This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 97/11/1173    most recent 01.RES.0000192146.85173.4bv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jiang, D. Articles by Chen, S. R. W. Search for Related Content PubMed PubMed Citation Articles by Jiang, D. Articles by Chen, S. R. W. Related Collections Related Article

(Circulation Research. 2005;97:1173.)
© 2005 American Heart Association, Inc.

Cellular Biology

Enhanced Store Overload–Induced Ca²⁺ Release and Channel Sensitivity to Luminal Ca²⁺ Activation Are Common Defects of RyR2 Mutations Linked to Ventricular Tachycardia and Sudden Death

Dawei Jiang, Ruiwu Wang, Bailong Xiao, Huihui Kong, Donald J. Hunt, Philip Choi, Lin Zhang, S. R. Wayne Chen

From the Cardiovascular Research Group, Departments of Physiology and Biophysics, and of Biochemistry and Molecular Biology, University of Calgary, Alberta, Canada.

Correspondence to Dr S.R. Wayne Chen, University of Calgary, Department of Physiology and Biophysics, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada. E-mail swchen{at}ucalgary.ca

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ventricular tachycardia (VT) is the leading cause of sudden death, and the cardiac ryanodine receptor (RyR2) is emerging as an important focus in its pathogenesis. RyR2 mutations have been linked to VT and sudden death, but their precise impacts on channel function remain largely undefined and controversial. We have previously shown that several disease-linked RyR2 mutations in the C-terminal region enhance the sensitivity of the channel to activation by luminal Ca²⁺. Cells expressing these RyR2 mutants display an increased propensity for spontaneous Ca²⁺ release under conditions of store Ca²⁺ overload, a process we referred to as store overload–induced Ca²⁺ release (SOICR). To determine whether common defects exist in disease-linked RyR2 mutations, we characterized 6 more RyR2 mutations from different regions of the channel. Stable inducible HEK293 cell lines expressing Q4201R and I4867M from the C-terminal region, S2246L and R2474S from the central region, and R176Q(T2504M) and L433P from the N-terminal region were generated. All of these cell lines display an enhanced propensity for SOICR. HL-1 cardiac cells transfected with disease-linked RyR2 mutations also exhibit increased SOICR activity. Single channel analyses reveal that disease-linked RyR2 mutations primarily increase the channel sensitivity to luminal, but not to cytosolic, Ca²⁺ activation. Moreover, the Ca²⁺ dependence of [³H]ryanodine binding to RyR2 wild type and mutants is similar. In contrast to previous reports, we found no evidence that disease-linked RyR2 mutations alter the FKBP12.6–RyR2 interaction. Our data indicate that enhanced SOICR activity and luminal Ca²⁺ activation represent common defects of RyR2 mutations associated with VT and sudden death. A mechanistic model for CPVT/ARVD2 is proposed.

Key Words: arrhythmia • Ca²⁺ handling • Ca²⁺ transients • heart failure • intracellular calcium • ryanodine receptor • sarcoplasmic reticulum • ventricular tachycardia

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
A leading cause of sudden death in patients with heart failure is ventricular tachycardia (VT). An increasing body of evidence indicates that abnormal sarcoplasmic reticulum (SR) Ca²⁺ handling is linked to VT^1,2; however, the exact defects underlying aberrant SR Ca²⁺ handling in failing hearts are not well defined. This is, in part, because of the complex nature of heart failure. Inherited genetic diseases have proven to be powerful models for studying complex syndromes. Hence we reason that inherited VT may provide an alternative approach to understanding VT in heart failure.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited VT associated with syncope and sudden death. It can be induced reproducibly by the infusion of catecholamines or by emotional or physical stress.^3,4 CPVT has been linked to 2 cardiac SR proteins, the cardiac ryanodine receptor (RyR2) and the cardiac calsequestrin (CASQ2). Mutations in RyR2 cause a dominant form of CPVT, whereas mutations in CASQ2 are linked to an autosomal recessive form. Mutations in RyR2 are also linked to arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2).^5–9 Interestingly, patients with CPVT apparently have functionally normal hearts.^3,4 The ECG of CPVT resembles that of digitalis-induced arrhythmia, which was the first clue that CPVT is caused by delayed afterdepolarizations (DADs) as a result of SR Ca²⁺ overload, the mechanism thought to underlie digitalis-induced arrhythmias. However, why patients with RyR2 mutations are more susceptible to SR Ca²⁺ overload–induced DADs and triggered arrhythmia is not clear.

RyR2 is a key component in cardiac excitation-contraction coupling. Under normal conditions, depolarization activates the L-type Ca²⁺ channels, leading to a small Ca²⁺ influx. This Ca²⁺ influx then activates RyR2, resulting in a large Ca²⁺ release from the SR and subsequent muscle contraction. This process is known as Ca²⁺-induced Ca²⁺ release (CICR).¹⁰ In addition to this depolarization-triggered Ca²⁺ release, spontaneous SR Ca²⁺ release via RyR2 can occur under the conditions of SR Ca²⁺ overload,¹¹ a process we have termed store-overload–induced Ca²⁺ release (SOICR).¹² It has long been recognized that SOICR can alter membrane potential by generating DADs, which can in turn lead to triggered arrhythmias.^13–16

To date &40 disease-linked RyR2 mutations have been identified. These mutations are largely clustered in the N-terminal, C-terminal, and central regions of RyR2 (Figure 1). The focus of current research is to understand how these mutations alter RyR2 function, leading to VT and sudden death. Given the link between SOICR and triggered arrhythmia, it is sensible to propose that CPVT/ARVD2 RyR2 mutations increase the susceptibility to arrhythmia by increasing the propensity for SOICR. In support of this hypothesis, we have recently shown that the CPVT RyR2 mutations N4104K, R4496C, and N4895D, located in the C-terminal region, enhance the channel sensitivity to activation by luminal Ca²⁺ and reduce the threshold for SOICR.¹² In line with this view, George et al have shown that disease-linked RyR2 mutants displayed an increased sensitivity to activation by caffeine or ß-adrenergic stimulation.^17,18 Interestingly, an ARVD2 RyR2 mutation, L433P, located in the N-terminal region, exhibited a marked reduction in channel response to caffeine.¹⁸ These observations suggest that not all disease-linked RyR2 mutations are gain-of-function.

View larger version (32K): [in this window] [in a new window]   Figure 1. Distribution of CPVT/ARVD2 mutations in the linear sequence of RyR2. CPVT/ARVD2 mutations are clustered in 3 regions: CPVT/ARVD2 I, II, and III. RyR2 mutations investigated in this study are indicated by a red dot. Regions important for FKBP12.6 and calmodulin binding, Ca2+ activation and conduction, sites for PKA phosphorylation, and the 3 most divergent regions among RyR isoforms are shown.

Wehrens et al propose a different mechanism for CPVT. It was reported that phosphorylation of RyR2 by PKA dissociated FKBP12.6, which led to an increased channel activity, and that CPVT RyR2 mutations reduced the binding affinity of FKBP12.6.¹⁹ As a result, CPVT RyR2 mutants displayed an enhanced channel activity on PKA phosphorylation as a result of an increased level of FKBP12.6 dissociation. However, George et al have demonstrated that CPVT RyR2 mutations augmented Ca²⁺ release in a manner independent of both FKBP12.6 and PKA phosphorylation.¹⁷ Both ourselves and others have since demonstrated that phosphorylation of RyR2 by PKA does not dissociate FKBP12.6 from RyR2.^20,21

In light of these controversies, it is necessary and important to determine whether enhanced SOICR activity and luminal Ca²⁺ activation are common features of disease-linked RyR2 mutations, and whether RyR2 mutations alter the FKBP12.6–RyR2 interaction. We characterized 6 CPVT/ARVD2 mutations, 2 from each region, and found that all these mutations increased SOICR activity and luminal Ca²⁺ activation. We also found that these mutations do not alter FKBP12.6 binding to RyR2. The observation that common mechanisms exist in disease-linked RyR2 mutations has important implications for the treatment of CPVT/ARVD2.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mutations were introduced into the mouse RyR2 by the overlap extension method. Stable inducible HEK293 cell lines expressing RyR2 wild type (wt) and mutants were generated using the Flp-In T-REx system from Invitrogen. HL-1 cardiac cells were transfected with RyR2 wt and mutant cDNAs using lipofectamine. Single cell Ca²⁺ imaging was used to investigate SOICR, while RyR2 activation by luminal Ca²⁺ was assessed in planar lipid bilayers. [³⁵S]FKBP12.6 was generated using the TNT Quick Coupled Transcription/Translation Kit from Promega and used for measuring FKBP12.6 binding to immunoprecipitated RyR2. A detailed Materials and Methods section can be found in the online data supplement available at http://circres.ahajournals.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CPVT/ARVD2 RyR2 Mutations From Different Regions of the Channel Enhance the Propensity for SOICR
To determine whether disease-linked RyR2 mutations from different regions of the channel alter SOICR in a similar manner, we generated stable inducible HEK293 cell lines expressing RyR2 wt and the CPVT/ARVD2 mutants Q4201R, I4867M, S2246L, R2474S, R176Q(T2504M), and L433P (Figure 1). Because the R176Q mutation was cosegregated with T2504M⁷, a double mutation R176Q(T2504M) was produced. To examine their SOICR properties, RyR2 wt or mutant cells were loaded with fura 2-AM. Store Ca²⁺ overload was induced by elevating the external Ca²⁺ concentration ([Ca²⁺]_o), and the occurrence of SOICR was monitored using single cell Ca²⁺ imaging. Figure 2A shows that Ca²⁺ oscillations occurred at lower [Ca²⁺]_o in cells expressing the C-terminal mutants, Q4201R and I4867M, as compared with those in wt cells. In addition, mutant cells displayed increased frequency and decreased amplitude of Ca²⁺ oscillations. Similar results were observed in cells expressing the central region mutants, S2246L and R2474S (Figure 2B), and the N-terminal mutants, R176Q(T2504M) and L433P (Figure 2C). In both wt and mutant cells, elevation of [Ca²⁺]_o increased the frequency of Ca²⁺ oscillations but had little effect on the amplitude of the oscillations, which remained relatively constant (Figure 2). These effects of [Ca²⁺]_o on SOICR are similar to those observed in cardiac myocytes.²² It should be noted that the levels of resting Ca²⁺ and RyR2 expression in wt and mutant cells are similar (Figure 2).

View larger version (43K): [in this window] [in a new window]   Figure 2. SOICR in HEK293 cells expressing RyR2 wt and CPVT/ARVD2 mutants. HEK293 cells expressing RyR2 wt or CPVT/ARVD2 mutants were grown on glass coverslips. Cells were induced with tetracycline for 24 hours and loaded with 5 µmol/L fura-2-AM in KRH buffer for 20 minutes at room temperature. Cells were perfused continuously with KRH buffer without (0 mmol/L) or with 0.1, 0.2, 0.3, 0.5, 1.0 mmol/L CaCl2, or 1.0 mmol/L CaCl2 plus 5 mmol/L caffeine. Fura-2 ratios of representative wt and mutant cells were determined using single cell Ca2+ imaging (A, B, and C). D, Western blot of RyR2 wt and mutants pulled down by GST-FKBP12.6 from the same amount of cell lysate using an anti-RyR antibody. It should be noted that HEK293 parental cells express no detectable level of RyR2 (not shown).

To assess their propensity for SOICR, we determined the fraction of wt and mutant cells that display Ca²⁺ oscillations at each [Ca²⁺]_o. A large number of cells were analyzed, including 1332 wt cells, 498 Q4201R, 417 I4867M, 276 S2246L, 242 R2474S, 560 R176Q(T2504M), and 390 L433P mutant cells. As seen in Figure 3, in the range of 0.1 to 0.3 mmol/L [Ca²⁺]_o, a greater fraction of mutant cells displayed Ca²⁺ oscillations as compared with wt cells. The frequency of Ca²⁺ oscillations and the level of store Ca²⁺ content at 1 mmol/L [Ca²⁺]_o were also analyzed. These analyses revealed that the RyR2 mutations located in either the N-terminal, central, or the C-terminal region increased the frequency of Ca²⁺ oscillations and decreased the store Ca²⁺ content (Figure 3B, 3D, and 3F). These observations are consistent with the notion that disease-linked RyR2 mutations reduce the threshold for SOICR. These results, together with those reported previously,¹² demonstrate that an enhanced propensity for SOICR is a common feature of CPVT/ARVD2 RyR2 mutations.

View larger version (41K): [in this window] [in a new window]   Figure 3. CPVT/ARVD2 RyR2 mutations enhance SOICR in HEK293 cells. The fraction of the RyR2 wt and CPVT/ARVD2 mutant cells that display Ca2+ oscillations at various [Ca2+]o are shown in panels A, C, and E. The frequency of Ca2+ oscillations and store Ca2+ levels in wt and mutant cells are shown in panels B, D, and F. The store Ca2+ content was determined by measuring the amplitude of caffeine (5 mmol/L) induced Ca2+ release, while the frequency of oscillations was estimated from the number of peaks in the presence of 1.0 mmol/L [Ca2+]o. Values were normalized to the wt level (100%). Data shown are mean±SEM from 3 to 7 separate experiments.

HL-1 Cardiac Cells Transfected With CPVT/ARVD2 RyR2 Mutants Display Enhanced SOICR Activity
To ascertain whether the impact of CPVT/ARVD2 RyR2 mutations on SOICR manifests in the context of cardiac cells, we transfected HL-1 cardiac cells, a mouse atrial cell line, with RyR2 wt and the RyR2 mutants R176Q/T2504M, R2474S, and Q4201R and monitored their SOICR activity. Figure 4 shows that HL-1 cells transfected with these CPVT/ARVD2 RyR2 mutants exhibit an increased propensity for SOICR compared with HL-1 cells transfected with RyR2 wt (Figure 4D). These results indicate that CPVT/ARVD2 RyR2 mutations can also alter SOICR in cardiac cells as they do in HEK293 (non-cardiac) cells. It should be noted that, compared with those seen in stable inducible HEK293 cells expressing RyR2 wt or mutants, the patterns of spontaneous Ca²⁺ oscillations in HL-1 cells transfected with RyR2 wt or mutants tend to be irregular, which makes quantitative analyses of the impact of the mutations on the amplitude and frequency of Ca²⁺ oscillations difficult. The reason for this irregularity is not clear, but is probably the result of Lipofectamine-mediated transfection.

View larger version (32K): [in this window] [in a new window]   Figure 4. CPVT/ARVD2 RyR2 mutations increase SOICR in HL-1 cardiac cells. HL-1 cells were transfected with the RyR2 wt, and the R176Q(T2504M) (A), R2474S (B), and Q4201R (C) mutants. Transfected cells were loaded with 5 mmol/L fura-2-AM, and their SOICR activities were monitored using single cell Ca2+ imagining. D, Fraction of HL-1 cells transfected with the RyR2 wt and CPVT/ARVD2 mutants that display Ca2+ oscillations at various [Ca2+]o. The total number of HL-1 cells analyzed was 423 for wt, 125 for R176Q/T2504M, 315 for R2474S, and 122 for Q4201R.

CPVT/ARVD2 Mutations Increase the Sensitivity of Single RyR2 Channels to Activation by Luminal Ca²⁺
We have previously demonstrated that the CPVT mutations N4104K, R4496C, and N4895D, located in the C-terminal region, augment SOICR by increasing the sensitivity of RyR2 to activation by luminal Ca²⁺¹². To determine whether this increased luminal Ca²⁺ activation is common to CPVT/ARVD2 RyR2 mutants, we assessed the luminal Ca²⁺ response of 6 more RyR2 mutants, 2 from each of the 3 mutation regions. Figure 5 shows that single mutant channels, Q4201R (Figure 5A) and I4867M (Figure 5B) from the C-terminal region and S2246L (Figure 5C) and R2474S (Figure 5D) from the central region, exhibited little activity at low cytosolic (45 nmol/L) and luminal (45 nmol/L) Ca²⁺ (panel a). Elevating the luminal Ca²⁺ concentration to 300 µmol/L markedly increased the open probability (Po) of these mutant channels (panel b). The average Po values were 0.414±0.080 (mean±SEM, n=7) for single Q4201R channels, 0.144±0.024 (n=5) for I4867M (Figure 5E), 0.142±0.046 (n=9) for S2246L, and 0.256±0.075 (n=5) for R2474S (Figure 5F)—considerably higher than that of single wt channels (0.023±0.007; n=22). The luminal Ca²⁺ responses of the N-terminal single R176Q(T2504M) and L433P mutant channels are shown in Figure 5H and 5I, respectively. At 0.6 mmol/L or higher luminal Ca²⁺, the activities of the mutant channels were enhanced compared with wt (Figure 5G). The average Po values of single R176Q(T2504M) and L433P channels at 1.2 mmol/L luminal Ca²⁺ were 0.126±0.030 (n=10) and 0.144±0.031 (n=8), respectively (Figure 5J). Taken together, these results directly demonstrate that CPVT/ARVD2 RyR2 mutations from different regions increase the channel sensitivity to luminal Ca²⁺ activation.

View larger version (45K): [in this window] [in a new window]   Figure 5. CPVT/ARVD2 mutations increase luminal Ca2+ activation. Single channel activities of the RyR2 mutants Q4201R (A), I4867M (B), S2246L (C), R2474S (D), R176Q/T2504M (H), and L433P (I), and wt (G) were recorded in a symmetrical recording solution containing 250 mmol/L KCl and 25 mmol/L Hepes (pH 7.4). The Ca2+ concentration on both the cytosolic and luminal face of the channel was adjusted to &45 nmol/L. The luminal Ca2+ concentration was then increased to various levels by the addition of aliquots of CaCl2 solution. The control single channel current traces for wt (Ga), and mutants (Aa, Ba, Ca, Da, Ha, and Ia) are shown in panel a, whereas single channel current traces at 300 µmol/L luminal Ca2+ (Ab, Bb, Cb, and Db) or 1.2 mmol/L luminal Ca2+ (Gb, Hb, and Ib) are depicted in panel b. The holding potential was –20 mV. Openings are downward. The open probability (Po), arithmetic mean open time (To), and the arithmetic mean closed time (Tc) are indicated on the top of each panel. The baselines are indicated. The relationships between Po and luminal Ca2+ concentrations of single wt and mutants Q4201R, I4867M (E), S2246L, R2474S (F), R176Q/T2504M, and L433P (J) are shown. Data points are mean±SEM from 5 to 22 single channels.

Effect of CPVT/ARVD2 Mutations on the Ca²⁺ Dependence of [³H]Ryanodine Binding
Figure 6A shows that the Ca²⁺ dependence of [³H]ryanodine binding to the C-terminal mutants Q4201R and I4867M, the central region mutants S2246L and R2474S, and the N-terminal mutants R176Q(T2504M) and L433P is similar to that of the wt. The EC50 values for Ca²⁺ activation of [³H]ryanodine binding were 0.28±0.02 µmol/L (n=15) for wt, 0.24±0.02 µmol/L (n=5) for Q4201R, 0.20±0.01 µmol/L (n=4) for I4867M, 0.24±0.02 µmol/L (n=4) for S2246L, 0.24±0.05 µmol/L (n=3) for R2474S, 0.19±0.01 µmol/L (n=3) for R176Q(T2504M), and 0.23±0.02 µmol/L (n=4) for L433P. These observations are consistent with our previous results, which showed that the CPVT mutations N4104K, R4496C, and N4895D from the C-terminal region did not markedly alter the Ca²⁺ dependence of [³H]ryanodine binding.¹²

View larger version (42K): [in this window] [in a new window]   Figure 6. Effects of CPVT/ARVD2 mutations on Ca2+ activation of RyR2. A, [3H]ryanodine binding to cell lysate prepared from HEK293 cells transfected with the RyR2 wt or mutants Q4201R, I4867M (a), S2246L, R2474S (b), R176Q/T2504M, and L433P (c) was performed at various Ca2+ concentrations (&0.2 nM to 0.1 mmol/L), 100 mmol/L KCl, and 5 nM [3H]ryanodine. Amounts of [3H]ryanodine binding at various Ca2+ concentrations were normalized to the maximal binding. Data points shown are mean±SEM from 3 to 15 experiments. B, Single channel activities of the RyR2 wt and mutants were recorded in the presence of &45 nmol/L luminal Ca2+ and various concentrations of cytosolic Ca2+. The relationships between Po and cytosolic Ca2+ concentrations (pCa) of single RyR2 wt channels (open circles) and single Q4201R (solid triangles; a), R2474S (solid diamond; b), and R176Q/T2504M (solid circles; c) mutant channels are shown. Data points shown are individual measurements obtained from 7 to 10 single RyR2 wt or mutant channels.

Effect of CPVT/ARVD2 Mutations on the Sensitivity of RyR2 to Cytosolic Ca²⁺ Activation
As the Ca²⁺ dependence of [³H]ryanodine binding largely reflects the response of the channel to cytosolic Ca²⁺ activation,²³ the results of our [³H]ryanodine binding studies (Figure 6A) suggest that CPVT/ARVD2 RyR2 mutations have little effect on the channel sensitivity to cytosolic Ca²⁺ activation. To test this possibility directly, we determined the sensitivity of single RyR2 wt and mutant channels to cytosolic Ca²⁺ activation in lipid bilayers in the near absence of luminal Ca²⁺ (45 nmol/L). As shown in Figure 6B, the responses to cytosolic Ca²⁺ of single RyR2 wt and mutant channels, R176Q/T2504M, R2474S, and Q4201R, are indistinguishable. Collectively, our [³H]ryanodine binding and single channel studies demonstrate that CPVT/ARVD2 RyR2 mutations primarily alter the sensitivity of the channel to luminal, but not to cytosolic, Ca²⁺ activation.

CPVT/ARVD2 Mutations Do Not Alter the FKBP12.6–RyR2 Interaction
Because HEK293 cells express no detectable level of FKBP12.6,²¹ the enhanced SOICR observed in HEK293 cells expressing CPVT/ARVD2 RyR2 mutants is unlikely to be dependent on the FKBP12.6–RyR2 interaction. However, this does not necessarily mean that CPVT/ARVD2 RyR2 mutations do not affect the FKBP12.6-RyR2 interaction. To address this possibility, we examined ³⁵S-labeled FKBP12.6 binding to RyR2 wt and the CPVT/ARVD2 mutants. As shown in Figure 7, the concentration dependence of [³⁵S]FKBP12.6 binding to RyR2 wt and the mutants Q4201R, I4867M, S2246L, R2474S, R176Q(T2504M), and L433P at 4°C is virtually identical. Similar results were obtained at 37°C. These data indicate that the CPVT/ARVD2 RyR2 mutations from different regions of the channel do not alter the FKBP12.6–RyR2 interaction.

View larger version (34K): [in this window] [in a new window]   Figure 7. Effects of CPVT/ARVD2 mutations on FKBP12.6 binding. Cell lysate prepared from HEK293 cells transfected with the RyR2 wt or mutants Q4201R, I4867M (A), S2246L, R2474S (B), R176Q/T2504M, or L433P (C) was incubated with various concentrations of [35S]FKBP12.6 (0.1 to 60 nmol/L) at 4°C for 18 hours or with 10 nmol/L [35S]FKBP12.6 at 37°C for 90 minutes (D). The [35S]FKBP12.6–RyR2 complex was immunoprecipitated with an anti-RyR antibody. The bound [35S]FKBP12.6 was normalized to the maximal binding obtained at 60 nmol/L [35S]FKBP12.6. All binding was performed in duplicate. Data shown are mean±SEM from 3 separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
To date &40 mutations in RyR2 have been associated with CPVT/ARVD2. They are mostly located in the N-terminal, central, and C-terminal regions of the channel. We have recently demonstrated that 3 CPVT mutations in the C-terminal region reduce the threshold for SOICR by increasing the sensitivity of the channel to luminal Ca²⁺ activation.¹² Despite these observations, an important question remained: do enhanced SOICR and luminal Ca²⁺ activation represent common defects of disease-linked RyR2 mutations? To address this question, we characterized 6 more CPVT/ARVD2 RyR2 mutations, 2 from each region, and found that each of these mutations augments SOICR activity and increases channel sensitivity to activation by luminal Ca²⁺ (Figures 2, 3, and 5). Importantly, the impact of CPVT/ARVD2 RyR2 mutations on SOICR was also observed in the context of cardiac cells (Figure 4). These data indicate that enhanced SOICR and luminal Ca²⁺ activation are common characteristics of CPVT/ARVD2 RyR2 mutations.

How Do RyR2 Mutations From Different Regions Alter Luminal Ca²⁺ Activation?
Activation of RyR2 by luminal Ca²⁺ is likely to be mediated by a luminal Ca²⁺ sensor, but the exact location of this putative luminal Ca²⁺ sensor has yet to be defined. It has recently been shown that CASQ2, together with triadin and junctin, confers luminal Ca²⁺ sensitivity to RyR2, suggesting that CASQ2 may serve as a luminal Ca²⁺ sensor.²⁴ Alternatively, data from our mutational studies using recombinant RyR2 channels expressed in HEK293 cells and results from other groups using purified native RyR2 channels suggest that the luminal Ca²⁺ sensor lies within the primary structure of RyR2.^25,26 It is possible that a macromolecular complex, including RyR2, CASQ2, triadin, junctin, or other RyR2 associated proteins, is involved in luminal Ca²⁺ sensing.

We reason that mutations in RyR2 may alter the channel sensitivity to luminal Ca²⁺ activation by affecting (1) the binding of Ca²⁺ to the luminal Ca²⁺ sensor, (2) conformational changes induced by Ca²⁺ binding to the sensor, or (3) the gating of the channel. The putative luminal Ca²⁺ sensor is almost certainly located in a region accessible to luminal Ca²⁺, either on the luminal side of the channel or within the channel pore. Hence, mutations in the C-terminal region of the channel, which is thought to encompass the channel pore and the luminal region of the channel, may interfere with the binding of Ca²⁺ to the luminal sensor or with channel gating that is coupled to the luminal Ca²⁺ sensor. On the other hand, mutations in the N-terminal and central regions of the channel are unlikely to directly affect the binding of Ca²⁺ to the luminal Ca²⁺ sensor. It has been proposed that the N-terminal and central regions of the channel may be located in close proximity in the 3D structure of RyR and physically interact with each other.²⁷ If so, the N-terminal and central region RyR2 mutations may weaken the domain–domain interactions that stabilize the channel, facilitating the conformational changes induced by binding of Ca²⁺ to the luminal Ca²⁺ sensor and enhancing luminal Ca²⁺ activation.

Roles of FKBP12.6 in CPVT/ARVD2
FKBP12.6 is believed to play an important role in RyR2 function. However, its involvement in CPVT/ARVD2 is controversial. Tiso et al reported that the N2386I and Y2392C mutations, located in the central region, reduced the affinity of FKBP12.6 binding, whereas the R2474S mutation, located in the same region, markedly increased the affinity of FKBP12.6 binding.²⁸ In contrast to these observations from Tiso et al, Wehrens et al reported that the R2474S mutation decreased the affinity of FKBP12.6 binding.¹⁹ A decreased FKBP12.6 binding affinity was also observed with the mutants S2246L and P2328S, located in the central region, and the mutants Q4201R, R4496C, and V4653F, located in the C-terminal region. Furthermore, it has been shown that these mutant channels display enhanced channel activity only on treatment with PKA.^19,29

Based on their previous finding that the phosphorylation of RyR2 by PKA causes the dissociation of FKBP12.6 from RyR2, Wehrens et al proposed that CPVT/ARVD2 mutant channels are more susceptible to PKA-phosphorylation–induced dissociation of FKBP12.6 as a result of a reduced affinity for FKBP12.6, thus leading to a more active channel on phosphorylation by PKA.¹⁹ However, studies by Gorge et al demonstrated that CPVT mutations enhance RyR2 channel activity in a manner independent of FKBP12.6 binding and PKA phosphorylation.¹⁷ We and others have also shown that complete phosphorylation of either recombinant or native RyR2 by PKA does not dissociate either coexpressed or endogenous FKBP12.6.^20,21 In the present study, we show that CPVT/ARVD2 RyR2 mutations have no effect on FKBP12.6 binding (Figure 7). The reason for this discrepancy is not clear but is most likely because of differences in experimental conditions. Further detailed understanding of the biochemistry of interaction between FKBP12.6 and RyR2 is essential to resolve this controversy.

Functional Heterogeneity of RyR2 Mutations
Although most of the disease-linked RyR2 mutations enhance channel activity-gain-of-function, variable impacts of RyR2 mutations on channel function have been reported.^18,30 For instance, it has been shown that the ARVD2 mutation L433P, located in the N-terminal region, exhibited a reduced sensitivity to activation by caffeine,¹⁸ which seemed to contradict our findings that the L433P mutation increased SOICR activity and the sensitivity of the channel to luminal Ca²⁺ activation. To further understand the impact of this mutation, we investigated the sensitivity of this mutant to activation by caffeine. We found that the L433P mutation increased, rather than decreased, the sensitivity of the RyR2 channel to caffeine activation (supplemental Figure I). One potential explanation for this discrepancy may be related to the DNA constructs used. In the study of Thomas et al, the human RyR2 wt and mutants were tagged at the N terminus with enhanced green fluorescence protein (GFP).¹⁸ This insertion of GFP into the N terminus may interfere with the action of the L433P mutation. More work is needed to resolve this discrepancy.

A Proposed Mechanism for CPVT/ARVD2 Linked to RyR2 Mutations
Based on our data, we suggest that CPVT/ARVD2 RyR2 mutations reduce the threshold for SOICR by increasing the sensitivity of the channel to activation by luminal Ca²⁺, thus enhancing the propensity for DADs and triggered arrhythmias under conditions of SR Ca²⁺ overload (Figure 8). Because CPVT/ARVD2 mutations do not alter cytosolic Ca²⁺ activation (Figure 6), which is thought to underlie the mechanism of CICR, it is expected that CPVT/ARVD2 mutations would not impair normal EC coupling in the absence of SR Ca²⁺ overload. Consistent with this prediction, patients with CPVT/ARVD2 mutations have apparently normal hearts at rest. However, under conditions in which the SR Ca²⁺ content is abruptly increased, such as during exercise, emotional stress, or on the infusion of catecholamines, SOICR will be more likely to occur in the CPVT/ARVD2 SR than in the normal SR, because of the reduced SOICR threshold of the former. The resulting large SR Ca²⁺ spillover can activate the Na/Ca²⁺ exchanger, leading to DADs, which can in turn result in triggered arrhythmia. Therefore, the sensitivity of RyR2 to luminal Ca²⁺ activation is a key determinant of SOICR, and, consequently, DADs and cardiac arrhythmia.

View larger version (32K): [in this window] [in a new window]   Figure 8. A proposed model for RyR2-associated CPVT/ARVD2. The mechanisms of Ca2+-induced Ca2+ release (CICR; left) and store overload–induced Ca2+ release (SOICR; right) are schematically shown. Some major proteins involved in cardiac Ca2+ cycling, including the L-type Ca2+ channel, the RyR2 channel complex, the Na/Ca2+ exchanger, the SR Ca2+ pump, phospholamban (PLB), and calsequestrin (CASQ2) are indicated. The threshold for SOICR (depicted by a red bar), which is primarily determined by the RyR2 channel complex, is reduced in the CPVT/ARVD2 SR as compared with that in the normal SR (depicted by a red dash-line) because of RyR2 mutations. The SR free Ca2+ level, which is predominantly determined by CASQ2, is represented by the blue area. Under conditions of emotional and physical stresses or on infusion of catecholamines, ß-adrenergic receptors are activated, leading to activation of PKA, which in turn phosphorylates the L-type Ca2+ channel and PLB. This PKA phosphorylation increases both Ca2+ influx and SR Ca2+ uptake, resulting in an abrupt increase in SR free Ca2+ (depicted by the yellow area). Because of its reduced threshold, SOICR will be more likely to occur from the CPVT/ARVD2 SR during SR Ca2+ loading. The resulting large SR Ca2+ spillover can lead to DAD and triggered arrhythmia.

Implication for CPVT Linked to CASQ2 Mutations
In addition to mutations in RyR2, CPVT is also linked to mutations in CASQ2. Given their virtually identical phenotypes, RyR2-associated CPVT and CASQ2-linked CPVT may share a common causal mechanism. CASQ2, a low affinity, high capacity Ca²⁺ binding protein, is a major SR Ca²⁺ buffering protein. Mutations in CASQ2 are believed to diminish SR Ca²⁺ buffering capacity by reducing either the expression level or the Ca²⁺ binding capability of CASQ2. From the perspective of our SOICR model, a reduction in SR Ca²⁺ buffering would be expected to increase the rate of SR free Ca²⁺ elevation, thus reducing the time to reach the threshold SR free Ca²⁺ that is required to initiate SOICR. Consistent with this view, Kubalova et al have recently shown that increasing the level of CASQ2 expression prolonged the period for SR free Ca²⁺ recovery after SR Ca²⁺ release, whereas reducing the level of CASQ2 expression shortened the period for SR free Ca²⁺ recovery.³¹ Interestingly, alterations in the expression level of CASQ2 or loading the cell with citrate, a low affinity Ca²⁺ chelator, have no effect on the threshold SR free Ca²⁺ level that is required for the initiation of Ca²⁺ waves. Ca²⁺ waves were always initiated when the SR free Ca²⁺ reached a certain threshold level.³¹ These observations, together with our data, indicate that either reducing the threshold for SOICR through mutations in RyR2 or increasing the rate of SR free Ca²⁺ elevation as a result of mutations in CASQ2^32,33 can enhance the propensity for SOICR and, consequently, DADs and triggered arrhythmias. Hence, enhanced SOICR as a result of increased luminal Ca²⁺ activation of RyR2 may be a common mechanism underlying both RyR2- and CASQ2-linked CPVT.

	Acknowledgments

 
This work was supported by research grants from the Canadian Institutes of Health Research (CIHR) and the Heart and Stroke Foundation of Alberta, Northwest Territories, and Nunavut (HSFA) to S.R.W.C. D.J., B.X., and H.K. are recipients of the Alberta Heritage Foundation for Medical Research (AHFMR) Studentship Award, and S.R.W.C. is a Senior Scholar of the AHFMR. D.J. is also a recipient of the Canada Graduate Scholarships (CGS) Doctoral Awards from CIHR. The authors thank Dr Jonathan Lytton for helpful discussions and the use of the single cell Ca²⁺ imaging facility and Jeff Bolstad for critical reading of the manuscript.

	Footnotes

 
Original received April 21, 2005; resubmission received September 26, 2005; accepted October 12, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Janse M. Electrophysiological changes in heart failure and their relationship to arrhythmogenesis. Cardiovasc Res. 2004; 61: 208–217.[Abstract/Free Full Text]

2. Pogwizd SM, Bers DM. Cellular basis of triggered arrhythmias in heart failure. Trends Cardiovasc Med. 2004; 14: 61–66.[CrossRef][Medline] [Order article via Infotrieve]

3. Leenhardt A, Lucet V, Denjoy I, Grau F, Ngoc DD, Coumel P. Catecholaminergic polymorphic ventricular tachycardia in children. A 7-year follow-up of 21 patients. Circulation. 1995; 91: 1512–1519.[Abstract/Free Full Text]

4. Laitinen P, Swan H, Piippo K, Viitasalo M, Toivonen L, Kontula K. Genes, exercise and sudden death: molecular basis of familial catecholaminergic polymorphic ventricular tachycardia. Ann Med. 2004; 36 (Suppl 1): 81–6.[CrossRef][Medline] [Order article via Infotrieve]

5. Priori SG, Napolitano C, Memmi M, Colombi B, Drago F, Gasparini M, DeSimone L, Coltorti F, Bloise R, Keegan R, Cruz Filho FE, Vignati G, Benatar A, DeLogu A. Clinical and molecular characterization of patients with catecholaminergic polymorphic ventricular tachycardia. Circulation. 2002; 106: 69–74.[Abstract/Free Full Text]

6. Laitinen PJ, Brown KM, Piippo K, Swan H, Devaney JM, Brahmbhatt B, Donarum EA, Marino M, Tiso N, Viitasalo M, Toivonen L, Stephan DA, Kontula K. Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic ventricular tachycardia. Circulation. 2001; 103: 485–490.[Abstract/Free Full Text]

7. Tiso N, Stephan DA, Nava A, Bagattin A, Devaney JM, Stanchi F, Larderet G, Brahmbhatt B, Brown K, Bauce B, Muriago M, Basso C, Thiene G, Danieli GA, Rampazzo A. Identification of mutations in the cardiac ryanodine receptor gene in families affected with arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2). Hum Mol Genet. 2001; 10: 189–194.[Abstract/Free Full Text]

8. Lahat H, Pras E, Olender T, Avidan N, Ben-Asher E, Man O, Levy-Nissenbaum E, Khoury A, Lorber A, Goldman B, Lancet D, Eldar M. A missense mutation in a highly conserved region of CASQ2 is associated with autosomal recessive catecholamine-induced polymorphic ventricular tachycardia in Bedouin families from Israel. Am J Hum Genet. 2001; 69: 1378–1384.[CrossRef][Medline] [Order article via Infotrieve]

9. Postma AV, Denjoy I, Hoorntje TM, Lupoglazoff J-M, Da Costa A, Sebillon P, Mannens MMAM, Wilde AAM, Guicheney P. Absence of calsequestrin 2 causes severe forms of catecholaminergic polymorphic ventricular tachycardia. Circ Res. 2002; 91: e21–e26.[CrossRef][Medline] [Order article via Infotrieve]

10. Bers DM. Excitation-Contraction Coupling and Cardiac Contractile Force, Second Edition. Dordrecht, The Netherlands: Kluwer Academic Publishers; 2001.

11. Lakatta EG. Functional implications of spontaneous sarcoplasmic reticulum Ca²⁺ release in the heart. Cardiovasc Res. 1992; 26: 193–214.[Free Full Text]

12. Jiang D, Xiao B, Yang D, Wang R, Choi P, Zhang L, Cheng H, Chen SRW. RyR2 mutations linked to ventricular tachycardia and sudden death reduce the threshold for store-overload-induced Ca²⁺ release (SOICR). Proc Natl Acad Sci U S A. 2004; 101: 13062–13067.[Abstract/Free Full Text]

13. Kass RS, Tsien RW. Fluctuations in membrane current driven by intracellular calcium in cardiac Purkinje fibers. Biophys J. 1982; 38: 259–269.[Medline] [Order article via Infotrieve]

14. Orchard C, Eisner D, Allen D. Oscillations of intracellular Ca²⁺ in mammalian cardiac muscle. Nature. 1983; 304: 735–738.[CrossRef][Medline] [Order article via Infotrieve]

15. Stern M, Kort A, Bhatnagar G, Lakatta E. Scattered-light intensity fluctuations in diastolic rat cardiac muscle caused by spontaneous Ca++-dependent cellular mechanical oscillations. J Gen Physiol. 1983; 82: 119–153.[Abstract/Free Full Text]

16. Wier W, Kort A, Stern M, Lakatta E, Marban E. Cellular calcium fluctuations in mammalian heart: direct evidence from noise analysis of aequorin signals in Purkinje fibers. Proc Natl Acad Sci U S A. 1983; 80: 7367–7371.[Abstract/Free Full Text]

17. George CH, Higgs GV, Lai FA. Ryanodine receptor mutations associated with stress-induced ventricular tachycardia mediate increased calcium release in stimulated cardiomyocytes. Circ Res. 2003; 93: 531–540.[Abstract/Free Full Text]

18. Thomas NL, George CH, Lai FA. Functional heterogeneity of ryanodine receptor mutations associated with sudden cardiac death. Cardiovasc Res. 2004; 64: 52–60.[Abstract/Free Full Text]

19. Wehrens X, Lehnart S, Huang F, Vest J, Reiken S, Mohler P, Sun J, Guatimosim S, Song L, Rosemblit N, D’Armiento J, Napolitano C, Memmi M, Priori S, Lederer W, Marks A. FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death. Cell. 2003; 113: 829–840.[CrossRef][Medline] [Order article via Infotrieve]

20. Stange M, Xu L, Balshaw D, Yamaguchi N, Meissner G. Characterization of recombinant skeletal muscle (Ser-2843) and cardiac muscle (Ser-2809) ryanodine receptor phosphorylation mutants. J Biol Chem. 2003; 278: 51693–51702.[Abstract/Free Full Text]

21. Xiao B, Sutherland C, Walsh MP, Chen SRW. Protein kinase A phosphorylation at serine-2808 of the cardiac Ca²⁺-release channel (ryanodine receptor) does not dissociate 12.6-kDa FK506-binding protein (FKBP12.6). Circ Res. 2004; 94: 487–495.[Abstract/Free Full Text]

22. Diaz M, Trafford A, O’Neill S, Eisner D. Measurement of sarcoplasmic reticulum Ca²⁺ content and sarcolemmal Ca²⁺ fluxes in isolated rat ventricular myocytes during spontaneous Ca²⁺ release. J Physiol (Lond). 1997; 501: 3–16.[Abstract/Free Full Text]

23. Jiang D, Xiao B, Zhang L, Chen SR. Enhanced basal activity of a cardiac Ca²⁺ release channel (ryanodine receptor) mutant associated with ventricular tachycardia and sudden death. Circ Res. 2002; 91: 218–225.[Abstract/Free Full Text]

24. Gyorke I, Hester N, Jones LR, Gyorke S. The Role of Calsequestrin, Triadin, and Junctin in Conferring Cardiac Ryanodine Receptor Responsiveness to Luminal Calcium. Biophys J. 2004; 86: 2121–2128.[Medline] [Order article via Infotrieve]

25. Sitsapesan R, Williams A. Regulation of the gating of the sheep cardiac sarcoplasmic reticulum Ca(²⁺)-release channel by luminal Ca²⁺. J Membr Biol. 1994; 137: 215–226.[Medline] [Order article via Infotrieve]

26. Xu L, Meissner G. Regulation of cardiac muscle Ca²⁺ release channel by sarcoplasmic reticulum lumenal Ca²⁺. Biophys J. 1998; 75: 2302–2312.[Medline] [Order article via Infotrieve]

27. Ikemoto N, Yamamoto T. Postulated role of inter-domain interaction within the ryanodine receptor in Ca(²⁺) channel regulation. Trends Cardiovasc Med. 2000; 10: 310–316.[CrossRef][Medline] [Order article via Infotrieve]

28. Tiso N, Salamon M, Bagattin A, Danieli G, Argenton F, Bortolussi M. The binding of the RyR2 calcium channel to its gating protein FKBP12.6 is oppositely affected by ARVD2 and VTSIP mutations. Biochem Biophys Res Commun. 2002; 299: 594–598.[CrossRef][Medline] [Order article via Infotrieve]

29. Lehnart SE, Wehrens XHT, Laitinen PJ, Reiken SR, Deng S-X, Cheng Z, Landry DW, Kontula K, Swan H, Marks AR. Sudden Death in familial polymorphic ventricular tachycardia associated with calcium release channel (ryanodine receptor) leak. Circulation. 2004; 109: 3208–3214.[Abstract/Free Full Text]

30. Thomas NL, Lai FA, George CH. Differential Ca²⁺ sensitivity of RyR2 mutations reveals distinct mechanisms of channel dysfunction in sudden cardiac death. Biochem Biophys Res Commun. 2005; 331: 231–238.[CrossRef][Medline] [Order article via Infotrieve]

31. Kubalova Z, Gyorke I, Terentyeva R, Viatchenko-Karpinski S, Terentyev D, Williams SC, Gyorke S. Modulation of cytosolic and intra-sarcoplasmic reticulum calcium waves by calsequestrin in rat cardiac myocytes. J Physiol (Lond). 2004; 561: 515–524.[Abstract/Free Full Text]

32. Terentyev D, Viatchenko-Karpinski S, Gyorke I, Volpe P, Williams SC, Gyorke S. Calsequestrin determines the functional size and stability of cardiac intracellular calcium stores: Mechanism for hereditary arrhythmia. Proc Natl Acad Sci U S A. 2003; 100: 11759–11764.[Abstract/Free Full Text]

33. Viatchenko-Karpinski S, Terentyev D, Gyorke I, Terentyeva R, Volpe P, Priori SG, Napolitano C, Nori A, Williams SC, Gyorke S. Abnormal calcium signaling and sudden cardiac death associated with mutation of calsequestrin. Circ Res. 2004; 94: 471–477.[Abstract/Free Full Text]

Related Article:

Intracellular Calcium Handling Dysfunction and Arrhythmogenesis: A New Challenge for the Electrophysiologist

Silvia G. Priori and Carlo Napolitano
Circ. Res. 2005 97: 1077-1079. [Extract] [Full Text] [PDF]

This article has been cited by other articles:

				D. H. MacLennan and S. R. W. Chen Store overload-induced Ca2+ release as a triggering mechanism for CPVT and MH episodes caused by mutations in RYR and CASQ genes J. Physiol., July 1, 2009; 587(13): 3113 - 3115. [Full Text] [PDF]

				W. Chen, J. A. Wasserstrom, and Y. Shiferaw Role of coupled gating between cardiac ryanodine receptors in the genesis of triggered arrhythmias Am J Physiol Heart Circ Physiol, July 1, 2009; 297(1): H171 - H180. [Abstract] [Full Text] [PDF]

				H. Tateishi, M. Yano, M. Mochizuki, T. Suetomi, M. Ono, X. Xu, H. Uchinoumi, S. Okuda, T. Oda, S. Kobayashi, et al. Defective domain-domain interactions within the ryanodine receptor as a critical cause of diastolic Ca2+ leak in failing hearts Cardiovasc Res, February 15, 2009; 81(3): 536 - 545. [Abstract] [Full Text] [PDF]

				D. R. Laver and B. N. Honen Luminal Mg2+, A Key Factor Controlling RYR2-mediated Ca2+ Release: Cytoplasmic and Luminal Regulation Modeled in a Tetrameric Channel J. Gen. Physiol., October 1, 2008; 132(4): 429 - 446. [Abstract] [Full Text] [PDF]

				D. Jiang, W. Chen, J. Xiao, R. Wang, H. Kong, P. P. Jones, L. Zhang, B. Fruen, and S. R. W. Chen Reduced Threshold for Luminal Ca2+ Activation of RyR1 Underlies a Causal Mechanism of Porcine Malignant Hyperthermia J. Biol. Chem., July 25, 2008; 283(30): 20813 - 20820. [Abstract] [Full Text] [PDF]

				S. Ozdemir, V. Bito, P. Holemans, L. Vinet, J.-J. Mercadier, A. Varro, and K. R. Sipido Pharmacological Inhibition of Na/Ca Exchange Results in Increased Cellular Ca2+ Load Attributable to the Predominance of Forward Mode Block Circ. Res., June 6, 2008; 102(11): 1398 - 1405. [Abstract] [Full Text] [PDF]

				N. Liu and S. G. Priori Disruption of calcium homeostasis and arrhythmogenesis induced by mutations in the cardiac ryanodine receptor and calsequestrin Cardiovasc Res, January 15, 2008; 77(2): 293 - 301. [Abstract] [Full Text] [PDF]

				S. Gyorke and D. Terentyev Modulation of ryanodine receptor by luminal calcium and accessory proteins in health and cardiac disease Cardiovasc Res, January 15, 2008; 77(2): 245 - 255. [Abstract] [Full Text] [PDF]

				J. Xiao, X. Tian, P. P. Jones, J. Bolstad, H. Kong, R. Wang, L. Zhang, H. J. Duff, A. M. Gillis, S. Fleischer, et al. Removal of FKBP12.6 Does Not Alter the Conductance and Activation of the Cardiac Ryanodine Receptor or the Susceptibility to Stress-induced Ventricular Arrhythmias J. Biol. Chem., November 30, 2007; 282(48): 34828 - 34838. [Abstract] [Full Text] [PDF]

				D. Jiang, W. Chen, R. Wang, L. Zhang, and S. R. W. Chen Loss of luminal Ca2+ activation in the cardiac ryanodine receptor is associated with ventricular fibrillation and sudden death PNAS, November 13, 2007; 104(46): 18309 - 18314. [Abstract] [Full Text] [PDF]

				B. Xiao, X. Tian, W. Xie, P. P. Jones, S. Cai, X. Wang, D. Jiang, H. Kong, L. Zhang, K. Chen, et al. Functional Consequence of Protein Kinase A-dependent Phosphorylation of the Cardiac Ryanodine Receptor: SENSITIZATION OF STORE OVERLOAD-INDUCED Ca2+ RELEASE J. Biol. Chem., October 12, 2007; 282(41): 30256 - 30264. [Abstract] [Full Text] [PDF]

				Z. A. Bhuiyan, M. P. van den Berg, J. P. van Tintelen, M. T.E. Bink-Boelkens, A. C.P. Wiesfeld, M. Alders, A. V. Postma, I. van Langen, M. M.A.M. Mannens, and A. A.M. Wilde Expanding Spectrum of Human RYR2-Related Disease: New Electrocardiographic, Structural, and Genetic Features Circulation, October 2, 2007; 116(14): 1569 - 1576. [Abstract] [Full Text] [PDF]

				P. J. Mohler and X. H. T. Wehrens Mechanisms of Human Arrhythmia Syndromes: Abnormal Cardiac Macromolecular Interactions Physiology, October 1, 2007; 22(5): 342 - 350. [Abstract] [Full Text] [PDF]

				N. Chopra, P. J. Kannankeril, T. Yang, T. Hlaing, I. Holinstat, K. Ettensohn, K. Pfeifer, B. Akin, L. R. Jones, C. Franzini-Armstrong, et al. Modest Reductions of Cardiac Calsequestrin Increase Sarcoplasmic Reticulum Ca2+ Leak Independent of Luminal Ca2+ and Trigger Ventricular Arrhythmias in Mice Circ. Res., September 14, 2007; 101(6): 617 - 626. [Abstract] [Full Text] [PDF]

				D. Terentyev, S. Viatchenko-Karpinski, S. Vedamoorthyrao, S. Oduru, I. Gyorke, S. C. Williams, and S. Gyorke Protein protein interactions between triadin and calsequestrin are involved in modulation of sarcoplasmic reticulum calcium release in cardiac myocytes J. Physiol., August 15, 2007; 583(1): 71 - 80. [Abstract] [Full Text] [PDF]

				R. Wang, W. Chen, S. Cai, J. Zhang, J. Bolstad, T. Wagenknecht, Z. Liu, and S. R. W. Chen Localization of an NH2-terminal Disease-causing Mutation Hot Spot to the "Clamp" Region in the Three-dimensional Structure of the Cardiac Ryanodine Receptor J. Biol. Chem., June 15, 2007; 282(24): 17785 - 17793. [Abstract] [Full Text] [PDF]

				R. P. Katra, T. Oya, G. S. Hoeker, and K. R. Laurita Ryanodine receptor dysfunction and triggered activity in the heart Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2144 - H2151. [Abstract] [Full Text] [PDF]

				H. E. D. J. ter Keurs and P. A. Boyden Calcium and Arrhythmogenesis Physiol Rev, April 1, 2007; 87(2): 457 - 506. [Abstract] [Full Text] [PDF]

				V. Iyer, R. J. Hajjar, and A. A. Armoundas Mechanisms of Abnormal Calcium Homeostasis in Mutations Responsible for Catecholaminergic Polymorphic Ventricular Tachycardia Circ. Res., February 2, 2007; 100(2): e22 - e31. [Abstract] [Full Text] [PDF]

				L. A. Venetucci, A. W. Trafford, and D. A. Eisner Increasing Ryanodine Receptor Open Probability Alone Does Not Produce Arrhythmogenic Calcium Waves: Threshold Sarcoplasmic Reticulum Calcium Content Is Required Circ. Res., January 5, 2007; 100(1): 105 - 111. [Abstract] [Full Text] [PDF]

				D. M. Bers Altered Cardiac Myocyte Ca Regulation In Heart Failure. Physiology, December 1, 2006; 21(6): 380 - 387. [Abstract] [Full Text] [PDF]

				D. M. Bers The Beat Goes On: Diastolic Noise That Just Won't Quit Circ. Res., October 27, 2006; 99(9): 921 - 923. [Full Text] [PDF]

				P. J. Kannankeril, B. M. Mitchell, S. A. Goonasekera, M. G. Chelu, W. Zhang, S. Sood, D. L. Kearney, C. I. Danila, M. De Biasi, X. H. T. Wehrens, et al. Mice with the R176Q cardiac ryanodine receptor mutation exhibit catecholamine-induced ventricular tachycardia and cardiomyopathy PNAS, August 8, 2006; 103(32): 12179 - 12184. [Abstract] [Full Text] [PDF]

				D.A. Eisner, L.A. Venetucci, and A.W. Trafford Life, Sudden Death, and Intracellular Calcium Circ. Res., August 4, 2006; 99(3): 223 - 224. [Full Text] [PDF]

				N. Liu, B. Colombi, M. Memmi, S. Zissimopoulos, N. Rizzi, S. Negri, M. Imbriani, C. Napolitano, F. A. Lai, and S. G. Priori Arrhythmogenesis in Catecholaminergic Polymorphic Ventricular Tachycardia: Insights From a RyR2 R4496C Knock-In Mouse Model Circ. Res., August 4, 2006; 99(3): 292 - 298. [Abstract] [Full Text] [PDF]

				I. Jona and P. P. Nanasi Cardiomyopathies and sudden cardiac death caused by RyR2 mutations: Are the channels the beginning and the end? Cardiovasc Res, August 1, 2006; 71(3): 416 - 418. [Full Text] [PDF]

				M. Fernandez-Velasco, A. M. Gomez, and S. Richard Unzipping RyR2 in adult cardiomyocytes: Getting closer to mechanisms of inherited ventricular arrhythmias? Cardiovasc Res, June 1, 2006; 70(3): 407 - 409. [Full Text] [PDF]

				D. Terentyev, A. Nori, M. Santoro, S. Viatchenko-Karpinski, Z. Kubalova, I. Gyorke, R. Terentyeva, S. Vedamoorthyrao, N. A. Blom, G. Valle, et al. Abnormal Interactions of Calsequestrin With the Ryanodine Receptor Calcium Release Channel Complex Linked to Exercise-Induced Sudden Cardiac Death Circ. Res., May 12, 2006; 98(9): 1151 - 1158. [Abstract] [Full Text] [PDF]

				S. G. Priori and C. Napolitano Intracellular Calcium Handling Dysfunction and Arrhythmogenesis: A New Challenge for the Electrophysiologist Circ. Res., November 25, 2005; 97(11): 1077 - 1079. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 97/11/1173    most recent 01.RES.0000192146.85173.4bv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jiang, D. Articles by Chen, S. R. W. Search for Related Content PubMed PubMed Citation Articles by Jiang, D. Articles by Chen, S. R. W. Related Collections Related Article