Download Mutant ryanodine receptors in catecholaminergic polymorphic

Clinical research
European Heart Journal (2007) 28, 1135–1142
doi:10.1093/eurheartj/ehl543
Arrhythmia/electrophysiology
Mutant ryanodine receptors in catecholaminergic
polymorphic ventricular tachycardia generate
delayed afterdepolarizations due to increased
propensity to Ca21 waves
Jere Paavola1, Matti Viitasalo2, Päivi J. Laitinen-Forsblom3, Michael Pasternack1, Heikki Swan2,
Ilkka Tikkanen1,3, Lauri Toivonen2, Kimmo Kontula3, and Mika Laine1,2*
1
Minerva Foundation Institute for Medical Research, FIN-00290, Helsinki, Finland; 2Department of Cardiology, Helsinki
University Central Hospital, University of Helsinki, FIN-00290, Haartmaninkatu 4, PO Box 340, HUS 00029, Helsinki, Finland;
and 3Department of Medicine, University of Helsinki, FIN-00290, Helsinki, Finland
Received 7 October 2006; revised 9 January 2007; accepted 25 January 2007; online publish-ahead-of-print 8 March 2007
See page 1054 for the editorial comment on this article (doi:10.1093/eurheartj/ehm068)
KEYWORDS
Arrhythmia;
Calcium;
Mutation;
cAMP;
HEK293 cells;
Heart failure;
Sudden death
Aims Mutations in cardiac ryanodine receptors (RyR2s) are linked to catecholaminergic polymorphic
ventricular tachycardia (CPVT), characterized by risk of polymorphic ventricular tachyarrhythmias
and sudden death during exercise. Arrhythmias are caused by gain-of-function defects in RyR2, but
cellular arrhythmogenesis remains elusive.
Methods and results We recorded endocardial monophasic action potentials (MAPs) at right ventricular
septum in 15 CPVT patients with a RyR2 mutation (P2328S, Q4201R, and V4653F) and in 12 control
subjects both at baseline and during epinephrine infusion (0.05 mg/kg/min). At baseline 3 and
during epinephrine infusion, four CPVT patients, but none of the control subjects, showed delayed
afterdepolarizations (DADs) occasionally coinciding with ventricular premature complexes. In order to
study the underlying mechanisms, we expressed two types of mutant RyR2 (P2328S and V4653F)
causing CPVT as well as wild-type RyR2 in HEK 293 cells. Confocal microscopy of Fluo-3 loaded cells
transfected with any of the three RyR2s showed no spontaneous subcellular Ca2þ release events at
baseline. Membrane permeable cAMP analogue (Dioctanoyl-cAMP) triggered subcellular Ca2þ release
events as Ca2þ sparks and waves. Cells expressing mutant RyR2s showed spontaneous Ca2þ release
events at lower concentrations of cAMP than cells transfected with wild-type RyR2.
Conclusion CPVT patients show DADs coinciding with premature action potentials in MAP recordings.
Expression studies suggest that DADs are caused by increased propensity of abnormal RyR2s to generate
spontaneous Ca2þ waves in response to cAMP stimulation. Increased sensitivity of mutant RyR2s to cAMP
may explain the occurrence of arrhythmias during exercise or emotional stress in CPVT.
Introduction
Mutations in the cardiac ryanodine receptor (RyR2) gene
have been reported to underlie an autosomally dominantly
inherited tachycardia syndrome called catecholaminergic
polymorphic ventricular tachycardia (CPVT).1,2 CPVT is
characterized by exercise- or stress-related ventricular
tachycardia episodes in the absence of structural heart
disease.3 Patients have syncopal events and a high probability of sudden cardiac death, with a mortality of 30%
by the age of 30 years. Arrhythmias include multifocal ventricular premature beats, bidirectional ventricular tachycardias, and polymorphic VTs.3 Although the molecular events
underlying CPVT have been studied earlier in detail,4,5 the
* Corresponding author. Tel: þ358 40 5245735; fax: þ358 9 471 74574.
E-mail address: Mika.Laine@hus.fi
exact electrophysiological and cellular mechanisms of ventricular arrhythmias remain elusive.
RyR2s are expressed on the sarcoplasmic reticulum (SR) of
cardiomyocytes, where they form intracellular Ca2þ release
channels that play a central role in excitation–contraction
(EC) coupling.6 We have earlier2 reported three
unrelated families with missense mutations in RyR2s
(RyR2-P2328S, RyR2-Q4201R, and RyR2-V4653F). All the
identified mutations co-segregated with the clinical phenotype of a family history of sudden death, frequent ventricular premature depolarizations, or polymorphic ventricular
arrhythmias.
In single-channel recordings, all three mutant RyR2s
showed a significant gain-of-function abnormality due to
the decreased binding affinity of the stabilizing FKBP 12.6
protein.5 However, the stabilizing role of FKBP 12.6 in
CPVT has not been universally accepted.7,8 As an alternative
& The European Society of Cardiology 2007. All rights reserved. For Permissions, please e-mail: [email protected]
1136
J. Paavola et al.
explanation for arrhythmias, Jiang et al.9 have suggested
that RyR2 mutations have increased sensitivity to luminal
Ca2þ activation and increased propensity for spontaneous
Ca2þ release from the SR. Thus, during exercise sympathetic
nervous system activation increases cAMP dependent
protein kinase A (PKA) activity, resulting in increased SR
Ca2þ load and arrhythmia triggering.
Although the molecular mechanism in CPVT is known to be
the gain-of-function mutation in RyR2, the exact mechanistic relationship linking increased RyR2 activity and arrhythmias is not clear. Based on earlier results from
experimental animal studies,10 we and others have hypothesized that CPVT patients have delayed afterdepolarizations
(DADs) due to uncontrolled release of Ca2þ during
diastole.7,8
In the present study, this hypothesis was tested by
measurement of monophasic ventricular action potentials
(MAPs) in the patients carrying RyR2 mutations. In addition,
two of the mutant (RyR2-P2328S and RyR2-V4653F) as well
as wild-type RyR2 (RyR2-wt) were expressed in human
embryonic kidney (HEK 293) cells to verify the defect in
Ca2þ release. Ca2þ signals were recorded with confocal
imaging to clarify how mutant RyR2s respond to PKA stimulation in comparison with the wild-type channel.
Methods
Study subjects
We studied 15 CPVT patients and 12 age-matched controls. For this
study, we reviewed all three Finnish families with a known RyR2
mutation. There were 22 affected live family members in these
families who showed evidence of the disease and were 10 years or
older. One patient had another serious disease and six patients
refused the electrophysiology study. MAP recording was performed
in the remaining 15 patients, each carrying one of the three
(P2328S, Q4201R, or V4653F) different RyR2 mutations. No patient
was excluded. Twelve patients had either exercise related
syncope or cardiac arrest in their medical history (Table 1). In the
three families, the incidence of sudden cardiac death by the age
of 30 years ranged from 30 to 35%.
All subjects had normal findings on echocardiography, and none
showed electrolyte abnormalities or were taking any medications
affecting the QT interval. The CPVT patients performed a bicycle
ergometer exercise test with continuous ECG recording. The initial
load was 30 W, followed by increments of the load by 15 W each
Table 1 Clinical characteristics of the study subjects
Age, y
Men/women
Baseline QTc interval (ms)
Cardiac events, n (%)
Cardiac arrest
Syncope
Exercise stress test
Patients with ventricular
Arrhythmias, n (%)
Heart rate by appearance of
ventricular arrhythmias
CPVT
patients
(n ¼ 15)
Control
subjects
(n ¼ 12)
34 +16
6/9
430+18
12 (80)
3
9
38 +11
6/6
402+17
0
15 (100)
127+20
minute until exhaustion or appearance of polymorphic ventricular
tachycardia. The heart rate at which ventricular bigeminy first
appeared was recorded. Next day, the CPVT patients underwent
right cardiac catheterization with a protocol of MAP recording.
The treatment with beta-adrenergic blocking agents was discontinued at least five drug half-lives before the exercise test until the
MAP recording was completed.
We recruited control subjects from patients undergoing an electrophysiology study to assess the efficacy of previous catheter ablation of concealed atrioventricular accessory pathways during the
same time period as patients carrying RyR2 mutations were
studied. We included 18- to 50-year-old patients with normal QT
interval and no structural heart disease and who did not have
ß-blockers or any other medication known to influence cardiac repolarization. Using these criteria, 12 consecutive patients were
included. The MAP recording protocol was performed at the end
of the routine electrophysiological examination.
The research protocol had been reviewed and approved by the
Ethics Review Committee of the Department of Medicine, University
of Helsinki, and written informed consent had been obtained from
all study subjects. The procedures followed were in accordance
with institutional guidelines.
Recording of monophasic action potentials
and measuring of DADs
A bipolar silver–silver chloride catheter (model 006248, Bard Inc.,
Lowell, MA, USA) was introduced through a femoral vein and
advanced into the right ventricle against the right ventricular
septum under fluoroscopic guidance to record MAPs.11 Three quadripolar electrophysiological catheters (Bard Electrophysiology,
Lowell, MA, USA) were placed in the right atrium, atrioventricular
junction, and the right ventricular apex. Blood pressure was
measured using a fluid manometer from the femoral artery. MAP
signals amplified and filtered at a frequency from 0.05 to 250 Hz,
standard 12-lead ECG and femoral arterial pressure were stored
digitally at a sample rate of 1 kHz (Cardiolab, Prucka Engineering,
Sugar Land, TX, USA).
MAPs were obtained during both sinus rhythm and during atrial
pacing at a constant cycle length of 600 ms after placement of
the catheter electrode in a position providing continuous recordings
with a stable amplitude, smooth configuration, and isopotential
diastolic baseline. Once the catheter was stabilized, stable MAPs
were obtained in all patients. Study data were obtained at baseline
and during infusions of epinephrine. For safety reasons, we limited
epinephrine infusion to a target rate of 0.05 mg/kg of body weight
per minute, which was reached stepwise while arrhythmias were
carefully monitored.
DAD was defined as an abnormal, low-amplitude afterdepolarization occurring after repolarization is completed.12 The amplitude of
a DAD was required to be .0.2 mV and .3% of the action potential
amplitude in five consecutive beats to be included in the measurements. The coupling interval of the DAD was defined as the interval
from the upstroke of the preceding action potential to the peak of
the DAD.13 We also recorded the diastolic upslope of the DAD by
measuring the mean rate of rise (dV/dt) of the ascending limb of
the DAD13 as a mean of three consecutive beats.
Site-directed mutagenesis
Point mutations P2328S and V4653F were generated in small
fragments of RYR2 cDNA with Chameleon Double-Stranded, SiteDirected Mutagenesis Kit (Stratagene, La Jolla, CA, California).
The P2328S mutation was induced into a fragment comprising the
nucleotides 2472–7678, and V4653F into a fragment consisting of
the nucleotides 11756–15257. The following mutagenesis primers
were used: P2328S, 50 -aga ttg ctc att cgg cga tcg gag tgt ttt ggt
cct g-3’ (includes conservative substitutions to generate a restriction site); V4653F, 50 -gg gac aaa ttt gtt aaa aga aag ttc atg gat
DADs and arrhythmogenesis in CPVT
1137
aaa tat gga gag ttc tac ggc cg-30 . The fragments with the desired
mutations were subcloned back to their original positions in a fulllength RyR2 cDNA in the pCMV5 expression vector with BstEII and
KpnI (P2328S), or by two steps with XhoI and PpuMI, and NheI and
SalI (V4653F).
Ryanodine receptor expression
HEK 293 cells were grown in DMEM medium (Biological Industries,
Israel) supplemented with 10% fetal calf serum, penicillin (50 IU/
mL) þ streptomycin (50 mg/mL) (ICN Biomedicals Inc., Aurora,
Ohio, USA), HEPES (15 mM), sodium pyruvate (1 mM), and L-glutamine (2 mM). Cells were maintained in 100 mm tissue culture
dishes at þ378C under 5% carbon dioxide and they were split
every second day. RyR2 cDNA was centrifuged and removed from
the buffer solution. Transfection was carried out by a Ca2þ phosphate precipitation method14 using 2 mg of RyR2 cDNA for each
40 mm diameter round glass coverslip. The coverslips were
treated with polylysine (1 mg/100 mL) for 1 h to allow attachment
of the cells on the surface of the glass.
Intracellular Ca21 recordings
Confocal Ca2þ measurements with HEK 293 cells were performed
24–30 h after the transfection. Experiments were carried out at
room temperature (20–238C). Cells were loaded on coverslips with
2 mM Fluo-3-AM (Molecular Probes, Leiden, The Netherlands) in
buffer solution (150 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM
CaCl2, 5 mM HEPES) for 30 min in dark, followed by a 20 min
de-esterification period in the same buffer solution. After the
loading, the coverslip was mounted on the stage of a Nikon
Eclipse TE300 inverted microscope attached to an Ultra View confocal imaging system (PerkinElmer Life Sciences, Cambridge, UK) with
a 40x objective. Fluo-3 was excited using a 488 nM laser line and the
emitted fluorescence was collected at wavelengths .505 nM. Confocal images were acquired using a CCD-camera at an image frequency of 10 frames/s. For the recordings a view of 10–20 cells
was selected. After recording the baseline activity, cells were
stimulated with caffeine (5 mM) or cAMP analogue (DioctanoylcAMP) by changing rapidly the external solution. The recordings
were analysed off-line (NIH Image) and absolute values for Ca2þ
were calculated as described earlier.15 A Kd value of 810 nM of
fluo-3 was used for the calculations.
Statistical analysis
Continuous variables are presented as means+SD or median (min–
max). Differences in the normally distributed variables were
assessed using the t-test and the paired t-test for dependent variables. Wilcoxon Signed Ranks test was used as a nonparametric
test. All tests were two-sided and a probability value of P , 0.05
was considered statistically significant. No corrections were used
to account for the potential inflation due to multiple testing. Categorical variables were assessed by the x2 test. The data were analysed using Systat statistical software version 9.
Results
Patient characteristics
The two study groups did not differ with regard to age or
gender. The mean age of the CPVT patients was 34+ 16
years and that of the control patients 38+ 11 years; 6/15
of the CPVT patients and 6/12 of the control patients
were male. Systolic blood pressure was also similar in the
study groups (Table 2). There was a tendency to longer QT
intervals in the CPVT patient group (434+ 20 ms) in comparison with the control patients (418 +28 ms). All patients
exhibited polymorphic ventricular extrasystoles or short
runs of polymorphic ventricular tachycardia during the
exercise test (Table 1).
Monophasic action potential recordings
The duration and the amplitude of MAPs did not differ
between the study groups (Table 2). Epinephrine infusion
increased heart rate in both study groups, but systolic
blood pressure remained unchanged (Table 2). Epinephrine
tended to shorten the duration of MAPs in the CPVT
patient group (Table 2). The MAP amplitude tended to be
lower during epinephrine infusion than during baseline, presumably because MAPs have a tendency to decrease their
amplitude over time.16
During baseline MAP recordings, DADs were observed in 3
out of 15 (20%) CPVT patients. DADs were recorded in two
patients carrying the RyR2-P2328S mutation and in one
patient carrying the RyR2-Q4201R mutation. Figure 1
shows a distinct DAD in the MAP recording of a CPVT
Table 2 Effects of epinephrine on systolic blood pressure, sinus cycle length, and monophasic action potential
CPVT patients (n ¼ 15)
Systolic blood pressure (mmHg)
Baseline
Epinephrine
Sinus cycle length (ms)
Baseline
Epinephrine
P-value
Duration of monophasic action potential during atrial
pacing at a cycle length of 600 ms (ms)
Baseline
Epinephrine
P-value
Amplitude of monophasic action potential (mV)
Baseline
Epinephrine
Control subjects (n ¼ 12)
P-value
146+29
144+25
139+21
138+21
0.49
0.58
825+104
692+63
4.9 1025
918+143
670+130
5.3 1027
0.10
0.64
306+18
298+18
0.06
292+17
293+18
0.68
0.12
0.65
11.8+6
9.0+4
12.5+4
8.7+4
0.75
0.89
1138
J. Paavola et al.
Table 3 Effects of epinephrine on DADs in patients with CPVT
DAD measure
Baseline
Epinephrine
P-value
Incidence, n (%)
Coupling interval
(ms)
Amplitude (mV)
Amplitude, % of
MAP amplitude
Slope (mV/s)
3/15 (20)
580 (500–580)
4/15 (27)
440 (380–520)
0.11
0.48 (0–0.94)
6 (0–10)
0.8 (0.2–1.1)
14 (3–16)
0.14
0.07
6.7 (0–12.1)
11 (3.7–18.2)
0.07
Figure 1 Simultanous ECG and MAP recorded from right ventricular septum
in a CPVT patient carrying RyR2 mutation (P2328S). Epicardial ECG (A) reveals
separate late appearing U waves (arrow), and monophasic action potentials
(B) show DADs. (C ) Invasively measured aortic pressure.
Figure 3 MAPs from right ventricular septum in a CPVT patient carrying
RyR2 mutation (P2328S) before (A) and after low-dose epinephrine infusion
(B) for 15 min (0.05 mg/kg/min). Epinephrine infusion increases the amplitude of DADs. (C ) Magnification of DADs in baseline and after epinephrine
infusion.
Figure 2 MAP recording in a CPVT patient carrying RyR2 mutation (P2328S).
Upper trace shows MAP recording of repetitive afteroscillations with significant beat-to-beat variation during epinephrine infusion (0.05 mg/kg/min).
Lower trace represents simultaneously recorded right atrial signal.
patient carrying the P2328S mutation. In the three CPVT
patients, the DADs were continuously present showing
minor beat-to-beat variation of the amplitude. One
patient exhibited occasional double and triple oscillations
of the DADs (Figure 2). We observed no DADs in any of the
12 control subjects. The three CPVT patients with DADs in
MAP recordings at baseline had a significantly lower
threshold heart rate for ventricular premature beats in exercise stress testing than the patients with no DADs (107 +21
vs. 131 +16 beats per minute, respectively, P , 0.05).
During the low dose epinephrine infusion four out of the
15 (27%) CPVT patients showed DADs. DADs were observed
in the same three patients who exhibited DADs during baseline and in one additional patient carrying the RyR2-P2328S
mutation. We were not able to demonstrate DADs in patients
carrying the RyR-V4653F mutation during baseline or after
the low dose epinephrine infusion. Epinephrine tended to
increase the relative amplitude and the slope of DADs
(Table 3, Figure 3). We observed occasional premature ventricular beats on DADs (Figure 4). Patients carrying
RyR2-P2328S and RyR2-V4653F mutations started to have
ventricular premature beats in exercise stress testing at
similar threshold heart rates, 130 +19 beats per minute
and 130 +15 beats per minute, respectively. Epinephrine
infusion did not induce DADs in any of the control subjects.
Verification of RyR2 expression in
HEK 293 cells by caffeine
Expression vectors for RyR2-wt, RyR2-P2328S, and
RyR2-V4653F were transfected into HEK 293 cells, followed
by an examination of the cells by confocal Ca2þ imaging.
Functional activity of expressed RyR2s was verified by intracellular Ca2þ release by caffeine, an activator of RyR2 Ca2þ
release channels. HEK 293 cells do not have endogenous
RyRs,14 and therefore any observed Ca2þ release in response
to caffeine exposure is supposed to originate from transfected RyR2s. We found no caffeine-induced Ca2þ release
in nontransfected cells, which is in agreement with the
lack of endogenous RyR expression in HEK 293 cells.14
Sarcoplasmic reticulum Ca2þ loading was investigated by
examining the characteristics of caffeine-induced Ca2þ
release. HEK 293 cells transfected with RyR2-wt,
RyR2-P2328S, and RyR2-V4653F responded to caffeine with
a rapid global increase of cytoplasmic [Ca2þ]. The peak of
the caffeine-induced Ca2þ release was not significantly
different in the cells transfected with RyR2-wt,
DADs and arrhythmogenesis in CPVT
1139
RyR2-P2328S, or RyR2-V4653F (363+ 68, 299+ 59, and
274+ 33 nM, respectively) when stimulated with a 5 mM
caffeine solution at the end of each experiment. Both wtand mutant RyR2 plasmids had transfection efficiencies of
50%. These results suggest that the studied RyR2 mutations
did not cause a significant change in SR Ca2þ content when
compared with wild-type RyR2s.
At baseline recordings (1.8 mmol/L extracellular [Ca2þ]),
confocal imaging of fluo-3-loaded cells transfected with any
of the RyR2 plasmids displayed an extremely low number of
spontaneous Ca2þ transients, and we found no difference in
the occurrence of Ca2þ release events between the cells
transfected with wild-type or mutant RyR2s. Only two isolated Ca2þ sparks were observed in the baseline recording
of 43 cells. These events localized to a small region in the
cell, similar to what has been reported earlier with RyR1
and RyR3 expression in HEK 293 cells.14 No spontaneous or
caffeine stimulated subcellular Ca2þ release events were
observed in non-transfected control cells.
Sensitivity of transfected HEK 293 cells
to dioctanoyl-cAMP
Figure 4 Simultaneous ECG and MAP recording from right ventricular
septum in a CPVT patient carrying a RyR2 mutation (Q4201R) the MAP recording shows DADs. A premature ventricular complex occurs on a DAD and
coincides with the separate late-appearing U wave distinct from an early U
wave merging with the T wave best seen in the post extrasystolic beat.
The characteristic induction of arrhythmias in CPVT patients
is thought to occur through stimulation of the adrenergic
system. To simulate sympathetic stimulation, cells were
stimulated with dioctanoyl-cAMP (di-cAMP). di-cAMP is a cell
permeable cAMP analogue that mimics the action of endogenous cAMP with approximately 100 times higher activity.
Figure 5 shows representative Ca2þ imaging recordings
of HEK 293 cells transfected with wild-type and mutant
Figure 5 Spontaneous Ca2þ release events (Ca2þ sparks and waves) in HEK 293 cells transfected with plasmids carrying RyR2 wild type (A), RyR2 P2328S (B), or
RyR2 V4653 (C ). Ca2þ traces show signals recorded from three different cells during extracellular stimulation with increasing (a–c) concentrations (1, 10, and
50 mM) of cell permeable cAMP (di-cAMP).
1140
Figure 6 Quantitative analysis of spontaneous Ca2þ release events in HEK
293 cells transfected with wild-type (n ¼ 209) or mutant (P2328S, n ¼ 170
and V4653F, n ¼ 211). RyR2s. Bars represent percentage of cells showing
Ca2þ waves in response to the addition of di-cAMP (1, 10 , and 50 mM).
RyR2s. The cells transfected with wt-RyR2s showed no clear
increase in the frequency of Ca2þ waves at low concentrations of di-cAMP (1 and 10 mM), and there was no
change in baseline cytosolic [Ca2þ] in response to di-cAMP
(102 +20 vs. 113 +22 and 119+ 16 nM). Only the highest
concentration of di-cAMP (50 mM) triggered Ca2þ waves
together with a mean 50 +30 nM elevation (P , 0.05) in
baseline cytosolic [Ca2þ]. In contrast, the cells transfected
with mutant RyR2s showed Ca2þ sparks and waves at all of
the di-cAMP concentrations used (1, 10, and 50 mM). The
increase in the number of Ca2þ -transients with 1 and
10 mM di-cAMP was observed without an increase in the
baseline Ca2þ level in cells transfected with RyR2-P2328S
(103 +12 vs. 103 +4 and 108+ 20 nM) or RyR2-V4653F
(102 +8 vs. 109 +20 and 107+ 9 nM). The highest di-cAMP
concentration increased the basal cytosolic Ca2þ level by
50 + 38 nM in RyR2-V4653F and 27+ 14 nM in RyR2-P2328S
transfected cells (P , 0.05). None of the untransfected
cells showed any Ca2þ events in response to stimulation
with di-cAMP.
Quantitative analysis of n ¼ 590 cells transfected with
RyR2-wt or mutant RyR2 is illustrated in Figure 6. A significantly larger amount of cells transfected with mutant
RyR2s responded with Ca2þ waves when compared with
cells transfected with wild-type RyR2s when stimulated
with 10 or 50 mM di-cAMP (P , 0.001).
Discussion
Main findings
Our results show that CPVT patients carrying a RyR2
mutation display DADs in right ventricular endocardial
MAP recordings whereas healthy control subjects do not
exhibit corresponding DADs at similar conditions. The DADs
coincided with separate late-appearing U waves in the
surface ECG, and a premature ventricular beat occasionally
occurred on a DAD. Cells transfected with two diseasecausing missense RyR2 mutations showed spontaneous Ca2þ
release events at lower concentrations of di-cAMP than
cells transfected with a wild-type RyR2 receptor.
J. Paavola et al.
DAD in arrhythmogenesis
DADs are caused by nonelectrically driven Ca2þ events
depolarizing the membrane potential during diastole by
activating the Na/Ca exchanger and Cl2 currents.17,18
When the membrane potential reaches the threshold, a
premature action potential is triggered initiating the
arrhythmia. DAD-mediated triggered activity is believed to
play an important role in arrhythmias associated with
strong sympathetic stimulation,19 heart failure,20 and RyR2
mutations.21 In the clinic, patients with a RyR2 mutation
typically show multiform ventricular premature complexes
and relatively slow polymorphic ventricular tachycardia
during exercise, as was also observed in our patients. The
polymorphic ventricular tachycardia is expected to be
caused by multiple spontaneous Ca2þ release events
causing DAD-induced firing of multiple sites in the ventricles, as recently reported by Katra and Laurita.22 However,
the transition of the slow polymorphic ventricular tachycardia to ventricular fibrillation may be related to other
mechanisms.10
The fact that DADs measured at one endocardial site in
our study was observed in only three of 15 CPVT patients
at baseline is consistent with an experimental finding, indicating a regional distribution of DAD development.10 Our
MAP recording may show a far field signal of DAD; therefore,
the regional distribution of DAD development may also
explain why no amplification of DAD was observed at the
time of premature ventricular beat. On the other hand,
abnormal Ca22 handling may not regularly manifest in all
CPVT patients during resting conditions. Furthermore,
patients having DADs at baseline showed premature ventricular complex at a lower heart rate than others. This
finding advocates that observation of DADs in CPVT patients
at resting conditions is associated with an increased likelihood of ventricular premature beats and polymorphic ventricular tachycardia.
Molecular background of DADs
Reported RyR2 mutations cluster in the following three areas
of RyR2: N-terminal (amino acids 176–433), central (2246–
2504), and C-terminal region (4104–4653). The three RyR2
mutations studied here are from three unrelated families
and result in a structural and functional phenotype with an
increased open probability and gating consistent with a
gain-of-function of the RyR2 receptor phenotype.23 In the
present study, DADs were recorded from three patients carrying a P2328S mutation and one patient carrying a Q4201R
mutation, but we were not able to show DADs in V4653F
mutation carriers.
The P2328S missense mutation is located near the binding
site of the regulatory protein calstabin 2, whereas the
V4653F mutation is in the pore region of the RyR2. It
remains unclear whether this difference in the location of
the mutation results in a different phenotype between
P2328S and V4653F carriers. Both P2328S and V4653F
mutation carriers have similar mortality rates of 30–33% by
the age of 35 years.5 During exercise testing, V4653F
mutation carriers had ventricular premature beats in ECG
at the same threshold heart rate as P2328S carriers. When
expressed in HEK 293 cells, both mutations had an increased
propensity to Ca2þ waves during PKA stimulation. It is probable that the low-dose adrenaline infusion in the present
study was not sufficient to produce DADs in V4653F mutation
DADs and arrhythmogenesis in CPVT
carriers or that our approach to record MAP at one endocardial site is not sufficient to expose DADs occurring at different locations. An alternative explanation would be that
arrhythmias in V4653F carriers are generated by another
mechanism than DADs.
In order to directly compare the impact of RyR2-P2328S
and V4653F mutations on Ca2þ handling, plasmids carrying
mutant and wild-type receptors were transfected into HEK
293 cells. At baseline, both the wild-type and the RyR2s carrying CPVT mutations showed an extremely low frequency of
spontaneous Ca2þ release events. This was in contrast to an
earlier report of increased basal activity of RyR2s carrying a
gain-of-function mutation.24 The lower baseline activity in
our experiments may be explained by differences in the molecular characteristics of the studied mutations. Loading of
the Ca2þ stores at baseline may also affect the results.
Stimulation with a cAMP analogue unmasked the defect in
RyR2s, and cells transfected with either RyR2-P2328S or
V4653F plasmids showed an increased number of spontaneous Ca2þ release events such as Ca2þ sparks and
waves when compared with cells transfected with wild-type
RyR2.
An increase in baseline Ca2þ levels were detected only
after the highest level of PKA stimulation, and a similar
rise in cytosolic Ca2þ was measured in the cells transfected
with native and mutant channels. These results suggest that
Ca2þ oscillations are not caused by an increase in baseline
Ca2þ due to a ‘leaky’ channel, but rather by an increased
propensity to Ca2þ waves in response to cAMP stimulation.
cAMP may increase RyR2 receptor opening probability
either by dissociating the stabilizing protein FKBP 12.64,5
or by increasing SR Ca2þ load. It was recently demonstrated
that RyR2s carrying CPVT mutations demonstrate increased
sensitivity to luminal Ca2þ.9,25 cAMP stimulation increases
Ca2þ load in the SR and may hence trigger arrhythmias in
patients carrying a CPVT mutation.
Abnormal luminal Ca2þ activation might explain the
observations of multiple repetitive Ca2þ waves with declining amplitude in HEK 293 cells transfected with mutant
RyR2s causing CPVT. Reflecting this phenomenon, repetitive
DADs were also displayed in MAP recordings (Figure 2). The
multiple DADs occurred with decreasing amplitude similarly
to what has been previously reported in single cell studies
provoked by Ca2þ overload.26 In cardiac myocytes, repetitive Ca2þ release may occur when luminal Ca2þ reaches a
certain threshold during Ca2þ removal from the cytosol.
Study limitations
Due to safety reasons, we used only a relatively low dose
(0.05 mg/kg/min) of epinephrine infusion resulting in an
average heart rate of 87 beats per minute in CPVT patients
and 90 beats per minute in control patients. Thus, the heart
rates achieved remained well below the clinical target rate
of 127 beats per minute before premature ventricular complexes appeared during the exercise test. This might have
explained why DADs and premature ventricular complexes
were not observed in all patients carrying RyR2 mutation.
We recorded DADs at one endocardial site only. The
observed DADs were of low amplitude and the ventricular
premature complexes recorded on DADs may thus also be
coincidental. We are aware of the mechanical artifacts
which may cause oscillations in MAP recordings resembling
DADs. Our previous experience in long QT syndrome27 and
1141
the fact that our control subjects did not exhibit DADs
support the idea that the DADs observed in the CPVT
patients are real events.
In cardiac myocytes, RyR2s are grouped in the SR membrane near the T-tubule system. HEK293 cells lack a
T-tubule system, which may have an effect on the organization and function of the channels. Furthermore, Ca2þ
events in HEK293 are much slower in comparison with
cardiac myocytes. Additional exploration of RyR2 mutations
in a cardiac myocyte model is therefore implicated in future
studies.
Conclusions
In conclusion, our results show that patients carrying an
inherited gain-of-function defect in RyR2s display DADs
coinciding with premature action potentials. By expressing
RyR2s in a cell model, we showed that mutations increase
the probability of spontaneous Ca2þ waves during adrenergic
stimulation. We suggest that arrhythmias are caused by Ca2þ
oscillations generating DADs rather than by an increase in
the basal cytosolic Ca2þ level. The increased sensitivity of
RyR2s to cAMP may explain the occurrence of clinical
arrhythmias in CPVT during exercise or emotional stress.
Acknowledgements
This work was supported by grants from the Finnish Foundation for
Cardiovascular Research and the Juselius Foundation.
Conflict of interest: none declared.
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