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(Circulation Research. 2004;94:471.)
© 2004 American Heart Association, Inc.

Cellular Biology

Abnormal Calcium Signaling and Sudden Cardiac Death Associated With Mutation of Calsequestrin

Serge Viatchenko-Karpinski, Dmitry Terentyev, Inna Györke, Radmila Terentyeva, Pompeo Volpe, Silvia G. Priori, Carlo Napolitano, Alessandra Nori, Simon C. Williams, Sandor Györke

From the Departments of Physiology (S.V.-K., D.T., I.G., R.T., S.G.) and Cell Biology and Biochemistry (S.C.W.), Texas Tech University Health Sciences Center, and Southwest Cancer Center at University Medical Center (S.C.W.), Lubbock, Tex; Dipartimento di Scienze Biomediche Sperimentali dell’Universita degli Studi di Padova (P.V., A.N.), Padova, Italy; and Department of Molecular Cardiology (S.G.P., C.N.), Fondazione Salvatore Maugeri, IRCCS, Pavia, Italy.

Correspondence to Sandor Gyorke, Department of Physiology, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430-6551. E-mail sandor.gyorke{at}ttuhsc.edu

	Abstract

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Mutations in human cardiac calsequestrin (CASQ2), a high-capacity calcium-binding protein located in the sarcoplasmic reticulum (SR), have recently been linked to effort-induced ventricular arrhythmia and sudden death (catecholaminergic polymorphic ventricular tachycardia). However, the precise mechanisms through which these mutations affect SR function and lead to arrhythmia are presently unknown. In this study, we explored the effect of adenoviral-directed expression of a canine CASQ2 protein carrying the catecholaminergic polymorphic ventricular tachycardia–linked mutation D307H (CASQ2^D307H) on Ca²⁺ signaling in adult rat myocytes. Total CASQ2 protein levels were consistently elevated 4-fold in cells infected with adenoviruses expressing either wild-type CASQ2 (CASQ2^WT) or CASQ2^D307H. Expression of CASQ2^D307H reduced the Ca²⁺ storing capacity of the SR. In addition, the amplitude, duration, and rise time of macroscopic I_Ca-induced Ca²⁺ transients and of spontaneous Ca²⁺ sparks were reduced significantly in myocytes expressing CASQ2^D307H. Myocytes expressing CASQ2^D307H also displayed drastic disturbances of rhythmic oscillations in [Ca²⁺]_i and membrane potential, with signs of delayed afterdepolarizations when undergoing periodic pacing and exposed to isoproterenol. Importantly, normal rhythmic activity was restored by loading the SR with the low-affinity Ca²⁺ buffer, citrate. Our data suggest that the arrhythmogenic CASQ2^D307H mutation impairs SR Ca²⁺ storing and release functions and destabilizes the Ca²⁺-induced Ca²⁺ release mechanism by reducing the effective Ca²⁺ buffering inside the SR and/or by altering the responsiveness of the Ca²⁺ release channel complex to luminal Ca²⁺. These results establish at the cellular level the pathological link between CASQ2 mutations and the predisposition to adrenergically mediated arrhythmias observed in patients carrying CASQ2 defects.

Key Words: excitation-contraction coupling • calcium-induced calcium release • catecholaminergic polymorphic ventricular tachycardia • sarcoplasmic reticulum • calsequestrin

	Introduction

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Catecholaminergic polymorphic ventricular tachycardia (CPVT; Online Mendelian Inheritance in Man database, No. 604772) is a familial arrhythmogenic disorder characterized by adrenergically mediated polymorphic ventricular tachyarrhythmias leading to syncope and sudden cardiac death.¹ Clinical manifestations of the disease typically occur in young children during physical activity or emotional stress. It has been suggested that arrhythmias in CPVT are mediated by delayed afterdepolarizations (DADs),^2,3 oscillations of the membrane potential associated with Ca²⁺ overload, but no conclusive evidence exists to support this hypothesis.

Two genetic variants of CPVT have been identified, one transmitted as an autosomal dominant trait caused by mutations in the gene encoding the cardiac ryanodine receptor (RyR2)^3,4 and one recessive form caused by mutations in the cardiac-specific isoform of the calsequestrin gene (CASQ2).^5,6 RyR2 and CASQ2 are key components of the excitation-contraction (EC) coupling machinery. Both proteins are part of a supramolecular Ca²⁺ signaling complex in the junctional sarcoplasmic reticulum (SR) that also contains triadin 1 and junctin, among other proteins.^7–9 RyR2 serves as a Ca²⁺ release channel in the SR. During the EC coupling process, RyR2 channels are activated by Ca²⁺ that enters the cell through voltage-dependent L-type Ca²⁺ channels, causing the release of Ca²⁺ from the SR into the cytosol, a mechanism known as Ca²⁺-induced Ca²⁺ release (CICR).^10,11 Increased cytosolic Ca²⁺ levels activate the contractile apparatus. Ca²⁺ release is terminated when SR luminal [Ca²⁺] falls below a threshold level, causing a decline in RyR2 activity via a mechanism termed luminal Ca²⁺-dependent deactivation.^12,14 CASQ2 is a high-capacity Ca²⁺-binding protein whose primary function is to store the releasable Ca²⁺ within the SR.^11,13 Additionally, CASQ2 seems to play an important role in regulation of SR Ca²⁺ release by controlling the local luminal [Ca²⁺] in the vicinity of the RyR2 channels¹⁴ and possibly also by serving as a luminal Ca²⁺ sensor for RyR2.¹⁵

The recessive form of CPVT was positionally mapped in several Bedouin families to the region of chromosome 1 (1p 13-21) in which the CASQ2 gene is located.¹⁶ Subsequent sequence analysis of CASQ2 genes from these individuals identified a missense point mutation in a highly conserved region of CASQ2.⁵ This mutation (referred to here as CASQ2^D307H) converts a negatively charged aspartic acid into a histidine in a putative Ca²⁺ chelating region of CASQ2. Lahat et al⁵ proposed that this mutation exerts its effects by disrupting Ca²⁺ binding to CASQ2, but the specific mechanisms whereby the D307H mutation causes CPVT remain unknown. In the present study, we used an adenoviral-mediated gene transfer strategy to express a canine CASQ2^D307H protein in cardiac myocytes and explored the effects of this mutation on intracellular Ca²⁺ handling using Ca²⁺ imaging and patch-clamp techniques. Our results establish a pathological link between the expression of CASQ2^D307H and the clinical phenotype observed in patients carrying this mutation.

	Materials and Methods

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Construction of Recombinant Adenoviruses
The construction of Ad-CSQ2^WT and Ad-Control was described previously.¹⁴ The D307H mutation was introduced into the full-length canine CASQ2 cDNA using the Quikchange Site-Directed Mutagenesis Kit (Stratagene). The CASQ2^D307H coding region was transferred into the Adeno-X Viral DNA, and recombinant adenoviruses were generated according to the instructions of the manufacturer (Clontech).

Adenoviral Gene Transfer
Ventricular myocytes were obtained from adult male Sprague-Dawley rat hearts by enzymatic dissociation, infected with adenoviruses at a multiplicity of infection of 100, and maintained in a CO₂ incubator at 5% CO₂ and 95% air at 37°C, as described.¹⁴ All experiments were performed 48 to 56 hours after infection of myocytes with the adenoviral constructs.

Western Blotting
Normal and mutant CASQ2 protein levels were determined by immunoblot analysis as described previously.¹⁴ Briefly, 10 µg of cell lysate proteins was subjected to 12% SDS-PAGE, blotted onto PVDF membranes (Santa Cruz Biotechnology, Inc), and probed with antibodies specific for CASQ2 (1:2500, PA1-913, Affinity Bioreagents). Blots were developed with Super Signal West Pico (PIERCE) and quantified using a Visage 2000 Blot Scanning and Analysis system (BioImage Systems Corporation).

Electrophysiological Recordings
Whole-cell patch-clamp recordings of transmembrane ionic currents were performed as described previously.¹² The voltage-clamp protocol consisted of 400-ms-long voltage pulses to specified membrane potentials applied from a holding potential of -50 mV at 1-minute intervals. The external solution contained (in mmol/L) NaCl 140, KCl 5.4, CaCl₂ 1.0, MgCl₂ 0.5, HEPES 10, and glucose 5.6, pH 7.3. Patch pipettes with tip resistance of 1 to 3 M were pulled from borosilicate glass (Sutter Instrument Co) and filled with a solution that contained (in mmol/L) Cs-aspartate 90, CsCl 50, Na₂ATP 3, MgCl₂ 3.5, HEPES 10, and Fluo-3 K⁺-salt 0.05, pH 7.3. In some experiments, the cells were stimulated periodically (2 Hz) and the membrane potential was recorded in the current-clamp configuration. In these experiments the pipette solution contained K⁺-aspartate instead of Cs-aspartate. Citrate K⁺-salt (5 mmol/L) was added into the pipette solution by replacing osmotically equivalent amounts of K⁺-aspartate. Rapid applications of caffeine were used to measure SR Ca²⁺ content (10 mmol/L). The amount of Ca²⁺ released was assessed by integration of the Na⁺-Ca²⁺ exchange current and from the peak amplitude of the caffeine-induced Ca²⁺ transients.

Confocal Ca²⁺ Measurements
Intracellular Ca²⁺ imaging was performed using a Bio-Rad Laser Scanning Confocal System (Bio-Rad MRC-1024ES interfaced to an Olympus IX-70 inverted microscope and equipped with an Olympus 60x1.4 NA oil objective), as described.¹² Fluo-3 was excited by the 488-nm beam of an argon-ion laser, and the fluorescence was acquired at wavelengths >515 nm in the line scan mode of the confocal system at rate of 2 or 6 ms per scan. The magnitude of fluorescent signals was quantified in terms of F/F₀, where F₀ is baseline fluorescence. Assuming that the basal cytosolic [Ca²⁺] is 100 nmol/L and a K_d for Fluo-3 Ca²⁺ binding of 1.1 µmol/L,¹⁷ the theoretical maximum for F/F₀ is 12. Ca²⁺ spark parameters were quantified with a detection/analysis computer algorithm.¹²

Statistics
All values were expressed as mean±SEM. Statistical comparisons were made using unpaired Student’s t test or 2-way ANOVA for repeated measurements (P<0.05).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Conclusions References

 
Adenoviral-Mediated Expression of Mutant CASQ2 in Isolated Rat Myocytes
The dog and human CASQ2 proteins display 91% sequence identity overall, and the D307H mutation is located in a region that is highly conserved among CASQ2 orthologues from various vertebrate species (Figures 1A and 1B). Interestingly, this residue is also located within a highly conserved region of CASQ1 proteins, suggesting that it may be crucial for a shared function of CASQ proteins (Figure 1B). The D307H mutation was introduced into the coding sequence of canine CASQ2 by site-directed mutagenesis. The canine CASQ2^D307H coding sequence was inserted into an adenoviral vector to generate Ad-CASQ2^D307H for subsequent gene transfer into adult rat ventricular myocytes. In addition, two adenoviruses containing the WT canine CASQ2 (Ad-CASQ2^WT) and the CASQ2 coding sequence with a stop codon inserted after amino acid 70 (Ad-Control) were used as controls for viral infection and CASQ2 expression. Our infection protocol results in infection efficiencies of nearly 100% at a multiplicity of infection of 100.¹⁴ Expression of both endogenous and recombinant proteins was examined in infected myocytes by Western blotting (Figures 1C and 1D). Infection of myocytes with either Ad-CASQ2^WT or Ad-CASQ2^D307H consistently resulted in equivalent 4-fold increases in total CASQ2 protein, and CASQ2 levels were unchanged in cells infected with Ad-Control.

View larger version (51K): [in this window] [in a new window]   Figure 1. Structure and expression of wild-type and mutant CASQ2 proteins. A, Schematic representation of functional domains in the CASQ2 monomer. SS indicates signal sequence; A and B, putative regions involved in oligomerization (by analogy with CASQ1; Reference 32); and D/E-rich, domain rich in aspartic and glutamic acid residues. Amino acid numbering refers to the mature protein after signal sequence removal. B, Comparison of sequence around the D307H mutation in CASQ2 and the related CASQ1 proteins. The mutation was named according to the amino acid number in the unprocessed protein and occurs at Asp288 in the mature protein. Amino acids identical in all CASQ2 and CASQ1 orthologues are indicated in red. C, Western analysis of CASQ2 protein levels in cardiac myocytes infected with the control adenovirus or adenoviruses expressing wild-type CASQ2 or CASQ2D307H. D, Intensities of the bands detected in 4 independent Western blots were measured by densitometry and are expressed relative to the control cells.

CASQ2^D307H Overexpression Decreases the SR Ca²⁺ Storage Capacity
Caffeine applications (10 mmol/L) were used to assess the changes in the total SR Ca²⁺ content in each group of adenovirus-infected myocytes. The relative amounts of Ca²⁺ released from the SR after caffeine administration were assessed from changes in both Fluo-3 fluorescence and Na⁺-Ca²⁺ exchange current (I_NCX) in myocytes dialyzed with the Ca²⁺ indicator Fluo-3 (Figure 2). The amplitude of caffeine-induced Ca²⁺ transients increased 2.2-fold in CASQ2^WT-overexpressing myocytes compared with control cells. In contrast, expression of CASQ2^D307H reduced the amplitude of the caffeine-induced fluorescence signal to 41% of control (Figure 2B). These changes in fluorescence signals in Ad-CASQ2^WT and Ad-CASQ2^D307H myocytes were paralleled by changes in the Na⁺-Ca²⁺ exchange current. The integral of I_NCX was 2.3-fold higher in Ad-CASQ2^WT myocytes and decreased to 36% of control in Ad-CASQ2^D307H myocytes (Figure 2C). Thus, ectopic expression of CASQ2^D307H suppressed the ability of SR to store Ca²⁺.

View larger version (31K): [in this window] [in a new window]   Figure 2. Effects of expressing the WT and mutant CASQ2 on SR Ca2+ content in rat ventricular myocytes. A, Caffeine-induced [Ca2+]i transients (upper traces) and INCX (lower traces) in myocytes infected with Ad-Control, Ad-CASQ2WT, and Ad-CASQ2D307H vectors. SR Ca2+ release was induced by application of 10 mmol/L caffeine. B and C, Pooled data (mean±SEM) on changes in SR Ca2+ content inferred from the peak amplitude of the Ca2+ transients (B) and from the integral of INCX (C) on application of caffeine (n=6, P<0.05).

Macroscopic Ca²⁺ Transients and I_Ca
The effects of expressing CASQ2^WT and CASQ2^D307H on I_Ca and intracellular [Ca²⁺] transients in patch-clamped myocytes are illustrated in Figure 3. There were no apparent changes in the parameters of I_Ca in myocytes expressing either CASQ2^WT or CASQ2^D307H (Figure 3A, bottom, and Figure 3B; Table 1). The peak amplitude of I_Ca was nearly identical for all groups of cells (Table 1). In addition, the time course of I_Ca decay was similar (Table 1). Thus, expression of CASQ2^WT or CASQ2^D307H did not change the characteristics of the Ca²⁺ trigger for Ca²⁺ release from the SR.

View larger version (24K): [in this window] [in a new window]   Figure 3. Effects of expressing the WT and mutant CASQ2 on EC coupling. A, Traces of ICa (lower traces) and Ca2+ transients (upper traces) induced by depolarization from -50 to 0 mV in cardiomyocytes infected with Ad-Control, Ad-CASQ2WT, and Ad-CASQ2D307H vectors. B and C, Voltage dependence of ICa (B) and Ca2+ transients (C) in myocytes infected with Ad-Control, Ad-CASQ2WT, and Ad-CASQ2D307H vectors (n for each point ranged from 5 to 18).

View this table: [in this window] [in a new window]   Table 1. Parameters of ICa and Ca2+ Transients

Overexpressing CASQ2^WT caused a dramatic increase in the magnitude and overall duration of Ca²⁺ transients (Figure 3A, top, and Figure 3C; Table 1). Importantly, the duration of the rising phase was increased by 90%, consistent with the hypothesis that CASQ2 modulates SR Ca²⁺ release by prolonging the duration of the Ca²⁺ flux from the SR to the cytosol.¹⁴ In contrast, in myocytes expressing CASQ2^D307H, the Ca²⁺ transients were drastically reduced in size and duration. Furthermore, the rise time of Ca²⁺ transients in Ad-CASQ2^D307H myocytes was shortened significantly compared with control (by 39%). Thus, expressing CASQ2^D307H inhibited active SR Ca²⁺ release, apparently by shortening Ca²⁺ release duration.

Ca²⁺ Sparks in Permeabilized Myocytes
We next examined the impact of expressing CASQ2^D307H on properties of focal fluorescence signals, ie, Ca²⁺ sparks, in permeabilized myocytes. Myocytes were permeabilized with saponin, and Ca²⁺ sparks were recorded at a constant cytosolic [Ca²⁺] of 100 nmol/L.¹⁸ Representative line-scan images of sparks acquired in myocytes infected with Ad-Control, Ad-CASQ2^WT, and Ad-CASQ2^D307H are shown in Figure 4A, and surface plots of sparks obtained by averaging multiple individual events¹² acquired in the same three groups of myocytes are illustrated in Figure 4B. The impact of expression of CASQ2^WT and CASQ2^D307H on parameters of Ca²⁺ sparks is documented in Table 2. Overexpression of CASQ2^WT resulted in a dramatic increase in the overall magnitude and spatiotemporal spread of sparks. In addition, the duration of the rising phase of sparks was increased in Ad-CASQ2^WT myocytes (172% of control). However, when CASQ2^D307H was expressed, the Ca²⁺ sparks were smaller and briefer and had rise times shorter than in control (73% of control). Thus, expressing CASQ2^D307H resulted in focal release events of reduced size and abbreviated duration.

View larger version (60K): [in this window] [in a new window]   Figure 4. Effects of expressing the WT and mutant CASQ2 on properties of spontaneous Ca2+ sparks in permeabilized myocytes. A, Representative line-scan images of Ca2+ sparks acquired in myocytes infected with Ad-Control, Ad-CASQ2WT, and Ad-CASQ2D307H vectors. B, Surface plots of averaged Ca2+ sparks in myocytes infected with Ad-Control (30 events), Ad-CASQ2WT (24 events), and Ad-CASQ2D307H (29 events) vectors.

View this table: [in this window] [in a new window]   Table 2. Parameters of Ca2+ Sparks

Ca²⁺ Cycling in Rhythmically Paced Myocytes
The effects of isoproterenol (ISO) treatment (1 µmol/L) on periodic Ca²⁺ transients in control myocytes and in myocytes expressing either CASQ2^WT or CASQ2^D307H is illustrated in Figure 5. The myocytes were stimulated at 2 Hz, and membrane potential (MP) changes were recorded in the current-clamp mode. In control myocytes, exposure to ISO caused an increase in the amplitude of Ca²⁺ transients without any apparent disturbances in periodic Ca²⁺ cycling (results are representative of six myocytes, Figure 5A). In myocytes overexpressing CASQ2^WT, the amplitude of Ca²⁺ transients was increased with respect to control, consistent with measurements under resting conditions. ISO treatment caused an additional augmentation of Ca²⁺ signals, again without disrupting rhythmicity (data are representative of nine myocytes, Figure 5B). In myocytes expressing CASQ2^D307H, the amplitude of Ca²⁺ transients was reduced with respect to control, consistent with measurements in resting myocytes. ISO application caused profound disturbances in Ca²⁺ cycling manifested by extrasystolic, spontaneous Ca²⁺ transients. As seen in the line-scan images (Figure 5C), spontaneous release usually originated locally and then propagated through the cell as a regenerative Ca²⁺ wave. Importantly, the MP traces showed clear signs of DADs at the time of spontaneous Ca²⁺ transients. These oscillations in MP are thought to be the underlying causes of arrhythmia.^11,19,20 Similar results were obtained in five other myocytes. These results indicate that expression of CASQ2^D307H not only reduces the amount of Ca²⁺ released from the SR but also destabilizes the Ca²⁺ release mechanism, leading to spontaneous, premature discharges of SR Ca²⁺ stores in myocytes undergoing periodic pacing.

View larger version (74K): [in this window] [in a new window]   Figure 5. Disturbances in rhythmic [Ca2+]i transients and membrane potential induced by ISO in myocytes expressing CASQ2D307H. Recordings of membrane potential (upper traces), along with line-scan images (middle) and time-dependent profiles of [Ca2+]i (lower traces) in myocytes infected with Ad-Control (A), Ad-CASQ2WT (B), and Ad-CASQ2D307H (C) vectors before and after exposure of the myocytes to 1 µmol/L ISO. Myocytes were stimulated at 2 Hz in the current-clamp mode.

Restoration of Normal Periodic Ca²⁺ Transients by Increasing SR Ca²⁺ Buffering Capacity With Low-Affinity Ca²⁺ Buffers
We have recently proposed that CASQ2 regulates the functional size and stability of SR Ca²⁺ stores by serving as a buffer for Ca²⁺ in the SR lumen.¹⁴ To test the hypothesis that disruption of Ca²⁺ cycling observed in cells expressing CASQ2^D307H is attributable to abnormal intra-SR Ca²⁺ buffering, we carried out experiments using the low-affinity exogenous Ca²⁺ buffer, citrate. Citrate was loaded into the SR of CASQ2^D307H-expressing myocytes by dialyzing them with a citrate-containing pipette solution.¹² Sequestration of citrate into the SR occurs substantially slower (20 to 30 minutes) than equilibration of Fluo-3 into the cytosol (5 to 10 minutes),¹² thus permitting determination of the effects of intra-SR citrate on Ca²⁺ cycling in the same individual myocytes (Figure 6). Again, application of ISO produced characteristic disturbances in the periodic Ca²⁺ transients and MP in CASQ2^D307H-expressing myocytes (Figure 6, left and middle). Continuous dialysis of the myocytes with citrate-containing solution for 20 minutes led to a substantial increase in the magnitude of Ca²⁺ transients. Importantly, citrate dialysis also normalized Ca²⁺ cycling by eliminating the spontaneous, extrasystolic Ca²⁺ transients (Figure 6, right). Similar results were obtained in all five independent cells examined.

View larger version (28K): [in this window] [in a new window]   Figure 6. Restoration of normal rhythmic activity by intracellular dialysis with citrate in myocytes expressing CASQ2D307H. Recordings of membrane potential (upper traces), along with line-scan images (middle) and time-dependent profiles of [Ca2+]i (lower traces), in myocyte infected with Ad-CASQ2D307H vector before and after exposure of the myocyte to 1 µmol/L ISO. Myocytes were stimulated at 2 Hz in the current-clamp mode. Pipette solution contained 5 mmol/L of SR-permeable low-affinity Ca2+ buffer citrate. Recordings on the left and in the middle were made 5 minutes after whole-cell configuration was established. Recordings on the right were made 15 minutes later.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Conclusions References

 
It has been reported recently that certain recessive forms of CPVT are associated with mutations in the cardiac CASQ2 gene^5,6; however, the mechanisms by which CASQ2 mutations cause the clinical phenotype have not been established. The objective of the present study was to investigate the functional characteristics of the missense mutation D307H identified in the first large pedigree affected by CPVT linked to mutations in CASQ2.⁵ Adenoviral-mediated expression of CASQ2^D307H diminished the Ca²⁺ storing and releasing capabilities of the SR in rat ventricular myocytes, thus resulting in pronounced dominant-negative effects on SR Ca²⁺ handling. Relevant to the arrhythmogenic consequence of the D307H mutation, our data demonstrated that myocytes expressing CASQ2^D307H develop abnormal intracellular [Ca²⁺] oscillations that cause DADs specifically during ß-adrenergic stimulation. Therefore, within the limits of an in vitro cell model, we establish for the first time a pathological link between a specific, clinically relevant mutation in the CASQ2 gene and arrhythmogenic behavior underlying CPVT. The dominant-negative effects of the mutant protein on intracellular Ca²⁺ signaling were unexpected considering the recessive mode of inheritance of the disease. They provide new clues for understanding the structure-functional relationships within the junctional Ca²⁺ signaling complex.

Effects of D307H on CASQ2 Function
Based on the analysis of the amino acid sequence and crystal structure of calsequestrin, Asp³⁰⁷, which harbors this arrhythmogenic mutation, is localized to a putative Ca²⁺ binding region between the second and third thioredoxin-like domains of the protein.^5,21 Consequently, it has been hypothesized that the pathology of CPVT may involve disrupted Ca²⁺ binding by CASQ2.⁵ Our finding that expression of CASQ2^D307H diminished SR Ca²⁺ releasing and storing capabilities despite the presence of normal levels of the endogenous wild-type protein suggests that the effect of this mutation on CASQ2 function may be more complex than merely altering Ca²⁺ binding by CASQ2 monomers. Previous studies performed with both the skeletal and cardiac isoforms of the protein demonstrated that calsequestrin oligomerizes in a Ca²⁺-dependent fashion and that the formation of calsequestrin polymers is important for high-capacity Ca²⁺ binding.^22,23 Although the precise mechanisms of Ca²⁺-dependent aggregation of calsequestrin monomers and of Ca²⁺ sequestration by the calsequestrin complex are not known, it has been proposed that calsequestrin polymers provide an electrostatically charged surface onto which Ca²⁺ can be absorbed.^21,23 Thus, structural defects in individual CASQ2 monomers could reduce high-capacity Ca²⁺ binding by disrupting Ca²⁺-dependent CASQ2 polymerization. This possibility is consistent with reduced SR Ca²⁺ storing capacity of myocytes expressing CASQ2^D307H (Figure 2). It is also possible that the effects of the mutation are attributable to abnormal interactions of CASQ2 with other components of the SR Ca²⁺ release machinery. CASQ2 has been proposed to be actively involved in regulation of Ca²⁺ release through protein-protein interactions with RyR2, junctin, and triadin.^8,24 Recent results obtained in our laboratory suggest that RyR2 complexed with junctin and triadin is inhibited by CASQ2 at low luminal [Ca²⁺] and that this inhibition is relieved at high luminal [Ca²⁺].¹⁵ If the D307H mutation were to affect the ability of CASQ2 to interact with the RyR2 complex, this could lead to RyR2 channels with abnormally high activity. In this case, the diminished SR Ca²⁺ storing and releasing functions in CASQ2^D307H-expressing myocytes could reflect the compromised ability of the SR to retain Ca²⁺ due to hyperactive, ie, leaky, RyR2 channels. Regardless of the specific molecular alterations, our results indicate that Asp³⁰⁷ resides in a region of the protein that is critical for normal function of CASQ2 in cardiac SR.

It is interesting to note that whereas the CASQ2^D307H protein exerted clear dominant-negative effects in infected myocytes in our experiments, in the clinical setting, this mutation causes an abnormal phenotype only when 100% of the CASQ2 protein is abnormal (ie, in homozygous carriers of the mutation).⁵ This apparent discrepancy could be ascribed to relative levels of the wild-type and mutant CASQ2 proteins in our experiments, where the ratio of mutant to wild-type protein was 3:1 in myocytes infected with Ad-CASQ2^D307H (Figure 1). Indeed, when the mutant CASQ2 was expressed at levels similar to those of the endogenous protein (ie, at a ratio of 1:1), the myocytes showed no reduction in the amplitude of depolarization- and caffeine-induced Ca²⁺ transients with respect to control (see the online data supplement, available at http://circres.ahajournals.org). Because a 2-fold increase in total CASQ2 level did not lead to any gain in function, our results still imply that the function of CASQ2^D307H should be impaired in heterozygous carriers of this mutation (50% of normal protein present). It is likely, however, that the clinical phenotype of D307H is influenced by various adaptive changes in cellular Ca²⁺ handling mechanisms such as increased expression of other luminal Ca²⁺ binding proteins (eg, calreticulin) or CASQ1 isoform transition.

Molecular Mechanisms of CPVT
Our results provide a plausible explanation for the cellular mechanism by which the D307H mutation of CASQ2 causes catecholaminergic tachycardia. Exposure of CASQ2^D307H myocytes to ß-adrenergic stimulation induced extrasystolic, spontaneous Ca²⁺ transients and resulted in the development of DADs (Figure 5C). It is known that generation of DADs involves Ca²⁺-dependent inward Ca²⁺ currents and that these deflections of the membrane potential can trigger arrhythmias.^19,25–28 Using the same experimental system as in the present study (ie, adult rat ventricular myocytes), we recently demonstrated that CASQ2 plays an important role in termination and restitution of CICR by influencing luminal Ca²⁺-dependent gating of the RyR2 channels.¹⁴ Furthermore, reduction of CASQ2 levels to 30% of wild-type levels (achieved using an antisense RNA approach) led to profound disturbances in rhythmic Ca²⁺ cycling attributable to an accelerated functional recovery of the release sites from a luminal Ca²⁺-dependent refractory state. The effects of expressing CASQ2^D307H on specific parameters of both cell-averaged Ca²⁺ transients and Ca²⁺ sparks (eg, amplitude and rise-time duration; Figures 2 through 4 and Tables 1 and 2 ) were similar to those we observed on reducing CASQ2 protein levels.¹⁴ Thus, our previous results and present findings suggest a mechanism whereby reduced buffering of Ca²⁺ in the SR lumen by CASQ2 (and/or disrupted interactions of CASQ2 with the RyR2 channel complex) leads to altered regulation of the Ca²⁺ release mechanism by luminal Ca²⁺. Within this mechanistic framework, the role of adrenergic stimulation in promoting the pathologic signs of disease can be ascribed to an accelerated recharging of the SR Ca²⁺ store (as a result of enhanced SR Ca²⁺ uptake by the CaATPase) and hence further contributing to the premature functional restitution of the RyR2s.¹⁴ Our finding that normal Ca²⁺ cycling in CASQ2^D307H-expressing myocytes can be restored by loading their SR with low-affinity exogenous Ca²⁺ buffers (Figure 6) provides a strong support for the proposed role of CASQ2 and luminal Ca²⁺ in the pathogenesis of CPVT.

Our results are also relevant for understanding the cellular mechanisms of genetically distinct forms of CPVT with similar clinical manifestations. Recent studies have indicated that RyR2 mutations associated with CPVT result in abnormal RyR2 channel activity, although the precise mechanisms underlying these changes remain controversial.^29–31 Given our findings that abnormal luminal Ca²⁺ regulation of SR Ca²⁺ release accounts for the arrhythmic behavior of myocytes, it is logical to propose that CPVT associated with RyR2 channel mutations^3,4 is a consequence of the compromised ability of the channel to sense or respond to changes in luminal Ca²⁺. In addition, it is possible that some forms of CPVT are caused by mutations in proteins such as junctin and triadin that are also part of the RyR2 complex⁸ and might be involved in sensing or communicating the luminal Ca²⁺ signal to the RyR2.

	Conclusions

Top Abstract Introduction Materials and Methods Results Discussion Conclusions References

 
In conclusion, we have established a pathological link between the D307H mutation in the CASQ2 gene and the clinical phenotype observed in CPVT patients carrying this mutation. The pathological chain seems to involve the following steps. First, the mutation in Asp³⁰⁷ compromises the ability of CASQ2 to form high-capacity Ca²⁺-binding oligomers and/or to interact with the RyR2 channel complex. Second, altered free [Ca²⁺] dynamics near the luminal phase of the RyR2 (as a result of impaired intra-SR Ca²⁺ buffering) and/or disrupted ability of the channel to respond to changes in luminal [Ca²⁺] (as a result of abnormal interactions of the mutant proteins with the RyR2 channel) cause premature functional restitution of the RyR2 Ca²⁺ release channels after each release. Third, premature functional recovery of the RyR channels leads to spontaneous, extrasystolic Ca²⁺ transients, which in turn trigger arrhythmogenic DADs. Thus, characterization of the effects of CASQ2^D307H in rat myocytes has elucidated the potential mechanisms by which mutations in CASQ2 determine the arrhythmia-prone substrate observed in CPVT patients. Future studies will focus on determining the precise effects of the mutation on intermolecular interactions between proteins comprising the Ca²⁺ release machinery.

	Acknowledgments

 
This work was supported by American Heart Association grant 0245088N (to S.C.W.) and NIH grants HL-74045 and HL-63043 (to S.G.). Additional support was provided by Leducq Foundation, Telethon P0227/01, CARIPLO 2001.3009/10.9079, FIRB-RBNE01XMP4_006, COFIN 2001067817 (to S.G.P.), and Telethon grant 1274 (to P.V.).

	Footnotes

 
Original received October 3, 2003; revision received December 18, 2003; accepted December 23, 2003.

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