Diagnosis and rescue of malignant hyperthermia induced by anesthesia during radical surgery in a cervical cancer patient using the National Remote Emergency System: A case report.

RATIONALE: Malignant hyperthermia (MH) is a rare yet serious medical complication that typically arises following general anesthesia or the administration of specific anesthetics. Due to the infrequency of MH, anesthesiologists often lack sufficient expertise in identifying and managing it, leading to misdiagnosis and inappropriate treatment. There is an urgent need to enhance the diagnosis and management of MH through the utilization of relevant tools. PATIENT CONCERNS: In this case, a 52-year-old woman underwent radical cervical cancer surgery under general anesthesia, with no family or significant medical history. She experienced a gradual increase in end-tidal carbon dioxide (ETCO2) to a maximum of 75 mm Hg and a rise in body temperature from 36.5 to 37.5 °C in a very short period, as well as a blood gas analysis showing a pH of 7.217. DIAGNOSIS: The anesthesiologist immediately used The WeChat applet-based National Remote Emergency System for Malignant Hyperthermia (MH-NRES), and the score was 40, which indicated that the patient was very likely to have MH. INTERVENTIONS: We immediately discontinued sevoflurane and switched total intravenous anesthesia to maintain general anesthesia, with a rapid intravenous infusion of dantrolene sodium. OUTCOMES: The ETCO2 and the temperature quickly dropped to normal, followed by successful completion of the surgery, and the patient was discharged 8 days after surgery. LESSONS: The experience can provide a basis use of MH-NRES and improve the ability of anesthesiologists to deal with intraoperative MH as well as increase the survival probability of patients.

Anaesthesia-related morbidity associated with recumbent, low-field magnetic resonance imaging of horses.

CASE HISTORY: Medical records from 2009 to 2021 from a private equine referral hospital in Rochester, NH, USA were analysed for cases that underwent general anaesthesia for low-field MRI of the distal limb. These were used to determine peri-anaesthetic morbidity and mortality. CLINICAL FINDINGS AND OUTCOME: Two hundred and forty-three anaesthetic episodes were recorded in horses undergoing low-field MRI. The peri-anaesthetic complication rate prior to discharge was 6.2% (15/243). No patients experienced a fatal complication. Ninety two of the 243 patients had multiple sites imaged, 90/243 received pre-anaesthetic dantrolene, 134/243 received intra-anaesthetic dobutamine, and 15/243 were positioned in dorsal recumbency. Complications included: abdominal discomfort ("colic"; 9/243), myopathy (4/243), hyphaema (1/243) and carpal fracture (1/243). At the time of discharge, 14/15 complications had resolved. Of 135 horses for which data were available 55 became hypotensive during the procedure (lowest mean arterial pressure < 65 mmHg). Median body weight was 553 (min 363, max 771) kg. Horses were anaesthetised for a median of 150 (min 45, max 210) minutes. There was no evidence of an association between higher body weight (p = 0.051) or longer duration of anaesthesia (p = 0.421) and development of an anaesthetic complication. For categorical variables (dantrolene administration pre-anaesthesia, dobutamine administration during anaesthesia, hypotension (mean < 65 mmHg) during anaesthesia, dorsal vs. lateral recumbency, and imaging of single vs. multiple sites), the 95% CI for the OR included 1, indicating a lack of effect of the variable on the odds of complication. CLINICAL RELEVANCE: The cases included in this series suggest that low-field MRI under general anaesthesia is a viable option for diagnostic imaging in otherwise healthy horses. Complications occur, but most resolve before discharge.

The novel rapid formulation of intravenous dantrolene (NPJ5008) versus standard dantrolene (Dantrium®): A clinical part-randomised phase 1 study in healthy volunteers.

BACKGROUND: Delays in treating anaesthesia-induced malignant hyperthermia increase risks of complications and death. NPJ5008 is a novel formulation of the indicated treatment, dantrolene sodium, developed to shorten preparation and administration times compared with the reference formulation Dantrium®. The two formulations have been compared preclinically. OBJECTIVES: Assess bioequivalence of overall dantrolene (free acid) exposure of NPJ5008 versus Dantrium® and ascertain similarities in their pharmacokinetics and safety/tolerability profiles. Evaluate preparation/administration time savings for the new formulation. DESIGN: Part 1 of this open-label trial in humans was a 1 : 1 randomised crossover study; part 2 was a single-arm study. Trial pharmacy data and laboratory simulations assessed preparation/administration step timings. SETTING: Single clinical centre in the UK, April to July 2021. PARTICIPANTS: Twenty-one healthy male and female individuals. INTERVENTIONS: Part 1: single intravenous 60 mg dose of NPJ5008 or Dantrium®, sequentially. Part 2: single intravenous 120 mg dose of NPJ5008. Simulation: five vials per formulation using paediatric and adult cannulas. MAIN OUTCOME MEASURES: Overall drug exposure to last measurable concentration (AUC 0 to last ) and extrapolated to infinity (AUC 0 to ∞ ) were primary endpoints. Other pharmacokinetic, clinical and muscle-function parameters, and adverse events, were monitored. RESULTS: Adjusted geometric mean ratios of NPJ5008 versus Dantrium® were 90.24 and 90.44% for AUC 0 to last and AUC 0 to ∞ , respectively, with the 90% confidence intervals (CI) within the 80 to 125% acceptance interval, establishing bioequivalence. No new safety issues emerged: any adverse events were of a similar magnitude across treatments and related to pharmacological properties of dantrolene. Pharmacy and simulation data revealed that every step in preparation and administration was 26 to 69% faster for NPJ5008 than Dantrium®. CONCLUSION: NPJ5008 showed comparable pharmacokinetic and safety profiles to Dantrium®, while reducing dantrolene dose preparation/administration times, potentially reducing patient complications/healthcare resourcing in malignant hyperthermia. TRIAL REGISTRATION: EudraCT Number: 2020-005719-35, MHRA approval.

Effects of Intranasal Dantrolene Nanoparticles on Brain Concentration and Behavior in PS19 Tau Transgenic Mice.

Background: Repurposing dantrolene to treat Alzheimer's disease has been shown to be effective in amyloid transgenic mouse models but has not been examined in a model of tauopathy. Objective: The effects of a nanoparticle intranasal formulation, the Eagle Research Formulation of Ryanodex (ERFR), in young adult and aged wild type and PS19 tau transgenic mice was investigated. Methods: The bioavailability of intranasal ERFR was measured in 2 and 9-11-month-old C57BL/6J mice. Blood and brain samples were collected 20 minutes after a single ERFR dose, and the plasma and brain concentrations were analyzed. Baseline behavior was assessed in untreated PS19 tau transgenic mice at 6 and 9 months of age. PS19 mice were treated with intranasal ERFR, with or without acrolein (to potentiate cognitive dysfunction), for 3 months, beginning at 2 months of age. Animal behavior was examined, including cognition (cued and contextual fear conditioning, y-maze), motor function (rotarod), and olfaction (buried food test). Results: The dantrolene concentration in the blood and brain decreased with age, with the decrease greater in the blood resulting in a higher brain to blood concentration ratio. The behavioral assays showed no significant changes in cognition, olfaction, or motor function in the PS19 mice compared to controls after chronic treatment with intranasal ERFR, even with acrolein. Conclusions: Our studies suggest the intranasal administration of ERFR has higher concentrations in the brain than the blood in aged mice and has no serious systemic side effects with chronic use in PS19 mice.

Post-Anesthesia Cognitive Dysfunction in Mice Is Associated with an Age-Related Increase in Neuronal Intracellular [Ca2+]-Neuroprotective Effect of Reducing Intracellular [Ca2+]: In Vivo and In Vitro Studies.

Background: Postoperative cognitive dysfunction (POCD) is a common disorder after general anesthesia in elderly patients, the precise mechanisms of which remain unclear. Methods: We investigated the effect of isoflurane with or without dantrolene pretreatment on intracellular calcium concentration ([Ca2+]i), reactive oxygen species (ROS) production, cellular lactate dehydrogenase (LDH) leak, calpain activity, and cognitive function using the Morris water maze test of young (3 months), middle-aged (12-13 months), and aged (24-25 months) C57BL6/J mice. Results: Aged cortical and hippocampal neurons showed chronically elevated [Ca2+]i compared to young neurons. Furthermore, aged hippocampal neurons exhibited higher ROS production, increased LDH leak, and elevated calpain activity. Exposure to isoflurane exacerbated these markers in aged neurons, contributing to increased cognitive deficits in aged mice. Dantrolene pretreatment reduced [Ca2+]i for all age groups and prevented or significantly mitigated the effects of isoflurane on [Ca2+]i, ROS production, LDH leak, and calpain activity in aged neurons. Dantrolene also normalized or improved age-associated cognitive deficits and mitigated the cognitive deficits caused by isoflurane. Conclusions: These findings suggest that isoflurane-induced cytotoxicity and cognitive decline in aging are linked to disruptions in neuronal intracellular processes, highlighting the reduction of [Ca2+]i as a potential therapeutic intervention.

Optimization of CHARMM force field parameters for ryanodine receptor inhibitory drug dantrolene using FFTK and FFParam.

CONTEXT: Ryanodine receptors (RyRs) are large intracellular ligand-gated calcium release ion channels. Mutations in human RyR1 in combination with a volatile anesthetic or muscle relaxant are known to cause leaky RyRs resulting in malignant hyperthermia (MH). This has long been primarily treated with the RyR inhibitory drug dantrolene. Alternatives to dantrolene as a RyR inhibitor may be found through computer-aided drug design. Additionally, molecular dynamics (MD) studies of dantrolene interacting with RyRs may reveal its full mechanism of action. The availability of accurate force field parameters is important for the success of both. METHODS: In this study, force field parameters for dantrolene were obtained from the CHARMM General Force Field (CGenFF) program and optimized using the force field toolkit (FFTK) and FFParam programs. The obtained parameters were then validated by a comparison between calculated and experimental IR spectra and normal mode analysis, among other techniques.

Ryanodine receptor 2 inhibition reduces dispersion of cardiac repolarization, improves contractile function, and prevents sudden arrhythmic death in failing hearts.

Sudden cardiac death (SCD) from ventricular tachycardia/fibrillation (VT/VF) is a leading cause of death, but current therapies are limited. Despite extensive research on drugs targeting sarcolemmal ion channels, none have proven sufficiently effective for preventing SCD. Sarcoplasmic ryanodine receptor 2 (RyR2) Ca2+ release channels, the downstream effectors of sarcolemmal ion channels, are underexplored in this context. Recent evidence implicates reactive oxygen species (ROS)-mediated oxidation and hyperactivity of RyR2s in the pathophysiology of SCD. We tested the hypothesis that RyR2 inhibition of failing arrhythmogenic hearts reduces sarcoplasmic Ca2+ leak and repolarization lability, mitigates VT/VF/SCD and improves contractile function. We used a guinea pig model that replicates key clinical aspects of human nonischemic HF, such as a prolonged QT interval, a high prevalence of spontaneous arrhythmic SCD, and profound Ca2+ leak via a hyperactive RyR2. HF animals were randomized to receive dantrolene (DS) or placebo in early or chronic HF. We assessed the incidence of VT/VF and SCD (primary outcome), ECG heart rate and QT variability, echocardiographic left ventricular (LV) structure and function, immunohistochemical LV fibrosis, and sarcoplasmic RyR2 oxidation. DS treatment prevented VT/VF and SCD by decreasing dispersion of repolarization and ventricular arrhythmias. Compared to placebo, DS lowered resting heart rate, preserved chronotropic competency during transient ß-adrenergic challenge, and improved heart rate variability and cardiac function. Inhibition of RyR2 hyperactivity with dantrolene mitigates the vicious cycle of sarcoplasmic Ca2+ leak-induced increases in diastolic Ca2+ and ROS-mediated RyR2 oxidation, thereby reducing repolarization lability and protecting against VT/VF/SCD. Moreover, the consequent increase in sarcoplasmic Ca2+ load improves contractile function. These potentially life-saving effects of RyR2 inhibition warrant further investigation, such as clinical studies of repurposing dantrolene as a potential new therapy for heart failure and/or SCD.

Each year, more than 300,000 people experience cardiac arrest or sudden cardiac death. Sudden cardiac death is caused by irregular heartbeats known as ventricular tachycardia or ventricular fibrillation, which prevent the heart from pumping blood. During a regular heart rhythm, the heart muscles contract and relax, regulated by a coordinated rise and fall of calcium ions within heart cells. In the cells of diseased hearts, on the other hand, calcium leaks out of a compartment known as the sarcoplasmic reticulum in an uncontrolled manner. This happens because an ion channel in the membrane of the sarcoplasmic reticulum known as ryanodine receptor 2 becomes hyperactive and releases calcium in an uncontrolled manner. This abnormal calcium release leads to irregular calcium waves, which can make the heart's electrical properties unstable, causing ventricular tachycardia, ventricular fibrillation and sudden cardiac death. Joshi et al. tested whether dantrolene, a molecule that blocks ryanodine receptor 2, can stop calcium leaks from the sarcoplasmic reticulum and prevent lethal arrhythmias and sudden cardiac death in failing hearts. To investigate this, Joshi et al. induced heart failure in guinea pigs that have abnormal heart calcium signalling similar to human heart failure, and then treated the animals with either dantrolene or a placebo. The results indicate that blocking ryanodine receptor 2 hyperactivity with dantrolene prevents lethal arrhythmias and sudden cardiac death by blocking calcium leaks and by preventing the instability of the electrical properties of the heart. Additionally, Joshi et al. found that dantrolene also improved the diseased heart's ability to pump adequate amounts of blood, allowing failing hearts to meet increased cardiovascular demands, and thereby improving the heart's overall function. The proposed studies come from a strong clinical need to improve bad outcomes in people who keep having fatal heart rhythm episodes despite getting the best medical care. Many heart failure patients are plagued by recurrent defibrillator shocks to abort sudden cardiac death from relentless lethal heart rhythms. These shocks are painful, injure the heart, and worsen the quality of life. Unfortunately, management options are extremely limited for these patients. The findings of Joshi et al. indicate that dantrolene may be a potential treatment for people with fatal heart rhythms who are at risk of sudden cardiac death and could have a positive impact on these people's quality of life. However, before this can happen, dantrolene will first have to be thoroughly tested to ensure effectivity and safety in humans. In any case, Joshi et al. have opened a new avenue in the search for medications to treat deadly arrhythmias and sudden cardiac death.

Oral Dantrolene Reduces Myalgia and Hyperckemia in a Child with RYR1-Related Exertional Myalgia/Rhabdomyolysis.

RYR1-related exertional myalgia/rhabdomyolysis (ERM) is an underrecognized condition, which can cause limiting muscle symptoms, and may account for more than one-third of undiagnosed rhabdomyolysis cases. Dantrolene has shown promising results in controlling muscle symptoms in individuals with ERM, however, its use in children remains poorly documented. This case report presents the successful treatment of a 5-year-old patient with ERM using oral dantrolene. The patient experienced notable improvements, including a reduction in the frequency and intensity of myalgia episodes, no hospitalizations due to rhabdomyolysis, a substantial decrease in creatine phosphokinase (CPK) levels, and enhanced performance on the 6-minute walk test. The use of dantrolene was well-tolerated, and no significant adverse effects were observed. This report adds to the existing evidence supporting the effectiveness of oral dantrolene in managing ERM, and, to the best of our knowledge, this is the first report of the use of dantrolene in a pediatric patient for controlling anesthesia-independent muscle symptoms.

Function of a mutant ryanodine receptor (T4709M) linked to congenital myopathy.

Physiological muscle contraction requires an intact ligand gating mechanism of the ryanodine receptor 1 (RyR1), the Ca2+-release channel of the sarcoplasmic reticulum. Some mutations impair the gating and thus cause muscle disease. The RyR1 mutation T4706M is linked to a myopathy characterized by muscle weakness. Although, low expression of the T4706M RyR1 protein can explain in part the symptoms, little is known about the function RyR1 channels with this mutation. In order to learn whether this mutation alters channel function in a manner that can account for the observed symptoms, we examined RyR1 channels isolated from mice homozygous for the T4709M (TM) mutation at the single channel level. Ligands, including Ca2+, ATP, Mg2+ and the RyR inhibitor dantrolene were tested. The full conductance of the TM channel was the same as that of wild type (wt) channels and a population of partial open (subconductive) states were not observed. However, two unique sub-populations of TM RyRs were identified. One half of the TM channels exhibited high open probability at low (100 nM) and high (50 µM) cytoplasmic [Ca2+], resulting in Ca2+-insensitive, constitutively high Po channels. The rest of the TM channels exhibited significantly lower activity within the physiologically relevant range of cytoplasmic [Ca2+], compared to wt. TM channels retained normal Mg2+ block, modulation by ATP, and inhibition by dantrolene. Together, these results suggest that the TM mutation results in a combination of primary and secondary RyR1 dysfunctions that contribute to disease pathogenesis.

Suspected Malignant Hyperthermia in a Brain-Dead Donor During Anesthesia for Organ Procurement Surgery: A Case Report.

We report an unusual case of highly suspected malignant hyperthermia after inducing anesthesia in a brain-dead 18-year-old male patient undergoing organ procurement surgery. The patient was administered desflurane (3 vol%) and rocuronium bromide (50 mg) to induce and maintain general anesthesia. He experienced hypercapnia and tachycardia within 5 minutes of anesthesia induction; however, his body temperature rapidly rose only after 15 minutes. The volatile anesthetic was discontinued, and dantrolene was administered at a low dose (1 mg/kg) to avert possible hepatotoxic effects on the donor liver. Fortunately, the clinical course of the brain-dead donor until the organs were harvested and the liver transplantation outcome of the recipient was favorable. A comprehensive understanding of the pathophysiology of brain death, organ transplantation, and malignant hyperthermia is essential to respond promptly and appropriately. Based on our experience, low-dose dantrolene may be clinically used in brain-dead donors while accounting for its potential hepatotoxic effects.

Ryanodine receptor inhibition with acute dantrolene treatment reduces arrhythmia susceptibility in human hearts.

Ryanodine receptor 2 (RyR2) hyperactivity is observed in structural heart diseases that are a result of ischemia or heart failure. It causes abnormal calcium handling and calcium leaks that cause metabolic, electrical, and mechanical dysfunction, which can trigger arrhythmias. Here, we tested the antiarrhythmic potential of dantrolene (RyR inhibitor) in human hearts. Human hearts not used in transplantation were obtained, and right ventricular outflow tract (RVOT) wedges and left ventricular (LV) slices were prepared. Pseudo-ECGs were recorded to determine premature ventricular contraction (PVC) incidences. Optical mapping was performed to determine arrhythmogenic substrates. After baseline optical recordings, tissues were treated with 1) isoproterenol (250 nM), 2) caffeine (200 mM), and 3) dantrolene (2 or 10 mM). Optical recordings were obtained after each treatment. Isoproterenol and caffeine treatment increased PVC incidence, whereas dantrolene reduced the PVC burden. Isoproterenol shortened action potential duration (APD) in the RV, RVOT, and LV regions and shortened calcium transient duration (CaTD) in the LV. Caffeine further shortened APD in the RV, did not modulate APD in the RVOT, and prolonged APD in the LV. In addition, in the LV, CaTD prolongation was also observed. More importantly, adding dantrolene did not alter APD in the RV or RVOT regions but produced a trend toward APD prolongation and significant CaTD prolongation in the LV, restoring these parameters to baseline values. In conclusions, dantrolene treatment suppresses triggers and reverses arrhythmogenic substrates in the human heart and could be a novel antiarrhythmic therapy in patients with structural heart disease.NEW & NOTEWORTHY Ryanodine receptor 2 hyperactivity is observed in structural heart diseases caused by ischemia or heart failure. It causes abnormal calcium leaks, which can trigger arrhythmias. To prevent arrhythmias, we applied dantrolene in human hearts ex vivo. Isoproterenol and caffeine treatment increased PVC incidence, whereas dantrolene reduced the PVC burden. Dantrolene treatment suppresses triggers and reverses arrhythmogenic substrates and could be a novel antiarrhythmic therapy in patients with structural heart disease.

Dantrolene inhibition of ryanodine channels (RyR2) in artificial lipid bilayers depends on FKBP12.6.

Dantrolene is a neutral hydantoin that is clinically used as a skeletal muscle relaxant to prevent overactivation of the skeletal muscle calcium release channel (RyR1) in response to volatile anesthetics. Dantrolene has aroused considerable recent interest as a lead compound for stabilizing calcium release due to overactive cardiac calcium release channels (RyR2) in heart failure. Previously, we found that dantrolene produces up to a 45% inhibition RyR2 with an IC50 of 160 nM, and that this inhibition requires the physiological association between RyR2 and CaM. In this study, we tested the hypothesis that dantrolene inhibition of RyR2 in the presence of CaM is modulated by RyR2 phosphorylation at S2808 and S2814. Phosphorylation was altered by incubations with either exogenous phosphatase (PP1) or kinases; PKA to phosphorylate S2808 or endogenous CaMKII to phosphorylate S2814. We found that PKA caused selective dissociation of FKBP12.6 from the RyR2 complex and a loss of dantrolene inhibition. Rapamycin-induced FKBP12.6 dissociation from RyR2 also resulted in the loss of dantrolene inhibition. Subsequent incubations of RyR2 with exogenous FKBP12.6 reinstated dantrolene inhibition. These findings indicate that the inhibitory action of dantrolene on RyR2 depends on RyR2 association with FKBP12.6 in addition to CaM as previously found.

RyR2 inhibition with dantrolene is antiarrhythmic, prevents further pathological remodeling, and improves cardiac function in chronic ischemic heart disease.

Diastolic Ca2+ leak due to cardiac ryanodine receptor (RyR2) hyperactivity has been widely documented in chronic ischemic heart disease (CIHD) and may contribute to ventricular tachycardia (VT) risk and progressive left-ventricular (LV) remodeling. Here we test the hypothesis that targeting RyR2 hyperactivity can suppress VT inducibility and progressive heart failure in CIHD by the RyR2 inhibitor dantrolene. METHODS AND RESULTS: CIHD was induced in C57BL/6 J mice by left coronary artery ligation. Four weeks later, mice were randomized to either acute or chronic (6 weeks via implanted osmotic pump) treatment with dantrolene or vehicle. VT inducibility was assessed by programmed stimulation in vivo and in isolated hearts. Electrical substrate remodeling was assessed by optical mapping. Ca2+ sparks and spontaneous Ca2+ releases were measured in isolated cardiomyocytes. Cardiac remodeling was quantified by histology and qRT-PCR. Cardiac function and contractility were measured using echocardiography. Compared to vehicle, acute dantrolene treatment reduced VT inducibility. Optical mapping demonstrated reentrant VT prevention by dantrolene, which normalized the shortened refractory period (VERP) and prolonged action potential duration (APD), preventing APD alternans. In single CIHD cardiomyocytes, dantrolene normalized RyR2 hyperactivity and prevented spontaneous intracellular Ca2+ release. Chronic dantrolene treatment not only reduced VT inducibility but also reduced peri-infarct fibrosis and prevented further progression of LV dysfunction in CIHD mice. CONCLUSIONS: RyR2 hyperactivity plays a mechanistic role for VT risk, post-infarct remodeling, and contractile dysfunction in CIHD mice. Our data provide proof of concept for the anti-arrhythmic and anti-remodeling efficacy of dantrolene in CIHD.

Feedback contributions to excitation-contraction coupling in native functioning striated muscle.

Skeletal and cardiac muscle excitation-contraction coupling commences with Nav1.4/Nav1.5-mediated, surface and transverse (T-) tubular, action potential generation. This initiates feedforward, allosteric or Ca2+-mediated, T-sarcoplasmic reticular (SR) junctional, voltage sensor-Cav1.1/Cav1.2 and ryanodine receptor-RyR1/RyR2 interaction. We review recent structural, physiological and translational studies on possible feedback actions of the resulting SR Ca2+ release on Nav1.4/Nav1.5 function in native muscle. Finite-element modelling predicted potentially regulatory T-SR junctional [Ca2+]TSR domains. Nav1.4/Nav1.5, III-IV linker and C-terminal domain structures included Ca2+ and/or calmodulin-binding sites whose mutations corresponded to specific clinical conditions. Loose-patch-clamped native murine skeletal muscle fibres and cardiomyocytes showed reduced Na+ currents (INa) following SR Ca2+ release induced by the Epac and direct RyR1/RyR2 activators, 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate and caffeine, abrogated by the RyR inhibitor dantrolene. Conversely, dantrolene and the Ca2+-ATPase inhibitor cyclopiazonic acid increased INa. Experimental, catecholaminergic polymorphic ventricular tachycardic RyR2-P2328S and metabolically deficient Pgc1ß-/- cardiomyocytes also showed reduced INa accompanying [Ca2+]i abnormalities rescued by dantrolene- and flecainide-mediated RyR block. Finally, hydroxychloroquine challenge implicated action potential (AP) prolongation in slowing AP conduction through modifying Ca2+ transients. The corresponding tissue/organ preparations each showed pro-arrhythmic, slowed AP upstrokes and conduction velocities. We finally extend discussion of possible Ca2+-mediated effects to further, Ca2+, K+ and Cl-, channel types. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Excitatory cholinergic responses in mouse primary bronchial smooth muscle require both Ca2+ entry via l-type Ca2+ channels and store operated Ca2+ entry via Orai channels.

Malfunctions in airway smooth muscle Ca2+-signalling leads to airway hyperresponsiveness in asthma and chronic obstructive pulmonary disease. Ca2+-release from intracellular stores is important in mediating agonist-induced contractions, but the role of influx via l-type Ca2+ channels is controversial. We re-examined roles of the sarcoplasmic reticulum Ca2+ store, refilling of this store via store-operated Ca2+ entry (SOCE) and l-type Ca2+ channel pathways on carbachol (CCh, 0.1-10 µM)-induced contractions of mouse bronchial rings and intracellular Ca2+ signals of mouse bronchial myocytes. In tension experiments, the ryanodine receptor (RyR) blocker dantrolene (100 µM) reduced CCh-responses at all concentrations, with greater effects on sustained rather than initial components of contraction. 2-Aminoethoxydiphenyl borate (2-APB, 100 µM), in the presence of dantrolene, abolished CCh-responses, suggesting the sarcoplasmic reticulum Ca2+ store is essential for contraction. The SOCE blocker GSK-7975A (10 µM) reduced CCh-contractions, with greater effects at higher (e.g. 3 and 10 µM) CCh concentrations. Nifedipine (1 µM), abolished remaining contractions in GSK-7975A (10 µM). A similar pattern was observed on intracellular Ca2+-responses to 0.3 µM CCh, where GSK-7975A (10 µM) substantially reduced Ca2+ transients induced by CCh, and nifedipine (1 µM) abolished remaining responses. When nifedipine (1 µM) was applied alone it had less effect, reducing tension responses at all CCh concentrations by 25% - 50%, with greater effects at lower (e.g. 0.1 and 0.3 µM) CCh concentrations. When nifedipine (1 µM) was examined on the intracellular Ca2+-response to 0.3 µM CCh, it only modestly reduced Ca2+ signals, while GSK-7975A (10 µM) abolished remaining responses. In conclusion, Ca2+-influx from both SOCE and l-type Ca2+ channels contribute to excitatory cholinergic responses in mouse bronchi. The contribution of l-type Ca2+ channels was especially pronounced at lower doses of CCh, or when SOCE was blocked. This suggests l-type Ca2+ channels might be a potential target for bronchoconstriction under certain circumstances.

Pediatric fulminant malignant hyperthermia with severe electroencephalographic abnormality and brain damage: a case report.

BACKGROUND: Malignant hyperthermia is an extremely dangerous condition that can occur with exposure to volatile inhalant anesthetics and depolarizing muscle relaxants, and that requires immediate intervention. Neurological complications have rarely been reported, with no reports of electroencephalographic abnormalities or encephalopathy. Here, we report a case of severe electroencephalographic abnormality in the acute phase of malignant hyperthermia that eventually led to diffuse cerebral cortical damage. CASE PRESENTATION: A 15-month-old Japanese boy underwent a Rastelli procedure to correct a double-outlet right ventricle and pulmonary atresia. Sevoflurane was used for induction and maintenance of anesthesia during surgery. After withdrawal from the heart-lung machine, his body temperature rose at a rate of 0.1 â„ƒ/minute, and when he left the operating room, his core body temperature had reached 42 â„ƒ. After admission to the intensive care unit, tachycardia, high PaCO2, and progressive metabolic acidosis were observed. A clinical grading scale score of 63 indicated malignant hyperthermia, and dantrolene was administered. The pupils were dilated, and the electroencephalogram showed persistent generalized continuous multifocal spikes. Midazolam, levetiracetam, and fosphenytoin were administered without improvement, and thiamylal and ketamine were infused continuously. After the electroencephalogram shifted to burst suppression, the epileptic firing gradually decreased, and the background electroencephalogram became lower in amplitude. Magnetic resonance imaging of the head performed after the patient was hemodynamically stable suggested diffuse cerebral cortical damage. Severe mental retardation, hypertonia, and quadriplegia were observed as neurological complications. CONCLUSIONS: In this case, despite the use of high-dose anticonvulsants, the patient showed severe electroencephalogram abnormality, resulting in diffuse cortical damage. Hyperthermia is known to damage the central nervous system by causing increased brain pressure and cerebral edema, which may have triggered the severe neuronal excitation that we observed in this case. The presence of systemic inflammatory response syndrome and the patient's background, including young age and ethnicity, might also have been factors. Malignant hyperthermia can be complicated by encephalopathy, and continuous electroencephalogram monitoring should be considered.

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.