Skip Navigation

Cardiovascular Research 2006 70(3):475-485; doi:10.1016/j.cardiores.2006.03.001
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Corrigendum (v71,p606)
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Yang, Z.
Right arrow Articles by Steele, D. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, Z.
Right arrow Articles by Steele, D. S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2006, European Society of Cardiology

The RyR2 central domain peptide DPc10 lowers the threshold for spontaneous Ca2+ release in permeabilized cardiomyocytes

Zhaokang Yanga, Noriaki Ikemotob, Graham D. Lambc and Derek S. Steelea,*

aSchool of Biomedical Sciences, University Of Leeds, Leeds, LS2 9JT, UK
bBoston Biomedical Research Institute, Watertown, MA 02472, USA
cDepartment of Zoology, La Trobe University, Bundoora, Victoria 3086, Australia

* Corresponding author. Tel.: +44 1133432912. Email address: d.steele{at}leeds.ac.uk

Received 1 December 2005; revised 6 February 2006; accepted 2 March 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Objective In vitro experiments have shown that the ryanodine receptor-2 (RyR2) central domain peptide DPc10 (Gly2460-Pro2495) mimics channel dysfunction associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) by acting competitively to reduce stabilizing interactions between the N-terminal and central domains. In the present study, DPc10 was used as a tool to establish an adult cell model of the disease and to analyse the underlying mechanisms.

Methods Rat ventricular myocytes were permeabilized with saponin and perfused with solutions approximating the intracellular milieu containing fluo-3. Sarcoplasmic reticulum (SR) Ca2+ release was detected using confocal microscopy. DPc10 (10 or 50µM) was compared with 0.2mM caffeine, which is known to activate RyR2 and to facilitate Ca2+-induced Ca2+ release (CICR).

Results Introduction of DPc10 induced a transient increase in spark frequency and a sustained rise in resting [Ca2+]. Under conditions causing initial Ca2+ overload of the SR, DPc10 reduced the frequency and amplitude of spontaneous, propagated Ca2+ release (SPCR). Following equilibration with 10µM DPc10, the cytosolic [Ca2+] threshold for SPCR was markedly reduced and the proportion of spontaneously active cells increased. Caffeine induced a similar, transient increase in spark frequency and a reduction in the [Ca2+] threshold for SPCR. However, unlike DPc10, caffeine increased SPCR frequency and had no sustained effect on resting [Ca2+]. These results suggest that the net effect of DPc10 (and CPVT mutations) on RyR2 function in situ is not only to increase the sensitivity to CICR as caffeine does, but also to potentiate Ca2+ leakage from the SR. As SPCR can trigger delayed after-depolarisations, the decrease in [Ca2+] threshold may contribute to arrhythmias in CPVT patients during exercise or stress.

KEYWORDS Sarcoplasmic reticulum; Ryanodine receptor; Mutation; Ca2+; Sparks; Autoregulation

This article is referred to in the Editorial by Fernadez-Velasco et al. (pages 407–409) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Missense mutations in RyR2 have been implicated in a number of cardiomyopathies, including CPVT and type-2 arrhythmogenic right ventricular cardiomyopathy (ARVD2) [1-3]. Affected patients experience tachyarrhythmias during exercise or stress and are at increased risk of sudden cardiac death. The identified RyR2 mutations are clustered in the same three regions of the channel as the RyR1 mutations that underlie malignant hyperthermia (MH) and central core disease in skeletal muscle (Fig. 1) [4]. Functional studies suggest that RyR mutations linked to MH, CPVT and ARVD2 exhibit increased sensitivity to agonists and enhanced CICR [5-8]. This can readily explain the development of MH, where hypersensitivity of RyR1 to volatile anaesthetics leads to a sustained rise in [Ca2+]i and subsequent contracture. However, the link between RyR2 mutations and myocardial dysfunction is less clear, as previous work suggests that facilitation of CICR induces only transient changes in systolic or diastolic Ca2+ release [9,10]. Indeed, this autoregulation of SR Ca2+ release has led to the proposal that RyR2 modulators cannot induce maintained functional changes in cardiac cells [9].


Figure 1
View larger version (53K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 RyR2 mutations associated with CPVT/ARVD2 and peptide probe method. (A) RyR2 mutation hotspots associated with the N-terminal, central and pore-forming domains. ARVD2 mutations are indicated in bold and CPVT mutations in plain text (except R2474S, red). The structures of DPc10-mut and DPc10 peptides are indicated below. (B) The proposed interaction between the N-terminal and central domains (left). The postulated domain UnzippingIs caused either by the R2474S mutation or a competitive interaction with DPc10 (right). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

 
A number of studies have characterised the properties of RyR2 mutations expressed in cultured cells. In HEK293 or HL-1 cells, RyR2 mutations associated with CPVT or ARVD2 exhibited increased sensitivity to activation by caffeine [7,8]. In further studies, expression of RyR2 mutations associated with CPVT lowered the [Ca2+] threshold for SPCR [11,12]. However, it is not clear to what extent these results obtained in cultured cells are applicable to adult myocytes. Expression of both wild type and mutant channels was associated with an increase in the maximum SR/ER Ca2+ content [7,8]. This may have occurred due to compensatory changes in the expression of other proteins involved in Ca2+ regulation (e.g. SERCA) [13], which complicates interpretation of these data. The physiological regulation of RyR2 is also known to involve numerous accessory proteins, which may be absent in RyR-deficient cell lines. Finally, it is not known whether autoregulation of Ca2+ release occurs in cultured cell models.

Recent work has shown that the functional characteristics of specific RyR mutations can be induced by exposure to peptides corresponding to regions of the N-terminal or central domains (Fig. 1A). Briefly, it has been proposed that (i) close contact between the N-terminal and central domains stabilizes the closed state of the channel and (ii) ‘hyper-activation’ occurs because RyR mutations decrease the interaction between these domain pairs [14,15]. Based on this proposal, a peptide containing the wild-type sequence would be expected to interact competitively to weaken the normal domain–domain interaction, thereby mimicking the disease state (Fig. 1B). Of equal importance, a peptide containing a cardiac disease mutation should lack the ability to interact with the wild-type domain pair and have little effect on channel gating. In support of this hypothesis, a peptide corresponding to the Gly2460-Pro2495 region of the RyR2 central domain (DPc10) enhanced ryanodine binding and increased the sensitivity of the channel to activating Ca2+ in SR vesicles, while a similar peptide (DPc10-mut) containing a point mutation linked to CPVT was without effect [16]. In a more recent study, DPc10 decreased the systolic Ca2+ transient in intact myocytes, consistent with SR Ca2+ depletion [15]. However, it is not clear why such an effect should precipitate arrhythmias in CPVT patients.

In the present study, the effects of DPc10 and DPc10-mut were characterised in permeabilized ventricular myocytes. The effects of these peptides were compared with those of caffeine, which has been used widely to facilitate CICR and to study the properties of autoregulation. The results are considered in relation to CPVT and previous studies involving cultured cell models of the disease.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1 Myocyte isolation and permeabilization
The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Adult Wistar rats (220–250g) were sacrificed and ventricular myocytes isolated by collagenase digestion as described previously [17]. Cells were permeabilized by exposure to saponin (10µg/ml) in a mock intracellular solution for 6min, before centrifugation and re-suspension. Unless otherwise stated, chemicals were obtained from the Sigma Chemical Corporation, UK.

2.2 Solutions
Permeabilized cells were perfused with weakly Ca2+-buffered solutions approximating to the intracellular milieu and SR Ca2+ release was detected using fluo-3. The basic solution contained (mM): KCl, 100; HEPES, 25; EGTA, 0.05–0.36; phosphocreatine 10; ATP, 5 and fluo-3, 0.001, pH 7.0, 22°C. MgCl2 was added (from 1M stock solution) to produce a free concentration of 1.0mM. The free [Ca2+] was adjusted by addition of CaCl2. 5mM sodium azide was included in the solutions to inhibit mitochondrial activity.

The peptides DPc10 (2460GFCPDHKAAMVLFLDRVYGIEVQDFLLHLLEVGFLP2495) and DPc10-mut containing Arg2474-Ser2474 mutation (2460GFCPDHKAAMVLFLDSVYGIEVQDFLLHLLEVGFLP2495) were synthesized on an Applied Biosystems model 431A synthesizer employing Fmoc (N-(9-fluorenyl)methoxycarbonyl) as the {alpha}-amino protecting group (see Fig. 1). The peptides were cleaved and de-protected with 95% trifluoroacetic acid and purified by reversed phase high-pressure liquid chromatography.

2.3 Confocal Ca2+ measurement
The apparatus used for [Ca2+] measurement has been described previously [17]. Briefly, the cells were placed in a cylindrical bath (5mm diameter) in a Perspex block. The bottom of the bath was formed by attaching a coverslip to the underside of the block. A drop of solution containing cells was placed at the bottom of the bath and a tightly fitting Perspex column inserted into the well until the lower surface was close to myocytes resting on the coverslip. Perfusion was achieved by pumping solution (0.3ml/min) down a narrow bore running longitudinally through the column.

The chamber was placed on the stage of a Nikon Diaphot Eclipse TE2000 inverted microscope and cells were viewed using a confocal laser-scanning unit (Microradiance, Bio-Rad, Herts, UK) via a 60 x water immersion lens (Plan Apo,NA 1.2). The dye was excited at 488nm and emitted fluorescence was measured at >515nm. Image processing and analysis were done using IDL (Research Systems Inc., Boulder, CO, USA) and Laserpix (Bio-Rad, Herts, UK).

2.4 Data analysis and statistics
Data are presented as mean values±S.E.M. Statistical significance was determined using a paired t-test, except for the data shown in Fig. 5, where a Chi-squared test was used. Significance levels were calculated using Origin (Microcal, MA, USA) software. p<0.05 was considered significant.


Figure 5
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Effects of DPc10 and caffeine on the [Ca2+] threshold for SPCR. (A) Permeabilized myocytes were exposed to stepwise increases in bathing [Ca2+] over the range 44–240nM in solutions containing 0.2mM EGTA. Representative records of fluo-3 fluorescence are shown from cells under control conditions or in the presence of 10µM DPc10, 10µM DPc10-mut or 0.2mM caffeine. (B) Accumulated data illustrating the percentage of cells exhibiting SPCR under each condition, as a function of bathing [Ca2+]. Values significantly different from corresponding controls are indicated (*p<0.05, **p<0.02, ***p<0.01, n=11–19).

 

   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1 Effects of DPc10 or caffeine on spontaneous Ca2+ sparks
The effects of DPc10 on Ca2+ sparks were studied in permeabilized myocytes perfused with a solution containing 220nM Ca2+ and 0.36mM EGTA. Under these conditions, the Ca2+ buffer capacity is sufficient to prevent SPCR [18]. Line scan images were obtained under control conditions and during a subsequent 2min exposure to 50µM DPc10 (Fig. 2A, upper). In this example, occasional spontaneous Ca2+ sparks were apparent under control conditions. Introduction of 50µM DPc10 caused a rapid increase in spark frequency, which peaked after approximately 30s. During the following 90s exposure, the frequency returned towards the control level. Throughout exposure to 50µM DPc10, spark amplitude declined (without a significant change in width), while the line scan image became brighter, consistent with a diffuse Ca2+ leak from the SR. In contrast, 50µM DPc10-mut had no significant effect on spark frequency (Fig. 2A, middle). Exposure to 0.2mM caffeine also induced a transient increase in spark frequency. However, unlike DPc10, there was no evidence of a sustained, diffuse rise in [Ca2+].


Figure 2
View larger version (59K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Effects of DPc10 and DPc10-mut on spontaneous Ca2+ sparks. (A) Line scan images obtained from a permeabilized myocyte under control conditions and 5, 30, 60 and 90s after introduction of 50µM DPc10 or DPc10-mut or 0.2mM caffeine. (B) Accumulated data showing the effects of 50µM DPc10 (green), 10µM DPc10 (blue) and 50µM DPc10-mut (orange) and 0.2mM caffeine (brown) on the frequency, amplitude and duration of spontaneous Ca2+ sparks. The effects of DPc10 and caffeine on resting [Ca2+] is also shown (lower right). The free [Ca2+] of the perfusing solution was 200nM throughout, EGTA 0.36mM. Responses are expressed relative to the mean control values obtained in the absence of DPc10, DPc10-mut or caffeine. The duration was measured at half maximal amplitude. Error bars represent the mean±S.E.M. (n=6–11). Values differing significantly from control (*p<0.05; **p<0.02, ***p<0.005) are indicated.

 
Accumulated data showing the effects of DPc10, DPc10-mut and caffeine on the properties of Ca2+ sparks and on resting [Ca2+] is given in Fig. 2B. Spark frequency increased markedly within 5s of exposure to 50µM DPc10 and peaked after ~30s. Thereafter, spark frequency decreased and was not significantly different from controls after 120s. Throughout exposure to 50µM DPc10, spark amplitude declined and the duration increased slightly. Resting [Ca2+] increased during exposure to 50µM DPc10, although the rate of increase slowed markedly after 120s, suggesting progression towards a new equilibrium state. The effects of 10µM DPc10 were qualitatively similar, although the increase in spark frequency was less pronounced and the effects developed more slowly. In contrast, 50µM DPc10-mut had no influence on resting [Ca2+] or spark properties. Caffeine transiently increased the frequency of Ca2+ sparks and the peak value similar to that induced by 10µM DPc10, although the effect developed more rapidly and there was no sustained effect on resting [Ca2+].

3.2 Effects of DPc10 or caffeine on SPCR
In the presence of 220nM Ca2+ and 0.05mM EGTA, permeabilized myocytes exhibited repeated SPCR. In the example shown in Fig. 3A (upper left), SPCR occurred at ~40s intervals. Introduction of 50µM DPc10 induced a slow diffuse increase in resting [Ca2+] and a decrease in SPCR frequency, followed by complete cessation of SPCR. While resting [Ca2+] declined slightly following removal of 50µM DPc10, SPCR was not restored after 3–4min. However, following much longer washout periods (>20min), cells exhibited localised SPCR (not shown).


Figure 3
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Effects of DPc10 and caffeine on SPCR. (A) Fluorescence records from permeabilized myocytes perfused with a solution containing 220nM Ca2+, 0.05mM EGTA. Each transient increase in fluorescence reflects a spontaneous release of Ca2+ from the SR. Introduction of 50µM DPc10 caused a slow diffuse increase in resting [Ca2+] followed by abolition of SPCR. Introduction of 10µM DPc10 caused a slow diffuse rise in resting [Ca2+] and a decrease in the SPCR frequency. DPc10-mut had no effect on the frequency or amplitude of SPCR. 0.2mM caffeine increased the frequency and decreased the amplitude of SPCR, but had no effect on resting [Ca2+]. (B) Accumulated data showing the relative changes in the frequency and amplitude of SPCR in the presence of 10µM DPc10 or 50µM DPc10-mut and 0.2mM caffeine. Error bars represent the mean±S.E.M. Values differing significantly from control (*p<0.05; ***p<0.005, n=8–12).

 
Similar changes occurred following introduction of 10µM DPc10, although SPCR was not abolished (upper right). In contrast, 50µM DPc10-mut had no effect on resting [Ca2+] or SPCR (lower left). Introduction of 0.2mM caffeine was associated with a rapid decrease in the amplitude of the spontaneous Ca2+ transients (lower right). However, caffeine increased the release frequency and had no apparent effect on resting [Ca2+]. Accumulated data illustrating the effect of DPc10, DPc10-mut and caffeine on the frequency and amplitude of SPCR is shown in Fig. 3B. In the presence of 10µM DPc10, the frequency and amplitude decreased by 49.6±15.2% (n=8) and 54.5±3.3% (n=8) respectively. There was no significant effect of 50µM DPc10-mut on either the frequency or amplitude of SPCR (n=12, p>0.05). Caffeine significantly increased the frequency and decreased the amplitude of spontaneous Ca2+ release to 151.1±7.2% and 77.1±3.4% respectively.

3.3 Effects of DPc10 or caffeine on the SR Ca2+ content
Figs. 2 and 3Go show that DPc10 induces both a sustained, diffuse increase in [Ca2+] and a transient increase in the frequency of spontaneous Ca2+ sparks. Therefore, experiments were carried out to assess the consequent effects on the SR Ca2+ content. In Fig. 4A, a cell was perfused with a solution containing 220nM Ca2+ and 0.36mM EGTA. Caffeine (20mM) was applied rapidly at 30s intervals and the amplitude of the resulting Ca2+ transient was used as an index of the SR Ca2+ content. Introduction of 10µM or 50µM DPc10 induced a concentration-dependent decrease in the amplitude of the caffeine-induced Ca2+ transient. It is noteworthy that the abolition of SPCR in 50µM DPc10 occurred with only an ~20% reduction in the amplitude of the caffeine-induced transient. The small size of this reduction in SR Ca2+ content can be explained if re-accumulation of Ca2+ via SERCA largely compensates for the sustained diastolic [Ca2+] leak induced by DPc10. Increased SERCA activity is also suggested by the significantly larger (by 38.3±11.5%, n=9, p<0.05) undershoot in each Ca2+ transient following introduction of 50µM DPc10(inset). This possibility was further investigated by exposure of the cells to 2µM cyclopiazonic acid (CPA), which is sufficient to partially inhibit SERCA [17]. Inhibition of SERCA will impair the ability of the SR to re-accumulate any Ca2+ leak.


Figure 4
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Effects of DPc10 on the SR Ca2+ content. (A) Permeabilized cells were perfused with solutions containing 200nM Ca2+ (0.36mM EGTA) and 20mM caffeine was briefly applied at 30s intervals. The amplitude of the caffeine-induced transient was used as an index of the SR Ca2+ content. After 4 control responses to caffeine, the solution was changed to one containing 10µM DPc10 (upper, left), 50µM DPc10 (upper right) or 50µM DPc10-mut (lower left). Inset shows superimposed undershoots in the fluorescence transient before and after addition of 50µM DPc10. In the final record (lower right), SERCA was partially inhibited with 2µM CPA before introduction of 50µM DPc10. (B) Accumulated data showing the amplitude of the caffeine-induced transient under each condition, expressed as a percentage of the preceding control responses. Also shown is the effect of 2µM CPA, 0.2mM caffeine (in the presence or absence of 2µM CPA) and 0.2mM CPA+50µM DPc10-mut. Error bars represent the mean±S.E.M. Values differing significantly from control (*p<0.05; **p<0.02, ***p<0.005; n.s., not significant) are indicated (n=5–6).

 
Fig. 4A (lower right) shows a series of responses to rapid application of 20mM caffeine following equilibration with a solution containing 2µM CPA. In this example, the CPA caused a small (~13%) decrease in the caffeine-induced Ca2+ transients (not shown). However, in the presence of CPA, introduction of 50µM DPc10 resulted in a profound decrease in the caffeine-induced response (Fig. 4A, lower right).

The cumulative data (Fig. 4B) show that the amplitude of the caffeine-induced Ca2+ transient decreased by 8.5±1.9% (n=6) and 21.3±4.1% (n=6) in the presence of 10 and 50µM DPc10 respectively, while DPc10-mut was without effect. On average, 2µM CPA or 0.2mM caffeine alone reduced the amplitude of the transients by only 13.8±2%, (n=5) or 7.6±2% (n=8) respectively. In combination, CPA and 0.2mM caffeine, or CPA and 50µM DPc10-mut decreased the transient amplitude by 27.1±3.4% (n=5) and 21.2±3.1% (n=5) respectively. This contrasts with the much greater effect of CPA and 50µM DPc10, which together decreased the caffeine-induced Ca2+ transient by 62±2.7% (n=5).

3.4 Effects of DPc10 or caffeine on the threshold for SPCR
The effect of DPc10 on the [Ca2+] threshold for SPCR was studied in permeabilized ventricular myocytes. Under control conditions, cells were exposed to stepwise increases in [Ca2+] over the range 44–240nM (Fig. 5A). SPCR typically occurred when the bathing [Ca2+] was increased to 140nM. Further rises in [Ca2+] were associated with corresponding increases in the release frequency, while the amplitude declined. Following equilibration with 10µM DPc10, SPCR was apparent at a lower cytosolic [Ca2+]. In contrast, 10µM DPc10-mut had no effect on the threshold [Ca2+] for SPCR. The effects of 10µM DPc10 were compared with caffeine, which is known to increase the sensitivity of RyR2 to cytoplasmic Ca2+ but with its net effect on triggered Ca2+ release being offset in the longer term by the resulting reduction in SR Ca2+ content, in a process referred to as autoregulation [9]. In the presence of 0.2mM caffeine, most cells also developed SPCR at a lower [Ca2+] than controls.

The accumulated data show the percentage of cells exhibiting SPCR as a function of cytosolic [Ca2+] under each condition (Fig. 5B). Control cells were consistently quiescent in the presence of 44–92nM Ca2+. SPCR was apparent in 19% of cells in the presence of 120nM Ca2+ and progressively raising [Ca2+] to 140nM and 176nM increased the percentage of spontaneously active cells to 52.9% and 64.7% respectively. Further increases in [Ca2+] to 207nM or 240nM had no effect on the number of spontaneously active cells. In the presence of 10µM DPc10, the relationship was steeper and shifted towards lower [Ca2+], while the maximum number of spontaneously active cells increased to 91%. In the presence of 0.2mM caffeine, the proportion of spontaneously active cells at [Ca2+]leg120nM increased significantly. However, there was no significant effect of caffeine at 92nM [Ca2+]. DPc10-mut (10µM) had no significant effect on SPCR.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
4.1 Transient vs. sustained changes in SR Ca2+ release
Introduction of caffeine was associated with a transient increase in the frequency of spontaneous Ca2+ sparks. This is consistent with studies describing autoregulation of diastolic Ca2+ release in response to caffeine and other modulators of CICR [10]. Autoregulation occurs because the initial increase in the open probability of RyR2 is counteracted by the fall in SR luminal [Ca2+], which progressively inhibits RyR2 gating. In the continued presence of caffeine, spark frequency returns to control levels, where Ca2+ efflux is again balanced by uptake (Fig. 6A). Introduction of DPc10 was associated with a similar, transient increase in Ca2+ spark frequency, suggesting that the peptide may also facilitate CICR (Fig. 2). Nevertheless, differences between the effects of DPc10 and caffeine suggest that modulation of CICR may not be the only action of the peptide. Unlike caffeine, DPc10 induced a sustained, diffuse increase in resting [Ca2+]. As sparks reflect the synchronised activation of RyR2 clusters [19], the diffuse release probably involves Ca2+ efflux via smaller units, or single channels. The sustained nature of the diffuse release indicates that (i) it is not tightly dependent on the SR Ca2+ content and (ii) it does not require self-reinforcement by CICR (which is subject to autoregulation). Thus, the binding of DPc10 likely initially increases spark frequency by sensitizing CICR, but the resulting SR Ca2+ depletion gradually counteracts this action, leaving a tonic hyperactivating effect of the peptide.


Figure 6
View larger version (64K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Effects of caffeine and DPc10 on SR Ca2+ uptake and release fluxes. (A) Ca2+ fluxes across the SR membrane under steady-state conditions (left), following introduction of 0.2mM caffeine (middle) and after a new steady state is achieved in the continued presence of caffeine (right). Note that Ca2+ release and uptake fluxes are identical in the presence or absence of caffeine. (B) Ca2+ fluxes under steady-state conditions in the presence of DPc10 (left). Note that in (A) the effect of partial SERCA inhibition will have a similar effect under control conditions (left) and after equilibration with 0.2mM caffeine (right). In (B), partial inhibition of SERCA will have a greater effect following equilibration with DPc10 (right) than under control conditions.

 
Experiments on cells exhibiting SPCR also suggest such differences between the actions of caffeine and DPc10. SPCR occurs when the SR Ca2+ content increases to a point where the gain of the CICR mechanism can support propagation between Ca2+ release sites [20]. As described previously, introduction of 0.2mM caffeine increased the SPCR frequency and decreased the amplitude [10]. This can be explained if caffeine sensitizes the CICR mechanism, such that the threshold for release propagation occurs at a lower luminal [Ca2+], resulting in a smaller transient. DPc10 also reduced the amplitude of the SPCR transient, consistent with a reduced threshold for propagated Ca2+ release. However, unlike caffeine, 10µM DPc10 decreased SPCR frequency and 50µM DPc10 abolished release completely. Importantly, this is unlikely to reflect the relative sensitization of CICR induced by caffeine and DPc10. Increasing the caffeine concentration above 0.2mM results in progressively higher frequency and lower amplitude Ca2+ release events before eventual abolition of SPCR at levels >1–2mM [21]. Indeed, we have never observed a decrease in SPCR frequency with any intervention that facilitates CICR. However, a decrease in SPCR frequency can be induced by either partial inhibition of SERCA [17] or introduction of a Ca2+ ionophore, which causes a sustained unregulated SR Ca2+ leak (unpublished observations). Both of these interventions reduce the net Ca2+ uptake rate, thereby increasing the time to reach the threshold for SPCR. This suggests that (i) the decrease in SPCR frequency induced by DPc10 is a consequence of the sustained, diffuse Ca2+ leak and (ii) this latter phenomenon is not mediated by sensitization of CICR.

The existence of a substantial, sustained leak in DPc10 was also shown from the effects of SERCA inhibition. Although 50µM DPc10 reduced the caffeine-induced Ca2+ transient by only 21.2%, this decrease in SR Ca2+ content reflects the balance between the Ca2+ leak and re-uptake via SERCA. After partial inhibition of SERCA, 50µM DPc10 reduced the SR Ca2+ content by >60%. In contrast, the combined effect of 0.2mM caffeine and CPA reduced the SR Ca2+ content by <30% (Fig. 4). The much smaller effect in the case of caffeine reflects the absence of a sustained release component and the fact that steady-state RyR2 mediated Ca2+ leak is the same with the presence and absence of the drug (Fig. 6A). In contrast, when the SR Ca2+ fluxes approach a new steady-state following addition of DPc10, there is evidently a sustained increase in the RyR2 mediated Ca2+ leak, which is balanced by an increased Ca2+ uptake via SERCA, supported by a local rise in [Ca2+] (Figs. 4 and 6GoB).

The properties of the diffuse Ca2+ leak appear consistent with single channel experiments on another CPVT mutant (R4497C), which exhibited enhanced Ca2+-independent basal activity [6]. The same study also reported an increase in the sensitivity of RyR2 to cytosolic Ca2+, which could contribute to the transient increase in spark frequency and the decrease in SPCR amplitude. More recently, it has been suggested that CPVT mutations exhibit enhanced CICR due to a primary defect in the regulation of RyR2 by SR luminal Ca2+ [12]. However, given that regulation of RyR2 by cytosolic and luminal Ca2+ are highly interdependent under physiological conditions, such a clear distinction may not be appropriate.

4.2 The cytosolic [Ca2+] threshold for SPCR
In the present study, 0.2mM caffeine induced a sustained decrease in the [Ca2+] threshold for SPCR (Fig. 6). This is not at variance with previous studies suggesting that caffeine produces only transient effects on SR Ca2+ regulation: Although systolic and diastolic Ca2+ release return to control levels in the presence of caffeine, this is accompanied by a sustained decrease in the SR Ca2+ content [9,10]. Therefore, factors which facilitate CICR actually cause a maintained decrease in the SR Ca2+ content required to support a given spark frequency (or systolic Ca2+ release). Consistent with this interpretation, a decrease in the threshold for spontaneous release reflects a maintained reduction in the minimum SR Ca2+ content necessary for propagation between Ca2+ release sites. Hence, although autoregulation ensures that factors that increase (moderately) the open probability of RyR2 have no functional consequences when [Ca2+]i is low, detrimental effects will become apparent when [Ca2+]i rises.

Like caffeine, 10µM DPc10 decreased the cytosolic [Ca2+] threshold for SPCR. Indeed, this effect was significantly greater than that which occurred with 0.2mM caffeine, despite a similar potentiation of spark frequency (Fig. 2), which is an index of CICR potentiation. One reason for the greater effect seen with DPc10 is that the accompanying diastolic Ca2+ leak induced by DPc10 quite likely contributes to the overall effect of the peptide on spontaneous release. Recent data on RyR clusters, incorporated into lipid bilayers, suggest that Ca2+ released by one RyR can facilitate the activation of neighbouring channels [22]. Therefore, in the present study, a tonic Ca2+ leak mediated by some RyRs would be expected to influence the gating of neighbouring channels and in particular, their response to progressively rising levels of cytosolic [Ca2+].

4.3 Clinical relevance and relationship to previous studies
Since the original proposal of the inter-domain hypothesis [14], a number of independent studies have provided evidence that (i) interaction between the N-terminal and central domains in the clamp region of RyR2 regulates channel activity and (ii) binding of DPc10 to the N-terminal domain of RyR2 induces ‘unzipping’ of the central and N-terminal domains, which mimics the disease mutation [15,23-25]. Therefore, the remaining discussion will assume that on binding DPc10, the gating characteristics of wild-type RyR2 channels are rapidly changed to that of the R2474S mutant (Fig. 1).

Previous studies on intact cultured cells have shown that RyR2 mutations associated with CPVT increase the probability of SPCR in circumstances where extracellular Ca2+ is raised [11,12]. The present study supports and extends these conclusions by showing that DPc10 decreases the intracellular [Ca2+] threshold for SPCR and the proportion of spontaneously active cells in an adult ventricular myocyte model, where the RyRs remain in situ and the normal features of autoregulation can be demonstrated. The ineffectiveness of DPc10-mut is of equal importance as it provides a direct link to abnormal Ca2+ regulation in CPVT: DPc10 and DPc10-mut differ only in a single Arg2474 to Ser2474 substitution, suggesting that the corresponding CPVT mutation lacks the structural characteristics necessary for a strong interaction with the N-terminal domain. The use of cell permeabilization enabled the cytosolic [Ca2+] to be controlled directly (as opposed to changing the extracellular [Ca2+]), which simplifies interpretation of these data [11]. The rapid effect of the peptide also avoided complications associated with compensatory changes in protein expression, which have been shown to occur in cultured cell models [13]. Furthermore, the ability to use each cell as its own control revealed, for the first time, evidence of a sustained diastolic Ca2+ leak, which persists despite autoregulation of Ca2+ sparks.

In addition to domain–domain interactions, protein–protein interactions may play an important part in RyR2 abnormalities associated with cardiac disease. In particular, it has been proposed that in heart failure, PKA-mediated hyper-phosphorylation of RyR2 causes dissociation of FKBP12.6, resulting in abnormal Ca2+ release and associated dysfunction [26]. Recent work suggests that unzipping of the N-terminal and central domains by DPc10 (or the R2474S mutation) is not associated with loss of FKBP12.6 per se [12,15]. However, once destabilized by DPc10, hyper-phosphorylation of RyR2 may have a greater effect on FKBP12.6 dissociation, providing a possible link between the two mechanisms [15]. In the present study, PKA mediated phosphorylation was not altered and consequently, the effects of DPc10 are likely to reflect domain unzipping rather than loss of FKBP12.6.

Patients affected by CPVT are typically asymptomatic at rest, but exhibit arrhythmias in response to exercise or stress. The response to exercise or stress involves an increase in heart rate, diastolic cell length and β-adrenergic stimulation, all of which result in a net gain of [Ca2+]i. The present study suggests that in patients with the R2474S RyR2 mutation, SPCR will occur at a lower [Ca2+]i than normal. This is of clinical importance, because SPCR is known to activate a transient inward current, resulting in delayed after-depolarisations and triggered arrhythmias. This primary defect may be further exacerbated by the effects of β-adrenergic stimulation on regulatory proteins such as FKBP12.6 [15,26]. Finally, when considering the design of drugs for the treatment of CPVT, the present study suggests that a useful approach may be to try to inactivate the sustained component of SR Ca2+ release that was revealed using the peptide probe technique.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
Both DPc10 and caffeine transiently increase the frequency of spontaneous Ca2+ sparks and lower the cytosolic [Ca2+] threshold for SPCR. However, unlike caffeine, DPc10 also induces a sustained Ca2+ efflux from the SR, which seems likely to contribute to the effects of the peptide on SR Ca2+ regulation. Given that DPc10 appears to mimic the R2474S RyR2 mutation, the increased propensity for SPCR would be expected to contribute to arrhythmias observed in CPVT patients during stress or exercise.


   Notes
 
Time for primary review 33 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

  1. Laitinen P.J., Brown K.M., Piippo K., Swan H., Devaney J.M., Brahmbhatt B., et al. Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic ventricular tachycardia. Circulation (2001) 103:485–490.[Abstract/Free Full Text]
  2. Priori S.G., Napolitano C., Tiso N., Memmi M., Vignati G., Bloise R., et al. Mutations in the cardiac ryanodine receptor gene (hRyR2) underlie catecholaminergic polymorphic ventricular tachycardia. Circulation (2001) 103:196–200.[Abstract/Free Full Text]
  3. Priori S.G., Napolitano C., Memmi M., Colombi B., Drago F., Gasparini M., et al. Clinical and molecular characterization of patients with catecholaminergic polymorphic ventricular tachycardia. Circulation (2002) 106:69–74.[Abstract/Free Full Text]
  4. Ikemoto N., Yamamoto T. Regulation of calcium release by interdomain interaction within ryanodine receptors. Front Biosci (2002) 7:d671–d683.[Web of Science][Medline]
  5. Valdiva H.H., Hogan K., Coronado R. Altered binding site for Ca2+ in the ryanodine receptor of human malignant hyperthermia. Am J Physiol (1991) 261:C235–C245.
  6. Jiang D., Xiao B., Zhang L., Chen S.R. Enhanced basal activity of a cardiac Ca2+ release channel (ryanodine receptor) mutant associated with ventricular tachycardia and sudden death. Circ Res (2002) 91:218–225.[Abstract/Free Full Text]
  7. George C.H., Higgs G.V., Lai F.A. Ryanodine receptor mutations associated with stress-induced ventricular tachycardia mediate increased calcium release in stimulated cardiomyocytes. Circ Res (2003) 93:531–540.[Abstract/Free Full Text]
  8. Lowri T.N., George C.H., Anthony L.F. Functional heterogeneity of ryanodine receptor mutations associated with sudden cardiac death. Cardiovasc Res (2004) 64:52–60.[Abstract/Free Full Text]
  9. Trafford A.W., Diaz M.E., Sibbring G.C., Eisner D.A. Modulation of CICR has no maintained effect on systolic Ca2+: simultaneous measurements of sarcoplasmic reticulum and sarcolemmal Ca2+ fluxes in rat ventricular myocytes. J Physiol (2000) 522:259–270.[Abstract/Free Full Text]
  10. Lukyanenko V., Viatchenko-Karpinski S., Smirnov A., Wiesner T.F., 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.[Web of Science][Medline]
  11. Jiang D., Xiao B., Yang D., Wang R., Choi P., Zhang L., et al. RyR2 mutations linked to ventricular tachycardia and sudden death reduce the threshold for store-overload-induced Ca2+ release (SOICR). Proc Natl Acad Sci USA (2004) 101:13062–13067.[Abstract/Free Full Text]
  12. Jiang D., Wang R., Xiao B., Kong H., Hunt D.J., Choi P., et al. 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 (2005) 97:1177–1179.
  13. Tong J., McCarthy T.V., MacLennan D.H. 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]
  14. Ikemoto N., Yamamoto T. Postulated role of inter-domain interaction within the ryanodine receptor in Ca2+ channel regulation. Trends Cardiovasc Med (2000) 10:310–316.[CrossRef][Web of Science][Medline]
  15. Oda T., Yano M., Yamamoto T., Tokuhisa T., Okuda S., Doi M., et al. Defective regulation of interdomain interactions within the ryanodine receptor plays a key role in the pathogenesis of heart failure. Circulation (2005) 111:3400–3410.[Abstract/Free Full Text]
  16. Yamamoto T., Ikemoto N. Peptide probe study of the critical regulatory domain of the cardiac ryanodine receptor. Biochem Biophys Res Commun (2002) 291:1102–1108.[CrossRef][Web of Science][Medline]
  17. Yang Z., Steele D.S. Effects of cytosolic ATP on spontaneous and triggered Ca2+-induced Ca2+ release in permeabilised rat ventricular myocytes. J Physiol (2000) 523:29–44.[Abstract/Free Full Text]
  18. Yang Z., Steele D.S. Effects of cytosolic ATP on Ca2+ sparks and SR Ca2+ content in permeabilized cardiac myocytes. Circ Res (2001) 89:526–533.[Abstract/Free Full Text]
  19. Franzini-Armstrong C., Protasi F., Ramesh V. Shape, size, and distribution of Ca2+ release units and couplons in skeletal and cardiac muscles. Biophys J (1999) 77:1528–1539.[Web of Science][Medline]
  20. Cheng H., Lederer M.R., Lederer W.J., Cannell M.B. Ca2+ sparks and [Ca2+]i waves in cardiac myocytes. Am J Physiol (1996) 270:C148–C159.[Web of Science][Medline]
  21. Nieman C.J., Eisner D.A. Effects of caffeine, tetracaine and ryanodine on calcium-dependent oscillations in sheep cardiac purkinje fibers. J Gen Physiol (1985) 86:877–889.[Abstract/Free Full Text]
  22. Laver D.R., O'Neill E.R., Lamb G.D. Luminal Ca2+-regulated Mg2+ inhibition of skeletal RyRs reconstituted as isolated channels or coupled clusters. J Gen Physiol (2004) 124:741–758.[Abstract/Free Full Text]
  23. Liu Z., Zhang J., Sharma M.R., Li P., Chen S.R., Wagenknecht T. Three-dimensional reconstruction of the recombinant type 3 ryanodine receptor and localization of its amino terminus. Proc Natl Acad Sci U S A (2001) 98:6104–6109.[Abstract/Free Full Text]
  24. Liu Z., Wang R., Zhang J., Chen S.R., Wagenknecht T. Localization of a disease-associated mutation site on the three-dimensional structure of cardiac ryanodine receptor. J Biol Chem (2005) 280:37941–37947.[Abstract/Free Full Text]
  25. Serysheva I.I., Hamilton S.L., Chiu W., Ludtke S.J. Structure of Ca2+ release channel at 14 A resolution. J Mol Biol (2005) 345:427–431.[CrossRef][Web of Science][Medline]
  26. Marx S.O., Reiken S., Hisamatsu Y., Jayaraman T., Burkhoff D., Rosemblit N., et al. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell (2000) 101:365–376.[CrossRef][Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J Am Coll CardiolHome page
S. Kobayashi, M. Yano, T. Suetomi, M. Ono, H. Tateishi, M. Mochizuki, X. Xu, H. Uchinoumi, S. Okuda, T. Yamamoto, et al.
Dantrolene, a therapeutic agent for malignant hyperthermia, markedly improves the function of failing cardiomyocytes by stabilizing interdomain interactions within the ryanodine receptor.
J. Am. Coll. Cardiol., May 26, 2009; 53(21): 1993 - 2005.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Corrigendum (v71,p606)
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Yang, Z.
Right arrow Articles by Steele, D. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, Z.
Right arrow Articles by Steele, D. S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?