Abnormal Calcium Signaling and Sudden Cardiac Death Associated With Mutation of Calsequestrin
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 (CASQ2D307H) 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 (CASQ2WT) or CASQ2D307H. Expression of CASQ2D307H reduced the Ca2+ storing capacity of the SR. In addition, the amplitude, duration, and rise time of macroscopic ICa-induced Ca2+ transients and of spontaneous Ca2+ sparks were reduced significantly in myocytes expressing CASQ2D307H. Myocytes expressing CASQ2D307H 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 CASQ2D307H 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.
- excitation-contraction coupling
- calcium-induced calcium release
- catecholaminergic polymorphic ventricular tachycardia
- sarcoplasmic reticulum
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 CASQ2D307H) 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 CASQ2D307H 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 CASQ2D307H and the clinical phenotype observed in patients carrying this mutation.
Materials and Methods
Construction of Recombinant Adenoviruses
The construction of Ad-CSQ2WT 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 CASQ2D307H 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.
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).
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 MΩ 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 60×1.4 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 Kd 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
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).
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 CASQ2D307H coding sequence was inserted into an adenoviral vector to generate Ad-CASQ2D307H for subsequent gene transfer into adult rat ventricular myocytes. In addition, two adenoviruses containing the WT canine CASQ2 (Ad-CASQ2WT) 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-CASQ2WT or Ad-CASQ2D307H consistently resulted in equivalent ≈4-fold increases in total CASQ2 protein, and CASQ2 levels were unchanged in cells infected with Ad-Control.
CASQ2D307H 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 (INCX) in myocytes dialyzed with the Ca2+ indicator Fluo-3 (Figure 2). The amplitude of caffeine-induced Ca2+ transients increased 2.2-fold in CASQ2WT-overexpressing myocytes compared with control cells. In contrast, expression of CASQ2D307H reduced the amplitude of the caffeine-induced fluorescence signal to 41% of control (Figure 2B). These changes in fluorescence signals in Ad-CASQ2WT and Ad-CASQ2D307H myocytes were paralleled by changes in the Na+-Ca2+ exchange current. The integral of INCX was 2.3-fold higher in Ad-CASQ2WT myocytes and decreased to 36% of control in Ad-CASQ2D307H myocytes (Figure 2C). Thus, ectopic expression of CASQ2D307H suppressed the ability of SR to store Ca2+.
Macroscopic Ca2+ Transients and ICa
The effects of expressing CASQ2WT and CASQ2D307H on ICa and intracellular [Ca2+] transients in patch-clamped myocytes are illustrated in Figure 3. There were no apparent changes in the parameters of ICa in myocytes expressing either CASQ2WT or CASQ2D307H (Figure 3A, bottom, and Figure 3B; Table 1). The peak amplitude of ICa was nearly identical for all groups of cells (Table 1). In addition, the time course of ICa decay was similar (Table 1). Thus, expression of CASQ2WT or CASQ2D307H did not change the characteristics of the Ca2+ trigger for Ca2+ release from the SR.
Overexpressing CASQ2WT 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 CASQ2D307H, the Ca2+ transients were drastically reduced in size and duration. Furthermore, the rise time of Ca2+ transients in Ad-CASQ2D307H myocytes was shortened significantly compared with control (by 39%). Thus, expressing CASQ2D307H inhibited active SR Ca2+ release, apparently by shortening Ca2+ release duration.
Ca2+ Sparks in Permeabilized Myocytes
We next examined the impact of expressing CASQ2D307H 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-CASQ2WT, and Ad-CASQ2D307H 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 CASQ2WT and CASQ2D307H on parameters of Ca2+ sparks is documented in Table 2. Overexpression of CASQ2WT 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-CASQ2WT myocytes (172% of control). However, when CASQ2D307H was expressed, the Ca2+ sparks were smaller and briefer and had rise times shorter than in control (73% of control). Thus, expressing CASQ2D307H resulted in focal release events of reduced size and abbreviated duration.
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 CASQ2WT or CASQ2D307H 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 CASQ2WT, 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 CASQ2D307H, 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 CASQ2D307H 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.
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 CASQ2D307H 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 CASQ2D307H-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 CASQ2D307H-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.
It has been reported recently that certain recessive forms of CPVT are associated with mutations in the cardiac CASQ2 gene5,6; 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 CASQ2D307H 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 CASQ2D307H develop abnormal intracellular [Ca2+] oscillations that cause DADs specifically during β-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 CASQ2D307H 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 CASQ2D307H (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 CASQ2D307H-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 CASQ2D307H 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-CASQ2D307H (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 CASQ2D307H 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 CASQ2D307H myocytes to β-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 CASQ2D307H 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 CASQ2D307H-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 mutations3,4 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.
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 CASQ2D307H 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.
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.).
Original received October 3, 2003; revision received December 18, 2003; accepted December 23, 2003.
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