Circulation: Arrhythmia and Electrophysiology. 2008;1:93-102
Published online before print April 30, 2008,
doi: 10.1161/CIRCEP.107.754788
CLINICAL PERSPECTIVE
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 and
Stanley Nattel, MD
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.
Correspondence: Correspondence to Stanley Nattel, MD, 5000 Belanger St E, Montreal, Quebec, Canada, H1T 1C8. E-mail stanley.nattel{at}icm-mhi.org
Received November 22, 2007; accepted February 29, 2008.
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Abstract
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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 (Ca
2+) handling contributes
to the CHF-related atrial arrhythmogenic substrate.
Methods and Results— CHF was induced in dogs by ventricular tachypacing (240 bpm x2 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.
Key Words: atrial fibrillation congestive heart failure delayed afterdepolarization calcium sarcoplasmic reticulum
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Introduction
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Congestive 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 (Ca
2+)-handling abnormalities, reduced
Ca
2+ transients, and cardiac ryanodine receptor (RyR2) dysfunction.
7,8 Ca
2+-handling abnormalities may play a role in CHF-related AF,
but there are no published descriptions of atrial Ca
2+ handling
in patients or clinically relevant animal models with CHF-associated
AF substrates. Accordingly, the present study was designed to
evaluate atrial cardiomyocyte Ca
2+ handling, and related protein
expression and phosphorylation, in a canine CHF model.
Editorial see p 77
Clinical Perspective see p 103
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Methods
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A detailed description of materials and methods used in the
study is available in the online Data Supplement.
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.
Cardiomyocyte Isolation
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 µmol/L Ca2+.
Measurement of Cell Contraction and Ca2+ 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 µmol/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-µm aperture focused at the cardiomyocyte center.
Ratiometric Ca2+i measurements were converted into intracellular Ca2+ concentration ([Ca2+]i) with the formula [Ca2+]i=Kdxβx(R400/500 –Rmin)/(Rmax–R400/500).10 Experimentally determined Rmin, Rmax, and β 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 µg/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.
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Results
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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 Ca2+ 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 Ca
2+ transients.
Cell shortening was significantly decreased in CHF cells (
Figure 1D),
whereas Ca
2+ transients were larger.

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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).
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Figure 2 shows detailed analyses of LA and RA [Ca
2+]
i transients.
CHF increased diastolic [Ca
2+]
i and [Ca
2+]
i transients at all
frequencies from 0.1 to 2 Hz (
Figure 2A through 2D). The time
to peak and decay time constants of [Ca
2+]
i transient were similar
for CHF and control (online Data Supplement Figure IIA through
IIB). APD prolongation due to CHF could contribute to Ca
2+i loading, so we performed AP-clamp experiments, imposing the
control and CHF waveforms at 2 Hz in control atrial myocytes.
[Ca
2+]
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 [Ca
2+]
i transient amplitudes (
Figure 1E,
right).

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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 caffeine-induced Ca2-transient amplitudes of LA (E) and RA (F) (n=12/group; **P<0.01, ***P<0.001).
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SR Ca2+ Content
To assess changes in SR Ca
2+ content, we paced cells for 1 minute
at 1 Hz and then rapidly applied 10 mmol/L caffeine. Caffeine-induced
Ca
2+ transients are illustrated in
Figure 2E (LA) and 2F (RA).
CHF increased caffeine-induced Ca
2+ transients by

85% in LA
and

50% in RA (
Figure 2E and 2F insets). In ventricular cardiomyocytes,
[Ca
2+]
i decay depends mainly on Ca
2+ 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).
Spontaneous Ca2+ Transient Events, Delayed Afterdepolarizations, Triggered APs, and Pharmacological Effects
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 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.

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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).
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To elucidate the basis of diastolic membrane-potential oscillations
and triggered APs occurring after the Ca
2+ 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 Ca
2+ release channel inhibitor, induced quiescence
in this and 5 other similar CHF cells. The Na
+/Ca
2+-exchanger
(NCX) suppression with Na
+- and Ca
2+-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 Ca
2+ loading (online Data Supplement
Figure III). These spontaneous events were completely eliminated
by 10 µmol/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 Ca
2+ release through ryanodine
receptors and associated arrhythmogenic Na
+- and Ca
2+-exchange
currents.

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Figure 4. Postpacing diastolic membrane potential oscillations and triggered activity in CHF cells before (left) and after (right) administration of 10 µmol/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).
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Ca2+ Handling and Myofilament Proteins
To gain insights into potential abnormalities of Ca
2+ 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
Ca
2+ 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, whereas fractional
CaMKII phospholamban phosphorylation increased by

120%. CHF
decreased SERCA2a protein expression (by

35%).

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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).
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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 Ca
2+ 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%.

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Figure 6. A, Top: Examples of Tn-I, Ser23/24-P-Tn-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-P-MyBP-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).
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The data in
Figures 5 and 6
show potentially important CHF-induced
changes in phosphorylation, a critical regulator of protein
function,
8,12,13 for a variety of Ca
2+ 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 PKA
C, whereas PKAc (PKA catalytic
subunit) and PKA
IIa (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 protein-phosphatase 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).

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Figure 7. A, Top: Examples of total CaMKII , Thr287-CaMKII (autophosphorylated), and GAPDH immunoblots. Top bands (58 kDa) represent CaMKII B 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).
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Discussion
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Main Findings
We have shown that atrial Ca
2+ handling is significantly disturbed
in dogs with experimental CHF. Ca
2+ overload was manifested
as increased diastolic Ca
2+ concentrations, Ca
2+ transient amplitudes,
and caffeine-releasable SR Ca
2+. These alterations resulted
in a predisposition to spontaneous Ca
2+ release, abnormal diastolic
membrane potential oscillations, and triggered activity. Despite
larger Ca
2+i transients, CHF cells displayed reduced contractility.
These findings were accompanied by altered expression and phosphorylation
of key Ca
2+ 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 Ca
2+-loaded state.
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 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 atrial-triggered 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 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 SERCA-mediated 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.

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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 stasis-related thrombogenesis which can lead to thromboembolism and stroke. For further details, see Discussion.
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In addition to the increased SR Ca
2+ loading, we found a significant
reduction in calsequestrin expression. Calsequestrin function–impairing
mutations are associated with catecholaminergic polymorphic
ventricular tachycardia and increased Ca
2+-spark and -wave generation.
19–21 Calsequestrin-knockout mice show spontaneous Ca
2+-release events
that are enhanced by isoproterenol.
15 Even moderate downregulation
of calsequestrin can increase SR Ca
2+ leak and arrhythmia susceptibility.
22 Therefore, the reduced calsequestrin expression that we observed
may contribute to spontaneous SR Ca
2+-release events independently
of absolute Ca
2+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 possibilities 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 Ca2+ 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+ accumulation 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 Ca2+ 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 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 Ca2+ homeostasis likely account for focal atrial tachyarrhythmias that contribute to atrial arrhythmogenesis in CHF.
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Acknowledgments
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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
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 Atrial-Fibrillation Research Alliance) from Fondation Leducq.
Disclosures
None.
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References
|
|---|
- Nattel S. New ideas about atrial fibrillation 50 years on. Nature. 2002; 415: 219–226.[CrossRef][Medline]
- Li D, Fareh S, Leung TK, Nattel S. Promotion of atrial fibrillation by heart failure in dogs: atrial remodeling of a different sort. Circulation. 1999; 100: 87–95.[Abstract/Free Full Text]
- Fenelon G, Shepard RK, Stambler BS. Focal origin of atrial tachycardia in dogs with rapid ventricular pacing-induced heart failure. J Cardiovasc Electrophysiol. 2003; 14: 1093–1102.[CrossRef][Medline]
- Stambler BS, Fenelon G, Shepard RK, Clemo HF, Guiraudon CM. Characterization of sustained atrial tachycardia in dogs with rapid ventricular pacing-induced heart failure. J Cardiovasc Electrophysiol. 2003; 14: 499–507.[CrossRef][Medline]
- Ryu K, Schroff SC, Sahadevan J, Martovitz NL, Khrestian CM, Stambler BS. Mapping of atrial activation during sustained atrial fibrillation in dogs with rapid ventricular pacing induced heart failure: evidence for a role of driver regions. J Cardiovasc Electrophysiol. 2005; 16: 1348–1358.[Medline]
- Boyden PA, Tilley LP, Albala A, Liu SK, Fenoglio JJ Jr, Wit AL. Mechanisms for atrial arrhythmias associated with cardiomyopathy: a study of feline hearts with primary myocardial disease. Circulation. 1984; 69: 1036–1047.[Abstract/Free Full Text]
- Sun H, Gaspo R, Leblanc N, Nattel S. Cellular mechanisms of atrial contractile dysfunction caused by sustained atrial tachycardia. Circulation. 1998; 98: 719–727.[Abstract/Free Full Text]
- Vest JA, Wehrens XH, Reiken SR, Lehnart SE, Dobrev D, Chandra P, Danilo P, Ravens U, Rosen MR, Marks AR. Defective cardiac ryanodine receptor regulation during atrial fibrillation. Circulation. 2005; 111: 2025–2032.[Abstract/Free Full Text]
- Cha TJ, Ehrlich JR, Zhang L, Shi YF, Tardif JC, Leung TK, Nattel S. Dissociation between ionic remodeling and ability to sustain atrial fibrillation during recovery from experimental congestive heart failure. Circulation. 2004; 109: 412–418.[Abstract/Free Full Text]
- Coutu P, Chartier D, Nattel S. Comparison of Ca2+-handling properties of canine pulmonary vein and left atrial cardiomyocytes. Am J Physiol Heart Circ Physiol. 2006; 291: H2290–H2300.[Abstract/Free Full Text]
- Popov N, Schmitt M, Schulzeck S, Matthies N. Reliable micromethod for determination of the protein content in tissue homogenates. Acta Biol Med Ger. 1975; 34: 1441–1446.[Medline]
- El-Armouche A, Boknik P, Eschenhagen T, Carrier L, Knaut M, Ravens U, Dobrev D. Molecular determinants of altered Ca2+ handling in human chronic atrial fibrillation. Circulation. 2006; 114: 670–680.[Abstract/Free Full Text]
- Boknik P, Fockenbrock M, Herzig S, Knapp J, Linck B, Luss H, Muller FU, Muller T, Schmitz W, Schroder F, Neumann J. Protein phosphatase activity is increased in a rat model of long-term beta-adrenergic stimulation. Naunyn Schmiedebergs Arch Pharmacol. 2000; 362: 222–231.[CrossRef][Medline]
- Hobai IA, O'Rourke B. Enhanced Ca2+-activated Na+-Ca2+ exchange activity in canine pacing-induced heart failure. Circ Res. 2000; 87: 690–698.[Abstract/Free Full Text]
- Knollmann BC, Chopra N, Hlaing T, Akin B, Yang T, Ettensohn K, Knollmann BE, Horton KD, Weissman NJ, Holinstat I, Zhang W, Roden DM, Jones LR, Franzini-Armstrong C, Pfeifer K. Casq2 deletion causes sarcoplasmic reticulum volume increase, premature Ca2+ release, and catecholaminergic polymorphic ventricular tachycardia. J Clin Invest. 2006; 116: 2510–2520.[CrossRef][Medline]
- Ehrlich JR, Nattel S, Hohnloser SH. Atrial fibrillation and congestive heart failure: specific considerations at the intersection of two common and important cardiac disease sets. J Cardiovasc Electrophysiol. 2002; 13: 399–405.[CrossRef][Medline]
- Okuyama Y, Miyauchi Y, Park AM, Hamabe A, Zhou S, Hayashi H, Miyauchi M, Omichi C, Pak HN, Brodsky LA, Mandel WJ, Fishbein MC, Karagueuzian HS, Chen PS. High resolution mapping of the pulmonary vein and the vein of Marshall during induced atrial fibrillation and atrial tachycardia in a canine model of pacing-induced congestive heart failure. J Am Coll Cardiol. 2003; 42: 348–360.[Abstract/Free Full Text]
- Overend CL, O'Neill SC, Eisner DA. The effect of tetracaine on stimulated contractions, sarcoplasmic reticulum Ca2+ content and membrane current in isolated rat ventricular myocytes. J Physiol. 1998; 507 (pt 3): 759–769.[Abstract/Free Full Text]
- Kubalova Z, Gyorke I, Terentyeva R, Viatchenko-Karpinski S, Terentyev D, Williams SC, Gyorke S. Modulation of cytosolic and intra-sarcoplasmic reticulum calcium waves by calsequestrin in rat cardiac myocytes. J Physiol. 2004; 561 (pt 2): 515–524.[Abstract/Free Full Text]
- Song L, Alcalai R, Arad M, Wolf CM, Toka O, Conner DA, Berul CI, Eldar M, Seidman CE, Seidman JG. Calsequestrin 2 (CASQ2) mutations increase expression of calreticulin and ryanodine receptors, causing catecholaminergic polymorphic ventricular tachycardia. J Clin Invest. 2007; 117: 1814–1823.[CrossRef][Medline]
- Terentyev D, Nori A, Santoro M, Viatchenko-Karpinski S, Kubalova Z, Gyorke I, Terentyeva R, Vedamoorthyrao S, Blom NA, Valle G, Napolitano C, Williams SC, Volpe P, Priori SG, Gyorke S. Abnormal interactions of calsequestrin with the ryanodine receptor calcium release channel complex linked to exercise-induced sudden cardiac death. Circ Res. 2006; 98: 1151–1158.[Abstract/Free Full Text]
- Chopra N, Kannankeril PJ, Yang T, Hlaing T, Holinstat I, Ettensohn K, Pfeifer K, Akin B, Jones LR, Franzini-Armstrong C, Knollmann BC. Modest reductions of cardiac calsequestrin increase sarcoplasmic reticulum Ca2+ leak independent of luminal Ca2+ and trigger ventricular arrhythmias in mice. Circ Res. 2007; 101: 617–626.[Abstract/Free Full Text]
- Guo T, Zhang T, Mestril R, Bers DM. Ca2+/Calmodulin-dependent protein kinase II phosphorylation of ryanodine receptor does affect calcium sparks in mouse ventricular myocytes. Circ Res. 2006; 99: 398–406.[Abstract/Free Full Text]
- Wehrens XH, Lehnart SE, Marks AR. Ryanodine receptor–targeted anti-arrhythmic therapy. Ann N Y Acad Sci. 2005; 1047: 366–375.[Abstract/Free Full Text]
- Akar FG, Tomaselli GF. Ion channels as novel therapeutic targets in heart failure. Ann Med. 2005; 37: 44–54.[CrossRef][Medline]
- Sadayappan S, Gulick J, Osinska H, Martin LA, Hahn HS, Dorn GW II, Klevitsky R, Seidman CE, Seidman JG, Robbins J. Cardiac myosin-binding protein-C phosphorylation and cardiac function. Circ Res. 2005; 97: 1156–1163.[Abstract/Free Full Text]
- Stelzer JE, Patel JR, Moss RL. Protein kinase A-mediated acceleration of the stretch activation response in murine skinned myocardium is eliminated by ablation of cMyBP-C. Circ Res. 2006; 99: 884–890.[Abstract/Free Full Text]
- Pohlmann L, Kroger I, Vignier N, Schlossarek S, Kramer E, Coirault C, Sultan KR, El-Armouche A, Winegrad S, Eschenhagen T, Carrier L. Cardiac myosin-binding protein C is required for complete relaxation in intact myocytes. Circ Res. 2007; 101: 928–938.[Abstract/Free Full Text]
- Stelzer JE, Patel JR, Walker JW, Moss RL. Differential roles of cardiac myosin-binding protein C and cardiac troponin I in the myofibrillar force responses to protein kinase A phosphorylation. Circ Res. 2007; 101: 503–511.[Abstract/Free Full Text]
- Grimm M, Haas P, Willipinski-Stapelfeldt B, Zimmermann WH, Rau T, Pantel K, Weyand M, Eschenhagen T. Key role of myosin light chain (MLC) kinase–mediated MLC2a phosphorylation in the
1-adrenergic positive inotropic effect in human atrium. Cardiovasc Res. 2005; 65: 211–220.[Abstract/Free Full Text] - Beuckelmann DJ, Nabauer M, Erdmann E. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation. 1992; 85: 1046–1055.[Abstract/Free Full Text]
- Capasso JM, Li P, Anversa P. Cytosolic calcium transients in myocytes isolated from rats with ischemic heart failure. Am J Physiol. 1993; 265: H1953–H1964.[Medline]
- Bers DM. Altered cardiac myocyte Ca regulation in heart failure. Physiology (Bethesda). 2006; 21: 380–387.[CrossRef][Medline]
- Hobai IA, O'Rourke B. Decreased sarcoplasmic reticulum calcium content is responsible for defective excitation-contraction coupling in canine heart failure. Circulation. 2001; 103: 1577–1584.[Abstract/Free Full Text]
- Armoundas AA, Rose J, Aggarwal R, Stuyvers BD, O'rourke B, Kass DA, Marban E, Shorofsky SR, Tomaselli GF, William Balke C. Cellular and molecular determinants of altered Ca2+ handling in the failing rabbit heart: primary defects in SR Ca2+ uptake and release mechanisms. Am J Physiol Heart Circ Physiol. 2007; 292: H1607–H1618.[Abstract/Free Full Text]
- Hasenfuss G, Schillinger W, Lehnart SE, Preuss M, Pieske B, Maier LS, Prestle J, Minami K, Just H. Relationship between Na+-Ca2+-exchanger protein levels and diastolic function of failing human myocardium. Circulation. 1999; 99: 641–648.[Abstract/Free Full Text]
- Ai X, Curran JW, Shannon TR, Bers DM, Pogwizd SM. Ca2+/calmodulin-dependent protein kinase modulates cardiac ryanodine receptor phosphorylation and sarcoplasmic reticulum Ca2+ leak in heart failure. Circ Res. 2005; 97: 1314–1322.[Abstract/Free Full Text]
- Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell. 2000; 101: 365–376.[CrossRef][Medline]
- Hove-Madsen L, Llach A, Bayes-Genis A, Roura S, Rodriguez Font E, Aris A, Cinca J. Atrial fibrillation is associated with increased spontaneous calcium release from the sarcoplasmic reticulum in human atrial myocytes. Circulation. 2004; 110: 1358–1363.[Abstract/Free Full Text]
- Nattel S, Maguy A, Le Bouter S, Yeh YH. Arrhythmogenic ion-channel remodeling in the heart: heart failure, myocardial infarction, and atrial fibrillation. Physiol Rev. 2007; 87: 425–456.[Abstract/Free Full Text]
- Schotten U, Greiser M, Benke D, Buerkel K, Ehrenteidt B, Stellbrink C, Vazquez-Jimenez JF, Schoendube F, Hanrath P, Allessie M. Atrial fibrillation–induced atrial contractile dysfunction: a tachycardiomyopathy of a different sort. Cardiovasc Res. 2002; 53: 192–201.[Abstract/Free Full Text]
- Saba S, Janczewski AM, Baker LC, Shusterman V, Gursoy EC, Feldman AM, Salama G, McTiernan CF, London B. Atrial contractile dysfunction, fibrosis, and arrhythmias in a mouse model of cardiomyopathy secondary to cardiac-specific overexpression of tumor necrosis factor-{alpha}. Am J Physiol Heart Circ Physiol. 2005; 289: H1456–H1467.[Abstract/Free Full Text]
- George CH, Jundi H, Thomas NL, Fry DL, Lai FA. Ryanodine receptors and ventricular arrhythmias: emerging trends in mutations, mechanisms and therapies. J Mol Cell Cardiol. 2007; 42: 34–50.[CrossRef][Medline]
- Carter S, Colyer J, Sitsapesan R. Maximum phosphorylation of the cardiac ryanodine receptor at serine-2809 by protein kinase a produces unique modifications to channel gating and conductance not observed at lower levels of phosphorylation. Circ Res. 2006; 98: 1506–1513.[Abstract/Free Full Text]
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 x2 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.
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Footnotes
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The online-only Data Supplement can be found at http://circep.ahajournals.org/cgi/content/full/CIRCEP.107.754788/DC1.
*Drs Yeh and Wakili contributed equally to this work. 
Drs Nattel and Dobrev share senior authorship.
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