Ryanodine Receptor Blocker Research Articles

Dantrolene paradoxically exacerbates short-term brain glucose hypometabolism, hippocampal damage and neuroinflammation induced by status epilepticus in the rat lithium-pilocarpine model.

Status epilepticus (SE) is a neurologic emergency characterized by prolonged or rapidly recurring seizures. Increased intracellular calcium concentration ([Ca2+]i) occurring after SE is a key mediator of excitotoxicity that contributes to the brain damage associated with the development of epilepsy. Accumulated evidence indicates that dantrolene, a ryanodine receptor (RyR) blocker may have protective effects against the SE-induced damage. We evaluated whether dantrolene (10mg/kg, i.p.) administered twice, 5min and 24h after the lithium-pilocarpine-induced SE in rats, had neuroprotective effects. Dantrolene by itself had no effects on control rats. However, it exacerbated the signs of damage in rats that underwent SE, increasing brain glucose hypometabolism as measured by PET neuroimaging 3 days after SE. Likewise, the neurohistochemical studies revealed that dantrolene aggravated signs of hippocampal neurodegeneration, neuronal death and microglia-induced neuroinflammation. Besides, the damaging effects were reflected by severe body weight loss. Overall, our results point towards a deleterious effect of dantrolene in the lithium-pilocarpine-induced SE model. Nonetheless, our results are in opposition to the reported neuroprotective effects of dantrolene. Whether the mechanisms underlying [Ca2+]i increase might significantly differ depending on the particularities of the model of epilepsy used and general experimental conditions need further studies. Besides, it is yet to be determined which isoform of RyRs significantly contributes to Ca2+-induced excitotoxicity in the lithium-pilocarpine SE rat model.

Atrial cardiomyopathy resulting from loss of plakophilin-2 expression: Response to adrenergic stimulation and implications for the exercise response.

Atrial arrhythmias occur in 20-40% of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) and are associated with an increased risk of sustained ventricular arrhythmias and inappropriate implantable cardioverter-defibrillator shocks. The pathophysiology of atrial arrhythmias in ARVC remains unclear. Most cases of gene-positive ARVC are linked to pathogenic variants in the desmosomal gene plakophilin-2 (PKP2). Here, we test the hypothesis that loss of PKP2 expression leads to pro-arrhythmic changes in atrial cardiomyocytes. Atrial cells/tissue were obtained from a cardiac-specific, tamoxifen-activated model of PKP2 deficiency (PKP2cKO). By contrast to PKP2cKO ventricular myocytes, PKP2cKO atrial cardiomyocytes presented no significant differences in intracellular calcium (Ca2+ i) transient dynamics, sarcoplasmic reticulum load or action potential morphology. PKP2cKO atrial cardiomyocytes showed elevated reactive oxygen species levels, increased frequency and amplitude of Ca2+ sparks, and increased diastolic [Ca2+]i compared to control; the latter two parameters were further increased by isoproterenol exposure and reversed by exposure to ryanodine receptor blocker dantrolene. We speculate that these isoproterenol-dependent effects may impact on the exercise-related atrial arrhythmia risk in ARVC patients. Despite absence of changes in Ca2+ i transient dynamics, PKP2cKO atrial cardiomyocytes showed enhanced sarcomere shortening and impaired sarcomere relaxation. Orthogonal transcriptomic analysis of human(GTEx) and PKP2cKO atrial tissue led to identification of 41 transcripts depending on PKP2 expression. Biochemical follow-up confirmed reduced abundance of sarcomeric protein myosin binding protein C, potentially playing a role in cellular shortening and relaxation changes observed. Our findings provide novel insights into the role of PKP2 in atrial myocardium with potential implications to therapeutic management of atrial fibrillation in patients with PKP2-related ARVC. KEY POINTS: Atrial arrhythmias occur in a large group of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), a cardiac disease mostly caused by pathogenic variants in the desmosomal gene plakophilin-2 (PKP2). Exercise is considered to be an independent risk factor for arrhythmias consequent to PKP2 deficiency. Weshow that loss of PKP2 expression affects cellular calcium handling and electrophysiology differently in left atrial vs. ventricular myocardium and causes extensive atrial fibrosis. PKP2-deficient atrial cardiomyocytes present increased spontaneous sarcoplasmic reticulum calcium release events, furtherenhancedby isoproterenol exposure and reversiblebya ryanodine receptor blocker(dantrolene). In addition, PKP2-deficient atrial myocytes exhibit impaired relaxation and enhanced sarcomere shortening, most probably related to reduced abundance of myosin binding protein C. We speculate that cellular effects reported upon isoproterenol impact on the exercise-related atrial arrhythmia risk in ARVC patients. We further propose that therapeutic approaches aimed at mitigating ventricular damage may be effective to treat the atrial disease in ARVC.

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.

Critical Link between Calcium Regional Heterogeneity and Atrial Fibrillation Susceptibility in Sheep Left Atria.

Atrial fibrillation is the most sustained form of arrhythmia in the human population that leads to important electrophysiological and structural cardiac remodeling as it progresses into a chronic form. Calcium is an established key player of cellular electrophysiology in the heart, yet to date, there is no information that maps calcium signaling across the left atrium. The aim of this study is to determine whether calcium signaling is homogenous throughout the different regions of the left atrium. This work tests the hypothesis that differences across the healthy left atrium contribute to a unique, region-dependent calcium cycling and participates in the pro-arrhythmic activity during atrial fibrillation. An animal model relevant to human cardiac function (the sheep) was used to characterize both the electrical activity and the calcium signaling of three distinct left atrium regions (appendage, free wall and pulmonary veins) in control conditions and after acetylcholine perfusion (5 μM) to induce acute atrial fibrillation. High-resolution dual calcium-voltage optical mapping on the left atria of sheep was performed to explore the spatiotemporal dynamics of calcium signaling in relation to electrophysiological properties. Action potential duration (at 80% repolarization) was not significantly different in the three regions of interest for the three pacing sites. In contrast, the time to 50% calcium transient decay was significantly different depending on the region paced and recorded. Acetylcholine perfusion and burst pacing-induced atrial fibrillation when pulmonary veins and appendage regions were paced but not when the free wall region was. Dantrolene (a ryanodine receptor blocker) did not reduce atrial fibrillation susceptibility. These data provide the first evidence of heterogenous calcium signaling across the healthy left atrium. Such basal regional differences may be exacerbated during the progression of atrial fibrillation and thus play a crucial role in focal arrhythmia initiation without ryanodine receptor gating modification.

Dantrolene reduces platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell proliferation and neointimal formation following vascular injury in mice

Dantrolene is a ryanodine receptor blocker that is used clinically for treatment of malignant hyperthermia. This study was conducted using murine aortic vascular smooth muscle cells (MOVAS) and a mouse arterial injury model to investigate the inhibitory effect of dantrolene on smooth muscle cell proliferation and migration.We investigated whether dantrolene suppressed platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell proliferation and migration in vitro. The effect of dantrolene on smooth muscle phenotype was evaluated using immunostaining. In addition, smooth muscle cell proliferation and phenotype switching were tested by applying dantrolene around blood vessels using a mouse arterial injury model. Dantrolene inhibited PDGF-induced cell proliferation and migration of MOVAS. Dantrolene also inhibited the switch from contractile to synthetic phenotype both in vitro and in vivo.Dantrolene is effective at inhibiting vascular smooth muscle cell proliferation, migration, and neointimal formation following arterial injury in mice.

Dantrolene Significantly Improves Cerebral Blood Flow In Diabetic Rats With Cerebral Vasospasms

Cerebral vasospasm (CVSP) is a potent vasoconstriction of the cerebral vasculature, and the primary cause of morbidity and mortality following a hemorrhagic stroke. Diabetics have a greater predisposition for developing CVSPs than nondiabetics, even when under glycemic control. The middle cerebral artery (MCA) is one of the most common vessels affected by CVSPs. Experimental evidence shows that concomitant administration of dantrolene (a ryanodine receptor blocker) and nimodipine (a Ca+2 channel blocker) synergistically reduces vasospasms in aortic rings from streptozotocin (STZ)‐induced diabetic rats. To determine if the effects observed in the systemic vasculature extend to the cerebral circulation, we investigated the effects of intravenous administration of dantrolene (2.5 mg/kg) and nimodipine (1 mg/kg and 2 mg/kg) on MCA blood flow velocity (BFV) seven days after the induction of CVSP in diabetic rats. Vasospasms were induced by bathing the external surface of the left common carotid artery with autologous whole blood. BFV was measured with a PeriFlux 5000 Laser Doppler System before, and sixty minutes after administering the drugs. Morphometric evaluations of the common carotid arteries were also performed to assess vascular alterations secondary to the induction of vasospasms. BFV was reduced by 30% with dantrolene alone (from 495.90 ±21.38 to 345.50±31.46 perfusion units, n=6, p≤0.05), however, nimodipine alone (1mg/kg or 2 mg/kg) did not affect this variable. The combination of dantrolene with 1 mg/kg nimodipine decreased BFV by 22% (from 518.90±31.49 to 405.50±23.84 perfusion units, n=7, p≤0.05), while a 32% reduction was obtained with the combination of dantrolene and 2 mg/kg nimodipine (from 504.60±15.38 to 344.90±36.98 perfusion units, n=6, p≤0.05). Furthermore, seven days after the induction of vasospasms, the 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 findings suggest that a period of seven days after the induction of vasospasms, was sufficient to cause vascular remodeling. Thus, our results indicate that, in diabetic rats, 2.5 mg/kg dantrolene alone was as effective as the combination of dantrolene and the highest dose of nimodipine in reducing BFV in the MCA. Therefore, dantrolene may provide a promising alternative therapy for the treatment of CVSP in the diabetic population.

Exercise Causes Arrhythmogenic Remodeling of Intracellular Calcium Dynamics in Plakophilin-2-Deficient Hearts.

Exercise training, and catecholaminergic stimulation, increase the incidence of arrhythmic events in patients affected with arrhythmogenic right ventricular cardiomyopathy correlated with plakophilin-2 (PKP2) mutations. Separate data show that reduced abundance of PKP2 leads to dysregulation of intracellular Ca2+ (Ca2+i) homeostasis. Here, we study the relation between excercise, catecholaminergic stimulation, Ca2+i homeostasis, and arrhythmogenesis in PKP2-deficient murine hearts. Experiments were performed in myocytes from a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout murine line (PKP2cKO). For training, mice underwent 75 minutes of treadmill running once per day, 5 days each week for 6 weeks. We used multiple approaches including imaging, high-resolution mass spectrometry, electrocardiography, and pharmacological challenges to study the functional properties of cells/hearts in vitro and in vivo. In myocytes from PKP2cKO animals, training increased sarcoplasmic reticulum Ca2+ load, increased the frequency and amplitude of spontaneous ryanodine receptor (ryanodine receptor 2)-mediated Ca2+ release events (sparks), and changed the time course of sarcomeric shortening. Phosphoproteomics analysis revealed that training led to hyperphosphorylation of phospholamban in residues 16 and 17, suggesting a catecholaminergic component. Isoproterenol-induced increase in Ca2+i transient amplitude showed a differential response to β-adrenergic blockade that depended on the purported ability of the blockers to reach intracellular receptors. Additional experiments showed significant reduction of isoproterenol-induced Ca2+i sparks and ventricular arrhythmias in PKP2cKO hearts exposed to an experimental blocker of ryanodine receptor 2 channels. Exercise disproportionately affects Ca2+i homeostasis in PKP2-deficient hearts in a manner facilitated by stimulation of intracellular β-adrenergic receptors and hyperphosphorylation of phospholamban. These cellular changes create a proarrhythmogenic state that can be mitigated by ryanodine receptor 2 blockade. Our data unveil an arrhythmogenic mechanism for exercise-induced or catecholaminergic life-threatening arrhythmias in the setting of PKP2 deficit. We suggest that membrane-permeable β-blockers are potentially more efficient for patients with arrhythmogenic right ventricular cardiomyopathy, highlight the potential for ryanodine receptor 2 channel blockers as treatment for the control of heart rhythm in the population at risk, and propose that PKP2-dependent and phospholamban-dependent arrhythmogenic right ventricular cardiomyopathy-related arrhythmias have a common mechanism.

Effects of HOCl oxidation on excitation–contraction coupling: Implications for the pathophysiology of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a fatal X-linked genetic disease characterized by progressive loss of skeletal muscle. The mechanisms underlying the DMD pathology likely involve the complex interaction between reactive oxygen species (ROS) impaired Ca2+ handling and chronic inflammation, characterized by the presence of immune cells such as neutrophils. Hypochlorous acid (HOCl) is a highly reactive form of ROS produced endogenously via the actions of myeloperoxidase, an enzyme secreted by neutrophils. Myeloperoxidase activity is significantly elevated in dystrophic muscle. This study aimed to determine the effect of HOCl exposure on excitation-contraction coupling and its potential contribution to the dystrophic pathology. Isolated extensor digitorum longus (EDL) muscles and single fibers from C57 (wild type) and mdx (dystrophic) mice were used to investigate the effects of HOCl on whole muscle function, intracellular Ca2+ handling, and myofilament force production. HOCl exposure significantly decreased maximum specific force in isolated EDL muscles by 26% and 49%, respectively, in C57 and mdx mice (P < 0.0001). In single interosseous fibers, HOCl exposure significantly increased resting intracellular Ca2+ concentration by ∼17-19% (P < 0.05) and decreased the amplitude of electrically induced Ca2+ transients by ∼45% and 50%, respectively, in C57 and mdx fibers (C57, P < 0.05; mdx, P < 0.01). These effects of HOCl on resting Ca2+ could be blocked via application of tetracaine (ryanodine receptor blocker) or Gd3+ (stretch-activated channel blocker; C57, P < 0.01; mdx, P < 0.01 for both). The effect of HOCl on Ca2+ transient amplitude was significantly reduced by Gd3+ (C57, P < 0.05; mdx, P < 0.01). In chemically skinned EDL fibers, HOCl exposure decreased maximum Ca2+-activated force by ∼40% in both C57 and mdx fibers (P < 0.001). These results indicate that HOCl potently affects excitation-contraction coupling via impaired Ca2+ handling and myofilament force production. Hence, HOCl potentially links the chronic inflammation, oxidative stress, and impaired Ca2+ handling that underlies the dystrophic pathology.

Calcium dysregulation and compensation in cortical pyramidal neurons of the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is a fatal, hereditary neurodegenerative disorder that predominantly affects striatal medium-sized spiny neurons and cortical pyramidal neurons (CPNs). It has been proposed that perturbations in Ca2+ homeostasis could play a role in CPN alterations. To test this hypothesis, we used the R6/2 mouse model of juvenile HD at different stages of disease progression; presymptomatic, early symptomatic, and late symptomatic. We combined whole-cell patch-clamp recordings of layer 2/3 CPNs with two-photon laser scanning microscopy to image somatic and dendritic Ca2+ transients associated with evoked action potentials (APs). We found that the amplitude of AP-induced Ca2+ transients recorded at the somata of CPNs was significantly reduced in presymptomatic and late symptomatic R6/2 mice compared with wild-type (WT) littermates. However, reduced amplitudes were compensated by increases in decay times, so that Ca2+ transient areas were similar between genotypes. AP-induced Ca2+ transients in CPN proximal dendrites were variable and differences did not reach statistical significance, except for reduced areas in the late symptomatic group. In late symptomatic mice, a specific store-operated Ca2+ channel antagonist, EVP4593, reduced somatic Ca2+ transient amplitude similarly in WT and R6/2 CPNs. In contrast, dantrolene, a ryanodine receptor (RyR) antagonist, and nifedipine, an L-type Ca2+ channel blocker, significantly reduced both somatic Ca2+ transient amplitude and area in R6/2 but not WT CPNs. These findings demonstrate that perturbations of Ca2+ homeostasis and compensation occur in CPNs before and after the onset of overt symptoms, and suggest RyRs and L-type Ca2+ channels as potential targets for therapeutic intervention.NEW & NOTEWORTHY We used two-photon microscopy to examine calcium influx induced by action potentials in cortical pyramidal neurons from a mouse model of Huntington's disease (HD), the R6/2. The amplitude of somatic calcium transients was reduced in R6/2 mice compared with controls. This reduction was compensated by increased decay times, which could lead to reduced calcium buffering capacity. L-type calcium channel and ryanodine receptor blockers reduced calcium transient area in HD neurons, suggesting new therapeutic avenues.

Role of Serosal TRPV4-Constituted SOCE Mechanism in Secretagogues-Stimulated Intestinal Epithelial Anion Secretion.

As little is known about the role of calcium (Ca2+) signaling mediating the small intestinal epithelial anion secretion, we aimed to study its regulatory role in secretagogue-stimulated duodenal anion secretion and the underlying molecular mechanisms. Therefore, intestinal anion secretion from native mouse duodenal epithelia was examined with Ussing chambers to monitor PGE2-, 5-HT-, and CCh-induced short-circuit currents (I sc). PGE2 (10 μM) and 5-HT (10 μM) induced mouse duodenal I sc, markedly attenuated by serosal Ca2+-free solution and selective blockers of store-operated Ca2+ channels on the serosal side of the duodenum. Furthermore, PGE2- and 5-HT-induced duodenal I sc was also inhibited by ER Ca2+ chelator TPEN. However, dantrolene, a selective blocker of ryanodine receptors, inhibited PGE2-induced duodenal I sc, while LiCl, an inhibitor of IP3 production, inhibited 5-HT-induced I sc. Moreover, duodenal I sc response to the serosal applications of both PGE2 and 5-HT was significantly attenuated in transient receptor potential vanilloid 4 (TRPV4) knockout mice. Finally, mucosal application of carbachol (100 μM) also induced duodenal I sc via selective activation of muscarinic receptors, which was significantly inhibited in serosal Ca2+-free solution but neither in mucosal Ca2+-free solution nor by nifedipine. Therefore, the serosal TRPV4-constituted SOCE mechanism is likely universal for the most common and important secretagogues-induced and Ca2+-dependent intestinal anion secretion. These findings will enhance our knowledge about gastrointestinal (G.I.) epithelial physiology and the associated G.I. diseases, such as diarrhea and constipation.

The combination of dantrolene and nimodipine effectively reduces 5-HT-induced vasospasms in diabetic rats

Diabetics have a higher risk of developing cerebral vasospasms (CVSP) after subarachnoid hemorrhagic stroke than non-diabetics. Serotonin (5-HT) is one of the key vasoconstrictors released in the hemorrhagic blood and an important contributor to the etiology of CVSP. The combination of the ryanodine receptor blocker dantrolene and the Ca2+ channel blocker nimodipine significantly reduces phenylephrine (PHE)-induced vascular contraction in both diabetic and nondiabetic rats, but the effectiveness of this drug combination in reducing 5-HT-induced contraction is unknown. Dose–response curves for the 5-HT-induced contraction (from 0.1 nM to 100 µM) were performed on aortic rings from diabetic and non-diabetic rats after a 30-min incubation period with dantrolene, nimodipine, and both drugs in combination. In diabetic rats, 10 μM of dantrolene alone failed to reduce 5-HT-induced maximal contraction (Emax), but 50 μM reduced this parameter by 34% (n = 7, p < 0.05). In non-diabetic rats, by contrast, dantrolene did not modify the vascular response to 5-HT. 50 nM of nimodipine alone, however, reduced this parameter by 57% in diabetic rats (n = 10, p < 0.05), and by 34% in non-diabetic rats (n = 10, p < 0.05). In addition, concomitant administration of dantrolene and nimodipine reduced vascular reactivity to a similar extent in both diabetic (~ 60% reduction, n = 10, p < 0.05) and non-diabetic rats (~ 70% reduction, n = 10, p < 0.05). Moreover, the combination of nimodipine with the higher concentration of dantrolene significantly increased the EC50 values for the 5-HT-induced contraction curves in both diabetics (from 10.31 ± 1.17 µM to 19.26 ± 2.82; n = 10, p < 0.05) and non-diabetic rats (5.93 ± 0.54 µM to 15.80 ± 3.24; n = 10, p < 0.05). These results suggest that simultaneous administration of dantrolene and nimodipine has a synergistic effect in reducing 5-HT-induced vascular contraction under both diabetic and non-diabetic conditions. If our findings with rats are applicable to humans, concomitant administration of these drugs may represent a promising alternative for the management of CVSP in both diabetics and non-diabetics.

Concomitant administration of dantrolene and nimodipine significantly improves cerebral blood flow perfusion in a rat model of cerebral‐induced vasospasm

A cerebral vasospasm (CVSP) is a potent vasoconstriction of the cerebral vasculature, and the primary cause of morbidity and mortality following a hemorrhagic stroke. The middle cerebral artery (MCA) is one of the most common vessels affected by CVSPs. Experimental evidence shows that concomitant administration of dantrolene (a ryanodine receptor blocker) and nimodipine (a Ca+2 channel blocker) has synergistic effects in reducing 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). Using a PeriFlux 5000 Laser Doppler System, we measured BFV before and after administration of the drugs 7 days after induction of cerebral vasospasms in Sprague Dawley rats. A reduction in blood flow velocity indicates that vascular reactivity decreases and blood perfusion increases. Mean blood pressure (MBP) and heart rate (HR) were also measured. Individual administration of these drugs resulted in similar reductions in BFV (34% with dantrolene, n=6, p<0.05; 32% with nimodipine 2 mg/kg, n=6, p<0.05). Moreover, dantrolene combined with 1mg/kg of nimodipine decreased BFW by 48% (from 435.70 ± 21.50 to 284.30 ± 26.10 perfusion units, n=6, p<0.05). Similarly, dantrolene combined with 2 mg/kg of nimodipine, reduced BFW by 54% (from 536.00 ± 32.60 to 367.80 ± 40.90 perfusion units, n=6, p<0.05). Although when combined with 2 mg/kg of nimodipine, dantrolene significantly increases HR, the combination of dantrolene and 1mg/kg of nimodipine, did not alter MBP or HR. Thus, our results indicate that concurrent administration of 2.5 mg/kg dantrolene and 1 mg/kg nimodipine significantly reduce BFV in the MCA without altering systemic hemodynamic parameters. By reducing vascular reactivity synergistically, these drugs may improve cerebral blood perfusion. Therefore, adding dantrolene to current standard pharmacological therapies with calcium channel blockers (CCB) may allow a dosage reduction of CCB and minimize the systemic secondary effects of these drugs. Furthermore, if our findings with rats are applicable to humans, the combined use of dantrolene and nimodipine at optimal doses may be effective in reducing CVSPs.

Optical mapping of Ca transient and voltage in left atria uncover critical links between Ca regional heterogeneity and atrial fibrillation susceptibility

Atrial fibrillation (AF) is the most sustained form of arrhythmia in human population. Although electrophysiological and structural abnormalities are known to be involved in AF development, it has not been established whether cardiac remodelling, including calcium (Ca) signalling dysregulation, is homogenous throughout the different regions of the left atrium (LA). We hypothesised that differences across healthy LA may contribute to a unique, region-dependent Ca signalling and thus be involved in the pro-arrhythmic activity associated with electrical dysfunctions observed during AF. The study aimed to characterise, in a physiological relevant model (adult sheep), the electrical activity and the Ca signalling of 3 distinct LA regions (appendage, free wall and pulmonary veins), in control conditions and after acetylcholine perfusion (5 μM, ACH) to induce acute AF. Sheep ( n = 16) were killed by pentobarbital overdose. Hearts were excised, LA separated and Langendorff-perfused after suturing leaky branches. Blebbistatin was used to eliminate motion artefact. Simultaneous Vm and intracellular Ca fluorescent signals were recorded with 2 CMOS cameras after loading preparations with RH237 and Rhod-2AM. LA were paced in the 3 LA regions and ECG was continuously recorded. In some experiments, ACH and burst pacing were used to trigger AF, with or without Dantrolene (a ryanodine receptor (RyR) blocker). Analysis was performed in all 3 regions. Action potential duration (at 80% repolarization) was not significantly different in the 3 regions (for the 3 pacing areas). In contrast, time to 50% Ca transient decay was significantly different (depending of the region recorded and paced). ACH perfusion and burst pacing induced AF (8/16). Dantrolene did not reduce AF susceptibility. These data provide evidence that Ca regional heterogeneity in LA can play a crucial role in focal arrhythmia initiation, without RyR gating modification.

Ryanodine receptor- and sodium-calcium exchanger-mediated spontaneous calcium activity in immature oligodendrocytes in cultures

Myelination in the central nervous system depends on interactions between axons and oligodendrocyte precursor cells (OPCs). Action potentials in an axon can be followed by release of biologically active substances, like glutamate, which can instruct OPCs to start myelination. Myelin Basic Protein (MBP) is an “executive molecule of myelin” required for the formation of compact myelin. As cells of the oligodendrocyte lineage (OLCs) are capable of producing MBP in pure oligodendrocyte cultures, i.e. without neurons, we investigated Ca2+ signaling in developing OLCs in cultures. We show that spontaneous Ca2+ transients (CTs) occur at very low frequency in both bipolar OPCs and mature oligodendrocytes. In contrast immature OLCs (imOLCs), cells with several thick processes, demonstrate a relatively high frequency of CTs. Moreover, CT frequency in imOLC processes is much higher as compared with the somatic CT frequency. Somatic CTs are almost completely blocked by thapsigargin, an antagonist of sarco-(endo-) plasmic reticulum Ca2+ ATPase, and ryanodine, a blocker of ryanodine receptors, indicating an involvement of Ca2+ release from the endoplasmic reticulum. Ryanodine strongly reduces CT frequency in imOLC processes. Ouabain, an antagonist of Na+, K+-ATPase (NKA), applied at low concentration increases CT frequency, while KB-R7943, a blocker of reverse mode of Na+, Ca2+ exchanger (NCX), decreases CT frequency. We suggest that local RyR-NCX-(NKA?) interaction might underlie the generation of CTs in imOLC in the absence of neurons, and this activity influences oligodendrocyte maturation.

Loss of tumor susceptibility gene 101 (TSG101) perturbs endoplasmic reticulum structure and function

Tumor susceptibility gene 101 (TSG101), an ESCRT-I protein, is implicated in multiple cellular processes and its functional depletion can lead to blocked lysosomal degradation, cell cycle arrest, demyelination and neurodegeneration. Here, we show that loss of TSG101 results in endoplasmic reticulum (ER) stress and this causes ER membrane remodelling (EMR). This correlates with an expansion of ER, increased vacuolation, altered relative distribution of the rough and smooth ER and disruption of three-way junctions. Blocked lysosomal degradation due to TSG101 depletion leads to ER stress and Ca2+ leakage from ER stores, causing destabilization of actin cytoskeleton. Inhibiting Ca2+ release from the ER by blocking ryanodine receptors (RYRs) with Dantrolene partially rescues the ER stress phenotypes. Hence, in this study we have identified the involvement of TSG101 in modulating ER stress mediated remodelling by engaging the actin cytoskeleton. This is significant because functional depletion of TSG101 effectuates ER-stress, perturbs the structure, mobility and function of the ER, all aspects closely associated with neurodegenerative diseases. Summary statementWe show that tumor susceptibility gene (TSG) 101 regulates endoplasmic reticulum (ER) stress and its membrane remodelling. Loss of TSG101 perturbs structure, mobility and function of the ER as a consequence of actin destabilization.

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.

Dopamine induces release of calcium from internal stores in layer II lateral entorhinal cortex fan cells.

The entorhinal cortex plays an important role in temporal lobe processes including learning and memory, object recognition, and contextual information processing. The alteration of the strength of synaptic inputs to the lateral entorhinal cortex may therefore contribute substantially to sensory and mnemonic functions. The neuromodulatory transmitter dopamine exerts powerful effects on excitatory glutamatergic synaptic transmission in the entorhinal cortex. Interestingly, inputs from midbrain dopamine neurons appear to specifically target clusters of excitatory cells located in the superficial layers of the entorhinal cortex. We have previously demonstrated that dopamine facilitates synaptic transmission through the activation of D1-like receptors. This facilitation of synaptic transmission is dependent on both activation of classical D1-like-receptors, and upon activation of dopamine receptors linked to increases in phospholipase C, inositol triphosphate (IP3), and intracellular calcium. In the present study we combined electrophysiological recordings of evoked excitatory postsynaptic currents with imaging of intracellular calcium using the fluorescent indicator fluo-4 to monitor calcium transients evoked by dopamine in electrophysiologically identified putative fan and pyramidal cells of the lateral entorhinal cortex. Bath application of dopamine (1 μM), or the phosphatidylinositol (PI)-linked D1-like-receptor agonist SKF83959 (5 μM), induced reliable and reversible increases in fluo-4 fluorescence and excitatory postsynaptic currents in fan cells, but not in pyramidal cells. In contrast, application of the classical D1-like-receptor agonist SKF38393 (10 μM) did not result in significant increases in fluorescence. Blocking release of calcium from internal stores by loading cells with the IP3 receptor blocker heparin (1 mM) or the ryanodine receptor blocker dantrolene (20 μM) abolished both the calcium transients and the facilitation of evoked synaptic currents induced by dopamine. Dopamine also induced calcium transients in fan cells when calcium was excluded from the extracellular medium, further indicating that the calcium transients are linked to release from internal stores. These results indicate that following D1-like-receptor binding, dopamine selectively induces transient elevations in intracellular calcium via activation of IP3 and ryanodine receptors, and that these elevations are linked to the facilitation of synaptic responses in putative layer II entorhinal cortex fan cells.

Reduction of Vascular Reactivity with Dantrolene in Type 1 Diabetic Rats is Dose‐dependent

Diabetics have a greater predisposition for developing cerebral vasospasms (CVSPs) than nondiabetics, even when under glycemic control. The etiology of CVSPs is associated with increased vascular tone, secondary to augmented intracellular Ca+2 in vascular smooth muscle. Current pharmacological treatments are ineffective in reducing CVSPs. The combination of the ryanodine receptor blocker dantrolene (50 μM) and the Ca+2 channel blocker nimodipine significantly decreases vascular reactivity in streptozotocin (STZ)‐induced diabetic rats. In order to determine if the beneficial effects observed at 50 μM are also maintained at a much lower concentration with less potentially harmful secondary effects, we evaluated the vascular effects of 10 μM dantrolene, alone and in combination with 50 nM nimodipine. Dose‐response curves for the phenylephrine (PHE)‐induced contraction (0.1 nM to 10 μM) and acetylcholine (ACh)‐induced relaxation (1 nM to 10 μM) were performed on aortic rings from STZ‐diabetic rats, both before and after a 30‐minute incubation period with dantrolene alone or in combination with nimodipine. Whereas 50 μM dantrolene reduced PHE‐induced contraction by 47% in diabetic rats and 29% in controls, 10 μM dantrolene failed to reduce this parameter in either group. Moreover, 50 μM dantrolene reduced PHE‐induced contraction by about 80% in both diabetic and controls when combined with nimodipine. The combination of 10 μM dantrolene and nimodipine, however, does not increase the reduction of 35% in diabetic rats and 47% in controls obtained with nimodipine alone. In addition, only 50 μM dantrolene increased ACh‐induced relaxation by 50% (N=9, P&lt;0.05) in diabetic rats. These results suggest that both, improved endothelial‐dependent relaxation and reduced vascular contraction with dantrolene are dosedependent. Thus, careful titration should be performed in order to achieve a significant reduction in hyper‐reactivity in both diabetics and nondiabetics.Support or Funding InformationThis work was supported by Grants from the National Institute of Health (MBRS‐RISE Grant R25GM061838), the NIMHD‐RCMI Program (Grant G12‐MD007600), and the Anesthesiology Department of the University of Puerto Rico‐School of Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synergistic effects of Dantrolene and Nimodipine on the Phenylephrine‐induced Contraction and ACh‐induced Relaxation in Aortic Rings from Diabetic Rats

Diabetics have a higher risk of developing cerebral vasospasms (CVSP) than non‐diabetics. The addition of the ryanodine receptor (RyR) blocker dantrolene to standard therapies reduces vasospasms in non‐diabetics. Whether diabetics with CVSP also benefit from this drug, however, is unknown. We evaluated the effects of a 30‐min incubation with dantrolene (50 μM), nimodipine (50 nM), and both drugs in combination, on phenylephrine (PHE)‐induced contraction, and on acetylcholine (ACh)–induced relaxation in aortic rings from streptozotocin (STZ) diabetic rats. Age‐matched, non‐diabetic rats served as controls. The oxidative‐stress markers malondialdehyde (MDA) and 4‐hydroxyalkenal (4‐HAE) were also evaluated in the presence and absence of dantrolene and nimodipine. The combination of these two drugs acted synergistically to reduce the PHE‐induced contraction by 80% in both diabetics and controls. In contrast, it increased the Emax value for ACh‐induced relaxation (from 56.46 ± 5.14% to 96.21 ± 7.50 %; n=6, P &lt; 0.05), and it decreased MDA +4‐HAE values in diabetic rats only. These results suggest that the combination of dantrolene and nimodipine benefits both diabetics and non‐diabetics by decreasing arterial tone synergistically.Support or Funding InformationSupported by NIH (MBRS‐RISE Grant R25GM061838), and NIMHD‐RCMI (Grant G12‐MD007600).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Atrial Infarction-Induced Spontaneous Focal Discharges and Atrial Fibrillation in Sheep: Role of Dantrolene-Sensitive Aberrant Ryanodine Receptor Calcium Release.

The mechanisms underlying spontaneous atrial fibrillation (AF) associated with atrial ischemia/infarction are incompletely elucidated. Here, we investigate the mechanisms underlying spontaneous AF in an ovine model of left atrial myocardial infarction (LAMI). LAMI was created by ligating the atrial branch of the left anterior descending coronary artery. ECG loop recorders were implanted to monitor AF episodes. In 7 sheep, dantrolene-a ryanodine receptor blocker-was administered in vivo during the 8-day observation period (LAMI-D, 2.5 mg/kg, IV, BID). LAMI animals experienced numerous spontaneous AF episodes during the 8-day monitoring period that were suppressed by dantrolene (LAMI, 26.1±5.1; sham, 4.3±1.1; LAMI-D, 2.8±0.8; mean±SEM episodes per sheep, P<0.01). Optical mapping showed spontaneous focal discharges (SFDs) originating from the ischemic/normal-zone border. SFDs were calcium driven, rate dependent, and enhanced by isoproterenol (0.03 µmol/L, from 210±87 to 3816±1450, SFDs per sheep) but suppressed by dantrolene (to 55.8±32.8, SFDs per sheep, mean±SEM). SFDs initiated AF-maintaining reentrant rotors anchored by marked conduction delays at the ischemic/normal-zone border. NOS1 (NO synthase-1) protein expression decreased in ischemic zone myocytes, whereas NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) oxidase and xanthine oxidase enzyme activities and reactive oxygen species (DCF [6-carboxy-2',7'-dichlorodihydrofluorescein diacetate]-fluorescence) increased. CaM (calmodulin) aberrantly increased [3H]ryanodine binding to cardiac RyR2 (ryanodine receptors) in the ischemic zone. Dantrolene restored the physiological binding of CaM to RyR2. Atrial ischemia causes spontaneous AF episodes in sheep, caused by SFDs that initiate reentry. Nitroso-redox imbalance in the ischemic zone is associated with intense reactive oxygen species production and altered RyR2 responses to CaM. Dantrolene administration normalizes the CaM response, prevents LAMI-related SFDs, and AF initiation. These findings provide novel insights into the mechanisms underlying ischemia-related atrial arrhythmias.