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Cardiovascular Research 2002 54(2):416-426; doi:10.1016/S0008-6363(02)00274-2
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Remodeling of Ca2+-handling by atrial tachycardia: evidence for a role in loss of rate-adaptation

James Kneller1, Hui Sun1, Normand Leblanc and Stanley Nattel*

Departments of Medicine and Physiology, Research Center, Montreal Heart Institute, University of Montreal, and Department of Pharmacology, McGill University, 5000 Belanger Street East, Montreal, Quebec, Canada H1T 1C8

* Corresponding author. Tel.: +1-514-376-3330x3990; fax: +1-514-376-1355 nattel{at}icm.umontreal.ca

Received 15 August 2001; accepted 21 January 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: Loss of rate-dependent action potential (AP) duration (APD) adaptation is a characteristic feature of atrial tachycardia-induced remodeling (ATR). ATR causes sarcolemmal ion-channel remodeling (ICR) and changes in Ca2+-handling. The present studies were designed to quantify Ca2+-handling changes and then to apply a mathematical AP model to assess the contributions of Ca2+-handling abnormalities and ICR to loss of APD rate-adaptation. Methods: Indo-1 fluorescence was used to measure intracellular Ca2-transients and whole-cell patch-clamp to record APs in atrial myocytes from control dogs and dogs subjected to atrial pacing at 400/min for 6 weeks. A previously developed ionic model of the canine atrial AP was modified to reproduce measured Ca2+-transients of control and ATR myocytes. Results: In control, APD to 95% repolarization (APD95) decreased by 91 ms experimentally and by 88 ms in the model over the 1–6 Hz range. In ATR myocytes, APD95 failed to decrease over the 1–6 Hz range. Ca2+-handling abnormalities in ATR myocytes included slowed upstroke, decreased amplitude and strong single-beat post-rest potentiation. Unaltered Ca2+-handling properties included caffeine-releasable Ca2+-stores and Ca2+-transient relaxation before and after exposure to the sarcoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor cyclopiazonic acid (CPA). Including ICR alone in the model accounted for loss of APD50 rate-adaptation; however, KR alone reduced APD95 rate-adaptation by only 19% to 71 ms. When both ICR and Ca2+-handling changes were incorporated, APD95 rate-adaptation decreased to 6 ms, accounting for experimental observations. Conclusion: ICR alone does not fully account for loss of APD rate-adaptation with atrial remodeling: Ca2+-handling changes appear to contribute to this clinically significant phenomenon.

KEYWORDS Arrhythmia (mechanisms); Calcium (cellular); Remodeling; SR (function); Supraventr. arrhythmia


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Atrial fibrillation (AF) is the most common arrhythmia in clinical practice [1]. Investigators have recently developed realistic animal models of AF related to the phenomenon of atrial tachycardia-induced remodeling, by which ‘AF begets AF’ [2,3]. Remodeling plays an important role in the pathophysiology of AF [4]. Loss of APD and refractoriness rate-adaptation are among the most universally recognized functional changes caused by atrial tachycardia-induced remodeling (ATR) [2–6].

Chronic tachypacing induces specific ion channel remodeling (ICR). L-type Ca2+ current (ICa.L) and transient outward current (Ito) are down-regulated by rapid pacing, with other plateau currents remaining unaltered [7–9]. Inhibition of ICa.L with nifedipine reproduces the AP-shortening and loss of rate-adaptation caused by remodeling [7]; however, the concentration of nifedipine used (10 µM) reduces ICa.L by over 90%, whereas atrial tachycardia decreases ICa.L by about 69% [7]. The intracellular Ca2+-transient, resulting from sarcolemmal Ca2+ entry and Ca2+-triggered Ca2+-release from the sarcoplasmic reticulum (SR), is also reduced in atrial myocytes of rapidly-paced dogs [10], and there is experimental evidence that tachycardia-induced changes in Ca2+-handling contribute to AP abnormalities [11]. We recently developed an ionically based mathematical model of the canine atrial AP and found that pacing-induced ICR fails to account fully for the loss of rate-adaptation produced by remodeling [12]. The Ca2+-handling properties of the latter model, which we will refer to as the Ramirez–Nattel–Courtemanche (RNC) model, were based on data from the literature, rather than direct measurements. The failure of ICR to reproduce lack of rate-adaptation in the RNC model may have been due to inaccuracies in the Ca2+-handling representation, to the lack of a representation of tachycardia-induced Ca2+-handling abnormalities, or to other missing elements. The present study was designed to obtain data with which atrial Ca2+-handling could be more precisely defined, to modify the RNC model to reproduce experimental Ca2+-transients, and to evaluate the role of tachycardia-induced ICR and changed cellular Ca2+-handling in APD rate-maladaptation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animal model, cell isolation and solutions
Animal-handling procedures followed guidelines of the Canadian Council on Animal Care and conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Under sterile conditions, a tined pacemaker lead was inserted in the right atrial appendage, connected to a pacemaker in a subcutaneous pocket in the neck and used to pace the atria at 400/min for 6 weeks to produce ATR [7]. On study days, dogs were anesthetized with 30 mg/kg pentobarbital i.v., the heart removed via right lateral thoracotomy and left atrial myocytes isolated from six control and six paced dogs as previously described [7,10]. Cells were superfused with Tyrode's solution containing (mM) NaCl 136, KCl 5.4, MgCl2 1.0, CaCl2 1.8, NaH2PO4 0.33, glucose 10, and HEPES 10, pH adjusted to 7.4 with NaOH at 36 °C. APs were recorded in current-clamp mode [7].

2.2 Recording of intracellular Ca2+-transients
Myocytes were incubated in indo-1 acetoxymethyl ester (Molecular Probes, 5 µM) for 10–12 min in 100 µM Ca2+-containing Tyrode's solution at room temperature. Myocytes were then superfused with Indo-free solution for >40 min to wash out extracellular indicator and allow for intracellular deesterification. Background and cell autofluorescence were cancelled by zeroing the output of the photomultiplier tubes using cells without indo-1 loading. Cell exposure to ultraviolet light from a mercury-arc lamp was controlled by an electronic shutter (Optikon T132, Vincent Associates) between the arc lamp and epifluorescence attachment of a Nikon Diaphot epifluorescence microscope. Only a portion of the cell (~15 µm diameter) was exposed to UV light. The dye was excited at 340 nm and the emitted fluorescent light (<380 nm) was relayed to the microscope and processed by a spectral microfluorometer (Sycamore Scientific) equipped with a charge-coupled camera (Pulnix America TM-440). The emitted light was split by dichroic mirrors and passed through narrow band-pass (±10 nm) filters centered at 400 and 500 nm. Light intensity was detected with matched photomultiplier tubes (Hamamatsu R2560HA). The ratio of the two fluorescent signals (400/500 nm) was filtered at 60 Hz and digitized at 1 kHz (TL-1-125 LabMaster, Axon). In this study, Indo-1 ratios were converted to Ca2+ concentrations ([Ca2+]i) by performing an in vivo calibration using the following relationship [13]:

Formula (1)
where Kd (844 nM) is the in vivo dissociation constant of Indo-1 [14]. Rmin and Rmax are, respectively, the minimum and maximum Indo-1 ratios determined by exposing atrial myocytes to a Tyrode's solution containing 100 nM ionomycin, and either 10 mM EGTA (no added Ca2+) or 5 mM Ca2+ (saturating Ca solution), and F0/Fs is the ratio of the maximum to minimum fluorescent intensities measured at 500 nm. In five myocytes, Rmin=0.755, Rmax=2.933, and F0/Fs=1.452.

A 1-min rest period separated episodes of field stimulation and Ca2+-transient recording. In some experiments, Ca2+-transients were induced by applying 10 mM caffeine with a temperature-controlled fast-flow system after stopping stimulation at 2 Hz following the attainment of steady state. Only one cell was studied for each cell aliquot added to the bath. In other experiments, Ca2+-transients were studied before and after the addition of 100 µM cyclopiazonic acid (CPA) to inhibit SR Ca2+-ATPase.

2.3 Model development and implementation
Ionic currents were modeled as detailed in Ramirez et al. [12]. The time-derivative of the membrane potential (V) is given by:

Formula (2)
where Iion and Istim are transmembrane total ionic and stimulus currents, respectively, and Cm is membrane capacitance.

Formula (3)
where INa, IK1, IKur,d, IKr, IKs, ICa are sodium, inward-rectifier, ultra-rapid delayed rectifier, rapid and slow delayed-rectifier and Ca2+ currents, respectively, Ip,Ca is sarcolemmal calcium pump, INaK is sodium–potassium pump, INaCa is sodium–calcium exchanger, ICl,Ca is calcium-activated chloride, Ib,Na, Ib,Ca, Ib,Cl are background sodium, calcium and chloride currents, respectively. The model constantly monitors intracellular [Na+], [K+], [Ca2+] and [Cl–].

Sarcoplasmic reticulum (SR) Ca2+-handling is described as illustrated in Fig. 1, with network SR (NSR) subserving Ca2+-uptake and junctional SR (JSR) governing release. Model parameters include cytoplasmic Ca2+-buffering by calmodulin ([Cmdn]max) and troponin ([Trpn]max) and JSR Ca2+-buffering by calsequestrin ([Csqn]max). An uptake flux ({Phi}up) moves Ca2+ from the cytosolic Ca2+ pool to the NSR. {Phi}up depends on [Ca2+]i, and is governed by a maximal value ({Phi}up(max)) and half-saturation constant (Kup). {Phi}leak depends on NSR [Ca2+] ([Ca2+]NSR), and is a function of {Phi}up(max) and maximal [Ca2+]NSR ([Ca2+]NSR(max)). A transfer flux ({Phi}tr) moves Ca2+ from NSR to JSR. {Phi}tr depends on the inter-compartment [Ca2+] gradient ([Ca2+]NSR–[Ca2+]JSR) and is governed by a transfer time-constant ({tau}tr). A release flux ({Phi}rel) corresponds to Ca2+-release from JSR to cytoplasm. {Phi}rel is closely coupled to sarcolemmal Ca2+ channels and is activated by Ca2+ flux into the cytoplasm with activation time constant {tau}rel. {Phi}rel also depends on the [Ca2+] gradient between JSR and cytoplasm ([Ca2+]JSR–[Ca2+]i), and has a maximal release rate (krel). {Phi}rel inactivation gating is both flux and voltage dependent. The parameters defining Ca2+-handling were altered from the RNC model as described below and shown in Fig. 2, to create agreement with experimental Ca2+-transient recordings. Numerical integration of Eq. (3) was carried out using a fixed time step of 5 µs. All simulations were performed using double-precision arithmetic on Unix PC workstations.


Figure 1
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  		Fig. 1 Schematic representation of key components of the Ca2+-handling model (bold). For description and definition of abbreviations, see text. Model constants involved in each process are shown next to each.

 

Figure 2
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  		Fig. 2 Ca2+-handling parameters in RNC model, alterations made to agree with experimental Ca2+-transient recordings (r-Ctl) and alterations in SR Ca2+-handling required to reproduce altered Ca2+ transients in remodeled cells.

 
Group data are expressed as mean±S.D.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Experimental Ca2+-transients and results of the RNC model
Fig. 3 shows representative recordings of Ca2+-transients measured in cells from a control dog (panel A) and a dog subjected to rapid pacing (panel B) stimulated at 2 Hz following a 1-min rest period. Both control and paced cells showed post-rest potentiation, but the control cell showed a biphasic staircase in contrast to low uniform-amplitude Ca2+ signals in the paced cell. For comparison, the RNC model was paced from rest at 2 Hz under control conditions (panel C) and after incorporating known pacing-induced ICR (panel D). Post-rest potentiation was observed in both cases while subsequent Ca2+-transients declined monotonically. The RNC post-rest pulse was reduced by ~200 nM and steady-state peak concentrations were 2.5-fold lower than mean control data (Fig. 4C). After ICR, the RNC post-rest pulse was ~300 nM greater than in ATR (Fig. 6E), while the 60th peak agreed well with experiment.


Figure 3
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  		Fig. 3 Sequential Ca2+ transients from a control (A) and remodeled (B) cell. RNC model simulations of a control cell (C) and a cell incorporating pacing-induced ICR (D). The short horizontal marker at left of CaT simulations in panel C indicates an intracellular Ca2+ level of 500 nM.

 

Figure 4
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  		Fig. 4 Ca2+-transients from a control cell (A), simulations with the r-Ctl model (B) and (C) comparison of Ca2+-transients (CaTs) measured in five control cells (mean±S.D.) and corresponding model simulation (curve). The short horizontal marker at left of CaT simulations in panel B indicates an intracellular Ca2+ level of 500 nM.

 

Figure 6
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  		Fig. 6 Ca2+-transients recorded in an ATR cell (A) compared with SR-only (B), ICR-only (C) and ICR+SR (D) models. (E) Experimental Ca2+-transients from 5 ATR cells (mean±S.D.) along with simulations (curves). The short horizontal marker at left of CaT simulations in panel C indicates an intracellular Ca2+ level of 500 nM.

 
The RNC Ca2+-handling model adopts SR buffer concentrations and binding constants from earlier models of guinea pig [15] and bullfrog [16] cardiac APs, and clearly fails to reproduce Ca2+-transients in dog atrial myocytes. Our first objective was therefore to produce a revised control model (designated ‘r-Ctl’) to match experimental observations. SR constants were adjusted to reproduce quantitatively the biphasic post-rest staircase in control cells (Fig. 4A). Modifications (Fig. 2) included: (for uptake) 20% increase in {Phi}up(max); 60% increase in [Ca2+]NSR(max); 86% reduction of Kup; (for transfer) 33% reduction in {tau}tr; and (for release) 40% increase in {tau}rel. Consistent with routine experimental procedure, all simulations were preceded by 2 min of pacing at 2 Hz followed by a 1-min rest period. The Ca2+-transients generated by the r-Ctl model during pulsing at 2 Hz are shown in Fig. 4B. The r-Ctl Ca2+ peaks differed from mean experimental data by less than 5% at each pulse (Fig. 4C). These adjustments required small modifications of K+ currents to maintain physiological APD50 (a 62% reduction in Ito) and APD95 (a 20% increase in IK1). In the original RNC model, ICa.L was decreased by 70% from experimental values in order to produce physiological APDs [12], but K+ currents were left at experimental values. Therefore, an adjustment in K+ currents of an order less than those of ICa.L was deemed reasonable.

3.2 Effects of rapid pacing on Ca2+-handling and model parameters
To determine the parameter modifications of r-Ctl necessary to reproduce the Ca2+-handling abnormalities caused by remodeling, we studied several specific aspects of Ca2+-handling in paced myocytes. First, we examined SR Ca2+-stores by rapid application of 10 mM caffeine. Fig. 5A shows representative Ca2+-transients at 2 Hz. At steady-state, a caffeine puff was applied via a fast-flow system that exchanged the extracellular milieu of the cell within 500 ms. Mean caffeine-induced [Ca2+] peaks averaged 958±17 nM in five control cells and 887±21 nM in seven paced cells (P=NS), indicating no change in releasable Ca2+. Fig. 5B shows the kinetics of Ca2+-transient rise and decay in a control and remodeled myocyte. The rate of Ca2+-rise was clearly decreased by pacing (from 0.016 to 0.012/ms in the cells shown). The relaxation time-course (quantified as the time for 50% decrease in the Ca2+-transient based on an exponential fit to transient decay) was not altered, measuring 207 and 212 ms in the cells shown. Similar results were obtained in a total of five control and five paced cells. Thus, Ca2+-release was slowed by remodeling, but the kinetics of Ca2+ removal were unaltered. We then studied Ca2+-transient decay kinetics before and after 100-µM CPA. Fig. 5C shows Ca2+-transients at 0.5 Hz before and after CPA in representative control and paced cells. CPA slowed Ca2+-transient relaxation, but there were no relaxation kinetic differences between control and remodeled cells. The 50% relaxation time averaged 244±13 and 257±16 ms in five control and five paced cells (P=NS) before CPA and 486±44 vs. 505±40 ms, respectively (P=NS) after CPA exposure. These results suggest that Na+, Ca2+ exchange (NCX) function and SERCA are unaltered by chronic tachycardia, consistent with previous molecular studies [17,18].


Figure 5
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  		Fig. 5 Examples of caffeine-induced Ca2+-transients (A), Ca2+-transients during steady-state 0.1-Hz stimulation (B) and Ca2+-transients before and after CPA (C).

 
Based on the observations in remodeled myocytes, we determined the parameter alterations of the r-Ctl model needed to reproduce Ca2+-transient reductions in paced cells, assuming that releasable SR Ca2+ stores, Ca2+-uptake rate, NCX and SERCA function are unaltered and Ca2+-release rate is reduced. The required changes are summarized in Fig. 2, and included a 76.3% reduction in [Ca2+]NSR(max), a 328.6% increase in {tau}tr, and a 128.6% increase in {tau}rel. Slowing of {Phi}rel kinetics were needed to match experimentally observed decreases in the Ca2+-transient rise rate. This alteration may reflect disruption of the close coupling of L-type Ca2+-channels and JSR Ca2+ release channels, or of channels selective to monovalent ions countering Ca2+-release [19–21]. [Ca2+]NSR(max) reduction accounted for the abolished positive post-rest staircase, and implies changed JSR leak. While this change appears as a decrease in maximum SR Ca2+-uptake capacity in the model, it may also represent reduced leakiness of ryanodine receptors in the disease state. The large increase in {tau}tr was primarily responsible for the reduced Ca2+-transient amplitude. These changes are consistent with observations of cardiac myolysis and SR fragmentation resulting from AF [22]. The slowing in intra-SR transfer may represent reduced recycling of intra-SR Ca2+, thought to be associated with impaired SR function and the conversion of rest-potentiation to rest-depression in canine myocytes as discussed by Hryshko et al. [23] and depicted schematically in Fig. 9 of Ref. [24].

The model incorporating pacing-induced Ca2+-handling alterations will be referred to as the SR-only model, and the model incorporating only ICR will be termed the ICR-only model. The model incorporating both will be termed the ICR+SR model. Fig. 6 shows beat-to-beat changes in Ca2+-transients recorded from a paced cell (Fig. 6A), along with simulations incorporating Ca2+-handling abnormalities in the SR-only model (Fig. 6B), the ICR-only model (Fig. 6C), and the ICR+SR model (Fig. 6D). Fig. 6E shows mean experimental data from five ATR cells, along with quantitative representations of simulation results. ICR-only fails to account for pacing-induced Ca2+-transient changes. The agreement was better with the SR-only model, but remained imperfect. Steady-state ICR-only and SR-only peaks were approximately 80 and 41% of r-Ctl, respectively, consistent with the findings of Bers et al. [25], where NCX was estimated to account for about 30% of Ca2+ removal from the cytoplasm during relaxation, with SR-uptake accounting for the remaining 70%. In the model, both are ~10% higher than these estimates as each also contributes to the other. In the ICR-only model, reduced trigger Ca2+ from remodeled ICa was nevertheless sufficient to induce a large response from the normal SR, while in the SR-only model, normal ICa was unable to elicit a substantial release from the dysfunctional SR. This property of the ICR-only model contributes both to the persistence of a positive staircase in Fig. 6, and to the persistence of AP rate-adaptation in the absence of SR abnormalities (Fig. 8). The combined ICR+SR model agreed well with experimental data, differing by less than 11% at each pulse.


Figure 8
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  		Fig. 8 Examples of APs recorded from control (A) and remodeled (B) cells at 1 and 6 Hz, along with simulated APs with r-Ctl (C) and ICR+SR (D) models. (E–H) Experimental mean data and corresponding model simulations. Markers at left indicate 0 mV.

 
Fig. 7A and B show individual normalized experimental Ca2+-transients at 1 Hz under control and remodeled conditions. Fig. 7C and D show corresponding simulations with the r-Ctl model and the combined ICR+SR model, respectively. There is clearly good agreement with recordings for both Ca2+-transient amplitude and kinetics, unlike the ICR-only model (Fig. 7E).


Figure 7
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  		Fig. 7 Examples of single Ca2+-transients recorded from control (A) and remodeled (B) cells, and results of simulations with r-Ctl (C), ICR+SR (D), ICR-only (E) and SR-only (F) models. Markers at left indicate 50% of control amplitude.

 
3.3 Tachycardia-induced changes in APD rate-adaptation
Fig. 8 shows representative APs at 1 and 6 Hz from a control (panel A) and ATR (panel B) cell. The control AP displayed a prominent plateau and significant rate-adaptation, while the paced cell showed the triangular morphology, APD abbreviation, and abolished APD rate-adaptation characteristic of tachycardia-induced remodeling. These morphologic features and associated properties were reproduced in the r-Ctl (panel C) and ICR+SR (panel D) models, respectively. Panels E–F show APD to 50% (APD50) repolarization from 1 to 6 Hz (n=25 cells each group). APD50 in the r-Ctl model paralleled experimental results at all rates. APD50 rate-adaptation over 1–6 Hz was 24 ms in the r-Ctl model compared to 32 ms in experiment (panel E). APD50 rate-adaptation was abolished in paced cells and reduced to 5 ms in the ICR+SR model (panel F). ICR-only accounted for 79% of the diminished APD50 rate-adaptation, as ICa reduction was responsible for much of the triangular AP morphology. Rate-adaptation was preserved in the SR-only model because the APD50-shortening effect of rate-dependent ICa inactivation remained, although somewhat offset by potentiation of ICa secondary to decreased Ca2+i-induced inactivation. However, in combination with ICR, SR dysfunction contributed importantly to the ICR+SR result.

Panels G–H show APD to 95% (APD95) repolarization from 1 to 6 Hz. APD95 in the r-Ctl model closely matched experimental data at all rates but was slightly prolonged at 6 Hz (panel G). APD95 rate-adaptation over 1–6 Hz was 91 ms in control, compared to 88 ms in the r-Ctl model. APD95 rate-adaptation was abolished in paced cells and reduced by over 90% (to 6 ms) in the ICR+SR model, which closely approximated the paced group (panel H). APD95 was reduced in the ICR- and SR-only models (panel H), but rate-adaptation persisted, with shortening by 71 and 20 ms, respectively, over the 1–6 Hz range. APD95 shortening in the ICR-only model was due to persistent rate-dependent ICa reduction, since ICa was incompletely down-regulated by ICR. In the SR-only model, AP morphology changes occurred because of reduced Ca2+i-dependent inactivation of ICa. This raised the plateau voltage and prolonged the plateau, thereby increasing IKr by ~61% relative to control, and had a net effect to decrease APD95 rate-adaptation. The ICR-only and SR-only models accounted for 19 and 78% of APD95 rate-adaptation reduction, respectively. Loss of rate-adaptation in ICR+SR arose from the balance of decreased ICa-related effects on the APD in ICR and plateau ICa potentiation by reduced Ca2+i-dependent ICa inactivation with SR abnormalities and resulting IKr recruitment. These results indicate that both ICR and SR dysfunction contribute to ATR-induced loss of APD rate-adaptation, and taken together can account fully for loss of rate-adaptation.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
We developed a mathematical model of the canine atrial AP by modifying Ca2+-handling terms based on experimental Ca2+-transient recordings in control and remodeled cells. The model reproduces changes in APD rate-adaptation in remodeling, suggesting that Ca2+-handling changes contribute to tachycardia-induced atrial repolarization abnormalities.

4.1 Significance and mechanisms of tachycardia-induced loss of APD rate-adaptation
A reduction in atrial refractoriness rate-adaptation is a finding characteristic of tachycardia-induced atrial remodeling [2,26] and is observed in patients with AF [5,6]. Loss of rate-adaptation causes marked refractory period reduction at the long basic cycle lengths of sinus rhythm [2,5,26]. Clinical AF is generally initiated by premature complexes during sinus rhythm, making refractory periods at longer cycle lengths a critical determinant of vulnerability to AF [27]. Thus, APD reduction and loss of APD rate-adaptation are potentially important contributors to the enhanced vulnerability for AF following remodeling.

Li et al. [28] demonstrated a central role of transmembrane Ca2+ current in human atrial APD rate-adaptation. In their human AP model, Courtemanche et al. [29] showed that rate-adaptation arose from a synergistic interaction between ICa and IK. At fast rates, available ICa was decreased while IK was increased, lowering the plateau and accelerating late repolarization, respectively [29]. The central role of ICa was further supported by evidence that rate-adaptation in normal canine atrial cells is abolished by 10 µM nifedipine [7]. Loss of rate-adaptation was reproduced in the RNC and Courtemanche models when ICa was reduced by 90%, simulating pharmacological blockade [12,29]. When ICa is strongly decreased, the plateau level is lowered to the point where IK activation is greatly reduced and can no longer contribute to rate-adaptation [29]. Tachycardia-induced ICa reduction in canine atria [7] is quantitatively similar (69% decrease) to the 63% atrial Ca2+ current density reduction in AF patients [8]. Both are less than the 90% ICa reduction that abolishes APD rate-adaptation in experimental [7] and modeling studies [12,29], indicating that additional mechanisms contribute to loss of rate-adaptation. Hara et al. [11] showed that ryanodine restores the AP plateau in remodeled atria, suggesting that changes in SR Ca2+-handling may play a significant role. The present study suggests that tachycardia-induced ICR is insufficient in itself to account for loss of rate-adaptation, but that the combination of ICR and changes in SR Ca2+-handling does explain loss of APD rate-dependence.

4.2 Comparison of Ca2+-handling abnormalities in remodeled atria and other cardiac pathologies
Congestive heart failure (CHF) is also well-known to cause AP remodeling [30,31] and Ca2+-handling abnormalities. Ventricular Ca2+-transients are prolonged, exhibiting reduced amplitude, slowed relaxation, and blunted frequency-dependence [32] while tissues [30,31,33] and cells [34,35] from failing human ventricles exhibit AP prolongation. Sarcolemmal NCX mRNA and protein levels are increased in CHF [32,36,37]. Ventricular ryanodine receptor mRNA decreases have been noted in some studies of terminal CHF [38,39], but no change in receptor protein level has been demonstrated [40]. Reduced SERCA2a mRNA [36,41–45] expression has been a common finding in CHF. Phospholamban mRNA is consistently reduced [32,41,46], but not necessarily phospholamban protein [37,41,47]. Winslow et al. [48] developed a mathematical model of the failing human ventricular AP with a detailed representation of subcellular Ca2+-handling. They found that changes in Ca2+-handling contribute importantly to AP prolongation in heart failure.

Although some features of Ca2+-transients in atrial remodeling resemble those in CHF (reduced amplitude, loss of positive staircase), others are quite different (minimal or no change in Ca2+-transient relaxation in atrial remodeling). Whereas NCX is prominently up-regulated in CHF, it is unchanged by atrial pacing [17,18,49]. SERCA expression is reduced in some clinical studies of AF patients [49–51] and unchanged in others [18]. Ryanodine receptors, phospholamban and calsequestrin are unaltered in AF [18,49,51]. The present results indicate that, as in CHF, Ca2+-handling abnormalities in remodeled atrial cells contribute to physiologically relevant changes in AP properties. For both pathologies, diminished [Ca2+]i-induced inactivation of L-type Ca2+ current was an important determinant of AP properties.

4.3 Novel findings and potential significance
The present study is the first to evaluate quantitatively the respective roles of ICR and Ca2+-handling abnormalities in the tachycardia-induced loss of atrial APD rate-adaptation. In order to achieve these objectives, we formulated the first atrial AP model incorporating Ca2+-handling formulations based on direct recordings of Ca2+-transients. Our results point to the importance of Ca2+-handling in governing the canine atrial AP and in understanding the electrophysiological basis of tachycardia-mediated changes in AP properties. Our results are consistent with experimental AP studies in atrial tachycardia remodeling [7,11], and give insights into underlying mechanisms. Because of the clinical importance of remodeling for AF [52,53], these insights have potential clinical relevance.

4.4 Potential limitations
The focus of this study was on the role of Ca2+-transient changes in AP properties. A key objective of the model was to reproduce quantitative experimental measurements of Ca2+-transients under control and paced conditions. This was done in a realistic mathematical model of the canine atrial AP that implements a widely accepted representation of the SR. It was recognized that the idea of ‘uptake’ and ‘release’ compartments is a hypothetical construct used to explain the delay between relaxation due to Ca2+ sequestration by the SR and availability of Ca2+ for release [54]. We acknowledge that Ca2+ leak from the SR may arise from the ryanodine receptor itself, allowing more Ca2+ to flow during diastole. What was modeled as a change in JSR leakage may correspond to altered release processes. These may also be thought of as SR release channels recovering from inactivation [54]. A more sophisticated representation would require much more extensive mechanistic and quantitative experimental studies including single Ca2+ channel analysis, beyond the scope of this study. Despite these limitations, the present approach to model development was consistent with known mechanisms. Solutions were unique and optimized to faithfully reproduce experimentally recorded Ca2+-transients under a variety of conditions. The AP is a sarcolemmal phenomenon arising from trans-membrane processes, and the modulation of these processes produced by the Ca2+-transient depends on the cytosolic transient per se, not on the specifics of the Ca2+-handling process that produce the measured Ca2+-transient. Therefore, even if the same Ca2+-transients were produced by a different set of Ca2+-handling alterations, the results would not change our conclusions, which depend on altered Ca2+-transient effects on APs and not how Ca2+-transient changes are achieved. The validity of the results is evinced by the quantitative agreement between experimental and model Ca2+-transients and APD in both control and ATR conditions.

Time for primary review 36 days.


   Acknowledgements
 
This work was supported by the Canadian Institutes of Health Research (CIHR), the Heart and Stroke Foundation of Quebec and the Mathematics of Information Technology and Complex Systems (MITACS) Network. Dr Leblanc is a Fonds de la Recherche en Sante du Quebec Research Scholar. James Kneller is supported by a CIHR MD, PhD Studentship and by a Merck Pharmacology Fellowship. The authors thank Diane Campeau for secretarial assistance.


   Notes
 
1 Both authors contributed equally. Back


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

  1. Kannel W.B., Wolf P.A. Atrial fibrillation: mechanisms and management. Falk R.H., Podrid P.J., eds. (1992) New York: Raven Press. 81–92.
  2. Wijffels M.C., Kirchhof C.J., Dorland R., et al. Atrial fibrillation begets atrial fibrillation. A study in awake chronically instrumented goats. Circulation (1995) 92:1954–1968.[Abstract/Free Full Text]
  3. Wijffels M.C., Kirchhof C.J., Dorland R., et al. Electrical remodeling due to atrial fibrillation in chronically instrumented conscious goats: roles of neurohumoral changes, ischemia, atrial stretch, and high rate of electrical activation. Circulation (1997) 96:3710–3720.[Abstract/Free Full Text]
  4. Nattel S., Li D., Yue L. Basic mechanisms of atrial fibrillation—very new insights into very old ideas. Annu Rev Physiol (2000) 62:51–77.[CrossRef][Web of Science][Medline]
  5. Attuel P., Childers R., Cauchemez B., et al. Failure in the rate adaptation of the atrial refractory period: its relationship to vulnerability. Int J Cardiol (1982) 2:179–197.[CrossRef][Web of Science][Medline]
  6. Boutjdir M., Le Heuzey J.Y., Lavergne T., et al. Inhomogeneity of cellular refractoriness in human atrium: factor of arrhythmia? Pacing Clin Electrophysiol (1986) 9:1095–1100.[CrossRef][Medline]
  7. Yue L., Feng J., Gaspo R., et al. Ionic remodeling underlying action potential changes in a canine model of atrial fibrillation. Circ Res (1997) 81:512–525.[Abstract/Free Full Text]
  8. Van Wagoner D.R., Pond A.L., Lamorgese M., et al. Atrial L-type Ca2+ currents and human atrial fibrillation. Circ Res (1999) 85:428–436.[Abstract/Free Full Text]
  9. Bosch R.F., Zeng X., Grammer J.B., et al. Ionic mechanisms of electrical remodeling in human atrial fibrillation. Cardiovasc Res (1999) 44:121–131.[Abstract/Free Full Text]
  10. Sun H., Gaspo R., Leblanc N., et al. Cellular mechanisms of atrial contractile dysfunction caused by sustained atrial tachycardia. Circulation (1998) 98:719–727.[Abstract/Free Full Text]
  11. Hara M., Shvilkin A., Rosen M.R., et al. Steady-state and nonsteady-state action potentials in fibrillating canine atrium: abnormal rate adaptation and its possible mechanisms. Cardiovasc Res (1999) 42:455–469.[Abstract/Free Full Text]
  12. Ramirez R.J., Nattel S., Courtemanche M. Mathematical analysis of canine atrial action potentials: rate, regional heterogeneity, and electrical remodeling. Am J Physiol (2000) 279:H1767–H1785.[Web of Science]
  13. Grynkiewicz G., Poenie M., Tsien R. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem (1985) 260:3440–3450.[Abstract/Free Full Text]
  14. Bassani J.W.M., Bassani R.A., Bers D.M. Calibration of indo-1 and resting intracellular [Ca]i in intact rabbit cardiac myocytes. Biophys J (1995) 68:1453–1460.[Web of Science][Medline]
  15. Luo C.H., Rudy Y. A dynamic model of the cardiac ventricular action potential. II. Afterdepolarizations, triggered activity, and potentiation. Circ Res (1994) 74:1097–1113.[Abstract/Free Full Text]
  16. Rasmusson R.L., Clark J.W., Giles W.R., et al. A mathematical model of electrophysiological activity in a bullfrog atrial cell. Am J Physiol (1990) 259:H370–H389.[Web of Science][Medline]
  17. Yue L., Melnyk P., Gaspo R., et al. Molecular mechanisms underlying ionic remodeling in a dog model of atrial fibrillation. Circ Res (1999) 84:776–784.[Abstract/Free Full Text]
  18. Van Gelder I.C., Brundel B.J., Henning R.H., et al. Alterations in gene expression of proteins involved in the calcium handling in patients with atrial fibrillation. J Cardiovasc Electrophysiol (1999) 10:552–560.[Web of Science][Medline]
  19. Hals G.D., Stein P.G., Palade P.T. Single channel characteristics of a high conductance anion channel in ‘sarcoballs’. J Gen Physiol (1989) 93:385–410.[Abstract/Free Full Text]
  20. Coronado R., Rosenberg R.L., Miller C. Ionic selectivity, saturation, and block in a K+-selective channel from sarcoplasmic reticulum. J Gen Physiol (1980) 76:425–446.[Abstract/Free Full Text]
  21. Coronado R., Miller C. Decamethonium and hexamethonium block K+ channels of sarcoplasmic reticulum. Nature (1980) 288:495–497.[CrossRef][Medline]
  22. Ausma J., Wijffels M., Thone F., et al. Structural changes of atrial myocardium due to sustained atrial fibrillation in the goat. Circulation (1997) 96:3157–3163.[Abstract/Free Full Text]
  23. Hryshko L.V., Kobayashi T., Bose D. Possible inhibition of canine ventricular sarcoplasmic reticulum by BAY K 8644. Am J Physiol (1989) 257:H407–H414.[Web of Science][Medline]
  24. Hryshko L.V., Bouchard R., Chau T., Bose D. Inhibition of rest potentiation in canine ventricular muscle by BAY K 8644: comparison with caffeine. Am J Physiol (1989) 257:H399–406.[Web of Science][Medline]
  25. Bers D.M., Bridge J.H.B., Spitzer K.W. Intracellular Ca transients during rapid cooling contractures in guinea-pig ventricular myocytes. J Physiol (1989) 417:537–553.[Abstract/Free Full Text]
  26. Gaspo R., Bosch R.F., Talajic M., et al. Functional mechanisms underlying tachycardia-induced sustained atrial fibrillation in a chronic dog model. Circulation (1997) 96:4027–4035.[Abstract/Free Full Text]
  27. Wang J., Liu L., Feng J., et al. Regional and functional factors determining induction and maintenance of atrial fibrillation in dogs. Am J Physiol (1996) 271:H148–H158.[Web of Science][Medline]
  28. Li G.R., Nattel S. Properties of transmembrane Ca2+ current at physiologic temperatures relevant to the action potential in human atrial myocytes. Am J Physiol (1997) 272:H227–H235.[Web of Science][Medline]
  29. Courtemanche M., Ramirez R.F., Nattel S. Ionic mechanisms underlying human atrial action potential properties: insights from a mathematical model. Am J Physiol (1998) 275:H301–H321.[Web of Science][Medline]
  30. Coltart D.J., Meldrum S.J. Intracellular action potential in hypertrophic obstructive cardiomyopathy. Br Heart J (1972) 34:204.[Medline]
  31. Gwathmey J.K., Copelas L., MacKinnon R., et al. Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ Res (1987) 61:70–76.[Abstract/Free Full Text]
  32. O'Rourke B., Kass D.A., Tomaselli G.F., et al. Mechanisms of altered excitation–contraction coupling in canine tachycardia-induced heart. I. Experimental studies. Circ Res (1999) 84:562–570.[Abstract/Free Full Text]
  33. Vermeulen J.T., McGuire M.A., Opthof T., et al. Triggered activity and automaticity in ventricular trabeculae of failing human and rabbit hearts. Cardiovasc Res (1994) 28:1547–1554.[Abstract/Free Full Text]
  34. Beuckelmann D.J., 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]
  35. Beuckelmann D.J., Nabauer M., Erdmann E. Alterations of K+ currents in isolated human ventricular myocytes from patients with terminal heart failure. Circ Res (1993) 73:279–285.
  36. Flesch M., Schwinger R.H., Schiffer F., et al. Evidence for functional relevance of an enhanced expression of the Na(+)–Ca2+ exchanger in failing human myocardium. Circulation (1996) 94:992–1002.[Abstract/Free Full Text]
  37. Reinecke H., Studer R., Vetter R., et al. Cardiac Na+/Ca2+ exchange activity in patients with end-stage heart failure. Cardiovasc Res (1996) 31:48–54.[Abstract/Free Full Text]
  38. Brillantes A.M., Allen P., Takahashi T., et al. Differences in cardiac calcium release channel (ryanodine receptor) expression in myocardium from patients with end-stage heart failure caused by ischemic versus dilated cardiomyopathy. Circ Res (1992) 71:18–26.[Abstract/Free Full Text]
  39. Go L.O., Moschella M.C., Watras J., et al. Differential regulation of two types of intracellular calcium release channels during end-stage heart failure. J Clin Invest (1995) 95:888–894.[Web of Science][Medline]
  40. Meyer M., Schillinger W., Pieske B., et al. Alterations of sarcoplasmic reticulum proteins in failing human dilated cardiomyopathy. Circulation (1995) 92:778–784.[Abstract/Free Full Text]
  41. Schwinger R.H., Bohm M., Schmidt U., et al. Unchanged protein levels of SERCA II and phospholamban but reduced Ca2+ uptake and Ca(2+)-ATPase activity of cardiac sarcoplasmic reticulum from dilated cardiomyopathy patients compared with patients with nonfailing hearts. Circulation (1995) 92:3220–3228.[Abstract/Free Full Text]
  42. Feldman A.M., Weinberg E.O., Ray P.E., et al. Selective changes in cardiac gene expression during compensated hypertrophy and the transition to cardiac decompensation in rats with chronic aortic banding. Circ Res (1993) 73:184–192.[Abstract]
  43. Zarain-Herzberg A., Afzal N., Elimban V., et al. Decreased expression of cardiac sarcoplasmic reticulum Ca(2+)-pump ATPase in congestive heart failure due to myocardial infarction. Mol Cell Biochem (1996) 163–164:285–290.
  44. Hasenfuss G., Reinecke H., Studer R., et al. Relation between myocardial function and expression of sarcoplasmic reticulum Ca(2+)-ATPase in failing and nonfailing human myocardium. Circ Res (1994) 75:434–442.[Abstract/Free Full Text]
  45. Kiss E., Ball N.A., Kranias E.G., et al. Differential changes in cardiac phospholamban and sarcoplasmic reticular Ca(2+)-ATPase protein levels. Effects on Ca2+ transport and mechanics in compensated pressure-overload hypertrophy and congestive heart failure. Circ Res (1995) 77:759–764.[Abstract/Free Full Text]
  46. Linck B., Boknik P., Eschenhagen T., et al. Messenger RNA expression and immunological quantification of phospholamban and SR-Ca(2+)-ATPase in failing and nonfailing human hearts. Cardiovasc Res (1996) 31:625–632.[Abstract/Free Full Text]
  47. Movsesian M.A., Karimi M., Green K., et al. Ca(2+)-transporting ATPase, phospholamban, and calsequestrin levels in nonfailing and failing human myocardium. Circulation (1994) 90:653–657.[Abstract/Free Full Text]
  48. Winslow R.L., Rice J., Jafri S., et al. Mechanisms of altered excitation–contraction coupling in canine tachycardia-induced heart failure. II. Model studies. Circ Res (1999) 84:571–586.[Abstract/Free Full Text]
  49. Brundel B.J., van Gelder I.C., Henning R.H., et al. Gene expression of proteins influencing the calcium homeostasis in patients with persistent and paroxysmal atrial fibrillation. Cardiovasc Res (1999) 42:443–454.[Abstract/Free Full Text]
  50. Ohkusa T., Ueyama T., Yamada J., et al. Alterations in cardiac sarcoplasmic reticulum Ca2+ regulatory proteins in the atrial tissue of patients with chronic atrial fibrillation. J Am Coll Cardiol (1999) 34:255–263.[Abstract/Free Full Text]
  51. Lai L.P., Su M.J., Lin J.L., et al. Down-regulation of L-type calcium channel and sarcoplasmic reticular Ca(2+)-ATPase mRNA in human atrial fibrillation without significant change in the mRNA of ryanodine receptor, calsequestrin and phospholamban: an insight into the mechanism of atrial electrical remodeling. J Am Coll Cardiol (1999) 33:1231–1237.[Abstract/Free Full Text]
  52. Zipes D.P. Atrial fibrillation. A tachycardia-induced atrial cardiomyopathy. Circulation (1997) 95:562–564.[Free Full Text]
  53. Nattel S. Atrial electrophysiological remodeling caused by rapid atrial activation: underlying mechanisms and clinical relevance to atrial fibrillation. Cardiovasc Res (1999) 42:298–308.[Abstract/Free Full Text]
  54. Yue D.T., Burkhoff D., Franz M.R., Hunter W.C., Sagawa K. Postextrasystolic potentiation of the isolated canine left ventricle. Circ Res (1985) 56:340–350.[Abstract/Free Full Text]

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