Enhanced Store OverloadeCInduced Ca2+ Release and Channel Sensitivity to Luminal Ca2+ Activation Are Common Defects of RyR2 Mutations Linked
http://www.100md.com
循环研究杂志 2005年第11期
the Cardiovascular Research Group, Departments of Physiology and Biophysics
of Biochemistry and Molecular Biology, University of Calgary, Alberta, Canada.
Abstract
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 overloadeCinduced 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 FKBP12.6eCRyR2 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
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 VT1,2; 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).5eC9 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+ overloadeCinduced 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-overloadeCinduced 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.13eC16
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 -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.
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 FKBP12.6eCRyR2 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
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
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).
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.
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.
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 eol/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.
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 eol/L (n=15) for wt, 0.24±0.02 eol/L (n=5) for Q4201R, 0.20±0.01 eol/L (n=4) for I4867M, 0.24±0.02 eol/L (n=4) for S2246L, 0.24±0.05 eol/L (n=3) for R2474S, 0.19±0.01 eol/L (n=3) for R176Q(T2504M), and 0.23±0.02 eol/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
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 FKBP12.6eCRyR2 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 FKBP12.6eCRyR2 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 FKBP12.6eCRyR2 interaction.
Discussion
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 domaineCdomain 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-phosphorylationeCinduced 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.
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 CASQ232,33 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.
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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: 215eC226.
Xu L, Meissner G. Regulation of cardiac muscle Ca2+ release channel by sarcoplasmic reticulum lumenal Ca2+. Biophys J. 1998; 75: 2302eC2312.
Ikemoto N, Yamamoto T. Postulated role of inter-domain interaction within the ryanodine receptor in Ca(2+) channel regulation. Trends Cardiovasc Med. 2000; 10: 310eC316.
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: 594eC598.
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: 3208eC3214.
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: 231eC238.
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: 515eC524.
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: 11759eC11764.
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: 471eC477.(Dawei Jiang, Ruiwu Wang, )
of Biochemistry and Molecular Biology, University of Calgary, Alberta, Canada.
Abstract
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 overloadeCinduced 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 FKBP12.6eCRyR2 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
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 VT1,2; 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).5eC9 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+ overloadeCinduced 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-overloadeCinduced 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.13eC16
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 -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.
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 FKBP12.6eCRyR2 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
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
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).
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.
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.
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 eol/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.
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 eol/L (n=15) for wt, 0.24±0.02 eol/L (n=5) for Q4201R, 0.20±0.01 eol/L (n=4) for I4867M, 0.24±0.02 eol/L (n=4) for S2246L, 0.24±0.05 eol/L (n=3) for R2474S, 0.19±0.01 eol/L (n=3) for R176Q(T2504M), and 0.23±0.02 eol/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
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 FKBP12.6eCRyR2 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 FKBP12.6eCRyR2 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 FKBP12.6eCRyR2 interaction.
Discussion
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 domaineCdomain 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-phosphorylationeCinduced 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.
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 CASQ232,33 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.
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