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Cardiovascular Research 2002 54(2):405-415; doi:10.1016/S0008-6363(02)00279-1
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Density and function of inward currents in right atrial cells from chronically fibrillating canine atria

Takuya Yagi, Jielin Pu1, Parag Chandra, Motoki Hara2, Peter Danilo, Jr., Michael R Rosen and Penelope A Boyden*

Departments of Pharmacology and Pediatrics, Center for Molecular Therapeutics, Columbia University, New York, NY 10032, USA

* Corresponding author. Department of Pharmacology, Columbia College of Physicians and Surgeons, 630 West 168th St., New York, NY 10032, USA. Tel.: +1-212-305-7907; Fax: +1-212-305-0529 pab4{at}columbia.edu

Received 16 July 2001; accepted 4 February 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: To determine whether INa and ICaL are altered in function/density in right atrial (RA) cells from dogs with chronic atrial fibrillation (cAF dogs, episodes lasting at least 6 days) and whether the changes that occur differ from those in dogs with nonsustained or brief episodes of fibrillation (nAF dogs). Methods: Using whole cell voltage clamp, sodium and calcium current density and function were determined in disaggregated RA cells from nAF, cAF and control atria (Con). Ca2+ currents were studied with either Ca2+ or Ba2+ as charge carrier, as well as with either EGTA or BAPTA as the internal solution Ca2+ chelator. Results: After rapid atrial pacing, dogs can either fibrillate for short periods of time (nAF) or longer, more sustained periods (cAF). Both the Na+ and Ca2+ current decrease in cells of the nAF atria. Na+ current density remains reduced in cAF cells with some slowing of recovery kinetics. Ca2+ current density does not further decrease with persistent atrial fibrillation (cAF cells) remaining significantly different from Con cells. However, the difference in density of Ca2+ currents between nAF and Con cells is negligible when Ba2+ is charge carrier and when Cai is quickly and effectively chelated with BAPTA. On the contrary, cAF IBaL densities remain significantly reduced compared to Con and nAF values when Ba2+/BAPTA conditions are used. Conclusions: Na+ current density/function does not recover to Con values in cAF. Further these enhanced Ca2+-dependent inactivation processes contribute significantly to the reduction of ICaL density observed in nAF cells while reduction of Ca2+ currents in cAF atria is probably by another mechanism

KEYWORDS Arrhythmia (mechanisms); Na-channel; Ca-channel; Remodeling; Supraventr. arrhythmia


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Rapid atrial rates induce significant electrophysiological remodeling in animal and human atria. This remodeling is characterized by a decrease in atrial effective refractory period with little change in conduction velocity (e.g., Refs. [1,2]) and by reduced duration of atrial action potentials. Furthermore, there is a loss in the slow phase of APD adaptation to rate changes in atrial fibers from dogs in chronic atrial fibrillation (cAF, episodes lasting 6–10 days to 9 months) and dogs that are paced but have only brief episodes or nonsustained atrial fibrillation (nAF) [3].

Reductions in Ca2+ current density have been reported in atrial cells from patients with persistent atrial fibrillation [4,5]. However, it is not clear whether the rate, alone, of persistent AF alters ion channel function in this setting or whether other uncontrolled factors (e.g., underlying disease, concurrent medications, etc.) contribute to the current changes. Reductions in both inward currents have been reported in the canine rapid pacing model of AF. However, the AF episodes in these studies were less than or equal to 45 min [6,7]. Thus, we do not know whether the rate-induced reductions in INa and ICaL observed continue to decline during persistent episodes of AF (<6 days) or whether function/densities of these currents recover toward control pre-rapid pace values.

Thus, the goal of these studies was to determine whether INa and ICaL are altered in function/density in right atrial (RA) cells from nAF dogs and whether any further changes occur in cAF dogs. Animals used in the study are identical or equivalent to animals used to perform the cellular electrophysiology studies of Hara et al. [3].


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animal preparation
This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US NIH Pub. No. 85-23, 1996.

Mongrel female dogs weighing 20–25 kg were anesthetized with thiopental sodium (17 mg/kg, i.v.) and ventilated with isoflurane, 1.5–2%, and O2, 2 l/min. Morphine sulfate, 0.15 mg/kg, was injected into the epidural space to reduce the pain after awaking from anesthesia. Using sterile techniques, Medtronic active fixation leads were attached to the right atrial appendage and the right ventricular free wall, tunneled subcutaneously and then connected to a Thera 8962 pacemaker (Medtronic, Minneapolis, MN). A bipolar stimulating and recording electrode was also attached to the RA appendage for the induction of atrial fibrillation (AF). Complete AV conduction block was produced by injection of 0.1–0.3 ml of 40% formaldehyde into the His bundle, usually resulting in an idioventricular escape rhythm of 30–50 bpm. The ventricular pacemaker was programmed as: rate, 60 bpm; pulse amplitude, 3.3–5 V; pulse width, 0.35–0.5 ms; sensitivity, 2.5 V; refractory period, 300 ms. After the incisions were closed and the dogs recovered from anesthesia they were maintained for 2 days in the recovery room before moving to routine care. The dogs were given cefazolin, 25 mg/kg i.m. prophylactically once before surgery and for 2 days after surgery. After recovery for at least 2 weeks, atrial pacing was instituted (rate, 400–800 bpm; amplitude, 2.5–4 V; pulse width, 0.2–0.4 ms, Itrel 7424 or MINIX 8340) and maintained for 5–7 weeks. At the time of terminal study, dogs were anesthetized with pentobarbital, 30 mg/kg, and the hearts removed.

Only sections of RA freewall were excised for myocyte studies to eliminate heterogeneity in ion channel function that has been reported for normal canine atria [8]. From adjacent tissue, RA trabeculae were removed for cellular electrophysiological studies. Trabecular data were reported by Hara et al. [3].

Three groups of dogs were studied. Dogs in nonsustained atrial fibrillation (nAF dogs) (n=15) had been paced but when AF was induced, it was short-lived. Dogs were monitored intermittently in the laboratory 3–5 days/week and for several hours each time. nAF was defined as an animal having all episodes of AF <6 days duration (usually <24 h). nAF animals could have multiple episodes of AF. Pacemakers of nAF dogs were stopped on the day of sacrifice. Dogs in chronic atrial fibrillation (cAF) (n=15) were paced as above, but when AF was induced, it persisted for at least 6 days. cAF dogs were sacrificed during AF. Age-matched animals (n=12) were used for control RA freewall myocytes (Con).

2.2 Myocyte preparation
Single atrial cells were dispersed from the RA sections using a modification of our previously described method [9]. Briefly, the tissue was rinsed twice in a Ca2+-free solution which contains (mM): NaCl, 115; KCl, 5; sucrose, 35; dextrose, 10; Hepes, 10; taurine, 4; pH, 6.95, to remove blood. Then it was triturated in 20 ml of enzyme containing solution (collagenase TYPE II from Worthington Biochemical, 0.38 mg/ml; 36–37 °C) for 30 min, after which the solution was decanted and discarded. The second trituration was discarded after 30 min. The next six to seven triturations were each done for 15 min. Each time the solution was centrifuged at 500 RPM for 3 min to collect the supernatant and dispersed cells. Resuspension solution was changed every 30 min for solutions containing increasing concentrations of Ca2+. With this procedure, the living atrial cell yield was approximately 30–40%. Only rod-shaped cells with staircase ends, clear cross striations and surface membranes free from blebs were used for study.

2.3 Experimental conditions
2.3.1 Na+ current studies
For study, an aliquot of cells was transferred onto a glass coverslip placed at the bottom of a chamber mounted on the stage of a Nikon inverted microscope (Nikon Diaphot, Tokyo, Japan). Myocytes were continuously superfused (2–3 ml/min) with normal Tyrode's solution containing (in mM): NaCl, 137; NaHCO3 24, NaH2PO4 1.8, MgCl2 0.5, CaCl2 2.0, KCl 4.0 and dextrose 5.5 (pH 7.4). The solution was bubbled with 5% CO2–95% O2, maintained at 19.0±0.5 °C for proper voltage control.Patch pipette resistances ranged between 0.6 and 0.9 M{Omega} when filled with an internal solution which had the following composition (mmol/l): CsOH 125, aspartic acid 125, tetraethylammonium chloride 20, Hepes 10, Mg-ATP 5, EGTA 10, and phosphocreatine 3.6 (pH 7.3 with CsOH). After the formation of the gigaohm seal, the stray capacitance was electronically nulled. The cell membrane under the pipette tip was then ruptured by a brief increase in suction, forming the whole cell recording configuration. Seven minutes were allowed for dialysis to begin before switching to the low sodium extracellular recording solution (mmol/l): NaCl 5, MgCl2 1.2, CaCl2 1.8, CsCl 5, tetraethylammonium chloride 125, Hepes 20, glucose 11, 4-aminopyridine (4-AP) 3, and MnCl2 2 (pH 7.3 with CsOH). With these solutions, INa would be of manageable size and isolated from other possible contaminating currents.

2.3.1.1 Voltage-clamp and recording techniques
Whole-cell Na+ currents (INa) were recorded from atrial cells using previously described whole-cell patch clamp techniques [10,11]. Membrane capacity (in pF) of each cell was measured in the Cs+ rich solution by integrating the area under a capacitative transient induced by a 10-mV hyperpolarizing clamp step (from –80 to –90 mV) and dividing this area by the voltage step. Current amplitude data of each cell was then normalized to its capacitance (current density (pA/pF)). For this series of experiments averaged cell capacitances were 53.0±5.0, 60.7±8.5 and 86.3±10.0 pF for Con (n=10), nAF(n=7) and cAF (n=9) (P<0.05). The average time constant of decay of the capacitive transient was 0.103±0.01, 0.151±0.03, 0.106±0.02 ms in Con, nAF and cAF, respectively. Therefore, the residual series resistance for each cell was calculated to be 2.03, 2.68 and 1.25 M{Omega} in Con,nAF and cAF. For consideration of the voltage control, we lowered extracellular Na+ concentration to 5 mM, maintained the temperature at 19±0.5 °C, used patch pipettes only with resistances lower than 1 M{Omega}. Whole cell INa was obtained by subtracting the traces elicited with comparable voltage steps containing no current from the raw current traces. In this way, the cell capacitance and linear leakage, if present, were subtracted.

To examine peak current density in cells from the different groups, voltage steps (50 ms duration) from holding potential (VH) of –100 mV were given from –70 to 5 mV (5-s intervals) [10]. The maximal peak current was divided by cell capacitance to obtain a peak current density (pA/pF) for each cell. Steady-state availability curves (I/Imax) and the time course of recovery of INa from inactivation were assessed in each cell as previously described [10]. The Boltzmann equation was used to fit normalized data to obtain V0.5, the voltage at half-maximal inactivation, and k, the slope factor. For recovery time constants, INa elicited by each test pulse was then normalized to the maximal current value obtained at the interpulse interval (IPI)=3000 ms. A biexponential function was fitted to the normalized values for each cell.

2.3.2 Calcium–barium current studies
For these studies,cells were initially superfused with normal Tyrode's (see above). Patch pipettes had resistances of 1–2 M{Omega} when filled with the following solution (in mM): CsOH 125, aspartic acid 125, TEACl 20, Hepes 10, Mg-ATP 5, EGTA 10, PCr 3.6, pH 7.3, with CsOH. After gigaohm seal formation and cell membrane rupture, 5–10 min were allowed for intracellular dialysis before switching to a Na+- and K+-free solution containing (in mM): CaCl2 3, TEACl 140, MgCl2 0.5, dextrose 10, Hepes 12, pH 7.3, with CsOH [12,13]. In all experiments, 2 mM 4-aminopyridine and ryanodine (2 µM) were added to the external solution to block current flow through voltage-dependent transient outward K+ channel (Ito) and to inhibit Ca2+ release from the SR. Thus, these recording conditions permitted quantification of the changes in properties of Ca2+ channels in the different cell types under similar conditions.

ICaL magnitudes were normalized by each cell's membrane capacitance (pF) and expressed as current density (pA/pF). Cell capacitances here averaged 66±4 pF in Con (n=43) and 90±7 pF in nAF (n=26) and cAF 84±5 pF (n=28) in this series. Voltages were not corrected for liquid junction potentials between the bath and pipette solutions (~–10 mV). Currents displayed are original tracings with no corrections for linear leakage currents or whole cell capacitance. When myocytes are dialyzed during whole cell recordings, there is ‘rundown’ of peak ICaL with time [14,15]. In this study, we started data acquisition at similar times after membrane rupture after determining that rundown was similar in all cells (average rate of rundown (pA/pF per min), Ca2+ solutions: Con, 0.07, nAF, 0.08, cAF 0.11, P>0.05. Ba2+ solutions: Con 0.14, nAF 0.11, cAF 0.12, P>0.05).

Peak ICaL at various test voltages (Vt) was measured as the difference between the maximal inward peak and the current level at the end of the 250-ms voltage clamp step [13]. As described by others [12,13,16,17], time course of ICaL decay was best fit using a biexponential function. Steady-state inactivation variables of peak ICaL were determined using a double-pulse protocol [18]. The Boltzmann equation was used to fit data as above. These values were used to determine the differences between Con, nAF and cAF cells. The time course of recovery from inactivation of ICaL was examined using a double pulse protocol (delivered every 8 s) consisting of a 1000-ms prepulse from VH=–50 mV to Vt=20 mV followed by a similar test pulse (250 ms duration) delivered at a progressively increasing interpulse interval (IPI) ranging from 2 to 5000 ms as described [13].

Since our initial studies using Ca2+ as the charge carrier suggested differences in density of Ca2+ currents in nAF and cAF cells, we determined whether the changes persist when barium was the charge carrier. Ba2+ studies were completed in different subsets of cells from animals from each group. Internal and external pipette solutions remained similar to above except equimolar Ba2+ was substituted for Ca2+ in external solutions and in one series, 10 mM bis-(o-amino-phenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) was substituted for 10 mM EGTA. Peak IBaL was taken as the difference between peak current and zero current. The time course of decay of Ba currents was determined using exponential functions but results were disappointing since often slow current decays were underestimated and some currents decayed with a monoexponential and others not (in nAF and cAF groups). Therefore, we compared the magnitude of the Ba2+ current at the end of the clamp step (I250) normalized to the peak amplitude (Imax) as well as the time to 80, 70, 60 and 50% of peak during the decay (T0.8max, T0.7max, T0.6max, and T0.5max, respectively) in cells from each group. Thus, for currents that decay quickly, the portion of current remaining at the end of the step would be small compared to that of the peak, and T0.5max should be short.

2.4 Statistics
Data are presented as mean±S.E.M. All data were tested using ANOVA for multiple comparisons. When F values permitted, group means were compared using Bonferroni's method. P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Na+ currents
Fig. 1A shows a family of capacitance and leak subtracted INa amplitudes for cells from RA Con, nAF and cAF dogs. There was a decrease in amplitude of INa in nAF and cAF. Furthermore, when amplitude values were normalized to cell capacitance (Fig. 1B), both nAF and cAF INa densities were significantly different from Con. These differences were unrelated to significant differences in Erev of INa or to INa activation relations (Table 1) nor to the effects of ‘rundown’ on the peak INa [19–21] since all values measured were from data acquired at similar times after establishing whole cell configuration (Con; 23±1.4 min, nAF 22±1.2 min, cAF 26±1 min, P>0.05).


Figure 1
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  		Fig. 1 (A) Average current voltage relations of peak INa in Con (n=10), nAF (n=7) and cAF (n=9) cells using protocol shown on left. (B) Average current density relations of peak INa in Con, nAF and cAF cells. *P<0.05 nAF, cAF versus Con. (C,D) Capacitance-subtracted peak INa in a typical Con and cAF cell. Holding potential, –100 mV.

 

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  		Table 1 Activation parameters of INa

 
The time course of peak INa decay during a maintained depolarization was voltage dependent in all groups and average {tau} fast values at Vt=–20 mV (Con, 1.67±0.08 ms; nAF, 1.94±0.2 ms; cAF, 1.79±0.05 ms) did not differ (P>0.05). Further, while the average maximally available INa was significantly different from Con in nAF and cAF, INa availability relations were not different from Con (Table 2). An altered time course of recovery of INa after inactivation could further contribute to Na+ current depression, particularly at rapid rates. Therefore we compared this process across cell groups and found this kinetic parameter of INa to be reduced in nAF and cAF (Fig. 2B). Con INa recovered from inactivation with two time constants as previously shown (Fig. 2A). In nAF, the first time constant is increased significantly (Table 3) such that I/Imax has only recovered 46% of its maximal value by the 40-ms IPI (interpulse interval) and 62% by the 100-ms IPI interval (Fig. 2B). In cAF, 54% of INa is available at 40-ms IPI interval and 70% INa is available at 100-ms IPI. These latter IPI values do not differ from Con.


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  		Table 2 Steady-state availability parameters of INa

 

Figure 2
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  		Fig. 2 Time course of recovery from inactivation of INa using the protocol shown in inset (B). (A) Recovery for cells in Con, nAF and cAF. INa at a certain interpulse interval (IPI) is normalized to Imax (at IPI=3000 ms) and plotted against IPI. (B) Average recovery curves for all groups for short IPIs. Note the slow recovery at short IPIs in both nAF and cAF cells. *P<0.05 Con versus nAF or Con versus cAF.

 

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  		Table 3 Time course of recovery from inactivation of INa

 
Hence, the altered INa recovery kinetics characteristic of nAF cells are partially reversed in cAF cells and Na+ current amplitudes tend to return toward Con values (Fig. 1A). However, since cAF cell capacitance increases, cAF INa density remains significantly lower than Con.

3.2 Ca2+ currents
Under our initial recording conditions (Ca2+/EGTAi=10 mM), peak ICaL density in RA cells from nAF and cAF hearts was decreased (Fig. 3A,B). There were no significant changes in voltage dependence of activation or inactivation, or recovery from inactivation (data not shown). Although peak ICaL currents in nAF and cAF cells differed from Con ICaL (Fig. 3B), our data and data previously reported [7] show that peak currents in all three groups decay with similar time courses (Fig. 3C). This is unexpected in that atrial Ca2+ currents have a strong current-dependent inactivation process [18,22]. Thus we had anticipated that the large currents of Con cells would decay more quickly than the smaller currents (and hence less Ca2+ influx) of the nAF and cAF cells (Fig. 3C inset). This was not found. Thus we determined in a subset of cells from each group the amplitudes and kinetics of these currents when Ba2+ was the charge carrier. Here Ca2+-dependent inactivation processes should be minimized.


Figure 3
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  		Fig. 3 (A) Currents tracings in Con (a), nAF (b), and cAF (c) cell under conditions of these experiments: Ca2+ 3 mM–EGTAi 10 mM; ryanodine 2 µM; holding voltage, –70 mV to various test voltages. Arrow indicates zero current level. (B) Average peak ICaL density in Con (n=25), nAF (n=11) and cAF (n=6) cells. *P<0.05 versus Con. All data collected at same time after establishing whole cell configuration (Con, 15.0±0.5 min; nAF, 16.5±0.7 min; and cAF, 17.5±0.8 min P>0.05). (C) Average first and second time constants ({tau}1 and {tau}2) of decay of peak ICaL in all cells of the three groups. A1/Atotal were 0.75±0.02, 0.79±0.03, 0.76±0.04 in Con, nAF and cAF cells, respectively (P>0.05). Inset shows first 150 ms of tracings from Con, nAF and cAF cells, each normalized to peak and superimposed to illustrate time course of peak current decay.

 
Under these recording conditions (Ba2+/EGTAi), peak IBaL in RA cells from Con group were larger than those when Ca2+ was the charge carrier (Fig. 4A). Thus, using Ba2+ as the charge carrier, Ca2+-dependent inactivation was minimized. Under these recording conditions, IBaL in nAF and cAF cells (3.87±0.92, n=5; 3.32±0.37, n=7 pA/pF, respectively) remained somewhat reduced compared to Con (6.55±0.95, n=6 pA/pF) but only cAF IBaL were significantly lower than Con (data not shown). Further, for all peak IBaL currents a majority of current (A1/Atotal=60–70%) still inactivated relatively quickly (Table 4). Thus, when the time to 0.8 max (T0.8max) to time to 0.5 max (T0.5max) was determined in cells of each group (Fig. 4B), Tmax values are greater in Con but not different from nAF or cAF at any value. When the fraction of Ba2+ current remaining at the end of the step was assessed relative to peak current (I250/Imax), there was no difference among the different groups (Fig. 4C). Thus the use of Ba2+/EGTAi may have reduced Ca2+-dependent processes.


Figure 4
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  		Fig. 4 (A) Currents tracings in Con (a), nAF (b), and cAF (c) cell under conditions of these experiments; Ba2+ 3 mM–EGTAi 10 mM, ryanodine 2 µM; holding voltage, –70 mV to various test voltages. Arrow indicates zero current. (B) Inset shows peak IBaL tracings during step depolarization in a Con, nAF and cAF cell each normalized to peak and superimposed to illustrate differences in current decay in cells of each group. Bar graph illustrates the average time for current decay to 80, 70, 60 and 50% maximum (T0.8max, T0.7max, T0.6max, T0.5max, respectively) for Con (n=6), nAF (n=5), cAF(n=7). There were no significant differences among groups. (C) Fraction of Ba2+ current remaining at I250 normalized to Imax current for cells of the three groups. Height of bar denotes average value for each of three groups. There was no significant difference in I250/Imax of peak currents under these recording conditions even though peak current densities differed.

 

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  		Table 4 Time course of decay of peak IBaL(EGTA)

 
Therefore, in the next subset of cells we measured IBaL currents and included a fast Ca2+ chelator in the internal solution (BAPTA 10 mM). Here Ba2+ currents were large (Fig. 5A) and there was no difference between Con and nAF. However, peak IBaL in cAF remained significantly less than Con and nAF peak IBaL (Fig. 5B). While there were slight differences in the time constants of decay of peak currents in nAF and cAF cells (Fig. 5C) (Table 5), we assessed T0.6max to T0.8max in each group (Fig. 5D). Clearly differences between intergroup values become larger as time from peak current increases. There is a significant decrease in T0.7max and T0.6max in cAF versus Con or nAF cells (Fig. 5D). Furthermore the fraction of Ba2+ current remaining at end of clamp pulse (I250/Imax) is less in cAF (Fig. 6A) consistent with significantly more current being inactivated during a depolarizing pulse in cAF cells. Finally, cAF acceleration of Ba2+ current decay was present at all test voltages (Fig. 6B) and not accompanied by significant changes in voltage dependence of inactivation or recovery from inactivation in cAF cells (Tables 6 and 7Go).


Figure 5
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  		Fig. 5 (A) Currents tracings in Con (a), nAF (b), and cAF (c) cell under conditions of these experiments; Ba2+ 3 mM/BAPTAi 10 mM; ryanodine, holding voltage, –70 mV to various test voltages. Arrow indicates zero current. (B) Average peak IBaL density voltage relations in Con (n=16), nAF (n=5) and cAF (n=12) cells. Data were all obtained at same time after establishing whole cell configuration (Con, 18.53±0.58 min, nAF, 19.8±1.2 min and cAF 17.9±0.5 min). Note that Con and nAF have comparable peak IBaL densities under Ba2+/BAPTA conditions while cAF density remains different from Con and nAF. *P<0.05 cAF versus Con, nAF. (C) Typical Con, nAF and cAF peak IBaL tracings under Ba2+/BAPTA conditions. Each has been normalized and superimposed to emphasize the differences in current decay between cells from the different groups. Note that the small Ba2+ currents of cAF cells decay more completely during the step depolarization. In (D) the bar graph illustrates the average time for peak current decay to T0.8max, T0.7max, T0.6max, for cells in each group. *P<0.05 Con versus cAF, **P<0.05 nAF versus cAF. Note one Con cell never decayed to 0.6 max during the step depolarization and therefore its value was not included in average at T0.6max.

 

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  		Table 5 Time course of decay of peak IBaL(BAPTA)

 

Figure 6
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  		Fig. 6 (A) The fraction of current remaining at the end of the clamp step (I250) normalized to the peak current (Imax) for peak Ba2+ currents in the three different groups. Height of bar denotes average I250 normalized to Imax. I250/Imax is significantly less in cAF group when compared to either Con or nAF values suggesting that Ba2+ current decay is accelerated in cAF cells (see Fig. 5C). (B) The average I250/Imax for several test voltages (Vt) under these same recording conditions for cells in each group. P<0.05 cAF versus Con curve.

 

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  		Table 6 Steady-state availability parameters of IBa BAPTA

 

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  		Table 7 Time course of recovery from Inactivation of IBa BAPTA

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Consistent with the results of others [6,7], we have shown that after approximately 42 days of rapid atrial pacing, dogs either fibrillate for short periods of time (nAF) or for longer, more sustained periods (cAF), and both the fast Na+ and Ca2+ currents decrease in cells of the nAF atria.

However in addition to a decrease in INa density (by 58%) in nAF cells we observed a slowing of recovery time course, particularly at short interpulse intervals. This is consistent with the reduced INa described by Gaspo et al. [6] for cells from atria paced for 42 days and having short episodes of AF [6]. Further, we now report that in the presence of cAF in dogs, INa density remains significantly reduced (by 59%). We attribute this latter finding to both an increase in cell capacitance and reduced INa amplitude in cAF cells. However, the increase in cell size does not explain the changes we observed in the slowed recovery of INa in both nAF and cAF cells. Thus since both nAF and cAF cells show similar Na current differences, changes in Na current function reported here most likely do not contribute to further ‘domestication of AF’ as it progresses from the nonsustained (nAF) to sustained form (cAF).

An important finding of our study is that significant changes in Ca2+ current density occurring in nAF and cAF cells appear to result from different mechanisms. We state this for several reasons: the Ca2+ current neither recovers to Con values nor continues to decrease with persistent atrial fibrillation (cAF). Furthermore, the difference in Ca2+ current density between nAF and Con is negligible when Ba2+ is the charge carrier and when Cai is quickly and effectively chelated with BAPTA. This suggests that enhanced Ca2+-dependent inactivation processes contribute significantly to the reduction of ICaL density in nAF cells. On the contrary, cAF IBaL densities remain significantly reduced compared to Con and nAF values even when Ba2+/BAPTAi was used.

4.1 Experimental considerations
Atrial cell Na+ and Ca2+ currents exhibit ‘rundown’ or loss in current amplitude with time after whole cell membrane rupture. Although we have noted significant differences in ion channel densities among cells of the different groups, we cannot assign these differences to rundown since we carefully controlled the time after establishing whole cell conditions when data were collected for our comparisons. Importantly, for recording we minimized this time but still allowed the time required for completion of intracellular dialysis and equilibration with the chosen external recording solutions. This time is essential to remove the possibility of overlapping contaminating currents (e.g., Na–Ca exchanger, Ca2+-dependent chloride currents) in our records.

We compared Ca2+ currents using either Ca2+ or Ba2+ as the charge carrier. Here we used EGTA (10 mM) to chelate intracellular Ca2+ levels, removing Cai-dependent inactivation processes. As reported elsewhere [7] with Ca2+/EGTAi, we found a reduction in ICaL in nAF cells and no further reduction in density in cAF cells. These findings are consistent with the loss of the plateau of the action potentials of the RA trabeculae isolated from the same nAF and cAF atria [3].

One possible mechanism for the decrease in ICaL in nAF and cAF cells is a direct effect of Cai on ICaL. In normal ventricular cells, intracellular dialysis with increased Ca2+ [23] or flash photolysis of Ca2+ [24] produces a decrease in ICaL. When EGTA and ryanodine were used, we assumed there to be adequate chelation of bulk Ca2+ since there were no visible signs of cellular contraction. However, we cannot rule out a contribution of Ca2+ accumulation in the subsarcolemmal spaces to changes in channel closing or inactivation [25]. Clearly Ca2+-dependent inactivation of the cardiac L-type Ca2+ channel involves interactions of Ca2+ ions with several different Ca2+ binding proteins and the C terminus of the {alpha} subunit of the channel protein [26,27].

We also used Ba2+ as the charge carrier along with a fast chelator of Ca2+ ions, BAPTA [28,29]. We expect in these experiments that the subsarcolemmal free Ca2+ in the vicinity of the L-type Ca2+ channel protein as well as the bulk free Ca2+ would be affected by the chelator. In fact, we found that Ba2+/BAPTA currents in Con cells were larger when compared to Con Ca2+/EGTA currents, consistent with further minimization of Cai-dependent processes with the BAPTA containing internal solutions.

We did not expect that with the use of BAPTA, Ba2+ currents in nAF cells would be similar to those in Con cells. Thus we hypothesize that in nAF cells the reduced ICaL measured in Ca2+/EGTA or Ba2+/EGTA solutions is largely due to augmented Ca2+-dependent processes that inactivate the Ca2+ channel. The reduced ICaL in cAF cells measured in Ca2+/EGTA or Ba2+/EGTA persisted in Ba2+/BAPTA solutions. This is consistent with a decreased number of functional Ca2+ channel proteins in cAF cells and with clinical reports of an inverse relationship between the amount of mRNA of the {alpha} subunit of the L-type Ca2+ channel and the duration of AF [30]. However, other studies of human AF and the rapidly paced canine AF model, transcriptional down regulation of the {alpha} subunit of the L-type Ca2+ channel parallels the reduced density of the current [31–33]. Still there is a report of no change in mRNA or protein [34] in AF.

There remains an acceleration in current decay in cAF cells in the presence of Ba2+ (Fig. 5C) which cannot be adequately explained by a reduction in number of channels in cAF cells alone. Thus there may be additional changes involving alterations in the inactivation processes of Ca2+ channels in cAF cells. Such a lesion could result from changes in {alpha} subunit interaction with auxiliary proteins and/or β subunits which are known to modulate kinetics of Ca2+ current decay [35–37]. Alternatively, Ba2+/BAPTA solutions may have effectively removed Ca2+-dependent inhibition of adenylyl cyclase [38,39] to restore peak currents in Con and nAF cells, but not in cAF cells. This would suggest a difference in integration of Ca2+ and cAMP signaling in cAF cells.

4.2 Implications of findings in relation to the pharmacology of atrial fibrillation
The changes in ion channel function described here may facilitate the occurrence of persistent AF (see Ref. [40]).Computer modeling studies by Ramirez et al. [41] of a canine atrial cell action potential have suggested that down regulation of ICaL contributes significantly to APD shortening [3]. Our studies suggest that although there remains a significant decrease of the L type Ca2+ currents in cAF cells, this effect evolves in a different manner from that in nAF cells, suggesting differences in mechanism. In nAF cells, Ba2+ currents were similar to Con suggesting that Cai-dependent inactivation of Ca2+ channels dominated the reduction of ICaL in nAF cells. Thus pharmacological agents effective in preventing or terminating nAF may be those which minimize intracellular Cai-dependent inactivation processes of the L-type Ca2+ channel.

While similar agents may be somewhat effective in restoring some Ca2+ channel function in cAF cells (or persistent AF), we suggest they would be of limited efficacy since even with nearly maximal chelation of Cai and its dependent processes in cAF cells, Ca2+ channel function remained decreased. Perhaps a more effective approach when treating persistent AF would be to prevent the critical, irreversible Ca2+ channel down regulation that apparently evolves between the nAF and cAF period.

Time for primary review 22 days.


   Acknowledgements
 
Supported by grants HL53956 AND HL 67449 from NHLBI Bethesda, Maryland and by grant from the Pfizer Company.


   Notes
 
2 Present address: Department of Medicine Keio University, Tokyo, Japan. Back

1 Present address: Department of Medicine University of Wisconsin, Madison, WI, USA. Back


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

  1. Morillo C.A, Klein G.J, Jones D.L, Guiraudon C.M. Chronic atrial pacing: structural, functional and electrophysiological characteristics of a new model of sustained atrial fibrillation. Circulation (1995) 91:1588–1595.[Abstract/Free Full Text]
  2. Wijffels M.C.E.F, Kirchhof C, Dorland R, Allessie M.A. Atrial fibrillation begets atrial fibrillation; a study in awake,chronically instrumented goats. Circulation (1995) 92:1954–1968.[Abstract/Free Full Text]
  3. Hara M, Shvilkin A, Rosen M.R, Danilo P Jr., Boyden P.A. 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]
  4. Van Wagoner D.R, Pond A, Lamorgese M, Rossie S, McCarthy P.M, Nerbonne J.M. Atrial L-type Ca2+ currents and human atrial fibrillation. Circ Res (1999) 85:428–436.[Abstract/Free Full Text]
  5. Bosch R, Zeng X, Grammer J.B, Popovic K, Mewis C, Kuhlkamp V. Ionic mechanisms of electrical remodeling in human atrial fibrillation. Cardiovasc Res. (1999) 44:121–131.[Abstract/Free Full Text]
  6. Gaspo R, Bosch R.F, Bou-Abboud E, Nattel S. Tachycardia-induced changes in Na+ current in a chronic dog model of atrial fibrillation. Circ Res (1997) 81:1045–1052.[Abstract/Free Full Text]
  7. Yue L, Feng J, Gaspo R, Li G.-R, Wang Z, Nattel S. Ionic remodeling underlying action potential changes in a canine model of atrial fibrillation. Circ Res (1997) 81:512–525.[Abstract/Free Full Text]
  8. Feng J, Yue L, Wang Z, Nattel S. Ionic mechanisms of regional action potential heterogeneity in the canine right atrium. Circ Res (1998) 83:541–551.[Abstract/Free Full Text]
  9. Lue W.-M, Boyden P.A. Abnormal electrical properties of myocytes from chronically infarcted canine heart. Alterations in Vmax and the transient outward current. Circulation (1992) 85:1175–1188.[Abstract/Free Full Text]
  10. Pu J, Boyden P.A. Alterations of Na+ currents in myocytes from epicardial border zone of the infarcted heart. A possible ionic mechanism for reduced excitability and postrepolarization refractoriness. Circ Res (1997) 81:110–119.[Abstract/Free Full Text]
  11. Pu J, Balser J, Boyden P.A. Lidocaine action on sodium currents of ventricular myocytes from the epicardial border zone of the infarcted heart. Circ Res (1998) 83:431–440.[Abstract/Free Full Text]
  12. Tseng G.-N, Boyden P.A. Multiple types of Ca currents in single canine Purkinje myocytes. Circ Res (1989) 65:1735–1750.[Abstract/Free Full Text]
  13. Aggarwal R, Boyden P.A. Diminished calcium and barium currents in myocytes surviving in the epicardial border zone of the 5 day infarcted canine heart. Circ Res (1995) 77:1180–1191.[Abstract/Free Full Text]
  14. Belles B, Malecot C.O, Heschler J, Trautwein W. ‘Run down’ of the Ca Current during long whole-cell recordings in guinea pig heart cells: role of phosphorylation and intracellular calcium. Pflugers Arch (1988) 411:353–360.[CrossRef][Web of Science][Medline]
  15. Kameyama M, Heschler J, Hofmann F, Trautwein W. Modulation of Ca current during the phosphorylation cycle in the guinea pig heart. Pflugers Arch (1986) 407:123–128.[CrossRef][Web of Science][Medline]
  16. Hirano Y, Fozzard H.A, January C.T. Characteristics of L- and T-type Ca2+ currents in canine cardiac Purkinje cells. Am J Physiol (1989) 256:H1478–H1492.[Web of Science][Medline]
  17. Rose W.C, Balke C.W, Wier W.G, Marban E. Macroscopic and unitary properties of physiological ion flux through L type Ca channels in guinea pig heart cells. J Physiol (1992) 456:267–284.[Abstract/Free Full Text]
  18. Campbell D.L, Giles W.R, Hume J.R, Shibata E.F. Inactivation of calcium current in bull-frog atrial myocytes. J Physiol (1988) 403:287–315.[Abstract/Free Full Text]
  19. Fenwick E.M, Marty A, Neher E. Sodium and calcium channels in bovine chromaffin cells. J Physiol (1982) 331:599–635.[Abstract/Free Full Text]
  20. Kimitsuki T, Mitsuiye T, Noma A. Negative shift of cardiac Na channel kinetics in cell attached patch recordings. Am J Physiol (1990) 258:H247–H254.[Web of Science][Medline]
  21. Hanck D.A, Sheets M.F. Time-dependent changes in kinetics of Na current in single canine Purkinje cells. Am J Physiol (1992) 262:H1197–H1207.[Web of Science][Medline]
  22. Sun H, Le Blanc N, Nattel S. Mechanisms of inactivation of L-type calcium channels in human atrial myocytes. Am J Physiol (1997) 272:H1625–H1635.[Web of Science][Medline]
  23. Tseng G.-N, Boyden P.A. Different effects of intracellular Ca2+ and a phorbol ester on the T and L types Ca2+ currents in ventricular and Purkinje cells. Am J Physiol (1991) 261:H364–H379.[Web of Science][Medline]
  24. Hadley R.W, Lederer W.J. Ca2+ and voltage inactivate Ca2+ channels in guinea pig myocytes through independent mechanisms. J Physiol (1991) 444:257–268.[Abstract/Free Full Text]
  25. Imredy J.P, Yue D.T. Submicroscopic calcium diffusion mediates inhibitory coupling between individual calcium channels. Neuron (1992) 9:197–207.[CrossRef][Web of Science][Medline]
  26. Hering S, Berjukow S, Sokolov S, Marksteiner R, WeiB R.G, Krasu R, Timin E.N. Molecular determinants of inactivation in voltage gated Ca2+ channels. J Physiol (2000) 528:237–249.[Abstract/Free Full Text]
  27. Anderson M.E. Ca2+ Dependent regulation of cardiac L type Ca2+ channels: is a unifying mechanism at hand? JMCC (2001) 33:639–650.
  28. Lattanzio F.A, Bartschat D.K. The effect of pH on rate constants, ion selectivity and thermodynamic properties of fluroescent calcium and magnesium indicators. Biochem Biophys Res Commun (1991) 177:184–191.[CrossRef][Web of Science][Medline]
  29. Smith P.D, Liesegang R.L, Berger R.L, Czerlinski G, Podolshy R.J. A stopped flow investigation of calcium ion binding by EGTA. Anal Biochem (1984) 143:188–195.[CrossRef][Web of Science][Medline]
  30. Brundel B.J.J.M, VanGelder I.C, Henning R.H, Tuinenburg A.E, Deelman L.E, Tieleman R.G, Grandjean J.G, VanGilst W.H, Crijns H.J.G.M. 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]
  31. Lai L.P, Su M.J, Lin J.L, Lin F.Y, Tsai C.H, Chen Y.S, Huang S.K, Tseng Y.Z, Lien W.P. 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]
  32. van Gelder I.C, Brundel B.J, Henning R.H, Tuinenburg A.E, Tieleman R.G, Deelman L, Grandjean J.G, de Kam P.J, Van Gilst W.H, Crijns H.J. 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]
  33. Yue L, Melnyk P, Gaspo R, Wang Z, Nattel S. Molecular mechanisms underlying ionic remodeling in a dog model of atrial fibrillation. Circ Res (1999) 84:776–784.[Abstract/Free Full Text]
  34. Grammer J.B, Zeng X, Bosch R.F, Kuhlkamp V. Atrial L-type Ca2+-channel, beta-adrenorecptor, and 5-hydroxytryptamine type 4 receptor mRNAs in human atrial fibrillation. Bas Res Cardiol. (2001) 96:82–90.[CrossRef][Web of Science][Medline]
  35. Birnbaumer L, Qin N, Olcese R, Tareilus E, Platano D, Costantin J, Stefani E. Structure and functions of calcium channel β subunits. J Bioenerg Biomembr (2000) 30(4):357–375.
  36. Wei S.-K, Colecraft H.M, DeMaria C.D, Peterson B.Z, Zhang R, Kohout T.A, Rogers T.B, Yue D.T. Ca2+ channel modulation by recombinant auxiliary β subunits expressed in young adult heart cells. Circ Res (2000) 86:175–184.[Abstract/Free Full Text]
  37. Perez Garcia M.T, Kamp T.J, Marban E. Functional properties of cardiac L type calcium channels transiently expressed in HEK293 cells. Role of alpha 1 and beta subunit. J Gen Physiol (1995) 105:289–305.[Abstract/Free Full Text]
  38. Shen J.B, Pappano A.J. Carbachol inhibits the L-type Ca2+ current augmented by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in guinea pig ventricular myocytes: calcium-sensitivity hypothesis for muscarinic inhibition. J Pharmacol Exp Ther (2001) 298:857–864.[Abstract/Free Full Text]
  39. Gu C, Cooper M.S. Ca2+, Sr2+ and Ba2+ identify distinct regulatory sites on adenylyl cyclase (AC) types VI and VIII and consolidate the apposition of capacitative cation entry channels and Ca2+ sensitive ACs. J Biol Chem (2001) 275:6980–6986.[CrossRef][Web of Science]
  40. Allessie M.A, Boyden P.A, Camm A.J, Kleber A.G, Lab M, Legato M.J, Rosen M.R, Schwartz P.J, Spooner P.M, Van Wagoner D.R, Waldo A.L. Pathophysiology and prevention of atrial fibrillation. Circulation (2001) 103:769–777.[Free Full Text]
  41. Ramirez R.J, Nattel S, Courtemanche M. Mathematical analysis of canine atrial action potentials: rate, regional factors and electrical remodeling. Am J Physiol (2000) 279:H1767–H1785.[Web of Science]

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