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.

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.

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.

A dual-targeted drug inhibits cardiac ryanodine receptor Ca2+ leak but activates SERCA2a Ca2+ uptake.

In the heart, genetic or acquired mishandling of diastolic [Ca2+] by ryanodine receptor type 2 (RyR2) overactivity correlates with risks of arrhythmia and sudden cardiac death. Strategies to avoid these risks include decrease of Ca2+ release by drugs modulating RyR2 activity or increase in Ca2+ uptake by drugs modulating SR Ca2+ ATPase (SERCA2a) activity. Here, we combine these strategies by developing experimental compounds that act simultaneously on both processes. Our screening efforts identified the new 1,4-benzothiazepine derivative GM1869 as a promising compound. Consequently, we comparatively studied the effects of the known RyR2 modulators Dantrolene and S36 together with GM1869 on RyR2 and SERCA2a activity in cardiomyocytes from wild type and arrhythmia-susceptible RyR2R2474S/+ mice by confocal live-cell imaging. All drugs reduced RyR2-mediated Ca2+ spark frequency but only GM1869 accelerated SERCA2a-mediated decay of Ca2+ transients in murine and human cardiomyocytes. Our data indicate that S36 and GM1869 are more suitable than dantrolene to directly modulate RyR2 activity, especially in RyR2R2474S/+ mice. Remarkably, GM1869 may represent a new dual-acting lead compound for maintenance of diastolic [Ca2+].

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.

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.

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.

RyR2-targeting therapy prevents left ventricular remodeling and ventricular tachycardia in post-infarction heart failure.

BACKGROUND: Dantrolene binds to the Leu601-Cys620 region of the N-terminal domain of cardiac ryanodine receptor (RyR2), which corresponds to the Leu590-Cys609 region of the skeletal ryanodine receptor, and suppresses diastolic Ca2+ leakage through RyR2. OBJECTIVE: We investigated whether the chronic administration of dantrolene prevented left ventricular (LV) remodeling and ventricular tachycardia (VT) after myocardial infarction (MI) by the same mechanism with the mutation V3599K of RyR2, which indicated that the inhibition of diastolic Ca2+ leakage occurred by enhancing the binding affinity of calmodulin (CaM) to RyR2. METHODS AND RESULTS: A left anterior descending coronary artery ligation MI model was developed in mice. Wild-type (WT) were divided into four groups: sham-operated mice (WT-Sham), sham-operated mice treated with dantrolene (WT-Sham-DAN), MI mice (WT-MI), and MI mice treated with dantrolene (WT-MI-DAN). Homozygous V3599K RyR2 knock-in (KI) mice were divided into two groups: sham-operated mice (KI-Sham) and MI mice (KI-MI). The mice were followed for 12 weeks. Survival was significantly higher in the WT-MI-DAN (73%) and KI-MI groups (70%) than the WT-MI group (40%). Echocardiography, pathological tissue, and epinephrine-induced VT studies showed that LV remodeling and VT were prevented in the WT-MI-DAN and KI-MI groups compared to the WT-MI group. An increase in diastolic Ca2+ spark frequency and a decrease in the binding affinity of CaM to the RyR2 were observed at 12 weeks after MI in the WT-MI group, although significant improvements in these values were observed in the WT-MI-DAN and KI-MI groups. CONCLUSIONS: Pharmacological or genetic stabilization of RyR2 tetrameric structure improves survival after MI by suppressing LV remodeling and proarrhythmia.

Dantrolene Significantly Improves Cerebral Blood Flow in a Rat Model of Hemorrhagic Vasospasms.

INTRODUCTION: A cerebral vasospasm (CVSP) is a potent vasoconstriction of the cerebral vasculature and the primary cause of morbidity and mortality following a subarachnoid hemorrhage. The middle cerebral artery (MCA) is commonly affected by CVSPs. Concomitant administration of dantrolene and nimodipine synergistically reduces vasospasms in aortic rings from Sprague Dawley rats. To determine if the effects observed in the systemic vasculature extend to the cerebral circulation, we investigated the effect of intravenous administration of dantrolene (2.5 mg/kg) and nimodipine (1 mg/kg and 2 mg/kg) on MCA blood flow velocity (BFV) 7 days after the induction of CVSPs. METHODS: Vasospasms were induced by bathing the left common carotid artery with autologous whole blood. Age-matched sham rats were used as controls. BFV, mean arterial pressure (MAP), and heart rate (HR) were measured with a PeriFlux 5000 Laser Doppler System, and a CODA non-invasive blood pressure system, before and after administering the drugs. Morphometric evaluations were also performed to assess vascular alterations. RESULTS: BFV was reduced by 37% with dantrolene alone (n = 6, p ≤ 0.05) and by 27% with 2 mg/kg nimodipine (n = 6, p < 0.05), while it was not affected by 1 mg/kg nimodipine. The combination of 1 mg/kg nimodipine with dantrolene, however, decreased BFV by 35% (from 435.70 ± 21.53 to 284.30 ± 23.13 perfusion units, n = 7, p ≤ 0.05). A similar reduction (31%) was obtained with dantrolene and 2 mg/kg nimodipine (from 536.00 ± 32.61 to 367.80 ± 40.93 perfusion units, n = 6, p ≤ 0.05). Neither MAP nor HR was affected by dantrolene or nimodipine alone. The combination of dantrolene with 2 mg/kg nimodipine, however, decreased MAP and increased HR. Furthermore, 7 days after the induction of vasospasms, lumen area of the left common carotid artery decreased, whereas media thickness and the wall-to-lumen ratio increased when compared to contralateral controls. The latter finding suggests that vascular remodeling was present at this stage. CONCLUSION: Altogether, our results indicate that 2.5 mg/kg dantrolene significantly reduces BFV in the MCA without altering systemic hemodynamic parameters to a similar extent than the highest dose of nimodipine or the combination of dantrolene and the lowest dose of nimodipine. Therefore, dantrolene may provide a promising alternative to lower the risk, or partially revert, CVSP.

Dantrolene and ryanodine receptors in COVID-19: The daunting task and neglected warden.

Dantrolene (DTN) is a ryanodine receptor (RyR) antagonist that inhibits Ca2+ release from stores in the sarcoplasmic reticulum. DTN is mainly used in the management of malignant hyperthermia. RyRs are highly expressed in immune cells and are involved in different viral infections, including severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), because Ca2+ is necessary for viral replication, maturation and release. DTN can inhibit the proliferation of SARS-CoV-2, indicating its potential role in reducing entry and pathogenesis of SARS-CoV-2. DTN may increase clearance of SARS-CoV-2 and promote coronavirus disease 2019 (COVID-19) recovery by shortening the period of infection. DTN inhibits N-methyl-D-aspartate (NMDA) mediated platelets aggregations and thrombosis. Therefore, DTN may inhibit thrombosis and coagulopathy in COVID-19 through suppression of platelet NMDA receptors. Moreover, DTN has a neuroprotective effect against SARS-CoV-2 infection-induced brain injury through modulation of NMDA receptors, which are involved in excitotoxicity, neuronal injury and the development of neuropsychiatric disorders. In conclusion, DTN by inhibiting RyRs may attenuate inflammatory disorders in SARS-CoV-2 infection and associated cardio-pulmonary complications. Therefore, DNT could be a promising drug therapy against COVID-19. Preclinical and clinical studies are warranted in this regards.

Design, synthesis, and evaluation of hydrazones as dual inhibitors of ryanodine receptors and acetylcholinesterases for Alzheimer's disease.

Alzheimer's disease (AD) implicates neuronal loss, plaque and neurofibrillary tangle formation, and disturbed neuronal Ca2+ homeostasis, which leads to severe dementia, memory loss, as well as thinking and behavioral perturbations that could ultimately lead to death. Calcium dysregulation and low acetylcholine levels are two main mechanisms implicated in Alzheimer's disease progression. Simultaneous inhibition of calcium oscillations (store overload-induced Ca2+ release [SOICR]) and acetylcholinesterase (AChE) by a single molecule may bring a new breath of hope for AD treatment. Here, we described some dantrolene derivatives as dual inhibitors of the ryanodine receptor and AChE. Two series of acylhydrazone/sulfonylhydrazone derivatives with aromaticgroup were designed and synthesized. In this study, the target compounds were evaluated for their ability to inhibit SOICR and AChE in vitro, using dantrolene and donepezil as positive controls. Compound 22a exhibited excellent and balanced inhibitory potency against SOICR (inhibition (%) = 90.1, IC50 = 0.162 µM) and AChE (inhibition (%) = 93.5, IC50 = 0.372 µM). Docking simulations showed that several preferred compounds could bind to the active sites of both the proteins, further validating the rationality of the design strategy. Potential therapeutic effects in AD were evaluated using the Barnes maze and Morris water maze tests, which demonstrated that compound 22a significantly improved memory and cognitive behavior in AD model mice. Moreover, it was also found that compound 22a could enhance synaptic strength by measuring hippocampal long-term potentiation (LTP) in brain slices. These results suggested that the introduction of a sulfonyl-hydrazone scaffold and aromatic substitution to dantrolene derivatives provided a useful template for the development of potential chemical entities against AD.

Dantrolene sodium fails to reverse robust brain hyperthermia induced by MDMA and methamphetamine in rats.

RATIONALE: Hyperthermia induced by psychomotor stimulants may cause leakage of the blood-brain barrier, vasogenic edema, and lethality in extreme cases. Current treatments such as whole-body cooling are only symptomatic and a clear need to develop pharmacological interventions exists. Dantrolene sodium, a peripheral muscle relaxant used in the treatment of malignant hyperthermia, has been proposed as potentially effective to treat MDMA-hyperthermia in emergency rooms. However, debate around its efficacy for this indication persists. OBJECTIVES: To investigate dantrolene as a treatment for illicit hyperthermia induced by psychomotor stimulant drugs, we examined how Ryanodex®, a concentrated formulation of dantrolene sodium produced by Eagle Pharmaceuticals, influences 3,4-methylenedioxymethamphetamine (MDMA)- and methamphetamine (METH)-induced hyperthermia in awake freely moving rats. We injected rats with moderate doses of MDMA (9 mg/kg) and METH (9 mg/kg) and administered Ryanodex® intravenously (6 mg/kg) after the development of robust hyperthermia (>2.5 °C) mimicking clinical acute intoxication. We conducted simultaneous temperature recordings in the brain, temporal muscle, and skin to determine the basic mechanisms underlying temperature responses. To assess the efficacy of dantrolene in attenuating severe hyperthermia, we administered MDMA to rats maintained in a warm ambient environment (29 °C), conditions which produce robust brain and body hyperthermia (>40 °C) and lethality. RESULTS: Dantrolene failed to attenuate MDMA- and METH-induced hyperthermia, though locomotor activity was significantly reduced. All animals maintained at warm ambient temperatures that received dantrolene during severe drug-induced hyperthermia died within or soon after the recording session. CONCLUSIONS: Our results suggest that dantrolene sodium formulations are not mechanistically suited to treat MDMA- and METH-induced hyperthermia.

Stabilizing cardiac ryanodine receptor with dantrolene treatment prevents left ventricular remodeling in pressure-overloaded heart failure mice.

Dantrolene (DAN) directly binds to cardiac ryanodine receptor 2 (RyR2) through Leu601-Cys620 in the N-terminal domain and subsequently inhibits diastolic Ca2+ leakage through RyR2. We previously reported that therapy using RyR2 V3599K mutation, which inhibits diastolic Ca2+ leakage by enhancing calmodulin (CaM) binding ability to RyR2, prevents left ventricular (LV) remodeling in transverse aortic constriction (TAC) heart failure. Here, we examined whether chronic administration of DAN prevents LV remodeling in TAC heart failure via the same mechanism as genetic therapy. A pressure-overloaded hypertrophy mouse model was developed using TAC. Wild-type (WT) mice were divided into three groups: sham-operated mice (Sham group), TAC mice (TAC group), and TAC mice treated with DAN (TAC-DAN group, 20 mg/kg/day, i.p.). They were then followed up for 8 weeks. The survival rate was higher in the TAC-DAN group (83%) than in the TAC group (49%), and serial echocardiography studies and pathological tissue analysis showed that LV remodeling was significantly prevented in the TAC-DAN group compared to the TAC group. An increase in the diastolic Ca2+ spark frequency and a decrease in the binding affinity of CaM to RyR2 were observed at 8 weeks in the TAC group but not in the TAC-DAN group. Stabilization of RyR2 with DAN prevented LV remodeling and improved survival after TAC by enhancing CaM binding to RyR2 and inhibiting RyR2-mediated diastolic Ca2+ leakage.

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.

Dantrolene, a stabilizer of the ryanodine receptor, prevents collagen-induced arthritis.

Dantrolene inhibits Ca2+ leakage from destabilized ryanodine receptors and therefore may serve as a therapeutic agent against endoplasmic reticulum stress-associated diseases. However, its effectiveness in treating autoimmune diseases remains unclear. Here, we investigated the effect of dantrolene on collagen-induced arthritis (CIA) in mice. Oral administration of dantrolene resulted in significantly lower arthritic scores in both male and female CIA mice than in the control mice. Micro-computed tomographic and histological analyses showed that dantrolene suppressed bone and chondral destruction. The serum levels of anti-type II collagen (CII) IgG were positively correlated with the arthritic scores (r = 0.704, p < 0.01). In addition, the serum levels of anti-CII IgG were significantly lower in the dantrolene group than in the control group (p < 0.05). These results demonstrate that oral administration of dantrolene to CIA mice inhibits the production of serum anti-CII IgG and consequently prevents arthritis. Therefore, dantrolene may be a potential anti-rheumatic drug.