Calcium Channelopathies: Structural Insights into Disorders of the Muscle Excitation-Contraction Complex.

Ion channels are membrane proteins responsible for the passage of ions down their electrochemical gradients and across biological membranes. In this, they generate and shape action potentials and provide secondary messengers for various signaling pathways. They are often part of larger complexes containing auxiliary subunits and regulatory proteins. Channelopathies arise from mutations in the genes encoding ion channels or their associated proteins. Recent advances in cryo-electron microscopy have resulted in an explosion of ion channel structures in multiple states, generating a wealth of new information on channelopathies. Disease-associated mutations fall into different categories, interfering with ion permeation, protein folding, voltage sensing, ligand and protein binding, and allosteric modulation of channel gating. Prime examples of these are Ca2+-selective channels expressed in myocytes, for which multiple structures in distinct conformational states have recently been uncovered. We discuss the latest insights into these calcium channelopathies from a structural viewpoint.

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.

Molecular mechanism of the severe MH/CCD mutation Y522S in skeletal ryanodine receptor (RyR1) by cryo-EM.

Ryanodine receptors (RyRs) are main regulators of intracellular Ca2+ release and muscle contraction. The Y522S mutation of RyR1 causes central core disease, a weakening myopathy, and malignant hyperthermia, a sudden and potentially fatal response to anesthetics or heat. Y522 is in the core of the N-terminal subdomain C of RyR1 and the mechanism of how this mutation orchestrates malfunction is unpredictable for this 2-MDa ion channel, which has four identical subunits composed of 15 distinct cytoplasmic domains each. We expressed and purified the RyR1 rabbit homolog, Y523S, from HEK293 cells and reconstituted it in nanodiscs under closed and open states. The high-resolution cryogenic electron microscopic (cryo-EM) three-dimensional (3D) structures show that the phenyl ring of Tyr functions in a manner analogous to a "spacer" within an α-helical bundle. Mutation to the much smaller Ser alters the hydrophobic network within the bundle, triggering rearrangement of its α-helices with repercussions in the orientation of most cytoplasmic domains. Examining the mutation-induced readjustments exposed a series of connected α-helices acting as an ∼100 Å-long lever: One end protrudes toward the dihydropyridine receptor, its molecular activator (akin to an antenna), while the other end reaches the Ca2+ activation site. The Y523S mutation elicits channel preactivation in the absence of any activator and full opening at 1.5 µM free Ca2+, increasing by ∼20-fold the potency of Ca2+ to activate the channel compared with RyR1 wild type (WT). This study identified a preactivated pathological state of RyR1 and a long-range lever that may work as a molecular switch to open the channel.

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.

Epidemiological and clinical features of malignant hyperthermia: A scoping review.

Malignant hyperthermia (MH) is a potentially fatal inherited pharmacogenetic disorder related to pathogenic variants in the RYR1, CACNA1S, or STAC3 genes. Early recognition of the occurrence of MH and prompt medical treatment are indispensable to ensure a positive outcome. The purpose of this study was to provide valuable information for the early identification of MH by summarizing epidemiological and clinical features of MH. This scoping review followed the methodological framework recommended by Arksey and O'Malley. PubMed, Embase, and Web of science databases were searched for studies that evaluated the epidemical and clinical characteristics of MH. A total of 37 studies were included in this review, of which 26 were related to epidemiology and 24 were associated with clinical characteristics. The morbidity of MH varied from 0.18 per 100 000 to 3.9 per 100 000. The mortality was within the range of 0%-18.2%. Identified risk factors included sex, age, disorders associated with MH, and others. The most frequent initial clinical signs included hyperthermia, sinus tachycardia, and hypercarbia. The occurrence of certain signs, such as hypercapnia, delayed first temperature measurement, and peak temperature were associated with poor outcomes. The epidemiological and clinical features of MH varied considerably and some risk factors and typical clinical signs were identified. The main limitation of this review is that the treatment and management strategies were not assessed sufficiently due to limited information.

Myopathic manifestations across the adult lifespan of patients with malignant hyperthermia susceptibility: a narrative review.

Malignant hyperthermia susceptibility (MHS) designates individuals at risk of developing a hypermetabolic reaction triggered by halogenated anaesthetics or the depolarising neuromuscular blocking agent suxamethonium. Over the past few decades, beyond the operating theatre, myopathic manifestations impacting daily life are increasingly recognised as a prevalent phenomenon in MHS patients. At the request of the European Malignant Hyperthermia Group, we reviewed the literature and gathered the opinion of experts to define MHS-related myopathy as a distinct phenotype expressed across the adult lifespan of MHS patients unrelated to anaesthetic exposure; this serves to raise awareness about non-anaesthetic manifestations, potential therapies, and management of MHS-related myopathy. We focused on the clinical presentation, biochemical and histopathological findings, and the impact on patient well-being. The spectrum of symptoms of MHS-related myopathy encompasses muscle cramps, stiffness, myalgias, rhabdomyolysis, and weakness, with a wide age range of onset mainly during adulthood. Histopathological analysis can reveal nonspecific abnormalities suggestive of RYR1 involvement, while metabolic profiling reflects altered energy metabolism in MHS muscle. Myopathic manifestations can significantly impact patient quality of life and lead to functional limitations and socio-economic burden. While currently available therapies can provide symptomatic relief, there is a need for further research into targeted treatments addressing the underlying pathophysiology. Counselling early after establishing the MHS diagnosis, followed by multidisciplinary management involving various medical specialties, is crucial to optimise patient care.

Caution for psychiatrists: malignant hyperthermia risks with the anesthetic agent succinylcholine (Suxamethonium) during electroconvulsive therapy.

BACKGROUND: Malignant hyperthermia is a potentially lethal condition triggered by specific anesthetic drugs, especially a depolarizing muscle relaxant of succinylcholine (Suxamethonium). Despite the frequent use of succinylcholine with electroconvulsive therapy (ECT), there has been no reported case of potentially lethal malignant hyperthermia following ECT. In addition, the time interval between the administration of succinylcholine and the onset of malignant hyperthermia has not been outlined in the context of ECT. CASE PRESENTATION: We present the case of a 79-year-old woman suffering from severe depression, who experienced severe malignant hyperthermia due to succinylcholine administration during an ECT session. She presented with a high fever of 40.2 °C, tachycardia of 140/min, hypertension with a blood pressure exceeding 200 mmHg, significant muscle rigidity, and impaired consciousness. These symptoms emerged two hours after ECT, which occurred in a psychiatric ward rather than an operating room, and reached their peak in less than 24 h. She was given 60 mg of dantrolene, which quickly reduced the muscular rigidity. Subsequently, she received two additional doses of 20 mg and 60 mg of dantrolene, which brought her fever down to 36.2 °C and completely eased her muscle rigidity within two days after ECT. CONCLUSIONS: This is the first reported case of potentially lethal malignant hyperthermia after ECT. In addition, it highlights the delayed onset of malignant hyperthermia following an ECT procedure, emphasizing the necessity for psychiatrists to recognize its onset even after the treatment. In the light of potentially lethal consequences of malignant hyperthermia, it is critically important for psychiatrists to closely monitor both intraoperative and postoperative patient's vital signs and characteristic physical presentations, promptly identify any symptomatic emergence, and treat it immediately with dantrolene.

Malignant hyperthermia safety - A nationwide survey of publicly funded Swedish healthcare.

BACKGROUND: Malignant hyperthermia (MH) is a rare pharmacogenetic disorder that can lead to a life-threatening reaction during general anaesthesia with triggering agents. Prompt life-saving treatment includes the immediate administration of the antidote dantrolene. This study investigated Swedish healthcare providers' awareness and adherence to guidelines and recommendations with respect to MH and whether adherence to safe MH-praxis varies with hospital care-complexity level and private versus public management form. METHOD: Agreements and procurement specifications between all 21 Swedish County Councils and privately run surgical care providers were reviewed alongside with questionnaire-aided collection of information from 62 publicly funded health care providers (both privately and publicly run). RESULTS: No procurement requirement specification or contract contained requirements on anaesthesia or aspects of MH. All publicly run hospitals stocked dantrolene and 28 out of 52 (54%) stocked the recommended amount. Seven out of nine (78%) of the privately run institutions stocked dantrolene, and one stocked the recommended amount. Publicly run hospitals adhered to recommendations to a greater extent than privately run institutions, both with respect to stocking of dantrolene (p = .02) and to stocking the recommended amount (p = .03). CONCLUSIONS: Contracts between Swedish county councils and private surgical care subcontractors rarely outline expectations of standards for the safe practice of anaesthesia such as preparedness to handle a life-threatening MH reaction. Among Swedish publicly funded anaesthesia providers there is room for improvement in adherence to the EMHG guideline on dantrolene availability. Publicly run hospitals seem to have better compliance with these recommendations than privately run institutions. Raising awareness about current guidelines is important to improve safety for known and unknown MH-susceptible individuals.

An Association between OXPHOS-Related Gene Expression and Malignant Hyperthermia Susceptibility in Human Skeletal Muscle Biopsies.

Malignant hyperthermia (MH) is a pharmacogenetic condition of skeletal muscle that manifests in hypermetabolic responses upon exposure to volatile anaesthetics. This condition is caused primarily by pathogenic variants in the calcium-release channel RYR1, which disrupts calcium signalling in skeletal muscle. However, our understanding of MH genetics is incomplete, with no variant identified in a significant number of cases and considerable phenotype diversity. In this study, we applied a transcriptomic approach to investigate the genome-wide gene expression in MH-susceptible cases using muscle biopsies taken for diagnostic testing. Baseline comparisons between muscle from MH-susceptible individuals (MHS, n = 8) and non-susceptible controls (MHN, n = 4) identified 822 differentially expressed genes (203 upregulated and 619 downregulated) with significant enrichment in genes associated with oxidative phosphorylation (OXPHOS) and fatty acid metabolism. Investigations of 10 OXPHOS target genes in a larger cohort (MHN: n = 36; MHS: n = 36) validated the reduced expression of ATP5MD and COQ6 in MHS samples, but the remaining 8 selected were not statistically significant. Further analysis also identified evidence of a sex-linked effect in SDHB and UQCC3 expression, and a difference in ATP5MD expression across individuals with MH sub-phenotypes (trigger from in vitro halothane exposure only, MHSh (n = 4); trigger to both in vitro halothane and caffeine exposure, MHShc (n = 4)). Our data support a link between MH-susceptibility and dysregulated gene expression associated with mitochondrial bioenergetics, which we speculate plays a role in the phenotypic variability observed within MH.

A novel RYR1 variant in an infant with a unique fetal presentation of central core disease.

Ryanodine receptor type 1-related disorder (RYR1-RD) is the most common subgroup of congenital myopathies with a wide phenotypic spectrum ranging from mild hypotonia to lethal fetal akinesia. Genetic testing for myopathies is imperative as the diagnosis informs counseling regarding prognosis and recurrence risk, treatment options, monitoring, and clinical management. However, diagnostic challenges exist as current options are limited to clinical suspicion prompting testing including: single gene sequencing or familial variant testing, multi-gene panels, exome, genome sequencing, and invasive testing including muscle biopsy. The timing of diagnosis is of great importance due to the association of RYR1-RD with malignant hyperthermia (MH). MH is a hypermetabolic crisis that occurs secondary to excessive calcium release in muscles, leading to systemic effects that can progress to shock and death if unrecognized. Given the association of MH with pathogenic variants in RYR1, a diagnosis of RYR1-RD necessitates an awareness of medical team to avoid potentially triggering agents. We describe a case of a unique fetal presentation with bilateral diaphragmatic eventrations who had respiratory failure, dysmorphic facial features, and profound global hypotonia in the neonatal period. The diagnosis was made at several months of age, had direct implications on her clinical care related to anticipated need to long-term ventilator support, and ultimately death secondary an arrhythmia as a result of suspected MH. Our report reinforces the importance of having high suspicion for a genetic syndrome and pursuing early, rapid exome or genome sequencing as first line testing in critically ill neonatal intensive care unit patients and further evaluating the pathogenicity of a variant of uncertain significance in the setting of a myopathic phenotype.

Distinct pathophysiological characteristics in developing muscle from patients susceptible to malignant hyperthermia.

BACKGROUND: Most patients with malignant hyperthermia susceptibility diagnosed by the in vitro caffeine-halothane contracture test (CHCT) develop excessive force in response to halothane but not caffeine (halothane-hypersensitive). Hallmarks of halothane-hypersensitive patients include high incidence of musculoskeletal symptoms at rest and abnormal calcium events in muscle. By measuring sensitivity to halothane of myotubes and extending clinical observations and cell-level studies to a large group of patients, we reach new insights into the pathological mechanism of malignant hyperthermia susceptibility. METHODS: Patients with malignant hyperthermia susceptibility were classified into subgroups HH and HS (positive to halothane only and positive to both caffeine and halothane). The effects on [Ca2+]cyto of halothane concentrations between 0.5 and 3 % were measured in myotubes and compared with CHCT responses of muscle. A clinical index that summarises patient symptoms was determined for 67 patients, together with a calcium index summarising resting [Ca2+]cyto and spontaneous and electrically evoked Ca2+ events in their primary myotubes. RESULTS: Halothane-hypersensitive myotubes showed a higher response to halothane 0.5% than the caffeine-halothane hypersensitive myotubes (P<0.001), but a lower response to higher concentrations, comparable with that used in the CHCT (P=0.055). The HH group had a higher calcium index (P<0.001), but their clinical index was not significantly elevated vs the HS. Principal component analysis identified electrically evoked Ca2+ spikes and resting [Ca2+]cyto as the strongest variables for separation of subgroups. CONCLUSIONS: Enhanced sensitivity to depolarisation and to halothane appear to be the primary, mutually reinforcing and phenotype-defining defects of halothane-hypersensitive patients with malignant hyperthermia susceptibility.

What is malignant hyperthermia susceptibility?

The molecular mechanisms of susceptibility to malignant hyperthermia are complex. The malignant hyperthermia susceptibility phenotype should be reserved for patients who have a personal or family history consistent with malignant hyperthermia under anaesthesia and are subsequently demonstrated through diagnostic testing to be at risk.

CaV1.1 Calcium Channel Signaling Complexes in Excitation-Contraction Coupling: Insights from Channelopathies.

In skeletal muscle, excitation-contraction (EC) coupling relies on the mechanical coupling between two ion channels: the L-type voltage-gated calcium channel (CaV1.1), located in the sarcolemma and functioning as the voltage sensor of EC coupling, and the ryanodine receptor 1 (RyR1), located on the sarcoplasmic reticulum serving as the calcium release channel. To this day, the molecular mechanism by which these two ion channels are linked remains elusive. However, recently, skeletal muscle EC coupling could be reconstituted in heterologous cells, revealing that only four proteins are essential for this process: CaV1.1, RyR1, and the cytosolic proteins CaVß1a and STAC3. Due to the crucial role of these proteins in skeletal muscle EC coupling, any mutation that affects any one of these proteins can have devastating consequences, resulting in congenital myopathies and other pathologies.Here, we summarize the current knowledge concerning these four essential proteins and discuss the pathophysiology of the CaV1.1, RyR1, and STAC3-related skeletal muscle diseases with an emphasis on the molecular mechanisms. Being part of the same signalosome, mutations in different proteins often result in congenital myopathies with similar symptoms or even in the same disease.

Rebounds of sevoflurane concentration during simulated trigger-free pediatric and adult anesthesia.

BACKGROUND: In trigger-free anesthesia a volatile anesthetic concentration of 5 parts per million (ppm) should not be exceeded. According to European Malignant Hyperthermia Group (EMHG) guideline, this may be achieved by removing the vapor, changing the anesthetic breathing circuit and renewing the soda lime canister followed by flushing with O2 or air for a workstation specific time. Reduction of the fresh gas flow (FGF) or stand-by modes are known to cause rebound effects. In this study, simulated trigger-free pediatric and adult ventilation was carried out on test lungs including ventilation maneuvers commonly used in clinical practice. The goal of this study was to evaluate whether rebounds of sevoflurane develop during trigger-free anesthesia. METHODS: A Dräger® Primus® was contaminated with decreasing concentrations of sevoflurane for 120 min. Then, the machine was prepared for trigger-free anesthesia according to EMHG guideline by changing recommended parts and flushing the breathing circuits using 10 or 18 l⋅min- 1 FGF. The machine was neither switched off after preparation nor was FGF reduced. Simulated trigger-free ventilation was performed with volume-controlled ventilation (VCV) and pressure-controlled ventilation (PCV) including various ventilation maneuvers like pressure support ventilation (PSV), apnea, decreased lung compliance (DLC), recruitment maneuvers, prolonged expiration and manual ventilation (MV). A high-resolution ion mobility spectrometer with gas chromatographic pre-separation was used to measure sevoflurane in the ventilation gas mixture in a 20 s interval. RESULTS: Immediately after start of simulated anesthesia, there was an initial peak of 11-18 ppm sevoflurane in all experiments. The concentration dropped below 5 ppm after 2-3 min during adult and 4-18 min during pediatric ventilation. Other rebounds of sevoflurane > 5 ppm occurred after apnea, DLC and PSV. MV resulted in a decrease of sevoflurane < 5 ppm within 1 min. CONCLUSION: This study shows that after guideline-compliant preparation for trigger-free ventilation anesthetic machines may develop rebounds of sevoflurane > 5 ppm during typical maneuvers used in clinical practice. The changes in rate and direction of internal gas flow during different ventilation modes and maneuvers are possible explanations. Therefore, manufacturers should provide machine-specific washout protocols or emphasize the use of active charcoal filters (ACF) for trigger-free anesthesia.

A WeChat applet-based national remote emergency system for malignant hyperthermia in China: a usability study.

BACKGROUND: Malignant hyperthermia (MH) is a rare anesthetic emergency with a high mortality rate in China. We developed a WeChat applet-based National Remote Emergency System for Malignant Hyperthermia (MH-NRES) to provide a real-time emergency system to help Chinese anesthesiologists deal with MH crises. However, it is imperative that close attention should be paid to the usability of the applet. PURPOSE: The objectives of this study were to (1) evaluate the usability of the applet-based MH-NRES for anesthesiologists; and (2) to test the validity and reliability of a modified mHealth app usability questionnaire. METHODS: A modified User Version of the Mobile Application Rating Scale (uMARS) was designed. Together with System Usability Scale (SUS) and Post-Study System Usability Questionnaire (PSSUQ), another two well-validated questionnaires, uMARS were then used to evaluate the usability of MH-NRES. The Cronbach alpha of the total score and the subscales of uMARS was calculated to evaluate the internal consistency. The correlation coefficients among three questionnaires were calculated. RESULTS: In this study, 118 anesthesiologists provided responses to the questionnaire. The overall mean uMARS score was 4.43 ± 0.61, which ranged from 3 to 5. The mean PSSUQ score were in good to excellent range with mean of 6.02 ± 0.97, which ranged from 3.19 to 7. The overall SUS score was 76.0 ± 17.6, which ranged from 45 to 100. The total uMARS score had excellent internal consistency (Cronbach alpha = 0.984). uMARS and its subscales were strongly correlated with PSSUQ (coefficient 0.758-0.819, P < 0.001) and SUS (coefficient 0.535-0.561, P < 0.001), respectively. CONCLUSIONS: Data obtained from the usability evaluation questionnaires in this study indicated a high quality of the MH-NRES on the ease of use, satisfaction and perceived usefulness, which suggest this system might be a useful tool for anesthesiologists' education and management of MH crises. Future feedback from high-fidelity simulation and clinical scenarios are need for further usability evaluation of this system.

Comparison of Transcriptomic Changes in Survivors of Exertional Heat Illness with Malignant Hyperthermia Susceptible Patients.

Exertional heat illness (EHI) is an occupational health hazard for athletes and military personnel-characterised by the inability to thermoregulate during exercise. The ability to thermoregulate can be studied using a standardised heat tolerance test (HTT) developed by The Institute of Naval Medicine. In this study, we investigated whole blood gene expression (at baseline, 2 h post-HTT and 24 h post-HTT) in male subjects with either a history of EHI or known susceptibility to malignant hyperthermia (MHS): a pharmacogenetic condition with similar clinical phenotype. Compared to healthy controls at baseline, 291 genes were differentially expressed in the EHI cohort, with functional enrichment in inflammatory response genes (up to a four-fold increase). In contrast, the MHS cohort featured 1019 differentially expressed genes with significant down-regulation of genes associated with oxidative phosphorylation (OXPHOS). A number of differentially expressed genes in the inflammation and OXPHOS pathways overlapped between the EHI and MHS subjects, indicating a common underlying pathophysiology. Transcriptome profiles between subjects who passed and failed the HTT (based on whether they achieved a plateau in core temperature or not, respectively) were not discernable at baseline, and HTT was shown to elevate inflammatory response gene expression across all clinical phenotypes.

Molecular Aspects Implicated in Dantrolene Selectivity with Respect to Ryanodine Receptor Isoforms.

Dantrolene is an intra-cellularly acting skeletal muscle relaxant used for the treatment of the rare genetic disorder, malignant hyperthermia (MH). In most cases, MH susceptibility is caused by dysfunction of the skeletal ryanodine receptor (RyR1) harboring one of nearly 230 single-point MH mutations. The therapeutic effect of dantrolene is the result of a direct inhibitory action on the RyR1 channel, thus suppressing aberrant Ca2+ release from the sarcoplasmic reticulum. Despite the almost identical dantrolene-binding sequence exits in all three mammalian RyR isoforms, dantrolene appears to be an isoform-selective inhibitor. Whereas RyR1 and RyR3 channels are competent to bind dantrolene, the RyR2 channel, predominantly expressed in the heart, is unresponsive. However, a large body of evidence suggests that the RyR2 channel becomes sensitive to dantrolene-mediated inhibition under certain pathological conditions. Although a consistent picture of the dantrolene effect emerges from in vivo studies, in vitro results are often contradictory. Hence, our goal in this perspective is to provide the best possible clues to the molecular mechanism of dantrolene's action on RyR isoforms by identifying and discussing potential sources of conflicting results, mainly coming from cell-free experiments. Moreover, we propose that, specifically in the case of the RyR2 channel, its phosphorylation could be implicated in acquiring the channel responsiveness to dantrolene inhibition, interpreting functional findings in the structural context.

Severe Diabetic Ketoacidosis with Malignant Hyperthermia Like Syndrome and Rhabdomyolysis Treated with ECMO: Unusual Severity and a Rare Occurrence.

How to cite this article: Accamma K, Shamarao S, Ram A, Devananda NS, Krishna M, Bandagi LS, et al. Severe Diabetic Ketoacidosis with Malignant Hyperthermia Like Syndrome and Rhabdomyolysis Treated with ECMO: Unusual Severity and a Rare Occurrence. Indian J Crit Care Med 2023;27(11):859-860.

Functional Calsequestrin-1 Is Expressed in the Heart and Its Deficiency Is Causally Related to Malignant Hyperthermia-Like Arrhythmia.

BACKGROUND: Calsequestrins (Casqs), comprising the Casq1 and Casq2 isoforms, buffer Ca2+ and regulate its release in the sarcoplasmic reticulum of skeletal and cardiac muscle, respectively. Human inherited diseases associated with mutations in CASQ1 or CASQ2 include malignant hyperthermia/environmental heat stroke (MH/EHS) and catecholaminergic polymorphic ventricular tachycardia. However, patients with an MH/EHS event often experience arrhythmia for which the underlying mechanism remains unknown. METHODS: Working hearts from conventional (Casq1-KO) and cardiac-specific (Casq1-CKO) Casq1 knockout mice were monitored in vivo and ex vivo by ECG and electric mapping, respectively. MH was induced by 2% isoflurane and treated intraperitoneally with dantrolene. Time-lapse imaging was used to monitor intracellular Ca2+ activity in isolated mouse cardiomyocytes or neonatal rat ventricular myocytes with knockdown, overexpression, or truncation of the Casq1 gene. Conformational change in both Casqs was determined by cross-linking Western blot analysis. RESULTS: Like patients with MH/EHS, Casq1-KO and Casq1-CKO mice had faster basal heart rate and ventricular tachycardia on exposure to 2% isoflurane, which could be relieved by dantrolene. Basal sinus tachycardia and ventricular ectopic electric triggering also occurred in Casq1-KO hearts ex vivo. Accordingly, the ventricular cardiomyocytes from Casq1-CKO mice displayed dantrolene-sensitive increased Ca2+ waves and diastole premature Ca2+ transients/oscillations on isoflurane. Neonatal rat ventricular myocytes with Casq1-knockdown had enhanced spontaneous Ca2+ sparks/transients on isoflurane, whereas cells overexpressing Casq1 exhibited decreased Ca2+ sparks/transients that were absent in cells with truncation of 9 amino acids at the C terminus of Casq1. Structural evaluation showed that most of the Casq1 protein was present as a polymer and physically interacted with ryanodine receptor-2 in the ventricular sarcoplasmic reticulum. The Casq1 isoform was also expressed in human myocardium. Mechanistically, exposure to 2% isoflurane or heating at 41 °C induced Casq1 oligomerization in mouse ventricular and skeletal muscle tissues, leading to a reduced Casq1/ryanodine receptor-2 interaction and increased ryanodine receptor-2 activity in the ventricle. CONCLUSIONS: Casq1 is expressed in the heart, where it regulates sarcoplasmic reticulum Ca2+ release and heart rate. Casq1 deficiency independently causes MH/EHS-like ventricular arrhythmia by trigger-induced Casq1 oligomerization and a relief of its inhibitory effect on ryanodine receptor-2-mediated Ca2+ release, thus revealing a new inherited arrhythmia and a novel mechanism for MH/EHS arrhythmogenesis.

Phosphodiesterases S-sulfhydration contributes to human skeletal muscle function.

The increase in intracellular calcium is influenced by cyclic nucleotides (cAMP and cGMP) content, which rating is governed by phosphodiesterases (PDEs) activity.Despite it has been demonstrated a beneficial effect of PDEs inhibitors in different pathological conditions involving SKM, not much is known on the role exerted by cAMP-cGMP/PDEs axis in human SKM contractility. Here, we show that Ssulfhydration of PDEs modulates human SKM contractility in physiological and pathological conditions. Having previously demonstrated that, in the rare human syndrome Malignant Hyperthermia (MH), there is an overproduction of hydrogen sulfide (H2S) within SKM contributing to hyper-contractility, here we have used MH negative diagnosed biopsies (MHN) as healthy SKM, and MH susceptible diagnosed biopsies (MHS) as a pathological model of SKM hypercontractility. The study has been performed on MHS and MHN human biopsies after diagnosis has been made and on primary SKM cells derived from both MHN and MHS biopsies. Our data demonstrate that in normal conditions PDEs are S-sulfhydrated in both quadriceps' biopsies and primary SKM cells. This post translational modification (PTM) negatively regulates PDEs activity with consequent increase of both cAMP and cGMP levels. In hypercontractile biopsies, due to an excessive H2S content, there is an enhanced Ssulfhydration of PDEs that further increases cyclic nucleotides levels contributing to SKM hyper-contractility. Thus, the identification of a new endogenous PTM modulating PDEs activity represents an advancement in SKM physiopathology understanding.