Store Overload-induced Ca Release Research Articles

A gain of function ryanodine receptor 2 mutation (R1760W-RyR2) in catecholaminergic polymorphic ventricular tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome associated with Ca2+ leak predominantly caused by ryanodine receptor 2 (RyR2) mutations. We identified a R1760W-RyR2 mutation located between the N-terminal domain and the central domain of RyR2 in a CPVT patient by DNA sequencing. Recombinant mutant RyR2-2801mcherry plasmid generated by the overlap extension polymerase chain reaction and seamless cloning was transfected in HEK293 cells for the cell model. Single-cell luminal and cytosolic Ca2+ imaging was measured by endoplasmic reticulum (ER) luminal Ca2+ -sensitive protein D1ER and Fura-2 AM on a confocal laser scanning microscope, respectively. We found that in RyR2 mutant cells, the propensity for store-overload-induced Ca2+ release (SOICR) was enhanced representing increased Ca2+ oscillations, reduced activation and termination thresholds of spontaneous Ca2+ release; and the sensitivity to cytosolic Ca2+ activation was increased manifesting reduced steady state ER Ca2+ levels. Our results indicated that R1760W-RyR2 mutation induced calcium leak, representing a gain of function. Further, antiarrhythmic drugs propafenone and flecainide significantly suppressed SOICR caused by the R1760W-RyR2 mutation at a concentration of 20 μM, which was lower than the concentration at which carvedilol suppressed SOICR.

Identification of loss-of-function RyR2 mutations associated with idiopathic ventricular fibrillation and sudden death.

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.

Design, synthesis, and biological activity of novel semicarbazones as potent Ryanodine receptor1 inhibitors of Alzheimer’s disease

Ryanodine receptors (RyRs) are important ligand-gated Ca2+ channels; their excessive activation leads to Ca2+ leakage in the sarcoplasmic reticulum that may cause neurological diseases. In this study, three series of novel potent RyR1 inhibitors based on dantrolene and bearing semicarbazone and imidazolyl moieties were designed and synthesized, and their biological activity was evaluated. Using a single-cell calcium imaging method, the calcium overload inhibitory activities of 26 target compounds were tested in the R614C cell line, using dantrolene as a positive control. The preliminary investigation showed that compound 12a suppressed Ca2+ release as evidenced by store overload-induced Ca2+release (SOICR) (31.5 ± 0.1%, 77.2 ± 0.1%, 93.7 ± 0.2%) at 0.1 μM, 3 μM and 10 μM, respectively. Docking simulation results showed that compound 12a could bind at the active site of the RyR1 protein. The Morris water-maze test showed that compound 12a significantly improved the cognitive behavior of AD-model mice. Further studies on the structural optimization of this series of derivatives are currently underway in our laboratory.

"Phosphorylation of cardiac ryanodine receptors by protein kinase G: Evaluation of its impact on store overload-induced calcium release

Cardiac Ryanodine receptors (RyR2) are Ca2+ channels located within the sarcoplasmic reticulum (SR) membrane of cardiac myocytes and play a key role in excitation-contraction coupling. RyR2 can also open spontaneously in a SR store dependent manner, a process known as Store Overload-Induced Ca2+ Release (SOICR). We and others previously demonstrated PKG-dependent phosphorylation of RyR2 at Ser2808 in cardiac myocytes but its functional impact on SOICR remains unknown. Our objective is to study the effects promoted by PKG-dependent phosphorylation of the RyR2, with respect to SOICR frequency and amplitude.

Abnormal RyR2 in arrhythmogenic disorders and CICR

Ca2+ induced Ca2+ release (CICR) via ryanodine receptor 2 (RyR2) plays a central role in E-C coupling in cardiac cells, i.e., Ca2+ influx via L-type Ca2+ channels during action potential (AP) activates RyR2 to release Ca2+ from the ER and causes muscle contractions. Many arrhythmogenic mutations in RyR2 are reported to increase AP-independent spontaneous Ca2+ release from ER that often lead to arrhythmia. Two explanations have been proposed for the increased propensity of spontaneous Ca2+ release: (1) CPVT mutations increase the cytoplasmic Ca2+ sensitivity of RyR2 to enhance CICR, or (2) mutations decrease threshold for store-overload induced Ca2+ release (SOICR) by reducing luminal Ca2+ sensitivity of RyR2. To understand the underlying mechanism for the increased spontaneous Ca2+ release by the mutations, we performed quantitative evaluation of CICR activity and cytoplasmic and ER Ca2+ signals in HEK293 cells expressing mutant RyR2s. Furthermore, the effects of RyR2 inhibitors, which had been found by recently established high-throughput screening method, were examined on Ca2+ signals in RyR2-HEK cells and cardiomyocytes from adult mice. Our results indicate that CICR plays critical role in generation of spontaneous Ca2+ release and that regulation of CICR is important in suppression of arrhythmia.

P3492Carvedilol and its analogue VK-II-86 attenuate stretch-induced manifestations of mechanoelectric feedback

Abstract Background Mechanical stretch modifies Ca2+ handling and myocardial electrophysiology, favoring arrhythmogenesis. The store-overload-induced Ca2+ release (SOICR) through the ryanodine receptor (RyR2) seems to be implicated in this deleterious effect. Carvedilol and its analogue VK-II-86 (which does not have significant beta-blocking effects) suppress SOCIR by directly reducing the open duration of the cardiac RyR2, and could modulate calcium-related changes produced by myocardial stretch. Purpose The aim of this study was to investigate, by the ventricular fibrillation (VF) spectral analysis, whether carvedilol and VK-II-86 prevents stretch-induced arrhythmogenic effects. Methods The VF modifications induced by acute stretch were studied in Langendorff-perfused rabbit hearts using epicardial multiple electrodes under control conditions (n=10), during carvedilol infusion (0.1 and 1 μM) (n=10) and during VK-II-86 infusion (0.1 and 1 μM) (n=10). Spectral techniques were used to establish the baseline and stretch characteristics in the three series above mentioned: VF dominant frequency (DF) and VF spectral concentration (SpC) were determined. A two-factor ANOVA test was used and significance was reached when p<0.05. Results Myocardial stretch significantly increased DF with respect to pre-stretch values in control conditions (13.3±1.2 vs. 16.1±3.0 Hz, p<0.05) and during the perfusion of 0.1μM carvedilol (11.6±1.5 vs. 13.6±2.9 Hz, p<0.05) and 0.1μM VK-II-86 (13.2±2.2 vs. 16.2±4.1 Hz, p<0.05). However, the maximum concentration of both drugs (1μM) abolished this stretch-induced VF acceleration (carvedilol: 6.9±2.2 vs. 7.3±2.6 Hz, ns; VK-II-86: 7.0±1.4 vs. 7.1±0.6 Hz, ns). The significant stretch-induced decrease in SpC in control conditions (31.4±8.6 vs. 23.2±6.4%, p<0.01) was attenuated under 0.1μM carvedilol (0.1μM: 27.6±8.4 vs. 23.5±6.1%, ns) and 0.1μM VK-II-86 (24.7±5.4 vs. 21.2±3.9%, ns), but not under 1μM of both drugs (carvedilol: 41.3±11.3 vs. 30.3±5.7%, p<0.05; VK-II-86: 34.7±7.2 vs. 28.3±3.9%, p<0.01). Nevertheless, during stretch, arrhythmia regularity and organization was greater (higher SpC) under the highest concentration of carvedilol (p<0.01) and VK-II-86 (p<0.05) than in control conditions (control: 23.2±6.4%, carvedilol 1μM: 30.3±5.7%, VK-II-86 1μM: 28.3±3.9%). Conclusion Carvedilol and its analogue VK-II-86 abolished the changes in VF activation frequency produced by myocardial stretch at the highest studied concentration, and also attenuated the stretch-induced activation heterogeneity at the lowest concentration. The similarity in the effects of both drugs on the stretch-induced alterations would imply that its protective effect is due to its ability to inhibit store-overload-induced calcium release.

Diminished inhibition and facilitated activation of RyR2‐mediated Ca2+ release is a common defect of arrhythmogenic calmodulin mutations

A number of calmodulin (CaM) mutations cause severe cardiac arrhythmias, but their arrhythmogenic mechanisms are unclear. While some of the arrhythmogenic CaM mutations have been shown to impair CaM-dependent inhibition of intracellular Ca2+ release through the ryanodine receptor type 2 (RyR2), the impact of a majority of these mutations on RyR2 function is unknown. Here, we investigated the effect of 14 arrhythmogenic CaM mutations on the CaM-dependent RyR2 inhibition. We found that all the arrhythmogenic CaM mutations tested diminished CaM-dependent inhibition of RyR2-mediated Ca2+ release and increased store-overload induced Ca2+ release (SOICR) in HEK293 cells. Moreover, all the arrhythmogenic CaM mutations tested either failed to inhibit or even promoted RyR2-mediated Ca2+ release in permeabilized HEK293 cells with elevated cytosolic Ca2+ , which was markedly different from the inhibitory action of CaM wild-type. The CaM mutations also altered the Ca2+ -dependency of CaM binding to the RyR2 CaM-binding domain. These results demonstrate that diminished inhibition, and even facilitated activation, of RyR2-mediated Ca2+ release is a common defect of arrhythmogenic CaM mutations.

Activation of RyR2 by class I kinase inhibitors.

Kinase inhibitors are a common treatment for cancer. Class I kinase inhibitors that target the ATP-binding pocket are particularly prevalent. Many of these compounds are cardiotoxic and can cause arrhythmias. Spontaneous release of Ca2+ via cardiac ryanodine receptors (RyR2), through a process termed store overload-induced Ca2+ release (SOICR), is a common mechanism underlying arrhythmia. We explored whether class I kinase inhibitors could modify the activity of RyR2 and trigger SOICR to determine if this contributes to the cardiotoxic nature of these compounds. The impact of class I and II kinase inhibitors on SOICR was studied in HEK293 cells and ventricular myocytes using single-cell Ca2+ imaging. A specific effect on RyR2 was confirmed using single channel recordings. Ventricular myocytes were also used to determine if drug-induced changes in SOICR could be reversed using anti-SOICR agents. Class I kinase inhibitors increased the propensity of SOICR. Single channel recording showed that this was due to a specific effect on RyR2. Class II kinase inhibitors decreased the activity of RyR2 at the single channel level but had little effect on SOICR. The promotion of SOICR mediated by class I kinase inhibitors could be reversed using the anti-SOICR agent VK-II-86. Part of the cardiotoxicity of class I kinase inhibitors can be assigned to their effect on RyR2 and increase in SOICR. Compounds with anti-SOICR activity may represent an improved treatment option for patients.

CPVT-associated cardiac ryanodine receptor mutation G357S with reduced penetrance impairs Ca2+ release termination and diminishes protein expression.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is one of the most lethal inherited cardiac arrhythmias mostly linked to cardiac ryanodine receptor (RyR2) mutations with high disease penetrance. Interestingly, a novel RyR2 mutation G357S discovered in a large family of more than 1400 individuals has reduced penetrance. The molecular basis for the incomplete disease penetrance in this family is unknown. To gain insights into the variable disease expression in this family, we determined the impact of the G357S mutation on RyR2 function and expression. We assessed spontaneous Ca2+ release in HEK293 cells expressing RyR2 wildtype and the G357S mutant during store Ca2+ overload, also known as store overload induced Ca2+ release (SOICR). We found that the G357S mutation reduced the percentage of RyR2-expressing cells that showed SOICR. However, in cells that displayed SOICR, G357S reduced the thresholds for the activation and termination of SOICR. Furthermore, G357S decreased the thermal stability of the N-terminal domain of RyR2, and markedly reduced the protein expression of the full-length RyR2. On the other hand, the G357S mutation did not alter the Ca2+ activation of [3H]ryanodine binding or the Ca2+ induced release of Ca2+ from the intracellular stores in HEK293 cells. These data indicate that the CPVT-associated G357S mutation enhances the arrhythmogenic SOICR and reduces RyR2 protein expression, which may be attributable to the incomplete penetrance of CPVT in this family.

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.

Nebivolol suppresses cardiac ryanodine receptor-mediated spontaneous Ca2+ release and catecholaminergic polymorphic ventricular tachycardia.

β-Blockers are a standard treatment for heart failure and cardiac arrhythmias. There are ∼30 commonly used β-blockers, representing a diverse class of drugs with different receptor affinities and pleiotropic properties. We reported that among 14 β-blockers tested previously, only carvedilol effectively suppressed cardiac ryanodine receptor (RyR2)-mediated spontaneous Ca2+ waves during store Ca2+ overload, also known as store overload-induced Ca2+ release (SOICR). Given the critical role of SOICR in arrhythmogenesis, it is of importance to determine whether there are other β-blockers that suppress SOICR. Here, we assessed the effect of other commonly used β-blockers on RyR2-mediated SOICR in HEK293 cells, using single-cell Ca2+ imaging. Of the 13 β-blockers tested, only nebivolol, a β-1-selective β-blocker with nitric oxide synthase (NOS)-stimulating action, effectively suppressed SOICR. The NOS inhibitor (N-nitro-l-arginine methyl ester) had no effect on nebivolol's SOICR inhibition, and the NOS activator (histamine or prostaglandin E2) alone did not inhibit SOICR. Hence, nebivolol's SOICR inhibition was independent of NOS stimulation. Like carvedilol, nebivolol reduced the opening of single RyR2 channels and suppressed spontaneous Ca2+ waves in intact hearts and catecholaminergic polymorphic ventricular tachycardia (CPVT) in the mice harboring a RyR2 mutation (R4496C). Interestingly, a non-β-blocking nebivolol enantiomer, (l)-nebivolol, also suppressed SOICR and CPVT without lowering heart rate. These data indicate that nebivolol, like carvedilol, possesses a RyR2-targeted action that suppresses SOICR and SOICR-evoked VTs. Thus, nebivolol represents a promising agent for Ca2+-triggered arrhythmias.

FKBPs facilitate the termination of spontaneous Ca2+ release in wild-typeRyR2 but not CPVT mutant RyR2.

FK506-binding proteins 12.6 (FKBP12.6) and 12 (FKBP12) tightly associate with the cardiac ryanodine receptor (RyR2). Studies suggest that dissociation of FKBP12.6 from mutant forms of RyR2 contributes to store overload-induced Ca(2+) release (SOICR) and Ca(2+)-triggered arrhythmias. However, these findings are controversial. Previous studies focused on the effect of FKBP12.6 on the initiation of SOICR and did not explore changes in the termination of Ca(2+) release. Less is known about FKBP12. We aimed to determine the effect of FKBP12.6 and FKBP12 on the termination of SOICR. Using single-cell imaging, in cells expressing wild-type RyR2, we found that FKBP12.6 and FKBP12 significantly increase the termination threshold of SOICR without changing the activation threshold of SOICR. This effect, dependent on the association of each FKBP with RyR2, reduced the magnitude of Ca(2+) release but had no effect on the propensity for SOICR. In contrast, neither FKBP12.6 nor FKBP12 was able to regulate an arrhythmogenic variant of RyR2, despite a conserved protein interaction. Our results suggest that both FKBP12.6 and FKBP12 play critical roles in regulating RyR2 function by facilitating the termination of SOICR. The inability of FKBPs to mediate a similar effect on the mutant RyR2 represents a novel mechanism by which mutations within RyR2 lead to arrhythmia.

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.

Carvedilol inhibits cADPR- and IP3-induced Ca2+ release.

Spontaneous Ca2+ waves, also termed store-overload-induced Ca2+ release (SOICR), in cardiac cells can trigger ventricular arrhythmias especially in failing hearts. SOICR occurs when RyRs are activated by an increase in sarcoplasmic reticulum (SR) luminal Ca2+. Carvedilol is one of the most effective drugs for preventing arrhythmias in patients with heart failure. Furthermore, carvedilol analogues with minimal β-blocking activity also block SOICR showing that SOICR-inhibiting activity is distinct from that for β-block. We show here that carvedilol is a potent inhibitor of cADPR-induced Ca2+ release in sea urchin egg homogenate. In addition, the carvedilol analog VK-II-86 with minimal β-blocking activity also suppresses cADPR-induced Ca2+ release. Carvedilol appeared to be a non-competitive antagonist of cADPR and could also suppress Ca2+ release by caffeine. These results are consistent with cADPR releasing Ca2+ in sea urchin eggs by sensitizing RyRs to Ca2+ involving a luminal Ca2+ activation mechanism. In addition to action on the RyR, we also observed inhibition of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release by carvedilol suggesting a common mechanism between these evolutionarily related and conserved Ca2+ release channels.

Inhibition of Rac1 reduces store overload-induced calcium release and protects against ventricular arrhythmia.

Rac1 is a small GTPase and plays key roles in multiple cellular processes including the production of reactive oxygen species (ROS). However, whether Rac1 activation during myocardial ischaemia and reperfusion (I/R) contributes to arrhythmogenesis is not fully understood. We aimed to study the effects of Rac1 inhibition on store overload‐induced Ca2+ release (SOICR) and ventricular arrhythmia during myocardial I/R. Adult Rac1f/f and cardiac‐specific Rac1 knockdown (Rac1ckd) mice were subjected to myocardial I/R and their electrocardiograms (ECGs) were monitored for ventricular arrhythmia. Myocardial Rac1 activity was increased and ventricular arrhythmia was induced during I/R in Rac1f/f mice. Remarkably, I/R‐induced ventricular arrhythmia was significantly decreased in Rac1ckd compared to Rac1f/f mice. Furthermore, treatment with Rac1 inhibitor NSC23766 decreased I/R‐induced ventricular arrhythmia. Ca2+ imaging analysis showed that in response to a 6 mM external Ca2+ concentration challenge, SOICR was induced with characteristic spontaneous intracellular Ca2+ waves in Rac1f/f cardiomyocytes. Notably, SOICR was diminished by pharmacological and genetic inhibition of Rac1 in adult cardiomyocytes. Moreover, I/R‐induced ROS production and ryanodine receptor 2 (RyR2) oxidation were significantly inhibited in the myocardium of Rac1ckd mice. We conclude that Rac1 activation induces ventricular arrhythmia during myocardial I/R. Inhibition of Rac1 suppresses SOICR and protects against ventricular arrhythmia. Blockade of Rac1 activation may represent a new paradigm for the treatment of cardiac arrhythmia in ischaemic heart disease.

Leaky ryanodine receptors delay the activation of store overload-induced Ca2+ release, a mechanism underlying malignant hyperthermia-like events in dystrophic muscle.

The mouse model of Duchenne muscular dystrophy, the mdx mouse, displays changes in Ca(2+)homeostasis that may lead to the pathology of the muscle. Here we examine the activation of store overload-induced Ca(2+)release (SOICR) in mdx muscle. The activation of SOICR is associated with the depolymerization of the sarcoplasmic reticulum (SR) Ca(2+)buffer calsequestrin and the reduction of SR Ca(2+)buffering power (BSR). The role of SOICR in healthy and dystrophic muscle is unclear. Using skinned fibers we show that lowering the Mg(2+)concentration can activate discrete Ca(2+)release events that did not necessarily lead to activation of SOICR. However, SOICR waves could propagate into these fiber segments. The average delay to activation of SOICR in mdx fibers was longer than in wild-type (WT) fibers. In the lowered Ca(2+)-buffered environment following large SOICR events, brief waves in mdx fibers displayed a low amplitude and propagation rate, in contrast to WT fibers that showed a range of amplitudes correlated with wave propagation rate. The distinct properties of SOICR in mdx fibers were consistent with a ryanodine receptor (RyR) that was leakier to Ca(2+)than in WT. The consequence of delayed SOICR and leaky RyRs is prolonged high BSRand a reduction in free Ca(2+)concentration inside the SR as total SR calcium drops. We present a hypothesis that SOICR activation is required in healthy muscle and that this mechanism works suboptimally in mdx fibers to fail to limit the activation of store-operated Ca(2+)entry.

The EF-hand Ca2+ Binding Domain Is Not Required for Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor

Activation of the cardiac ryanodine receptor (RyR2) by elevating cytosolic Ca2+ is a central step in the process of Ca2+-induced Ca2+ release, but the molecular basis of RyR2 activation by cytosolic Ca2+ is poorly defined. It has been proposed recently that the putative Ca2+ binding domain encompassing a pair of EF-hand motifs (EF1 and EF2) in the skeletal muscle ryanodine receptor (RyR1) functions as a Ca2+ sensor that regulates the gating of RyR1. Although the role of the EF-hand domain in RyR1 function has been studied extensively, little is known about the functional significance of the corresponding EF-hand domain in RyR2. Here we investigate the effect of mutations in the EF-hand motifs on the Ca2+ activation of RyR2. We found that mutations in the EF-hand motifs or deletion of the entire EF-hand domain did not affect the Ca2+-dependent activation of [3H]ryanodine binding or the cytosolic Ca2+ activation of RyR2. On the other hand, deletion of the EF-hand domain markedly suppressed the luminal Ca2+ activation of RyR2 and spontaneous Ca2+ release in HEK293 cells during store Ca2+ overload or store overload-induced Ca2+ release (SOICR). Furthermore, mutations in the EF2 motif, but not EF1 motif, of RyR2 raised the threshold for SOICR termination, whereas deletion of the EF-hand domain of RyR2 increased both the activation and termination thresholds for SOICR. These results indicate that, although the EF-hand domain is not required for RyR2 activation by cytosolic Ca2+, it plays an important role in luminal Ca2+ activation and SOICR.

Arrhythmogenic Calmodulin Mutations Affect the Activation and Termination of Cardiac Ryanodine Receptor-mediated Ca2+ Release

The intracellular Ca(2+) sensor calmodulin (CaM) regulates the cardiac Ca(2+) release channel/ryanodine receptor 2 (RyR2), and mutations in CaM cause arrhythmias such as catecholaminergic polymorphic ventricular tachycardia (CPVT) and long QT syndrome. Here, we investigated the effect of CaM mutations causing CPVT (N53I), long QT syndrome (D95V and D129G), or both (CaM N97S) on RyR2-mediated Ca(2+) release. All mutations increased Ca(2+) release and rendered RyR2 more susceptible to store overload-induced Ca(2+) release (SOICR) by lowering the threshold of store Ca(2+) content at which SOICR occurred and the threshold at which SOICR terminated. To obtain mechanistic insights, we investigated the Ca(2+) binding of the N- and C-terminal domains (N- and C-domain) of CaM in the presence of a peptide corresponding to the CaM-binding domain of RyR2. The N53I mutation decreased the affinity of Ca(2+) binding to the N-domain of CaM, relative to CaM WT, but did not affect the C-domain. Conversely, mutations N97S, D95V, and D129G had little or no effect on Ca(2+) binding to the N-domain but markedly decreased the affinity of the C-domain for Ca(2+). These results suggest that mutations D95V, N97S, and D129G alter the interaction between CaM and the CaMBD and thus RyR2 regulation. Because the N53I mutation minimally affected Ca(2+) binding to the C-domain, it must cause aberrant regulation via a different mechanism. These results support aberrant RyR2 regulation as the disease mechanism for CPVT associated with CaM mutations and shows that CaM mutations not associated with CPVT can also affect RyR2. A model for the CaM-RyR2 interaction, where the Ca(2+)-saturated C-domain is constitutively bound to RyR2 and the N-domain senses increases in Ca(2+) concentration, is proposed.

Role of Cys3602 in the function and regulation of the cardiac ryanodine receptor

The cardiac Ca²⁺ release channel [ryanodine receptor type 2 (RyR2)] is modulated by thiol reactive agents, but the molecular basis of RyR2 modulation by thiol reagents is poorly understood. Cys³⁶³⁵ in the skeletal muscle RyR1 is one of the most hyper-reactive thiols and is important for the redox and calmodulin (CaM) regulation of the RyR1 channel. However, little is known about the role of the corresponding cysteine residue in RyR2 (Cys³⁶⁰²) in the function and regulation of the RyR2 channel. In the present study, we assessed the impact of mutating Cys³⁶⁰² (C³⁶⁰²A) on store overload-induced Ca²⁺ release (SOICR) and the regulation of RyR2 by thiol reagents and CaM. We found that the C³⁶⁰²A mutation suppressed SOICR by raising the activation threshold and delayed the termination of Ca²⁺ release by reducing the termination threshold. As a result, C³⁶⁰²A markedly increased the fractional Ca²⁺ release. Furthermore, the C³⁶⁰²A mutation diminished the inhibitory effect of N-ethylmaleimide on Ca²⁺ release, but it had no effect on the stimulatory action of 4,4'-dithiodipyridine (DTDP) on Ca²⁺ release. In addition, Cys³⁶⁰² mutations (C³⁶⁰²A or C³⁶⁰²R) did not abolish the effect of CaM on Ca²⁺-release termination. Therefore, RyR2-Cys³⁶⁰² is a major site mediating the action of thiol alkylating agent N-ethylmaleimide, but not the action of the oxidant DTDP. Our data also indicate that residue Cys³⁶⁰² plays an important role in the activation and termination of Ca²⁺ release, but it is not essential for CaM regulation of RyR2.

Roles of the NH2-terminal Domains of Cardiac Ryanodine Receptor in Ca2+ Release Activation and Termination

The NH2-terminal region (residues 1-543) of the cardiac ryanodine receptor (RyR2) harbors a large number of mutations associated with cardiac arrhythmias and cardiomyopathies. Functional studies have revealed that the NH2-terminal region is involved in the activation and termination of Ca(2+) release. The three-dimensional structure of the NH2-terminal region has recently been solved. It is composed of three domains (A, B, and C). However, the roles of these individual domains in Ca(2+) release activation and termination are largely unknown. To understand the functional significance of each of these NH2-terminal domains, we systematically deleted these domains and assessed their impact on caffeine- or Ca(2+)-induced Ca(2+) release and store overload-induced Ca(2+) release (SOICR) in HEK293 cells. We found that all deletion mutants were capable of forming caffeine- and ryanodine-sensitive functional channels, indicating that the NH2-terminal region is not essential for channel gating. Ca(2+) release measurements revealed that deleting domain A markedly reduced the threshold for SOICR termination but had no effect on caffeine or Ca(2+) activation or the threshold for SOICR activation, whereas deleting domain B substantially enhanced caffeine and Ca(2+) activation and lowered the threshold for SOICR activation and termination. Conversely, deleting domain C suppressed caffeine activation, abolished Ca(2+) activation and SOICR, and diminished protein expression. These results suggest that domain A is involved in channel termination, domain B is involved in channel suppression, and domain C is critical for channel activation and expression. Our data shed new insights into the structure-function relationship of the NH2-terminal domains of RyR2 and the action of NH2-terminal disease mutations.