This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 91/3/218    most recent 01.RES.0000028455.36940.5Ev1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jiang, D. Articles by Chen, S.R. W. Search for Related Content PubMed PubMed Citation Articles by Jiang, D. Articles by Chen, S.R. W. Related Collections Animal models of human disease Gene expression Genetically altered mice

(Circulation Research. 2002;91:218.)
© 2002 American Heart Association, Inc.

Molecular Medicine

Enhanced Basal Activity of a Cardiac Ca²⁺ Release Channel (Ryanodine Receptor) Mutant Associated With Ventricular Tachycardia and Sudden Death

Dawei Jiang, Bailong Xiao, Lin Zhang, S.R. Wayne Chen

From the Cardiovascular Research Group, Departments of Physiology and Biophysics and Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada.

Correspondence to S.R. Wayne Chen, 3330 Hospital Dr NW, Calgary, Alberta, Canada, T2N 4N1. E-mail swchen{at}ucalgary.ca

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mutations in the human cardiac Ca²⁺ release channel (ryanodine receptor, RyR2) gene have recently been shown to cause effort-induced ventricular arrhythmias. However, the consequences of these disease-causing mutations in RyR2 channel function are unknown. In the present study, we characterized the properties of mutation R4496C of mouse RyR2, which is equivalent to a disease-causing human RyR2 mutation R4497C, by heterologous expression of the mutant in HEK293 cells. [³H]ryanodine binding studies revealed that the R4496C mutation resulted in an increase in RyR2 channel activity in particular at low Ca²⁺ concentrations. This increased basal channel activity remained sensitive to modulation by caffeine, ATP, Mg²⁺, and ruthenium red. In addition, the R4496C mutation enhanced the sensitivity of RyR2 to activation by Ca²⁺ and by caffeine. Single-channel analysis showed that single R4496C mutant channels exhibited considerable channel openings at low Ca²⁺ concentrations. HEK293 cells transfected with mutant R4496C displayed spontaneous Ca²⁺ oscillations more frequently than cells transfected with wild-type RyR2. Substitution of a negatively charged glutamate for the positively charged R4496 (R4496E) further enhanced the basal channel activity, whereas replacement of R4496 by a positively charged lysine (R4496K) had no significant effect on the basal activity. These observations indicate that the charge and polarity at residue 4496 plays an essential role in RyR2 channel gating. Enhanced basal activity of RyR2 may underlie an arrhythmogenic mechanism for effort-induced ventricular tachycardia.

Key Words: ryanodine receptor • Ca²⁺ release • excitation-contraction coupling • mutations • ventricular arrhythmias

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
An increasing body of evidence indicates that defects in cardiac ion channels can lead to ventricular arrhythmias, the major cause of sudden death.¹ To date, 6 ion channel genes have been linked to cardiac arrhythmias. These include genes encoding the potassium channels, KVLQT1, HERG, minK, and MiRP1, the sodium channel, SCN5A, and the cardiac Ca²⁺ release channel (ryanodine receptor, RyR2). Mutations in the potassium channels can cause long-QT syndrome, whereas mutations in the sodium channel can lead to both long-QT syndrome and familial ventricular fibrillation.¹

Mutations in the human RyR2 gene have recently been shown to cause different forms of cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia (PMVT) and arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2).^2–4 However, the molecular and cellular mechanisms underlying these forms of ventricular arrhythmias are not clear. Catecholaminergic PMVT is an autosomal-dominant, genetic arrhythmogenic disorder associated with syncope and sudden death.⁵ This disorder is characterized by stress-, emotion-, or physical exercise-induced bidirectional ventricular tachycardia. Patients with catecholaminergic PMVT apparently have functionally and structurally normal hearts and display normal QT intervals at rest and during exercise. Interestingly, the ECG pattern of catecholaminergic PMVT resembles that of digitalis-induced arrhythmia.⁶ Based on these observations, it has been proposed that catecholaminergic PMVT and digitalis-induced arrhythmia may share a similar arrhythmogenic mechanism, which is believed to be Ca²⁺ overload-induced delayed afterdepolarizations.^2,7 How mutations in the RyR2 gene could lead to Ca²⁺ overload-induced delayed afterdepolarizations is, however, unclear.

RyR2 is a key component involved in excitation-contraction (EC) coupling that is thought to occur in cardiac muscle via a mechanism known as Ca²⁺-induced Ca²⁺ release.^8,9 In this process, the RyR2 Ca²⁺ release channel located in the sarcoplasmic reticulum (SR) is activated by a small influx of Ca²⁺ through the voltage-dependent L-type Ca²⁺ channel, leading to a large Ca²⁺ release from the SR. In addition to this stimulated Ca²⁺ release during normal EC coupling, Ca²⁺ release from SR can also occur spontaneously as a result of spontaneous openings of the RyR2 channels. Under normal Ca²⁺ loading conditions, spontaneous Ca²⁺ release can be detected as localized Ca²⁺ transients known as "Ca²⁺ sparks."^10,11 On the other hand, when cardiac cells are overloaded with Ca²⁺, Ca²⁺ sparks lead to propagating Ca²⁺ waves.¹² It has been well established that propagating Ca²⁺ waves cause delayed afterdepolarizations via Ca²⁺-activated inward currents, which in turn could lead to triggered arrhythmias.^13–16 Alterations in RyR2 channel function are likely to influence the properties of spontaneous Ca²⁺ release and propagating Ca²⁺ waves, and thus the occurrence of triggered arrhythmias.

In the present study, we have expressed a mouse RyR2 mutant R4496C, corresponding to an arrhythmogenic human RyR2 mutant R4497C, in HEK293 cells and characterized the mutant by using [³H]ryanodine binding assay, single-channel analysis, and single-cell Ca²⁺ imaging. Our results show, for the first time, that mutation R4496C enhances the basal channel activity and the propensity for spontaneous Ca²⁺ release. These observations shed important insights into the molecular basis of catecholaminergic PMVT.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Site-Directed Mutagenesis and DNA Transfection
Point mutations were generated by the overlap extension method using the polymerase chain reaction as described previously.^17,18 HEK293 cells grown on 100-mm tissue culture dishes were transfected with wild-type or mutant RyR cDNA using Ca²⁺ phosphate precipitation.¹⁹

Preparation of Cell Lysate
Cell lysate was prepared as described previously.¹⁸

[³H]Ryanodine Binding
Equilibrium [³H]ryanodine binding to cell lysate (30 µL, 3 to 5 µg/µL) was performed as described previously.¹⁸

Single-Channel Recordings
Single-channel recordings were performed as described previously.¹⁸ Free Ca²⁺ concentrations were calculated using the computer program of Fabiato and Fabiato.²⁰

Single-Cell Ca²⁺ Imaging
Ca²⁺ transients in transfected HEK293 cells were measured using single-cell Ca²⁺ imaging and the fluorescence Ca²⁺ indicator dye fura-2-AM.

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mutation R4496C Enhances the Basal Level of [³H]Ryanodine Binding
To investigate the impacts of PMVT mutations on RyR2 channel function, we have generated a mutation, R4496C, in mouse RyR2 equivalent to the disease-causing mutation R4497C in human RyR2.² The mouse R4496C mutant RyR2 was expressed in HEK293 cells and characterized by [³H]ryanodine binding. Figure 1A shows [³H]ryanodine binding to wild-type (wt) and R4496C mutant mouse RyR2 in the presence of a wide range of Ca²⁺ concentrations. Analysis of the Ca²⁺ dependence of [³H]ryanodine binding to the wt channels by the Hill equation yielded an EC₅₀ of 0.26±0.04 µmol/L and a Hill coefficient of 2.41±0.15 (n=4), similar to those reported previously.¹⁸ Different from the wt channels, the R4496C mutant channels were activated by Ca²⁺ with a decreased EC₅₀ of 0.19±0.02 µmol/L (P<0.03) and a decreased Hill coefficient of 1.82±0.21 (n=5). In addition to the slightly leftward shift in Ca²⁺ dependence, the R4496C mutant channels exhibited an elevated level of [³H]ryanodine binding particularly at low Ca²⁺ concentrations. For instance, at Ca²⁺ concentrations between 0.2 to 20 nmol/L, a considerable level of [³H]ryanodine binding (10.6±1.4% maximum binding; n=4) was detected in mutant R4496C, significantly higher than the 2.11±0.74% (n=4) observed in the wt channels (P<0.001) (Figure 1A). These results indicate that mutation R4496C increases the basal level and decreases the threshold for Ca²⁺ activation of [³H]ryanodine binding to RyR2.

View larger version (25K): [in this window] [in a new window]   Figure 1. Ca2+ dependence and affinity of [3H]ryanodine binding to wt and R4496C mutant RyR2. [3H]ryanodine binding to cell lysate prepared from HEK293 cells transfected with mRyR2 (wt) (filled circles) or R4496C mutant (open circles) cDNA was carried out at various concentrations of Ca2+ (0.2 nmol/L to 0.1 mmol/L) and 5 nmol/L [3H]ryanodine (A) and at various concentrations of [3H]ryanodine (0.1 to 30 nmol/L) and 100 µmol/L Ca2+ (B). Data shown are from representative experiments each repeated 3 to 5 times. Inset in B shows a Scatchard plot.

Mutation R4496C Does Not Alter the Affinity of [³H]Ryanodine Binding to RyR2
The increased basal [³H]ryanodine binding activity observed in mutant R4496C may result from alterations in the properties of [³H]ryanodine binding. To test this possibility, we carried out [³H]ryanodine binding to the R4496C mutant and wt RyR2 in the presence of different concentrations of [³H]ryanodine. Scatchard analysis of the binding data revealed that mutant R4496C exhibited a binding affinity (K_d) of 2.5±0.1 nmol/L and a maximum binding capacity (B_max) of 0.98±0.04 pmol/mg (n=3) (Figure 1B). The [³H]ryanodine binding properties of mutant R4496C were very similar to those of the wt RyR2 (K_d=2.8 nmol/L, B_max=1.01 pmol/mg) (Figure 1B).¹⁸ Therefore, mutation R4496C does not appear to alter the intrinsic properties of [³H]ryanodine binding to RyR2.

Increased Basal [³H]Ryanodine Binding Activity Is Sensitive to Modulation
It is also possible that the increased basal [³H]ryanodine binding activity of mutant R4496C may be due to nonspecific binding. Accordingly, we examined the effects of various channel modulators on [³H]ryanodine binding to mutant R4496C. As shown in Figure 2a, at 3 nmol/L Ca²⁺ concentration (pCa=8.49), mutant R4496C showed 10% maximum [³H]ryanodine binding. This basal activity was activated by caffeine and ATP and was inhibited by Mg²⁺ and ruthenium red (Figure 2a). Furthermore, the responses of mutant R4496C to various modulators at this Ca²⁺ concentration were similar to those observed at a higher Ca²⁺ concentration (100 nmol/L, pCa=7.02) (Figure 2b). Thus, the basal [³H]ryanodine binding activity observed in mutant R4496C is sensitive to regulation by various channel modulators and does not result from nonspecific [³H]ryanodine binding.

View larger version (20K): [in this window] [in a new window]   Figure 2. Effect of channel modulators on [3H]ryanodine binding to mutant R4496C. [3H]ryanodine binding to cell lysate prepared from HEK293 cells transfected with the R4496C mutant cDNA was performed at 2 different Ca2+ concentrations: 3 nmol/L, pCa=8.49 (a); 100 nmol/L, pCa=7.02 (b). At each Ca2+ concentration, [3H]ryanodine binding was determined in the absence (Cont) or the presence of 2.5 mmol/L caffeine, 2.5 mmol/L ATP, 5 mmol/L MgCl2, or 30 µmol/L ruthenium red. Amounts of bound [3H]ryanodine under each condition are presented as percentages of the maximum binding (100%) determined at pCa=4.2 (63 µmol/L). Data shown are mean±SEM from 3 separate experiments.

Single R4496C Mutant Channels Exhibit Enhanced Channel Activity at Nanomolar Ca²⁺ Concentrations
[³H]ryanodine binding is thought to reflect channel openings of RyR, as ryanodine binds to only the open state of the channel.²¹ Our observation that mutant R4496C displayed increased [³H]ryanodine binding at nanomolar Ca²⁺ concentrations (Figure 1A) suggests that mutant R4489C may exhibit considerable channel openings in the near absence of Ca²⁺. To examine this possibility directly, we incorporated single wt and R4496C mutant channels into planar lipid bilayers and examined their channel activities at low Ca²⁺ concentrations. As shown in Figure 3A, a single wt channel was almost completely inhibited by the addition of 0.1 mmol/L EGTA, which would reduce the free Ca²⁺ concentration to less than 5 nmol/L (assuming that the contaminated level of Ca²⁺ in the recording solution is less than 5 µmol/L). The average open probability (Po) of single wt channels under this condition was 1.6x10^-5±4.7x10^-6 (n=30). In contrast, under the same condition, considerable opening events were detected in single R4496C mutant channels (Figure 3b). The average Po of the EGTA-inhibited single R4496C mutant channels was 1.1x10^-4±3.3x10^-5 (n=28), significantly greater than that observed with the wt channels (P<0.005). These observations are consistent with the view that mutant R4496C displays increased basal channel activity.

View larger version (72K): [in this window] [in a new window]   Figure 3. Basal activity and Ca2+ dependence of single wt and R4496C mutant RyR2 channels. Single-channel activities of the mRyR2 (wt) (A) and R4496C mutant RyR2 (B and C) were recorded in a symmetrical recording solution containing 250 mmol/L KCl and 25 mmol/L HEPES (pH 7.4). The trans chamber was connected to the input of the headstage amplifier, and the cis chamber was held at virtual ground. Addition of 0.1 mmol/L EGTA to the cis chamber inhibited the single-channel activities of both the mRyR2 (wt) (A-b) and mutant R4496C (B-b). C-a through C-d, Current traces of single R4496C mutant RyR2 channel at various cytoplasmic free Ca2+ concentrations. Holding potential was +20 mV. Open probability (Po), arithmetic mean open time (To), and the arithmetic mean closed time (Tc) are indicated on the top of each panel. A short line to the right of each current trace indicates the baseline. Openings are upward. Relationships between open probability (Po) and Ca2+ concentrations (pCa) of single wild-type (open triangles) and single R4496C mutant (open circles) RyR2 channels are shown in C-e. Data points shown are individual measurements obtained from 7 single wt RyR2 channels and 14 single R4496C mutant channels. Curves shown represent fits using the Hill equation.

[³H]ryanodine binding assay is very sensitive for monitoring RyR channel activities when Po of the channel is relatively low (less than 0.05). However, when Po is greater than 0.3, [³H]ryanodine binding becomes saturated and relatively insensitive to changes in channel activities.²¹ In order to characterize the overall Ca²⁺ dependence of the R4496C mutant channels, we determined the Po of single R4496C mutant channels at a wide range of Ca²⁺ concentrations. As shown in Figure 3C, single R4496C mutant channels were activated by 100 nmol/L Ca²⁺ and was inhibited by 10 mmol/L Ca²⁺ (Figures 3C-a through 3C-e). At Ca²⁺ concentrations between 1 µmol/L and 1000 µmol/L, the R4496C mutant channels were maximally activated with Po near unity. This bell-shaped Ca²⁺ response of single R4496C mutant channels (n=14) could be described by an EC₅₀ of 0.46 µmol/L and a Hill coefficient of 2.8 for Ca²⁺ activation, and an IC₅₀ of 6.3 mmol/L and a Hill coefficient of 0.9 for Ca²⁺ inactivation. Under the same conditions, single wt RyR2 channels (n=7) were activated by Ca²⁺ with an EC₅₀ of 0.42 µmol/L and a Hill coefficient of 2.2, and were inactivated by Ca²⁺ with an IC₅₀ of 7.0 mmol/L and a Hill coefficient of 1.1. These analyses indicate that the overall Ca²⁺ response of single R4496C mutant channels is similar to that of single wt channels. Taken together, it appears that mutation R4496C affects RyR2 channel gating mainly at low Ca²⁺ concentrations.

Mutation R4496C Increases the Sensitivity of RyR2 to Activation by Caffeine
To examine whether mutation R4496C affects the caffeine sensitivity of RyR2, we determined [³H]ryanodine binding to the R4496C mutant and wt RyR2 at various concentrations of caffeine. As shown in Figure 4, the wt RyR2 channels were activated by caffeine with an EC₅₀ of 2.64±0.05 mmol/L and a Hill coefficient of 1.99±0.11 (n=3). On the other hand, under the same condition, the R4496C mutant channels were activated by caffeine with an EC₅₀ of 1.44±0.01 mmol/L, significantly lower than that of the wt (P<0.0001) (n=3), and a Hill coefficient of 1.66±0.31. Therefore, mutation R4496C enhances the sensitivity of RyR2 to activation by caffeine.

View larger version (22K): [in this window] [in a new window]   Figure 4. Effect of mutation R4496C on the sensitivity of RyR2 to activation by caffeine. [3H]ryanodine binding was performed in the presence of 43 nmol/L Ca2+ (pCa=7.37) plus different concentrations of caffeine (0.01 to 20 mmol/L). A, Amounts of [3H]ryanodine binding to wt (solid circles) or mutant R4496C (open circles) at various caffeine concentrations, expressed in percentages of the maximum [3H]ryanodine binding (100%) determined at pCa=4.2 (63 µmol/L). B, Percentage increases in [3H]ryanodine binding activated by different concentrations of caffeine. Curves shown represent fits using the Hill equation. Data shown are from representative experiments each repeated 3 times.

Effects of Mutations R4496E, R4496A, and R4496K on [³H]Ryanodine Binding
To investigate whether removal of the positive charge at residue 4496 accounts for the increased basal channel activity seen in mutant R4496C, we mutated R4496 to a negatively charged glutamate (R4496E), an uncharged nonpolar alanine (R4496A), and a positively charged lysine (R4496K). The effects of these mutations on the Ca²⁺ dependence of activation were determined by [³H]ryanodine binding. As shown in Figure 5, the Ca²⁺ dependence of [³H]ryanodine binding to mutant R4496E displayed a considerable leftward shift as compared with that of the wt channels. The R4496E mutant channels were activated with an EC₅₀ of 0.12±0.01 µmol/L and a Hill coefficient of 1.70±0.38 (n=4). On the other hand, the Ca²⁺ responses of [³H]ryanodine binding to mutants R4496A and R4496K were found to be similar to that of the wt. The values of EC₅₀ and Hill coefficient were 0.24±0.03 µmol/L and 2.41±0.44 (n=3) for mutant R4496A, and 0.33±0.01 µmol/L and 2.91±0.37 (n=3) for mutant R4496K, respectively.

View larger version (19K): [in this window] [in a new window]   Figure 5. Ca2+ dependence of [3H]ryanodine binding to the R4496E, R4496A, or R4496K mutant RyR2. [3H]ryanodine binding to the R4496E (filled circles), R4496A (open circles), or R4496K (filled triangles) mutant was performed at various Ca2+ concentrations (0.2 nmol/L to 0.1 mmol/L). Concentration of [3H]ryanodine used was 5 nmol/L. Data shown are from representative experiments each repeated 3 to 4 times. Ca2+ response curves for mRyR2 (wt) (dashed line) and mutant R4496C (dotted line) were taken from Figure 1 for direct comparison.

The mutations also affect the basal level of [³H]ryanodine binding to different extents. At nanomolar Ca²⁺ concentrations (0.2 to 20 nmol/L), mutant R4496K exhibited a basal level of [³H]ryanodine binding of 1.43±0.65% maximum binding (n=3), similar to the 2.11±0.74% observed with the wt, whereas mutant R4496A showed a basal level of [³H]ryanodine binding of 5.30±1.55% (n=3), which is slightly higher than that of the wt. On the other hand, mutant R4496E displayed a basal level of [³H]ryanodine binding of 30.0±3.84% (n=4), much higher than that of the wt. Thus, these results indicate that the charge and polarity at residue 4496 have a strong influence on RyR2 channel gating, particularly at low Ca²⁺ concentrations. It should be noted that the marked increase in basal [³H]ryanodine binding activity observed in mutant R4496E does not result from nonspecific [³H]ryanodine binding. [³H]ryanodine binding to mutant R4496E at both low (3 nmol/L, pCa=8.49) (Figure 6a) and high (100 nmol/L, pCa=7.02) (Figure 6b) Ca²⁺ concentrations was inhibited by Mg²⁺ and ruthenium red, and was activated by caffeine and ATP, similar to the ligand responses seen with the R4496C mutant channel (Figure 2).

View larger version (22K): [in this window] [in a new window]   Figure 6. Effect of channel modulators on [3H]ryanodine binding to mutant R4496E. [3H]ryanodine binding to mutant R4496E was performed as described in the legend to Figure 2. Amounts of bound [3H]ryanodine at each condition are expressed as percentages of the maximum binding (100%) determined at pCa=4.2 (63 µmol/L). Data shown are mean±SEM from 3 separate experiments.

HEK293 Cells Transfected With Mutant R4496C Exhibit Frequent Spontaneous Ca²⁺ Oscillations
To test the possibility that mutation R4496C may lead to an increased propensity for spontaneous Ca²⁺ release, we measured intracellular Ca²⁺ transients in intact transfected HEK293 cells using fluorescent Ca²⁺ indicator fura-2-AM and single-cell Ca²⁺ imaging. These measurements revealed that HEK293 cells transfected with mutant R4496C displayed spontaneous Ca²⁺ oscillations much more frequently than HEK293 cells transfected with wt RyR2. In the absence of caffeine, about 16% (27 out of 169 cells from 4 separate experiments) caffeine (2 mmol/L)-sensitive R4496C-transfected HEK293 cells showed spontaneous Ca²⁺ oscillations (Figure 7A), whereas under the same conditions, only about 2% (3/177) caffeine (2 mmol/L)-sensitive wt RyR2-transfected cells exhibited spontaneous Ca²⁺ oscillations (Figure 7D). Addition of low concentrations of caffeine increased the number of oscillating cells. About 25% (43/169) caffeine-sensitive R4496C-transfected cells displayed Ca²⁺ oscillations in the presence of 0.1 mmol/L caffeine (Figures 7A and 7B), and about 30% (51/169) in the presence of 0.2 mmol/L caffeine (Figures 7A through 7C). On the other hand, about 6% (10/177) of caffeine-sensitive wt transfected cells showed Ca²⁺ oscillations in the presence of 0.1 mmol/L caffeine (Figures 7D and 7E), and about 11% (20/177) in the presence of 0.2 mmol/L caffeine (Figures 7D through 7F). It should be noted that HEK293 cells (>190) transfected with the expression plasmid pCDNA3 did not respond to caffeine, but responded to ATP (5 mmol/L) (Figure 7G). These data demonstrate that mutation R4496C increases the propensity of RyR2 for spontaneous Ca²⁺ release when expressed in HEK293 cells.

View larger version (42K): [in this window] [in a new window]   Figure 7. Spontaneous Ca2+ oscillations in mutant R4496C- and wt RyR2-transfected HEK293 cells. HEK293 cells transfected with R4496C (A through C), RyR2 (wt) (D through F), and pcDNA3 (G) were loaded with 5 µmol/L fura-2-AM in Krebs-Ringer-HEPES (KRH) buffer for 40 to 50 minutes at room temperature. Cells were perfused continuously with KRH buffer without or with 0.1 mmol/L (open box), 0.2 mmol/L (hatched box), or 2 mmol/L (filled box) caffeine as indicated. 169 caffeine (2 mmol/L)-sensitive R4496C-transfected HEK293 cells, 177 caffeine-sensitive RyR2 (wt)-transfected cells, and 190 ATP-sensitive pcDNA3-transfected cells from 4 separate experiments were analyzed for Ca2+ oscillations. Fura-2 ratios of representative cells that displayed Ca2+ oscillations in KRH buffer without and with caffeine (A and D), only in the presence of 0.1 mmol/L and 0.2 mmol/L caffeine (B and E), and only in the presence of 0.2 mmol/L caffeine (C and F) are shown. G, Fura-2 ratio of a representative wt transfected cell. No spontaneous Ca2+ oscillations or caffeine response were observed in pCDNA3-transfected cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
RyRs are a family of intracellular Ca²⁺ channels that govern the release of Ca²⁺ from the sarco(endo)plasmic reticulum of muscle or nonmuscle cells.^22,23 Given the essential roles of RyRs in excitation-contraction coupling in muscles and Ca²⁺ signaling in a variety of cells,²⁴ one would expect that alterations in RyR function would lead to various disease states. Indeed, mutations in the skeletal muscle RyR (RyR1) have been shown to cause two skeletal muscle diseases, malignant hyperthermia (MH) and central core disease (CCD).^25,26 Consistent with this view, mutations in RyR2 have also been shown recently to cause PMVT and ARVD2.^2–4

Genetic studies and mutational analyses have identified a number of MH and CCD mutations in RyR1. Almost all the MH/CCD mutations are clustered in 3 conserved regions of RyR1: the NH₂ terminal region, a region near the center (2162 to 2458), and the COOH terminal region.^25,26 Interestingly, all arrhythmogenic mutations found to date are located in the corresponding regions in RyR2.^2–4 Expression studies have shown that MH/CCD mutants exhibit an increased sensitivity to caffeine and halothane.²⁷ In addition, HEK293 cells, myocytes, or lymphocytes expressing the MH/CCD mutants showed reduced sizes of intracellular Ca²⁺ stores. These observations have led to the conclusion that MH/CCD mutations result in spontaneous Ca²⁺ leaks.^28–30 The consequences of PMVT and ARVD2 mutations in RyR2 channel function, however, are unclear. Based largely on their similar locations as the MH/CCD mutations, it has been hypothesized that the disease-associated RyR2 mutations are likely to cause the channel to open spontaneously⁴ or to increase the sensitivity of the channel to activation by Ca²⁺.²

Consistent with these hypotheses, the results of our present study demonstrate directly that mutation R4496C enhances the basal channel activity as assessed by [³H]ryanodine binding, single-channel recordings, and single-cell Ca²⁺ imaging. Mutant R4496C shows a considerable level of [³H]ryanodine binding (Figure 1) and single-channel openings (Figure 3) at nanomolar Ca²⁺ concentrations. HEK293 cells expressing the R4496C mutant exhibit frequent spontaneous Ca²⁺ oscillations (Figure 7). Like most of the MH/CCD mutants, the R4496C mutant also shows an increased sensitivity to activation by caffeine (Figures 4). Interestingly, the overall Ca²⁺ dependence of single R4496C mutant channels is indistinguishable from that of single wt channels (Figure 3), although a slight increase in the sensitivity to Ca²⁺ activation was detected by [³H]ryanodine binding assay (Figure 1). The increased Ca²⁺ sensitivity was, however, observed mainly at low Ca²⁺ concentrations. Hence, it is most likely that enhanced basal activity is the main manifestation of mutation R4496C.

The mechanism by which mutation R4496C leads to increased basal channel activity is unclear. Replacing the positively charged arginine (4496) with lysine, a relatively smaller but still positively charged amino acid, did not have significant impact on basal activity. On the other hand, replacement with a neutral amino acid, alanine, led to an increase in basal activity. Mutation R4496C, in which a positive charge was replaced with an uncharged but negatively polar group, resulted in a further increase in basal activity. A more dramatic increase in basal activity was observed when R4496 was replaced by a negatively charged glutamate (Figure 5). Thus, a positive charge at this position appears to be important for stabilizing the closed state of the channel at low Ca²⁺ concentrations, suggesting that electrostatic interactions may be involved in channel gating.

The molecular basis of how an increased basal channel activity of RyR2 could lead to catecholaminergic PMVT is also unclear. In cardiac muscle, normal Ca²⁺ release is triggered by the Ca²⁺ current through the L-type Ca²⁺ channel via the Ca²⁺-induced Ca²⁺ release mechanism.^8,9 The amount of Ca²⁺ released from the SR is controlled by the level of Ca²⁺ entry, the activity of RyR2, and the level of Ca²⁺ content in the SR.³¹ It has been shown that moderate activation of RyR2 by channel activators such as caffeine or moderate inhibition by inhibitors such as tetracaine did not cause maintained effect on the resting cytoplasmic Ca²⁺ level or the stimulated Ca²⁺ release in cardiac myocytes.³² This is because small changes in RyR2 channel activity could be compensated by the SR Ca²⁺ content. For instance, a small increase in RyR2 activity due to activation by low concentrations of caffeine would result in an increase in Ca²⁺ leak and a decreased SR Ca²⁺ content, which in turn would reduce the channel activity as a result of luminal Ca²⁺ regulation of RyR2.^33,34 Thus, in analogy, an enhanced basal channel activity observed in the R4496C mutant channel would likely be compensated by a reduced SR Ca²⁺ content and would be unlikely to result in maintained changes in Ca²⁺ transients and excitation-contraction coupling. This explanation appears to be consistent with the observation that patients with catecholaminergic PMVT show structurally and functionally normal heart and normal electrocardiograms.^5,6

Although moderate alterations in RyR2 activity may not have maintained effects on stimulated Ca²⁺ release, they can influence the properties of spontaneous Ca²⁺ release and propagating Ca²⁺ waves induced by Ca²⁺ overload. It has been shown that an increase in RyR2 activity (for instance by low concentrations of caffeine) lowered the critical SR Ca²⁺ concentration at which spontaneous Ca²⁺ release and propagating Ca²⁺ waves occur, and increased the frequency of propagating Ca²⁺ waves.³⁴ It is of interest to know that the effects on channel function of mutation R4496C resemble those of low concentrations of caffeine, and that HEK293 cells expressing the R4496C mutation show frequent spontaneous Ca²⁺ oscillations. However, whether cardiac myocytes expressing the R4496C mutation or other PMVT and ARVD2 mutations are prone to spontaneous Ca²⁺ oscillations has yet to be determined. It has long been recognized that spontaneous Ca²⁺ release can induce inward currents and delayed afterdepolarization, which could lead to triggered arrhythmia.^13–16 An increased propensity for spontaneous Ca²⁺ release and propagating Ca²⁺ waves under the condition of Ca²⁺ overload may underlie an arrhythmogenic mechanism of catecholaminergic PMVT.

It has now become clear that alterations in RyR2 channel function as a result of genetic defects can cause ventricular arrhythmias.^2–4 These findings raise a question as to whether acquired dysfunction of RyR2 could lead to arrhythmia susceptibility. Ventricular arrhythmias are common in various cardiac conditions such as heart failure,³⁵ hypertrophy,³⁶ and ischemic heart disease.³⁷ Cardiac myocytes or muscles of failing or hypertrophied hearts are known to be vulnerable to Ca²⁺ overload-induced delayed afterdepolarizations and triggered activity. It has been suggested that abnormal intracellular Ca²⁺ handling may give rise to arrhythmias in these settings.³⁸ In particular, alterations in RyR2 function may be involved. It has been shown that cardiac muscle of congestive heart failure exhibited enhanced spontaneous Ca²⁺ release,³⁹ and that RyR2 from failing hearts displayed an increased sensitivity to Ca²⁺ activation.⁴⁰ Further genetic and molecular studies of RyR2-associated arrhythmias should provide a better understanding of the role of RyR2 in cardiac arrhythmias.

	Acknowledgments

 
This work was supported by research grants from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Alberta, NWT, and Nunavut to S.R.W.C. D.J. was the recipient of Alex W. Church Graduate Student Award. S.R.W.C. is a Senior Scholar of the Alberta Heritage Foundation for Medical Research. The authors sincerely appreciate Dr Jonathan Lytton and Jeremy Dunn’s suggestions, discussions, and assistance in performing the single-cell Ca²⁺ imaging experiments and data analysis. The authors would like to thank Dr Wayne R. Giles and the Ion Channels and Transporters Group for continued support and encouragement, Dr Henk E.D.J. ter Keurs for helpful discussions, and Jeff Bolstad for critical reading of the manuscript.

Received February 7, 2002; revision received June 21, 2002; accepted June 24, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Keating MT, Sanguinetti MC. Molecular and cellular mechanisms of cardiac arrhythmias. Cell. 2001; 104: 569–580.[CrossRef][Medline] [Order article via Infotrieve]

2. Priori SG, Napolitano C, Tiso N, Memmi M, Vignati G, Bloise R, Sorrentino VV, Danieli GA. Mutations in the cardiac ryanodine receptor gene (hRyR2) underlie catecholaminergic polymorphic ventricular tachycardia. Circulation. 2001; 103: 196–200.[Abstract/Free Full Text]

3. Laitinen PJ, Brown KM, Piippo K, Swan H, Devaney JM, Brahmbhatt B, Donarum EA, Marino M, Tiso N, Viitasalo M, Toivonen L, Stephan DA, Kontula K. Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic ventricular tachycardia. Circulation. 2001; 103: 485–490.[Abstract/Free Full Text]

4. Tiso N, Stephan DA, Nava A, Bagattin A, Devaney JM, Stanchi F, Larderet G, Brahmbhatt B, Brown K, Bauce B, Muriago M, Basso C, Thiene G, Danieli GA, Rampazzo A. Identification of mutations in the cardiac ryanodine receptor gene in families affected with arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2). Hum Mol Genet. 2001; 10: 189–194.[Abstract/Free Full Text]

5. Passman R, Kadish A. Polymorphic ventricular tachycardia, long Q-T syndrome, and torsades de pointes. Med Clin North Am. 2001; 85: 321–341.[CrossRef][Medline] [Order article via Infotrieve]

6. Leenhardt A, Lucet V, Denjoy I, Grau F, Ngoc DD, Coumel P. Catecholaminergic polymorphic ventricular tachycardia in children: a 7-year follow-up of 21 patients. Circulation. 1995; 91: 1512–1519.[Abstract/Free Full Text]

7. Priori SG, Corr PB. Mechanisms underlying early and delayed afterdepolarizations induced by catecholamines. Am J Physiol. 1990; 258: H1796–H1805.[Medline] [Order article via Infotrieve]

8. Fabiato A. Time and calcium dependence of activation and inactivation of calcium- induced release of calcium from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell. J Gen Physiol. 1985; 85: 247–289.[Abstract/Free Full Text]

9. Bers DM. Excitation-Contraction Coupling, and Cardiac Contractile Force. Dordrecht, The Netherlands: Kluwer Academic Publisher; 1991.

10. Cheng H, Lederer MR, Xiao RP, Gomez AM, Zhou YY, Ziman B, Spurgeon H, Lakatta EG, Lederer WJ. Excitation-contraction coupling in heart: new insights from Ca²⁺ sparks. Cell Calcium. 1996; 20: 129–140.[CrossRef][Medline] [Order article via Infotrieve]

11. Cheng H, Lederer WJ, Cannell MB. Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science. 1993; 262: 740–744.[Abstract/Free Full Text]

12. Cheng H, Lederer MR, Lederer WJ, Cannell MB. Calcium sparks and [Ca²⁺]_i waves in cardiac myocytes. Am J Physiol. 1996; 270: C148–C159.[Medline] [Order article via Infotrieve]

13. Kass RS, Tsien RW. Fluctuations in membrane current driven by intracellular calcium in cardiac Purkinje fibers. Biophys J. 1982; 38: 259–269.[Medline] [Order article via Infotrieve]

14. Allen DG, Eisner DA, Orchard CH. Characterization of oscillations of intracellular calcium concentration in ferret ventricular muscle. J Physiol. 1984; 352: 113–128.[Abstract/Free Full Text]

15. Lakatta EG, Guarnieri T. Spontaneous myocardial calcium oscillations: are they linked to ventricular fibrillation? J Cardiovasc Electrophysiol. 1993; 4: 473–489.[Medline] [Order article via Infotrieve]

16. Marban E, Robinson SW, Wier WG. Mechanisms of arrhythmogenic delayed and early afterdepolarizations in ferret ventricular muscle. J Clin Invest. 1986; 78: 1185–1192.[Medline] [Order article via Infotrieve]

17. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989; 77: 51–59.[CrossRef][Medline] [Order article via Infotrieve]

18. Li P, Chen SR. Molecular basis of Ca²⁺ activation of the mouse cardiac Ca²⁺ release channel (ryanodine receptor). J Gen Physiol. 2001; 118: 33–44.[Abstract/Free Full Text]

19. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.

20. Fabiato A, Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris). 1979; 75: 463–505.

21. Tanna B, Welch W, Ruest L, Sutko JL, Williams AJ. Interactions of a reversible ryanoid (21-amino-9alpha-hydroxy-ryanodine) with single sheep cardiac ryanodine receptor channels. J Gen Physiol. 1998; 112: 55–69.[Abstract/Free Full Text]

22. Franzini-Armstrong C, Protasi F. Ryanodine receptors of striated muscles: a complex channel capable of multiple interactions. Physiol Rev. 1997; 77: 699–729.[Abstract/Free Full Text]

23. Shoshan-Barmatz V, Ashley RH. The structure, function, and cellular regulation of ryanodine-sensitive Ca²⁺ release channels. Int Rev Cytol. 1998; 183: 185–270.[Medline] [Order article via Infotrieve]

24. Berridge MJ, Bootman MD, Lipp P. Calcium: a life and death signal. Nature. 1998; 395: 645–648.[CrossRef][Medline] [Order article via Infotrieve]

25. McCarthy TV, Quane KA, Lynch PJ. Ryanodine receptor mutations in malignant hyperthermia and central core disease. Hum Mutat. 2000; 15: 410–417.[CrossRef][Medline] [Order article via Infotrieve]

26. Loke J, MacLennan DH. Malignant hyperthermia and central core disease: disorders of Ca²⁺ release channels. Am J Med. 1998; 104: 470–486.[CrossRef][Medline] [Order article via Infotrieve]

27. Tong J, Oyamada H, Demaurex N, Grinstein S, McCarthy TV, MacLennan DH. Caffeine and halothane sensitivity of intracellular Ca²⁺ release is altered by 15 calcium release channel (ryanodine receptor) mutations associated with malignant hyperthermia and/or central core disease. J Biol Chem. 1997; 272: 26332–26339.[Abstract/Free Full Text]

28. Tong J, McCarthy TV, MacLennan DH. Measurement of resting cytosolic Ca²⁺ concentrations and Ca²⁺ store size in HEK-293 cells transfected with malignant hyperthermia or central core disease mutant Ca²⁺ release channels. J Biol Chem. 1999; 274: 693–702.[Abstract/Free Full Text]

29. Avila G, Dirksen RT. Functional effects of central core disease mutations in the cytoplasmic region of the skeletal muscle ryanodine receptor. J Gen Physiol. 2001; 118: 277–290.[Abstract/Free Full Text]

30. Tilgen N, Zorzato F, Halliger-Keller B, Muntoni F, Sewry C, Palmucci LM, Schneider C, Hauser E, Lehmann-Horn F, Muller CR, Treves S. Identification of four novel mutations in the C-terminal membrane spanning domain of the ryanodine receptor 1: association with central core disease and alteration of calcium homeostasis. Hum Mol Genet. 2001; 10: 2879–2887.[Abstract/Free Full Text]

31. Bers DM, Li L, Satoh H, McCall E. Factors that control sarcoplasmic reticulum calcium release in intact ventricular myocytes. Ann N Y Acad Sci. 1998; 853: 157–177.[CrossRef][Medline] [Order article via Infotrieve]

32. Eisner DA, Trafford AW, Diaz ME, Overend CL, O’Neill SC. The control of Ca release from the cardiac sarcoplasmic reticulum: regulation versus autoregulation. Cardiovasc Res. 1998; 38: 589–604.[Abstract/Free Full Text]

33. Lukyanenko V, Viatchenko-Karpinski S, Smirnov A, Wiesner TF, Gyorke S. Dynamic regulation of sarcoplasmic reticulum Ca²⁺ content and release by luminal Ca²⁺-sensitive leak in rat ventricular myocytes. Biophys J. 2001; 81: 785–798.[Medline] [Order article via Infotrieve]

34. Trafford AW, Sibbring GC, Diaz ME, Eisner DA. The effects of low concentrations of caffeine on spontaneous Ca release in isolated rat ventricular myocytes. Cell Calcium. 2000; 28: 269–276.[CrossRef][Medline] [Order article via Infotrieve]

35. Pogwizd SM, Schlotthauer K, Li L, Yuan W, Bers DM. Arrhythmogenesis and contractile dysfunction in heart failure: roles of sodium-calcium exchange, inward rectifier potassium current, and residual ß-adrenergic responsiveness. Circ Res. 2001; 88: 1159–1167.[Abstract/Free Full Text]

36. Meszaros J, Khananshvili D, Hart G. Mechanisms underlying delayed afterdepolarizations in hypertrophied left ventricular myocytes of rats. Am J Physiol Heart Circ Physiol. 2001; 281: H903–H914.[Abstract/Free Full Text]

37. Opie LH, Clusin WT. Cellular mechanism for ischemic ventricular arrhythmias. Annu Rev Med. 1990; 41: 231–238.[CrossRef][Medline] [Order article via Infotrieve]

38. Missiaen L, Robberecht W, van den Bosch L, Callewaert G, Parys JB, Wuytack F, Raeymaekers L, Nilius B, Eggermont J, De Smedt H. Abnormal intracellular Ca²⁺ homeostasis and disease. Cell Calcium. 2000; 28: 1–21.[CrossRef][Medline] [Order article via Infotrieve]

39. Davidoff AW, Boyden PA., ter Keurs HEDJ. Force development in trabeculae from rats with a large myocardial infarction. Circulation. 1997; 96: I-517. Abstract.

40. Marks AR. Ryanodine receptors/calcium release channels in heart failure and sudden cardiac death. J Mol Cell Cardiol. 2001; 33: 615–624.[CrossRef][Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				N. S. Ghais, Y. Zhang, A. A. Grace, and C. L.-H. Huang Arrhythmogenic actions of the Ca2+ channel agonist FPL-64716 in Langendorff-perfused murine hearts Exp Physiol, February 1, 2009; 94(2): 240 - 254. [Abstract] [Full Text] [PDF]

				N. Liu and S. G. Priori Disruption of calcium homeostasis and arrhythmogenesis induced by mutations in the cardiac ryanodine receptor and calsequestrin Cardiovasc Res, January 15, 2008; 77(2): 293 - 301. [Abstract] [Full Text] [PDF]

				D. Jiang, W. Chen, R. Wang, L. Zhang, and S. R. W. Chen Loss of luminal Ca2+ activation in the cardiac ryanodine receptor is associated with ventricular fibrillation and sudden death PNAS, November 13, 2007; 104(46): 18309 - 18314. [Abstract] [Full Text] [PDF]

				M. Cerrone, S. F. Noujaim, E. G. Tolkacheva, A. Talkachou, R. O'Connell, O. Berenfeld, J. Anumonwo, S. V. Pandit, K. Vikstrom, C. Napolitano, et al. Arrhythmogenic Mechanisms in a Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia Circ. Res., November 9, 2007; 101(10): 1039 - 1048. [Abstract] [Full Text] [PDF]

				P. J. Mohler and X. H. T. Wehrens Mechanisms of Human Arrhythmia Syndromes: Abnormal Cardiac Macromolecular Interactions Physiology, October 1, 2007; 22(5): 342 - 350. [Abstract] [Full Text] [PDF]

				S. A. Goonasekera, N. A. Beard, L. Groom, T. Kimura, A. D. Lyfenko, A. Rosenfeld, I. Marty, A. F. Dulhunty, and R. T. Dirksen Triadin Binding to the C-Terminal Luminal Loop of the Ryanodine Receptor is Important for Skeletal Muscle Excitation Contraction Coupling J. Gen. Physiol., September 24, 2007; 130(4): 365 - 378. [Abstract] [Full Text] [PDF]

				J. Paavola, M. Viitasalo, P. J. Laitinen-Forsblom, M. Pasternack, H. Swan, I. Tikkanen, L. Toivonen, K. Kontula, and M. Laine Mutant ryanodine receptors in catecholaminergic polymorphic ventricular tachycardia generate delayed afterdepolarizations due to increased propensity to Ca2+ waves Eur. Heart J., May 1, 2007; 28(9): 1135 - 1142. [Abstract] [Full Text] [PDF]

				H. E. D. J. ter Keurs and P. A. Boyden Calcium and Arrhythmogenesis Physiol Rev, April 1, 2007; 87(2): 457 - 506. [Abstract] [Full Text] [PDF]

				L. Hove-Madsen, C. Prat-Vidal, A. Llach, F. Ciruela, V. Casado, C. Lluis, A. Bayes-Genis, J. Cinca, and R. Franco Adenosine A2A receptors are expressed in human atrial myocytes and modulate spontaneous sarcoplasmic reticulum calcium release Cardiovasc Res, November 1, 2006; 72(2): 292 - 302. [Abstract] [Full Text] [PDF]

				N. Liu, B. Colombi, M. Memmi, S. Zissimopoulos, N. Rizzi, S. Negri, M. Imbriani, C. Napolitano, F. A. Lai, and S. G. Priori Arrhythmogenesis in Catecholaminergic Polymorphic Ventricular Tachycardia: Insights From a RyR2 R4496C Knock-In Mouse Model Circ. Res., August 4, 2006; 99(3): 292 - 298. [Abstract] [Full Text] [PDF]

				M. Fernandez-Velasco, A. M. Gomez, and S. Richard Unzipping RyR2 in adult cardiomyocytes: Getting closer to mechanisms of inherited ventricular arrhythmias? Cardiovasc Res, June 1, 2006; 70(3): 407 - 409. [Full Text] [PDF]

				Z. Yang, N. Ikemoto, G. D. Lamb, and D. S. Steele The RyR2 central domain peptide DPc10 lowers the threshold for spontaneous Ca2+ release in permeabilized cardiomyocytes Cardiovasc Res, June 1, 2006; 70(3): 475 - 485. [Abstract] [Full Text] [PDF]

				C. H. George, H. Jundi, N. Walters, N. L. Thomas, R. R. West, and F. A. Lai Arrhythmogenic Mutation-Linked Defects in Ryanodine Receptor Autoregulation Reveal a Novel Mechanism of Ca2+ Release Channel Dysfunction Circ. Res., January 6, 2006; 98(1): 88 - 97. [Abstract] [Full Text] [PDF]

				S. G. Priori and C. Napolitano Intracellular Calcium Handling Dysfunction and Arrhythmogenesis: A New Challenge for the Electrophysiologist Circ. Res., November 25, 2005; 97(11): 1077 - 1079. [Full Text] [PDF]

				D. Jiang, R. Wang, B. Xiao, H. Kong, D. J. Hunt, P. Choi, L. Zhang, and S. R. W. Chen Enhanced Store Overload-Induced Ca2+ Release and Channel Sensitivity to Luminal Ca2+ Activation Are Common Defects of RyR2 Mutations Linked to Ventricular Tachycardia and Sudden Death Circ. Res., November 25, 2005; 97(11): 1173 - 1181. [Abstract] [Full Text] [PDF]

				I. Zahradnik, S. Gyorke, and A. Zahradnikova Calcium Activation of Ryanodine Receptor Channels--Reconciling RyR Gating Models with Tetrameric Channel Structure J. Gen. Physiol., October 31, 2005; 126(5): 515 - 527. [Abstract] [Full Text] [PDF]

				R. Balasubramaniam, S. Chawla, A. A. Grace, and C. L.-H. Huang Caffeine-induced arrhythmias in murine hearts parallel changes in cellular Ca2+ homeostasis Am J Physiol Heart Circ Physiol, October 1, 2005; 289(4): H1584 - H1593. [Abstract] [Full Text] [PDF]

				K. Kontula, P. J. Laitinen, A. Lehtonen, L. Toivonen, M. Viitasalo, and H. Swan Catecholaminergic polymorphic ventricular tachycardia: Recent mechanistic insights Cardiovasc Res, August 15, 2005; 67(3): 379 - 387. [Abstract] [Full Text] [PDF]

				G.-B. Nam, A. Burashnikov, and C. Antzelevitch Cellular Mechanisms Underlying the Development of Catecholaminergic Ventricular Tachycardia Circulation, May 31, 2005; 111(21): 2727 - 2733. [Abstract] [Full Text] [PDF]

				S. R. Houser Can Novel Therapies for Arrhythmias Caused by Spontaneous Sarcoplasmic Reticulum Ca2+ Release be Developed Using Mouse Models? Circ. Res., May 27, 2005; 96(10): 1031 - 1032. [Full Text] [PDF]

				M. Cerrone, B. Colombi, M. Santoro, M. R. di Barletta, M. Scelsi, L. Villani, C. Napolitano, and S. G Priori Bidirectional Ventricular Tachycardia and Fibrillation Elicited in a Knock-In Mouse Model Carrier of a Mutation in the Cardiac Ryanodine Receptor Circ. Res., May 27, 2005; 96(10): e77 - e82. [Abstract] [Full Text] [PDF]

				A. M. Gomez and S. Richard Mutant cardiac ryanodine receptors and ventricular arrhythmias: is 'gain-of-function' obligatory? Cardiovasc Res, October 1, 2004; 64(1): 3 - 5. [Full Text] [PDF]

				N. Lowri Thomas, C. H. George, and F. Anthony Lai Functional heterogeneity of ryanodine receptor mutations associated with sudden cardiac death Cardiovasc Res, October 1, 2004; 64(1): 52 - 60. [Abstract] [Full Text] [PDF]

				D. Jiang, B. Xiao, D. Yang, R. Wang, P. Choi, L. Zhang, H. Cheng, and S. R. W. Chen RyR2 mutations linked to ventricular tachycardia and sudden death reduce the threshold for store-overload-induced Ca2+ release (SOICR) PNAS, August 31, 2004; 101(35): 13062 - 13067. [Abstract] [Full Text] [PDF]

				X. H.T. Wehrens, S. E. Lehnart, S. R. Reiken, and A. R. Marks Ca2+/Calmodulin-Dependent Protein Kinase II Phosphorylation Regulates the Cardiac Ryanodine Receptor Circ. Res., April 2, 2004; 94(6): e61 - e70. [Abstract] [Full Text] [PDF]

				S. Viatchenko-Karpinski, D. Terentyev, I. Gyorke, R. Terentyeva, P. Volpe, S. G. Priori, C. Napolitano, A. Nori, S. C. Williams, and S. Gyorke Abnormal Calcium Signaling and Sudden Cardiac Death Associated With Mutation of Calsequestrin Circ. Res., March 5, 2004; 94(4): 471 - 477. [Abstract] [Full Text] [PDF]

				P.D. Allen Not All Sudden Death Is the Same Circ. Res., September 19, 2003; 93(6): 484 - 486. [Full Text] [PDF]

				C. H. George, G. V. Higgs, and F. A. Lai Ryanodine Receptor Mutations Associated With Stress-Induced Ventricular Tachycardia Mediate Increased Calcium Release in Stimulated Cardiomyocytes Circ. Res., September 19, 2003; 93(6): 531 - 540. [Abstract] [Full Text] [PDF]

				T. Yang, T. A. Ta, I. N. Pessah, and P. D. Allen Functional Defects in Six Ryanodine Receptor Isoform-1 (RyR1) Mutations Associated with Malignant Hyperthermia and Their Impact on Skeletal Excitation-Contraction Coupling J. Biol. Chem., July 3, 2003; 278(28): 25722 - 25730. [Abstract] [Full Text] [PDF]

				M Scoote, P A Poole-Wilson, and A J Williams The therapeutic potential of new insights into myocardial excitation-contraction coupling Heart, April 1, 2003; 89(4): 371 - 376. [Abstract] [Full Text] [PDF]

				M. Scoote and A. J Williams The cardiac ryanodine receptor (calcium release channel): Emerging role in heart failure and arrhythmia pathogenesis Cardiovasc Res, December 1, 2002; 56(3): 359 - 372. [Abstract] [Full Text] [PDF]

				P.D. Allen Leaky "Feet" and Sudden Death Circ. Res., August 9, 2002; 91(3): 181 - 182. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 91/3/218    most recent 01.RES.0000028455.36940.5Ev1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jiang, D. Articles by Chen, S.R. W. Search for Related Content PubMed PubMed Citation Articles by Jiang, D. Articles by Chen, S.R. W. Related Collections Animal models of human disease Gene expression Genetically altered mice