RyR2 Activity Research Articles

KChIP2 regulates the cardiac Ca2+ transient and myocyte contractility by targeting ryanodine receptor activity.

Pathologic electrical remodeling and attenuated cardiac contractility are featured characteristics of heart failure. Coinciding with these remodeling events is a loss of the K+ channel interacting protein, KChIP2. While, KChIP2 enhances the expression and stability of the Kv4 family of potassium channels, leading to a more pronounced transient outward K+ current, Ito,f, the guinea pig myocardium is unique in that Kv4 expression is absent, while KChIP2 expression is preserved, suggesting alternative consequences to KChIP2 loss. Therefore, KChIP2 was acutely silenced in isolated guinea pig myocytes, which led to significant reductions in the Ca2+ transient amplitude and prolongation of the transient duration. This change was reinforced by a decline in sarcomeric shortening. Notably, these results were unexpected when considering previous observations showing enhanced ICa,L and prolonged action potential duration following KChIP2 loss, suggesting a disruption of fundamental Ca2+ handling proteins. Evaluation of SERCA2a, phospholamban, RyR, and sodium calcium exchanger identified no change in protein expression. However, assessment of Ca2+ spark activity showed reduced spark frequency and prolonged Ca2+ decay following KChIP2 loss, suggesting an altered state of RyR activity. These changes were associated with a delocalization of the ryanodine receptor activator, presenilin, away from sarcomeric banding to more diffuse distribution, suggesting that RyR open probability are a target of KChIP2 loss mediated by a dissociation of presenilin. Typically, prolonged action potential duration and enhanced Ca2+ entry would augment cardiac contractility, but here we see KChIP2 fundamentally disrupts Ca2+ release events and compromises myocyte contraction. This novel role targeting presenilin localization and RyR activity reveals a significance for KChIP2 loss that reflects adverse remodeling observed in cardiac disease settings.

Basal ryanodine receptor activity suppresses autophagic flux

The inositol 1,4,5-trisphosphate receptors (IP3Rs) and intracellular Ca2+ signaling are critically involved in regulating different steps of autophagy, a lysosomal degradation pathway. The ryanodine receptors (RyR), intracellular Ca2+-release channels mainly expressed in excitable cell types including muscle and neurons, have however not yet been extensively studied in relation to autophagy. Yet, aberrant expression and excessive activity of RyRs in these tissues has been implicated in the onset of several diseases including Alzheimer’s disease, where impaired autophagy regulation contributes to the pathology. In this study, we determined whether pharmacological RyR inhibition could modulate autophagic flux in ectopic RyR-expressing models, like HEK293 cells and in cell types that endogenously express RyRs, like C2C12 myoblasts and primary hippocampal neurons. Importantly, RyR3 overexpression in HEK293 cells impaired the autophagic flux. Conversely, in all cell models tested, pharmacological inhibition of endogenous or ectopically expressed RyRs, using dantrolene or ryanodine, augmented autophagic flux by increasing lysosomal turn-over (number of autophagosomes and autolysosomes measured as mCherry-LC3 punctae/cell increased from 70.37±7.81 in control HEK RyR3 cells to 111.18±7.72 and 98.14±7.31 after dantrolene and ryanodine treatments, respectively). Moreover, in differentiated C2C12 cells, transmission electron microscopy demonstrated that dantrolene treatment decreased the number of early autophagic vacuoles from 5.9±2.97 to 1.8±1.03 per cellular cross section. The modulation of the autophagic flux could be linked to the functional inhibition of RyR channels as both RyR inhibitors efficiently diminished the number of cells showing spontaneous RyR3 activity in the HEK293 cell model (from 41.14%±2.12 in control cells to 18.70%±2.25 and 9.74%±2.67 after dantrolene and ryanodine treatments, respectively). In conclusion, basal RyR-mediated Ca2+-release events suppress autophagic flux at the level of the lysosomes.

Calmodulin Regulation of Ryanodine Receptors (RyR2) Depends on RyR2 Phosphorylation at Residues S2808 and S2814

Introduction: Calmodulin (CaM) is a calcium-binding protein that regulates target proteins directly or via their regulatory kinases. CaM binds RyR2 and forms part of the RyR2 macromolecular complex that includes Protein Kinase A (PKA), CaM-dependent kinase II (CaMKII) and the phosphatases, PP1 and PP2A. In the complex, CaM influences SR Ca2+ release either by directly binding RyR2 (inhibition) or by phosphorylation of the RyR2 at S2814 through CaMKII (activation). CaM binding to RyR2 is critical for normal cardiac function and loss of CaM inhibiting efficacy leads to pathological activation of RyR2 that produce spontaneous Ca2+ releases from the SR. These in themselves can trigger miss-timed APs, cardiac arrhythmias and sudden death. Several recent studies have reported that CaM binding affinity to RyR2 is reduced by oxidation of both CaM and RyR2. Here we test the hypothesis that the action of CaM on the activity of RyR2 also depends on the phosphorylation state of the channel. Methods and Results: CaM effect on Ca2+ sparks (and hence RyR2 activity) was tested on permeabilized cardiomyocytes from transgenic mice where CaMKII or PKA phosphorylation sites in RyR2 were ablated (S2814A and S2808A, respectively) or where CaMKII phosphorylation site in RyR2 was fully active (S2814D). RyR2 phosphorylation was confirmed by Western Blot. CaM only inhibited Ca2+ sparks frequency in cardiomyocytes with high phosphorylation levels of RyR2 in CaMKII site (S2814D), in PKA site (S2814A+cAMP or S2808A+cAMP) or in both residues (RyR2 S2814D+cAMP). In contrast, CaM did not have an inhibitory effect in cardiomyocytes with low phosphorylation levels of RyR2 (S2814A and S2808A). Conclusion: Our results show that CaM inhibitory efficacy is dependent on RyR2 phosphorylation in CaMKII and PKA sites S2814 and S2808 in mouse ventricular myocytes.

High-Throughput Screens to Discover Small-Molecule Modulators of Ryanodine Receptor Calcium Release Channels

Using time-resolved fluorescence resonance energy transfer (FRET), we have developed and validated the first high-throughput screening (HTS) method to discover compounds that modulate an intracellular Ca2+ channel, the ryanodine receptor (RyR), for therapeutic applications. Intracellular Ca2+ regulation is critical for striated muscle function, and RyR is a central player. At resting [Ca2+], an increased propensity of channel opening due to RyR dysregulation is associated with severe cardiac and skeletal myopathies, diabetes, and neurological disorders. This leaky state of the RyR is an attractive target for pharmacological agents to treat such pathologies. Our FRET-based HTS detects RyR binding of accessory proteins calmodulin (CaM) or FKBP12.6. Under conditions that mimic a pathological state, we carried out a screen of the 727-compound NIH Clinical Collection, which yielded six compounds that reproducibly changed FRET by >3 SD. Dose–response of FRET and [3H]ryanodine binding readouts reveal that five hits reproducibly alter RyR1 structure and activity. One compound increased FRET and inhibited RyR1, which was only significant at nM [Ca2+], and accentuated without CaM present. These properties characterize a compound that could mitigate RyR1 leak. An excellent Z′ factor and the tight correlation between structural and functional readouts validate this first HTS method to identify RyR modulators.

Novel Approach to Study SERCA Function In Situ

Previously used techniques to measure the sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA) function either are cell destructive or lack sensitivity. We designed a new approach that allows studying SERCA function in the cellular environment under well-controlled conditions. We used the genetically encoded Ca sensor CEPIA-1er to directly measure ER [Ca] ([Ca]ER) in HEK293 cells expressing SERCA2a. These cells were also expressing ryanodine receptor (RyR2) for pharmacological control of ER Ca content. The plasma membrane was permeabilized with saponin to control cytosolic [Ca] and energy supply. HEK293 cells expressing wt-SERCA2a manifested periodic [Ca]ER depletions (Ca waves) due to spontaneous activation of RyR2. Ca waves were rarely seen in cells expressing only RyR2, because endogenous SERCA was unable to increase [Ca]ER to a critical level that triggers spontaneous Ca-induced Ca release (CICR). Application of 10 mM caffeine completely depleted [Ca]ER. Once caffeine was removed, RyR2 inhibitors were applied to measure the rate of [Ca]ER recovery. Thus, ER Ca uptake can be analyzed throughout the whole physiological range of [Ca]ER. ER Ca uptake was progressively decreased with increasing cytosolic [ADP] and can be blocked by thapsigargin. At the end of each experiment, the CEPIA-1er signal was calibrated with ionomycin. Using this approach, we characterized properties of wt- and AAA-SERCA2a (partial loss-of-function SERCA2a mutant). ER Ca uptake as a function of [Ca]ER was analyzed to estimate maximum ER Ca uptake rate and maximum ER Ca load generated by wt and the SERCA mutant. We found that AAA-SERCA2a had slower Ca uptake rate than wt-SERCA2a, as a result HEK293 cells expressing AAA-SERCA2a were not effective to increase ER Ca load to the level that triggers Ca waves. Thus, this new approach can be used as a sensitive screening tool to study different drugs and mutations that affect SERCA function.

Recombinant expression of Intrepicalcin from the scorpion Vaejovis intrepidus and its effect on skeletal ryanodine receptors

BackgroundScorpion venoms contain toxins that modulate ionic channels, among which are the calcins, a small group of short, basic peptides with an Inhibitor Cystine Knot (ICK) motif that target calcium release channels/ryanodine receptors (RyRs) with high affinity and selectivity. Here we describe the heterologous expression of Intrepicalcin, identified by transcriptomic analysis of venomous glands from Vaejovis intrepidus. MethodsRecombinant Intrepicalcin was obtained in Escherichia coli BL21-DE3 (periplasm) by fusing the Intrepicalcin gene to sequences coding for signal-peptide, thioredoxin, His-tag and enterokinase cleavage site. Results[3H]Ryanodine binding, used as a functional index of RyR activity, revealed that recombinant Intrepicalcin activates skeletal RyR (RyR1) dose-dependently with Kd=17.4±4.0nM. Intrepicalcin significantly augments the bell-shaped [Ca2+]-[3H]ryanodine binding curve at all [Ca2+] ranges, as is characteristic of the calcins. In single channel recordings, Intrepicalcin induces the appearance of a subconductance state in RyR1 with a fractional value ∼55% of the full conductance state, very close to that of Vejocalcin. Furthermore, Intrepicalcin stimulates Ca2+ release at an initial dose=45.3±2.5nM, and depletes ~50% of Ca2+ load from skeletal sarcoplasmic reticulum vesicles. ConclusionsWe conclude that active recombinant Intrepicalcin was successfully obtained without the need of manual oxidation, enabling it to target RyR1s with high affinity. General significanceThis is the first calcin heterologously expressed in the periplasma of Escherichia coli BL21-DE3, shown to be pharmacologically effective, thus paving the way for the generation of Intrepicalcin variants that are required for structure-function relationship studies of calcins and RyRs.

Ryanodine receptor modification and regulation by intracellular Ca2 + and Mg2 + in healthy and failing human hearts

RationaleHeart failure is a multimodal disorder, of which disrupted Ca2+ homeostasis is a hallmark. Central to Ca2+ homeostasis is the major cardiac Ca2+ release channel – the ryanodine receptor (RyR2) – whose activity is influenced by associated proteins, covalent modification and by Ca2+ and Mg2+. That RyR2 is remodelled and its function disturbed in heart failure is well recognized, but poorly understood. ObjectiveTo assess Ca2+ and Mg2+ regulation of RyR2 from left ventricles of healthy, cystic fibrosis and failing hearts, and to correlate these functional changes with RyR2 modifications and remodelling. Methods and resultsThe function of RyR2 from left ventricular samples was assessed using lipid bilayer single-channel measurements, whilst RyR2 modification and protein:protein interactions were determined using Western Blots and co-immunoprecipitation. In all failing hearts there was an increase in RyR2 activity at end-diastolic cytoplasmic Ca2+ (100nM), a decreased cytoplasmic [Ca2+] required for half maximal activation (Ka) and a decrease in inhibition by cytoplasmic Mg2+. This was accompanied by significant hyperphosphorylation of RyR2 S2808 and S2814, reduced free thiol content and a reduced interaction with FKBP12.0 and FKBP12.6. Either dephosphorylation of RyR2 using PP1 or thiol reduction using DTT eliminated any significant difference in the activity of RyR2 from healthy and failing hearts. We also report a subgroup of RyR2 in failing hearts that were not responsive to regulation by intracellular Ca2+ or Mg2+. ConclusionDespite different aetiologies, disrupted RyR2 Ca2+ sensitivity and biochemical modification of the channel are common constituents of failing heart RyR2 and may underlie the pathological disturbances in intracellular Ca2+ signalling.

Sensitized signalling between L-type Ca2+ channels and ryanodine receptors in the absence or inhibition of FKBP12.6 in cardiomyocytes.

The heart contraction is controlled by the Ca2+-induced Ca2+ release (CICR) between L-type Ca2+ channels and ryanodine receptors (RyRs). The FK506-binding protein FKBP12.6 binds to RyR subunits, but its role in stabilizing RyR function has been debated for long. Recent reports of high-resolution RyR structure show that the HD2 domain that binds to the SPRY2 domain of neighbouring subunit in FKBP-bound RyR1 is detached and invisible in FKBP-null RyR2. The present study was to test the consequence of FKBP12.6 absence on the in situ activation of RyR2. Using whole-cell patch-clamp combined with confocal imaging, we applied a near threshold depolarization to activate a very small fraction of LCCs, which in turn activated RyR Ca2+ sparks stochastically. FKBP12.6-knockout and FK506/rapamycin treatments increased spark frequency and LCC-RyR coupling fidelity without altering LCC open probability. Neither FK506 nor rapamycin further altered LCC-RyR coupling fidelity in FKBP12.6-knockout cells. In loose-seal patch-clamp experiments, the LCC-RyR signalling kinetics, indexed by the delay for a LCC sparklet to trigger a RyR spark, was accelerated after FKBP12.6 knockout and FK506/rapamycin treatments. These results demonstrated that RyRs became more sensitive to Ca2+ triggers without FKBP12.6. Isoproterenol (1 μM) further accelerated the LCC-RyR signalling in FKBP12.6-knockout cells. The synergistic sensitization of RyRs by catecholaminergic signalling and FKBP12.6 dysfunction destabilized the CICR system, leading to chaotic Ca2+ waves and ventricular arrhythmias. FKBP12.6 keeps the RyRs from over-sensitization, stabilizes the potentially regenerative CICR system, and thus may suppress the life-threatening arrhythmogenesis.

Arrhythmogenic ATP-Based Kinase Inhibitors Directly Increase the Activity of RyR2

Spontaneous Ca2+ release via cardiac ryanodine receptors (RyR2) can occur under conditions of SR Ca2+ overload, a process termed store-overload–induced Ca2+ release (SOICR), and is known trigger for arrhythmia. ATP-based kinase inhibitors are a class of drugs increasingly used to treat cancer. Several of these compounds have been linked to arrhythmias, but the underlying mechanism is unknown. ATP is an agonist for RyR2, therefore we hypothesised that the arrhythmogenic nature of ATP-based kinase inhibitors is due to an “ATP-like” activation of RyR2 leading to SOICR.

From the Cover: BDE-47 and BDE-49 Inhibit Axonal Growth in Primary Rat Hippocampal Neuron-Glia Co-Cultures via Ryanodine Receptor-Dependent Mechanisms.

Polybrominated diphenyl ethers (PBDEs) are widespread environmental contaminants associated with adverse neurodevelopmental outcomes in children and preclinical models; however, the mechanisms by which PBDEs cause developmental neurotoxicity remain speculative. The structural similarity between PBDEs and nondioxin-like (NDL) polychlorinated biphenyls (PCBs) suggests shared toxicological properties. Consistent with this, both NDL PCBs and PBDEs have been shown to stabilize ryanodine receptors (RyRs) in the open configuration. NDL PCB effects on RyR activity are causally linked to increased dendritic arborization, but whether PBDEs similarly enhance dendritic growth is not known. In this study, we quantified the effects of individual PBDE congeners on not only dendritic but also axonal growth since both are regulated by RyR-dependent mechanisms, and both are critical determinants of neuronal connectivity. Neuronal-glial co-cultures dissociated from the neonatal rat hippocampus were exposed to BDE-47 or BDE-49 in the culture medium. At concentrations ranging from 20 pM to 2 µM, neither PBDE congener altered dendritic arborization. In contrast, at concentrations ≥ 200 pM, both congeners delayed neuronal polarization resulting in significant inhibition of axonal outgrowth during the first few days in vitro. The axon inhibitory effects of these PBDE congeners occurred independent of cytotoxicity, and were blocked by pharmacological antagonism of RyR or siRNA knockdown of RyR2. These results demonstrate that the molecular and cellular mechanisms by which PBDEs interfere with neurodevelopment overlap with but are distinct from those of NDL PCBs, and suggest that altered patterns of neuronal connectivity may contribute to the developmental neurotoxicity of PBDEs.

Enhanced Cytosolic Ca2+ Activation Underlies a Common Defect of Central Domain Cardiac Ryanodine Receptor Mutations Linked to Arrhythmias

Recent three-dimensional structural studies reveal that the central domain of ryanodine receptor (RyR) serves as a transducer that converts long-range conformational changes into the gating of the channel pore. Interestingly, the central domain encompasses one of the mutation hotspots (corresponding to amino acid residues 3778–4201) that contains a number of cardiac RyR (RyR2) mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) and atrial fibrillation (AF). However, the functional consequences of these central domain RyR2 mutations are not well understood. To gain insights into the impact of the mutation and the role of the central domain in channel function, we generated and characterized eight disease-associated RyR2 mutations in the central domain. We found that all eight central domain RyR2 mutations enhanced the Ca2+-dependent activation of [3H]ryanodine binding, increased cytosolic Ca2+-induced fractional Ca2+ release, and reduced the activation and termination thresholds for spontaneous Ca2+ release in HEK293 cells. We also showed that racemic carvedilol and the non-beta-blocking carvedilol enantiomer, (R)-carvedilol, suppressed spontaneous Ca2+ oscillations in HEK293 cells expressing the central domain RyR2 mutations associated with CPVT and AF. These data indicate that the central domain is an important determinant of cytosolic Ca2+ activation of RyR2. These results also suggest that altered cytosolic Ca2+ activation of RyR2 represents a common defect of RyR2 mutations associated with CPVT and AF, which could potentially be suppressed by carvedilol or (R)-carvedilol.

Suppression of ryanodine receptor function prolongs Ca2+ release refractoriness and promotes cardiac alternans in intact hearts.

Beat-to-beat alternations in the amplitude of the cytosolic Ca2+ transient (Ca2+ alternans) are thought to be the primary cause of cardiac alternans that can lead to cardiac arrhythmias and sudden death. Despite its important role in arrhythmogenesis, the mechanism underlying Ca2+ alternans remains poorly understood. Here, we investigated the role of cardiac ryanodine receptor (RyR2), the major Ca2+ release channel responsible for cytosolic Ca2+ transients, in cardiac alternans. Using a unique mouse model harboring a suppression-of-function (SOF) RyR2 mutation (E4872Q), we assessed the effect of genetically suppressing RyR2 function on Ca2+ and action potential duration (APD) alternans in intact hearts, and electrocardiogram (ECG) alternans in vivo We found that RyR2-SOF hearts displayed prolonged sarcoplasmic reticulum Ca2+ release refractoriness and enhanced propensity for Ca2+ alternans. RyR2-SOF hearts/mice also exhibited increased propensity for APD and ECG alternans. Caffeine, which enhances RyR2 activity and the propensity for catecholaminergic polymorphic ventricular tachycardia (CPVT), suppressed Ca2+ alternans in RyR2-SOF hearts, whereas carvedilol, a β-blocker that suppresses RyR2 activity and CPVT, promoted Ca2+ alternans in these hearts. Thus, RyR2 function is an important determinant of Ca2+, APD, and ECG alternans. Our data also indicate that the activity of RyR2 influences the propensity for cardiac alternans and CPVT in an opposite manner. Therefore, overly suppressing or enhancing RyR2 function is pro-arrhythmic.

An Extended Structure-Activity Relationship of Nondioxin-Like PCBs Evaluates and Supports Modeling Predictions and Identifies Picomolar Potency of PCB 202 Towards Ryanodine Receptors.

Nondioxin-like polychlorinated biphenyls (NDL PCBs) activate ryanodine-sensitive Ca2+ channels (RyRs) and this activation has been associated with neurotoxicity in exposed animals. RyR-active congeners follow a distinct structure-activity relationship and a quantitative structure-activity relationship (QSAR) predicts that a large number of PCBs likely activate the receptor, which requires validation. Additionally, previous structural based conclusions have been established using receptor ligand binding assays but the impact of varying PCB structures on ion channel gating behavior is not understood. We used [3H]Ryanodine ([3H]Ry) binding to assess the RyR-activity of 14 previously untested PCB congeners evaluating the predictability of the QSAR. Congeners determined to display widely varying potency were then assayed with single channel voltage clamp analysis to assess direct influences on channel gating kinetics. The RyR-activity of individual PCBs assessed in in vitro assays followed the general pattern predicted by the QSAR but binding and lipid bilayer experiments demonstrated higher potency than predicted. Of the 49 congeners tested to date, tetra-ortho PCB 202 was found to be the most potent RyR-active congener increasing channel open probability at 200 pM. Shifting meta-substitutions to the para-position resulted in a > 100-fold reduction in potency as seen with PCB 197. Non-ortho PCB 11 was found to lack activity at the receptor supporting a minimum mono-ortho substitution for PCB RyR activity. These findings expand and support previous SAR assessments; where out of the 49 congeners tested to date 42 activate the receptor demonstrating that the RyR is a sensitive and common target of PCBs.

Three residues in the luminal domain of triadin impact on Trisk 95 activation of skeletal muscle ryanodine receptors.

Triadin isoforms, splice variants of one gene, maintain healthy Ca2+ homeostasis in skeletal muscle by subserving several functions including an influence on Ca2+ release through the ligand-gated ryanodine receptor (RyR1) ion channels. The predominant triadin isoform in skeletal muscle, Trisk 95, activates RyR1 in vitro via binding to previously unidentified amino acids between residues 200 and 232. Here, we identify three amino acids that influence Trisk 95 binding to RyR1 and ion channel activation, using peptides encompassing residues 200-232. Selective alanine substitutions show that K218, K220, and K224 together facilitate normal Trisk 95 binding to RyR1 and channel activation. Neither RyR1 binding nor activation are altered by alanine substitution of K220 alone or of K218 and K224. Therefore K218, K220, and K224 contribute to a robust binding and activation site that is disrupted only when the charge on all three residues is neutralized. We suggest that charged pair interactions between acidic RyR1 residues D4878, D4907, and E4908 and Trisk 95 residues K218, K220, and K224 facilitate Trisk 95 binding to RyR1 and channel activation. Since K218, K220, and K224 are also required for CSQ binding to RyRs (Kobayashi et al. 17, J Biol Chem 275, 17639-17646), the results suggest that Trisk 95 may not simultaneously bind to RyR1 and CSQ, contrary to the widely held belief that triadin monomers form a quaternary complex with junctin, CSQ and RyR1. Therefore, the in vivo role of triadin monomers in modulating RyR1 activity is likely unrelated to CSQ.

Structural Basis for Gating and Activation of RyR1

The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca(2+)) release channel required for skeletal muscle contraction. Here, we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca(2+), ATP, and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca(2+) alone induces conformational changes in the cytoplasmic assembly ("priming"), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated withgating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement, and deformation of the S4-S5 linker and conformational changes in the pseudo-voltage-sensor domain.

Real-time intra-store confocal Ca2+ imaging in isolated mouse cardiomyocytes

To initiate the contraction of cardiomyocytes, Ca2+ is released from the SR to the cytosol via ryanodine receptors (RyRs), which are activated by the Ca2+–induced Ca2+ release mechanism (CICR). The activity of RyRs is regulated by both, cytosolic and SR luminal Ca2+. Deregulation of the CICR, by dysfunctional SR Ca2+ release or uptake, is frequently associated with cardiac pathologies (e.g. arrhythmias, CPVT, heart failure). Recently, the interest to directly measure changes of the free Ca2+ concentration within the SR ([Ca2+]SR) has led to the application of low affinity Ca2+ indicators (mag-fluo-4, Fluo-5N) to follow changes of [Ca2+]SR in cardiomyocytes from some species. However, direct measurement of Ca2+ signals from the SR have not been possible in freshly isolated mouse cardiomyocytes. Here, we show a new protocol optimized to measure changes of [Ca2+]SR in mouse cardiomyocytes using fluorescent Ca2+ indicators and confocal microscopy. The application of this protocol permits the design of experimental studies with direct evaluation of SR Ca2+ in real time in various mouse models of cardiac disease, including transgenic animals harboring mutants of RyRs or other Ca2+ signaling proteins. The technique, in combination with these models, will help to understand how these diseases and mutations affect Ca2+ signals within the SR and the Ca2+ sensitivity of the RyRs for cytosolic and SR luminal Ca2+, thereby contributing to arrhythmias or weak heart beat.

Calcium/calmodulin-dependent kinase II and nitric oxide synthase 1-dependent modulation of ryanodine receptors during β-adrenergic stimulation is restricted to the dyadic cleft.

Key points The dyadic cleft, where coupled ryanodine receptors (RyRs) reside, is thought to serve as a microdomain for local signalling, as supported by distinct modulation of coupled RyRs dependent on Ca2+/calmodulin‐dependent kinase II (CaMKII) activation during high‐frequency stimulation.Sympathetic stimulation through β‐adrenergic receptors activates an integrated signalling cascade, enhancing Ca2+ cycling and is at least partially mediated through CaMKII.Here we report that CaMKII activation during β‐adrenergic signalling is restricted to the dyadic cleft, where it enhances activity of coupled RyRs thereby contributing to the increase in diastolic events. Nitric oxide synthase 1 equally participates in the local modulation of coupled RyRs.In contrast, the increase in the Ca2+ content of the sarcoplasmic reticulum and related increase in the amplitude of the Ca2+ transient are primarily protein kinase A‐dependent.The present data extend the concept of microdomain signalling in the dyadic cleft and give perspectives for selective modulation of RyR subpopulations and diastolic events. In cardiac myocytes, β‐adrenergic stimulation enhances Ca2+ cycling through an integrated signalling cascade modulating L‐type Ca2+ channels (LTCCs), phospholamban and ryanodine receptors (RyRs). Ca2+/calmodulin‐dependent kinase II (CaMKII) and nitric oxide synthase 1 (NOS1) are proposed as prime mediators for increasing RyR open probability. We investigate whether this pathway is confined to the high Ca2+ microdomain of the dyadic cleft and thus to coupled RyRs. Pig ventricular myocytes are studied under whole‐cell voltage‐clamp and confocal line‐scan imaging with Fluo‐4 as a [Ca2+]i indicator. Following conditioning depolarizing pulses, spontaneous RyR activity is recorded as Ca2+ sparks, which are assigned to coupled and non‐coupled RyR clusters. Isoproterenol (ISO) (10 nm) increases Ca2+ spark frequency in both populations of RyRs. However, CaMKII inhibition reduces spark frequency in coupled RyRs only; NOS1 inhibition mimics the effect of CaMKII inhibition. Moreover, ISO induces the repetitive activation of coupled RyR clusters through CaMKII activation. Immunostaining shows high levels of CaMKII phosphorylation at the dyadic cleft. CaMKII inhibition reduces I CaL and local Ca2+ transients during depolarizing steps but has only modest effects on amplitude or relaxation of the global Ca2+ transient. In contrast, protein kinase A (PKA) inhibition reduces spark frequency in all RyRs concurrently with a reduction of sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude and relaxation. In conclusion, CaMKII activation during β‐adrenergic stimulation is restricted to the dyadic cleft microdomain, enhancing LTCC‐triggered local Ca2+ release as well as spontaneous diastolic Ca2+ release whilst PKA is the major pathway increasing global Ca2+ cycling. Selective CaMKII inhibition may reduce potentially arrhythmogenic release without negative inotropy.

Oxidation of RyR2 Has a Biphasic Effect on the Threshold for Store Overload-Induced Calcium Release

At the single-channel level, oxidation of the cardiac ryanodine receptor (RyR2) is known to activate and inhibit the channel depending on the level of oxidation. However, the mechanisms through which these changes alter the activity of RyR2 in a cellular setting are poorly understood. In this study, we determined the effect of oxidation on a common form of RyR2 regulation; store overload-induced Ca2+ release (SOICR). We found that oxidation resulted in concentration and time-dependent changes in the activation threshold for SOICR. Low concentrations of the oxidant H2O2 resulted in a decrease in the threshold for SOICR, which led to an increase in SOICR events. However, higher concentrations of H2O2, or prolonged exposure, reversed these changes and led to an increase in the threshold for SOICR. This increase in the threshold for SOICR in most cells was to such an extent that it led to the complete inhibition of SOICR. Acute exposure to high concentrations of H2O2 led to an initial decrease and then increase in the threshold for SOICR. In the majority of cells the increased threshold could not be reversed by the application of the reducing agent dithiothreitol. Therefore, our data suggest that low levels of RyR2 oxidation increase the channel activity by decreasing the threshold for SOICR, whereas high levels of RyR2 oxidation irreversibly increase the threshold for SOICR leading to an inhibition of RyR2. Combined, this indicates that oxidation regulates RyR2 by the same mechanism as phosphorylation, methylxanthines, and mutations, via changes in the threshold for SOICR.

Role of SERCA and the sarcoplasmic reticulum calcium content on calcium waves propagation in rat ventricular myocytes

In Ca2+-overloaded ventricular myocytes, SERCA is crucial to steadily achieve the critical sarcoplasmic reticulum (SR) Ca2+ level to trigger and sustain Ca2+ waves, that propagate at constant rate (ʋwave). High luminal Ca2+ sensitizes RyR2, thereby increasing Ca2+ sparks frequency, and the larger RyR2-mediated SR Ca2+ flux (dF/dt) sequentially activates adjacent RyR2 clusters. Recently, it was proposed that rapid SERCA Ca2+ reuptake, ahead of the wave front, further sensitizes RyR2, increasing ʋwave. Nevertheless, this is controversial because rapid cytosolic Ca2+ removal could instead impair RyR2 activation. We assessed whether rapid SR Ca2+ uptake enhances ʋwave by changing SERCA activity (ҡDecay) over a large range (∼175%). We used normal (Ctrl) and hyperthyroid rat (HT; reduced phospholamban by ∼80%) myocytes treated with thapsigargin or isoproterenol (ISO). We found that ʋwave and dF/dt had a non-linear dependency with ҡDecay, while Ca2+ waves amplitude was largely unaffected. Furthermore, SR Ca2+ also showed a non-linear dependency with ҡDecay, however, the relationships ʋwave vs. SR Ca2+ and ʋwave vs. dF/dt were linear, suggesting that high steady state SR Ca2+ determines ʋwave, while rapid SERCA Ca2+ uptake does not. Finally, ISO did not increase ʋwave in HT cells, therefore, ISO-enhanced ʋwave in Ctrl depended on high SR Ca2+.

Novel Anthranilic Diamide Scaffolds Containing N-Substituted Phenylpyrazole as Potential Ryanodine Receptor Activators.

To discover potent insecticides targeting ryanodine receptors (RyRs), a series of novel anthranilic diamides analogues (12a-12u) containing N-substituted phenylpyrazole were designed and synthesized. These compounds were characterized by (1)H NMR, (13)C NMR, and HRMS, and the structure of compound 12u was confirmed by X-ray diffraction. Their insecticidal activities indicated that these compounds displayed moderate to excellent activities. In particular, 12i showed 100 and 37% larvicidal activities against oriental armyworm (Mythimna separata) at 0.25 and 0.05 mg L(-1), equivalent to that of chlorantraniliprole (100%, 0.25 mg L(-1); and 33%, 0.05 mg L(-1)). The activity of 12i against diamondback moth (Plutella xylostella) was 95% at 0.05 mg L(-1), whereas the control was 100% at 0.05 mg L(-1). The calcium-imaging technique experiment results showed that the effects of 12i on the intracellular calcium ion concentration ([Ca(2+)]i) in neurons were concentration-dependent. After the central neurons of Helicoverpa armigera were dyed by loading with fluo-5N and treated with 12i, the free calcium released in endoplasmic reticulum indicated the target of compound 12i is RyRs or IP3Rs. The activation of RyRs by natural ryanodine completely blocked the calcium release induced by 12i, which indicated that RyRs in the central neurons of H.armigera third-instar larvae is the possible target of compound 12i.