Inhibition Of RyRs Research Articles

High-Throughput Screening Yields Allosteric Inhibitors of Leaky RyRs for Therapeutic Development

Using fluorescence lifetime detection of FRET, we have developed and validated high-throughput screening (HTS) assays to discover compounds that modulate the ryanodine receptor (RyR) calcium release channel, for therapeutic development. Intracellular calcium regulation is critical for striated muscle function, and RyR is a central player. Under resting calcium, 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 a high-value target for pharmacological agents to treat such pathologies. Our FRET-based HTS detects RyR binding of accessory proteins calmodulin or FKBP12.6. Under conditions that mimic a pathological state, we have carried out pilot screens of the 1280-compound library of pharmaceutically active compounds (LOPAC), and then progressed to larger screens of a 50k-compound library, to identify modulators of RyR in either skeletal or cardiac SR membrane preparations. These screens yielded compounds (hits) that are specific for either RyR1 (skeletal isoform) or RyR2 (cardiac isoform), or that act on both RyR1 and RyR2. Ongoing studies indicate good correlation between the FRET (structural) readout for the hits and their effects on RyR function in isolated sarcoplasmic reticulum, skeletal myofibers, and adult ventricular myocytes. This work is supported by NIH grants R01HL092097 and R01HL138539 (RLC/DMB) and R37AG26160 (DDT), and by American Heart Association Postdoctoral Fellowship 16POST31010019 (RTR).

Ablation of phospholamban rescues reperfusion arrhythmias but exacerbates myocardium infarction in hearts with Ca2+/calmodulin kinase II constitutive phosphorylation of ryanodine receptors.

Abnormal Ca2+ release from the sarcoplasmic reticulum (SR), associated with Ca2+-calmodulin kinase II (CaMKII)-dependent phosphorylation of RyR2 at Ser2814, has consistently been linked to arrhythmogenesis and ischaemia/reperfusion (I/R)-induced cell death. In contrast, the role played by SR Ca2+ uptake under these stress conditions remains controversial. We tested the hypothesis that an increase in SR Ca2+ uptake is able to attenuate reperfusion arrhythmias and cardiac injury elicited by increased RyR2-Ser2814 phosphorylation. We used WT mice, which have been previously shown to exhibit a transient increase in RyR2-Ser2814 phosphorylation at the onset of reperfusion; mice with constitutive pseudo-phosphorylation of RyR2 at Ser2814 (S2814D) to exacerbate CaMKII-dependent reperfusion arrhythmias and cardiac damage, and phospholamban (PLN)-deficient-S2814D knock-in (SDKO) mice resulting from crossbreeding S2814D with phospholamban knockout deficient (PLNKO) mice. At baseline, S2814D and SDKO mice had structurally normal hearts. Moreover none of the strains were arrhythmic before ischaemia. Upon cardiac I/R, WT, and S2814D hearts exhibited abundant arrhythmias that were prevented by PLN ablation. In contrast, PLN ablation increased infarct size compared with WT and S2814D hearts. Mechanistically, the enhanced SR Ca2+ sequestration evoked by PLN ablation in SDKO hearts prevented arrhythmogenic events upon reperfusion by fragmenting SR Ca2+ waves into non-propagated and non-arrhythmogenic events (mini-waves). Conversely, the increase in SR Ca2+ sequestration did not reduce but rather exacerbated I/R-induced SR Ca2+ leak, as well as mitochondrial alterations, which were greatly avoided by inhibition of RyR2. These results indicate that the increase in SR Ca2+ uptake is ineffective in preventing the enhanced SR Ca2+ leak of PLN ablated myocytes from either entering into nearby mitochondria and/or activating additional CaMKII pathways, contributing to cardiac damage. Our results demonstrate that increasing SR Ca2+ uptake by PLN ablation can prevent the arrhythmic events triggered by CaMKII-dependent phosphorylation of RyR2-induced SR Ca2+ leak. These findings underscore the benefits of increasing SERCA2a activity in the face of SR Ca2+ triggered arrhythmias. However, enhanced SERCA2a cannot prevent but rather exacerbates I/R cardiac injury.

Efficient High-Throughput Screening by Endoplasmic Reticulum Ca2+ Measurement to Identify Inhibitors of Ryanodine Receptor Ca2+-Release Channels.

Genetic mutations in ryanodine receptors (RyRs), Ca2+-release channels in the sarcoplasmic reticulum essential for muscle contractions, cause various skeletal muscle and cardiac diseases. Because the main underlying mechanism of the pathogenesis is overactive Ca2+ release by gain-of-function of the RyR channel, inhibition of RyRs is expected to be a promising treatment of these diseases. Here, to identify inhibitors specific to skeletal muscle type 1 RyR (RyR1), we developed a novel high-throughput screening (HTS) platform using time-lapse fluorescence measurement of Ca2+ concentrations in the endoplasmic reticulum (ER) ([Ca2+]ER). Because expression of RyR1 carrying disease-associated mutation reduces [Ca2+]ER in HEK293 cells through Ca2+ leakage from RyR1 channels, specific drugs that inhibit RyR1 will increase [Ca2+]ER by preventing such Ca2+ leakage. RyR1 carrying the R2163C mutation and R-CEPIA1er, a genetically encoded ER Ca2+ indicator, were stably expressed in HEK293 cells, and time-lapse fluorescence was measured using a fluorometer. False positives were effectively excluded by using cells expressing wild-type (WT) RyR1. By screening 1535 compounds in a library of well characterized drugs, we successfully identified four compounds that significantly increased [Ca2+]ER They include dantrolene, a known RyR1 inhibitor, and three structurally different compounds: oxolinic acid, 9-aminoacridine, and alexidine. All the hit compounds, except for oxolinic acid, inhibited [3H]ryanodine binding of WT and mutant RyR1. Interestingly, they showed different dose dependencies and isoform specificities. The highly quantitative nature and good correlation with the channel activity validated this HTS platform by [Ca2+]ER measurement to explore drugs for RyR-related diseases.

High-throughput Screening Yields Allosteric Inhibitors of Leaky RyRS for Therapeutic Applications

High-throughput Screening Yields Allosteric Inhibitors of Leaky RyRS for Therapeutic Applications

RyR2 Inhibition by Dantrolene Requires both Calmodulin and FKBP12.6

Dantrolene protects cardiac muscle from arrhythmia and heart failure by inhibiting Ca2+ release from the sarcoplasmic reticulum (SR). Single-channel recordings were obtained from SR calcium release channels (RyR2) isolated from sheep and human heart and incorporated into artificial lipid bilayers. Dantrolene inhibition was measured by exposing RyR2 both in the absence and presence of exogenous 100 nM CaM. In diastolic cytoplasmic [Ca2+] (100 nM), dantrolene (50 μM) reduced the mean open probability of RyR2 to 45 ± 6% in the presence of CaM (n = 7, p<0.05, Student's paired t-test) but had no significant effect when CaM was absent (95 ± 9%, n = 20, p = 0.24). Dantrolene exhibited a hyperbolic dose response in the presence of CaM with an IC50 of 0.16 ± 0.03 μM and with a saturating relative Po (Emax) of 52 ± 4%. Emax increased to 1 as cytoplasmic Ca2+ was increased to levels >1 μM. In saponin-permeablised mouse cardiomyocytes supplemented with 100 nM CaM, dantrolene reduced the frequency and amplitude of Ca2+ waves, with an IC50 of 0.3 μM. However, when cells were depleted of CaM, dantrolene had no effect. Thus, CaM is essential for inhibitory action of dantrolene. Incubation of RyR2 to rapamycin prior to single-channel recording, known to dissociate FKBP12.6 from RyR2, removed the inhibitory action of dantrolene. This could be restored by adding FKBP12.6 to the bathing media. We conclude that the dantrolene inhibition required the presence of both CaM and FKBP12.6 in the RyR2 complex.

Electropermeabilization of Inner and Outer Cell Membranes with Microsecond Pulsed Electric Fields: Quantitative Study with Calcium Ions

Microsecond pulsed electric fields (μsPEF) permeabilize the plasma membrane (PM) and are widely used in research, medicine and biotechnology. For internal membranes permeabilization, nanosecond pulsed electric fields (nsPEF) are applied but this technology is complex to use. Here we report that the endoplasmic reticulum (ER) membrane can also be electropermeabilized by one 100 µs pulse without affecting the cell viability. Indeed, using Ca2+ as a permeabilization marker, we observed cytosolic Ca2+ peaks in two different cell types after one 100 µs pulse in a medium without Ca2+. Thapsigargin abolished these Ca2+ peaks demonstrating that the calcium is released from the ER. Moreover, IP3R and RyR inhibitors did not modify these peaks showing that they are due to the electropermeabilization of the ER membrane and not to ER Ca2+ channels activation. Finally, the comparison of the two cell types suggests that the PM and the ER permeabilization thresholds are affected by the sizes of the cell and the ER. In conclusion, this study demonstrates that µsPEF, which are easier to control than nsPEF, can permeabilize internal membranes. Besides, μsPEF interaction with either the PM or ER, can be an efficient tool to modulate the cytosolic calcium concentration and study Ca2+ roles in cell physiology.

Two EF-hand motifs in ryanodine receptor calcium release channels contribute to isoform-specific regulation by calmodulin

The mammalian ryanodine receptor Ca2+ release channel (RyR) has a single conserved high affinity calmodulin (CaM) binding domain. However, the skeletal muscle RyR1 is activated and cardiac muscle RyR2 is inhibited by CaM at submicromolar Ca2+. This suggests isoform-specific domains are involved in RyR regulation by CaM. To gain insight into the differential regulation of cardiac and skeletal muscle RyRs by CaM, RyR1/RyR2 chimeras and mutants were expressed in HEK293 cells, and their single channel activities were measured using a lipid bilayer method. All RyR1/RyR2 chimeras and mutants were inhibited by CaM at 2μM Ca2+, consistent with CaM inhibition of RyR1 and RyR2 at micromolar Ca2+ concentrations. An RyR1/RyR2 chimera with RyR1 N-terminal amino acid residues (aa) 1–3725 and RyR2 C-terminal aa 3692–4968 were inhibited by CaM at <1μM Ca2+ similar to RyR2. In contrast, RyR1/RyR2 chimera with RyR1 aa 1–4301 and RyR2 4254–4968 was activated at <1μM Ca2+ similar to RyR1. Replacement of RyR1 aa 3726–4298 with corresponding residues from RyR2 conferred CaM inhibition at <1μM Ca2+, which suggests RyR1 aa 3726–4298 are required for activation by CaM. Characterization of additional RyR1/RyR2 chimeras and mutants in two predicted Ca2+ binding motifs in RyR1 aa 4081–4092 (EF1) and aa 4116–4127 (EF2) suggests that both EF-hand motifs and additional sequences in the large N-terminal regions are required for isoform-specific RyR1 and RyR2 regulation by CaM at submicromolar Ca2+ concentrations.

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.

Mg2+ Inhibits Cardiac SR Calcium Release and has Biphasic Effects on Calmodulin Binding to RyR2

Mg2+ is a potent inhibitor of RyR channel gating, as demonstrated in lipid bilayer and SR vesicles. Less is known about Mg2+ effects on RyR2 function in the cardiac myocyte environment or how it might alter RyR2 interaction with accessory proteins like calmodulin (CaM), which by itself binds to, and inhibits RyR2 opening. We used permeabilized mouse ventricular cardiomyocytes to study the effects of Mg2+ on spontaneous Ca2+ release and CaM affinity for RyR2. We recorded Ca sparks at three [Mg2+]: 0.1 mM (low), 1 mM (physiological), and 3 mM (high). We found that in the presence of endogenous WT-CaM, with increasing [Mg2+], Ca spark frequency (CaSpF) decreased monotonically, and was accompanied by an increase in Ca spark amplitude and SR Ca2+ load. To test whether Mg2+ also modulates RyR2 interaction with accessory proteins, we measured binding affinity of WT-CaM and two CPVT-linked CaM mutants (N54I and N98S). The apparent affinity of WT-CaM for RyR2 in myocytes was highest at 1 mM Mg2+ and decreased at both 0.1 mM and 3 mM Mg2+. The same pattern was found for N54I- and N98S-CaM. We conclude that in cardiomyocytes, Mg2+ decreases CaSpF and increases SR Ca2+ load. As [Mg2+] increases in the physiologically range (0.1 - 1 mM), part of the Ca spark inhibition may be due to enhanced CaM binding to RyR2. In contrast, pathophysiologically high [Mg2+] (3 mM) may (1) limit RyR2-CaM binding (and thus CaM inhibition of RyR2) and (2) still directly inhibit RyR2 opening.

The Arrhythmogenic Calmodulin p.Phe142Leu Mutation Impairs C-domain Ca2+ Binding but Not Calmodulin-dependent Inhibition of the Cardiac Ryanodine Receptor

A number of point mutations in the intracellular Ca2+-sensing protein calmodulin (CaM) are arrhythmogenic, yet their underlying mechanisms are not clear. These mutations generally decrease Ca2+ binding to CaM and impair inhibition of CaM-regulated Ca2+ channels like the cardiac Ca2+ release channel (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca2+ binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start-Met), which markedly reduces CaM Ca2+ binding. Surprisingly, CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca2+ release in HEK293 cells compared with CaM-WT. Furthermore, CaM-F142L enhanced CaM-dependent RyR2 inhibition at the single channel level compared with CaM-WT. This is in stark contrast to the actions of arrhythmogenic CaM mutations N54I, D96V, N98S, and D130G, which all diminish CaM-dependent RyR2 inhibition. Thermodynamic analysis showed that apoCaM-F142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover, NMR spectra revealed that the CaM-F142L-CaMBD interaction is structurally different from that of CaM-WT at low Ca2+. These data indicate a distinct interaction between CaM-F142L and the RyR2 CaMBD, which may explain the stronger CaM-dependent RyR2 inhibition by CaM-F142L, despite its reduced Ca2+ binding. Collectively, these results add to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca2+ release by manipulating the CaM-RyR2 interaction.

The GSTM2 C-Terminal Domain Depresses Contractility and Ca2+ Transients in Neonatal Rat Ventricular Cardiomyocytes.

The cardiac ryanodine receptor (RyR2) is an intracellular ion channel that regulates Ca2+ release from the sarcoplasmic reticulum (SR) during excitation–contraction coupling in the heart. The glutathione transferases (GSTs) are a family of phase II detoxification enzymes with additional functions including the selective inhibition of RyR2, with therapeutic implications. The C-terminal half of GSTM2 (GSTM2C) is essential for RyR2 inhibition, and mutations F157A and Y160A within GSTM2C prevent the inhibitory action. Our objective in this investigation was to determine whether GSTM2C can enter cultured rat neonatal ventricular cardiomyocytes and influence contractility. We show that oregon green-tagged GSTM2C (at 1 μM) is internalized into the myocytes and it reduces spontaneous contraction frequency and myocyte shortening. Field stimulation of myocytes evoked contraction in the same percentage of myocytes treated either with media alone or media plus 15 μM GSTM2C. Myocyte shortening during contraction was significantly reduced by exposure to 15 μM GSTM2C, but not 5 and 10 μM GSTM2C and was unaffected by exposure to 15 μM of the mutants Y160A or F157A. The amplitude of the Ca2+ transient in the 15 μM GSTM2C - treated myocytes was significantly decreased, the rise time was significantly longer and the decay time was significantly shorter than in control myocytes. The Ca2+ transient was not altered by exposure to Y160A or F157A. The results are consistent with GSTM2C entering the myocytes and inhibiting RyR2, in a manner that indicates a possible therapeutic potential for treatment of arrhythmia in the neonatal heart.

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.

Dantrolene Inhibition of Skeletal Muscle RYR in the Presence of CAM

Dantrolene is a muscle relaxant that has been used clinically as the treatment for malignant hyperthermia (MH). Our group recently reported that dantrolene inhibition of RyR1 and RyR2 required calmodulin (CaM)1. However, nothing more is known about the properties of dantrolene inhibition of RyR1 on single channels. Here, we characterized dantrolene inhibition on RyR1 in the presence of exogenous CaM at various cytosolic [Ca2+] and in the presence of other activators: ATP, halothane and domain peptide, DP4. RyR1 was isolated from rabbit skeletal muscle and incorporated into artificial lipid bilayers and their open probability, Po, was measured using single channel recording. Dantrolene caused inhibition of RyR1 and RyR2, but when CaM was presence in the cytoplasmic bath. In the presence of cytoplasmic [Ca2+] (100 nmol/l) and 2 mmol/l ATP, dantrolene inhibited both RyR1 and RyR2 with identical hyperbolic dose-responses with IC50 of 0.16 ± 0.03 μmol/l and with saturating Po of 52 ± 4 % of control. RyR1 has a bell-shaped cytoplasmic Ca2+ activation curve with half activation at 1.75 ± 0.62 μmol/l and half inhibition at 0.21 ± 0.02 mmol/l and a peak Po = 0.94 ± 0.06. Dantrolene reduced Po in sub-activating and inhibiting [Ca2+] but failed to reduce peak Po. Dantrolene caused 50-60% reduction in Po of RyR1 activated by cytoplasmic Ca2+ alone (100 nmol/l) or in conjunction with halothane (5 mmol/l) or DP4 (10 μmol/l). To conclude, dantrolene inhibits RyR1 activity by 1) decreasing RyR1 sensitivity to Ca2+ activation, 2) increasing sensitivity to Ca2+ inhibition and 3) decreasing channel activation halothane or by 4) reducing the effect of inter-domain disruption in the RyR1 protein.

Engineering an Anti-Arrhythmic Calmodulin

The cardiac ryanodine receptor (RYR2) is a critical Ca²+ handling protein. Several post-translational modifications, mutations, and altered interactions between RYR2 and its regulatory proteins can all increase the RYR2 open probability (P0), disrupting normal Ca²+ handling, leading to arrhythmia. There is a great need for therapeutic strategies that decrease RYR2 P0. Normally, calmodulin (CaM) decreases RYR2 P0, decreasing Ca²+ sparks and waves. Naturally occurring mutations in CaM have recently been identified that instead increase RYR2 P0 and cause arrhythmia. Using these CaM mutations as tools, we have discovered that the N-terminal rate of Ca²+ dissociation from the CaM-RYR2 complex is an important factor that modulates RYR2 inhibition by CaM. That is, the longer Ca²+ remains bound to the CaM-RYR2 complex the longer RYR2 remains refractory, ultimately influencing the frequency of cardiac myocyte sparks and waves. We hypothesize that slowing the N-terminal Ca²+ dissociation rate from CaM bound to RYR2 will decrease RYR2 P0 and prevent arrhythmias. Using both rational engineering (GSH M37Q CaM) and nature's design principles (soybean CaM4) to sensitize CaM to Ca²+, we are attempting to create therapeutic CaMs against defective RYR2 regulation. Excitingly, our therapeutic GSH M37Q CaM decreases the arrhythmogenic potential of myocytes isolated from the pro-arrhythmic calsequestrin knockout mouse. Thus, tuning CaM's regulation of RYR2 through Ca²+ dissociation is a viable therapeutic strategy to prevent arrhythmia.

Role of Ryanodine and NMDA Receptors in Tetrabromobisphenol A-Induced Calcium Imbalance and Cytotoxicity in Primary Cultures of Rat Cerebellar Granule Cells.

The study assessed the role of ryanodine receptors (RyRs) and NMDA receptors (NMDARs) in the Ca2+ transients and cytotoxicity induced in neurons by the brominated flame retardant tetrabromobisphenol A (TBBPA). Primary cultures of rat cerebellar granule cells (CGC) were exposed to 7.5, 10, or 25 µM TBBPA for 30 min, and cell viability was assessed after 24 h. Moreover, 45Ca uptake was measured, and changes in the intracellular Ca2+ concentration ([Ca2+]i) were studied using the fluo-3 probe. The involvement of NMDARs and RyRs was verified using the pertinent receptor antagonists, 0.5 µM MK-801 and 2.5 µM bastadin 12, which was co-applied with 200 µM ryanodine, respectively. The results show that TBBPA concentration-dependently induces an increase in [Ca2+]i. This effect was partly suppressed by the inhibitors of RyRs and NMDARs when administered separately, and completely abrogated by their combined application. A concentration-dependent activation of 45Ca uptake by TBBPA was prevented by MK-801 but not by RyR inhibitors. Application of ≥10 µM TBBPA concentration-dependently reduced neuronal viability, and this effect was only partially and to an equal degree reduced by NMDAR and RyR antagonists given either separately or in combination. Our results directly demonstrate that both the RyR-mediated release of intracellular Ca2+ and the NMDAR-mediated influx of Ca2+ into neurons participate in the mechanism of TBBPA-induced Ca2+ imbalance in CGC and play a significant, albeit not exclusive, role in the mechanisms of TBBPA cytotoxicity.

Essential Role of Calmodulin in RyR Inhibition by Dantrolene.

Dantrolene is the first line therapy of malignant hyperthermia. Animal studies suggest that dantrolene also protects against heart failure and arrhythmias caused by spontaneous Ca(2+) release. Although dantrolene inhibits Ca(2+) release from the sarcoplasmic reticulum of skeletal and cardiac muscle preparations, its mechanism of action has remained controversial, because dantrolene does not inhibit single ryanodine receptor (RyR) Ca(2+) release channels in lipid bilayers. Here we test the hypothesis that calmodulin (CaM), a physiologic RyR binding partner that is lost during incorporation into lipid bilayers, is required for dantrolene inhibition of RyR channels. In single channel recordings (100 nM cytoplasmic [Ca(2+)] + 2 mM ATP), dantrolene caused inhibition of RyR1 (rabbit skeletal muscle) and RyR2 (sheep) with a maximal inhibition of Po (Emax) to 52 ± 4% of control only after adding physiologic [CaM] = 100 nM. Dantrolene inhibited RyR2 with an IC50 of 0.16 ± 0.03 µM. Mutant N98S-CaM facilitated dantrolene inhibition with an IC50 = 5.9 ± 0.3 nM. In mouse cardiomyocytes, dantrolene had no effect on cardiac Ca(2+) release in the absence of CaM, but reduced Ca(2+) wave frequency (IC50 = 0.42 ± 0.18 µM, Emax = 47 ± 4%) and amplitude (IC50 = 0.19 ± 0.04 µM, Emax = 66 ± 4%) in the presence of 100 nM CaM. We conclude that CaM is essential for dantrolene inhibition of RyR1 and RyR2. Its absence explains why dantrolene inhibition of single RyR channels has not been previously observed.

Dyad content is reduced in cardiac myocytes of mice with impaired calmodulin regulation of RyR2.

In cardiac muscle, calmodulin (CaM) regulates the activity of several membrane proteins involved in Ca(2+) homeostasis (CaV1.2; RyR2, SERCA2, PMCA). Three engineered amino acid substitutions in the CaM binding site of the cardiac ryanodine receptor (RyR2) in mice (Ryr2 (ADA/ADA) ) strongly affect cardiac function, with impaired CaM inhibition of RyR2, reduced SR Ca(2+) sequestration, and early cardiac hypertrophy and death (Yamaguchi et al., J Clin Invest 117:1344-1353, 2007). We have examined the ultrastructure and RyR2 immunolocalization in WT and Ryr2 (ADA/ADA) hearts at ~10 days after birth. The myocytes show only minor evidence of structural damage: some increase in intermyofibrillar space, with occasional areas of irregular SR disposition and an increase in frequency of smaller myofibrils, despite an increase of about 15 % in average myocyte cross sectional area. Z line streaming, a sign of myofibrillar stress, is limited and fairly rare. Immunolabeling with an anti-RyR2 antibody shows that RyR-positive foci located at the level of the Z lines are less frequent in mutant hearts. A dramatic decrease in the frequency and size of dyads, accompanied by a decrease in occupancy of the gap by RyR2, but without obvious alterations in location and general structure is a notable ultrastructural feature. The data suggest that the uneven distribution of dyads or calcium release sites within the cells resulting from an overall reduction in RyR2 content may contribute to the poor cardiac performance and early death of Ryr2 (ADA/ADA) mice. An unusual fragmentation of mitochondria, perhaps related to imbalances in free cytoplasmic calcium levels, accompanies these changes.

MTOR signaling in mice with dysfunctional cardiac ryanodine receptor ion channel.

Simultaneous substitution of three amino acid residues in the calmodulin binding domain (W3587A/L3591D/F3603A, ADA) of the cardiac ryanodine receptor ion channel (RyR2) impairs calmodulin inhibition of RyR2 and causes cardiac hypertrophy and early death of Ryr2ADA/ADA mice. To determine the physiological significance of growth promoting signaling molecules, the protein and phosphorylation levels of Ser/Thr kinase mTOR and upstream and downstream signaling molecules were determined in hearts of wild-type and Ryr2ADA/ADA mice. Phosphorylation of mTOR at Ser-2448, and mTOR downstream targets p70S6 kinase at Thr-389, S6 ribosomal protein at Ser-240/244, and 4E-BP1 at Ser-65 were increased. However, there was no increased phosphorylation of mTOR upstream kinases PDK1 at Ser-241, AKT at Thr-308, AMPK at Thr-172, and ERK1/2 at Thr-202/Tyr204. To confirm a role for mTOR signaling in the development of cardiac hypertrophy, rapamycin, an inhibitor of mTOR, was injected into wild-type and mutant mice. Rapamycin decreased mouse heart-to-body weight ratio, improved cardiac performance, and decreased phosphorylation of mTOR and downstream targets p70S6K and S6 in 10-day-old Ryr2ADA/ADA mice but did not extend longevity. Taken together, the results link a dysfunctional RyR2 to an altered activity of signaling molecules that regulate cardiac growth and function.

Influence of RyR2 Inhibition Kinetics on Calcium Sparks and Waves in a 3D Model of a Cardiac Cell

Although excess Ca2+ release through RyR2 in diastole is a key factor in arrhythmia, not all RyR2 antagonists have an antiarrhythmic action. Flecainide, carvedilol and dantrolene are RyR2 inhibitors with an anti-arrhythmic blocking action whereas tetracaine is a RyR2 inhibitor with a pro-arrhythmic action. Block by these agents is known to have different RyR2 blocking kinetics and different effects on Ca2+ sparks and waves. However, it is not clear how the different RyR2 blocking kinetics produce different calcium release properties in intact cells.We have developed a 3D model of Ca2+ dynamics, which is based on realistic morphological parameters and RyR2 kinetics. By including different kinetic models for block of RyR2, we find that open state block of RyR2 stabilises Ca2+ release. In contrast, the closed state block of RyR2 has the effect of increasing the positive feedback of Ca2+ on SR Ca2+ release; the net result is an increase in the amplitude of Ca2+ sparks, destabilization of SR Ca2+ cycling.

Antiarrhythmic potential of drugs targeting the cardiac ryanodine receptor Ca2+ release channel: case study of dantrolene.

Driven by the limitations of the traditional antiarrhythmic pharmacology, current antiarrhythmic research is trying to identify new avenues for the development of specific and safe antiarrhythmic drugs. One of the most promising approaches in this field is the amelioration of the abnormal events in cellular Ca(2+) handling originating from the dysfunction of ryanodine receptor Ca(2+) release complex (RyR), which is an inevitable key factor in the pathology of myocardial dysfunction, remodeling and arrhythmogenesis. Accordingly, both in experimental and clinical situations, inhibition of abnormal activity of RyR, regardless of being the primary cause or a consequence during the pathogenesis appears to exert beneficial effect on disease outcome, including a marked antiarrhythmic defense. Considerable amount of our knowledge in this field originates from studies using dantrolene, a human drug with RyR stabilizing effect. Our review summarizes the cardiovascular pharmacology of dantrolene and the results of its use in experimental models of cardiac diseases, which emphasize a promising perspective for the possible antiarrhythmic application of RyR inhibition in the future.