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(Circulation Research. 2006;98:88.)
© 2006 American Heart Association, Inc.

Cellular Biology

Arrhythmogenic Mutation-Linked Defects in Ryanodine Receptor Autoregulation Reveal a Novel Mechanism of Ca²⁺ Release Channel Dysfunction

Christopher H. George, Hala Jundi, Nicola Walters, N. Lowri Thomas, Robert R. West, F. Anthony Lai

From the Department of Cardiology, Wales Heart Research Institute, Cardiff University School of Medicine, Cardiff, UK.

Correspondence to Dr Christopher H. George, Department of Cardiology, Wales Heart Research Institute, Cardiff University, Heath Park, Cardiff, UK CF14 4XN. E-mail georgech{at}cf.ac.uk

	Abstract

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Arrhythmogenic cardiac ryanodine receptor (RyR2) mutations are associated with stress-induced malignant tachycardia, frequently leading to sudden cardiac death (SCD). The causative mechanisms of RyR2 Ca²⁺ release dysregulation are complex and remain controversial. We investigated the functional impact of clinically-severe RyR2 mutations occurring in the central domain, and the C-terminal I domain, a key locus of RyR2 autoregulation, on interdomain interactions and Ca²⁺ release in living cells. Using high-resolution confocal microscopy and fluorescence resonance energy transfer (FRET) analysis of interaction between fusion proteins corresponding to amino- (N-) and carboxyl- (C-) terminal RyR2 domains, we determined that in resting cells, RyR2 interdomain interaction remained unaltered after introduction of SCD-linked mutations and normal Ca²⁺ regulation was maintained. In contrast, after channel activation, the abnormal Ca²⁺ release via mutant RyR2 was intrinsically linked to altered interdomain interaction that was equivalent with all mutations and exhibited threshold characteristics (caffeine >2.5 mmol/L; Ca²⁺ >150 nmol/L). Noise analysis revealed that I domain mutations introduced a distinct pattern of conformational instability in Ca²⁺ handling and interdomain interaction after channel activation that was absent in signals obtained from the central domain mutation. I domain–linked channel instability also occurred in intact RyR2 expressed in CHO cells and in HL-1 cardiomyocytes. These new insights highlight a critical role for mutation-linked defects in channel autoregulation, and may contribute to a molecular explanation for the augmented Ca²⁺ release following RyR2 channel activation. Our findings also suggest that the mutational locus may be an important mechanistic determinant of Ca²⁺ release channel dysfunction in arrhythmia and SCD.

Key Words: ryanodine receptor • mutations • interdomain interaction • arrhythmia

	Introduction

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To date, 60 arrhythmogenic mutations in ryanodine receptor (RyR2) have been reported to underlie stress- or exercise-induced malignant tachycardia, frequently leading to sudden cardiac death (SCD).^1–3 The mutations cluster in 3 discrete loci at the amino (N) terminus (15%), a central domain (25%), and at the carboxyl (C) terminus (60%). In a large number of mutations, their segregation into functionally distinct domains within the polypeptide and the complexity of the clinical phenotype may preclude a unifying mechanism of RyR2 Ca²⁺ channel dysfunction.^1–4 Currently, our understanding of the correlation between mutation location, phenotypic manifestation, and the molecular basis of defective Ca²⁺ release is incomplete. Several mechanisms underlying RyR2 channel dysfunction in SCD have been proposed, including altered sensitivity to luminal⁵ and cytoplasmic Ca²⁺,⁶ decreased Mg²⁺-dependent inhibition,⁷ and FKBP12.6-dependent^7,8 and FKBP12.6-independent mechanisms.^9–11 Despite persistent controversy surrounding the mechanistic basis of RyR2 dysregulation, there is a consensus that mutations functionally characterized to date mediate abnormal Ca²⁺ release after channel activation.²

Intramolecular interaction between discrete RyR domains is necessary for the proper folded channel architecture and is emerging as an important mode of channel autoregulation.^12,13 However, far from being static, intra-RyR domain interactions are dynamically reordered and normal channel activation is associated with large-scale structural rearrangement.¹⁴ Furthermore, factors promoting weakened domain interaction (termed domain unzipping) have been proposed to exacerbate Ca²⁺ release dysfunction¹⁵ and are implicated in the pathogenesis of heart failure.¹⁶ In the context of RyR2-dependent arrhythmia, mutation loci may correspond to key sites of interdomain interaction,^12,17 and peptides targeted to a central mutation-linked domain induced hypersensitive Ca²⁺ release through native RyR2 in vitro.¹⁸ However, a causative link between RyR2 mutations and defective domain interaction has not been conclusively demonstrated.

We previously identified the I domain, a hydrophobic RyR2 region (amino acids 3722 to 4610) that is postulated to transduce cytoplasmic events to regulate the Ca²⁺ pore-forming domain¹³ (see Figure 1), and a "hot-spot" for arrhythmia-linked RyR2 mutations (comprising more than one third of reported mutations).² In this study, we provide the first cell-based evidence to support the hypothesis that SCD-linked mutations occurring in the central domain (S2246L) and the I domain (N4104K and R4497C) directly cause RyR2 channel instability via defective interdomain interaction, resulting in Ca²⁺ release dysfunction. The magnitude of augmented Ca²⁺ release was intrinsically linked to the extent of defective conformational rearrangement, thereby implicating abnormal interdomain interaction as a fundamental event in mutant RyR2-mediated arrhythmogenesis. Noise analysis demonstrated that 2 I domain mutations were characterized by distinct patterns of postactivation Ca²⁺ signals and interdomain interaction, supporting a link between the mutation locus and the precise mode of channel dysfunction.

View larger version (21K): [in this window] [in a new window]   Figure 1. Schematic representation of fluorescent RyR2 proteins. Fluorescent RyR2 fusion proteins corresponding to the C terminus (RyR2C) and cytoplasmic N-terminal domains (amino acids 1 to 3772 [3722N] and 1 to 4610 [4610N], collectively referred to as RyR2N) containing either WT sequence or SCD-linked mutations (S2246L [RyR2N-SL], N4104K [RyR2C-NK], and R4497C [RyR2C-RC]) were constructed. Dotted and hatched bars represent eGFP and DsRed, respectively. The putative arrangement of transmembrane domains within RyR2C is from the model of Du et al.40 Inset, an illustration depicting our current model of I domain–mediated autoregulation of the intact RyR2 in which basal interactions within the I domain undergo conformational reordering after channel activation. The precise determinants of intra–I domain interaction remain to be determined.

	Materials and Methods

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Construction of RyR2 Domains Containing SCD-Linked Mutations
Oligonucleotide-directed mutagenesis (Quikchange, Stratagene) of the wild-type (WT) human RyR2 C-terminal Ca²⁺-pore forming domain (amino acid residues 3722 to 4967; RyR2^C-WT) or RyR2^N-WT, a collective term for N-terminal constructs encoding WT amino acids 1 to 3722 and 1 to 4610,¹³ were used to generate SCD-linked mutants R4497C (RyR2^C-RC), N4104K (RyR2^C-NK), and S2246L (RyR2^N-SL). All constructs were verified by automated DNA sequencing (ABI3700, Applied Biosystems). A schematic illustration of constructs is given in Figure 1.

Cellular Expression of RyR2 Domains and Intact RyR2
WT and mutant RyR2^C expressions were induced in Chinese hamster ovary (CHO) cells stably expressing the heterodimeric ecdysone receptor (VgRxR)¹³ using ponasterone A (PonA; 5 µmol/L, 24 hours). After induction, cells expressing eGFP-tagged RyR2^C were isolated using fluorescence-activated cell sorting (FACS) (MoFlo; Dako Cytomation) (see the online-only data supplement at http://circres.ahajournals.org) and were used as the background for RyR2^N-WT and RyR2^N-SL expression. Recombinant intact eGFP-tagged RyR2 was expressed in CHO and HL-1 cardiomyocytes as previously described.^9,13,19

Immunoblotting Analysis of Recombinant and Endogenous Protein Expression
Recombinant RyR2^N and RyR2^C domains were detected using rabbit polyclonal antibodies pAb2143 and pAb129 (RyR2 epitope: residues 91 to 105 and 4674 to 4697, respectively), and intact RyR2 was detected using pAb129. The endogenous expression levels of RyR2, FK506-binding proteins (FKBP12 and 12.6), calsequestrin (CSQ), sarco/endoplasmic reticulum Ca²⁺ ATPase isoform 2 (SERCA2), and Na⁺/Ca²⁺ exchanger (NCX) were determined using antibodies described in the data supplement and immunoblotting protocols described elsewhere.^9,19

Ca²⁺ and Fluorescence Resonance Energy Transfer Imaging
Intracellular Ca²⁺ measurement in resting (nonstimulated) or caffeine-stimulated cells was performed in Ca²⁺-crimson loaded cells.¹³ Fluorescence resonance energy transfer (FRET) analysis of protein–protein interaction between DsRed-tagged RyR2^N and eGFP-tagged RyR2^C fusion partners under nonstimulated conditions was performed using acceptor photobleaching²⁰ (see data supplement) and after caffeine activation (0.1 to 30 mmol/L) using ratiometric DsRed:eGFP imaging.¹³ All Ca²⁺ and FRET imaging was performed using a resonance-scanning confocal microscope (Leica RS2, Leica Microsystems).^9,13,21 In some experiments, cytoplasmic [Ca²⁺] ([Ca²⁺]_c) was clamped in streptolysin-O (SLO) permeabilized cells using known [Ca²⁺]-containing buffers (38 nmol/L to 1.35 µmol/L) and sarcoplasmic reticulum (SR) Ca²⁺ content was estimated after thapsigargin-induced (5 µmol/L) depletion of SR Ca²⁺ stores.^6,10,19

Noise Analysis of Ca²⁺ and FRET Signals: Calculation of the Relative Signal Variability (RSV)
Noise analysis was used to determine the amplitude and temporal aspects of signal variability (noise) in Ca²⁺ and FRET signals after addition of caffeine (0.1 to 30 mmol/L) to cells. Analysis was performed by both F ratio test on log-transformed data, as the mean signal levels before and after caffeine stimulation were very different, and by calculating the relative signal variability (RSV). The RSV represents the relative variability in amplitude and temporal patterning of postactivation Ca²⁺ and FRET signals (see data supplement for detailed description). All analysis was performed on data sampled at 5Hz.

	Results

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Normal Basal Interaction Between Mutant RyR2^N/RyR2^C Domains
The cytotoxicity associated with high intracellular levels of RyR2^C is incompatible with its permanent, constitutive overexpression.¹³ To negate this, we coupled an inducible cell system with FACS analysis to achieve high-level transient expression of eGFP-tagged WT and mutant RyR2^C in viable cells (supplemental Figure I). In the absence of RyR2^C, DsRed-tagged 3722^N and 4610^N were homogeneously distributed throughout the cytoplasm (Figure 2A, –). After PonA induction, RyR2^C were expressed to similar levels (RyR2^C-WT [100%], RyR2^C-NK [109±16%] and RyR2^C-RC [105±12%] based on normalized eGFP fluorescence intensity analysis, >30 cells) and were correctly targeted to the endoplasmic reticulum (ER) as determined by the lattice-like distribution (Figure 2A, +). RyR2^C expression induced a striking intracellular redistribution of 4610^N to the ER (Figure 2A, +) that remained unaffected after SCD-linked mutation (Figure 2B). In contrast, 3722^N was not sequestered to the ER after the coexpression of WT or mutant RyR2^C and remained in the bulk cytoplasm (Figure 2A), consistent with negligible interdomain interaction. In agreement with the pixel colocalization data, FRET analysis of DsRed-tagged RyR2^N and eGFP-tagged RyR2^C domain interaction (ie, occurring within 100Å) revealed a significant basal interaction between 4610^N/RyR2^C in nonstimulated cells that was not disrupted by SCD-linked mutation (Figure 2B). FRET analysis also confirmed the lack of 3722^N and RyR2^C interaction, as comparable data were obtained in control experiments using with RyR2^C and DsRed (Figure 2B), proteins that do not physically interact.¹³

View larger version (34K): [in this window] [in a new window]   Figure 2. SCD-linked mutations do not perturb RyR2N/RyR2C interaction in resting cells. A, The intracellular localization of eGFP-tagged RyR2C-RC and DsRed-tagged RyR2N-WT in the absence (–) or presence (+) of PonA-induction was determined using confocal microscopy. Scale bar represents 10 µm. Images and colocalization data of RyR2C-WT/RyR2N-WT interactions have been published previously.13 B, Analysis of RyR2N/RyR2C domain coincidence was performed using the proportion of eGFP-tagged RyR2C positive pixels that directly overlaid DsRed-tagged RyR2N pixels (white bars). The limit of spatial resolution (&30%) was estimated after determination of pixel coincidence between RyR2C and DsRed, proteins that do not physically interact13 (DsRed lane). Domain interactions occurring below the limit of light microscope resolution were analyzed using the FRET method of acceptor photobleaching20 (black bars). Data are given as mean±SEM (n=5 experiments, 8 to 12 cells analyzed per experiment). There was no significant difference (P>0.05) within groupings.

Expression of RyR2^C in CHO^RxR cells leads to a persistent elevation in cytoplasmic [Ca²⁺] ([Ca²⁺]_c) that is restored to normal levels (&100 nmol/L) via interaction with 4610^N.¹³ The 4610^N-mediated regulation of RyR2^C strongly suggested that the elevated [Ca²⁺]_c occurred via Ca²⁺ leak through RyR2^C, although a contributory role of other [Ca²⁺]_c regulatory components (eg, RyR2^C-induced Ca²⁺ influx) cannot be excluded. It might be expected that normal [Ca²⁺]_c would be gradually recovered via a new equilibrium between cytoplasmic and ER Ca²⁺ stores, yet this does not occur in CHO^RxR cells. Perhaps the expression profile of Ca²⁺ regulatory proteins in CHO^RxR may provide clues as to the molecular basis of the sustained [Ca²⁺]_c increase, although this issue remains to be fully resolved. Nevertheless, in the present study, WT and mutant 4610^N/RyR2^C interaction resulted in equivalent suppression of basal [Ca²⁺]_c in nonstimulated cells (in nmol/L: WT, 81.9±17.2; N4104K, 95.1±14.7; R4497C, 78.6±17.1; S2246L, 91.3±13.6) when compared with [Ca²⁺]_c in cells coexpressing noninteracting 3722^N/RyR2^C domains (in nmol/L: WT, 138.8±18.0; N4104K, 150.8±19.5; R4497C, 161.8±21.4; S2246L, 133.2±18.2) (P>0.05 within grouping; P<0.05 between grouping). Thus, RyR2 mutations did not perturb normal 4610^N interaction with RyR2^C in resting cells and normal [Ca²⁺]_c was maintained.

Mutation-Linked Defects in Interdomain Interactions Exhibit Threshold Characteristics
Under nonstimulated conditions, the comparable interaction between WT and mutant 4610^N/RyR2^C domains correlated with the appropriate regulation of [Ca²⁺]_c. In the majority of symptomatic RyR2 mutation carriers, however, cardiac abnormalities are unmasked after exposure to stress or exercise.^1–3 Consequently, we investigated the effect of SCD-linked mutation on RyR2 interdomain interaction and intracellular Ca²⁺ release after channel activation. Consistent with the lack of interaction (Figure 2), coexpression of WT and mutant 3722^N/RyR2^C did not reconstitute caffeine-sensitive Ca²⁺ release (Figure 3A). In contrast, 4610^N/RyR2^C formed caffeine-sensitive Ca²⁺ channels, and SCD-linked mutations significantly augmented the peak Ca²⁺ release when compared with the WT domain combination (Figures 3B and 4A). FRET analysis of mutant 4610^N/RyR2^C interaction indicated that greater conformational changes accompanied the increased Ca²⁺ release after channel activation when compared WT domain combinations (Figure 4B). These results concur with the large-scale structural rearrangements accompanying RyR closed:open transition,¹⁴ but importantly they reveal that after mutation of the central domain (S2246L) or I domain (N4104K and R4497C), the augmented Ca²⁺ release was intrinsically linked to perturbed intra-RyR2 domain interaction (Figures 3B and 4).

View larger version (31K): [in this window] [in a new window]   Figure 3. Investigating the impact of SCD-linked mutations on Ca2+ handling and interdomain interaction in living cells. Fluorescence emission spectra of DsRed-tagged RyR2N (583 nm) (gray traces) and eGFP-tagged RyR2C (507 nm) (black traces) after coexpression of WT and mutant RyR2C with 3722N (A) or 4610N (B) (upper panels) were used to derive the DsRed:eGFP FRET ratio (middle panels). Cytoplasmic [Ca2+] ([Ca2+]c) was measured in parallel experiments using Ca2+ crimson (lower panels) and is given in arbitrary fluorescence units (F.U.). Arrowhead represents addition of caffeine (30 mmol/L). Traces correspond to data obtained from single-cell Ca2+ and FRET imaging and are representative of at least 6 separate experiments (&8 cells per experiment).

View larger version (33K): [in this window] [in a new window]   Figure 4. Mutation-linked defects in Ca2+ release are associated with abnormal intra-RyR2 interaction and exhibit threshold characteristics. The peak change in Ca2+ (A) and FRET (B) after addition of caffeine (30 mmol/L) to cells coexpressing RyR2N and RyR2C was determined. Data are given as the percentage increase in the caffeine-induced change in Ca2+-dependent fluorescence of Ca2+-crimson (Ca) and FRET ratio (R), when compared with resting Ca2+ and FRET values (Ca0 and R0, respectively). Data are given as mean±SEM (n=6 experiments, 8 cells analyzed per experiment). * P<0.01 when compared within grouping. The effect of mutations N4104K, R4497C, or S2246L on Ca2+ release (C) and FRET (D) after caffeine activation (0.1 to 30 mmol/L) of coexpressed 4610N/RyR2C domains was determined. *P<0.01 for all mutations when compared with WT. E, Plotting data given in C and D reveal a close correlation between the magnitudes of the caffeine dose-dependent increase in Ca2+ release and the changes in intramolecular interaction (R2 values: WT, 0.9736; N4104K, 0.9798; R4497C, 0.9651; S2246L, 0.8909). Note that the mutation-linked augmentation of Ca2+ dysfunction and interdomain interaction occurred at [caffeine] > 2.5 mmol/L.

The profiles of caffeine-activated Ca²⁺ release and interdomain interaction revealed that the magnitude of Ca²⁺ release associated with RyR2 mutations were proportional to the extent of abnormal domain interaction (Figure 4E). Mutant 4610^N/RyR2^C combinations exhibited sensitized and augmented caffeine-induced Ca²⁺ release and interdomain reordering, in keeping with data from intact mutant RyR2 in cardiomyocytes (supplemental Table III). Importantly, the mutation-linked abnormalities in Ca²⁺ release and the associated defects in domain interaction were only unmasked after channel activation using caffeine at >2.5 mmol/L, whereas below this threshold, peak Ca²⁺ release and changes in interdomain interaction were indistinguishable from those of WT (Figure 4C and 4D). Caffeine is a useful and widely used pharmacological tool to investigate RyR2 function, but its quasi-physiological mechanism of RyR2 activation (via enhanced channel sensitivity to [Ca²⁺]_c) may be different from RyR2 activation in vivo via Ca²⁺-induced Ca²⁺ release. To this end, we investigated the effect of increased [Ca²⁺]_c on intra-RyR2 conformational changes using a permeabilized cell system.^6,9 Mutant 4610^N/RyR2^C exhibited sensitized and augmented Ca²⁺-dependent changes in interdomain interaction when compared with WT domain combinations (Figure 5). The ER Ca²⁺ content in SLO-permeabilized cells was maintained for the duration of our experiments (WT, 84±27%; N4104K, 76±32%; R4497C, 87±24%; S2246L, 82±31% at [Ca²⁺]_c &100 nmol/L; when compared with nonpermeabilized cells expressing WT RyR2 [100%]), and thus these experiments were performed against a background of comparable ER Ca²⁺ stores. Consistent with our data obtained after caffeine activation (Figure 4D), we also determined that the abnormal interdomain interactions associated with SCD-linked mutations displayed threshold characteristics ([Ca²⁺]_c > 150 nmol/L) (Figure 5). Taken together, our data show that mutation-linked abnormalities in Ca²⁺ handling and domain interaction exhibit threshold characteristics in response to caffeine and Ca²⁺ activation, consistent with the stress-induced nature of RyR2 mutation-dependent arrhythmias.

View larger version (24K): [in this window] [in a new window]   Figure 5. Mutations sensitize RyR2 to Ca2+-induced conformational changes. FRET analysis of 4610N/RyR2C interaction was determined in SLO-permeabilized cells pre-equilibrated with known [Ca2+]c (38 nmol/L to 1350 nmol/L) using DsRed photobleaching (see data supplement). Data represent mean±SEM (n=4 experiments, 12 cells analyzed per experiment). [Ca2+]c producing 50% maximal conformational changes (EC50) was calculated and is shown ± SEM. * P<0.05 when compared with WT.

Noise Analysis Reveals Mutational Locus-Specific Abnormalities of Interdomain Interaction
All mutations characterized in this study exhibited equivalent defects in Ca²⁺ release and interdomain interaction (Figure 4). However, it was apparent from the experimental traces that there was significant signal variability (noise) present in the post-caffeine activation Ca²⁺ and FRET signals after mutation (Figure 3). We performed a detailed noise analysis of the experimental Ca²⁺ and FRET traces and used the RSV to determine the amplitude and temporal aspects of the signal variability (see data supplement). I domain mutations (N4104K and R4497C) were associated with profoundly increased postactivation signal noise in Ca²⁺ and FRET traces that were absent in the traces obtained from the central domain mutation (S2246L) (Figure 6). There was a close correlation between the signal variability in the postactivation Ca²⁺ and FRET traces, suggesting a link between the noise in the Ca²⁺ signals and the mutation-linked instability in the activated channel. Notably, the augmented Ca²⁺- and FRET-signal noise resulting from I domain mutant activation arose solely as a result of increased variability in the postactivation signals (supplemental Table I), and were not attributed to increased noise in individual GFP or DsRed signals (see Figure 3). Moreover, the increased postactivation signal noise from mutant 4610^N/RyR2^C resulted from domain interaction, as there was negligible noise increase after analysis of noninteracting 3722^N/RyR2^C combinations (Figure 6A and 6B). The augmented Ca²⁺ and FRET signal noise exhibited by caffeine-activated I domain mutants also displayed threshold characteristics (caffeine >2.5 mmol/L), as the RSV values determined at lower caffeine concentrations were indistinguishable from WT (Figure 6C and 6D). Thus, despite mutants S2246L, N4104K, and R4497C exhibiting comparable defects in Ca²⁺ release and domain interaction abnormalities, more detailed noise analysis revealed distinct modes of Ca²⁺ release dysfunction and the accompanying channel instability that were dependent on the RyR2 mutation locus.

View larger version (37K): [in this window] [in a new window]   Figure 6. Noise analysis reveals mutation locus-specific differences in postactivation Ca2+ and FRET signals. The RSV in Ca2+ and FRET signals from coexpressed 4610N/RyR2C after maximum caffeine activation (30 mmol/L) (A and B, respectively), or in response to a range of caffeine activation (0.1 to 30 mmol/L) (C and D, respectively) were calculated as described in the data supplement. *P<0.05 when compared within grouping (A and B) and when N4104K and R4497C were compared with WT and S2246L (C and D).

I Domain Mutation-Linked Channel Instability Occurs in Intact RyR2
Data obtained using a fusion protein complementation approach suggested that I domain RyR2 mutations induced a distinct pattern of conformational instability after channel activation (Figure 6). However, it was necessary to determine whether this phenomenon could be reproduced in the full-length intact RyR2 and also to investigate the impact of intracellular protein environment on channel instability. We expressed intact WT and mutant RyR2 in CHO cells and HL-1 cardiomyocytes²² that differ markedly in their endogenous expression of Ca²⁺ regulatory proteins (CHO cells are RyR2-, FKBP12.6-, CSQ-, and NCX-deficient, whereas HL-1 cardiomyocytes express RyR2, FKBP12.6, CSQ, and NCX) (Figure 7). Caffeine-activated Ca²⁺ release in CHO and HL-1 cells exhibited profoundly different profiles (a sustained elevation of [Ca²⁺]_c versus transient Ca²⁺ release, respectively), and the apparent differences in the regulation of postactivation [Ca²⁺]_c may reflect the different Ca²⁺-regulatory protein expression profile in these cells (Figure 7). Although all SCD-linked mutations resulted in increased Ca²⁺ release in each cell type, the magnitude of the peak Ca²⁺ release (Figure 8A; supplemental Figure III) and postactivation signal noise (Figure 8B; supplemental Tables I and II) were significantly different between cell types (HL-1 > CHO). However, when Ca²⁺ release occurring through mutant RyR2 was normalized to WT channels in each cell type, we determined equivalent Ca²⁺ release dysfunction independent of both cellular environment and whether Ca²⁺ release was mediated by interacting fusion proteins or the intact RyR2 molecule (supplemental Figure III). Likewise, in agreement with our data obtained with 4610^N/RyR2^C, I domain mutations in the intact RyR2 were associated with increased postactivation Ca²⁺ signal noise in each cell type (HL-1 > CHO; Figure 8A and 8B) that was equivalent when normalized to the corresponding WT channels in each cell type (Figure 8C). Consequently, the expression of full-length intact RyR2 in CHO cells and HL-1 cardiomyocytes corroborated our findings obtained with the 4610^N/RyR2^C domain interaction. Furthermore, our data indicated that the similar patterns of postactivation channel instability associated with I domain mutations occurred in nonmyocytic (CHO) or myocytic (HL-1) environments and was thus independent of cell background.

View larger version (46K): [in this window] [in a new window]   Figure 7. Expression profile of Ca2+ regulatory proteins in CHO and HL-1 cells. Cellular lysates (250 µg) from untransfected CHO and HL-1 cells were immunoblotted for RyR2, FKBP12.6, CSQ, SERCA, and NCX using antibodies described in the data supplement. + indicates rabbit cardiac homogenates (100 µg, except for RyR2 [250 µg]). Molecular weight markers (kDal) are shown. Blots are representative of 3 separate experiments.

View larger version (32K): [in this window] [in a new window]   Figure 8. Intact RyR2 exhibits I domain mutation-linked channel instability independent of cell background. A, Caffeine-activated (30 mmol/L, arrowhead) Ca2+ release in CHO and HL-1 cells expressing intact WT and mutant eGFP-tagged RyR2 was determined. B, The RSV in postactivation Ca2+ signals in response to caffeine (0.1 to 30 mmol/L) in CHO and HL-1 cells expressing WT and mutant RyR2 was calculated. C, Maximal RSV values were normalized to post-caffeine signal variability occurring in WT RyR2 (assigned 100% for each cell type). Data are given as mean±SEM (n=3 experiments). B and C, * P<0.05 when N4104K and R4497C are compared with S2246L and WT channels; no significant differences between groupings.

	Discussion

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Abnormal RyR2 Interdomain Interaction Associated With Stress-Induced Ventricular Tachycardia
This study provides the first evidence that mutation-linked defects in interdomain interactions are intrinsically linked to Ca²⁺ release dysfunction associated with stress-induced ventricular tachycardia (VT), indicating that there may be a structural basis to the pathological RyR2 Ca²⁺ release at the onset of arrhythmogenesis. Furthermore, the mutation-linked defects in RyR2 Ca²⁺ release and interdomain interactions exhibited threshold characteristics, consistent with the stress-induced nature of the disease phenotype. It is intriguing that the abnormal domain interactions occurring in mutant 4610^N/RyR2^N were unmasked at a [Ca²⁺]_c level approximately 2-fold greater than the level of [Ca²⁺]_c occurring in resting cells (&100 nmol/L). The presently identified link between agonist-stimulated Ca²⁺ release dysfunction and conformational instability in RyR2 mutations may provide an important advance in the knowledge of how channel activation is structurally transduced into abnormal Ca²⁺ release, thereby triggering delayed after-depolarizations, a fundamental event in arrhythmogenesis.²³ Defective interdomain interaction between RyR2 N-terminal and central domains is proposed to underscore the abnormal Ca²⁺ handling in heart failure,¹⁶ and we extend these findings by showing that in the context of arrhythmia, there is an additional contributory role of altered C-terminal interaction in RyR2 dysfunction. The striking utility of FRET to analyze RyR2 domain interaction in living cells and in real time, coupled with parallel determination of intracellular Ca²⁺ release, permitted detailed amplitude and temporal analysis of the interdomain interaction and the functional impact of these interactions on Ca²⁺ handling in resting and stimulated cells. However, although we propose a link between mutant RyR2 Ca²⁺ release abnormalities and channel instability, the issue of whether RyR2 conformational instability results in defective Ca²⁺ release or vice versa remains to be conclusively determined.

Mutation Locus-Specific Defects in RyR2 Channel Regulation
Augmented Ca²⁺ release linked to defective channel interactions after agonist activation was a common feature of the central and C-terminal mutations characterized in this study. In recognition of the enormous structural and functional complexity of RyR2, however, the plausible speculation that all reported mutations cause RyR2 dysfunction via similar defects in interdomain interaction may represent an over-simplification. We used noise analysis, a powerful tool to elucidate mechanistically relevant information in the amplitude patterning of experimental traces,^21,24 to demonstrate that differences existed in the precise mode of Ca²⁺ release dysfunction and conformational instability arising from central or I domain mutations. I domain mutations exhibited postactivation channel instability (manifested as increased signal variability in Ca²⁺ release and interdomain interaction) (Figures 3, 6, and 8) that did not occur with central domain mutation. This finding supports our hypothesis that the mutational locus may be an important mechanistic determinant of channel dysfunction.¹⁰ The central mutation locus of RyR2 (containing S2246L) has been mapped to a bridge structure that undergoes structural rearrangement during channel opening,²⁵ thereby providing additional support for our finding that intramolecular interaction may be disrupted by the S2246L mutation. However, the present study suggests that the precise mode of conformational alterations arising from mutations in distinct RyR2 domains may be different. A model incorporating domain-specific arrhythmogenic mechanisms may have significant implications for RyR2-tailored therapy, possibly precluding a common therapeutic strategy to restore normal channel function.²

The Mechanistic Complexities of RyR2 Dysfunction
A picture of mechanistic complexity underlying stress-induced VT is emerging. The development of bidirectional and polymorphic VT (bVT and pVT, respectively), characteristic features of RyR2-linked arrhythmia,²⁶ may arise from multiple mechanisms,²⁷ and the spatial origin of Ca²⁺ dysfunction within the myocardium may predict the resultant electrical abnormality.²³ Furthermore, we do not know why a significant proportion of RyR2 mutation carriers are asymptomatic (&30%)^26,28 or why others die under nonstressed conditions.²⁹ Consequently, an improved understanding of the link between mutation locus, the resulting molecular basis of RyR2 dysfunction, and the clinical manifestation of the disease is crucial. Several laboratories, including our own, have proposed mechanisms to explain arrhythmogenic RyR2 dysregulation.^5–8,18 A mechanism linking RyR2 mutations with decreased affinity for a key regulatory coprotein, FKBP12.6, resulting in enhanced Ca²⁺ leak from the SR and increased arrhythmogenic propensity in affected individuals is controversial,⁸ and its pathophysiological relevance is disputed.^30,31 The present study strongly supports an FKBP12.6-independent mode of channel dysregulation, as both normal (WT) and abnormal (mutant) Ca²⁺ handling and channel stability were independent of the cellular levels of FKBP12.6. Thus, our findings broadly agree with those of Oda et al,¹⁶ who showed that in heart failure (HF), FKBP12.6 dissociation is a consequence rather than a cause of RyR2 instability. Furthermore, data obtained from CHO cells and HL-1 cardiomyocytes indicated inherent channel instability in the activated mutant RyR2 tetramer occurred independently of the cellular expression profile of RyR2 regulatory proteins (eg, FKBP12.6, CSQ). However, we must also consider that in the intact myocardium, mutant RyR2 dysfunction may be exacerbated by defective interaction with accessory proteins (eg, CSQ),³² possibly arising as a consequence of RyR2 channel instability. It will be important to determine the status of RyR2 interaction with accessory proteins in the transgenic model of RyR2 mutation-linked VT.³³ These future investigations should yield important insights into the feasibility of therapeutic strategies centered around RyR2 modulation via overexpression of regulatory proteins.³⁴

The transgenic mouse model of mutation-linked RyR2 dysfunction exhibited exercise-induced bVT³³ that was absent in FKBP12.6-deficient models of cardiopathology.^8,35 Consequently, cardiac disorders caused by decreased RyR2:FKBP12.6 interaction may be mechanistically and phenotypically distinct from the RyR2 mutation-linked arrhythmia. Thus, questions remain as to whether there are common mechanisms of RyR2 dysregulation in the pathogenesis of stress-induced VT and HF.³⁶ Clearly, there are similarities between the mutation-linked defects in Ca²⁺ release and interdomain interaction identified in the present study and those occurring in HF.¹⁶ However, the incomplete efficacy of ß-adrenoceptor blockade in preventing stress-induced VT in patients^26,37 and in the arrhythmogenic RyR2 mutant mouse model³³ is in contrast to the beneficial effects of ß-adrenoceptor blockade in restoring normal RyR2 channel functionality in HF,^38,39 suggesting that some of the underlying mechanisms of RyR2-dependent arrhythmia and HF are different.

	Acknowledgments

 
This work was funded by British Heart Foundation grants FS/2000020 and BS/04/02 (C.H.G.) and studentship FS/04/088 (H.J.), and a Cardiff University studentship (N.L.T.). We thank Dr Chris Pepper for help developing the FACS strategy.

	Footnotes

 
Original received July 20, 2005; resubmission received October 20, 2005; revised resubmission received November 15, 2005; accepted November 29, 2005.

	References

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