Dantrolene Research Articles

Development of a water-soluble ryanodine receptor 1 inhibitor

Ryanodine receptor 1 (RyR1) is a Ca2+-release channel expressed on the sarcoplasmic reticulum (SR) membrane. RyR1 mediates release of Ca2+ from the SR to the cytoplasm to induce muscle contraction, and mutations associated with overactivation of RyR1 cause lethal muscle diseases. Dantrolene sodium salt (dantrolene Na) is the only approved RyR inhibitor to treat malignant hyperthermia patients with RyR1 mutations, but is poorly water-soluble. Our group recently developed a bioassay system and used it to identify quinoline derivatives such as 1 as potent RyR1 inhibitors. In the present study, we focused on modification of these inhibitors with the aim of increasing their water-solubility. First, we tried reducing the hydrophobicity by shortening the N-octyl chain at the quinolone ring of 1; the N-heptyl compound retained RyR1-inhibitory activity, but the N-hexyl compound showed decreased activity. Next, we introduced a more hydrophilic azaquinolone ring in place of quinolone; in this case, only the N-octyl compound retained activity. The sodium salt of N-octyl azaquinolone 7 showed similar inhibitory activity to dantrolene Na with approximately 1,000-fold greater solubility in saline.

Stabilization of RyR2 maintains right ventricular function, reduces the development of ventricular arrhythmias, and improves prognosis in pulmonary hypertension.

Right ventricular (RV) dysfunction and its associated arrhythmias are recognized as important determinants of the prognosis of pulmonary arterial hypertension (PAH). Here, we aimed to investigate whether direct pharmacological intervention in the RV muscle with dantrolene (DAN), a stabilizer of the cardiac ryanodine receptor (RyR2), has a protective effect against RV dysfunction and arrhythmia in a monocrotaline (MCT)-induced PAH rat model. Male 8-week-old Sprague-Dawley rats were injected with MCT for the induction of PAH. Induction of ventricular tachycardia (VT) by catecholamines was also evaluated in association with RyR2-mediated Ca2+ release properties in isolated cardiomyocytes. A pulmonary artery-banding model has also been established to assess the independent effects of chronic pressure overload on RV morphology and function. In the MCT-induced PAH rat model, RV hypertrophy, dilation, and functional decline were observed, with a survival rate of 0% 2 months after MCT induction. In contrast, chronic DAN treatment improved all these RV parameters and increased survival by 80%. Chronic DAN treatment also prevented the dissociation of calmodulin from RyR2, thereby inhibiting Ca2+ sparks and spontaneous Ca2+ transients in MCT-induced hypertrophied RV cardiomyocytes. Epinephrine induced VT in more than 50% of rats with MCT-induced PAH, but complete suppression of VT was achieved by chronic DAN treatment. Stabilization of RyR2 by DAN has potential as a new therapeutic agent against the development of RV dysfunction and fatal arrhythmia associated with PAH.

Heliox Preconditioning Exerts Neuroprotective Effects on Neonatal Ischemia/Hypoxia Injury by Inhibiting Necroptosis Induced by Ca2+ Elevation.

Our previous studies have indicated that heliox preconditioning (HePC) may exert neuroprotective effects on neonatal hypoxic-ischemic encephalopathy (HIE). The present study was to investigate whether HePC alleviates neonatal HIE by inhibiting necroptosis and explore the potential mechanism. Seven-day-old rat pups were randomly divided into Sham group, HIE group, HIE + HePC group, HIE + Dantrolene (DAN) group, and HIE + Necrostatin-1 (Nec-1) group. HIE was induced by common carotid artery ligation and subsequent hypoxia exposure. The neurological function, brain injury, and molecular mechanism were evaluated by histological staining, neurobehavioral test, Western blotting, Ca2+, immunofluorescence staining, co-immunoprecipitation (Co-IP), and transmission electron microscopy (TEM). Results supported that the expression of necroptosis markers and p-RyR2 in the brain increased significantly after HIE. HePC, DAN, or Nec-1 was found to improve the neurological deficits after H/I and inhibit neuronal necroptosis. Interestingly, both HePC and DAN inhibited the increases in cytoplasmic Ca2+ and CaMK-II phosphorylation in the brain secondary to HIE, but Nec-1 failed to affect Ca2+. In conclusion, our results suggest HePC may alleviate cytoplasmic Ca2+ overload by regulating p-RyR2, which inhibits the necroptosis in the brain, exerting neuroprotective effects on HIE.

Abstract 13286: Ryanodine Receptor Inhibition Prevents Sudden Cardiac Death by Preserving Cardiac Repolarization Reserve in Stressed Arrhythmogenic Hearts

Introduction: Current therapies for preventing sudden cardiac death (SCD) are limited. Prior studies have shown that cardiac stress-induced ryanodine receptor (RyR2) hyperactivity increases sarcoplasmic calcium leak, leading to ventricular tachyarrythmias (VT) and SCD. We hypothesized that inhibition of RyR2 with dantrolene sodium (DS) reduces Ca 2+ leak and mitigates VT and SCD risk in pressure-overloaded arrhythmogenic hearts. Methods: We used an established guinea pig model that uniquely recapitulates key features of human SCD, including a high incidence of spontaneous VT in freely ambulating animals, randomized to placebo vs. DS treatment. We performed in vivo (e.g., VT/SCD incidence, ECG heart rate and QT variability, echocardiographic measures of cardiac function) and ex vivo (e.g., pressure-volume, electrophysiology) studies. We performed intention to treat analysis for 4-week VT-free survival. Results: About half the animals receiving placebo had SCD by 4 weeks. By contrast, dantrolene treatment reduced SCD incidence by 50%. Compared to placebo, dantrolene treatment preserved chronotropic competency during transient β-adrenergic challenge, and improved heart rate variability (SDNN: 11±1.1 vs 17±2.8 ms, p<0.05), reduced QT variability (0.52±0.13 vs 0.19±0.08 (log, ms, p<0.05), and improved cardiac function (fractional shortening: 26±1.9 vs 45±4.5 %, p<0.05). In perfused excised hearts, dantrolene treatment mitigated VT (induced by isoproterenol or burst pacing). Conclusion: Inhibition of RyR2 hyperactivity by dantrolene treatment prevents VT and SCD by increasing cardiac repolarization reserve. Dantrolene also improved overall cardiac electromechanical function in stressed arrhythmogenic hearts. Targeted inhibition of RyR2 hyperactivity may be an effective therapeutic strategy for preventing VT and SCD.

Malignant Hyperthermia-A Case From Camp Humphreys, South Korea, and Lessons for the Military Health System.

Although malignant hyperthermia (MH) is a well-known complication of anesthesia, it presents unique considerations in the military health system. In this case report, the authors present a 26-year-old male active duty service member who experienced an MH crisis during a routine bilateral sagittal split osteotomy. The case presented here, which occurred at Brian D Allgood Army Community Hospital at Camp Humphreys, South Korea, highlights the challenges presented when caring for these patients in minimally staffed environments with frequent turnover of staff. The authors discuss the challenges to the military system such as the importance of adequate documentation of MH-susceptible service members, the benefits of rapidly dissolving dantrolene sodium nanosuspension, and the necessity for frequent training of military medical staff in the recognition and management of MH.

Simultaneous determination of dantrolene and paracetamol in human plasma by liquid chromatography tandem mass spectrometry

A simple, sensitive, rapid and specific method based on ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) for the simultaneous quantification of dantrolene (DAN) and paracetamol (PAR) in real human plasma was developed and validated. The preparation of sample was achieved by liquid–liquid extraction with tertiary butyl methyl ether. The analysis was performed on a reversed-phase C18column (1.7 µm, 2.1 × 30 mm) using acetonitrile: 0.1% formic acid (80:20, v/v) as the mobile phase and pumped in an isocratic mode at a flow rate of 0.3 mL/min using citalopram (CIT) as an internal standard. Tandem mass spectrometric detection was carried out by both positive and negative electrospray ionization (ESI) in the multiple-reaction monitoring mode (MRM). The analysis was carried out within 1 min for each sample which made it possible to analyze more than 350 human samples per day. Validation of the method was performed according to FDA guidelines for bio-analytical method. The method was found to be linear in the range of 25–2500 ng/mL and 100–10,000 ng/mL for DAN and PAR, respectively. The method was applied successfully for the determination of the two analytes in the plasma after oral administration of Dantrelax® compound capsules to healthy volunteers. The study was accomplished after approval of the ethics committee.

Green spectrofluorimetric assay of dantrolene sodium via reduction method: application to content uniformity testing.

Green analysis has turned out to be of a great value in all areas, including pharmaceutical analysis. Thus, it is extremely important to consider the environmental influence of each step in developing any analysis technique. The present work illustrates a validated, simple and green spectrofluorimetric method for the analysis of dantrolene sodium (DAN). The developed process is characterized by being of high sensitivity as well as being relatively inexpensive. The suggested technique based on the formation of a highly fluorescent product of DAN via its reduction by the aid of Zn/HCl system. The resulting fluorophore showed a powerful fluorescence at λem 344 nm after excitation at λex 279 nm. Calibration graph revealed a great linear regression (r = 0.9998) within concentration ranging from 0.05 to 2.0 µg ml−1. The suggested method had very low detection and quantification limits of 0.010 and 0.031 µg ml−1, respectively. The applied technique was effectively used in the determination of DAN in its pharmaceutical preparations. The results were compared with those from using the official United States Pharmacopeia (USP) method and they were in a good agreement. Moreover, content uniformity testing of DAN in capsules was performed adopting the investigated technique with satisfying results. The greenness of the suggested technique was confirmed by the three standard assessment tools. Therefore, the developed technique can be used in the routine quality control analysis of DAN with minimum harmful impact on nature or individuals.

Anti‐contractile activity of SIRT1 in the perivascular adipose tissue

Background ˗ Perivascular adipose tissue (PVAT) reduces the contraction of blood vessel at least in part by secreting transferable relaxing factors. Nevertheless, the molecular mechanisms underlying PVAT-mediated anti-contractile activity remain uncharacterized. The present study examined the role of SIRT1, a longevity regulator, in modulating the anti-contractile effects of PVAT. Methods and Results ˗ Mesenteric arteries with or without the attached PVAT were isolated from wild type animals (WT), or mice with selective overexpression of human SIRT1 in adipose tissues (Adipo-SIRT1). Wire myography was performed to examine phenylephrine-induced arterial contractions. PVAT derived from Adipo-SIRT1 exhibited dose- and time-dependent anti-contractile activity, which was not affected by feeding the animals with high fat diet. Molecular analyses revealed that overexpression of SIRT1 decreased the expression of Triadin, an important component of the calcium-releasing ryanodine receptor (RyR) channels. Treatment with dantrolene sodium, an inhibitor of RyR, increased the anti-contractile activity of PVAT derived from WT. Mechanistically, SIRT1-triggered mitochondrial hormesis via modulating the calcium flux from endoplasmic reticulum. The increased production of mitochondrial reactive oxygen species (mtROS) was critically involved in SIRT1-mediated anti-contractile activity of PVAT. The beneficial function of SIRT1 in PVAT was abolished in transgenic mice with selective overexpression of Triadin in adipose tissues (Adipo-TRDN). Conclusions ˗Increased SIRT1 expression in PVAT promotes vascular anti-contraction at least partly through down regulation of Triadin-mediated calcium signaling.

Dantrolene Induces Mitigation of Myocardial Ischemia-Reperfusion Injury by Ryanodine Receptor Inhibition

The impairment of intracellular calcium homeostasis plays an essential role during ischemia-reperfusion injury. Calcium release from sarcoplasmic reticulum which is triggered by myocardial ischemia is mainly mediated by ryanodine receptors. Dantrolene sodium is a ryanodine receptor antagonist. The objective of the present study was to evaluate the in-vivo impact of dantrolene sodium on myocardial ischemia-reperfusion injury in swine models. An in vivo, experimental trial comparing 10 experimental animals which received dantrolene sodium with 9 control swine models was conducted. Their left anterior descending coronary artery was temporarily occluded for 75 minutes via a vessel tourniquet, which was then released. Myocardial reperfusion was allowed for 24 hours. Dantrolene was administered at the onset of the reperfusion period and levels of troponin, creatine phosphokinase and creatine kinase myocardial band between the two groups were compared. Additionally, various other hemodynamic parameters and left ventricular morphology and function were examined. There were significantly lower values of troponin, creatine phosphokinase and creatine kinase myocardial band in the dantrolene group indicating less ischemia-reperfusion injury. Moreover, the postischemic cardiac index was also greater in the dantrolene group, whereas viable myocardium was also better preserved. In conclusion, the in vivo cardioprotective role of dantrolene sodium against ischemia-reperfusion injury in swine models was indicated in this study. Therefore, dantrolene sodium administration could be a promising treatment against ischemia-reperfusion injury in humans. However, large randomized clinical studies should be firstly carried out to prove this hypothesis.

T lymphocytes from malignant hyperthermia-susceptible mice display aberrations in intracellular calcium signaling and mitochondrial function

Gain-of-function RyR1-p.R163C mutation in ryanodine receptors type 1 (RyR1) deregulates Ca2+ signaling and mitochondrial function in skeletal muscle and causes malignant hyperthermia in humans and mice under triggering conditions. We investigated whether T lymphocytes from heterozygous RyR1-p.R163C knock-in mutant mice (HET T cells) display measurable aberrations in resting cytosolic Ca2+ concentration ([Ca2+]i), Ca2+ release from the store, store-operated Ca2+ entry (SOCE), and mitochondrial inner membrane potential (ΔΨm) compared with T lymphocytes from wild-type mice (WT T cells). We explored whether these variables can be used to distinguish between T cells with normal and altered RyR1 genotype.HET and WT T cells were isolated from spleen and lymph nodes and activated in vitro using phytohemagglutinin P. [Ca2+]i and ΔΨm dynamics were examined using Fura 2 and tetramethylrhodamine methyl ester fluorescent dyes, respectively. Activated HET T cells displayed elevated resting [Ca2+]i, diminished responses to Ca2+ mobilization with thapsigargin, and decreased rate of [Ca2+]i elevation in response to SOCE compared with WT T cells. Pretreatment of HET T cells with ryanodine or dantrolene sodium reduced disparities in the resting [Ca2+]i and ability of thapsigargin to mobilize Ca2+ between HET and WT T cells. While SOCE elicited dissipation of the ΔΨm in WT T cells, it produced ΔΨm hyperpolarization in HET T cells. When used as the classification variable, the amplitude of thapsigargin-induced Ca2+ transient showed the best promise in predicting the presence of RyR1-p.R163C mutation. Other significant variables identified by machine learning analysis were the ratio of resting cytosolic Ca2+ level to the amplitude of thapsigargin-induced Ca2+ transient and an integral of changes in ΔΨm in response to SOCE.Our study demonstrated that gain-of-function mutation in RyR1 significantly affects Ca2+ signaling and mitochondrial fiction in T lymphocytes, which suggests that this mutation may cause altered immune responses in its carrier. Our data link the RyR1-p.R163C mutation, which causes inherited skeletal muscle diseases, to deregulation of Ca2+ signaling and mitochondrial function in immune T cells and establish proof-of-principle for in vitro T cell-based diagnostic assay for hereditary RyR1 hyperfunction.

117 Update: Dantrolene Sodium Suspension (250 Mg/5mL) in Patients with Exertional Heat Stroke

Exertional heat stroke (EHS) is a rare, sudden and unpredictable hyperthermic, hypermetabolic crisis that may affect anyone performing intense physical activity. EHS is one of the most severe forms of heat illness and a leading cause of death and disability in athletes, outdoor workers and military personnel. EHS is marked by severe hyperthermia (core body temp. ≥40°C) and CNS dysfunction (eg, seizures, coma, abnormal behavior). The sporadic, unpredictable and life-threatening nature of EHS limits the ability to conduct prospective controlled clinical trials. Current treatment is limited to efficient body cooling which is infrequently available and inconsistently administered. Up to 1/3 of survivors may have long-term neurologic sequelae despite rapid body cooling. The objective was to evaluate the efficacy and safety of dantrolene sodium for injectable suspension (Ryanodex®) (DSS) for the treatment of EHS, administered as an adjunct to body cooling (BC). We conducted two comparable clinical studies evaluating DSS+BC compared to BC alone in patients with EHS. Study 1 (ITT N=34, mITT N=24) and Study 2 (ITT N=17) were randomized, controlled, 2-arm parallel studies. Patients had a core temp. ≥40°C, impaired level of consciousness (LOC)(GCS score <13) and tachycardia. The primary efficacy endpoint in both studies was the cumulative incidence of recovery of LOC defined as a GCS score ≥13 at or prior to 90 minutes post-randomization. Safety and tolerability were assessed. As reported at the 2017 ACEP Research Forum, the mITT population in Study 1 excluded patients who were intubated at baseline (precluding full assessment of GCS). Study 2 ran from August 2018 to August 2019. These multicenter studies were conducted in a real-world emergency medicine setting in the same hospitals. Due to the limitations enrolling EHS patients and given the comparability of the studies, an integrated analysis of efficacy was warranted. Study 1 and Study 2 demonstrated consistent, clinically meaningful results. Demographics and baseline characteristics in the integrated analysis were balanced between the DSS+BC (n=19) and BC only (n=22) groups regarding age (median 45 vs 43.5 yrs.), baseline GCS (mean 7.1 vs 6.2) and baseline rectal temp. (mean 41.6 vs 41.3°C). 51% were male. A statistically significant number of patients in the DSS+BC group met the primary endpoint vs BC alone (47.4% vs 18.2%, p=0.0452). Safety and tolerability findings were comparable in both groups and consistent with the known safety profile in the approved indication. Conducting clinical studies for EHS is challenging due to its rare, unpredictable and life-threatening nature. We conducted two comparable studies showing consistent and clinically meaningful benefit from the addition of DSS to BC. Approximately twice as many DSS treated subjects in each study achieved the primary endpoint. The integrated analysis of efficacy demonstrates that DSS added to BC results in a clinically meaningful and statistically significant improvement of neurologic impairment in subjects with EHS within 90 minutes of administration.

Retrospective survey of compounded medications for children in Japan

Compounding of medications, such as crushing tablets and dispersing the contents of capsules, is a common practice in pharmacies and hospitals worldwide and is often done to provide age-appropriate formulations for oral use in pediatric patients. In the present study, a retrospective, descriptive, questionnaire-based survey was conducted to clarify the current status of drug compounding for pediatric patients in Japan. An electronic questionnaire was distributed to 740 hospitals in Japan with pediatric beds, and 208 (28.1%) of these hospitals responded. The total instances of compounding numbered 14,864 (9.6% of the total pediatric oral prescriptions) and comprised 266 active pharmaceutical ingredients (APIs), one-third of which (98 APIs) were compounded even though flexible dosage forms were available. The three most frequently compounded drugs were dantrolene sodium capsules (1152 prescriptions), ramelteon tablets (726 prescriptions), and hydrocortisone tablets (652 prescriptions), all of which were prescribed and administered in powder form. Although compounding of medications frequently varied by the patients’ age, steroids such as prednisolone, dexamethasone, and hydrocortisone were commonly compounded in all age groups. To ensure the quality and safety of these compounded medications, developing a standard protocol for compounding methods is urgently needed in Japan.

Pharmacological evidence for the involvement of ryanodine receptors in halothane-induced liver injury in mice

Drug-induced liver injury (DILI) is a major safety concern in drug development. Halothane (HAL), an inhaled anesthetic, induces severe and idiosyncratic liver injury. Ryanodine receptors (RyR) are major intracellular calcium release channels found on the plasma membrane of the endoplasmic reticulum (ER). It has been reported that disordered hepatic calcium homeostasis is a feature of HAL-induced liver injury (HILI) in guinea pigs. However, there are no reports on whether RyR could mediate the pathogenesis of HILI. The aim of the present study was to investigate the effect of RyR on HILI. Ryanodine (RYA, RyR agonist, 50 μg/kg, i.p.) was administered to BALB/c female mice 1 h before HAL administration (15 mmol/kg, i.p.), which significantly elevated plasma transaminase levels and induced severe hepatic inflammation and necrosis. In contrast, dantrolene sodium (DAN, RyR antagonist) treatment significantly suppressed HILI in a dose- and time-dependent manner and alleviated liver damage. The number of infiltrated neutrophils in the liver were higher in the group treated with HAL + RYA than in the group treated with HAL alone, while DAN treatment decreased neutrophil infiltration in HILI. The hepatic mRNA levels of proinflammatory cytokines; chemokines; and factors related to danger signals, neutrophils, oxidative and ER stress, pro-apoptosis, and RyR were significantly increased with RYA pretreatment, whereas these levels were decreased with DAN treatment. These results suggest that RYA exacerbates HILI, and DAN exerts a protective effect against HILI. Hence, our study provides a novel insight regarding the effect of RyR in the mechanism underlying HILI.

An enteric polymer mitigates the effects of gastric pH on oral absorption of poorly soluble weak acid drugs from supersaturable formulations: A case study with dantrolene

This study demonstrated that an enteric polymer can mitigate the effects of gastric pH on the oral absorption of a poorly water-soluble weak acid drug, dantrolene (DNT). An amorphous solid dispersion (ASD) of DNT with hydroxypropyl methylcellulose (HPMC) acetate succinate (ASD-HPMCAS) was prepared as the enteric released ASD (ER-SF). ASD with HPMC (ASD-HPMC) and DNT sodium salt were also used as immediate-release supersaturable formulations (IR-SFs) with and without water-soluble polymer, respectively. In vivo study with rats and in vitro study with a dissolution/permeation (D/P) system were performed to evaluate oral DNT absorption from each formulation under normal and high gastric pH conditions in rats and humans, respectively. The oral absorption of DNT from both IR-SFs in rats with a high gastric pH was significantly higher than that in rats with a normal gastric pH. In contrast, ASD-HPMCAS attenuated the difference in oral absorption between normal and high gastric pH conditions with significant improvement of DNT absorption. In vivo results implied that an enteric polymer delayed the onset of dissolution until after gastric emptying. ASD-HPMCAS generated supersaturation in the small intestine irrespective of gastric conditions, which was supported bythe in vitrostudy using the D/P system. This study suggested that an enteric polymer is useful to mitigate the inter- and intra-individual differences in oral absorption of poorly water-soluble weak acid drugs.

Overlapping Mechanisms of Exertional Heat Stroke and Malignant Hyperthermia: Evidence vs. Conjecture.

Exertional heat stroke (EHS) and malignant hyperthermia (MH) are life-threatening conditions, triggered by different environmental stimuli that share several clinical symptoms and pathophysiological features. EHS manifests during physical activity normally, but not always, in hot and humid environments. MH manifests during exposure to haloalkane anesthetics or succinylcholine, which leads to a rapid, unregulated release of calcium (Ca2+) within the skeletal muscles inducing a positive-feedback loop within the excitation-contraction coupling mechanism that culminates in heat stroke-like symptoms, if not rapidly recognized and treated. Rare cases of awake MH, independent of anesthesia exposure, occur during exercise and heat stress. It has been suggested that EHS and MH are mediated by similar mechanisms, including mutations in Ca2+ regulatory channels within the skeletal muscle. Rapid cooling, which is the most effective treatment for EHS, is ineffective as an MH treatment; rather, a ryanodine receptor antagonist drug, dantrolene sodium (DS), is administered to the victim to prevent further muscle contractions and hyperthermia. Whether DS can be an effective treatment for EHS victims remains uncertain. In the last decade, multiple reports have suggested a number of mechanistic links between EHS and MH. Here, we discuss aspects related to the pathophysiology, incidence, diagnosis and treatment. Furthermore, we present evidence regarding potential overlapping mechanisms between EHS and MH and explore current knowledge to establish what is supported by evidence or a lack thereof (i.e. conjecture).

739SERCA2 Cys674 modification lead to ventricular arrhythmia due to impaired sarcoplasmic reticulum Ca2+ handling

Abstract Background Sarcoplasmic reticulum Ca2+-ATPase2 (SERCA2) plays an important role in intracellular Ca2+ handling. Under pathological conditions, oxidative stress leads to irreversible oxidation of Cys674 on SERCA2 which causes intracellular Ca2+ overload. Intracellular Ca2+ overload is known as the cause of ventricular arrhythmia, but the relation between SERCA2 function and ventricular arrhythmia remains unclear. Purpose To investigate the role of Cys674 on SERCA2 in the intracellular Ca2+ handling and the induction of ventricular arrhythmia. Methods We employed SERCA2 Cys674Ser heterozygote knock-in mice (SKI) which mimics oxidative modification of Cys674 on SERCA2. Continuous infusion of angiotensin (ANG) (3mg/kg/day) or distilled water were performed both in wild type mice (WT) and SKI for a week. After 1 week, electrophysiological study and intracellular Ca2+ transient measurement were performed. Results ANG elevated blood pressure and represented cardiac hypertrophy with fibrosis similarly both in WT and SKI. The mRNA expression of calcium/calmodulin-dependent protein kinase-II (CaMKII), ryanodine receptor (RyR) and sodium-calcium exchanger (NCX) was increased in SKI heart compared with WT. QTc interval was prolonged in SKI compared with WT, which was markedly prolonged with ANG infusion. Under programmed electrical stimulation, only SKI with ANG showed high incidence of pacing induced ventricular arrhythmia (0/11 in WT/SKI control, 0/14 in WT with ANG vs. 8/14 in SKI with ANG, P &amp;lt; 0.01). In Ca2+ transient measurement, the peak Ca2+ transient amplitude (F/F0) was decreased (WT: 1.81 vs. SKI: 1.46, P &amp;lt; 0.01) and the time to 50% of cytosolic Ca2+ extrusion (T50) was prolonged (WT: 152.6ms vs. SKI: 202.3ms, P &amp;lt; 0.05) in SKI with ANG, suggesting decreased sarcoplasmic reticulum Ca2+ content and impaired SERCA2 activity in SKI with ANG. Intraperitoneal administration of dantrolene sodium (DAN) which inhibit Ca2+ leakage from RyR receptor normalized decreased F/F0 and prolonged T50 in SKI with ANG (Fig. 1 A, B). DAN also prevented QTc prolongation and decreased the incidence of ventricular arrhythmia in SKI with ANG (8/14 in SKI with ANG vs. 2/13 in SKI with ANG + DAN, P &amp;lt; 0.05) (Fig. 2). Conclusions The loss of thiol on Cys674 under pathological condition resulted in impaired Ca2+ handling and high incidence of ventricular arrhythmia which were ameliorated by inhibition of Ca2+ leakage through RyR. Oxidative modification of Cys674 on SERCA2 might contribute to Ca2+ mishandling and arrhythmogenesis. Abstract Figure.

Stabilizing cardiac ryanodine receptor prevents the development of cardiac dysfunction and lethal arrhythmia in Ca2+/calmodulin-dependent protein kinase IIδc transgenic mice

AimsCa2+/calmodulin-dependent protein kinase II (CaMKII) has been shown to induce aberrant Ca2+ release from the cardiac ryanodine receptor (RyR2) in various diseased hearts. However, the precise pathogenic mechanism remains to be elucidated. Here, we investigated the effect of dantrolene (DAN): a RyR2 stabilizer on local Ca2+ release, cardiac function, and lethal arrhythmia in CaMKIIδc transgenic (TG) mice. Methods and resultsThe TG mice showed an increase in left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) with a reduction in LV fractional shortening (LVFS). The phosphorylation levels of Ser2814 in RyR2 and Thr287 in CaMKII increased in TG mice. In TG cardiomyocytes, peak cell shortening (CS) decreased, and the frequency of spontaneous Ca2+ transients (sCaTs) increased. Endogenous RyR2-associated calmodulin (CaM) markedly decreased in TG cardiomyocytes. After chronic DAN treatment for 1 month, LVESD (but not LVEDD) decreased with an increase in LVFS. In the chronic DAN-treated cardiomyocytes, CS increased, sCaTs decreased, and the endogenous CaM binding to RyR2 normally restored. The phosphorylation levels of Ser2814 in RyR2 and Thr287 in CaMKII remained elevated even after DAN treatment. Moreover, in TG mice, chronic DAN treatment prevented sustained ventricular tachycardia induced by epinephrine. ConclusionsDefective association of CaM with RyR2 is most likely to be involved in the pathogenesis of CaMKII-mediated cardiac dysfunction and lethal arrhythmia.

Dantrolene Attenuates Cardiotoxicity of Doxorubicin Without Reducing its Antitumor Efficacy in a Breast Cancer Model

Dysregulation of calcium homeostasis is a major mechanism of doxorubicin (DOX)-induced cardiotoxicity. Treatment with DOX causes activation of sarcoplasmic reticulum (SR) ryanodine receptor (RYR) and rapid release of Ca2+ in the cytoplasm resulting in depression of myocardial function. The aim of this study was to examine the effect of dantrolene (DNT) a RYR blocker on both the cardiotoxicity and antitumor activity of DOX in a rat model of breast cancer. Female F344 rats with implanted MAT B III breast cancer cells were randomized to receive intraperitoneal DOX twice per week (12 mg/kg total dose), 5 mg/kg/day oral DNT or a combination of DOX + DNT for 3 weeks. Echocardiography and blood troponin I levels were used to measure myocardial injury. Hearts and tumors were evaluated for histopathological alterations. Blood glutathione was assessed as a measure of oxidative stress. The results showed that DNT improved DOX-induced alterations in the echocardiographic parameters by 50%. Histopathologic analysis of hearts showed reduced DOX induced cardiotoxicity in the group treated with DOX + DNT as shown by reduced interstitial edema, cytoplasmic vacuolization, and myofibrillar disruption, compared with DOX-only–treated hearts. Rats treated with DNT lost less body weight, had higher blood GSH levels and lower troponin I levels than DOX-treated rats. These data indicate that DNT is able to provide protection against DOX cardiotoxicity without reducing its antitumor activity. Further studies are needed to determine the optimal dosing of DNT and DOX in a tumor-bearing host.

Protective effects of dantrolene and methylprednisolone against spinal cord injury-induced early oxidative damage in rabbit bladder: A comparative experimental study.

Spinal cord injury (SCI) may cause dysfunction in the bladder and many distal organs due to systemic inflammatory response and oxidative stress-related injury. We investigated the preventive effects of dantrolene (DNT) and methylprednisolone (MP) on stress-induced tissue damage in rabbit bladder with SCI. A total of 35 rabbits were included in this study and they were divided into 5 groups: group 1 - control, group 2 - SCI only, group 3 - SCI and DNT, group 4 - SCI and MP, and group 5 - SCI and DNT+MP. Twenty-four hours after SCI, the bladders of these rabbits were removed and the histopathologic changes in the bladder were examined under a light microscope. Additionally, malondialdehyde (MDA), glutathione (GSH), and nitric oxide (NO) levels were evaluated as antioxidant agents both in bladder tissue and in blood. Compared to the control group, there was an increase in edema and congestion in all groups. The least amount of edema was observed in the group receiving DNT and the least amount of congestion was observed in the group receiving combined treatment (group 5). No superiority was found between the drug-receiving groups in terms of reducing MDA level in blood and tissue after SCI. The most successful group was the group receiving combined drug therapy in terms of increasing the blood GSH level, which was significantly decreased after SCI. After SCI, blood NO level increased significantly in all groups. Nitric oxide levels in the bladder tissue significantly decreased in the groups receiving DNT and combination therapy and fell in the control group. Dantrolene and MP may have potential benefits against oxidative damage in the bladder after SCIs because of their anti-inflammatory and antioxidant effects. In particular, the combined use of DNT and MP at different doses can be considered a treatment strategy.

Pharmacological Treatment of Malignant Hyperthermia: Update 2019

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle presenting as a hypermetabolic response to potent volatile anesthetics such as sevoflurane, desflurane, isoflurane and the depolarizing muscle relaxant succinylcholine. Following introduction of the hydantoin derivative dantrolene, the previously high mortality of fulminant MH episodes could be reduced from > 80% to less than 10%. For treatment of MH an initial intravenous bolus of 2.5 mg/kg dantrolene based on the actual body weight should be applied. If symptoms are not improving after the initial dose, up to 10 mg/kg dantrolene can be necessary within the first 24 h. In facilities where MH triggering anesthetics and depolarizing muscle relaxants are administered, at least 36 - 48 vials of dantrolene 20 mg should be stocked according to the recent German S1 guideline on MH. If none of these agents are ever used in the facility, the stockage of dantrolene is dispensable. Since dantrolene is not easily dissoluble, preparation requires time and manpower. Due to its pharmacological characteristics, ryanodex, a modern nanocrystalline dantrolene sodium suspension, might be a promising alternative in the treatment of MH.