点击显示 收起
the Department of Cardiology, Wales Heart Research Institute, Cardiff University School of Medicine, Cardiff, UK.
Abstract
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 domaineClinked 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
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).1eC3 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.1eC4 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-dependent7,8 and FKBP12.6-independent mechanisms.9eC11 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.
Materials and Methods
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; RyR2C-WT) or RyR2N-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 (RyR2C-RC), N4104K (RyR2C-NK), and S2246L (RyR2N-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 RyR2C expressions were induced in Chinese hamster ovary (CHO) cells stably expressing the heterodimeric ecdysone receptor (VgRxR)13 using ponasterone A (PonA; 5 eol/L, 24 hours). After induction, cells expressing eGFP-tagged RyR2C 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 RyR2N-WT and RyR2N-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 RyR2N and RyR2C 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 proteineCprotein interaction between DsRed-tagged RyR2N and eGFP-tagged RyR2C 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 eol/L) and sarcoplasmic reticulum (SR) Ca2+ content was estimated after thapsigargin-induced (5 eol/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
Normal Basal Interaction Between Mutant RyR2N/RyR2C Domains
The cytotoxicity associated with high intracellular levels of RyR2C 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 RyR2C in viable cells (supplemental Figure I). In the absence of RyR2C, DsRed-tagged 3722N and 4610N were homogeneously distributed throughout the cytoplasm (Figure 2A, eC). After PonA induction, RyR2C were expressed to similar levels (RyR2C-WT [100%], RyR2C-NK [109±16%] and RyR2C-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, +). RyR2C expression induced a striking intracellular redistribution of 4610N to the ER (Figure 2A, +) that remained unaffected after SCD-linked mutation (Figure 2B). In contrast, 3722N was not sequestered to the ER after the coexpression of WT or mutant RyR2C 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 RyR2N and eGFP-tagged RyR2C domain interaction (ie, occurring within 100) revealed a significant basal interaction between 4610N/RyR2C in nonstimulated cells that was not disrupted by SCD-linked mutation (Figure 2B). FRET analysis also confirmed the lack of 3722N and RyR2C interaction, as comparable data were obtained in control experiments using with RyR2C and DsRed (Figure 2B), proteins that do not physically interact.13
Expression of RyR2C in CHORxR cells leads to a persistent elevation in cytoplasmic [Ca2+] ([Ca2+]c) that is restored to normal levels (&100 nmol/L) via interaction with 4610N.13 The 4610N-mediated regulation of RyR2C strongly suggested that the elevated [Ca2+]c occurred via Ca2+ leak through RyR2C, although a contributory role of other [Ca2+]c regulatory components (eg, RyR2C-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 CHORxR cells. Perhaps the expression profile of Ca2+ regulatory proteins in CHORxR 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 4610N/RyR2C 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 3722N/RyR2C 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 4610N interaction with RyR2C 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 4610N/RyR2C 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.1eC3 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 3722N/RyR2C did not reconstitute caffeine-sensitive Ca2+ release (Figure 3A). In contrast, 4610N/RyR2C 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 4610N/RyR2C 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).
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 4610N/RyR2C 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 4610N/RyR2C 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.
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 4610N/RyR2C resulted from domain interaction, as there was negligible noise increase after analysis of noninteracting 3722N/RyR2C 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.
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 4610N/RyR2C, 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 4610N/RyR2C 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.
Discussion
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 4610N/RyR2N 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.5eC8,18 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 -adrenoceptor blockade in preventing stress-induced VT in patients26,37 and in the arrhythmogenic RyR2 mutant mouse model33 is in contrast to the beneficial effects of -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.
References
Kontula K, Laitinen P, Lehtonen A, Toivonen L, Viitasalo M, Swan H. Catecholaminergic polymorphic ventricular tachycardia: recent mechanistic insights. Cardiovasc Res. 2005; 67: 379eC387.
George CH, Thomas NL, Lai FA. Ryanodine receptor dysfunction in arrhythmia and sudden cardiac death. Future Cardiol. 2005; 1: 531eC541.
Priori SG, Napolitano C. Cardiac and skeletal muscle disorders caused by mutations in the intracellular Ca2+ release channel. J Clin Invest. 2005; 115: 2033eC2038.
Allen PD. Not all sudden death is the same. Circ Res. 2003; 93: 484eC486.
Jiang D, Xiao B, Yang D, Wang R, Choi P, Zhang L, Cheng H, Chen SRW. RyR2 mutations linked to ventricular tachycardia and sudden death reduce the threshold for store-overload-induced Ca2+ release (SOICR). Proc Natl Acad Sci U S A. 2004; 101: 13062eC13067.
Thomas NL, Lai FA, George CH. Differential Ca2+ sensitivity of RyR2 mutations reveals distinct mechanims of channel dysfunction in sudden cardiac death. Biochem Biophys Res Commun. 2005; 331: 231eC238.
Lehnart SE, Wehrens XHT, Laitinen PJ, Reiken SR, Deng SX, 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.
Wehrens XHT, Lehnart SE, Huang F, Vest JA, Reiken SR, Mohler PJ, Sun J, Guatimosim S, Song LS, Rosemblit N, D’Armiento JM, Napolitano C, Memmi M, Priori SG, Lederer WJ, Marks AR. FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death. Cell. 2003; 113: 829eC840.
George CH, Higgs GV, Lai FA. Ryanodine receptor mutations associated with stress-induced ventricular tachycardia mediate increased calcium release in stimulated cardiomyocytes. Circ Res. 2003; 93: 531eC540.
Thomas NL, George CH, Lai FA. Functional heterogeneity of ryanodine receptor mutations associated with sudden cardiac death. Cardiovasc Res. 2004; 64: 52eC60.
Jiang D, Xiao B, Zhang L, Chen SRW. Enhanced basal activity of a cardiac Ca2+ release channel (ryanodine receptor) mutant associated with ventricular tachycardia and sudden death. Circ Res. 2002; 91: 218eC225.
Ikemoto N, Yamamoto T. Regulation of calcium release by interdomain interaction within ryanodine receptors. Front Biosci. 2002; 7: d671eC683.
George CH, Jundi H, Thomas NL, Scoote M, Walters N, Williams AJ, Lai FA. Ryanodine receptor regulation by intramolecular interaction between cytoplasmic and transmembrane domains. Mol Biol Cell. 2004; 15: 2627eC2638.
Orlova EV, Serysheva II, van Heel M, Hamilton SL, Chiu W. Two structural configurations of the skeletal muscle calcium release channel. Na Struct Biol. 1996; 3: 547eC552.
Kobayashi S, Yamamoto T, Parness J, Ikemoto N. Antibody probe study of Ca2+ channel regulation by interdomain interaction within the ryanodine receptor. Biochem J. 2004; 380: 561eC569.
Oda T, Yano M, Yamamoto T, Tokuhisa T, Okuda S, Doi M, Ohkusa T, Ikeda Y, Kobayashi S, Ikemoto N, Matsuzaki M. Defective regulation of interdomain interactions within the ryanodine receptor plays a key role in the pathogenesis of heart failure. Circulation. 2005; 111: 3400eC3410.
George CH, Yin CC, Lai FA. Toward a molecular understanding of the structure:function of ryanodine receptor Ca2+ release channels: perspectives from recombinant expression systems. Cell Biochem Biophys. 2005; 42: 197eC222.
Yamamoto T, Ikemoto N. Peptide probe study of the critical regulatory domain of the cardiac ryanodine receptor. Biochem Biophys Res Commun. 2002; 291: 1102eC1108.
George CH, Sorathia R, Bertrand BMA, Lai FA. In situ modulation of the human cardiac ryanodine receptor (hRyR2) by FKBP12.6. Biochem J. 2003; 370: 579eC589.
Hoppe A, Christensen K, Swanson JA. Fluorescence resonance energy transfer-based stoichiometry in living cells. Biophys J. 2002; 83: 3652eC3664.
George CH, Higgs GV, Mackrill JJ, Lai FA. Dysregulated ryanodine receptors mediate cellular toxicity: restoration of normal phenotype by FKBP12.6. J Biol Chem. 2003; 278: 28856eC28864.
Claycomb WC, Lanson NA, Stallworth BS, Egeland DB, Delcarpio JB, Bahinski A, Izzo NJ. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci U S A. 1998; 95: 2979eC2984.
Katra RP, Laurita KR. Cellular mechanism of calcium-mediated triggered activity in the heart. Circ Res. 2005; 96: 535eC542.
Wier WG, Kort AA, Stern MD, Lakatta EG, Marban E. Cellular calcium fluctuations in mammalian heart: direct evidence from noise analysis of aequorin signals in Purkinje fibers. Proc Natl Acad Sci U S A. 1983; 80: 7367eC7371.
Liu Z, Wang R, Zhang J, Chen SRW, Wagenknecht T. Localization of a disease-associated mutation site on the three-dimensional structure of the cardiac ryanodine receptor. J Biol Chem. 2005; 280: 37941eC37947.
Priori SG, Napolitano C, Memmi M, Colombi B, Drago F, Gasparini M, DeSimeone L, Coltorti F, Bloise R, Keegan R, Cruz Filho FES, Vignati G, Benetar A, DeLogu A. Clinical and molecular characterization of patients with catecholaminergic polymorphic ventricular tachycardia. Circulation. 2002; 106: 69eC74.
Nam G-B, Burashnikov A, Antzelevitch C. Cellular mechanisms underlying the development of catecholaminergic ventricular tachycardia. Circulation. 2005; 111: 2727eC2733.
Bauce B, Rampazzo A, Basso C, Bagattin A, Daliento L, Tiso N, Turrini P, Thiene G, Danieli GA, Nava A. Screening for ryanodine receptor type 2 mutations in families with effort-induced polymorphic ventricular arrhythmias and sudden death: early diagnosis of asymptomatic carriers. J Am Coll Cardiol. 2002; 40: 341eC349.
Allouis M, Probst V, Jaafar P, Schott JJ, Le Marec H. Unusual clinical presentation in a family with catecholaminergic polymorphic ventricular tachycardia due to a G14876A ryanodine receptor gene mutation. Am J Cardiol. 2005; 95: 700eC702.
Xiao B, Sutherland C, Walsh MP, Chen SRW. Protein kinase A phosphorylation at serine-2808 of the cardiac Ca2+-release channel (ryanodine receptor) does not dissociate 12.6-kDa FK506-binding protein (FKBP12.6). Circ Res. 2004; 94: 487eC495.
Stange M, Xu L, Balshaw D, Yamaguchi N, Meissner G. Characterization of recombinant skeletal muscle (Ser-2843) and cardiac muscle (Ser-2809) ryanodine receptor phosphorylation mutants. J Biol Chem. 2003; 278: 51693eC51702.
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.
Cerrone M, Colombi B, Santoro M, Raffale di Barletta M, Scelsi M, Villani L, Napolitano C, Priori SG. Bidirectional ventricular tachycardia and fibrillation elicited in a knock-in mouse model carrier of a mutation in the cardiac ryanodine receptor (RyR2). Circ Res. 2005; 96: e77eCe82.
Hoshijima M. Gene therapy targeted at calcium handling as an approach to the treatment of heart failure. Pharmacol Ther. 2005; 105: 211eC218.
Xin HB, Senbonmatsu T, Cheng DS, Wang YX, Copello JA, Ji GJ, Collier ML, Deng KY, Jeyakumar LH, Magnuson MA, Inagami T, Kotlikoff MI, Fleischer S. Oestrogen protects FKBP12.6 null mice from cardiac hypertrophy. Nature. 2002; 416: 334eC337.
Houser SR. Can novel therapies for arrhythmias caused by spontaneous sarcoplasmic reticulum Ca2+ release be developed using mouse models Circ Res. 2005; 96: 1031eC1032.
Swan H, Laitinen P. Familial polymorphic ventricular tachycardia-intracellular calcium channel disorder. Cardiac Electrophys Rev. 2002; 6: 81eC87.
Doi M, Yano M, Kobayashi S, Kohno M, Tokuhisa T, Okuda S, Suetsugu M, Hisamatsu Y, Ohkusa T, Kohno M, Matsuzaki M. Propranolol prevents the development of heart failure by restoring FKBP12.6 mediated stabilization of ryanodine receptor. Circulation. 2002; 105: 1374eC1379.
Reiken S, Wehrens XHT, Vest JA, Barbone A, Klotz S, Mancini D, Burkhoff D, Marks AR. B-blockers restore calcium release channel function and improve cardiac muscle performance in human heart failure. Circulation. 2003; 107: 2459eC2466.
Du GG, Sandhu B, Khanna VK, Guo XH, MacLennan DH. Topology of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (RyR1). Proc Natl Acad Sci U S A. 2002; 99: 16725eC16730.