BACKGROUND: Chronic sympathetic stimulation drives desensitization and downregulation of β1 adrenergic receptor (β1AR) in heart failure. We aim to explore the differential downregulation subcellular pools of β1AR signaling in the heart.
RESULTS: We applied chronic infusion of isoproterenol to induced cardiomyopathy in male C57BL/6J mice. We applied confocal and proximity ligation assay to examine β1AR association with L-type calcium channel, ryanodine receptor 2, and SERCA2a ((Sarco)endoplasmic reticulum calcium ATPase 2a) and Förster resonance energy transfer-based biosensors to probe subcellular β1AR-PKA (protein kinase A) signaling in ventricular myocytes. Chronic infusion of isoproterenol led to reduced β1AR protein levels, receptor association with L-type calcium channel and ryanodine receptor 2 measured by proximity ligation (puncta/cell, 29.65 saline versus 14.17 isoproterenol, P<0.05), and receptor-induced PKA signaling at the plasma membrane (Förster resonance energy transfer, 28.9% saline versus 1.9% isoproterenol, P<0.05) and ryanodine receptor 2 complex (Förster resonance energy transfer, 30.2% saline versus 10.6% isoproterenol, P<0.05). However, the β1AR association with SERCA2a was enhanced (puncta/cell, 51.4 saline versus 87.5 isoproterenol, P<0.05), and the receptor signal was minimally affected. The isoproterenol-infused hearts displayed decreased PDE4D (phosphodiesterase 4D) and PDE3A and increased PDE2A, PDE4A, and PDE4B protein levels. We observed a reduced role of PDE4 and enhanced roles of PDE2 and PDE3 on the β1AR-PKA activity at the ryanodine receptor 2 complexes and myocyte shortening. Despite the enhanced β1AR association with SERCA2a, the endogenous norepinephrine-induced signaling was reduced at the SERCA2a complexes. Inhibiting monoamine oxidase A rescued the norepinephrine-induced PKA signaling at the SERCA2a and myocyte shortening.
CONCLUSIONS: This study reveals distinct mechanisms for the downregulation of subcellular β1AR signaling in the heart under chronic adrenergic stimulation.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) may cause sudden cardiac death (SCD) despite medical therapy. Therefore, implantable cardioverter-defibrillators (ICDs) are commonly advised. However, there is limited data on the outcomes of ICD use in children.
OBJECTIVE: The purpose of this study was to compare the risk of arrhythmic events in pediatric patients with CPVT with and without an ICD.
METHODS: We compared the risk of SCD in patients with RYR2 (ryanodine receptor 2) variants and phenotype-positive symptomatic CPVT patients with and without an ICD who were younger than 19 years and had no history of sudden cardiac arrest at phenotype diagnosis. The primary outcome was SCD; secondary outcomes were composite end points of SCD, sudden cardiac arrest, or appropriate ICD shocks with or without arrhythmic syncope.
RESULTS: The study included 235 patients, 73 with an ICD (31.1%) and 162 without an ICD (68.9%). Over a median follow-up of 8.0 years (interquartile range 4.3-13.4 years), SCD occurred in 7 patients (3.0%), of whom 4 (57.1%) were noncompliant with medications and none had an ICD. Patients with ICD had a higher risk of both secondary composite outcomes (without syncope: hazard ratio 5.85; 95% confidence interval 3.40-10.09; P < .0001; with syncope: hazard ratio 2.55; 95% confidence interval 1.50-4.34; P = .0005). Thirty-one patients with ICD (42.5%) experienced appropriate shocks, 18 (24.7%) inappropriate shocks, and 21 (28.8%) device-related complications.
CONCLUSIONS: SCD events occurred only in patients without an ICD and mostly in those not on optimal medical therapy. Patients with an ICD had a high risk of appropriate and inappropriate shocks, which may be reduced with appropriate device programming. Severe ICD complications were common, and risks vs benefits of ICDs need to be considered.

To increase the structural diversity of insecticides and meet the needs of effective integrated insect management, the structure of chlorantraniliprole was modified based on a previously established three-dimensional quantitative structure-activity relationship (3D-QSAR) model. The pyridinyl moiety in the structure of chlorantraniliprole was replaced with a 4-fluorophenyl group. Further modifications of this 4-fluorophenyl group by introducing a halogen atom at position 2 and an electron-withdrawing group (e.g., iodine, cyano, and trifluoromethyl) at position 5 led to 34 compounds with good insecticidal efficacy against Mythimna separata, Plutella xylostella, and Spodoptera frugiperda. Among them, compound IV f against M. separata showed potency comparable to that of chlorantraniliprole. IV p against P. xylostella displayed a 4.5 times higher potency than chlorantraniliprole. In addition, IV d and chlorantraniliprole exhibited comparable potencies against S. frugiperda. Transcriptome analysis showed that the molecular target of compound IV f is the ryanodine receptor. Molecular docking was further performed to verify the mode of action and insecticidal activity against resistant P. xylostella.

The diamondback moth Plutella xylostella, a global insect pest of cruciferous vegetables, has evolved resistance to many classes of insecticides including diamides. Three point mutations (I4790M, I4790K, and G4946E) in the ryanodine receptor of P. xylostella (PxRyR) have been identified to associate with varying levels of resistance. In this study, we generated a knockin strain (I4790K-KI) of P. xylostella, using CRISPR/Cas9 to introduce the I4790K mutation into PxRyR of the susceptible IPP-S strain. Compared to IPP-S, the edited I4790K-KI strain exhibited high levels of resistance to both anthranilic diamides (chlorantraniliprole 1857-fold, cyantraniliprole 1433-fold) and the phthalic acid diamide flubendiamide (>2272-fold). Resistance to chlorantraniliprole in the I4790K-KI strain was inherited in an autosomal and recessive mode, and genetically linked with the I4790K knockin mutation. Computational modeling suggests the I4790K mutation reduces the binding of diamides to PxRyR by disrupting key hydrogen bonding interactions within the binding cavity. The approximate frequencies of the 4790M, 4790K, and 4946E alleles were assessed in ten geographical field populations of P. xylostella collected in China in 2021. The levels of chlorantraniliprole resistance (2.3- to 1444-fold) in these populations were significantly correlated with the frequencies (0.017-0.917) of the 4790K allele, but not with either 4790M (0-0.183) or 4946E (0.017-0.450) alleles. This demonstrates that the PxRyR I4790K mutation is currently the major contributing factor to chlorantraniliprole resistance in P. xylostella field populations within China. Our findings provide in vivo functional evidence for the causality of the I4790K mutation in PxRyR with high levels of diamide resistance in P. xylostella, and suggest that tracking the frequency of the I4790K allele is crucial for optimizing the monitoring and management of diamide resistance in this crop pest.

UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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).
UNASSIGNED: 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.
UNASSIGNED: 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.

BACKGROUND: Endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) is crucial for maintaining intracellular calcium homeostasis due to the calcium-signaling-related proteins on its membrane. While ryanodine receptors (RyR) on insect ER/SR membranes are well-known as targets for diamide insecticides, little is known about other calcium channels. Given the resistance of diamide insecticides, the establishment of molecular screening models targeting RyR or sarco/endoplasmic reticulum calcium ATPase (SERCA) is conducive to the discovery of new insecticidal molecules.
RESULTS: The morphological features of Mythimna separata SR have closed vesicles with integrity and high density. The 282 proteins in the SR component contained RyR and SERCA. A measurement model for the release and uptake of calcium was successfully established by detecting calcium ions outside the SR membrane using a fluorescence spectrophotometer. In vitro testing systems using SR vesicles found that diamide insecticides could activate dose-dependently RyR, with EC50 values of 0.14 μM (Chlorantraniliprole), 0.21 μM (Flubendiamide), and 0.57 μM (Cyantraniliprole), respectively. However, dantrolene inhibited RyR-mediated calcium release with an IC50 value of 353.9 μM, suggesting that dantrolene can weakly antagonize RyR. Moreover, cyclopiazonic acid significantly reduced the enzyme activity and calcium uptake capacity of SERCA. On the contrary, CDN1163 markedly activated the enzyme activity and improved the calcium transport capacity of SERCA.
CONCLUSIONS: SR vesicles can be used to study the function of unknown proteins on the SR membranes, as well as for high-throughput screening of highly active compounds targeting RyR or SERCA. © 2024 Society of Chemical Industry.

Junctional ectopic tachycardia (JET) is a potentially fatal cardiac arrhythmia. Hcn4:shJph2 mice serve as a model of nodal arrhythmias driven by ryanodine type 2 receptor (RyR2)-mediated Ca2+ leak. EL20 is a small molecule that blocks RyR2 Ca2+ leak. In a novel in vivo model of JET, Hcn4:shJph2 mice demonstrated rapid conversion of JET to sinus rhythm with infusion of EL20. Primary atrioventricular nodal cells demonstrated increased Ca2+ transient oscillation frequency and increased RyR2-mediated stored Ca2+ leak which was normalized by EL20. EL20 was found to be rapidly degraded in mouse and human plasma, making it a potential novel therapy for JET.

Diamide insecticides have gained popularity due to their high efficacy and low toxicity to nontarget organisms. However, diamide-associated resistance has emerged recently, causing a significant reduction in their potency, thereby hindering sustainable agricultural development. Here, we explored novel diamide insecticide analogs and, using a structure-based approach, rationally designed and synthesized 28 nitrophenyl substituted anthranilic diamides. Most of the compounds showed moderate to good activity against Mythimna separata, Plutella xylostella, and Spodoptera frugiperda. Among them, compounds Ia and Im showed extraordinarily high activity and their mode of action was verified on isolated neurons. Additionally, Im exhibited over 10-fold greater potency than chlorantraniliprole in a HEK293 cell line stably expressing S. frugiperda ryanodine receptors (SfRyRs) containing the resistance mutations, G4891E and I4734M. The binding modes of Im in the SfRyRs were predicted using in silico molecular docking analysis. Our novel nitrophenyl substituted anthranilic diamide derivatives provide valuable insights for the design of insecticidal RyR-targeting compounds to effectively control both wild type and diamide insecticide-resistant lepidopteran pests.

Computer-aided molecular modeling was applied to design a series of Spodoptera frugiperda RyR agonists. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were used to generate 3D-QSAR models. MD simulations in the complex with S. frugiperda native, mutant RyR, and mammalian RyR1 under physiological conditions were used to validate the detailed binding mechanism. Binding free energy calculation by molecular mechanics generalized surface area (MM-GBSA) explained the role of key amino acid residues in ligand-receptor binding. Therefore, 14 new compounds were effectively designed and synthesized, and a bioassay indicated that compounds A-2 and A-3 showed comparable activity to that of chloranthraniliprole with LC50 values of 0.27, 0.18, and 0.20 mg L-1, respectively, against S. frugiperda. Most target compounds also displayed good activity against Mythinma separata at 0.1 mg L-1. Molecular docking and MM-GBSA calculations demonstrated that A-3 had a better binding capacity with native and mutant S. frugiperda RyRs.

Postoperative cognitive dysfunction (POCD) is characterized by impaired learning and memory. 6 h duration isoflurane anesthesia is an important factor to induce POCD, and the dysfunction of ryanodine receptor (RyR) in the hippocampus may be involved in this process. We investigated the expression of RyR3 in the hippocampus of mice after 6-h duration isoflurane anesthesia, as well as the improvement of RyR receptor agonist caffeine on POCD mice, while attempting to identify the underlying molecular mechanism.
We constructed a POCD model using 8-week-old male C57BL/6J mice that were exposed to 6-h duration isoflurane. Prior to the three-day cognitive behavioral experiment, RyR agonist caffeine were injected. Fear conditioning and location memory tests were used in behavioral studies. We also exposed the mouse neuroblastoma cell line Neuro-2a (N2A) to 6-h duration isoflurane exposure to simulate the conditions of in vivo cognitive dysfunction. We administered ryanodine receptor agonist (caffeine) and inhibitor (ryanodine) to N2a cells. Following that, we performed a series of bioinformatics analysis to discover proteins that are involved in the development of cognitive dysfunction. Rt-PCR and Western blot were used to assess mRNA level and protein expression.
6-h duration isoflurane anesthesia induced cognitive dysfunction and increased RyR3 mRNA levels in hippocampus. The mRNA levels of RyR3 in cultured N2a cells after anesthesia were comparable to those in vivo, and the RyR agonist caffeine corrected the expression of some cognitive-related phenotypic proteins that were disturbed after anesthesia. Intraperitoneal injection of RyR agonist caffeine can improve cognitive function after isoflurane anesthesia in mice, and bioinformatics analyses suggest that CaMKⅣ may be involved in the molecular mechanism.
Ryanodine receptor agonist caffeine may improve cognitive dysfunction in mice after isoflurane anesthesia.