Download Calcium-Handling Abnormalities Underlying Atrial

Calcium-Handling Abnormalities Underlying Atrial
Arrhythmogenesis and Contractile Dysfunction in Dogs
With Congestive Heart Failure
Yung-Hsin Yeh, MD;* Reza Wakili, MD;* Xiao-Yan Qi, PhD; Denis Chartier, BSc; Peter Boknik, PhD;
Stefan Kääb, MD; Ursula Ravens, MD; Pierre Coutu, PhD; Dobromir Dobrev, MD; Stanley Nattel, MD
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Background—Congestive heart failure (CHF) is a common cause of atrial fibrillation. Focal sources of unknown
mechanism have been described in CHF-related atrial fibrillation. The authors hypothesized that abnormal calcium
(Ca2⫹) handling contributes to the CHF-related atrial arrhythmogenic substrate.
Methods and Results—CHF was induced in dogs by ventricular tachypacing (240 bpm ⫻2 weeks). Cellular Ca2⫹-handling
properties and expression/phosphorylation status of key Ca2⫹ handling and myofilament proteins were assessed in control and
CHF atria. CHF decreased cell shortening but increased left atrial diastolic intracellular Ca2⫹ concentration ([Ca2⫹]i), [Ca2⫹]i
transient amplitude, and sarcoplasmic reticulum (SR) Ca2⫹ load (caffeine-induced [Ca2⫹]i release). SR Ca2⫹ overload was
associated with spontaneous Ca2⫹ transient events and triggered ectopic activity, which was suppressed by the inhibition of
SR Ca2⫹ release (ryanodine) or Na⫹/Ca2⫹ exchange. Mechanisms underlying abnormal SR Ca2⫹ handling were then studied.
CHF increased atrial action potential duration and action potential voltage clamp showed that CHF-like action potentials
enhance Ca2⫹i loading. CHF increased calmodulin-dependent protein kinase II phosphorylation of phospholamban by 120%,
potentially enhancing SR Ca2⫹ uptake by reducing phospholamban inhibition of SR Ca2⫹ ATPase, but it did not affect
phosphorylation of SR Ca2⫹-release channels (RyR2). Total RyR2 and calsequestrin (main SR Ca2⫹-binding protein)
expression were significantly reduced, by 65% and 15%, potentially contributing to SR dysfunction. CHF decreased
expression of total and protein kinase A–phosphorylated myosin-binding protein C (a key contractile filament regulator) by
27% and 74%, potentially accounting for decreased contractility despite increased Ca2⫹ transients. Complex phosphorylation
changes were explained by enhanced calmodulin-dependent protein kinase II␦ expression and function and type-1
protein-phosphatase activity but downregulated regulatory protein kinase A subunits.
Conclusions—CHF causes profound changes in Ca2⫹-handling and -regulatory proteins that produce atrial fibrillation–
promoting atrial cardiomyocyte Ca2⫹-handling abnormalities, arrhythmogenic triggered activity, and contractile
dysfunction. (Circ Arrhythmia Electrophysiol. 2008;1:93-102.)
Key Words: atrial fibrillation 䡲 congestive heart failure 䡲 delayed afterdepolarization 䡲 calcium
䡲 sarcoplasmic reticulum
C
ongestive heart failure (CHF) is a common cause of atrial
fibrillation (AF). Both reentrant and triggered mechanisms
have been implicated in AF.1 Although CHF induces a substrate
for atrial reentry,2 there is also evidence for a role of focal drivers
and triggered activity in CHF-related AF.3– 6 Dogs with atrial
tachycardia remodeling have calcium (Ca2⫹)-handling abnormalities, reduced Ca2⫹ transients, and cardiac ryanodine receptor
(RyR2) dysfunction.7,8 Ca2⫹-handling abnormalities may play a
role in CHF-related AF, but there are no published descriptions
of atrial Ca2⫹ handling in patients or clinically relevant animal
models with CHF-associated AF substrates. Accordingly, the
present study was designed to evaluate atrial cardiomyocyte
Ca2⫹ handling, and related protein expression and phosphorylation, in a canine CHF model.
Editorial see p 77
Clinical Perspective see p 102
Methods
A detailed description of materials and methods used in the study is
available in the online Data Supplement.
Received November 22, 2007; accepted February 29, 2008.
From the Department of Medicine and Research Center, Montreal Heart Institute and Université de Montréal (Y.H.Y., R.W., X.Q., D.C., P.C., S.N.),
Montreal, Canada; the Department of Pharmacology and Toxicology (R.W., U.R., D.D.), Dresden University of Technology, Dresden, Germany; Chang
Gung Memorial Hospital and Chang Gung University (Y.H.Y.), Tao-Yuan, Taiwan; the Department of Pharmacology and Toxicology (P.B.), University
of Münster, Münster, Germany; and Ludwig-Maximilians University, Department of Medicine I, Klinikum Grosshadern (R.W., S.K.), Munich, Germany.
The online-only Data Supplement can be found at http://circep.ahajournals.org/cgi/content/full/CIRCEP.107.754788/DC1.
Correspondence to Stanley Nattel, MD, 5000 Belanger St E, Montreal, Quebec, Canada, H1T 1C8. E-mail [email protected]
*Drs Yeh and Wakili contributed equally to this work.
Drs Nattel and Dobrev share senior authorship.
© 2008 American Heart Association, Inc.
Circ Arrhythmia Electrophysiol is available at http://circep.ahajournals.org
93
DOI: 10.1161/CIRCEP.107.754788
94
Circ Arrhythmia Electrophysiol
June 2008
Animal Model
The animal model was prepared as previously described.9 Forty adult
mongrel dogs (22 to 36 kg) were divided into 2 groups: (1) control
(n⫽20) and (2) 2-week ventricular tachypacing–induced CHF
(n⫽20). CHF dogs had unipolar pacing leads inserted fluoroscopically into the right ventricular apex, which were programmed at 240
bpm for 2 weeks.
On the days of study, dogs were anesthetized with morphine (2
mg/kg SC) and ␣-chloralose (120 mg/kg IV, followed by 29.25
mg/kg per hour) and ventilated mechanically. AF (irregular atrial
rhythm ⬎400 bpm) was induced by burst pacing. Mean AF
duration was determined on the basis of multiple AF inductions in
each dog, as an index of the AF-maintaining substrate (for details
see the Methods section in the online Data Supplement). AF
duration (mean⫾SEM) was then calculated for each experimental
group as an indicator of the ability of each group to sustain AF.
Hemodynamic data were obtained with fluid-filled catheters and
transducers.
Table.
Hemodynamic Data
Control
(n⫽20)
CHF
(n⫽20)
Systolic BP, mm Hg
132⫾5
Diastolic BP, mm Hg
82⫾4
114⫾3*
67⫾2*
LVSP, mm Hg
131⫾5
113⫾4*
LVEDP, mm Hg
4.9⫾0.5
15.3⫾1.0†
LAP, mm Hg
4.0⫾0.6
15.4⫾0.9†
RAP, mm Hg
2.7⫾0.5
DAF, s
26⫾8
9.6⫾0.4†
1564⫾106*
*P⬍0.01, †P⬍0.001 vs control.
BP indicates blood pressure; LVSP, LV systolic pressure; LVEDP, LV
end-diastolic pressure; LAP and RAP, LA and RA mean pressure; and DAF,
duration of AF.
Cardiomyocyte Isolation
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Right atrial (RA) and left atrial (LA) preparations were dissected and
coronary perfused at ⬇10 mL/min for cardiomyocyte isolation as
previously described.9 RA and LA cells were stored separately in
Tyrode solution with 200 ␮mol/L Ca2⫹.
Measurement of Cell Contraction and
Ca2ⴙ Fluorescence
Cell-shortening measurements, based on the average of 10 consecutive beats, were obtained from field-stimulated cardiomyocytes
with a video edge detector coupled to a charged-coupled device
camera. Edge-detection cursors were positioned at both cell ends to
measure whole-cell shortening.
[Ca2⫹]i transients were recorded with microfluorimetry.10 Cardiomyocytes were incubated with indo-1 AM (5 ␮mol/L) for 4 to 5
minutes. The cells were then superfused with Tyrode solution for 10
minutes to allow intracellular de-esterification. Cardiomyocytes
were excited with ultraviolet light (340 nm), and emission ratios
(R400/500) were measured through a 10-␮m aperture focused at the
cardiomyocyte center.
Ratiometric Ca2⫹i measurements were converted into intracellular
Ca2⫹ concentration ([Ca2⫹]i) with the formula [Ca2⫹]i⫽Kd⫻␤⫻(R400/500
⫺Rmin)/(Rmax⫺R400/500).10 Experimentally determined Rmin, Rmax, and ␤
averaged 0.43, 2.34, and 1.79, respectively. Sarcoplasmic reticulum
(SR) Ca2⫹ content was evaluated with 15-second 10-mmol/L caffeine
applications via a rapid-switching perfusion system.
For frequency-response measurements, a minimum of 4 Ca2⫹
transients (at 0.1 Hz) or a maximum of 20 Ca2⫹ transients (⬎1.0 Hz)
were time-averaged. The decay time constant (␶) was based on a
monoexponential fit to the [Ca2⫹]i decay curve.
Cellular Electrophysiology
LA cells were used for arrhythmogenic action potential (AP) studies
and RA tissue for biochemistry. APs were recorded with whole-cell
perforated-patch methods. Borosilicate glass electrodes (1.0 mm
outer diameter) had tip resistances between 3 and 5 M⍀. Pipette tips
were filled with nystatin-containing (60 ␮g/mL) intracellular solution.
Junction potentials averaged 15.9 mV and were corrected for APs. For
solution contents, see the online Data Supplement Methods section. All
recordings were obtained at 35⫾0.5°C.
The AP voltage-clamp (whole-cell perforated patch) technique
was used to study AP-dependent effects on [Ca2⫹]i transients. [Ca2⫹]i
transients were recorded from LA cardiomyocytes subjected to
typical AP waveforms from control and CHF cardiomyocytes at 2 Hz
for sequential 2-minute periods (in randomized order).
Western Blot and Phosphatase
Activity Measurements
RA tissue homogenates were prepared and protein concentrations
determined with Amido-black 10B.11 Proteins were fractionated on
SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Western blotting was performed with primary antibodies as
previously described.12 A detailed list of antibodies and sources is
presented in the online Data Supplement Methods section. Protein
bands were visualized by electrochemoluminescence.
Phosphatase activity was measured in atrial homogenates.13 Okadaic acid (3 nmol/L) was used to differentiate between PP1 and
PP2A activities.13
Data Analysis
Group data are presented as mean⫾SEM. Repeated-measures analyses were performed with 2-way analysis of variance (ANOVA),
followed by Bonferroni-corrected t tests for statistically significant ANOVAs. Nonpaired t tests were applied for single 2-group
comparisons and paired t tests for single repeated measures within
one group. Contingency analyses were measured by ␹2 test.
Two-tailed P⬍0.05 was considered statistically significant. The
authors had full access to the data and take responsibility for the
integrity of the data. All authors have read and agreed to the
manuscript as written.
Results
Hemodynamics and AF Duration
Hemodynamic indices and in vivo electrophysiology data are
shown in the Table. Arterial pressures were significantly
reduced in CHF dogs, whereas left ventricle end-diastolic and
LA and RA pressures were increased. CHF significantly
prolonged AF duration.
APs, Cell Shortening, and Ca2ⴙ Transients
CHF cardiomyocytes were enlarged, with a mean capacitance
of 120⫾6 pF, versus 91⫾3 pF for control (n⫽25/group,
P⬍0.01). APs obtained by averaging all available recordings
from each group at 1 Hz are shown in Figure 1A, with mean
action potential duration (APD) data provided in the inset.
APDs were significantly prolonged by CHF in both LA and
RA over a wide range of frequencies (online Data Supplement Figure I). Figure 1B and 1C show recordings of
steady-state cell shortening and Ca2⫹ transients. Cell shortening was significantly decreased in CHF cells (Figure 1D),
whereas Ca2⫹ transients were larger.
Figure 2 shows detailed analyses of LA and RA [Ca2⫹]i
transients. CHF increased diastolic [Ca2⫹]i and [Ca2⫹]i transients at all frequencies from 0.1 to 2 Hz (Figure 2A through
2D). The time to peak and decay time constants of [Ca2⫹]i
Ca2ⴙ Handling in Failing Atrium
A
300
***
***
150
0
B
CHF
Control
0
0
20 mV
APD90
APD50
Cell shortening (µm)
Yeh et al
0
CHF
-5
CTL
-15
0
C
1000
500
1500
2000 ms
1000
[Ca2+]i (nM)
800
CHF
CTL
Figure 1. A, AP waveforms obtained by
digitally averaging all AP recordings from
LA cardiomyocytes at 1 Hz from control
(CTL, left) and CHF (right) dogs. Inset:
Mean⫾SEM results at 1 Hz (n⫽16, n⫽37
for CTL and CHF, respectively;
***P⬍0.001). B and C, Recordings of cell
shortening (B) and Ca2⫹ transients (C)
from single control (CTL) and CHF cardiomyocytes. D, Mean⫾SEM cell shortening as a function of pacing frequency
(n⫽15 and 12 for control and CHF,
respectively; ***P⬍0.001, effect of group:
CHF vs control). E, AP-clamp results;
left, Ca2⫹ transient recordings obtained
with CHF and control waveforms in a
control LA cell; right, corresponding
mean⫾SEM. Ca2⫹ transient amplitudes
(n⫽6 cells, **P⬍0.01).
-10
APD (ms)
50 ms
600
1000
CHF-AP
800
[Ca2+]i (nM)
600
[Ca2+]i (nM)
400
AP-clamp
400
300
CTL-AP
200
100
900
800
700
600
500
400
300
200
100
0
**
CHF
200
CTL
0
D
CTL-AP CHF-AP
100 ms
CHF
Control
0
0
14
12
10
8
6
4
2
0
1000
500
1500
CTL
2000 ms
CHF
***
0.0
0.5
1.0
1.5
2.0
Hz
(LA) and 2F (RA). CHF increased caffeine-induced Ca2⫹
transients by ⬇85% in LA and ⬇50% in RA (Figure 2E and
2F insets). In ventricular cardiomyocytes, [Ca2⫹]i decay
depends mainly on Ca2⫹ extrusion by NCX.14 The decay time
constant of the caffeine-induced transient was comparable for
CHF and control in LA and RA (online Data Supplement
Figure IIC and IID).
transient were similar for CHF and control (online Data
Supplement Figure IIA through IIB). APD prolongation due
to CHF could contribute to Ca2⫹i loading, so we performed
AP-clamp experiments, imposing the control and CHF waveforms at 2 Hz in control atrial myocytes. [Ca2⫹]i transients
obtained in 1 cell with either AP waveform are shown in
Figure 1E (left). CHF APs induced a statistically significant
increase (⬇18%) in [Ca2⫹]i transient amplitudes (Figure 1E,
right).
SR Ca2ⴙ Content
Spontaneous Ca2ⴙ Transient Events, Delayed
Afterdepolarizations, Triggered APs, and
Pharmacological Effects
To assess changes in SR Ca2⫹ content, we paced cells for 1
minute at 1 Hz and then rapidly applied 10 mmol/L caffeine.
Caffeine-induced Ca2⫹ transients are illustrated in Figure 2E
The results in Figures 1 and 2 show larger SR Ca2⫹ loads and
Ca2⫹ transients in CHF atria versus control, with LA behaving
similarly to RA. We therefore determined whether CHF LA
400
600
***
***
200
0
0.0
0.5
1.0
1.5
400
***
***
0.5
1.0
1.5
CTL
*P<0.05
CHF
LA
∆[Ca2+]
Caffeine
(nM)
2000
1000
500
0
1200
800
RA
***
0
**
0.0
0.5
CTL
15 20
Caffeine
(nM)
i
**
1500
400
10
∆[Ca2+]
2000
CHF
25 30
[Sec]
*
1.0
1.5
2.0
600 *
** *
*
*
400
200
0
0.0
0.5
1.0
1.5
2.0
f (Hz)
**P<0.01
i
CTL CHF
***
f (Hz)
F
[Ca2+]i (nM)
2000
1600
5
***
200
2.0
1500
0
*
600
400
Amplitude RA
800
f (Hz)
f (Hz)
E
***
***
200
0
0.0
2.0
800
***
D
Amplitude LA
[Ca 2+]i (nM)
[Ca 2+ ]i (nM)
***
***
C
[Ca 2+]i (nM)
600 ***
Diastolic [Ca2+]i RA
B
Diastolic [Ca2+]i LA
[Ca 2+ ]i (nM)
A
[Ca2+]i (nM)
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Cell shortening (µm)
E
95
2000
1000
1600
500
0
1200
800
CTL CHF
CHF
400
CTL
0
5
10
15 20
25 30
[Sec]
Figure 2. Mean⫾SEM. Ca2⫹ transient indices. A
and B, [Ca2⫹]i diastolic levels in left (A) and in
right atrial (B) cells. C and D, Ca2⫹ transient
amplitude in left (C) and right atrial cells (D) (n⫽20
to 24 cells/group; *P⬍0.05, **P⬍0.01, ***P⬍0.001
CHF vs control). E and F, Caffeine-induced Ca2⫹
transients in control and CHF left (E) and right
atrial (F) cardiomyocytes. A 10-mmol/L local caffeine concentration was achieved in ⬍500 ms
with a laminar-flow rapid solution-switching system, producing Ca2⫹ transients, which are indicated by arrows. Insets: Mean⫾SEM caffeineinduced Ca2-transient amplitudes of LA (E) and
RA (F) (n⫽12/group; **P⬍0.01, ***P⬍0.001).
96
Circ Arrhythmia Electrophysiol
June 2008
CTL
A
B
CHF
Post-pacing follow-up 1 min
2 Hz 30 secs
Ca2+ transient events/run
14
** P<0.003
**
12
10
8
6
(28)
4
2
(19)
0
CTL
CHF
CTL
D
**
100
2 sec
CHF
Cells with events (%)
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80
60
40
20
0
-20
-40
-60
-80
80
60
40
20
0
-20
-40
-60
-80
50 mV
C
80
23/26
60
40
20
6/24
0
2 Hz 1 min
Post-pacing follow-up 30 sec
Post-pacing
APs
Figure 3. A, Spontaneous Ca2⫹ transient events after Ca2⫹ loading by 30 seconds of 2-Hz pacing. B, Mean⫾SEM postpacing Ca2⫹
transient events (cell numbers provided in brackets). C, Triggered APs (vertical arrows), after Ca2⫹ loading by 1 minute of pacing at 2
Hz. D, Percentage of cardiomyocytes showing triggered APs (**P⬍0.003).
cardiomyocytes are predisposed to abnormal Ca2⫹ release–
associated arrhythmic events. We first recorded spontaneous
Ca2⫹ transients after a 30-second period of pacing at 2 Hz.
The cessation of cell stimulation was followed by occasional
nonstimulated Ca2⫹ transients in control cells (Figure 3A,
top). In CHF (bottom), many more spontaneous Ca2⫹ transients were seen. Nonstimulated Ca2⫹ transients were ⬇5-fold
more frequent in CHF cells (Figure 3B). Figure 3C shows
whole-cell perforated-patch AP recordings immediately after
1-minute stimulation at 2 Hz. In control cells, triggered APs
were rare, whereas triggered activity was greatly increased in
CHF cells (Figure 3D). Triggered activity was accompanied
by prominent diastolic membrane oscillations that sometimes
appeared as delayed afterdepolarizations and at other times
transitioned smoothly into triggered APs, suggesting abnormal automaticity.
To elucidate the basis of diastolic membrane-potential
oscillations and triggered APs occurring after the Ca2⫹
loading by rapid pacing, we studied the response to pharmacological manipulations. Figure 4A (top) shows spontaneous
AP generation and diastolic oscillatory activity after 2-Hz
pacing in a CHF cardiomyocyte. Ryanodine, an SR Ca2⫹
release channel inhibitor, induced quiescence in this and 5
other similar CHF cells. The Na⫹/Ca2⫹-exchanger (NCX)
suppression with Na⫹- and Ca2⫹-free medium produced
similar responses (Figure 4B). No change in spontaneous
activity was seen on If inhibition with 2 mmol/L Cs⫹ (Figure
4C). Some CHF atrial cardiomyocytes presented spontaneous
activity and APs without pacing-induced Ca2⫹ loading (online
Data Supplement Figure III). These spontaneous events were
completely eliminated by 10 ␮mol/L ryanodine (n⫽4 cells),
whereas CsCl had no effect (n⫽4 cells). These results
indicate that CHF-induced diastolic membrane potential instability leads to triggered activity via mechanisms involving
SR Ca2⫹ release through ryanodine receptors and associated
arrhythmogenic Na⫹- and Ca2⫹-exchange currents.
Ca2ⴙ Handling and Myofilament Proteins
To gain insights into potential abnormalities of Ca2⫹ handling
and contractile protein systems in CHF atria, we performed
Western blots with antibodies that recognize total and phosphorylated forms. Protein-band intensities were normalized to
those of GAPDH on the same lanes (GAPDH intensities did
not differ between CHF and control atria). The expression of
total phospholamban was similar in CHF and control atria
(Figure 5A). Phospholamban phosphorylation by protein
kinase A (PKA) at Ser16 (Ser16-P) and by CaMKII at Thr17
(Thr17-P) functionally enhances SERCA2a Ca2⫹ uptake.
Ser16-P-phospholamban expression was unchanged in CHF,
but Thr17-P-phospholamban increased by ⬇85%. Fractional
phospholamban PKA phosphorylation (Ser16-P-phospholamban–to–total phospholamban ratio) was unchanged in CHF,
Yeh et al
Ca2ⴙ Handling in Failing Atrium
97
100 mV
A
Ryanodine 10 μM
B
Figure 4. Postpacing diastolic membrane
potential oscillations and triggered activity in
CHF cells before (left) and after (right) administration of 10 ␮mol/L ryanodine (A), Na⫹- and
Ca2⫹-free external solution (Na⫹ replaced by
equimolar Li⫹) (B), and 2 mmol/L CsCl (C)
(dashed lines represent 0 mV level). Similar
results were obtained in 6 experiments of each
type. The mean number of triggered APs per
run averaged 13.3⫾4.5 before and 13.0⫾3.1
after 2 mmol/L CsCl application (P⫽NS).
2 sec
Na+ free Ca2+ free
C
Cs+ 2 mM
post pacing follow-up 3 min
post pacing follow-up 3 min
2 Hz 1 min
CTL CTL CHF CHF
A
B
PLB-Ser16
7 kDa
PLB-Thr17
7 kDa
SERCA2a
565 kDa
RyR2-Ser2809
565 kDa
RyR2-Ser2815
565 kDa
CSQ
53 kDa
NCX1
120 kDa
37 kDa
CTL
CHF
200
150
100
*
100
* *
50
50
0
X1
0
C
Relative to CTL (A.U.)
150
GAPDH
yR
2
P2
80
9
P2
81
5
P2
R 80
yR 9
2
P
R 281
yR 5
2
C
SQ
Se
200
R
PL
r1 B
6
PL -P
L
Th B B
r1
7PL PL
B B
LB
7-
6P
r1
Th
Se
r1
PL
B
a
*
*
*
a2
We have shown that atrial Ca2⫹ handling is significantly
disturbed in dogs with experimental CHF. Ca2⫹ overload was
manifested as increased diastolic Ca2⫹ concentrations, Ca2⫹
Relative to CTL
(normalized to GAPDH)
*
rc
Main Findings
CTL CTL CHF CHF
37 kDa
CTL
CHF
Se
Discussion
105 kDa
GAPDH
350
300
250
200
150
100
50
0
of protein function,8,12,13 for a variety of Ca2⫹ handling and
contractile proteins. To assess the underlying mechanisms,
we analyzed the expression of key protein kinases and protein
phosphatases. CHF increased expression and autophosphorylation of the cytosolic CaMKII␦ isoform by 123% and
114%, respectively (Figure 7A). PKA expression was unchanged for PKAC, whereas PKAc (PKA catalytic subunit)
and PKAIIa (PKA regulatory subunit) was decreased by 72%
(Figure 7B). Because RyR2 and MyBP-C are dephosphorylated primarily by protein phosphatase (PP)1, their reduced
PKA phosphorylation may be caused by the increased PP1
activity. Consistent with this notion, total proteinphosphatase activity was 34% higher in CHF, PP1 activity
was increased by 83%, and PP2A activity was unchanged
(Figure 7C). Protein expression of PP1 and PP2A catalytic
subunits was unchanged in CHF (Figure 7D).
RyR2-Total
7 kDa
PLB-Total
Relative to CTL
(Normalized to GAPDH)
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whereas fractional CaMKII phospholamban phosphorylation
increased by ⬇120%. CHF decreased SERCA2a protein
expression (by ⬇35%).
CHF resulted in a 65% decrease in total RyR2 protein
expression (Figure 5B) and a 73% decrease in PKA-phosphorylated RyR2 (Ser2809-P). Fractional RyR2 phosphorylation (ratios of Ser2809-P-RyR2 and Ser2815-P-RyR2 to
total RyR2) was not significantly altered by CHF. Calsequestrin, a major SR Ca2⫹ buffer and regulator of RyR2 function,15 was reduced ⬇15% in CHF. No significant changes
were noted for NCX1.
Expression values for thin myofilament proteins troponin
(Tn)-I and Tn-C are shown in Figure 6A. Total Tn-I expression, PKA-phosphorylated (Ser23/24)–to–total Tn-I ratio,
and total Tn-C were unchanged by CHF. However, total
thick-myofilament myosin-binding protein C (MyBP-C) and
PKA-phosphorylated MyBP-C (Ser282-P) were significantly
decreased by ⬇27% and ⬇74%, respectively (Figure 6B).
The Ser282-P MyBP-C/total MyBP-C ratio was also reduced
by 67% in CHF, and phosphorylated myosin light chain-2a
protein (MLC2a) was decreased by 46%.
The data in Figures 5 and 6 show potentially important
CHF-induced changes in phosphorylation, a critical regulator
N
2 Hz 1 min
Figure 5. A, Top: Examples of total
phospholamban (PLB), Ser16-P PLB,
and Thr17-P PLB, SERCA2a, and
GAPDH immunoblots. Bottom:
Mean⫾SEM protein-band intensities normalized to GAPDH, relative to control
(n⫽14 control and 10 CHF atria/analysis,
*P⬍0.05 vs control). B, Top: Examples of
total RyR2, Ser2809-P RyR2 and
Ser2815-P RYR, calsequestrin (CSQ),
NCX1, and GAPDH immunoblots. Bottom: Mean⫾SEM protein-band intensities of total RyR2, Ser2809-P RyR2, and
Ser2815-P RyR2 relative to control, and
CSQ and NCX1 normalized to GAPDH,
expressed relative to control (n⫽10 control and 6 CHF atria for RyR2, n⫽14
control and 10 CHF for calsequestrin,
n⫽10 control and 8 CHF for NCX1;
*P⬍0.05 vs control).
98
Circ Arrhythmia Electrophysiol
CTL CTL CHF CHF
A
June 2008
CTL CTL CHF CHF
B
MyBP-C
150 kDa
Tn-I
Ser23/24
30 kDa
MyBP-C-P
150 kDa
Tn-C-Total
21 kDa
MLC2a
21 kDa
GAPDH
37 kDa
GAPDH
37 kDa
200
Relative to CTL
(Normalized to GAPDH)
30 kDa
Relative to CTL
(Normalized to GAPDH)
Tn-I-Total
CTL, n=10
CHF, n=8
150
100
50
0
Tn-I
200
CTL, n=14
CHF, n=10
150
100
*
50
*
MyBP-C P282
P282 MLC2a-P
MyBP-C
afterdepolarizations in cardiomyopathic cats. Epicardial mapping suggests focal drivers during AF in CHF dogs,3,5,17 and
focal-driver ablation can terminate atrial tachyarrhythmias.3
In vivo pharmacological responses consistent with atrial
Ca2⫹-dependent triggered activity have been noted in animals
with CHF.4 Although atrial fibrosis favors atrial reentry,
reentrant activity is rarely triggered by single atrial extrasystoles, requiring bursts of rapid atrial activation for induction,2
a function that could be fulfilled by Ca2⫹-dependent ectopic
firing. Triggered activity could thus contribute to AF in 2
ways: (1) by producing atrial tachycardia bursts that trigger
reentrant AF in vulnerable substrates and (2) by providing
focal drivers that maintain AF.
Our studies have revealed potential mechanisms underlying the Ca2⫹-handling abnormalities that cause atrialtriggered activity in CHF (Figure 8). Changes that we
identified in these studies are color coded, with red representing increases, and blue, decreases. The central abnormality
Significance for Clinically Relevant Mechanisms
AF is very commonly associated with CHF.16 CHF-induced
atrial fibrosis impairs intra-atrial conduction and favors AF
by promoting atrial reentry. 2 However, focal atrial
tachyarrhythmias may also be important in CHF-related AF.
Boyden et al6 showed atrial triggered activity due to delayed
CaMKIIδδB
CTL CHF
CTL
CHF
58 kDa
CTL
B
56 kDa
PKAIIα
GAPDH
37 kDa
GAPDH
CTL, n=16
CHF, n=8
P
37 kDa
150
125
CTL, n=10
CHF, n=8
100
75
50
25
*
0
PKAc PKAIIα
C
P
C-
IIδ
K
51 kDa
K
aM
C
aM
C
54 kDa
IIδ
C
C-
K
IIδ
IIδ
aM
K
aM
C
C
CTL CTL CHF CHF
D
PP1
Phosphatase activity
300
CTL, n=10
CHF, n=11
*
200
*
100
0
Total
PP1 PP2A
37 kDa
PP2A
Relative to CTL
(Normalized to GAPDH)
C
*
*
CHF
40 kDa
Relative to CTL
(normalized to GAPDH)
Thr-287-CaMKIIδC
Relative to CTL
(normalized to GAPDH)
56 kDa
58 kDa
400
350
300
250
200
150
100
50
0
CTL CHF
PKAc
CaMKIIδC
Thr-287-CaMKIIδB
Relative to CTL
(nmol/mg/min)
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transient amplitudes, and caffeine-releasable SR Ca2⫹. These
alterations resulted in a predisposition to spontaneous Ca2⫹
release, abnormal diastolic membrane potential oscillations,
and triggered activity. Despite larger Ca2⫹i transients, CHF
cells displayed reduced contractility. These findings were
accompanied by altered expression and phosphorylation of
key Ca2⫹ handling, myofilament, and contractile proteins, as
well as changes in crucially important regulatory kinases and
phosphatases. CHF increased atrial APD and enhanced
CaMKII phospholamban phosphorylation, both of which
likely contributed to the Ca2⫹-loaded state.
A
*
*
0
Tn-C
Tn-I-P Tn-I-P
Tn-I
Figure 6. A, Top: Examples of Tn-I, Ser23/24-PTn-I, and Tn-C, along with GAPDH bands on the
same lanes. Bottom: Mean⫾SEM protein-band
intensities of total Tn-I, Ser23/24-P-Tn-I, and Tn-C
normalized to GAPDH, expressed relative to control (n⫽10 control and 8 CHF atria/analysis). B,
Top: Examples of total MyBP-C, Ser282-PMyBP-C, and phosphorylated MLC2a bands, along
with GAPDH on the same lanes. Bottom:
Mean⫾SEM protein-band intensities normalized to
GAPDH, expressed relative to control (n⫽14 control, 10 CHF atria/analysis; *P⬍0.05 vs control).
37 kDa
250
200
CTL, n=14
CHF, n=10
150
100
50
0
PP1 PP2A
Figure 7. A, Top: Examples of total CaMKII␦,
Thr287-CaMKII (autophosphorylated), and
GAPDH immunoblots. Top bands (58 kDa)
represent CaMKII␦〉 and bottom (56 kDa)
CaMKII␦C. Bottom: Mean⫾SEM protein-band
intensities normalized to GAPDH, expressed
relative to control (n⫽16 control and 8 CHF
atria/analysis, *P⬍0.05 vs control). B, Top:
Examples of PKAC, PKAII␣, and GAPDH
immmunoblots. The PKAII␣ antibody recognized bands at 51 and 54 kDa. Quantification
is based on the sum of the bands. Bottom:
Mean⫾SEM protein band intensities normalized to GAPDH, expressed relative to control
(n⫽10 control and 8 CHF atria/analysis,
*P⬍0.05 vs control). C, D, Serine/threonine
protein-phosphatase (PP) activity and corresponding protein expression in control and
CHF atria. C, PP activity assessed with
phosphorylase-A as substrate, quantified as
nanomoles of 32Pi released per milligram protein per minute (n⫽10 for control and 11
CHF atria/analysis). D, Representative examples and mean⫾SEM protein-band intensities
of PP1 and PP2A normalized to GAPDH,
expressed relative to control (n⫽14 control
and 10 CHF atria/analysis; *P⬍0.05 vs
control).
Ca2ⴙ Handling in Failing Atrium
Yeh et al
Atrial Fibrillation
Initiation / maintenance
Ectopic focus
CHF
Na+
ICa,L
NCX1
increased
increased
[Ca2+]i
Ca2+- influx
CTL
[Na+]i
●DADs
●Abnormal
automaticity
Ca2+ systolic
2+
CHF
Ca release
APD-prolongation
Triggered activity
diastolic
Ca2+ release
Ser2815
P
P
Ser2809
PP1
Ca2+
RyR
Ca2+
increased CaMKII
2+
Ca2+ [Ca ]SR
PLB
CSQ
Ca2+
Ca2+
Ca2+
Sarcoplasmic
reticulum
SERCA
Ser16
P
P Thr17
Tn-I
P
Ser23/24
Tn-C Ca2+ MyBP-C
Ca2+
Ca2+
99
282
P Ser
PP1
Ca2+
P
Ca2+
MLC-2a
Thromboembolism,
Stroke
Myofilaments
Embolus
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Atrial hypocontractility
Atrial thrombogenesis
that we observed is SR Ca2⫹ overload, likely resulting from 2
primary mechanisms: (1) increased transmembrane Ca2⫹
entry through ICaL during the prolonged CHF-induced AP
waveform and (2) increased SERCA2a-mediated SR Ca2⫹
uptake due to the reduced phospholamban inhibition of
SERCA2a caused by CaMKII phospholamban hyperphosphorylation. This effect of phospholamban hyperphosphorylation presumably overcomes the CHF downregulation of
SERCA2a expression to produce a net increase in SERCAmediated SR Ca2⫹ uptake. In addition, decreased ryanodine
receptor expression can promote increased SR Ca2⫹ content
by reducing SR Ca2⫹ release.18 SR Ca2⫹ overload increases
systolic Ca2⫹ release and also results in spontaneous diastolic
Ca2⫹-release events. The NCX responds to diastolic [Ca2⫹]i
transients by exchanging Ca2⫹ for Na⫹ in a 1:3 ratio,
producing depolarizing inward currents manifesting as delayed afterdepolarizations and abnormal automaticity that
cause triggered activity. Triggered activity is a strong candidate to underlie atrial ectopic foci that can initiate or maintain
AF.
In addition to the increased SR Ca2⫹ loading, we found a
significant reduction in calsequestrin expression. Calsequestrin function–impairing mutations are associated with catecholaminergic polymorphic ventricular tachycardia and increased Ca2⫹-spark and -wave generation.19 –21 Calsequestrinknockout mice show spontaneous Ca2⫹-release events that are
enhanced by isoproterenol.15 Even moderate downregulation
of calsequestrin can increase SR Ca2⫹ leak and arrhythmia
susceptibility.22 Therefore, the reduced calsequestrin expression that we observed may contribute to spontaneous SR
Ca2⫹-release events independently of absolute Ca2⫹i levels.23
In recent years, there has been a concerted effort to develop
novel, mechanistically based approaches to treating AF that
avoid the risks of traditional ion channel targets.1 The
identification of Ca2⫹-handling abnormalities as an important
participant in CHF-related AF opens up interesting possibil-
AF
Thrombus
Figure 8. Schematic representation of
our findings and their potential significance. Observed changes in protein
expression/phosphorylation or Ca2⫹handling function are indicated in red or
blue: Red indicates increases and blue
indicates decreases. Vertical solid black
arrows represent changes in phosphorylating or dephosphorylating enzyme
activity. Increased SR Ca2⫹ loading
causes diastolic Ca2⫹ release through SR
Ca2⫹-release channels (RyR), producing
electrogenic Na⫹,Ca2⫹-exchange currents. The resulting diastolic membrane
potential abnormalities may induce triggered activity-mediated ectopic firing
that can initiate or maintain AF.
Decreased phosphorylation of key contractile element proteins causes atrial cell
hypocontractility that promotes stasisrelated thrombogenesis which can lead
to thromboembolism and stroke. For further details, see Discussion.
ities for novel small molecule – and gene transfer– based
therapeutics.24,25
Impaired atrial contractility predisposes to atrial thromboembolism, one of the most significant complications of AF.
Our study is the first to analyze atrial myocyte contractility
changes and related mechanisms in the CHF setting. Prominent decreases in atrial cardiomyocyte contractility despite
enlarged systolic Ca2⫹ transients imply atrial contractile
protein dysfunction. We identified dramatically reduced
PKA-phosphorylation of MyBP-C at Ser-282 as a potential
underlying factor. MyBP-C PKA-phosphorylation is important for maximum force development, stretch-dependent augmentation of force generation, normal cross-bridge cycling
kinetics, and diastolic relaxation.26 –29 Reduced phosphorylation of MLC2a, another important regulator of myofilament
function,30 may also contribute to contractile dysfunction.
Anticoagulant therapy has been the traditional mainstay of
AF-related thromboembolism prophylaxis but is complex to
maintain and may cause bleeding complications. A better
recognition of the molecular basis of atrial hypocontractility
could lead to new and potentially safer approaches to combating thromboembolic risk.
Comparison With Previous Studies of Ca2ⴙ
Handling in Failing Ventricles and AF
Early studies of Ca2⫹ handling in ventricular cardiomyocytes
from humans31 and rats32 with CHF showed increased diastolic [Ca2⫹]i, reduced Ca2⫹ transients, and slowed Ca2⫹
transient decay. Subsequent work consistently showed reduced and slowed Ca2⫹ transients in failing ventricular
cardiomyocytes, with reduced SR Ca2⫹ stores and abnormal
Ca2⫹ uptake and release mechanisms.14,33–35 The molecular
basis of these ventricular Ca2⫹-handling abnormalities has
been studied extensively.33 Decreased SERCA2a expression
tends to reduce SR Ca2⫹ stores and slow Ca2⫹ transient
decay.34 –36 NCX upregulation attenuates diastolic Ca2⫹ accu-
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mulation due to the reduced SR Ca2⫹ uptake by SERCA2a
and minimizes diastolic dysfunction,36 but it further decreases
SR Ca2⫹ stores.33,35 Ca2⫹ release channel dysfunction caused
by CaMKII (Ser-2815) and/or PKA (Ser-2809) RyR2 hyperphosphorylation contributes to SR Ca2⫹ depletion and arrhythmogenesis by causing diastolic Ca2⫹ leak.35,37,38
The Ca2⫹-handling abnormalities we identified in atrial
cardiomyocytes from CHF dogs differ from previously reported CHF-induced ventricular changes.14,33–35,37 Instead of
reduced Ca2⫹ transients and SR Ca2⫹ stores, atrial cells show
increases in both. We were concerned by the discrepancies
between our atrial Ca2⫹-handling changes and previous reports in ventricular cardiomyocytes. We therefore subjected
paired ventricular and atrial cardiomyocytes from 2 dogs to
Ca2⫹ transient measurements. As shown in online Data
Supplement Figure IV, CHF-induced ventricular Ca2⫹handling changes resembled those in previous reports and
differed clearly from Ca2⫹-handling changes in the atria.
Several studies have examined the functional abnormalities
in Ca2⫹ handling of AF patients, generally without overt CHF.
Quantal Ca2⫹-release events and Ca2⫹ waves are more frequent in AF atria.39 PKA hyperphosphorylation of RyR2
promotes FKBP12.6 unbinding and Ca2⫹-release channel
opening.8 We observed no significant changes in fractional
RyR phosphorylation. This discrepancy may relate to the
specific pathophysiological properties of our model, which
involved recent-onset CHF, as opposed to clinical series
including patients with long-standing AF, a variety of underlying heart diseases, and various cardioactive medications.
Studies of atrial Ca2⫹-handling protein biochemistry in AF
patients have provided widely varying results, possibly because of variations in patient populations.40 Ca2⫹-handling
protein abnormalities in AF subjects reflect the effects of both
underlying cardiac diseases and changes induced by the
arrhythmia itself. Atrial tachycardia alters atrial cardiomyocyte Ca2⫹ handling, prominently decreasing Ca2⫹ transients.7,41 Mice with tumor necrosis factor-␣ overexpression
develop a cardiomyopathic phenotype with atrial fibrosis and
atrial reentrant arrhythmias.42 They also display reduced Ca2⫹
transients and caffeine-releasable Ca2⫹ stores in contrast to
the increased Ca2⫹ loading and Ca2⫹ release in our canine
model. Further studies in well-defined animal models and
clinical populations are needed to clarify the factors contributing to abnormal Ca2⫹ handling in AF.
Potential Limitations
Some of the changes we observed in protein expression and
steady-state phosphorylation appear to contradict our functional observations. Decreased SERCA2a protein expression
should reduce SR Ca2⫹ loading, yet we noted increased SR
Ca2⫹ loads. Increased Ca2⫹ influx due to prolonged atrial
APDs, decreased SR Ca2⫹ discharge due to reduced RyR2
expression, and SERCA function enhancement via phospholamban hyperphosphorylation may be sufficient to offset
decreased SERCA expression and increase SR Ca2⫹ content.
We observed statistically significant ⬇15% decreases in the
principal SR Ca2⫹-binding protein calsequestrin, which would
be expected to reduce SR Ca 2⫹ storage. However,
calsequestrin-knockout mice maintain normal SR Ca2⫹ stores,
apparently by increasing SR volume.15 The decreased RyR2
expression that we observed might be expected to reduce
[Ca2⫹]i transient amplitude by decreasing SR Ca2⫹ release.
Previous studies have shown that decreased RyR2 function
initially does decrease [Ca2⫹]i transients but results in SR
Ca2⫹ accumulation that eventually restores Ca2⫹ release.18
We observed complex changes in protein phosphorylation,
the functional result of which is difficult to predict. Phosphorylating enzymes showed varying changes: unchanged or
decreased expression of PKA subunits and unchanged or
increased expression of CaMKII components. Dephosphorylating enzyme changes also varied, including unchanged
PP2A and enhanced PP1 activity. Thus, for each Ca2⫹handling protein, alterations at specific phosphorylation sites
will depend on the net changes in phosphorylating and
dephosphorylating enzymes acting on that site. Previous
studies have reported varying results with regard to phosphorylation-dependent regulation of RyR2 in CHF, with increased
or decreased ventricular RyR2 Ser-2809 phosphorylation
having been observed (for review, see George et al43).
Ser-2809 is 75% maximally phosphorylated at rest, producing
a minimum basal activity level of RyR2 channels.44 Either
increased or reduced RyR2 phosphorylation enhances RyR2
channel activity.44 We observed a decrease in total RyR2
phosphorylation but no significant changes in fractional
RyR2 phosphorylation because decreased phosphorylated
RyR2 expression paralleled decreased total RyR2 levels.
Changes in expression and activity of ventricular kinases and
phosphatases within local macromolecular complexes do not
necessarily follow global changes in these enzymes,23,37
suggesting localized regulation of kinase or phosphatase
activity in cellular microdomains. Thus, local changes in
atrial macromolecular complexes containing RyR2, phospholamban, MyBP-C, and/or MLC2a may also have contributed
to CHF-induced alterations.
We found similar Ca2⫹-handling changes in LA and RA
free-wall cardiomyocytes, but we cannot be sure that our
results extend to other atrial sites. Having shown the similarity of LA and RA Ca2⫹-handling effects of CHF, we optimized the efficiency of dog usage by studying cellular
arrhythmias and their response in LA cells and analyzing
biochemical changes in RA tissue. We did not perform
detailed complementary studies to assess arrhythmogenic
changes in RA cells and biochemistry in LA tissue. We
estimate that such studies would have required us to euthanize at least 20 additional dogs (10 dogs per group), which we
felt was not justified because of very similar Ca2⫹-handling
changes with CHF in LA versus RA. We did have frozen LA
tissue samples from 6 additional dogs/group, which we
subjected to biochemical analyses. The results, shown in Data
Supplement Figures V through VIII, indicate that Ca2⫹handling protein changes in LA tissue were qualitatively
quite similar to those in RA (which are reproduced beside the
LA data for comparison).
Conclusions
Dogs with ventricular tachypacing–induced dilated cardiomyopathy display important abnormalities in atrial Ca2⫹
handling and in the expression, phosphorylation, and/or
Yeh et al
activity of key atrial Ca2⫹-handling, contractile, and regulatory proteins. They show signs of cellular Ca2⫹ overload,
which results in a predisposition to spontaneous diastolic
Ca2⫹ release, arrhythmogenic diastolic membrane potential
oscillations, and triggered activity. These abnormalities in
Ca 2⫹ homeostasis likely account for focal atrial
tachyarrhythmias that contribute to atrial arrhythmogenesis in
CHF.
Acknowledgments
The authors thank Nathalie L’Heureux, Chantal Maltais, Manja
Schöne, Annett Opitz, and Sabine Kirsch for technical support and
France Thériault for secretarial help with the manuscript.
Sources of Funding
Downloaded from http://circep.ahajournals.org/ by guest on May 11, 2017
The present study was supported by the Canadian Institutes of Health
Research, Quebec Heart and Stroke Foundation, German Federal
Ministry of Education and Research (Atrial Fibrillation Competence
Network grant 01Gi0204), German Research Foundation (grant BO
1263/9 –1), and a network grant (European-North American AtrialFibrillation Research Alliance) from Fondation Leducq.
Disclosures
None.
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CLINICAL PERSPECTIVE
Congestive heart failure (CHF) is a common cause of atrial fibrillation (AF). The therapeutic tools available for AF
management are suboptimal, and new insights into fundamental mechanisms underlying AF may permit the development
of new treatment approaches. The basis of spontaneous atrial ectopic activity that can trigger or drive AF is poorly
understood. This study focused on the potential role of Ca2⫹-handling abnormalities in CHF-related atrial ectopic activity.
CHF was induced in dogs by ventricular tachypacing (240 bpm ⫻2 weeks). Cellular Ca2⫹-handling properties and
expression/phosphorylation status of key Ca2⫹-handling and myofilament proteins were assessed. CHF increased atrial cell
Ca2⫹ loading, leading to spontaneous sarcoplasmic reticulum Ca2⫹ release (especially under Ca2⫹ entry– enhancing
conditions like tachycardia), which resulted in diastolic atrial membrane potential oscillation and ectopic firing. Ca2⫹
overload was due to multiple factors: (1) increased action potential duration, which enhanced Ca2⫹ entry during the action
potential plateau; (2) increased phosphorylation of phospholamban by Ca2⫹/calmodulin-dependent protein kinase II
(CaMKII), which reduces the phospholamban-dependent suppression of sarcoplasmic reticulum Ca2⫹ uptake through the
sarcoplasmic reticulum Ca2⫹ transporter (SERCA); and (3) decreased ryanodine receptor expression, which reduces
sarcoplasmic reticulum Ca2⫹ egress through Ca2⫹-release channels. Despite increased sarcoplasmic reticulum Ca2⫹ loading,
atrial contractility was reduced in CHF, likely because of decreased phosphorylation of 2 important contraction-regulating
proteins, myosin-binding protein C and myosin light chain-2a. Phosphorylation abnormalities in CHF were related to
altered expression and/or activity of phosphorylation-enhancing (CaMKII expression/activity upregulated) and phosphorylation-suppressing (protein phosphatase-1 activity upregulated) proteins. Thus, CHF causes profound changes in
phosphorylation and expression of key Ca2⫹-handling and myofilament regulatory proteins that cause atrial cardiomyocyte
Ca2⫹ homeostasis and contractile abnormalities, which in turn contribute to AF-promoting triggered activity and
thrombosis-promoting contractile dysfunction.
Calcium-Handling Abnormalities Underlying Atrial Arrhythmogenesis and Contractile
Dysfunction in Dogs With Congestive Heart Failure
Yung-Hsin Yeh, Reza Wakili, Xiao-Yan Qi, Denis Chartier, Peter Boknik, Stefan Kääb, Ursula
Ravens, Pierre Coutu, Dobromir Dobrev and Stanley Nattel
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Circ Arrhythm Electrophysiol. 2008;1:93-102; originally published online January 1, 2008;
doi: 10.1161/CIRCEP.107.754788
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