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Diamides, such as flubendiamide and chlorantraniliprole, belong to a new chemical class of insecticides that act as conformation-sensitive activators of insect ryanodine receptors (RyRs). Both compounds are registered for use against lepidopteran species such as the diamondback moth, Plutella xylostella , a notorious global pest of cruciferous crops. Recently acquired resistance to diamide insecticides in this species is thought to be due to a target-site mutation conferring an amino acid substitution (G4946E), located within the trans-membrane domain of the RyR, though the exact role of this mutation has not yet been fully determined. To address this we have cloned a full-length cDNA encoding the P. xylostella RyR and established clonal Sf9 cell lines stably expressing either the wildtype RyR or the G4946E variant, in order to test the sensitivity to flubendiamide and chlorantraniliprole on the recombinant receptor. We report that the efficacy of both diamides was dramatically reduced in clonal Sf9 cells stably expressing the G4946E modified RyR, providing clear functional evidence that the G4946E RyR mutation impairs diamide insecticide binding.

Resting skeletal muscle generates heat for endothermy in mammals but not amphibians, while both use the same Ca2+-handling proteins and membrane structures to conduct excitation–contraction coupling apart from having different ryanodine receptor (RyR) isoforms for Ca2+ release. The sarcoplasmic reticulum (SR) generates heat following Adenosine triphosphate (ATP) hydrolysis at the Ca2+ pump, which is amplified by increasing RyR1 Ca2+ leak in mammals, subsequently increasing cytoplasmic [Ca2+] ([Ca2+]cyto). For thermogenesis to be functional, rising [Ca2+]cyto must not interfere with cytoplasmic effectors of the sympathetic nervous system (SNS) that likely increase RyR1 Ca2+ leak; nor should it compromise the muscle remaining relaxed. To achieve this, Ca2+ activated, regenerative Ca2+ release that is robust in lower vertebrates needs to be suppressed in mammals. However, it has not been clear whether: i) the RyR1 can be opened by local increases in [Ca2+]cyto; and ii) downstream effectors of the SNS increase RyR Ca2+ leak and subsequently, heat generation. By positioning amphibian and malignant hyperthermia-susceptible human-skinned muscle fibers perpendicularly, we induced abrupt rises in [Ca2+]cyto under identical conditions optimized for activating regenerative Ca2+ release as Ca2+ waves passed through the junction of fibers. Only mammalian fibers showed resistance to rising [Ca2+]cyto, resulting in increased SR Ca2+ load and leak. Fiber heat output was increased by cyclic adenosine monophosphate (cAMP)-induced RyR1 phosphorylation at Ser2844 and Ca2+ leak, indicating likely SNS regulation of thermogenesis. Thermogenesis occurred despite the absence of SR Ca2+ pump regulator sarcolipin. Thus, evolutionary isolation of RyR1 provided increased dynamic range for thermogenesis with sensitivity to cAMP, supporting endothermy.

Awareness of new potential doping agents and the proactive implementation of detection methods are key aspects of preventive antidoping research. Ryanodine receptor‐1‐calstabin complex stabilizers (RYR‐stabilizers) are a novel class of drug candidates for the treatment of various diseases associated with leaky Ca2+ channels in the cardiac or skeletal muscle. Also, intense physical activity was shown to transiently cause leakage of skeletal muscle Ca2+ channels, and RYR‐stabilizers have been shown to restore normal activity and, thus, increase endurance performance. Consequently, such compounds are relevant targets in doping controls, and to date, in particular, compounds S107, JTV‐519, ARM 036, and ARM 210 have been subject of antidoping research. In this study, ARM 036 and ARM 210 as well as the commercially available compounds S107 and JTV‐519 were synthesized using a multistep approach. Subsequently, all compounds were investigated concerning their in vitro metabolic behavior, and various metabolites were identified. Selected metabolites were then chemically synthesized for comprehensive structure confirmation. The findings of this study will contribute to routine doping control analytical programs and allow for improving existing detection methods. RYR‐stabilizers such as S107, JTV‐519, ARM 036, and ARM 210 are emerging doping‐relevant compounds due to their performance‐enhancing effects on leaky skeletal muscle Ca2+ channels. To support proactive antidoping efforts, all four compounds were synthesized and their in vitro metabolism was systematically investigated. Multiple metabolites were identified, and selected structures were chemically synthesized for confirmation. These results expand analytical knowledge of RYR‐stabilizers and enable improved detection strategies for routine doping‐control programs.

Mutations in cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most CPVT RyR2 mutations characterized are gain-of-function (GOF), indicating enhanced RyR2 function as a major cause of CPVT. Loss-of-function (LOF) RyR2 mutations have also been identified and are linked to a distinct entity of cardiac arrhythmia termed RyR2 Ca2+ release deficiency syndrome (CRDS). Exercise stress testing (EST) is routinely used to diagnose CPVT, but it is ineffective for CRDS. There is currently no effective diagnostic tool for CRDS in humans. An alternative strategy to assess the risk for CRDS is to directly determine the functional impact of the associated RyR2 mutations. To this end, we have functionally screened 18 RyR2 mutations that are associated with idiopathic ventricular fibrillation (IVF) or sudden death. We found two additional RyR2 LOF mutations E4146K and G4935R. The E4146K mutation markedly suppressed caffeine activation of RyR2 and abolished store overload induced Ca2+ release (SOICR) in human embryonic kidney 293 (HEK293) cells. E4146K also severely reduced cytosolic Ca2+ activation and abolished luminal Ca2+ activation of single RyR2 channels. The G4935R mutation completely abolished caffeine activation of and [3H]ryanodine binding to RyR2. Co-expression studies showed that the G4935R mutation exerted dominant negative impact on the RyR2 wildtype (WT) channel. Interestingly, the RyR2-G4935R mutant carrier had a negative EST, and the E4146K carrier had a family history of sudden death during sleep, which are different from phenotypes of typical CPVT. Thus, our data further support the link between RyR2 LOF and a new entity of cardiac arrhythmias distinct from CPVT.

Ca2+ leak via ryanodine receptor type 2 (RyR2) can cause potentially fatal arrhythmias in a variety of heart diseases and has also been implicated in neurodegenerative and seizure disorders, making RyR2 an attractive therapeutic target for drug development. Here we synthesized and investigated the fungal natural product and known insect RyR antagonist (−)-verticilide and several congeners to determine their activity against mammalian RyR2. Although the cyclooligomeric depsipeptide natural product (−)-verticilide had no effect, its nonnatural enantiomer [ent-(+)-verticilide] significantly reduced RyR2-mediated spontaneous Ca2+ leak both in cardiomyocytes from wild-type mouse and from a gene-targeted mouse model of Ca2+ leak-induced arrhythmias (Casq2−/−). ent-(+)-verticilide selectively inhibited RyR2-mediated Ca2+ leak and exhibited higher potency and a distinct mechanism of action compared with the pan-RyR inhibitors dantrolene and tetracaine and the antiarrhythmic drug flecainide. ent-(+)-verticilide prevented arrhythmogenic membrane depolarizations in cardiomyocytes without significant effects on the cardiac action potential and attenuated ventricular arrhythmia in catecholamine-challenged Casq2−/− mice. These findings indicate that ent-(+)-verticilide is a potent and selective inhibitor of RyR2-mediated diastolic Ca2+ leak, making it a molecular tool to investigate the therapeutic potential of targeting RyR2 hyperactivity in heart and brain pathologies. The enantiomer-specific activity and straightforward chemical synthesis of (unnatural) ent-(+)-verticilide provides a compelling argument to prioritize ent-natural product synthesis. Despite their general absence in nature, the enantiomers of natural products may harbor unprecedented activity, thereby leading to new scaffolds for probe and therapeutic development.

The cardiac ryanodine receptor (RyR2) governs the release of Ca2+from the sarcoplasmic reticulum, which initiates muscle contraction. Mutations in RyR2 have been linked to ventricular tachycardia (VT) and sudden death, but the precise molecular mechanism is unclear. It is known that when the sarcoplasmic reticulum store Ca2+content reaches a critical level, spontaneous Ca2+release occurs, a process we refer to as store-overload-induced Ca2+release (SOICR). In view of the well documented arrhythmogenic nature of SOICR, we characterized the effects of disease-causing RyR2 mutations on SOICR in human embryonic kidney (HEK)293 cells and found that, at elevated extracellular Ca2+levels, HEK293 cells expressing RyR2 displayed SOICR in a manner virtually identical to that observed in cardiac cells. Using this cell model, we demonstrated that the RyR2 mutations linked to VT and sudden death, N4104K, R4496C, and N4895D, markedly increased the occurrence of SOICR. At the molecular level, we showed that these RyR2 mutations increased the sensitivity of single RyR2 channels to activation by luminal Ca2+and enhanced the basal level of [3H]ryanodine binding. We conclude that disease-causing RyR2 mutations, by enhancing RyR2 luminal Ca2+activation, reduce the threshold for SOICR, which in turn increases the propensity for triggered arrhythmia. Abnormal RyR2 luminal Ca2+activation likely contributes to the enhanced SOICR commonly observed in various cardiac conditions, including heart failure, and may represent a unifying mechanism for Ca2+overload-associated VT.

The core myopathies are a group of congenital myopathies with variable clinical expression – ranging from early-onset skeletal-muscle weakness to later-onset disease of variable severity – that are identified by characteristic ‘core-like’ lesions in myofibers and the presence of hypothonia and slowly or rather non-progressive muscle weakness. The genetic causes are diverse; central core disease is most often caused by mutations in ryanodine receptor 1 (RYR1), whereas multi-minicore disease is linked to pathogenic variants of several genes, including selenoprotein N (SELENON), RYR1 and titin (TTN). Understanding the mechanisms that drive core development and muscle weakness remains challenging due to the diversity of the excitation-contraction coupling (ECC) proteins involved and the differential effects of mutations across proteins. Because of this, the use of representative models expressing a mature ECC apparatus is crucial. Animal models have facilitated the identification of disease progression mechanisms for some mutations and have provided evidence to help explain genotype-phenotype correlations. However, many unanswered questions remain about the common and divergent pathological mechanisms that drive disease progression, and these mechanisms need to be understood in order to identify therapeutic targets. Several new transgenic animals have been described recently, expanding the spectrum of core myopathy models, including mice with patient-specific mutations. Furthermore, recent developments in 3D tissue engineering are expected to enable the study of core myopathy disease progression and the effects of potential therapeutic interventions in the context of human cells. In this Review, we summarize the current landscape of core myopathy models, and assess the hurdles and opportunities of future modeling strategies.

To analyze the copper-induced cross talk among calcium, nitric oxide (NO), and hydrogen peroxide (H₂O₂) and the calciumdependent activation of gene expression, the marine alga Ulva compressa was treated with the inhibitors of calcium channels, ned-19, ryanodine, and xestospongin C, of chloroplasts and mitochondrial electron transport chains, 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and antimycin A, of pyruvate dehydrogenase, moniliformin, of calmodulins, N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide, and of calcium-dependent protein kinases, staurosporine, as well as with the scavengers of NO, 2-(4-carboxypheny1)-4,4,5,5-tetramethylimidazoline-1-oxy1-3-oxide, and of H₂O₂, ascorbate, and exposed to a sublethal concentration of copper (10 µm) for 24 h. The level of NO increased at 2 and 12 h. The first peak was inhibited by ned-19 and 3-(2,3-dichloropheny1)-1, 1-dimethylurea and the second peak by ned-19 and antimycin A, indicating that NO synthesis is dependent on calcium release and occurs in organdíes. The level of H₂O₂ increased at 2, 3, and 12 h and was inhibited by ned-19, ryanodine, xestospongin C, and moniliformin, indicating that H₂O₂ accumulation is dependent on calcium release and Krebs cycle activity In addition, pyruvate dehydrogenase, 2-oxoxglutarate dehydrogenase, and isocitrate dehydrogenase activities of the Krebs cycle increased at 2, 3,12, and/or 14 h, and these increases were inhibited in vitro by EGTA, a calcium chelating agent. Calcium release at 2, 3, and 12 h was inhibited by 2-(4-carboxypheny1)-4,4,5,5-tetramethylimidazoline-1-oxy1-3-oxide and ascorbate, indicating activation by NO and H₂O₂. In addition, the level of antioxidant protein gene transcripts decreased with N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide and staurosporine. Thus, there is a copper-induced cross talk among calcium, H₂O₂, and NO and a calcium-dependent activation of gene expression involving calmodulins and calcium-dependent protein kinases.

The effect of the compound N 1 -(2,3,4-trimethoxy)-N 2 -{2-[(2,3,4-trimethoxybenzyl)amino]ethyl}-1,2-ethane-diamine (code ALM-802) on the amplitude of the Ca 2+ response in the cell was studied in in vitro experiments. The concentration of intracellular calcium was assessed using a Fura-2 two-wave probe. The experiments were performed on a culture of isolated rat hippocampal neurons. The effect of compound ALM-802 on the activity of ryanodine receptors (RyR2) was studied on an isolated strip of rat myocardium. The compound ALM-802 (69.8 μM) in hippocampal neurons causes a significant decrease in the amplitude of the Ca 2+ response induced by addition of KCl to the medium. Experiments performed on an isolated myocardial strip showed that compound ALM-802 (10 -5 M) almost completely blocked the positive inotropic reaction of the strip to the RyR2 agonist caffeine (5×10 -5 M). The data obtained indicate that the decrease in the concentration of Ca 2+ ions in the cell caused by ALM-802 is due to its ability to block RyR2 located on the membrane of the sarcoplasmic reticulum, which can be associated with the antiarrhythmic activity of the compound.

The nucleotide NAADP was recently discovered as a second messenger involved in the initiation and propagation of Ca²⁺ signaling in lymphoma T cells, but its impact on primary T cell function is still unknown. An optimized, synthetic, small molecule inhibitor of NAADP action, termed BZ194, was designed and synthesized. BZ194 neither interfered with Ca²⁺ mobilization by D-myo-inositol 1,4,5-trisphosphate or cyclic ADP-ribose nor with capacitative Ca²⁺ entry. BZ194 specifically and effectively blocked NAADP-stimulated [³H]ryanodine binding to the purified type 1 ryanodine receptor. Further, in intact T cells, Ca²⁺ mobilization evoked by NAADP or by formation of the immunological synapse between primary effector T cells and astrocytes was inhibited by BZ194. Downstream events of Ca²⁺ mobilization, such as nuclear translocation of \"nuclear factor of activated T cells\" (NFAT), T cell receptor-driven interleukin-2 production, and proliferation in antigen-experienced CD4⁺ effector T cells, were attenuated by the NAADP antagonist. Taken together, specific inhibition of the NAADP signaling pathway constitutes a way to specifically and effectively modulate T-cell activation and has potential in the therapy of autoimmune diseases.