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(Circulation Research. 2007;100:e22.)
© 2007 American Heart Association, Inc.

UltraRapid Communications

Mechanisms of Abnormal Calcium Homeostasis in Mutations Responsible for Catecholaminergic Polymorphic Ventricular Tachycardia

Vivek Iyer, Roger J. Hajjar, Antonis A. Armoundas

From the Cardiovascular Research Center, Massachusetts General Hospital, Charlestown.

Correspondence to Antonis A. Armoundas, PhD, Cardiovascular Research Center, Massachusetts General Hospital, 149 13th St, Charlestown, MA 02129. E-mail aarmoundas{at}partners.org

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Catecholaminergic polymorphic ventricular tachycardia is a heritable arrhythmia unmasked by exertion or stress and is characterized by triggered activity and sudden cardiac death. In this study, we simulated mutations in 2 genes linked to catecholaminergic polymorphic ventricular tachycardia, the first located in calsequestrin (CSQN2) and the second in the ryanodine receptor (RyR2). The aim of the study was to investigate the mechanistic basis for spontaneous Ca²⁺ release events that lead to delayed afterdepolarizations in affected patients. Sarcoplasmic reticulum (SR) luminal Ca²⁺ sensing was incorporated into a model of the human ventricular myocyte, and CSQN2 mutations were modeled by simulating disrupted RyR2 luminal Ca²⁺ sensing. In voltage-clamp mode, the mutant CSQN2 model recapitulated the smaller calcium transients, smaller time to peak calcium transient, and accelerated recovery from inactivation seen in experiments. In current clamp mode, in the presence of ß stimulation, we observed delayed afterdepolarizations, suggesting that accelerated recovery of RyR2 induced by impaired luminal Ca²⁺ sensing underlies the triggered activity observed in mutant CSQN2-expressing myocytes. In current-clamp mode, in a model of mutant RyR2 that is characterized by reduced FKBP12.6 binding to the RyR2 on ß stimulation, the impaired coupled gating characteristic of these mutations was modeled by reducing cooperativity of RyR2 activation. In current-clamp mode, the mutant RyR2 model exhibited increased diastolic RyR2 open probability that resulted in formation of delayed afterdepolarizations. In conclusion, these minimal order models of mutant CSQN2 and RyR2 provide plausible mechanisms by which defects in RyR2 gating may lead to the cellular triggers for arrhythmia, with implications for the development of targeted therapy.

Key Words: catecholaminergic polymorphic ventricular tachycardia • delayed afterdepolarization • ryanodine receptor • calsequestrin • calcium handling • simulation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a heritable arrhythmia unmasked by exertion or stress. Mutations in the cardiac ryanodine receptor 2 (RyR2, gene RYR2)^1–3 and the calcium buffer calsequestrin 2 (CSQN2, gene CASQ2)^4,5 have been reported in families affected by the disorder. More than 40 mutations have been identified thus far, with many additional mutants constantly being added to the spectrum of disease-linked gene products.^6–8

The rate of discovery of newly identified CPVT mutations has outpaced the experimental characterization of the function of the mutant gene products. Indeed, much controversy still exists regarding the cellular mechanisms by which these mutations cause arrhythmias. Hypotheses have been advanced on the basis of recent experimental characterizations in transgenic animal models,⁹ in ventricular myocytes,^10,11 and in lipid bilayers.¹² Evidence suggests that the RyR2 exists in a supramolecular release complex along with CSQN2 at junctional release sites.¹³ Mutations linked to CPVT are likely responsible for disrupted regulation of Ca²⁺ release from the sarcoplasmic reticulum (SR) through the RyR2, resulting in aberrant diastolic openings and diastolic oscillations in membrane potential (delayed afterdepolarizations [DADs]).

CSQN2 mutations responsible for CPVT are inherited in an autosomal dominant fashion, although an autosomal recessive mode has also been suggested.⁵ Studies have suggested that CSQN2 may function as a luminal Ca²⁺"sensor" of the RyR2 channel.^14,15 Experiments in ventricular myocytes expressing mutant D307H CSQN2¹¹ and under- and overexpressing CSQN2¹⁶ have suggested that impaired luminal Ca²⁺ sensing in CPVT promotes the spontaneous SR release events and DADs, which may underlie the ventricular tachyarrhythmias characteristic of the disease. However, the precise causative defects in RyR2 gating have not been clearly established.

Mutations in RyR2 cause an autosomal dominant form of CPVT. Analysis of the biophysical properties of the mutant RyR2 channels show that under nonstimulated, resting conditions, CPVT-mutant RyR2 channels are indistinguishable from normal (wild-type) channels.¹⁷ However, several CPVT-linked mutant RyR2 channels display abnormal single-channel function following phosphorylation by protein kinase A.¹² Although not universally demonstrated in all mutants,^8,18 mutant RyR2 channels also have been shown to exhibit decreased affinity for the channel-stabilizing molecule FKB12.6 (calstabin 2) compared with wild-type channels.^9,12 The function of FKBP12.6 in vivo is likely to both structurally and functionally couple adjacent RyR2 subunits.¹⁹ One hypothesis that has been advanced is that the disruption of FKBP12.6–RyR2 interaction is responsible for hyperactive "leaky" release receptors, which promote DADs.^9,12,20

At present, the precise mechanistic basis for the genesis of DADs in CSQN2 and RyR2 mutations has not been determined. We therefore used a detailed model of excitation-contraction coupling²¹ to investigate the hypothesis that impaired luminal Ca²⁺ sensing in CPVT promotes the spontaneous SR release underlying the DAD formation in this disease. Specifically, the aims of this study were (1) to evaluate the restitution properties of the RyR2 in CSQN2 mutations, (2) to examine the role of impaired FKBP12.6 binding in RyR2 mutations, and (3) to examine the role of each of these mechanisms in generating DADs at the whole-cell level under conditions that simulate stress.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
We developed models for CSQN2 and RyR2 mutations that were comprehensive, yet relatively simple to understand. These models involved minimum order alterations of the normal model, yet recapitulated the known experimental behavior of the mutants. All simulations were performed using a recently described biophysically detailed model of the human left ventricular myocyte.²¹

Briefly, the whole-cell model is composed of a system of 67 coupled nonlinear differential equations that are integrated numerically. Numerical integration was performed using the Livermore Solver for Ordinary Differential Equations integration package,²² with 10^–4 relative error tolerance. Use of these numerical methods ensures convergence of model equations and allows simulation of cell electrophysiological responses in real time.²¹

The RyR2 Model
The RyR2 channel is represented using a model developed by Keizer and Levine.²³ This model was developed to replicate open and dwell times of isolated RyR2 channels in vitro and in vivo, as well as measured peak and plateau open probabilities with Ca²⁺ or cesium (Cs²⁺) as the charge carrier. The latter measurements describe the adaptive behavior of the RyR2 channel. As originally described, adaptation is a property of the RyR2 in which, after rapid activation by a step increase in Ca²⁺, the channel undergoes a slow spontaneous decrease in open probability.²⁴ Closing of the RyR has also been attributed to inactivation.²⁵ In isolated bilayers, adaptation occurs within milliseconds, whereas inactivation occurs within a few seconds.²³

A state diagram of the RyR2 model is shown in Figure 1. This model has 2 open states (O1 and O2) and 2 closed states (C1 and C2). At rest, the channel resides primarily in the first closed state, C1. On an increase in Ca²⁺, the channel switches briefly to the first open state, O1, allowing Ca²⁺ to move through the channel, before it adapts by its transition to C2. On additional increases in Ca²⁺, the channel reopens by its transition to state O2, displaying the adaptive behavior seen experimentally.²⁴ In the original model,²⁶ the charge carrier could be Ca²⁺ or Cs²⁺, so that some transition rates depended on Ca²⁺ in the subspace around the channel, whereas others depended on the bulk cytosolic Ca²⁺. In the present model, the charge carrier is Ca²⁺, so all rates depend on the Ca²⁺ in the subspace.

View larger version (11K): [in this window] [in a new window]   Figure 1. Schematic of gating structure for the RyR2 receptor. Channels are initially in closed state C1 and open by gating to conducting state O1. Adaptation is incorporated through closure of channels into state C2, from which the receptor recovers into state O1 and state O2. Addition of luminal Ca2+ dependence is reflected by parameter klumen in the C1O1 and O1 C2 transitions (shown in red).

Furthermore, the Keizer–Levine model assumes that the RyR2 can be exposed to peak [Ca²⁺]_i values of 1.0 µmol/L²⁶; in the present model, the RyR2 is located in the subspace (SS), where it is exposed to [Ca²⁺]_ss in excess of 10.0 µmol/L^27–30; therefore, the rates have been modified such that they are scaled to depend on [Ca²⁺]_ss. In addition, because [Ca²⁺]_ss levels change more rapidly than [Ca²⁺]_i, resulting in saturating functions of the Ca²⁺ following Michaelis–Menten kinetics of the Keizer–Levine model,²⁶ the rate constants have been modified to adjust the channel sensitivity to Ca²⁺ such that the channel functions properly in the appropriate Ca²⁺ range.³¹ Transition rates between states are given by k_x and are provided by Jafri et al.³¹

The gating structure for the RyR2 channel is presented in Figure 1, and all parameter choices are shown in Table 1.

View this table: [in this window] [in a new window]   Table 1. Models Parameters

Incorporation of Luminal Ca²⁺ Dependence of SR Ca²⁺ Release
RyR2 channels are known to be activated by Ca²⁺ concentration on the dyadic side of the protein during calcium-induced calcium release (CICR). Furthermore, RyR2 channels are also activated by free Ca²⁺ on the luminal (intra-SR) side of the protein, as channel open probability has been shown to rise with increases in SR Ca²⁺ in lipid bilayers.^32–34 Recently, CSQN2 was shown to confer reduced open probability to partially assembled RyR2 release units, an inhibition that was relieved by increasing Ca²⁺ concentration on the luminal side of the receptor.^15,35

To reflect luminal Ca²⁺ dependence of RyR2 opening, the model C1O1 transition was multiplied by a Hill function of SR Ca²⁺ concentration, k_lumen, providing a graded increase in opening rate with increase in free SR Ca²⁺: equation

Furthermore, the mechanism(s) underlying termination of RyR2 release in the myocyte have not been precisely determined, but it appears that SR Ca²⁺ depletion,³⁶ in addition to adaptation (as implemented in the current model),²⁴ may be involved. The role of SR Ca²⁺ in termination of release is suggested by experiments in which the SR Ca²⁺ content was modulated with low-affinity Ca²⁺ buffers, resulting in altered Ca²⁺ spark durations and amplitudes.¹⁰ These findings suggest that RyR2 channels might enter a refractory state more quickly on reductions in luminal Ca²⁺, possibly by adjusting their responsiveness to cytosolic Ca²⁺ (see Györke and Györke³⁵). This behavior of the receptor was simulated by scaling the openadapted transition (O1C2) by 1/k_lumen, such that SR Ca²⁺ depletion accelerates termination of release.

Finally, restitution of RyR2 appears also to be dependent on restoration of SR load (reviewed elsewhere³⁷). Ventricular myocytes simultaneously loaded with imperatoxin A (an RyR2 activator³⁸), and intra-SR Ca²⁺ buffers showed increased spark frequency in the absence of SR buffering, suggesting enhanced recovery dynamics at higher luminal Ca²⁺.¹⁰ This finding was recently supported by a study using paired pulses of photolysis of caged Ca²⁺, in which by manipulating SR load, it was determined that SR Ca²⁺ directly determines the time course of restitution of the paired Ca²⁺ transient.³⁹ To reproduce this important experimental behavior, the model RyR2 recovery from the adapted state (transition C2O1) was also scaled by k_lumen (a parameter that controls the luminal Ca²⁺ sensitivity).

Initial parameter choices for k_lumen were taken from a recent simulation study⁴⁰ and were adjusted to ensure physiological transition scaling rates to the range of wild-type human myocyte SR Ca²⁺ content (from 0.5 to 2 Hz range from 0.2 to 0.6 mmol/L) by pacing the model at 1 Hz; the final parameter choices are listed in Table 1.

Modeling Impaired Luminal Ca²⁺ Sensing in CSQN2 Mutations
Regulation of RyR2 activity by luminal Ca²⁺ likely involves CSQN2 as a luminal Ca²⁺"sensor" (reviewed elsewhere^14,15). Recently, in a series of experiments, Györke and colleagues have shown that mutations in CSQN2¹¹ or reduced CSQN2 expression levels^11,16,41 affect properties of calcium release by altering the luminal Ca²⁺ dependent regulation of the RyR2. These changes were simulated in the mutant model through appropriate adjustments to k_lumen, the factor that confers luminal Ca²⁺ dependence to each RyR2 transition (see the online data supplement for details).

Viatchenko-Karpinski et al¹¹ demonstrated that mutant CSQN2 expressed in rat ventricular myocytes was associated with smaller elemental Ca²⁺ sparks with reduced time to peak amplitude, consistent with accelerated termination of release in the mutant compared with wild-type myocytes. These findings were in line with experiments underexpressing CSQN2 in ventricular myocytes.¹⁶ Accelerated termination of release was therefore incorporated in the mutant model, by increasing maximal k_lumen for the O1C2 adaptation transition.

Furthermore, Terentyev et al¹⁶ and Kubalova et al⁴¹ demonstrated that the kinetics of recovery of the RyR2 are also altered in CSQN2-underexpressing myocytes. These experiments showed (1) an increase in imperatoxin-induced spark frequency with mutant CSQN2¹⁶ and (2) shorter intervals between Ca²⁺ waves in myocytes expressing reduced CSQN2 levels,⁴¹ suggesting that the CSQN2 expression level might control the size of a reservoir of intra-SR Ca²⁺, whose recharging by SR repletion controls the RyR2 recovery. In this scheme, mutant CSQN2 or reduced levels of CSQN2 creates a lower threshold of SR Ca²⁺ repletion before store recharge.^11,16,41 To emulate this process, mutant RyR2 recovery was accelerated in the model by reducing the H₅₀"threshold" parameter (the SR Ca²⁺ concentration at which the magnitude of k_lumen is half maximal), for the transition C2O1.

Mutant CSQN2 has been shown to have reduced buffering capacity for SR Ca²⁺.⁴² This was simulated by adjusting the rapid buffering approximation of Wagner and Keizer⁴³ by scaling the effective CSQN2 concentration by the factor k_CSQN2.

We used parametric analysis to determine physiologically relevant parameters for the mutant CSQN2 model (for further details, please see the online data supplement). Overall, in investigating the mechanisms of DAD formation in the mutant CSQN2 model, the selection of a well-defined specific value for each of the mutant models was not a goal of the study (a specific value itself may have little physiological significance). Instead, we aimed to identify processes that would reproducibly recapitulate the experimental data including DAD formation, for a physiologically reasonable range of the mutant parameters.

Parameter adjustments reflecting the mutant CSQN2 are listed in Table 1.

Modeling Altered RyR2 Gating in RyR2 Mutations
Several experiments investigating impaired calcium cycling in CPVT mutant RyR2 have demonstrated reduced FKBP12.6 (calstabin) binding to RyR2 on protein kinase A phosphorylation.^9,12 FKBP12.6 is one component of the macromolecular complex that comprises the RyR2 receptor. FKBP12.6 binds adjacent RyR2 subunits and is responsible for the structural association of subunits and coupled gating between assembled channels,^19,44 causing linked RyR2 channels to act as a synchronous Ca²⁺ release unit.⁴⁵ Experiments using the immunophilin FK506 (which dissociates FKBP12.6 from assembled RyRs) have been shown to uncouple individual RyR2 channels functionally, but not structurally, as assessed by subconductance states unmasked with FK506 administration.¹⁹

Both reduced coupled gating and altered intersubunit interaction in RyR2 mutants is modeled, as recently described,^46,47 as a reduction in cooperativity of activation of RyR2, by scaling the parameters responsible for RyR2 cooperativity (m_coop and n_coop) by a constant k_RyR2.

Similar to the mutant CSQN2 model, we used parametric analysis to determine physiologically relevant parameters for the mutant RyR2 model that would recapitulate the experimental data including the DAD formation.

The Isoproterenol Model
The main effect of isoproterenol on Ca²⁺ cycling proteins was modeled as an increase in the density of the L-type calcium current and the rate of SR Ca²⁺ uptake by the SR Ca²⁺ ATPase by a factor k_beta.⁴⁰ The choice not to incorporate changes in peak RyR2 Ca²⁺ flux and the sodium–calcium exchanger (NCX) current resulting from isoproterenol stimulation was made on the basis of better isolating changes to RyR2 gating imposed by the mutant gene. However, additional simulations that include the effect of a comprehensive isoproterenol model are shown in Figure II in the online data supplement, and did not change the conclusions drawn from this simpler isoproterenol stimulation model.

Stimulation Protocols
To determine model parameters and facilitate comparisons with the experimental data,^11,16 voltage-clamp simulations of the mutant CSQN2 model were conducted using 400-ms-long pulses from a holding potential of –40 mV to a step potential of 0 mV, delivered at 0.5 Hz. Also, the virtual myocyte was stimulated at 2 Hz for all simulations in current-clamp mode, thus matching experimental pacing rates,^11,16 which also correspond to the heart rate in humans, above which ventricular arrhythmias occur most commonly.^12,48

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Luminal Ca²⁺ Dependence of RyR2 Gating
Figure 1 shows a schematic illustration of the RyR2 receptor model used in this study. Full details about the model state transitions and their dependence on luminal Ca²⁺, as well as the simulation protocols, are described in Materials and Methods.

We first incorporated SR Ca²⁺ dependence of RyR2 gating into the Keizer–Levine model²³ and observed its effects on features of whole-cell Ca²⁺ cycling. In Figure 2, we present simulation results of the whole-cell model, stimulated in voltage-clamp mode at 0.5 Hz. Figure 2A shows Ca²⁺ transients ([Ca²⁺]_i) produced by the original Keizer–Levine model without (black trace) and with (red trace) isoproterenol stimulation; Figure 2B shows [Ca²⁺]_i produced by the modified Keizer–Levine model in which luminal Ca²⁺ dependence has been added, without (blue trace) and with (green trace) isoproterenol stimulation. The upgraded Keizer–Levine model achieves steady state after short-term pacing and reproduces [Ca²⁺]_i with morphology and size typical of human voltage-clamped [Ca²⁺]_i.

View larger version (15K): [in this window] [in a new window]   Figure 2. The RyR2 luminal Ca2+-dependent model. Voltage-clamp simulations of the wild-type human cell model using the original Keizer–Levine RyR2 model (KL model) and the new model that includes luminal Ca2+ dependence; simulations were conducted by delivering 400-ms voltage steps to 0 mV from a holding potential of –40 mV until steady state was achieved at 0.5-Hz stimulation frequency. A, Ca2+ transients ([Ca2+]i) of the original RyR2 model (black trace, KL model; red trace, KL model and ISO; ISO indicates isoproterenol). B, Ca2+ transients of the KL model with luminal Ca2+ dependence at baseline (blue trace) and in the presence of isoproterenol (green trace). C, Pooled RyR2 open probability (states O1 plus O2) in the presence of isoproterenol, for the KL model without (red trace) and with luminal Ca2+ dependence (green trace).

In Figure 2C, we compare the behavior of the 2 models in voltage clamp and in the presence of simulated isoproterenol (red trace, the Keizer–Levine model+isoproterenol; green trace, the Keizer–Levine model with luminal Ca²⁺ dependence+isoproterenol). Incorporation of luminal Ca²⁺ dependence in the rate transitions between states (as shown in Figure 1) augments peak RyR2 open probability by 10%, consistent with the effect of higher SR Ca²⁺ on RyR2 activation. Furthermore, in the model that includes the luminal Ca²⁺ dependence, the initial depletion in SR Ca²⁺ results in earlier termination of Ca²⁺ release (accelerated transition from O1C2). Thus, the functional significance of these results is that in the upgraded model presented here, SR Ca²⁺ depletion now becomes an important mechanism in terminating SR Ca²⁺ release.

CSQN2 Modulation of [Ca²⁺]_i
The next set of simulations uses the mutant CSQN2 model to simulate the effect of CSQN2 under- and overexpression, as well as the altered RyR2 gating seen in D307H mutant CSQN2 gene transfer.¹¹

Figure 3A shows the overall evolution of [Ca²⁺]_i to repetitive voltage-clamp stimulation at 0.5 Hz. As shown in the figure, [Ca²⁺]_i is smaller in the mutant compared with the wild type, particularly in the initial several beats. On pacing to steady state, the difference in peak [Ca²⁺]_i becomes smaller, a finding that is consistent with previous studies.⁴⁰ Other experimental recordings of [Ca²⁺]_i in CSQN2 mutants similarly show smaller [Ca²⁺]_i; however, they remain small after long-term stimulation.¹¹ Possible reasons for this experimental difference at steady state are discussed further in Discussion.

View larger version (19K): [in this window] [in a new window]   Figure 3. Simulated effects of the mutant CSQN2 model. A, Evolution of voltage-clamped Ca2+ transients for the mutant CSQN2 model (red trace) and for the wild-type model (black trace). B, Enlarged view of the peak of the voltage-clamped Ca2+ transient for wild-type (black trace), CSQN2 mutant (red trace), and simulated CSQN2 overexpression (blue trace). C, Restitution of RyR2 for wild type (black trace) and CSQN2 mutant (red trace). Voltage-clamp steps were delivered at 0.2 Hz until steady state, followed by a test voltage clamp delivered after different intervals. The restitution curve was obtained by measuring peak RyR2 open probability during the test clamp and normalizing it to the steady-state open probability.

To closer examine the amplitude and kinetics of [Ca²⁺]_i, Figure 3B provides an expanded view of [Ca²⁺]_i in the vicinity of its peak amplitude. Compared with wild type, simulation of the mutant CSQN2 (in transition to steady state) shows a 36% smaller [Ca²⁺]_i amplitude and reduced time to peak, with a similar rate of rise of [Ca²⁺]_i. These results compare well with the faster time-to-peak and smaller amplitude [Ca²⁺]_i measured in rat ventricular myocytes subjected to CSQN2 underexpression (compare with Terentyev et al¹⁶) and gene transfer of D307H mutant CSQN2 (compare with Viatchenko-Karpinski et al¹¹).

The blue trace in Figure 3B shows the [Ca²⁺]_i peak in simulated CSQN2 overexpression (simulated by reversing the changes imposed in the mutant, ie, increasing CSQN2 buffering capacity, reducing peak adaptation rate, and shifting H₅₀ for recovery to higher SR Ca²⁺; see Materials and Methods). Similar to the features of [Ca²⁺]_i observed experimentally in CSQN2 overexpression,^11,16 the simulation shows that peak [Ca²⁺]_i is 130% larger than in the wild type, with a delayed, dome-like peak. The initial rate of rise of the [Ca²⁺]_i is unchanged in the mutant and CSQN2 overexpression simulations, similar to the experiments.^11,16

To assess the time course of recovery of Ca²⁺ release, a paired voltage-clamp stimuli protocol was applied in mutant CSQN2 and wild-type simulations. The time course of recovery was accelerated in the mutant simulations (Figure 3C), providing confirmation that global Ca²⁺ release recovers more quickly in the mutant CSQN2 model.

Features of experimental and model-derived [Ca²⁺]_i for mutant CSQN2 and CSQN2-underexpressing myocytes are presented in Table 2. Overall, the results presented in Figure 3 and Table 2 suggest that the mutant CSQN2 model reliably recapitulates the altered gating of the mutant CSQN2, reproducing the available experimental data in CSQN2-underexpressing and D307H mutant CSQN2-expressing ventricular myocytes.^11,16

View this table: [in this window] [in a new window]   Table 2. Experimental Features of [Ca2+]i for Mutant CSQN2 and CSQN2 Overexpression

DADs in Mutant CSQN2
It has been noted that rat ventricular myocytes expressing mutant CSQN2 demonstrate small-amplitude DADs on rhythmic pacing in the presence of isoproterenol.¹¹ To examine whether the implemented changes in luminal Ca²⁺ sensing in the mutant CSQN2 model result in DADs, the model was stimulated at 2 Hz in current-clamp mode.

As shown in Figure 4, isoproterenol administration provokes DADs (Figure 4A). These DADs are caused by aberrant Ca²⁺ release, seen as spontaneous oscillations in intracellular Ca²⁺ (Figure 4B) and SR Ca²⁺ (Figure 4C). The response of the model to ß-adrenergic stimulation and the shape and amplitude (5% of the action potential amplitude) of the DADs compare well with experimental results in rat ventricular myocytes expressing mutant CSQN2 (see Viatchenko-Karpinski et al,¹¹ in which the amplitude of the DADs is 8% of that of the action potential).

View larger version (17K): [in this window] [in a new window]   Figure 4. Features of aberrant Ca2+ handling in the mutant CSQN2 model stimulated in current-clamp mode at 2 Hz. The model was paced to steady state, at which point isoproterenol was added (as indicated by the bar, +ISO). A, Action potentials. B, Ca2+ transients ([Ca2+]i). C, SR free Ca2+ load. Arrows indicate DADs.

Use of a comprehensive isoproterenol model that also includes increases in the peak RyR2 Ca²⁺ flux, the density of delayed-rectifier K⁺ current (I_Ks) and the sodium–calcium exchanger current (see the online data supplement) results in triggered activity in the mutant myocyte, manifested by a premature action potential (see supplemental Figure II).

Similar to experiments in CSQN2-underexpressing myocytes,¹¹ in which loading the SR with the low-affinity Ca²⁺ buffer citrate abrogated DADs, citrate simulated at a SR concentration of 5 mmol/L (using the Wagner–Keizer rapid-buffering approximation⁴³ at a K_d of 0.47¹⁰) produced no DADs, even in the presence of ß stimulation (Figure 5). In these simulations, there is no evidence of aberrant Ca²⁺ release through pacing to steady state (action potential in Figure 5A and [Ca²⁺]_i in Figure 5B). Thus, it appears that stabilization of the SR Ca²⁺ by the buffer reduces the probability of spontaneous Ca²⁺ release.

View larger version (21K): [in this window] [in a new window]   Figure 5. Response of the mutant CSQN2 model in the presence of isoproterenol, with citrate simulated at an intra-SR concentration of 5 mmol/L. A, Action potentials. B, Ca2+ transients ([Ca2+]i).

Interestingly, the wild-type model that included luminal Ca²⁺ dependence and reduced CSQN2 concentration (to 5% of baseline value) resulted in DADs in the presence of ß stimulation. However, the same model in the presence of ß stimulation exhibited no DADs in the absence of RyR2 luminal Ca²⁺ dependence. Finally, the mutant CSQN2 model, but with normal CSQN2 concentration, exhibited no DADs in the presence of ß stimulation. These results suggest that because of the RyR2 luminal Ca²⁺ dependence, the buffering role of CSQN2 in the SR is critical in triggering DADs, as has been recently presented in a Casq2^–/– mouse model.⁴⁹

Thus in CSQN2 mutations, DADs form only in the presence of ß stimulation. DADs arise with increases in SR Ca²⁺, and the buffering capacity of CSQN2 is critical in stabilizing the SR Ca²⁺ release process.

DADs in Mutant RyR2
In Figure 6A, one sees that on pacing the mutant RyR2 model in the presence of ß-adrenergic stimulation at 2 Hz, a DAD is observed (after the third stimulus), which again corresponds to a spontaneous calcium release event.

View larger version (17K): [in this window] [in a new window]   Figure 6. Aberrant Ca2+ handling in RyR2 mutants in the presence of isoproterenol. A, Action potentials for mutant RyR2. B, The RyR2 state O1 occupancy over time for the mutant (red trace) and the wild type (black trace). C, Ca2+ transients ([Ca2+]i) for the mutant RyR2. Arrows indicate DADs.

The mechanism for the DAD becomes apparent from analysis of the RyR2 first open state O1 (Figure 6B), which exhibits higher initial and diastolic open probability and longer overall duration of release. In Figure 6B, at time 1400 ms during diastole, O1 is 0.0022 in the mutant model, which is nearly double the 0.0012 value in the wild-type model. The results demonstrate that mutant channels, in the presence of isoproterenol, exhibit abnormally high open probability during diastole. This increased open probability results in progressive accumulation of subspace Ca²⁺, which leads to a regenerative opening of the RyR2 channels and a premature spontaneous Ca²⁺ release in the diastolic interval (Figure 6C).

Thus, in a mutant RyR2 model, ß stimulation induced reduction in coupled gating and intersubunit interaction, manifested in the model by a reduction in cooperativity of the RyR2 activation, results in DADs through an increase in diastolic open probability.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study investigated the mechanisms of DAD generation in CPVT using a mathematical model of human excitation–contraction coupling. It used a simple RyR2 model that incorporates explicit representations of activation, adaptation, and recovery properties of the channel. We have found (1) that the accelerated termination and recovery of RyR2 release in CSQN2 mutants result in DAD formation in the presence of ß stimulation; (2) that the free SR Ca²⁺ content and rate of recovery from inactivation are the key parameters in controlling the stability of Ca²⁺ release from the RyR2s in CSQN2 mutations; and (3) that impaired FKBP12.6 binding is likely to generate DADs through hyperactive RyR2s that exhibit larger diastolic open probability. Overall, our results provide plausible mechanisms by which defects in RyR2 gating induced by CPVT-related gene mutations may lead to multiple cellular triggers of arrhythmias.

SR Ca²⁺ and [Ca²⁺]_i in the Mutant CSQN2 Model
An important aspect of the present study is the ability to reproduce experimental recordings in myocytes reflecting impaired calcium handling secondary to CSQN2 mutations. A minimal order model was formulated to account for the effects of the mutant CSQN2 (Figure 1 and Table 1), based on a series of experimental studies aimed at modulating functional levels of CSQN2 and analyzing features of the resulting Ca²⁺ sparks and Ca²⁺ waves.^11,16,41 Experiments using gene transfer of the mutant CSQN2 mutant D307H in rat ventricular myocytes show [Ca²⁺]_i with smaller amplitude and faster time to peak (despite equivalent rise rates)¹¹; these data were reproduced by our mutant CSQN2 model (Figure 3A and 3B of the current study and shown previously^11,16).

Our study shows that simulations in the mutant CSQN2 model, whereas in transition to steady state, [Ca²⁺]_i amplitude is 36% smaller than in wild-type simulations (a 41% reduction has been observed experimentally in mutant CSQN2-expressing myocytes¹¹). However, at steady state, the simulated [Ca²⁺]_i are only 10% smaller, whereas the experiments show larger persistent depressions in peak [Ca²⁺]_i. Similar to our findings, simulations of CSQN2 overexpression⁴⁰ and experiments using blockers of RyR2 release⁵⁰ have revealed only transient effects on [Ca²⁺]_i, which abate on long-term pacing. The present study supports the idea of a type of "autoregulation" of SR Ca²⁺ release,^40,50–52 whereby depressed SR Ca²⁺ release raises the SR Ca²⁺ content, which in turn increases SR Ca²⁺ release to baseline levels through an increase in luminal Ca²⁺ dependent RyR2 openings⁵² and a larger Ca²⁺ gradient across the SR membrane at the release site.

Although the reasons for the discrepancy in steady-state [Ca²⁺]_i^11,16 are thus still unclear, it is likely that the stimulation frequency used experimentally is partially responsible. The model was paced until steady state was achieved, at a cycle length of 2000 ms. However, in the experimental studies, myocytes were paced at a rate of 1 every 60 seconds. At this stimulation frequency, equilibration of intracellular Ca²⁺ pools may be difficult to achieve. Clearly, more experiments are needed to resolve the issue of steady-state Ca²⁺ dynamics in the CSQN2 mutant myocyte.

Mechanisms of DADs in the Mutant CSQN2 Model
A critical test of the mutant CSQN2 model is its ability to reproduce experimental data are not used to constrain the model. Thus, the demonstration that the mutant CSQN2 minimal order model (as a result of CSQN2-dependent altered RyR2 Ca²⁺ release termination and recovery) produced DADs only in the presence of isoproterenol provides evidence that the proposed mechanisms are plausible explanations for the aberrant Ca²⁺ release in the mutant.

How these DADs arise is a question that is particularly well suited for analysis using the model. To isolate the effect of each of the 2 modifications in the minimal model of CSQN2 mutant myocytes (ie, accelerated termination of release and accelerated recovery of release), additional simulations were conducted by (1) changing the rate of termination alone and holding the rate of recovery unchanged from that in the wild type and (2) changing the rate of recovery from inactivation alone and holding the rate of termination at wild type. Simulations in which only termination of Ca²⁺ release was incrementally accelerated reveals that DADs could be elicited as SR Ca²⁺ reached a critical level. This phenomenon is likely to be a consequence of autoregulation, whereby smaller release events raise free SR Ca²⁺, resulting in enhanced luminal Ca²⁺-dependent aberrant RyR2 openings. Thus, these simulations add further support to the central role of free SR Ca²⁺ in the genesis of DADs.

One interpretation of these findings suggests that restoration of SR free Ca²⁺ may function to promote DADs by disinhibiting CSQN2-bound closed RyR2 (ie, reactivating the receptor or facilitating transition C1O1 in Figure 1). Another possibility is that repletion of SR Ca²⁺ rescues RyR2 from a luminal Ca²⁺-dependent refractory state (ie, from state C2). This possibility was tested in simulations that only modified the C2O1, or recovery step, of the mutant. When the recovery step is accelerated sufficiently (in the absence of changes to the rate of termination of release), DADs result, as demonstrated in supplemental Figure IV. This finding suggests that primary modulation of RyR2 gating that provides premature recovery of the RyR2s from a luminal Ca²⁺-dependent refractory state is a plausible mechanism for producing spontaneous Ca²⁺ release events. It also provides theoretical support to the scheme presented by Györke and colleagues, who suggested that DADs in CSQN2 mutations might arise from altered RyR2 luminal Ca²⁺ sensitivity,¹¹ possibly through premature recovery of receptors associated with accelerated recharge of a "smaller" functional SR Ca²⁺ store.¹⁶ In this scheme, ß-adrenergic stimulation may unmask DADs by further enhancing the recharge of this smaller store.

With either scenario (premature recovery from adaptation or premature reactivation) free SR Ca²⁺ content (or its disrupted sensing by RyR2) appears to be a critical determinant of DAD formation in CPVT. Alternatively, because of the RyR2 luminal Ca²⁺ dependence, the buffering capacity of CSQN2 alone appears to play a critical role in triggering DADs, as has been recently presented in a Casq2^–/– mouse model.⁴⁹ Thus, interventions that reduce SR Ca²⁺ content, or alternatively that stabilize the release mechanism to respond at higher free SR Ca²⁺, can be expected to minimize the effect of the mutation. This was demonstrated by simulations (Figure 5) and experiments¹¹ in which citrate loaded into the SR stabilizes the release mechanism and restores rhythmic excitation in ventricular myocytes expressing mutant CSQN2.

Mechanisms of DADs in the Mutant RyR2 Model
The results of this investigation show that reduced RyR2 coupled gating as a consequence of reduced FKBP12.6 binding results in DADs (Figure 6). Common missense mutations linked to CPVT, when expressed in HEK293 cell lines, demonstrate reduced binding of FKBP12.6 to RyR2 in the presence of protein kinase A phosphorylation, as assessed by coimmunoprecipitation.¹² Most recently, FKBP12.6-null mouse myocytes were shown to exhibit higher diastolic SR Ca²⁺ leak and episodes of ventricular tachycardia.²⁰ These experiments suggest that RyR2 CPVT mutants are likely to give rise to DADs through reduced FKBP12.6 binding.

Each RyR2 subunit in the tetrameric release assembly binds 1 FKBP12.6 protein, coupling RyR2 activity^19,44 and stabilizing the closed conformation of the channel.^46,47,53 Recent simulation studies^37,53 have shown that increased coupling between adjacent RyR2 subunits in a "sticky cluster model" stabilizes the release mechanism, perhaps through a more synchronous activation of the subunits, thus shortening the overall release duration. Conversely, decreased RyR2 coupling increases heterogeneity of release across release sites, prolonging overall release duration, as confirmed by our study. We have found that in a whole-cell model of reduced FKBP12.6 binding that there was evidence of aberrant diastolic RyR2 openings, which are associated with increased diastolic open probability. This provides strong theoretical support to the idea that reduced FKBP12.6 binding in the presence of protein kinase A phosphorylation can lead to uncoupled, hyperactive RyR2 channels that are prone to triggered activity in CPVT.^9,12,54

It is likely that, given the genotypic heterogeneity of CPVT-linked mutations, there is also heterogeneity in the mechanisms whereby different mutations exert their effects.⁵⁵ Indeed, some studies have not been able to demonstrate reduced FKBP12.6 binding in RyR2 mutations linked to CPVT.^17,18,56 One explanation for this discrepancy is the use of different experimental preparations in these studies.⁷ The other possibility is that the DADs in these RyR2 mutants arise from FKBP12.6-independent mechanisms, such as impaired luminal Ca²⁺ sensing. Recently, Jiang et al proposed a scheme attributing DADs in RyR2 mutants to a reduced threshold for functional recovery of receptors, a process they have termed "store overload–induced calcium release."¹⁸

Single-channel analysis in these mutants¹⁸ reveals that the mechanism for these changes is increased sensitivity of mutant channels to luminal Ca²⁺, with little effect on features of cytosolic Ca²⁺ activation. To test this hypothesis, simulations were performed in which the sensitivity of channels to luminal Ca²⁺ was increased (by reducing the C2O1 H₅₀ recovery threshold parameter to 0.5). These simulations (presented in supplemental Figure IV) produce DADs, providing support for the store overload–induced calcium release mechanism in RyR2 mutants. Such a scheme is furthermore attractive because it provides a common mechanistic framework for understanding different mutations in CSQN2 and RyR2 that lead to a similar phenotype, ie, through enhanced sensitivity to luminal Ca²⁺.

Summary
In conclusion, in this study, we explored the mechanistic basis for aberrant Ca²⁺ homeostasis in 2 recently identified mutations linked to CPVT. Importantly, we developed minimal order models of mutant CSQN2 and RyR2 that recapitulated known experimental behavior of these gene products when expressed in ventricular myocytes. Using these models, we have demonstrated that CSQN2 mutations may give rise to DADs through accelerated recovery from inactivation and that free SR Ca²⁺ is an important variable in determining the propensity for DADs. We have further demonstrated that reduced cooperativity of RyR2 gating, as might be found with reduced FKBP12.6–RyR2 binding in CPVT mutants, can also plausibly cause DADs associated with the mutant RyR2s via hyperactive release channels. These findings can potentially be used to guide further experiments in characterizing these mutants and assist in the development of targeted therapies.

Limitations
The current work provides demonstration that the proposed mechanisms are plausible explanations for the triggered activity observed in CPVT mutations. Unfortunately, the study is limited by the lack of published experimental characterization of calcium handling in human ventricular myocytes carrying the relevant mutations, eg, from biopsy specimens of human CPVT patients. Such data might permit more quantitative, rigorous constraining of parametric changes to the model.

Furthermore, the majority of DADs observed in this investigation are of small amplitude (8% of the action potential peak amplitude), similar to those recorded experimentally.¹¹ However, as we show in the online data supplement, triggered premature action potentials do occur in the mutant myocyte using the comprehensive ß-stimulation model. Therefore, whereas the minimum-order model enabled us to relate alterations in the gating properties of the RyR2, to abnormal SR Ca²⁺ release and DAD formation, the observations (1) that the ß-stimulation effect in vivo is not precisely known and likely depends on the magnitude of the catecholaminergic surge in the exercising or stressed patient and (2) that ß stimulation is likely to differentially affect each of the Ca²⁺ regulatory genes (ie, L-type channel, RyR2, SERCA2a, and sodium–calcium exchanger) clearly provide plausible explanations about the variability in the DAD amplitude observed experimentally and in this study. Nevertheless, the current model serves as a valuable tool for unraveling mechanistic underpinnings of the disease and may become useful in assessing the effects of recently proposed therapeutic interventions.^12,57

	Acknowledgments

 
Sources of Funding

The work was supported by Beginning Grant-in-Aid (no. 0365304U) and a Scientist Development Grant (no. 0635127N), American Heart Association awards (to V.I. and A.A.A.), and by the NIH (R01 HL078691, HL071763, HL080498, and HL083156) and a Leducq Transatlantic (to R.J.H.).

Disclosures

None.

	Footnotes

 
Original received July 26, 2006; revision received September 6, 2006; resubmission received October 30, 2006; revised resubmission received January 5, 2007; accepted January 9, 2007.

	References

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