Catecholaminergic Polymorphic Ventricular Tachycardia Is Caused by Mutation-Linked Defective Conformational Regulation of the Ryanodine Receptor
Rationale: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by a single point mutation in a well-defined region of the cardiac type 2 ryanodine receptor (RyR)2. However, the underlying mechanism by which a single mutation in such a large molecule produces drastic effects on channel function remains unresolved.
Objective: Using a knock-in (KI) mouse model with a human CPVT-associated RyR2 mutation (R2474S), we investigated the molecular mechanism by which CPVT is induced by a single point mutation within the RyR2.
Methods and Results: The R2474S/+ KI mice showed no apparent structural or histological abnormalities in the heart, but they showed clear indications of other abnormalities. Bidirectional or polymorphic ventricular tachycardia was induced after exercise on a treadmill. The interaction between the N-terminal (amino acids 1 to 600) and central (amino acids 2000 to 2500) domains of the RyR2 (an intrinsic mechanism to close Ca2+ channels) was weakened (domain unzipping). On protein kinase A-mediated phosphorylation of the RyR2, this domain unzipping further increased, resulting in a significant increase in the frequency of spontaneous Ca2+ transients. cAMP-induced aberrant Ca2+ release events (Ca2+ sparks/waves) occurred at much lower sarcoplasmic reticulum Ca2+ content as compared to the wild type. Addition of a domain-unzipping peptide, DPc10 (amino acids 2460 to 2495), to the wild type reproduced the aforementioned abnormalities that are characteristic of the R2474S/+ KI mice. Addition of DPc10 to the (cAMP-treated) KI cardiomyocytes produced no further effect.
Conclusions: A single point mutation within the RyR2 sensitizes the channel to agonists and reduces the threshold of luminal [Ca2+] for activation, primarily mediated by defective interdomain interaction within the RyR2.
To date, more than 70 cardiac ryanodine receptor (RyR)2 missense mutations have been identified that are linked with 2 inherited forms of sudden cardiac death: catecholaminergic polymorphic ventricular tachycardia (CPVT)1 and arrhythmogenic right ventricular cardiomyopathy type 2.1 These mutations cluster in 3 well-defined regions of the RyR2 that correspond to malignant hyperthermia or the central core disease mutable regions, designated as the N-terminal domain (amino acids 1 to 600), central domain (amino acids 2000 to 2500), and the C-terminal transmembrane channel domain of the skeletal muscle-type ryanodine receptor (RyR1).1 This suggests that the RyR2 shares a common domain-mediated channel regulation mechanism with RyR1. Mutations at different positions in each of these domains result in the nearly identical phenotype of channel dysfunctions such as hyperactivation of the Ca2+ channel and hypersensitization to agonists. To account for these phenomena, Ikemoto et al2,3 proposed the so-called “domain switch hypothesis” and stated that in the resting or nonactivated state, the N-terminal domain and the central domain make close contact at several subdomains (domain zipping). Then, on physiological or pharmacological stimulation, these critical interdomain contacts are weakened, resulting in the loss of conformational constraints (domain unzipping), thus lowering the energy barrier for Ca2+ channel opening. Consistent with this hypothesis, single particle analysis of the 3D structure of the RyR2 molecule revealed that the N-terminal and central domains (located in domains 5 and 6 of the so-called clamp region, respectively) are in a close apposition to each other.4,5
Recent reports deal with 3 types of knock-in (KI) mice with human CPVT/arrhythmogenic right ventricular cardiomyopathy-associated RyR2 mutations: R4496C,6 R176Q,7 and R2474S.8 Injection of caffeine plus epinephrine or exercise induces ventricular tachycardia (VT) in these mice, indicating that these point mutations can cause lethal arrhythmias. However, the underlying mechanism by which a single mutation causes lethal arrhythmia remains unresolved. We recently reported that in failing hearts, defective interdomain interaction within the RyR2 (aberrant unzipping of the N-terminal/central domain pair and channel activation in an otherwise resting state) causes diastolic Ca2+ leakage and contractile dysfunction.9 As shown in our previous report,9 pathological conditions (diastolic Ca2+ leakage and contractile dysfunction) are reproduced in the otherwise normal system by adding DPc10, a central domain peptide (Gly2460-Pro2495) of the RyR2 that interferes with the interaction between the N-terminal and central domains of the RyR2 and causes defective domain unzipping. George et al10 showed that functional coupling between the cytoplasmic and transmembrane domains of the RyR2 is mediated by the 3722 to 4610 residue region, called the I-domain and that sudden cardiac death (SCD)-linked mutations occurring in the I-domain (N4104K and R4496C) caused channel instability and Ca2+ release dysfunction owing to defective interdomain interactions between the I-domain and the channel domain. These findings suggest that the weakened interdomain interaction at least in these 2 regions of the RyR2 (N-terminal domain/central domain and I-domain/channel domain) is the key mechanism underlying the pathogenesis of CPVT/arrhythmogenic right ventricular cardiomyopathy and heart failure.
Although the domain peptide approach provided important information regarding the underlying mechanism of the RyR2 abnormalities during heart failure and lethal arrhythmia, further in vivo studies with the mutation-linked disease model are required for a straightforward test of the interdomain interaction hypothesis. In the present study, using the KI mouse model with a human CPVT-associated RyR2 mutation, R2474S, we investigated the molecular mechanism by which CPVT is induced by a single point mutation within the RyR2. The data presented here suggested that the introduced mutation, in fact, causes defective interdomain interaction in the RyR2, reduces the threshold of luminal Ca2+-dependance for channel activation, sensitizes RyR2 to protein kinase (PK)A-dependent phosphorylation, and in turn leads to CPVT.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
This study conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH (NIH Publication No. 85-23, revised 1996). The care of the animals and the protocols used were in accordance with guidelines established by the Animal Ethics Committee of Yamaguchi University School of Medicine.
Paired or unpaired t tests were used for statistical comparisons of data obtained during the 2 different situations, whereas ANOVA with a post hoc Scheffe’s test was used for statistical comparison of concentration-dependent data. All data are expressed as means±SE. A probability value of <0.05 was considered statistically significant.
No Appreciable Change in Structural or Functional Characteristics of R2474S/+ KI Mice During the Resting (Nonactivated) State
In the absence of activation, there was no appreciable difference in the structural or functional features of the hearts between wild-type (WT) and KI mice. Thus, the cross-sectional view showed the identical features (Figure 1A). Echocardiography revealed no functional difference between WT and KI mice (Figure 1B). There was no appreciable change in the KI mice regarding the expression or phosphorylation levels of any of the sarcoplasmic reticulum (SR) proteins examined (Online Figure III).
Exercise or Drug (Epinephrine and Caffeine) Administration Induced VT in R2474S/+ KI Mice
In the resting conscious condition, we frequently observed polymorphic ventricular premature contractions in KI mice in response to even very weak stimuli, like light or sound, but not in WT mice (data not shown). Injection of caffeine plus epinephrine (IP) or exercise on a treadmill induced bidirectional or polymorphic VT in KI mice, but not in WT mice (Figure 2A). The duration of VT in most KI mice was less than 30 seconds (Figure 2B).
Seizures Were Not Observed in R2474S/+ KI Mice
To determine whether seizures occurred in KI mice, as previously reported,8 we monitored the behavior of mice by video recording for 1 week and assessed their susceptibility to seizures by pharmacological induction (see expanded Methods in the Online Data Supplement). Thus far, we have not observed spontaneous seizures in KI or WT mice. Furthermore, a pharmacological provocation test with 4-aminopyridine and caffeine showed no difference in the latency to the development of generalized tonic-clonic seizures (Online Figure IV).
Relaxation Phase of Cell Shortening and Ca2+ Transient Was Prolonged in the Isoproterenol-Activated R2474S/+ KI Mice
There was no statistically significant difference in the contour or kinetic parameters of Ca2+ transient and cell shortening at baseline between the WT and KI mice (Figure 3A and Online Table I). In response to isoproterenol; however, the time from the peak to 80% decline in cell shortening or Ca2+ transient was prolonged in KI cardiomyocytes, suggesting a delay in the inactivation of Ca2+ release and/or spontaneous Ca2+ release events. Moreover, the SR Ca2+ content, determined by caffeine application, was significantly lower in KI cardiomyocytes than in WT cardiomyocytes, both before and after the addition of isoproterenol (Figure 3B). SR Ca2+-ATPase (SERCA)2 activity could contribute to the slowed decay kinetics of the Ca2+ transient during heart failure. Thus, we next measured the SERCA2-mediated Ca2+ uptake by monitoring the time-dependent change in the intra-SR [Ca2+] (Online Figure V). As shown, there was no appreciable change in the time course of SR Ca2+ uptake.
RyR2 Ca2+ Channels of R2474S/+ KI Cardiomyocytes Were Hypersensitive to Channel Activation by Isoproterenol, PKA Phosphorylation, and Luminal Calcium
In KI cardiomyocytes, Ca2+ spark frequency (SpF) was higher than that in WT cardiomyocytes, both before and after the addition of isoproterenol (Figure 4A; for 3D images, see Online Figure VI, A). In the presence of a higher concentration of isoproterenol (100 nmol/L), the frequency of spontaneous Ca2+ waves was much higher in KI cardiomyocytes than in WT cardiomyocytes and the duration of local Ca2+ release provoked by the transmission of the Ca2+ waves was markedly prolonged (Online Figure VII). In both KI and WT cardiomyocytes, isoproterenol (10 nmol/L) increased the peak amplitude, full width at half maximum (FWHM), and full duration at half maximum (FDHM) (Online Table II). In KI cardiomyocytes, however, FDHM increased approximately twice as much as in WT cardiomyocytes (Online Table II). In response to isoproterenol (10 nmol/L), the level of PKA-dependent phosphorylation at Ser2808 of the RyR2 showed a rather modest increase that was slightly less than half of maximum phosphorylation (see Online Figure II). However, in this case, the extent of the increase was nearly the same in the KI and WT cardiomyocytes (Figure 4B). These results suggest that the sensitivity of the channel to both SR content (luminal [Ca2+]) and PKA phosphorylation is markedly increased in KI channels.
For further analysis of the increased sensitivity of KI channels to PKA phosphorylation and the SR Ca2+ content, we measured the PKA phosphorylation level at Ser2808 of RyR2 after addition of cAMP under the same conditions as the Ca2+ spark assay ([Ca2+]=30 nmol/L, buffered by 0.5 mmol/L EGTA). For this purpose, we added the Ca2+/calmodulin-dependent protein kinase (CaMK)II inhibitor KN-93 (1 μmol/L) to inhibit the effect of intrinsic CaMKII on the phosphorylation of the RyR2. We then measured both SpF and SR Ca2+ content in the absence and in the presence of 1 μmol/L cAMP in the saponin-permeabilized cardiomyocytes. There was no significant difference in the PKA phosphorylation level at Ser2808 between the KI and WT cardiomyocytes, both in the absence and the presence of cAMP (Figure 4C). Because there is marked variation in specificity among antibodies generated against Ser2808,11 we compared the phosphorylation level at Ser2808 of RyR2 by using another different antibody against Ser2808 (Badrilla, Leeds, UK). There was no significant difference in the PKA phosphorylation level at Ser2808 between KI and WT cardiomyocytes (Online Figure VIII).
To test the postulated possibility that destabilization of the RyR2 caused by the dissociation of FK506 binding protein 12.6 (FKBP12.6) from the RyR2 is a common pathogenic mechanism underlying heart failure and lethal arrhythmia,12,13 we determined the RyR2-bound FKBP12.6 by using a pull-down assay. As shown in Figure 4D, between the WT and KI mice, there was no significant difference in the RyR2-bound FKBP12.6 in the absence or the presence of cAMP (1 μmol/L).
Figure 5A shows the representative traces of Ca2+ sparks in the presence of various concentrations of cAMP (0.1 to 1 μmol/L) (for 3D images of Ca2+ sparks, see Online Figure VI, B). The dependence of SpF on the SR Ca2+ content is plotted in Figure 5B. To obtain the point at lower SR Ca2+ content, thapsigargin was added to the cardiomyocytes. Then, both Ca2+ sparks and SR Ca2+ content were measured 3, 5, and 10 minutes after the addition of thapsigargin. SpF was higher in KI than in WT cardiomyocytes, although there was a considerable reduction in the SR Ca2+ content in KI cardiomyocytes. As a result, there was a considerable left-shift of the SpF versus SR Ca2+ content plot in the case of KI cardiomyocytes (Figure 5B). The effect of cAMP on Ca2+ spark characteristics are summarized in Online Table III. Compared to the WT cardiomyocytes, both the peak amplitude and FWHM decreased whereas FDHM increased in KI cardiomyocytes, suggesting a delay in the inactivation of the RyR2. These results suggest that the threshold of luminal [Ca2+] for channel opening decreased considerably owing to the single R2474S CPVT mutation of the RyR2. To assess the maximum capacity of Ca2+ loading of SR, we added tetracaine (1 mmol/L) to inhibit Ca2+ release and then evaluated SR Ca2+ content by caffeine application. After treatment with the Ca2+ release blocker tetracaine (arrows, Online Figure IX), the SR Ca2+ content increased and reached nearly the same level (≈4 F/F0) in both WT and KI cardiomyocytes. This suggests that the reduced SR Ca2+ content in the KI cardiomyocytes was not attributable to reduced capacity of Ca2+ loading but to increased activation of the Ca2+ release flux relative to the influx.
PKA phosphorylation modulates not only RyR2 function but also SR Ca2+ load dependence of channel activation. Thus, to clarify the direct effect of PKA phosphorylation on RyR2 function, we measured the SpF and SR Ca2+ content in the presence of 0.3 μmol/L thapsigargin (inhibitor of SERCA2a activity) (Figure 5C). At comparable SR Ca2+ content, PKA phosphorylation increased SpF in KI cardiomyocytes, but not in WT cardiomyocytes. This suggests that PKA-dependent phosphorylation sensitized diastolic SR Ca2+ release, but only in CPVT, and not WT RyR2 channels.
Domain Unzipping Peptide DPc10 Mimics the Phenotype of KI Channels in WT Cardiomyocytes
As shown previously,9 DPc10 (a peptide corresponding to the 2460 to 2495 region of the central domain) mimics the channel disorder in the CPVT mutant (R2474S) by interfering with the channel-stabilizing interdomain interactions between the N-terminal and central domains (ie, domain unzipping). To assess the effect of domain unzipping on the dependence of SpF on the SR Ca2+ content, we added DPc10 to the WT saponin-permeabilized cardiomyocytes. Successful incorporation of DPc10 was confirmed by the intracellular fluorescence signal of Alexa fluor 488-labeled DPc10 (Online Figure X). DPc10 (50 μmol/L, applied externally) induced Ca2+ sparks in WT cardiomyocytes (Figure 5D, top). Importantly, addition of DPc10 caused a left-shift of the SpF/SR Ca2+ content relationship in WT cardiomyocytes (Figure 5D, bottom), resulting in the nearly identical SpF/SR Ca2+ profile as that of KI cardiomyocytes (Figure 5B). Similar to cAMP-treated KI cardiomyocytes, addition of DPc10 to WT cardiomyocytes prolonged FDHM (21.32±0.31 ms [n=27 cells] to 23.82±0.54 ms [n=48 cells]; P<0.01), but decreased the peak (1.74±0.03 to 1.69±0.01 arbitrary units; P=0.068) and FWHM (2.05±0.03 to 2.00±0.02 μm; P<0.01). Addition of DPc10 to the (cAMP-treated) KI cardiomyocytes produced no further effect (Figure 5E). To assess whether suppression of domain unzipping reversed the left-shift of the SpF/ SR Ca2+ content relationship in KI cardiomyocytes, we added dantrolene, which has corrected aberrant domain unzipping and prevented the development of heart failure.14 Interestingly, dantrolene shifted back the SpF/ SR Ca2+ content relationship to the right, almost toward the point corresponding to that of WT cardiomyocytes (Figure 5E). Dantrolene also prevented the DPc10-induced left-shift of the SpF/SR Ca2+ content relationship in WT cardiomyocytes (Figure 5D). Collectively, these findings indicate that the defective interdomain interaction within the RyR2 caused by either point mutation (R2474S) or domain unzipping peptide (DPc10) reduced the threshold of luminal [Ca2+] for channel activation, leading to the phenotype of CPVT (hyper-activation of the RyR2 channel). This suggests that as in the case of malignant hyperthermia, correction of the defective interdomain interaction by dantrolene may provide an effective method to treat CPVT.
Luminal [Ca2+] of SR in Saponin-Permeabilized WT and KI Cardiomyocytes
To confirm the above notion that the level of SR Ca2+ load was decreased in KI cardiomyocytes, we monitored luminal [Ca2+] in both cardiomyocytes using fluo-5N as a luminal Ca2+ probe. The average level of free diastolic SR Ca2+ decreased in KI cardiomyocytes, and on caffeine-induced discharge of luminal Ca2+, the luminal Ca2+ reached the same basal level in both WT and KI cardiomyocytes (Figure 6).
Spectroscopic Evidence That the Interdomain Interaction Is Defective in R2474S/+ KI Cardiomyocytes
To investigate whether the interdomain interaction was defective in KI mice, we used the fluorescence quench technique that permits spectroscopic monitoring of the state of the interdomain interaction (zipped or unzipped).15 The methylcoumarin acetamido (MCA) probe that has been attached to the critical domain would be inaccessible to a bulky fluorescence quencher (QSY-BSA conjugate) in a zipped configuration of the interacting domains, whereas it would become accessible to the quencher on unzipping. As in our previous study with DPc10,9 site-specific intense fluorescence labeling of the RyR2 band was achieved using DPc10 as a carrier (Figure 7A, left lane), but there was no MCA labeling when DPc10-mut was used as a carrier (middle lane). As shown in the “cold-chase” experiment (right lane), excess unlabeled DPc10 (10 mmol/L) prevented DPc10-mediated MCA labeling. DPc10 increased the slope of the Stern-Volmer plot (KQ), a measure of the extent of unzipping between the N-terminal (amino acids 1 to 600) and central (amino acids 2000 to 2500) domains in WT SR (Figure 7B, left). Addition of cAMP (1 μmol/L) had no effect (no unzipping) in WT SR (Figure 7B, left). In contrast, KI SR showed a high KQ value (Figure 7B, right). Addition of cAMP (1 μmol/L) further increased the KQ, which was comparable with the value of WT SR with added DPc10 (Figure 7B, right). Addition of DPc10 on the top of cAMP (1 μmol/L) produced no further increase in KQ. Interestingly, dantrolene (1 μmol/L) partially reversed the KQ that had been increased by cAMP (Figure 7B, right). Dantrolene also prevented the DPc10-induced unzipping in WT SR (Figure 7B, left). These KQ values are summarized in Figure 7C.
Arrhythmogenic Characteristics of the Membrane Potential in R2474S/+ KI Cardiomyocytes
Because altered membrane potential events represent an important landmark in arrhythmogenesis,16 we measured m,R 00–2500) a hegher the WT cardiomyocytes. organized Ca“eased SpF only in KI cardiomyocytes.ICR), membrane potentials of WT and KI cardiomyocytes by recording the di-8-ANEPPS fluorescence while pacing in the presence of 30 nmol/L isoproterenol (Online Figure XI). WT cardiomyocytes showed no spontaneous after potential (Figure 8A) and no spontaneous Ca2+ transient (Figure 8B). However, KI cardiomyocytes showed spontaneous Ca2+ transients and spontaneous after potentials in response to isoproterenol (30 nmol/L) when we increased the pacing rate from 1 to 5 Hz (Figure 8A and 8B). Interestingly, the spontaneous after potentials and Ca2+ transients disappeared in the presence of dantrolene (1 μmol/L), which corrected the defective interdomain interaction between the N-terminal and central domains (Figure 8A and 8B).
Many point mutations have been found in RyR2 in patients with CPVT. Several pieces of biochemical evidence suggest that these mutations cause defective channel gating, leading to diastolic Ca2+ leakage.17 More direct evidence that a single point mutation in the RyR2 is the primary cause of channel dysfunctions in CPVT patients have been obtained by recent studies with a KI mouse model.6–8 A single point mutation (R4496C) introduced in the RyR2 was found to cause bidirectional or polymorphic VT in the KI mice.6 In isolated cardiomyocytes from the R4496C KI mice, both delayed afterdepolarization (DAD) and triggered activity were induced on stimulation with isoproterenol.18 A more recent study using the same model19 showed that a dramatic increase in the Ca2+ sensitivity of the RyR2 channel resulted in the increased frequency of Ca2+ sparks and Ca2+ waves, which was further amplified by either isoproterenol or high pacing rates. Further studies7,8 confirmed mutation-linked dysfunction of RyR2, namely spontaneous Ca2+ release events and DAD. These studies show a close inter-relationship between the single point mutation, increased Ca2+ sensitivity of the channel gating, and the resulting lethal arrhythmia. However, the underlying mechanism by which a single mutation in such a large molecule causes drastic effects on cardiac function has remained unclear.
Defective Interdomain Interaction Is the Source Mechanism of Mutation-Linked Channel Disorder
The most important new aspect of the present study is the finding that introduction of the R2474S CPVT mutation into the central domain of RyR2 induced a defective interaction between the central domain and the N-terminal domain, as predicted from the “domain switch hypothesis,” and this caused channel dysfunction similar to that of CPVT patients in KI mice. The 3 lines of evidence are consistent with this. First, DPc10 (amino acids 2460 to 2495), which contains the mutable R2474 residue and is known to interfere with normal interdomain interaction between the N-terminal and central domains,9 reproduced the abnormal cellular Ca2+ events seen in the R2474S/+ KI mice (eg, increased frequency of Ca2+ sparks) in an otherwise normal system (ie, in cardiomyocytes isolated from WT mice). However, the addition of DPc10 to the (cAMP-treated) R2474S KI cardiomyocytes produced no further effect, suggesting that the defective interdomain interaction (aberrant domain unzipping) had already taken place in the KI cardiomyocytes. Second, The R2474S mutation, introduced into the central domain of RyR2 of KI mice, did produce defective interdomain interaction between the N-terminal and central domain (aberrant domain unzipping), as evidenced by the accessibility of the fluorescent probe MCA attached to the N-terminal domain to a high molecular weight fluorescence quencher QSY7-BSA was considerably higher in the KI RyR2 than the WT RyR2. Finally, dantrolene, which corrects aberrant domain unzipping, did suppress aberrant phenomena characteristic of CPVT KI mice, such as reduced threshold of luminal Ca2+ for channel activation, spontaneous Ca2+ sparks, and DAD.
PKA-Dependent Phosphorylation of the RyR2 at Ser2808 Facilitates Domain Unzipping Only in the CPVT Mutant Ryanodine Receptor
Interesting new finding in the present study is that the threshold of luminal [Ca2+] for activation of Ca2+ sparks was much lower in R2474S/+ KI mice than in WT mice. In other words, the sensitivity of the RyR2 channel to activation by luminal [Ca2+] was increased in R2474S/+ KI mice. More importantly, we could reproduce this sensitized channel gating to luminal [Ca2+] that is characteristic of the R2474S/+ KI mice, in WT cardiomyocytes by adding DPc10 (Figure 5D). This provides further support for the notion that the aberrant channel gating in R2474S/+ KI mice is produced by defective interdomain interaction between the N-terminal and central domains.
This study also showed that the level of PKA-dependent phosphorylation of the RyR2 at Ser2808 was virtually indistinguishable between KI and WT RyR2s (Figure 4B and 4C), yet PKA phosphorylation produced a much larger effect in increasing the frequency of Ca2+ sparks (SpF) in the KI cardiomyocytes than the WT myocytes (Figures 4A and 5⇑A). This suggests that the CPVT mutation also sensitizes the channel to PKA phosphorylation-dependent activation. In the 3D image of the RyR2, Ser2808, the site of PKA phosphorylation, has been localized in the vicinity of the boundary between the N-terminal (amino acids 1 to 600) and the central domains (amino acids 2000 to 2500).20 Earlier cryoelectron microscopy single particle study of RyR121 also showed that domain 5 (including the N-terminal domain) and domain 6 (including the central domain) at the clamp region are indeed in close apposition to each other in the resting state, whereas these domains become separated in the activated (channel-open) state (eg, in the presence of cAMP and activating Ca2+). Thus, it is tempting to suggest that PKA phosphorylation at Ser2808 accelerates domain unzipping in the KI channel, where domain unzipping has already progressed because of a weakened interdomain interaction caused by the CPVT mutation.
A New Molecular Mechanism for CPVT
Although recent studies using KI mouse models have demonstrated that CPVT is caused by mutation-linked dysregulation of intracellular Ca2+ events and membrane potential events (DAD and triggered activity), it is still unclear how a single mutation changes the conformational state of the RyR2, leading to leaky channel. We propose a new molecular mechanism underlying CPVT (Online Figure XII). In the normal channel, domain-domain interaction between the N-terminal (amino acids 1 to 600) and central (amino acids 2000 to 2500) domains is maintained in a zipped state, and thus, the channel is stabilized, preventing Ca2+ leakage and DAD at a physiological range of SR Ca2+ contents. In the mutant channel, the stabilized interdomain interaction is disrupted, causing aberrant domain unzipping; domain unzipping is further aggravated by the PKA phosphorylation of Ser2808, located at the boundary between the 2 domains at the clamp region.20 In turn, the threshold of luminal [Ca2+] for channel activation is decreased (cf, elsewhere17). Together, this results in SR Ca2+ leakage, DAD, and lethal arrhythmia. It should be noted, however, that DAD-triggered arrhythmia can also be induced by intracellular Ca2+ overload, for example, the toxic arrhythmogenic effects of the cardiac glycosides.22
In contrast to a previous report,8 the RyR2-FKBP12.6 link was not disrupted in R2474S/+ KI mice. In the present study, the level of PKA-dependent phosphorylation at Ser2808 of the RyR2 showed only a modest increase in response to 1 μmol/L cAMP (slightly less than half of maximum phosphorylation; see Online Figure II). This level of phosphorylation may not have been high enough to dissociate FKBP12.6 from RyR2. Nonetheless, the fact that such a modest increase in the PKA phosphorylation at Ser2808 induced domain unzipping and the resulting Ca2+ leakage in KI mice, suggests that the defective interdomain interaction within the RyR2 is the source mechanism of CPVT and that FKBP dissociation is not a direct cause of defective channel gating in CPVT. As we previously reported,9 however, it is also true that FKBP dissociation induces domain unzipping, which may account for the destabilized channel gating in heart failure. Thus, either a CPVT-type mutation or FKBP dissociation in heart failure commonly induces domain unzipping as an independent trigger, resulting in aberrant Ca2+ release in diseased hearts.
The reduction in SR Ca2+ content in heart failure may be partly attributable to increased Na+/Ca2+ exchanger function and the increased SR Ca2+ leakage (in addition to reduced SERCA function), causing contractile dysfunction as well as arrhythmia. We previously demonstrated that dantrolene corrects defective interdomain interactions within the RyR2 in failing hearts, inhibits spontaneous Ca2+ leakage, and in turn improves cardiomyocyte function in failing hearts.14 In this study, we showed that dantrolene was equally effective in the CPVT-type mutated RyR2 as in failing hearts. This indicates that channel dysfunction in CPVT and heart failure are caused by a common mechanism, that is, defective interdomain interaction within the RyR2.
According to a recent report by Lehnart et al,8 R2474S KI mice, which harbors the same mutation as those used in the present study, exhibited spontaneous generalized tonic-clonic seizures. In our KI mice, however, we did not observe spontaneous tonic-clonic seizures. We have no explanation for this difference. It may be ascribable to differences in the background of the mouse model. In generating the RyR2 KI mice, we used ES cells derived from C57BL/6J mouse (no difference in the background between ES cell line mice and KI mice), but Lehnart et al used those from 129 mice, followed by back-crossing with C57BL/6J mice.
In this, study, we evaluated the phosphorylation status only at Ser2808. However, because β-adrenergic stimulation has been reported to activate CaMKII,11,23 a further investigation is clearly needed to assess the role of other phosphorylation sites (eg, Ser2814 and Ser2030) on CPVT-type channel disorder.
In conclusion, a single point mutation within the RyR2 sensitizes the RyR2 channel to activation by luminal [Ca2+] (ie, a decreased threshold of luminal [Ca2+] for channel activation), and in turn induces spontaneous Ca2+ sparks and DAD, leading to CPVT. More importantly, this aberrant channel opening is primarily mediated through defective interdomain interaction between the N-terminal (amino acids 1 to 600) and central (amino acids 2000 to 2500) domains.
Sources of Funding
This work was supported by grants-in-aid for scientific research from the Ministry of Education in Japan (grants 20390226 to M.Y., 20590868 to T.Y., 20591805 to S.K., 19209030 to M.M.); a grant from Takeda Science Foundation (to M.Y.); and a grant from the NIH National Heart, Lung, and Blood Institute (HL072841 to N.I.).
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Novelty and Significance
What Is Known?
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by single point mutation in cardiac, type 2, ryanodine receptor (RyR2).
Aberrant Ca2+ release occurs in CPVT-type mutant RyR2.
What New Information Does This Article Contribute?
Defective interdomain interaction (namely, domain unzipping) within the RyR2 is a source mechanism of catecholamine-induced aberrant Ca2+ release in CPVT.
Correction of the defective interdomain interaction could be a new strategy against CPVT.
CPVT is known to be caused by single point mutation taking place in well defined regions of the RyR2. A single point mutation introduced in the RyR2 has been found to cause bidirectional or polymorphic VT in the knock-in mice, showing a close inter-relationship among the single point mutation and the resultant lethal arrhythmia. However, the underlying mechanism by which a single mutation in such a large molecule causes drastic effects on channel function has remained unresolved. Here, we report that introduction of R2474S CPVT mutation into the central domain of mouse RyR2 interferes with a normal tight interaction between the central domain (amino acids 2000 to 2500) and the N-terminal domain (amino acids 1 to 600), which reduces the threshold of luminal [Ca2+] for channel activation, sensitizes to the protein kinase A-dependent phosphorylation, and in turn leads to CPVT. Correction of the defective interdomain interaction by dantrolene stops the aberrant Ca2+ release and spontaneous after potential characteristic of CPVT. These results provide a new pathogenic mechanism of CPVT and a novel therapeutic strategy against CPVT.
Original received September 15, 2009; revision received February 9, 2010; accepted February 26, 2010.