents. Here, we show in muscle cells from MH-RyR1(R163C) knock-in mice that increased passive SR Ca(2+) leak causes an enlarged basal influx of sarcolemmal Ca(2+) that results in chronically elevated myoplasmic free Ca(2+) concentration ([Ca(2+)]i) at rest. We discovered that Gd(+3) and GsMTx-4 were more effective than BTP2 or expression of the dominant-negative Orai1(E190Q) in reducing both Ca(2+) entry and [Ca(2+)]i, implicating a non-STIM1/Orai1 SOCE pathway in resetting resting [Ca(2+)]i. Indeed, two nonselective cationic channels, TRPC3 and TRPC6, are overexpressed, and [Na]i is chronically elevated in MH-RyR1(R163C) muscle cells. [Ca(2+)]i and [Na(+)]i are persistently elevated in vivo and further increased by halothane in MH-RyR1(R163C/WT) muscle. These increases are markedly attenuated by local perfusion of Gd(+3) or GsMTx-4 and completely suppressed by dantrolene. These results contribute a new paradigm for understanding MH pathophysiology by demonstrating that nonselective sarcolemmal cation channel activity plays a critical role in causing myoplasmic Ca(2+) and Na(+) overload both at rest and during the MH crisis.-Eltit, J. M., Ding, X., Pessah, I. N., Allen, P. D., Lopez, J. R. Nonspecific sarcolemmal cation channels are critical for the pathogenesis of malignant hyperthermia.

The effect of succinylcholine on malignant hyperthermia events in susceptible swine.

BACKGROUND: While the impact of volatile anaesthetics to induce malignant hyperthermia (MH) is abundantly clear, the role of succinylcholine still remains controversial. To evaluate the influence of succinylcholine on porcine MH events, the authors investigated the hemodynamic and metabolic responses in MH susceptible (MHS) and non-susceptible (MHN) swine following either succinylcholine or halothane application alone or a combination of both substances. METHODS: With approval of the local animal care committee 27 MHS and 30 MHN pigs were anaesthetized and mechanically ventilated. Fiberoptic probes for continuous PCO2 measurement were inserted into the femoral vein and the triceps muscle. Group A received succinylcholine 4 mg/kg, group B incremental doses of halothane (0.5, 1.0 vol%) and group C succinylcholine and halothane simultaneously. Vital signs were recorded continuously. RESULTS: Prior to drug application measured values did not differ between MHS and MHN. While MHN pigs did not show relevant alterations, succinylcholine, halothane and the combination of both lead to significant hemodynamic and metabolic changes in MHS swine. CONCLUSIONS: Hemodynamic and metabolic alterations following succinylcholine were similar to halothane in MHS pigs. The combination of both pharmacological agents potentiated the observed effects. According to these results succinylcholine acted as an independent and supportive factor during onset of an MH episode.

Escobar syndrome with heterotaxia and esophageal atresia: case report.

Escobar syndrome (ES) or multiple pterygia syndrome (MIM#265000) is an infrequent condition characterized by facial dysmorphism, multiple webbing (pterygia), congenital contractures (arthrogryposis) and other internal anomalies. We describe an 8-days-old male newborn from consanguineous parents with ES who also presented heterotaxia syndrome and esophageal atresia, anomalies that not have been previously reported as associated to ES.

Intra-operative hyperthermia in a cat with a fatal outcome.

HISTORY: A four year old male neutered Domestic Short Hair cat presented for general anaesthesia for hind limb orthopaedic surgery. The cat had been anaesthetized four days previously with propofol and isoflurane and made an uneventful recovery. PHYSICAL EXAMINATION AND MANAGEMENT: On pre-anaesthetic examination the cat had a temperature of 38.9 °C and was otherwise healthy. After a premedication of acepromazine and pethidine, anaesthesia was induced with thiopental and maintained with isoflurane in oxygen 50% and nitrous oxide 50%. Increases in heart rate, respiratory rate, end tidal carbon dioxide tension and temperature were observed, occurring sequentially, from 110 to 175 minutes after anaesthetic induction. Despite ceasing all warming measures and attempting to cool the patient, body temperature continued to rapidly rise, reaching 42.5 °C and limb rigidity was observed. Isoflurane administration was stopped and esmolol was administered. Cardiac arrest occurred. Cardio-pulmonary cerebral resuscitation was commenced and a lateral thoracotomy was performed to allow cardiac compressions and internal defibrillation. Atropine, adrenaline, glucose and dopamine were administered and cold saline was instilled into the thoracic cavity. FOLLOW-UP: Resuscitation was unsuccessful and the cat died. CONCLUSIONS: A presumptive diagnosis of malignant hyperthermia was made. Malignant hyperthermia should be considered, even if prior exposure to volatile inhalational anaesthesia was uneventful, and prompt and aggressive therapy instituted.

Sequence capture and massively parallel sequencing to detect mutations associated with malignant hyperthermia.

BACKGROUND: Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder in which intracellular calcium homeostasis in the skeletal muscle of susceptible individuals is disrupted upon exposure to halogenated anaesthetics. While MH is linked to the ryanodine receptor (RYR1) on chromosome 19 and the α1S subunit of the voltage-dependent L-type calcium channel (CACNA1S) on chromosome 1, mutations have been found in only 50-70% of patients, and subsequently, there is a need for a more powerful screening tool. METHODS: Genomic DNA capture and next-generation sequencing was used to screen 32 genes involved in excitation-contraction coupling, skeletal muscle calcium homeostasis, or immune response in two MH patients. Lymphoblastoid cell lines were used to functionally characterize candidate RYR1 mutations in one family. RESULTS: Sequence analysis revealed two putative causative mutations in RYR1 in one patient. Segregation analysis and functional analysis support a causative role of the detected variants. The amount of Ca(2+) released after stimulation with 4-chloro-m-cresol from B lymphocytes of the MH-susceptible patients in the family was significantly greater compared with that of Ca(2+) released from cells of an MH-negative family member. In the other patient, no causative mutations were identified in the 32 genes screened. CONCLUSIONS: In this study, we successfully demonstrate the use of genomic DNA capture and next-generation sequencing for identification of putative mutations causing MH. We also suggest that whole exome sequencing may be necessary to identify MH causing mutations in patients where no mutations in RYR1 and CACNA1S have been identified thus far.