Abnormal Interactions of Calsequestrin With the Ryanodine Receptor Calcium Release Channel Complex Linked to Exercise-Induced Sudd
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Dmitry Terentyev, Alessandra Nori, Massi
参见附件。
the Department of Physiology and Cell Biology (D.T., S.V.-K., Z.K., I.G., R.T., S.V., S.G.), Heart and Lung Research Institute, Ohio State University, Columbus
Dipartimento di Scienze Biomediche Sperimentali dell’Università di Padova (A.N., G.V., P.V.), Istituto Interuniversitario di Miologia, Padova, Italy
Department of Molecular Cardiology (M.S., C.N., S.G.P.), Fondazione Salvatore Maugeri, IRCCS, Pavia, Italy
University Hospital Leiden (N.A.B), The Netherlands
Department of Cell Biology and Biochemistry (S.C.W.), Texas Tech University Health Science Center, Lubbock
Southwest Cancer Center at University Medical Center (S.C.W.), Lubbock, Texas.
Abstract
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic disorder associated with mutations in the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2) genes. Previous in vitro studies suggested that RyR2 and CASQ2 interact as parts of a multimolecular Ca2+-signaling complex; however, direct evidence for such interactions and their potential significance to myocardial function remain to be determined. We identified a novel CASQ2 mutation in a young female with a structurally normal heart and unexplained syncopal episodes. This mutation results in the nonconservative substitution of glutamine for arginine at amino acid 33 of CASQ2 (R33Q). Adenoviral-mediated expression of CASQ2R33Q in adult rat myocytes led to an increase in excitation–contraction coupling gain and to more frequent occurrences of spontaneous propagating (Ca2+ waves) and local Ca2+ signals (sparks) with respect to control cells expressing wild-type CASQ2 (CASQ2WT). As revealed by a Ca2+ indicator entrapped inside the sarcoplasmic reticulum (SR) of permeabilized myocytes, the increased occurrence of spontaneous Ca2+ sparks and waves was associated with a dramatic decrease in intra-SR [Ca2+]. Recombinant CASQ2WT and CASQ2R33Q exhibited similar Ca2+-binding capacities in vitro; however, the mutant protein lacked the ability of its WT counterpart to inhibit RyR2 activity at low luminal [Ca2+] in planar lipid bilayers. We conclude that the R33Q mutation disrupts interactions of CASQ2 with the RyR2 channel complex and impairs regulation of RyR2 by luminal Ca2+. These results show that intracellular Ca2+ cycling in normal heart relies on an intricate interplay of CASQ2 with the proteins of the RyR2 channel complex and that disruption of these interactions can lead to cardiac arrhythmia.
Key Words: calsequestrin ryanodine receptor sarcoplasmic reticulum Ca2+-induced Ca2+ release catecholaminergic polymorphic ventricular tachycardia
Introduction
Catecholaminergic polymorphic ventricular tachycardia (CPVT) (Online Mendelian Inheritance in Man no. 604772) is a familial arrhythmogenic disorder characterized by adrenergically mediated polymorphic ventricular tachyarrhythmias, leading to syncope and sudden cardiac death in individuals with structurally normal hearts.1 The episodes of tachyarrhythmia are typically triggered by physical exercise or emotional stress. Two genetic variants of the disease have been described: a recessive form associated with homozygous mutations in the gene encoding the cardiac isoform of calsequestrin (CASQ2)2,3 and a second form transmitted as an autosomal dominant trait associated with mutations in the gene encoding the cardiac ryanodine receptor (RyR2).4,5
The contractile machinery of cardiac myocytes becomes activated when Ca2+ enters the sarcoplasmic reticulum (SR) via L-type Ca2+ channels and triggers a process termed Ca2+-induced Ca2+ release (CICR) from the SR.6 Whereas CICR controls the release process from the cytosolic side, a second Ca2+-dependent mechanism controls the activity of the Ca2+-release channels from the SR lumen. Specifically, the decline of intra-SR [Ca2+] that accompanies the Ca2+-release process contributes to Ca2+-release termination, a mechanism referred to as luminal Ca2+-dependent deactivation.7–9 The Ca2+-release channel is present in the junctional SR membrane in the form of a quaternary complex composed of RyR2, triadin, junctin, and CASQ2.10,11 The integral membrane proteins triadin and junctin physically interact with RyR2 and link the Ca2+-binding protein CASQ2 to the complex. Ca2+-dependent interactions of CASQ2 with the RyR2–triadin complex are thought to provide a molecular basis for regulation of RyR2 channel by luminal Ca2+.12,13 In addition, CASQ2 monomers can form polymers with high Ca2+-binding capacities that are essential for the Ca2+ storage function of the SR.14,15
To date, 4 homozygous sequence variations in the CASQ2 gene have been identified in patients with CPVT (see Inherited Arrhythmias Database at http://pc4.fsm.it:81/cardmoc).2,3 The precise molecular basis for the alterations in Ca2+ handling in cells expressing CPVT-linked CASQ2 mutants remains to be determined. To date, the effect of only 1 of these mutations on CASQ2 activity and function has been examined.16,17 These studies focused on a CASQ2 mutant protein in which aspartate 307 is changed to histidine (CASQ2D307H) and suggested that the CASQ2D307H protein is compromised in its ability to facilitate the Ca2+ storing and releasing functions of the SR. These effects may be a consequence of a reduction in the Ca2+-binding capacity of the mutant protein or altered interactions between CASQ2D307H and components of the RyR2 channel complex.16,17 Furthermore, it is unknown how independent mutations in the CASQ2 and RyR2 genes result in similar clinical manifestations in CPVT. In the present study, we report the identification of a new mutation in the CASQ2 gene in a patient with CPVT. We also demonstrate that this mutation alters the functional interactions between CASQ2 and the RyR2 channel complex, resulting in abnormal luminal Ca2+-dependent regulation of the RyR2 channel.
Materials and Methods
CPVT in human patients was diagnosed using standard cardiologic tests. Genetic analyses of the CPVT patients were performed using a combination of methods of PCR, single-strand conformation polymorphism (SSCP) analysis, and denaturing high-performance liquid chromatography (DHPLC) (Wave Transgenomics). The cellular effects of the newly identified CPVT-linked CASQ2 mutation were studied in isolated adult rat ventricular myocytes infected with adenoviruses for expression of either the wild-type (WT) or mutant forms of CASQ2. Cytosolic and intra-SR [Ca2+] changes were monitored using confocal microscopy, and whole cell currents were recorded with the patch-clamp technique. In vitro single-RyR2 channel recordings and CASQ2 Ca2+-binding measurements were performed.
An expanded Materials and Methods section can be found in the online data supplement available at http://circres.ahajournals.org.
Results
Identification of a Novel CPVT-Associated Mutation in CASQ2
The CASQ2 coding sequence from a patient diagnosed with CPVT revealed the presence of a previously unidentified sequence alteration (online data supplement). This alteration changed codon 33 of CASQ2 from CGA to CAA and resulted in the nonconservative substitution of glutamine for arginine (R33Q). This residue is located within a conserved region of CASQ2, and this position in the related CASQ1 protein is also occupied by arginine in all CASQ2 and CASQ1 sequences available in public databases (Figure I in the online data supplement). Analysis of the domain structures of CASQ2 and particularly CASQ1 suggest that this residue is located in a domain involved in protein–protein interactions that may participate in the formation of CASQ polymers or interactions with other components of the junctional complex.10,15,18 Interestingly, a different mutation in this codon was previously reported that resulted in a stop codon in place of the arginine residue,3 suggesting it may represent a relatively frequently mutated genomic location.
Electrophysiological Recordings and Intracellular Ca2+ Transients in Myocytes Expressing CASQ2R33Q
CASQ2 is a major intracellular Ca2+-binding protein that plays a key role in cardiac excitation–contraction (EC) coupling. To test whether the R33Q substitution in CASQ2 caused substantial changes in EC coupling and intracellular Ca2+ handling, we examined the effects of overexpressing the CASQ2R33Q protein on a series of electrophysiological and intracellular Ca2+-handling parameters in rat ventricular myocytes. In these experiments, cultured myocytes were infected with adenoviral vectors engineered to direct the expression of either human CASQ2WT or CASQ2R33Q. An adenovirus containing a nontranslatable fragment of CASQ2 sequence was used as an infection control. We have previously used this experimental strategy to characterize the effects of a different CPVT-associated CASQ2 mutant protein on myocyte function.16 In agreement with our earlier studies, immunoblot analysis revealed that this infection protocol resulted in a 3-fold increase in total CASQ2 protein levels in cells infected with either the CASQ2WT or CASQ2R33Q adenovirus, whereas the control virus did not affect CASQ2 levels (Figure 1). The increase in total CASQ2 abundance was caused by expression of the mutant protein because endogenous protein levels remained unchanged in CASQ2R33Q cells, as determined by an antibody that recognizes the rat but not the human form of CASQ2.
Initially, the effects of CASQ2R33Q expression on SR Ca2+ handling and release were tested. The total SR Ca2+ content of control myocytes, or myocytes expressing CASQ2WT or CASQ2R33Q, was assessed from the amplitude of the Ca2+ transients and the integral of Na/Ca2+ exchange current (INCX) evoked by the application of caffeine. Although, ectopic expression of the WT protein resulted in a dramatic increase in SR Ca2+ content, no statistically significant changes in SR Ca2+ content were observed with expression of CASQ2R33Q (Figure 2A and 2B).
Next, the effects of overexpression of CASQWT and CASQ2R33Q on Ca2+ release during EC coupling were compared in myocytes undergoing voltage clamp stimulation. The amplitude of the ICa-induced Ca2+ transients was similarly increased 30% in myocytes overexpressing both forms of CASQ2 (Figure 3 and supplemental Table I). However, whereas the duration of the rising phase of the Ca2+ transients was slowed in CASQ2WT-overexpressing cells, Ca2+ transient rise was accelerated in CASQ2R33Q-expressing myocytes (supplemental Table I). Additionally, expression of CASQ2WT and CASQ2R33Q had opposite effects on the gain of CICR (ie, Ca2+-release rate for a given Ca2+ trigger and a given SR Ca2+ content), a term that characterizes the efficiency of ICa to elicit Ca2+ release. Whereas overexpression of WT CASQ2 resulted in a decreased gain of CICR, expression of the mutant form of the protein increased CICR gain (Figure 3C, inset). Therefore, expression of CASQ2R33Q enhanced the functional activity of the Ca2+-release mechanism with respect to both control myocytes and myocytes overexpressing the WT form of the protein. Because the potentiating effects of R33Q on the Ca2+-release mechanism occurred on the background of a full set of native CASQ, they can be qualified as "dominant positive" effects. In general, these effects strongly suggest that the mutant protein disrupts protein–protein interactions involved in control of the SR Ca2+-release process.
Periodic Pacing
CPVT is associated with ventricular tachycardia, particularly in response to adrenergic stimulation, and thus we next examined whether CASQ2R33Q expression would affect electrical and intracellular Ca2+ signals in rhythmically paced cardiac myocytes. Periodic Ca2+ transients and action potentials (APs) were compared in myocytes overexpressing CASQ2WT and CASQ2R33Q undergoing rhythmic stimulation in the absence or presence of isoproterenol (ISO). In control and CASQ2WT-overexpressing myocytes, we observed stable, rhythmic Ca2+ transients and APs both in the absence and presence of ISO (1 μmol/L; 8 and 6 experiments, respectively; not shown). In the absence of ISO, CASQ2R33Q myocytes also showed only regular AP-induced Ca2+ transients. However, following exposure to 0.01 to 1 μmol/L ISO, these cells developed characteristic disturbances in Ca2+ release and electrical activity manifested as extrasystolic Ca2+ transients, delayed afterdepolarizations (DADs), and irregular APs (Figure 4 and supplemental Figure IV). The percentage of cells exhibiting such disturbances increased with increasing ISO concentration (from 30% at 0.01 μmol/L to a maximum of 80% at 0.2 to 1 μmol/L ISO).
Ca2+ Sparks and Waves
To further understand the effects of CASQ2R33Q on the Ca2+-release mechanism, we measured spontaneous local (sparks) and global (waves) Ca2+ signals in saponin-permeabilized myocytes maintained at a constant cytosolic [Ca2+] (75 nmol/L). Consistent with earlier results, overexpression of CASQ2WT resulted in an increase in the magnitude and slowing of the kinetics of Ca2+ sparks without significantly changing the frequency of events (Figure 5A through 5D and supplemental Table II). In contrast, although overexpression of CASQR33Q did not alter the amplitude of Ca2+ sparks, it did increase their frequency (Figure 5A through 5D). The kinetics of the local events in R33Q myocytes, however, did not change with respect to control cells (Figure 5 and supplemental Table II).
Next, the consequences of overexpression of CASQ2WT and CASQ2R33Q on the periodic occurrence of spontaneous Ca2+ waves were compared in myocytes incubated in a bathing solution containing 75 nmol/L Ca2+ and 100 μmol/L EGTA. Under these conditions, overexpression of CASQ2WT dramatically reduced wave frequency, whereas overexpression of CASQ2R33Q caused an increase in Ca2+ wave occurrence (Figure 5E and supplemental Table III). As with ICa-induced Ca2+ transients (see Figure 3 and supplemental Table I), the amplitude of spontaneous Ca2+ transients was increased in both CASQ2WT and CASQ2R33Q-overexpressing myocytes, and the kinetics of Ca2+ transients were slowed in CASQ2WT but accelerated in CASQ2R33Q cells (Figure 5E and supplemental Table III). Of note, expression of CASQ2R33Q also resulted in an increase in the frequency of Ca2+ sparks and waves in intact myocytes loaded with fluo-3 acetoxymethyl ester (fluo-3 AM) (supplemental Table VI and supplemental Figure II), indicating that the observed CASQ2R33Q-induced changes in Ca2+ signals were not attributable to myocyte permeabilization. These results suggest that expression of CASQ2R33Q enhances the propensity for spontaneous Ca2+ release from the SR, apparently by increasing the functional activity of the RyR2 channels.
Intra-SR [Ca2+]
Given the 2 potential functions of CASQ2 (ie, as a Ca2+-binding protein and as a modulator of the RyR2 channel), expression of the R33Q mutant could influence the total amount of Ca2+ stored in the SR by changing SR Ca2+ buffering and/or by affecting Ca2+ leak through RyR2s. To distinguish between these mechanisms, we performed measurements of free [Ca2+] inside the SR ([Ca2+]SR). The total resting SR luminal [Ca2+] is determined by both Ca2+ transport across the SR membrane and Ca2+ binding to luminal buffers. On the other hand, owing to the finite nature of the SR Ca2+ store, the steady-state free SR [Ca2+] is independent of the concentration of intra-SR Ca2+-binding sites and is solely governed by a balance between Ca2+ leak and Ca2+ uptake across the SR membrane. Therefore, potential changes in free basal [Ca2+]SR should provide good indications for altered RyR2 activity. [Ca2+]SR was monitored by the low-affinity Ca2+ indicator fluo-5N loaded into the SR. The cytosolic Ca2+ signal was recorded simultaneously using the Ca2+ dye rhod-2. In comparison with control cells, the basal [Ca2+]SR was significantly reduced in CASQ2R33Q myocytes, as evidenced by the reduced intensity of the SR-entrapped fluo-5N (Figure 6). Additionally, the amplitudes of the Ca2+-depletion signals during waves were diminished in CASQ2R33Q-expressing myocytes (Figure 6 and supplemental IV). Importantly, fluo-5N fluorescence in both cell types was similar after depletion of the SR Ca2+ store by caffeine (supplemental Table IV), indicating that the changes in fluo-5N fluorescence in Ca2+-loaded SR reflected true changes in [Ca2+]SR. The reduced [Ca2+]SR is CASQ2R33Q myocytes suggests that SR Ca2+ leak was enhanced in these cells, consistent with the increased frequency of spontaneous Ca2+ sparks. Similar to intact myocytes (Figure 2), the amplitude of the cytosolic spontaneous and caffeine-induced Ca2+ transients was preserved in permeabilized CASQ2R33Q myocytes despite the reduction of [Ca2+]SR (Figure 6B through 6D). The ability of the CASQ2R33Q myocytes to maintain the amplitude of their Ca2+ transients despite the reduced [Ca2+]SR is attributable to increased intrastore Ca2+ buffering (ie, an increased concentration of Ca2+-binding sites that can bind and release Ca2+ on discharge of the store) in myocytes overexpressing CASQ2R33Q. Collectively, these results suggest that CASQ2R33Q expression resulted in both increased leak of Ca2+ through the RyR2 and increased intra-SR Ca2+-buffering capacity.
Effects of CASQWT and CASQ2R33Q on Single-RyR2 Channel Activity
CASQ2 has been shown to inhibit the functional activity of the RyR2 channel complex.12 To directly examine the effect of the R33Q mutation on the ability of CASQ2 to influence RyR2 behavior, we performed single-RyR2 channel recordings using the planar lipid bilayer technique (Figure 7). Cardiac SR vesicles were incorporated into planar lipid bilayers, and the activity of single-RyR2 channels was measured using Cs+ as the charge carrier.12 In these experiments, single RyR2s were stripped of endogenous CASQ2 by exposing the luminal side of the channel to 5 mmol/L Ca2+. This treatment promoted efficient dissociation of CASQ2, as evidenced by the enhanced RyR2 activity that persisted after the [Ca2+] in the trans (luminal) chamber was reduced to the initial low level. Consistent with our previous studies,12 addition of CASQ2WT to the RyR2 complex resulted in a reduction in RyR2 activity. In contrast, addition of CASQ2R33Q to the RyR2 complex did not produce a similar inhibitory effect. Interestingly, subsequent addition of CASQ2WT (in the continuous presence of CASQ2R33Q) did not restore RyR2 open probability (Po) to that observed with CASQ2WT alone (supplemental Figure V). Therefore, the substitution of glutamine for arginine at amino acid 33 of CASQ2 appears to compromise the ability of the protein to modulate the functional activity of the RyR2 channel. Moreover, the R33Q mutant seems to impair the functional interactions of the WT protein with the RyR2 complex, consistent with the dominant positive effects of the mutant on SR Ca2+ release.
Determination of Ca2+-Binding Affinities of Recombinant CASQ2WT and CASQ2R33Q
In principle, the pathological effects of the R33Q mutation could be attributable to alterations in the Ca2+-binding properties of the mutant protein. We, therefore, tested whether the CASQ2R33Q protein displayed altered Ca2+-binding affinities when compared with CASQ2WT using Ca2+ overlay experiments.19 Two kinetic parameters of Ca2+ binding, the Ca2+ affinity (Kd) and capacity (Bmax), were calculated and are shown in supplemental Table V. Both values were comparable for the two proteins and were in agreement with previous values reported for native CASQ2WT.20 Thus the effects of the R33Q mutation in CASQ2 function appears to be unrelated to changes in Ca2+ binding.
Discussion
Genetic defects in the SR Ca2+-handling proteins RyR2 and CASQ2 have been linked to CPVT, a familial disease that predisposes young individuals with structurally normal hearts to sudden cardiac death. In this study, we report on a novel CPVT-linked mutation in CASQ2 that results in the nonconservative substitution of glutamine for arginine at amino acid 33. Using a combination of cellular and in vitro techniques, we demonstrate that ectopic expression of the mutant protein in cardiac myocytes increased the functional activity of the RyR2 channel, thereby increasing the rate of Ca2+ leak from the SR and enhancing the propensity of SR Ca2+ release to be spontaneously activated.
Molecular Mechanisms of R33Q
The potentiatory effects of CASQ2 on the Ca2+-release channels were evidenced by the following findings. Expression of CASQ2R33Q resulted in a shortening of the activation kinetics of Ca2+ transients, and increased CICR gain compared with control myocytes or myocytes overexpressing CASQ2WT. Additionally, the frequency of spontaneous Ca2+ sparks and waves were increased in myocytes expressing CASQ2R33Q. These changes in focal and global cytosolic Ca2+ transients were accompanied by a dramatic decrease in intra-SR [Ca2+], consistent with an increase in the leak of Ca2+ through RyR2s in CASQ2R33Q-expressing cells. The consequences of expressing CASQ2R33Q on Ca2+ handling were clearly different from the effects of expressing the CASQ2D307H mutant protein, the only other CPVT-linked CASQ2 mutation that has been characterized at the cellular and molecular level thus far.16,17 In those earlier studies, ectopic expression of CASQ2D307H in myocytes led to decreases in both active SR Ca2+ release and SR Ca2+ content.16,17 These effects were attributed to disruptions of the CASQ2 polymerization16 that is required for high-capacity Ca2+ binding, although in vitro binding studies also indicated that the mutant protein interacted more weakly with triadin and junctin.17
Several key pieces of experimental data from our study suggest that CASQ2R33Q exerts its effects by disrupting protein–protein interactions within the RyR2 complex rather than by compromising the Ca2+-binding capacity of CASQ2. The free [Ca2+] in the SR lumen at steady state is determined by the balance of Ca2+ leakage and uptake across the SR membrane and should not be influenced by the concentration of Ca2+-binding sites inside the SR. Therefore, the reduced [Ca2+]SR combined with the increased spark frequency observed in CASQ2R33Q-expressing myocytes strongly suggests that RyR2 activity was enhanced independent of any changes in the intra SR Ca2+-buffering capacity. Planar lipid bilayer experiments provided further evidence for altered interactions of CASQ2R33Q with the RyR2 channel complex. In this system, the inclusion of CASQ2WT decreases the open probability of RyR2 channels, presumably via interactions with triadin or junctin (present study and others12,21). However, the R33Q mutation abolished the ability of CASQ2 to inhibit RyR2 activity.
At the same time, the total SR Ca2+ content (judged from the size of caffeine-induced Ca2+ transients) was preserved in cells expressing CASQ2R33Q, indicating that the concentration of Ca2+-binding sites in the SR increased, as would be expected if the mutant protein maintained its Ca2+-binding function. Similarly, the mutation did not affect the ability of CASQ2 to bind Ca2+ in vitro. Thus, it appears that the R33Q mutation alters intracellular Ca2+ handling by compromising interactions of CASQ2 with the RyR2 complex without affecting CASQ2 Ca2+-binding function. Consistent with this conclusion, the N-terminal region of CASQ2, which contains a high proportion of negatively and positively charged amino acids, has been proposed to interact with KEKE motifs in triadin and/or junctin by forming "polar zippers."10,18
Implications for Pathophysiology of CPVT
Similar to other genetic forms of CPVT,16,22 the cellular mechanisms of arrhythmia caused by the R33Q mutation involved spontaneous discharges of the SR Ca2+ stores followed by DADs and extrasystolic action potentials (Figure 4). Spontaneous SR Ca2+ release in cardiac myocytes is commonly associated with increased SR Ca2+ load23–25 and stimulatory effects of high luminal [Ca2+] on the open probability of RyR2 channels.26 Our results indicated that in CASQ2R33Q-expressing myocytes the predisposition of SR to spontaneous discharges was increased because of enhanced responsiveness of the release mechanism to luminal Ca2+.
It is interesting to note that although expression of CASQ2R33Q produced clear changes in Ca2+ handling and electrical activity in myocytes expressing the full set endogenous CASQ2, CPVT does not develop in the heterozygous carriers of the R33Q mutation; in fact, none of the heterozygous carriers in the study developed ventricular arrhythmias. This lack of a clinical phenotype in the heterozygous carriers could be attributable to the lower ratio of CASQ2R33Q to the WT protein in these human subjects (presumably 1:1) when compared with our myocyte experiments (2:1), leading to less-profound changes in Ca2+ handling than in myocytes. In support of this notion, expression of the mutant protein at levels similar to those of the endogenous protein (ie, at a ratio of 1:1; supplemental Figure III) did not result in changes in Ca2+ handling observed with higher mutant expression. However, we note that our rat myocyte model can be taken as only an approximate representation of the results of mutant protein expression during human disease. Species-related differences in intracellular Ca2+ handling and membrane excitability, the likely presence of compensatory mechanisms in human disease but not during the acute myocyte experiments, and differences in adrenergic stimulation are only some of the factors that may complicate such a comparison.
Abnormal Modulation of RyR2 Channels by Luminal Ca2+ as a Common Mechanism for Various Genetic Forms of CPVT
To date, 4 mutations in the CASQ2 gene have been linked to CPVT.2,3 In addition, a number of mutations in the RyR2 gene have been reported to be associated with CPVT.27 Although the primary molecular alterations caused by the various genetic defects differ, they are likely to converge on a common pathogenic pathway to cause CPVT. Growing evidence indicates that abnormal modulation of RyR2 by luminal Ca2+ might be a common pathogenic factor in these genetically distinct forms of CPVT; however, clear proof of such a common mechanism is lacking. Mutations in CASQ2 that compromise either CASQ2 expression or its Ca2+-binding ability reportedly act on RyR2 indirectly by altering the dynamics of free Ca2+ in the vicinity of the channel, hence accelerating the channel recovery from a luminal Ca2+-dependent refractory state.8,16,28 The effects of CPVT-associated RyR2 mutations have been ascribed to either dissociation of FKBP12.6 from the RyR2 causing changes in RyR2 gating29 (but see George et al30) or, more recently, to changes in RyR2 sensitivity to luminal Ca2+.31,32 Our present findings clearly show that the R33Q mutation disrupts interactions of CASQ2 with the RyR2 complex, thereby sensitizing the release mechanism to activation by luminal Ca2+. We propose that CPVT can be caused by genetic defects in any component of the luminal Ca2+-signaling pathway, including steps involved in (1) controlling and sensing free Ca2+ in the vicinity of RyR2, (2) transmitting the luminal Ca2+ change signal to RyR2, and (3) RyR2-gating conformations. Our results strongly support a concept of abnormal luminal regulation as a common mechanism for genetically-distinct forms of CPVT.
Conclusions
In conclusion, our results show that substitution of glutamine for arginine at amino acid 33 of CASQ2 is a naturally occurring mutation that leads to CPVT in homozygous carriers. The underlying molecular mechanism of this mutation appears to involve disrupted interactions of CASQ2 with the proteins of the RyR2 Ca2+-release complex, resulting in enhanced sensitivity of the RyR2 channel to activation by luminal Ca2+. The enhanced responsiveness of RyR2s to luminal Ca2+ in turn leads to the generation of extrasystolic spontaneous Ca2+ transients, DADs, and arrhythmogenic action potentials in myocytes expressing CASQ2R33Q. These results show that intracellular Ca2+ cycling in the normal heart relies on an intricate interplay of CASQ2 with the proteins of the RyR2 channel complex and that disruption of these interactions can lead to cardiac arrhythmias.
Acknowledgments
This work was supported by NIH grants HL-74045 and HL-63043 and by Telethon, Italy grant no. GGP04066 to P.V. and S.G.P.
Footnotes
Original received December 20, 2005; resubmission received March 14, 2006; accepted March 24, 2006.
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the Department of Physiology and Cell Biology (D.T., S.V.-K., Z.K., I.G., R.T., S.V., S.G.), Heart and Lung Research Institute, Ohio State University, Columbus
Dipartimento di Scienze Biomediche Sperimentali dell’Università di Padova (A.N., G.V., P.V.), Istituto Interuniversitario di Miologia, Padova, Italy
Department of Molecular Cardiology (M.S., C.N., S.G.P.), Fondazione Salvatore Maugeri, IRCCS, Pavia, Italy
University Hospital Leiden (N.A.B), The Netherlands
Department of Cell Biology and Biochemistry (S.C.W.), Texas Tech University Health Science Center, Lubbock
Southwest Cancer Center at University Medical Center (S.C.W.), Lubbock, Texas.
Abstract
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic disorder associated with mutations in the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2) genes. Previous in vitro studies suggested that RyR2 and CASQ2 interact as parts of a multimolecular Ca2+-signaling complex; however, direct evidence for such interactions and their potential significance to myocardial function remain to be determined. We identified a novel CASQ2 mutation in a young female with a structurally normal heart and unexplained syncopal episodes. This mutation results in the nonconservative substitution of glutamine for arginine at amino acid 33 of CASQ2 (R33Q). Adenoviral-mediated expression of CASQ2R33Q in adult rat myocytes led to an increase in excitation–contraction coupling gain and to more frequent occurrences of spontaneous propagating (Ca2+ waves) and local Ca2+ signals (sparks) with respect to control cells expressing wild-type CASQ2 (CASQ2WT). As revealed by a Ca2+ indicator entrapped inside the sarcoplasmic reticulum (SR) of permeabilized myocytes, the increased occurrence of spontaneous Ca2+ sparks and waves was associated with a dramatic decrease in intra-SR [Ca2+]. Recombinant CASQ2WT and CASQ2R33Q exhibited similar Ca2+-binding capacities in vitro; however, the mutant protein lacked the ability of its WT counterpart to inhibit RyR2 activity at low luminal [Ca2+] in planar lipid bilayers. We conclude that the R33Q mutation disrupts interactions of CASQ2 with the RyR2 channel complex and impairs regulation of RyR2 by luminal Ca2+. These results show that intracellular Ca2+ cycling in normal heart relies on an intricate interplay of CASQ2 with the proteins of the RyR2 channel complex and that disruption of these interactions can lead to cardiac arrhythmia.
Key Words: calsequestrin ryanodine receptor sarcoplasmic reticulum Ca2+-induced Ca2+ release catecholaminergic polymorphic ventricular tachycardia
Introduction
Catecholaminergic polymorphic ventricular tachycardia (CPVT) (Online Mendelian Inheritance in Man no. 604772) is a familial arrhythmogenic disorder characterized by adrenergically mediated polymorphic ventricular tachyarrhythmias, leading to syncope and sudden cardiac death in individuals with structurally normal hearts.1 The episodes of tachyarrhythmia are typically triggered by physical exercise or emotional stress. Two genetic variants of the disease have been described: a recessive form associated with homozygous mutations in the gene encoding the cardiac isoform of calsequestrin (CASQ2)2,3 and a second form transmitted as an autosomal dominant trait associated with mutations in the gene encoding the cardiac ryanodine receptor (RyR2).4,5
The contractile machinery of cardiac myocytes becomes activated when Ca2+ enters the sarcoplasmic reticulum (SR) via L-type Ca2+ channels and triggers a process termed Ca2+-induced Ca2+ release (CICR) from the SR.6 Whereas CICR controls the release process from the cytosolic side, a second Ca2+-dependent mechanism controls the activity of the Ca2+-release channels from the SR lumen. Specifically, the decline of intra-SR [Ca2+] that accompanies the Ca2+-release process contributes to Ca2+-release termination, a mechanism referred to as luminal Ca2+-dependent deactivation.7–9 The Ca2+-release channel is present in the junctional SR membrane in the form of a quaternary complex composed of RyR2, triadin, junctin, and CASQ2.10,11 The integral membrane proteins triadin and junctin physically interact with RyR2 and link the Ca2+-binding protein CASQ2 to the complex. Ca2+-dependent interactions of CASQ2 with the RyR2–triadin complex are thought to provide a molecular basis for regulation of RyR2 channel by luminal Ca2+.12,13 In addition, CASQ2 monomers can form polymers with high Ca2+-binding capacities that are essential for the Ca2+ storage function of the SR.14,15
To date, 4 homozygous sequence variations in the CASQ2 gene have been identified in patients with CPVT (see Inherited Arrhythmias Database at http://pc4.fsm.it:81/cardmoc).2,3 The precise molecular basis for the alterations in Ca2+ handling in cells expressing CPVT-linked CASQ2 mutants remains to be determined. To date, the effect of only 1 of these mutations on CASQ2 activity and function has been examined.16,17 These studies focused on a CASQ2 mutant protein in which aspartate 307 is changed to histidine (CASQ2D307H) and suggested that the CASQ2D307H protein is compromised in its ability to facilitate the Ca2+ storing and releasing functions of the SR. These effects may be a consequence of a reduction in the Ca2+-binding capacity of the mutant protein or altered interactions between CASQ2D307H and components of the RyR2 channel complex.16,17 Furthermore, it is unknown how independent mutations in the CASQ2 and RyR2 genes result in similar clinical manifestations in CPVT. In the present study, we report the identification of a new mutation in the CASQ2 gene in a patient with CPVT. We also demonstrate that this mutation alters the functional interactions between CASQ2 and the RyR2 channel complex, resulting in abnormal luminal Ca2+-dependent regulation of the RyR2 channel.
Materials and Methods
CPVT in human patients was diagnosed using standard cardiologic tests. Genetic analyses of the CPVT patients were performed using a combination of methods of PCR, single-strand conformation polymorphism (SSCP) analysis, and denaturing high-performance liquid chromatography (DHPLC) (Wave Transgenomics). The cellular effects of the newly identified CPVT-linked CASQ2 mutation were studied in isolated adult rat ventricular myocytes infected with adenoviruses for expression of either the wild-type (WT) or mutant forms of CASQ2. Cytosolic and intra-SR [Ca2+] changes were monitored using confocal microscopy, and whole cell currents were recorded with the patch-clamp technique. In vitro single-RyR2 channel recordings and CASQ2 Ca2+-binding measurements were performed.
An expanded Materials and Methods section can be found in the online data supplement available at http://circres.ahajournals.org.
Results
Identification of a Novel CPVT-Associated Mutation in CASQ2
The CASQ2 coding sequence from a patient diagnosed with CPVT revealed the presence of a previously unidentified sequence alteration (online data supplement). This alteration changed codon 33 of CASQ2 from CGA to CAA and resulted in the nonconservative substitution of glutamine for arginine (R33Q). This residue is located within a conserved region of CASQ2, and this position in the related CASQ1 protein is also occupied by arginine in all CASQ2 and CASQ1 sequences available in public databases (Figure I in the online data supplement). Analysis of the domain structures of CASQ2 and particularly CASQ1 suggest that this residue is located in a domain involved in protein–protein interactions that may participate in the formation of CASQ polymers or interactions with other components of the junctional complex.10,15,18 Interestingly, a different mutation in this codon was previously reported that resulted in a stop codon in place of the arginine residue,3 suggesting it may represent a relatively frequently mutated genomic location.
Electrophysiological Recordings and Intracellular Ca2+ Transients in Myocytes Expressing CASQ2R33Q
CASQ2 is a major intracellular Ca2+-binding protein that plays a key role in cardiac excitation–contraction (EC) coupling. To test whether the R33Q substitution in CASQ2 caused substantial changes in EC coupling and intracellular Ca2+ handling, we examined the effects of overexpressing the CASQ2R33Q protein on a series of electrophysiological and intracellular Ca2+-handling parameters in rat ventricular myocytes. In these experiments, cultured myocytes were infected with adenoviral vectors engineered to direct the expression of either human CASQ2WT or CASQ2R33Q. An adenovirus containing a nontranslatable fragment of CASQ2 sequence was used as an infection control. We have previously used this experimental strategy to characterize the effects of a different CPVT-associated CASQ2 mutant protein on myocyte function.16 In agreement with our earlier studies, immunoblot analysis revealed that this infection protocol resulted in a 3-fold increase in total CASQ2 protein levels in cells infected with either the CASQ2WT or CASQ2R33Q adenovirus, whereas the control virus did not affect CASQ2 levels (Figure 1). The increase in total CASQ2 abundance was caused by expression of the mutant protein because endogenous protein levels remained unchanged in CASQ2R33Q cells, as determined by an antibody that recognizes the rat but not the human form of CASQ2.
Initially, the effects of CASQ2R33Q expression on SR Ca2+ handling and release were tested. The total SR Ca2+ content of control myocytes, or myocytes expressing CASQ2WT or CASQ2R33Q, was assessed from the amplitude of the Ca2+ transients and the integral of Na/Ca2+ exchange current (INCX) evoked by the application of caffeine. Although, ectopic expression of the WT protein resulted in a dramatic increase in SR Ca2+ content, no statistically significant changes in SR Ca2+ content were observed with expression of CASQ2R33Q (Figure 2A and 2B).
Next, the effects of overexpression of CASQWT and CASQ2R33Q on Ca2+ release during EC coupling were compared in myocytes undergoing voltage clamp stimulation. The amplitude of the ICa-induced Ca2+ transients was similarly increased 30% in myocytes overexpressing both forms of CASQ2 (Figure 3 and supplemental Table I). However, whereas the duration of the rising phase of the Ca2+ transients was slowed in CASQ2WT-overexpressing cells, Ca2+ transient rise was accelerated in CASQ2R33Q-expressing myocytes (supplemental Table I). Additionally, expression of CASQ2WT and CASQ2R33Q had opposite effects on the gain of CICR (ie, Ca2+-release rate for a given Ca2+ trigger and a given SR Ca2+ content), a term that characterizes the efficiency of ICa to elicit Ca2+ release. Whereas overexpression of WT CASQ2 resulted in a decreased gain of CICR, expression of the mutant form of the protein increased CICR gain (Figure 3C, inset). Therefore, expression of CASQ2R33Q enhanced the functional activity of the Ca2+-release mechanism with respect to both control myocytes and myocytes overexpressing the WT form of the protein. Because the potentiating effects of R33Q on the Ca2+-release mechanism occurred on the background of a full set of native CASQ, they can be qualified as "dominant positive" effects. In general, these effects strongly suggest that the mutant protein disrupts protein–protein interactions involved in control of the SR Ca2+-release process.
Periodic Pacing
CPVT is associated with ventricular tachycardia, particularly in response to adrenergic stimulation, and thus we next examined whether CASQ2R33Q expression would affect electrical and intracellular Ca2+ signals in rhythmically paced cardiac myocytes. Periodic Ca2+ transients and action potentials (APs) were compared in myocytes overexpressing CASQ2WT and CASQ2R33Q undergoing rhythmic stimulation in the absence or presence of isoproterenol (ISO). In control and CASQ2WT-overexpressing myocytes, we observed stable, rhythmic Ca2+ transients and APs both in the absence and presence of ISO (1 μmol/L; 8 and 6 experiments, respectively; not shown). In the absence of ISO, CASQ2R33Q myocytes also showed only regular AP-induced Ca2+ transients. However, following exposure to 0.01 to 1 μmol/L ISO, these cells developed characteristic disturbances in Ca2+ release and electrical activity manifested as extrasystolic Ca2+ transients, delayed afterdepolarizations (DADs), and irregular APs (Figure 4 and supplemental Figure IV). The percentage of cells exhibiting such disturbances increased with increasing ISO concentration (from 30% at 0.01 μmol/L to a maximum of 80% at 0.2 to 1 μmol/L ISO).
Ca2+ Sparks and Waves
To further understand the effects of CASQ2R33Q on the Ca2+-release mechanism, we measured spontaneous local (sparks) and global (waves) Ca2+ signals in saponin-permeabilized myocytes maintained at a constant cytosolic [Ca2+] (75 nmol/L). Consistent with earlier results, overexpression of CASQ2WT resulted in an increase in the magnitude and slowing of the kinetics of Ca2+ sparks without significantly changing the frequency of events (Figure 5A through 5D and supplemental Table II). In contrast, although overexpression of CASQR33Q did not alter the amplitude of Ca2+ sparks, it did increase their frequency (Figure 5A through 5D). The kinetics of the local events in R33Q myocytes, however, did not change with respect to control cells (Figure 5 and supplemental Table II).
Next, the consequences of overexpression of CASQ2WT and CASQ2R33Q on the periodic occurrence of spontaneous Ca2+ waves were compared in myocytes incubated in a bathing solution containing 75 nmol/L Ca2+ and 100 μmol/L EGTA. Under these conditions, overexpression of CASQ2WT dramatically reduced wave frequency, whereas overexpression of CASQ2R33Q caused an increase in Ca2+ wave occurrence (Figure 5E and supplemental Table III). As with ICa-induced Ca2+ transients (see Figure 3 and supplemental Table I), the amplitude of spontaneous Ca2+ transients was increased in both CASQ2WT and CASQ2R33Q-overexpressing myocytes, and the kinetics of Ca2+ transients were slowed in CASQ2WT but accelerated in CASQ2R33Q cells (Figure 5E and supplemental Table III). Of note, expression of CASQ2R33Q also resulted in an increase in the frequency of Ca2+ sparks and waves in intact myocytes loaded with fluo-3 acetoxymethyl ester (fluo-3 AM) (supplemental Table VI and supplemental Figure II), indicating that the observed CASQ2R33Q-induced changes in Ca2+ signals were not attributable to myocyte permeabilization. These results suggest that expression of CASQ2R33Q enhances the propensity for spontaneous Ca2+ release from the SR, apparently by increasing the functional activity of the RyR2 channels.
Intra-SR [Ca2+]
Given the 2 potential functions of CASQ2 (ie, as a Ca2+-binding protein and as a modulator of the RyR2 channel), expression of the R33Q mutant could influence the total amount of Ca2+ stored in the SR by changing SR Ca2+ buffering and/or by affecting Ca2+ leak through RyR2s. To distinguish between these mechanisms, we performed measurements of free [Ca2+] inside the SR ([Ca2+]SR). The total resting SR luminal [Ca2+] is determined by both Ca2+ transport across the SR membrane and Ca2+ binding to luminal buffers. On the other hand, owing to the finite nature of the SR Ca2+ store, the steady-state free SR [Ca2+] is independent of the concentration of intra-SR Ca2+-binding sites and is solely governed by a balance between Ca2+ leak and Ca2+ uptake across the SR membrane. Therefore, potential changes in free basal [Ca2+]SR should provide good indications for altered RyR2 activity. [Ca2+]SR was monitored by the low-affinity Ca2+ indicator fluo-5N loaded into the SR. The cytosolic Ca2+ signal was recorded simultaneously using the Ca2+ dye rhod-2. In comparison with control cells, the basal [Ca2+]SR was significantly reduced in CASQ2R33Q myocytes, as evidenced by the reduced intensity of the SR-entrapped fluo-5N (Figure 6). Additionally, the amplitudes of the Ca2+-depletion signals during waves were diminished in CASQ2R33Q-expressing myocytes (Figure 6 and supplemental IV). Importantly, fluo-5N fluorescence in both cell types was similar after depletion of the SR Ca2+ store by caffeine (supplemental Table IV), indicating that the changes in fluo-5N fluorescence in Ca2+-loaded SR reflected true changes in [Ca2+]SR. The reduced [Ca2+]SR is CASQ2R33Q myocytes suggests that SR Ca2+ leak was enhanced in these cells, consistent with the increased frequency of spontaneous Ca2+ sparks. Similar to intact myocytes (Figure 2), the amplitude of the cytosolic spontaneous and caffeine-induced Ca2+ transients was preserved in permeabilized CASQ2R33Q myocytes despite the reduction of [Ca2+]SR (Figure 6B through 6D). The ability of the CASQ2R33Q myocytes to maintain the amplitude of their Ca2+ transients despite the reduced [Ca2+]SR is attributable to increased intrastore Ca2+ buffering (ie, an increased concentration of Ca2+-binding sites that can bind and release Ca2+ on discharge of the store) in myocytes overexpressing CASQ2R33Q. Collectively, these results suggest that CASQ2R33Q expression resulted in both increased leak of Ca2+ through the RyR2 and increased intra-SR Ca2+-buffering capacity.
Effects of CASQWT and CASQ2R33Q on Single-RyR2 Channel Activity
CASQ2 has been shown to inhibit the functional activity of the RyR2 channel complex.12 To directly examine the effect of the R33Q mutation on the ability of CASQ2 to influence RyR2 behavior, we performed single-RyR2 channel recordings using the planar lipid bilayer technique (Figure 7). Cardiac SR vesicles were incorporated into planar lipid bilayers, and the activity of single-RyR2 channels was measured using Cs+ as the charge carrier.12 In these experiments, single RyR2s were stripped of endogenous CASQ2 by exposing the luminal side of the channel to 5 mmol/L Ca2+. This treatment promoted efficient dissociation of CASQ2, as evidenced by the enhanced RyR2 activity that persisted after the [Ca2+] in the trans (luminal) chamber was reduced to the initial low level. Consistent with our previous studies,12 addition of CASQ2WT to the RyR2 complex resulted in a reduction in RyR2 activity. In contrast, addition of CASQ2R33Q to the RyR2 complex did not produce a similar inhibitory effect. Interestingly, subsequent addition of CASQ2WT (in the continuous presence of CASQ2R33Q) did not restore RyR2 open probability (Po) to that observed with CASQ2WT alone (supplemental Figure V). Therefore, the substitution of glutamine for arginine at amino acid 33 of CASQ2 appears to compromise the ability of the protein to modulate the functional activity of the RyR2 channel. Moreover, the R33Q mutant seems to impair the functional interactions of the WT protein with the RyR2 complex, consistent with the dominant positive effects of the mutant on SR Ca2+ release.
Determination of Ca2+-Binding Affinities of Recombinant CASQ2WT and CASQ2R33Q
In principle, the pathological effects of the R33Q mutation could be attributable to alterations in the Ca2+-binding properties of the mutant protein. We, therefore, tested whether the CASQ2R33Q protein displayed altered Ca2+-binding affinities when compared with CASQ2WT using Ca2+ overlay experiments.19 Two kinetic parameters of Ca2+ binding, the Ca2+ affinity (Kd) and capacity (Bmax), were calculated and are shown in supplemental Table V. Both values were comparable for the two proteins and were in agreement with previous values reported for native CASQ2WT.20 Thus the effects of the R33Q mutation in CASQ2 function appears to be unrelated to changes in Ca2+ binding.
Discussion
Genetic defects in the SR Ca2+-handling proteins RyR2 and CASQ2 have been linked to CPVT, a familial disease that predisposes young individuals with structurally normal hearts to sudden cardiac death. In this study, we report on a novel CPVT-linked mutation in CASQ2 that results in the nonconservative substitution of glutamine for arginine at amino acid 33. Using a combination of cellular and in vitro techniques, we demonstrate that ectopic expression of the mutant protein in cardiac myocytes increased the functional activity of the RyR2 channel, thereby increasing the rate of Ca2+ leak from the SR and enhancing the propensity of SR Ca2+ release to be spontaneously activated.
Molecular Mechanisms of R33Q
The potentiatory effects of CASQ2 on the Ca2+-release channels were evidenced by the following findings. Expression of CASQ2R33Q resulted in a shortening of the activation kinetics of Ca2+ transients, and increased CICR gain compared with control myocytes or myocytes overexpressing CASQ2WT. Additionally, the frequency of spontaneous Ca2+ sparks and waves were increased in myocytes expressing CASQ2R33Q. These changes in focal and global cytosolic Ca2+ transients were accompanied by a dramatic decrease in intra-SR [Ca2+], consistent with an increase in the leak of Ca2+ through RyR2s in CASQ2R33Q-expressing cells. The consequences of expressing CASQ2R33Q on Ca2+ handling were clearly different from the effects of expressing the CASQ2D307H mutant protein, the only other CPVT-linked CASQ2 mutation that has been characterized at the cellular and molecular level thus far.16,17 In those earlier studies, ectopic expression of CASQ2D307H in myocytes led to decreases in both active SR Ca2+ release and SR Ca2+ content.16,17 These effects were attributed to disruptions of the CASQ2 polymerization16 that is required for high-capacity Ca2+ binding, although in vitro binding studies also indicated that the mutant protein interacted more weakly with triadin and junctin.17
Several key pieces of experimental data from our study suggest that CASQ2R33Q exerts its effects by disrupting protein–protein interactions within the RyR2 complex rather than by compromising the Ca2+-binding capacity of CASQ2. The free [Ca2+] in the SR lumen at steady state is determined by the balance of Ca2+ leakage and uptake across the SR membrane and should not be influenced by the concentration of Ca2+-binding sites inside the SR. Therefore, the reduced [Ca2+]SR combined with the increased spark frequency observed in CASQ2R33Q-expressing myocytes strongly suggests that RyR2 activity was enhanced independent of any changes in the intra SR Ca2+-buffering capacity. Planar lipid bilayer experiments provided further evidence for altered interactions of CASQ2R33Q with the RyR2 channel complex. In this system, the inclusion of CASQ2WT decreases the open probability of RyR2 channels, presumably via interactions with triadin or junctin (present study and others12,21). However, the R33Q mutation abolished the ability of CASQ2 to inhibit RyR2 activity.
At the same time, the total SR Ca2+ content (judged from the size of caffeine-induced Ca2+ transients) was preserved in cells expressing CASQ2R33Q, indicating that the concentration of Ca2+-binding sites in the SR increased, as would be expected if the mutant protein maintained its Ca2+-binding function. Similarly, the mutation did not affect the ability of CASQ2 to bind Ca2+ in vitro. Thus, it appears that the R33Q mutation alters intracellular Ca2+ handling by compromising interactions of CASQ2 with the RyR2 complex without affecting CASQ2 Ca2+-binding function. Consistent with this conclusion, the N-terminal region of CASQ2, which contains a high proportion of negatively and positively charged amino acids, has been proposed to interact with KEKE motifs in triadin and/or junctin by forming "polar zippers."10,18
Implications for Pathophysiology of CPVT
Similar to other genetic forms of CPVT,16,22 the cellular mechanisms of arrhythmia caused by the R33Q mutation involved spontaneous discharges of the SR Ca2+ stores followed by DADs and extrasystolic action potentials (Figure 4). Spontaneous SR Ca2+ release in cardiac myocytes is commonly associated with increased SR Ca2+ load23–25 and stimulatory effects of high luminal [Ca2+] on the open probability of RyR2 channels.26 Our results indicated that in CASQ2R33Q-expressing myocytes the predisposition of SR to spontaneous discharges was increased because of enhanced responsiveness of the release mechanism to luminal Ca2+.
It is interesting to note that although expression of CASQ2R33Q produced clear changes in Ca2+ handling and electrical activity in myocytes expressing the full set endogenous CASQ2, CPVT does not develop in the heterozygous carriers of the R33Q mutation; in fact, none of the heterozygous carriers in the study developed ventricular arrhythmias. This lack of a clinical phenotype in the heterozygous carriers could be attributable to the lower ratio of CASQ2R33Q to the WT protein in these human subjects (presumably 1:1) when compared with our myocyte experiments (2:1), leading to less-profound changes in Ca2+ handling than in myocytes. In support of this notion, expression of the mutant protein at levels similar to those of the endogenous protein (ie, at a ratio of 1:1; supplemental Figure III) did not result in changes in Ca2+ handling observed with higher mutant expression. However, we note that our rat myocyte model can be taken as only an approximate representation of the results of mutant protein expression during human disease. Species-related differences in intracellular Ca2+ handling and membrane excitability, the likely presence of compensatory mechanisms in human disease but not during the acute myocyte experiments, and differences in adrenergic stimulation are only some of the factors that may complicate such a comparison.
Abnormal Modulation of RyR2 Channels by Luminal Ca2+ as a Common Mechanism for Various Genetic Forms of CPVT
To date, 4 mutations in the CASQ2 gene have been linked to CPVT.2,3 In addition, a number of mutations in the RyR2 gene have been reported to be associated with CPVT.27 Although the primary molecular alterations caused by the various genetic defects differ, they are likely to converge on a common pathogenic pathway to cause CPVT. Growing evidence indicates that abnormal modulation of RyR2 by luminal Ca2+ might be a common pathogenic factor in these genetically distinct forms of CPVT; however, clear proof of such a common mechanism is lacking. Mutations in CASQ2 that compromise either CASQ2 expression or its Ca2+-binding ability reportedly act on RyR2 indirectly by altering the dynamics of free Ca2+ in the vicinity of the channel, hence accelerating the channel recovery from a luminal Ca2+-dependent refractory state.8,16,28 The effects of CPVT-associated RyR2 mutations have been ascribed to either dissociation of FKBP12.6 from the RyR2 causing changes in RyR2 gating29 (but see George et al30) or, more recently, to changes in RyR2 sensitivity to luminal Ca2+.31,32 Our present findings clearly show that the R33Q mutation disrupts interactions of CASQ2 with the RyR2 complex, thereby sensitizing the release mechanism to activation by luminal Ca2+. We propose that CPVT can be caused by genetic defects in any component of the luminal Ca2+-signaling pathway, including steps involved in (1) controlling and sensing free Ca2+ in the vicinity of RyR2, (2) transmitting the luminal Ca2+ change signal to RyR2, and (3) RyR2-gating conformations. Our results strongly support a concept of abnormal luminal regulation as a common mechanism for genetically-distinct forms of CPVT.
Conclusions
In conclusion, our results show that substitution of glutamine for arginine at amino acid 33 of CASQ2 is a naturally occurring mutation that leads to CPVT in homozygous carriers. The underlying molecular mechanism of this mutation appears to involve disrupted interactions of CASQ2 with the proteins of the RyR2 Ca2+-release complex, resulting in enhanced sensitivity of the RyR2 channel to activation by luminal Ca2+. The enhanced responsiveness of RyR2s to luminal Ca2+ in turn leads to the generation of extrasystolic spontaneous Ca2+ transients, DADs, and arrhythmogenic action potentials in myocytes expressing CASQ2R33Q. These results show that intracellular Ca2+ cycling in the normal heart relies on an intricate interplay of CASQ2 with the proteins of the RyR2 channel complex and that disruption of these interactions can lead to cardiac arrhythmias.
Acknowledgments
This work was supported by NIH grants HL-74045 and HL-63043 and by Telethon, Italy grant no. GGP04066 to P.V. and S.G.P.
Footnotes
Original received December 20, 2005; resubmission received March 14, 2006; accepted March 24, 2006.
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