Malignant Hyperthermia Susceptibility Research Articles

In vitro effects of levosimendan on muscle of malignant hyperthermia susceptible and non-susceptible swine

BackgroundThe calcium sensitizer levosimendan is increasingly used to improve hemodynamics in patients with acutely decompensated heart failure. By binding to cardiac troponin C the conformation of the calcium-troponin C complex is stabilized, which leads to acceleration of actin-myosin crossbrigde formation and increased force generating capacity of muscle fibers. Besides indications in cardiac failure, beneficial effects of levosimendan in skeletal muscle disorders are currently evaluated. The aim of this study was to investigate differential effects of levosimendan on skeletal muscle of pigs with and without susceptibility to malignant hyperthermia (MH) in order to identify possible risks of this emerging drug for patients with predisposition to MH.MethodsMuscle bundles of 17 pigs (9 MH susceptible (MHS); 8 MH non-susceptible (MHN)) were excised under general anesthesia and examined in the tissue bath with increasing concentrations of levosimendan (0.065; 0.125; 0.5; 1.0; 10 and 50 μg/ml). Baseline tension and twitch force were monitored continuously. Data are presented as median and interquartile range. Statistical evaluation was performed using D’Agostino & Pearson test for normal distribution and student’s t test and 2-way ANOVA for differences between the groups. P < 0.05 was considered significant.ResultsThere were no differences between the groups concerning length, weight, initial twitch force and pre-drug resting tension of the investigated muscle strips. After an initial decrease in both groups, twitch amplitude was significantly higher in MHN (− 3.0 [− 5.2–0.2] mN) compared to MHS (− 7.5 [− 10.8- -4.5] mN) (p = 0.0034) muscle at an applied levosimendan concentration of 50 μg/ml. A marked increase in resting tension was detected following levosimendan incubation with 50 μg/ml in MHS muscle bundles (3.3 [0.9–6.1] mN) compared to MHN (− 0.7 [− 1.3–0.0] mN) (p < 0.0001).ConclusionsThis in vitro investigation revealed the development of significant contractures in muscle bundles of MHS pigs after incubation with levosimendan. However, the effect appeared only at supra-therapeutic concentrations and further research is needed to determine the impact of levosimendan on MHS individuals in vivo.

Association between known or strongly suspected malignant hyperthermia susceptibility and postoperative outcomes: an observational population-based study.

Whether current standards of care management for malignant hyperthermia (MH)-susceptible patients result in acceptable postoperative clinical outcomes at a population level is not known. Our objective was to determine if patients with susceptibility to MH experienced similar outcomes as patients without MH susceptibility after surgery under general anesthesia. This was a retrospective, population-based cohort study from 1 April 2009 until 31 March 2016 in the Canadian province of Ontario. Participants were adults who underwent common in- or outpatient surgeries under general anesthesia. The exposure studied was either known or strongly suspected MH susceptibility as determined by usage of a specific physician billing code. The primary outcome was a composite of all-cause death, hospital readmission, or major postoperative complications, all within 30 postoperative days. Separate analyses were employed, based on whether a patient had in- or outpatient surgery. Inverse probability of exposure weighting based on the propensity score was used to estimate adjusted exposure effects. The cohort included 957,876 patients (583,254 in- and 374,622 outpatients). There were 2,900 (0.3%) patients with a known or strong suspicion of MH susceptibility. For inpatients, the primary outcome occurred in 146,192 (25.1%) of the non-MH-susceptible group and in 337 (20.1%) of the MH-susceptible group (unadjusted risk difference [RD], -5.0%; 95% confidence interval [CI], -6.9 to -3.1%; P < 0.001). In outpatients, the primary outcome occurred in 9,146 (2.4%) of the non-MH-susceptible group and in 32 (2.6%) of the MH-susceptible group (RD, 0.2%; 95% CI, -0.7 to 1.1%; P = 0.72). After adjustment, MH susceptibility was not associated with the primary outcome in either the inpatients (adjusted risk difference [aRD], 1.2%; 95% CI, -1.3 to 3.6%; P = 0.35) or outpatients (aRD, -0.1%; 95% CI -1.0 to 0.9%; P = 0.90). Among adults in Ontario who underwent common surgeries under general anesthesia from 2009 to 2016, known or strongly suspected MH was not associated with a higher risk of adverse postoperative outcomes. These findings support the current standard of care management for MH-susceptible patients.

Comparison of Chlorantraniliprole and Flubendiamide Activity Toward Wild-Type and Malignant Hyperthermia-Susceptible Ryanodine Receptors and Heat Stress Intolerance.

Chlorantraniliprole (CP) and flubendiamide (FD) are widely used in agriculture globally to control lepidopteran pests. Both insecticides target ryanodine receptors (RyRs) and promote Ca2+ leak from sarcoplasmic reticulum (SR) within insect skeletal muscle yet are purportedly devoid of activity toward mammalian RyR1 and muscle. RyRs are ion channels that regulate intracellular Ca2+ release from SR during physiological excitation-contraction coupling. Mutations in RYR1 genes confer malignant hyperthermia susceptibility (MHS), a potentially lethal pharmacogenetic disorder in humans and animals. Compared with vehicle control, CP (10 µM) triggers a 65-fold higher rate of Ca2+ efflux from Ca2+-loaded mammalian WT-RyR1 SR vesicles, whereas FD (10 µM) produces negligible influence on Ca2+ leak. We, therefore, compared whether CP or FD differentially influence patterns of high-affinity [3H]ryanodine ([3H]Ry) binding to RyR1 isolated from muscle SR membranes prepared from adult C57BL/6J mice expressing WT, homozygous C-terminal MHS mutation T4826I, or heterozygous N-terminal MHS mutation R163C. Basal [3H]Ry binding differed among genotypes with rank order T4826I ≫R163C∼WT, regardless of [Ca2+] in the assay medium. Both CP and FD (0.01-100 µM) elicited concentration-dependent increase in [3H]Ry binding, although CP showed greater efficacy regardless of genotype or [Ca2+]. Exposure to CP (500 mg/kg; p.o) failed to shift intolerance to heat stress (38°C) characteristic of R163C and T4826I MHS mice, nor cause lethality in WT mice. Although nM-µM of either diamide is capable of differentially altering WT and MHS RyR1 conformation in vitro, human RyR1 mutations within putative diamide N- and C-terminal interaction domains do not alter heat stress intolerance (HSI) in vivo.

STAC3 variants cause a congenital myopathy with distinctive dysmorphic features and malignant hyperthermia susceptibility.

SH3 and cysteine-rich domain-containing protein 3 (STAC3) is an essential component of the skeletal muscle excitation-contraction coupling (ECC) machinery, though its role and function are not yet completely understood. Here, we report 18 patients carrying a homozygous p.(Trp284Ser) STAC3 variant in addition to a patient compound heterozygous for the p.(Trp284Ser) and a novel splice site change (c.997-1G>T). Clinical severity ranged from prenatal onset with severe features at birth, to a milder and slowly progressive congenital myopathy phenotype. A malignant hyperthermia (MH)-like reaction had occurred in several patients. The functional analysis demonstrated impaired ECC. In particular, KCl-induced membrane depolarization resulted in significantly reduced sarcoplasmic reticulum Ca2+ release. Co-immunoprecipitation of STAC3 with CaV 1.1 in patients and control muscle samples showed that the protein interaction between STAC3 and CaV 1.1 was not significantly affected by the STAC3 variants. This study demonstrates that STAC3 gene analysis should be included in the diagnostic work up of patients of any ethnicity presenting with congenital myopathy, in particular if a history of MH-like episodes is reported. While the precise pathomechanism remains to be elucidated, our functional characterization of STAC3 variants revealed that defective ECC is not a result of CaV 1.1 sarcolemma mislocalization or impaired STAC3-CaV 1.1 interaction.

Malignant hyperthermia: still an issue for neuromuscular diseases?

We will give an overview of neuromuscular disorders that can be linked with malignant hyperthermia or malignant hyperthermia-like reactions, and suggest an appropriate approach to interpret the risks. An increasing number of neuromuscular phenotypes have been linked to malignant hyperthermia susceptibility (MHS). This is for an important part due to the highly variable phenotype associated with mutations in the ryanodine receptor 1 gene (RYR1), the gene most frequently associated with MHS. A RYR1-mutation or a clinical RYR1-phenotype does not automatically translate in MHS, but precautions should be taken nonetheless. In addition, several other genes and phenotypes are now considered to be associated with MHS. In contrast, several neuromuscular diseases that were long thought to be linked to MHS are now known to cause malignant hyperthermia-like reactions instead of malignant hyperthermia. This is highly relevant as not only the given preoperative advice differs, but also acute treatment. This review provides a summary of current evidence linking certain neuromuscular diseases to malignant hyperthermia or malignant hyperthermia-like reactions. We provide a guide for the clinician, to determine which patients are at risk of malignant hyperthermia or malignant hyperthermia-like reactions perioperatively, and to ensure adequate treatment in case such a severe acute complication occurs.

Genetic epidemiology of malignant hyperthermia in the UK

BackgroundGaps in our understanding of genetic susceptibility to malignant hyperthermia (MH) limit the application and interpretation of genetic diagnosis of the condition. Our aim was to define the prevalence and role of variants in the three genes implicated in MH susceptibility in the largest comprehensively phenotyped MH cohort worldwide. MethodsWe initially included one individual from each positive family tested in the UK MH Unit since 1971 to detect variants in RYR1, CACNA1S, or STAC3. Screening for genetic variants has been ongoing since 1991 and has involved a range of techniques, most recently next generation sequencing. We assessed the pathogenicity of variants using standard guidelines, including family segregation studies. The prevalence of recurrent variants of unknown significance was compared with the prevalence reported in a large database of sequence variants in low-risk populations. ResultsWe have confirmed MH susceptibility in 795 independent families, for 722 of which we have a DNA sample. Potentially pathogenic variants were found in 555 families, with 25 RYR1 and one CACNA1S variants previously unclassified recurrent variants significantly over-represented (P<1×10−7) in our cohort compared with the Exome Aggregation Consortium database. There was genotype–phenotype discordance in 86 of 328 families suitable for segregation analysis. We estimate non-RYR1/CACNA1S/STAC3 susceptibility occurs in 14–23% of MH families. ConclusionsOur data provide current estimates of the role of variants in RYR1, CACNA1S, and STAC3 in susceptibility to MH in a predominantly white European population.

CASQ1 Gene Is an Unlikely Candidate for Malignant Hyperthermia Susceptibility in the North American Population: Erratum.

CASQ1 Gene Is an Unlikely Candidate for Malignant Hyperthermia Susceptibility in the North American Population: Erratum.

Mitochondrial Dysfunction in Malignant Hyperthermia Susceptible Skeletal Muscle

Malignant Hyperthermia (MH) is a pharmacogenetic condition primarily caused by mutations in RYR1 and CACNA1S which lead to dysregulation in intracellular calcium handling. Individuals with these mutations suffer from potentially fatal, hypermetabolic reactions when exposed to volatile anaesthetics and/or depolarizing muscle relaxants. Several studies have suggested a possible link between MH susceptibility and mitochondrial dysfunction. Here we investigated skeletal muscle mitochondrial function by measuring oxygen consumption rates (OCR) in six respiratory states through high resolution respirometry using the Oroboros Oxograph-2k analyser. Human skeletal muscle biopsies from MH susceptible (MHS) and MH normal (MHN) individuals confirmed by IVCT, and skeletal muscle from wild type and MHS RYR1G2435R knock-in mice were permeabilized and subjected to a substrate-inhibitor-titration (SUIT) protocol to analyze different stages of oxidative phosphorylation (OXPHOS). In comparison to MHN controls, human MHS skeletal muscle showed a significantly reduced OCR in two different respiratory states, both involving complex II (succinate dehydrogenase) of the electron transport chain. In contrast, when compared to wild type mice, MHS RYR1G2435R knock-in murine mitochondria showed evidence of defect in complex I (NADH dehydrogenase), in addition to an overall reduction in the maximum capacity of the electron transport system (ETS). We conclude that based on these data that there is clear evidence of reduced OXPHOS capacity in MHS human and MHS RYR1G2435R murine mitochondria using permeabilized skeletal muscle. The significantly reduced OCR seen in both datasets, provide additional evidence towards a connection between MH susceptibility and mitochondrial dysfunction, prompting the need for further research in this area.

Cross-Influence of Halothane and Voltage on Intracellular Ca2+ in Isolated Muscle Fibers of Mice Expressing Human RyR1 Mutation Y522S

We studied the effect of halothane and its interference with membrane voltage in malignant hyperthermia (MH) susceptible muscle by using enzymatically isolated single fibers (m. interosseus) of knock-in mice with the RyR1 mutation Y524S (human MH mutation Y522S). To investigate anesthetic- and voltage-triggered changes of Ca2+ concentration, we measured fluorescence from fibers loaded with Fura-2-AM. Similar as in the diagnostic in-vitro contracture test (IVCT) for MH susceptibility, cells were exposed to different halothane-containing solutions using a vaporizer system. The percentage of halothane in the gas phase was determined using infrared photometry. The temporal change of the halothane concentration in the recording chamber was checked using gas chromatography. In Krebs-Ringer's solution at room temperature, a step from 0 to 0.5% halothane in the gas phase caused a clear increase in resting Ca2+ concentration in most of the investigated fibers of mutant mice but very little response in fibers of WT littermates. The Ca2+ response was phasic: after reaching a peak the Ca2+ signal decreased at a rate of about 10%/min indicating either reduced sensitivity to halothane or inhibition of Ca2+ release after activation. A likely explanation is Ca2+-induced inactivation of RyR1. In further experiments, we applied halothane to single fibers which were voltage-clamped using two intracellular microelectrodes. Depolarizing pulses to different membrane potentials from a holding potential of −80 mV revealed a strong shift in the voltage threshold for activation of Ca2+ release to more negative potentials. Correspondingly, hyperpolarizing steps led to a rapid partial recovery from the halothane-induced increase in basal Ca2+ concentration. These results demonstrate reciprocal effects of a volatile anesthetic drug and membrane voltage on Ca2+ release in MH-susceptible skeletal muscle.

Genetic and functional analysis of the RYR1 mutation p.Thr84Met revealed a susceptibility to malignant hyperthermia.

The aim of this study was to analyze the genetic and functional role of a novel RYR1 variant c.251 C>T (p.Thr84Met) identified in a patient with muscle weakness demonstrating MH susceptibility. DNA testing of family members was conducted for assessment of pathogenicity of the genetic variant. For functional analysis, Ca2+ measurement using patient-derived myotubes and p.Thr84Met RYR1-transfected human embryonic kidney (HEK)-293 cells was performed to evaluate reactivity to RYR1 activators. The half-maximal effective concentration (EC50) values of two RYR1 activators, caffeine and 4-chloro-m-cresol (4CmC), were calculated from the acquired dose-response curves. The EC50 was compared between two groups: for myotubes, the control group and the patient, and for HEK-293 cells, WT and p.Thr84Met. Dose-response curves for caffeine and 4CmC were shifted to the left in both myotubes and HEK-293 cells compared to controls. The 50% effective concentration values for caffeine and 4CmC were significantly lower in both myotubes and HEK-293 cells compared to controls (P<0.001 for all comparisons). Our results of functional testing indicated RYR1 hypersensitivity to caffeine and 4CmC. We conclude that the genetic variant was associated with MH susceptibility.

Malignant Hyperthermia Susceptibility and Related Diseases.

This review identifies disease states associated with malignant hyperthermia susceptibility based on genotypic and phenotypic findings, and a framework is established for clinicians to identify a potentially malignant hyperthermia–susceptible patient.

Phenotype and genotype of muscle ryanodine receptor rhabdomyolysis-myalgia syndrome.

Rhabdomyolysis and myalgia are common conditions, and mutation in the ryanodine receptor 1 gene (RYR1) is suggested to be a common cause. Due to the large size of RYR1, however, sequencing has not been widely accessible before the recent advent of next-generation sequencing technology and limited phenotypic descriptions are therefore available. We present the medical history, clinical and ancillary findings of patients with RYR1 mutations and rhabdomyolysis and myalgia identified in Denmark, France and The Netherlands. Twenty-two patients with recurrent rhabdomyolysis (CK>10000) or myalgia with hyperCKemia (>1.5×ULN) and a RYR1 mutation were identified. One had mild wasting of the quadriceps muscle, but none had fixed weakness. Symptoms varied from being restricted to intense exercise to limiting ADL function. One patient developed transient kidney failure during rhabdomyolysis. Two received immunosuppressants on suspicion of myositis. None had episodes of malignant hyperthermia. Muscle biopsies were normal, but CT/MRI showed muscle hypertrophy in most. Delay from first symptom to diagnosis was 12years on average. Fifteen different dominantly inherited mutations were identified. Ten were previously described as pathogenic and 5 were novel, but rare/absent from the background population, and predicted to be pathogenic by in silico analyses. Ten of the mutations were reported to give malignant hyperthermia susceptibility. Mutations in RYR1 should be considered as a significant cause of rhabdomyolysis and myalgia syndrome in patients with the characteristic combination of rhabdomyolysis, myalgia and cramps, creatine kinase elevation, no weakness and often muscle hypertrophy.

Malignant Hyperthermia and Ryanodine Receptor Type 1 Gene (RyR1) Mutation in a Family in Singapore

Sporadic clinical episodes of malignant hyperthermia (MH) that develop during general anaesthesia (GA) have been reported in Singapore. However, there is no published local report of a confirmed case of MH susceptibility (MHS) by skeletal muscle contracture tests and/or molecular tests. We report 2 patients from an extended family who developed signs of clinical MH while under GA. The MH episodes were successfully treated with intravenous dantrolene sodium. Sequence analysis of the entire Ryanodine Receptor Type 1 (RyR1) coding gene was carried out in an index patient. The index patient was found to carry a c.7373G>A (p.Arg2458His) mutation in exon 46. This particular mutation satisfies the criteria for a MHS causative mutation. Hence, the index patient was considered to be MHS and did not need to undergo further muscle contracture testing. The same mutation was also found in 3 other members of his extended family. This is the first report of a Singaporean family with at least 4 members carrying a MH-causative mutation in RyR1 gene. This report serves to highlight the existence of the putative gene for MH in Singapore, and the need for clinical vigilance during anaesthesia involving the use of triggering agents.

Voltage modulates halothane-triggered Ca2+ release in malignant hyperthermia-susceptible muscle.

Malignant hyperthermia (MH) is a fatal hypermetabolic state that may occur during general anesthesia in susceptible individuals. It is often caused by mutations in the ryanodine receptor RyR1 that favor drug-induced release of Ca2+ from the sarcoplasmic reticulum. Here, knowing that membrane depolarization triggers Ca2+ release in normal muscle function, we study the cross-influence of membrane potential and anesthetic drugs on Ca2+ release. We used short single muscle fibers of knock-in mice heterozygous for the RyR1 mutation Y524S combined with microfluorimetry to measure intracellular Ca2+ signals. Halothane, a volatile anesthetic used in contracture testing for MH susceptibility, was equilibrated with the solution superfusing the cells by means of a vaporizer system. In the range 0.2 to 3%, the drug causes significantly larger elevations of free myoplasmic [Ca2+] in mutant (YS) compared with wild-type (WT) fibers. Action potential-induced Ca2+ signals exhibit a slowing of their time course of relaxation that can be attributed to a component of delayed Ca2+ release turnoff. In further experiments, we applied halothane to single fibers that were voltage-clamped using two intracellular microelectrodes and studied the effect of small (10-mV) deviations from the holding potential (-80 mV). Untreated WT fibers show essentially no changes in [Ca2+], whereas the Ca2+ level of YS fibers increases and decreases on depolarization and hyperpolarization, respectively. The drug causes a significant enhancement of this response. Depolarizing pulses reveal a substantial negative shift in the voltage dependence of activation of Ca2+ release. This behavior likely results from the allosteric coupling between RyR1 and its transverse tubular voltage sensor. We conclude that the binding of halothane to RyR1 alters the voltage dependence of Ca2+ release in MH-susceptible muscle fibers such that the resting membrane potential becomes a decisive factor for the efficiency of the drug to trigger Ca2+ release.

Functional and structural characterization of a novel malignant hyperthermia-susceptible variant of DHPR-β1a subunit (CACNB1).

Malignant hyperthermia (MH) susceptibility has been recently linked to a novel variant of β1a subunit of the dihydropyridine receptor (DHPR), a channel essential for Ca2+ regulation in skeletal muscle. Here we evaluate the effect of the mutant variant V156A on the structure/function of DHPR β1a subunit and assess its role on Ca2+ metabolism of cultured myotubes. Using differential scanning fluorimetry, we show that mutation V156A causes a significant reduction in thermal stability of the Src homology 3/guanylate kinase core domain of β1a subunit. Expression of the variant subunit in β1-null mouse myotubes resulted in increased sensitivity to caffeine stimulation. Whole cell patch-clamp analysis of β1a-V156A-expressing myotubes revealed a -2 mV shift in voltage dependence of channel activation, but no changes in Ca2+ conductance, current kinetics, or sarcoplasmic reticulum Ca2+ load were observed. Measurement of resting free Ca2+ and Na+ concentrations shows that both cations were significantly elevated in β1a-V156A-expressing myotubes and that these changes were linked to increased rates of plasmalemmal Ca2+ entry through Na+/Ca2+ exchanger and/or transient receptor potential canonical channels. Overall, our data show that mutant variant V156A results in instability of protein subdomains of β1a subunit leading to a phenotype of Ca2+ dysregulation that partly resembles that of other MH-linked mutations of DHPR α1S subunit. These data prove that homozygous expression of variant β1a-V156A has the potential to be a pathological variant, although it may require other gene defects to cause a full MH phenotype.

Mitochondrial-nuclear crosstalk, haplotype and copy number variation distinct in muscle fiber type, mitochondrial respiratory and metabolic enzyme activities

Genes expressed in mitochondria work in concert with those expressed in the nucleus to mediate oxidative phosphorylation (OXPHOS), a process that is relevant for muscle metabolism and meat quality. Mitochondrial genome activity can be efficiently studied and compared in Duroc and Pietrain pigs, which harbor different mitochondrial haplotypes and distinct muscle fiber types, mitochondrial respiratory activities, and fat content. Pietrain pigs homozygous-positive for malignant hyperthermia susceptibility (PiPP) carried only haplotype 8 and showed the lowest absolute mtDNA copy number accompanied by a decrease transcript abundance of mitochondrial-encoded subunits ND1, ND6, and ATP6 and nuclear-encoded subunits NDUFA11 and NDUFB8. In contrast, we found that haplotype 4 of Duroc pigs had significantly higher mitochondrial DNA (mtDNA) copy numbers and an increase transcript abundance of mitochondrial-encoded subunits ND1, ND6, and ATP6. These results suggest that the variation in mitochondrial and nuclear genetic background among these animals has an effect on mitochondrial content and OXPHOS system subunit expression. We observed the co-expression pattern of mitochondrial and nuclear encoded OXPHOS subunits suggesting that the mitochondrial-nuclear crosstalk functionally involves in muscle metabolism. The findings provide valuable information for understanding muscle biology processes and energy metabolism, and may direct use for breeding strategies to improve meat quality and animal health.

Idiopathic hyperCKemia and malignant hyperthermia susceptibility.

HyperCKemia is a persistent rise in serum creatine kinase (CK) levels of at least 1.5 times the normal value, as evidenced by a minimum of two measurements at 30-day intervals. One of the neuromuscular diseases associated with hyperCKemia is malignant hyperthermia (MH). This study investigated the susceptibility to MH in patients with hyperCKemia via in vitro contracture testing (IVCT) and a search of mutations in the RYR1 gene. Patients in an MH centre were followed from 1997-2012, and their epidemiologic, clinical, and laboratory data were analyzed, including IVCT, muscle histochemical analysis, and next-generation sequencing molecular analysis. There were nine patients (eight male) in our study with a mean (SD) age of 33 (12) yr. Four patients were Caucasian and five were African Brazilian. Most complained about myalgia or cramps, but all had a normal neurological examination. They persistently presented with hyperCKemia from three months to ten years, with a mean (SD) CK value of 788 (507) IU·L-1 ranging from 210-1,667 IU·L-1. These values corresponded to a 1.5- to nine-fold increase in the normal value (mean increase, 3.7-fold). Six patients were MH susceptible (MHS) after a positive IVCT. Histopathological muscular analysis disclosed unspecified changes in four of the MHS patients. Mitochondrial proliferation was observed in the other two MHS patients and in three MH negative patients. No pathogenic mutations were identified in the RYR1 gene in the five patients evaluated. When investigating patients with idiopathic hyperCKemia, susceptibility to MH should be taken into account, and guidance should be offered to prevent anesthetic complications in the family.

NG.O.9 - Dihydropyridine receptor (DHPR, CACNA1S) congenital myopathy

Muscle contraction upon nerve stimulation relies on excitation–contraction coupling (ECC) to promote the rapid and generalized release of calcium within myofibers. In skeletal muscle, ECC is performed by the direct coupling of a voltage-gated L-type Ca2+ channel (dihydropyridine receptor; DHPR) located on the T-tubule with a Ca2+ release channel (ryanodine receptor; RYR1) on the sarcoplasmic reticulum (SR) component of the triad. Here, we characterize a novel class of congenital myopathy at the morphological, molecular, and functional levels. We describe a cohort of 11 patients from 7 families presenting with perinatal hypotonia, severe axial and generalized weakness. Ophthalmoplegia is present in four patients. The analysis of muscle biopsies demonstrated a characteristic intermyofibrillar network due to SR dilatation, internal nuclei, and areas of myofibrillar disorganization in some samples. Exome sequencing revealed ten recessive or dominant mutations in CACNA1S (Cav1.1), the pore-forming subunit of DHPR in skeletal muscle. Both recessive and dominant mutations correlated with a consistent phenotype, a decrease in protein level, and with a major impairment of Ca2+ release induced by depolarization in cultured myotubes. While dominant CACNA1S mutations were previously linked to malignant hyperthermia susceptibility or hypokalemic periodic paralysis, our findings strengthen the importance of DHPR for perinatal muscle function in human. These data also highlight CACNA1S and ECC as therapeutic targets for the development of treatments that may be facilitated by the previous knowledge accumulated on DHPR.

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.

Skeletal Muscle Metabolic Dysfunction in Patients With Malignant Hyperthermia Susceptibility.

Malignant hyperthermia (MH), a pharmacogenetic disorder of skeletal muscle, presents with a potentially lethal hypermetabolic reaction to certain anesthetics. However, some MH-susceptible patients experience muscle weakness, fatigue, and exercise intolerance in the absence of anesthetic triggers. The objective of this exploratory study was to elucidate the pathophysiology of exercise intolerance in patients tested positive for MH with the caffeine-halothane contracture test. To this end, we used phosphorus magnetic resonance spectroscopy, blood oxygen level-dependent functional magnetic resonance imaging (MRI), and traditional exercise testing to compare skeletal muscle metabolism in MH-positive patients and healthy controls. Skeletal muscle metabolism was assessed using phosphorus magnetic resonance spectroscopy and blood oxygen level-dependent functional MRI in 29 MH-positive patients and 20 healthy controls. Traditional measures of physical capacity were employed to measure aerobic capacity, anaerobic capacity, and muscle strength. During 30- and 60-second exercise, MH-positive patients had significantly lower ATP production via the oxidative pathway compared to healthy controls. MH-positive patients also had a longer recovery time with blood oxygen level-dependent functional MRI compared to healthy controls. Exercise testing revealed lower aerobic and anaerobic capacity in MH-positive patients compared to healthy controls. Results of this exploratory study suggest that MH-positive patients have impaired aerobic metabolism compared to healthy individuals. This could explain the exercise intolerance exhibited in MH-susceptible patient population.