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Am J Physiol Heart Circ Physiol 291: H2290-H2300, 2006. First published June 23, 2006; doi:10.1152/ajpheart.00730.2005
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Comparison of Ca2+-handling properties of canine pulmonary vein and left atrial cardiomyocytes

Pierre Coutu, Denis Chartier, and Stanley Nattel

Department of Medicine and Research Center, Montreal Heart Institute and Université de Montréal, Montreal, Quebec, Canada

Submitted 8 July 2005 ; accepted in final form 7 June 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cardiac tissue in the pulmonary vein sleeves plays an important role in clinical atrial fibrillation. Mechanisms leading to pulmonary vein activity in atrial fibrillation remain unclear. Indirect experimental evidence points to pulmonary vein Ca2+ handling as a potential culprit, but there are no direct studies of pulmonary vein cardiomyocyte Ca2+ handling in the literature. We used the Ca2+-sensitive dye indo-1 AM to study Ca2+ handling in isolated canine pulmonary vein and left atrial myocytes. Results were obtained at 35°C and room temperature in cells from control dogs and in cardiomyocytes from dogs subjected to 7-day rapid atrial pacing. We found that basic Ca2+-transient properties (amplitude: 186 ± 28 vs. 216 ± 25 nM; stimulus to half-decay time: 192 ± 9 vs. 192 ± 9 ms; atria vs. pulmonary vein, respectively, at 1 Hz), beat-to-beat regularity, propensity to alternans, beta-adrenergic response (amplitude increase at 0.4 Hz: 96 ± 52 vs. 129 ± 61%), number of spontaneous Ca2+-transient events after Ca2+ loading (in normal Tyrode: 0.9 ± 0.2 vs. 1.3 ± 0.2; with 1 µM isoproterenol: 7.6 ± 0.3 vs. 5.1 ± 1.8 events/min), and caffeine-induced Ca2+-transient amplitudes were not significantly different between atrial and pulmonary vein cardiomyocytes. In an arrhythmia-promoting model (dogs subjected to 7-day atrial tachypacing), Ca2+-transient amplitude and kinetics were the same in cells from both pulmonary veins and atrium. In conclusion, the similar Ca2+-handling properties of canine pulmonary vein and left atrial cardiomyocytes that we observed do not support the hypothesis that intrinsic Ca2+-handling differences account for the role of pulmonary veins in atrial fibrillation.

arrhythmia; heart rhythm disorders; ion transport; supraventricular arrhythmia

SINCE THE PIONEERING WORK of Haissaguerre et al. (13, 17) in the late 1990s, it has become clear that the cardiac tissue in the pulmonary vein (PV) sleeves is a major source of ectopic activity in patients with paroxysmal atrial fibrillation (AF). PV ablation has become a widely accepted procedure to prevent AF recurrence (13, 17, 25, 27). However, the mechanisms by which PVs could play a role in the initiation and/or maintenance of AF are not fully understood.

The following three main hypotheses have been advanced to explain PV activity: 1) increased automaticity, 2) reentry, and 3) triggered activity (19, 24). Chen et al. (5–8) found myocytes exhibiting automaticity in PV sleeves, with action potentials (APs) resembling those found in the sinoatrial node, but several other groups failed to find such automaticity under physiological conditions in tissue preparations (1, 9, 15, 16, 22, 38). Reentry, the second mechanism, has been observed to occur in normal canine PVs (1) and apparently in PVs of patients with AF (20). Potential contributors to PV reentry may include heterogeneity in fiber orientation and conduction velocity, as well as in ionic currents and AP properties (1, 9, 14, 15, 37). The propensity for alternans behavior is also an important factor in promoting reentry and fibrillation (23, 30). Cellular Ca2+ handling may be more important than electrophysiological restitution in determining alternans behavior (32). In addition, Ca2+-handling properties are important determinants of triggered activity (the 3rd mechanism postulated to explain PV activity), with spontaneous Ca2+ release known to activate the Na+/Ca2+ exchanger (NCX), which can generate delayed afterdepolarizations (DADs) sufficient to initiate ectopic activation (34). Chen et al. (5, 6) showed evidence of triggered activity in PV cardiomyocytes, and PVs showed triggered activity under conditions favoring enhanced Ca2+ release (16). Thus a variety of lines of evidence point to abnormalities in cellular Ca2+-handling intrinsic to PVs (i.e., present in normal PVs) as a potential mechanism underlying arrhythmic PV activity. To our knowledge, there are no reports in the literature describing Ca2+-handling property measurements in PVs, nor any studies comparing Ca2+ handling of PVs with that of atrial tissue.

Accordingly, the present study was designed to investigate the fundamental Ca2+-handling properties of PV cardiomyocytes isolated from canine hearts and to compare PV properties with those of atrial cardiomyocytes in the same animals. In addition, we examined two Ca2+-handling-related mechanisms mentioned above that could contribute to arrhythmias, Ca2+-transient alternans, and spontaneous coordinated release of Ca2+ (which could underlie triggered activity).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Myocyte isolation. Myocyte isolation was performed as described previously (9). Briefly, adult mongrel dogs (17 males and 9 females; 27.5 ± 1.1 kg) were anesthetized using pentobarbital sodium (30 mg/kg iv). All animal-handling procedures were approved by the Animal Research Ethics Committee of the Montreal Heart Institute and were consistent with National Institutes of Health guidelines and the principles of the American Physiological Society. We also studied an additional group of four dogs (22.4 ± 1.6 kg) that had been subjected to 1 wk of rapid atrial pacing (400 beats/min) according to previously described methods (35). Hearts were removed and placed in oxygenated normal Tyrode (NT) solution (for composition, see ref. 9) with 2 mM Ca2+. The left atrium (LA) was removed along with the PV sleeves, and the PVs were marked with silk thread before digestion. The LA/PV tissue was then perfused at 35°C via the left circumflex coronary artery with 2 mM Ca2+-NT (~10–20 min), then with NT without Ca2+ (15 min), and finally with the enzyme-containing solution (~25–40 min) until the tissue appeared digested. The enzyme solution consisted of NT without Ca2+, collagenase type II (85–105 U/ml; Worthington), and BSA (0.1%; Sigma). LA and PV sleeve myocytes were then removed, triturated, and transferred to separate petri dishes, each containing NT with 200 µM Ca2+, and were kept at room temperature until use.

Ca2+ fluorescence measurements and experimental protocols. Before each set of measurements in intact cells, the myocytes were incubated with the Ca2+ indicator indo-1 AM [5 µM (Molecular Probes) in 100 µM pluronic F-127 (Molecular Probes) and 0.5% DMSO (Sigma)] for 4–5 min. The cells were then superfused with NT containing 1.8 mM Ca2+ for 5–10 min at room temperature to allow intracellular deesterification and at 35°C for the rest of the experiment unless otherwise specified. A new aliquot of cells was added to the bath for application of each of the three protocols described below. The myocytes were excited with ultraviolet light at 340 nm, and the emission ratio (400 nm/500 nm; denoted as R or R400/500) was measured through a 10-µm-diameter aperture focusing at the center of the myocyte with a sampling frequency of 200 Hz on a previously described system (36). Before each measurement, the chamber background fluorescence was removed by adjusting the 400- and 500-nm channels to zero over an empty field of view near the cell.

For the majority of the experiments (Figs. 1–8), field stimulation in nonvoltage-clamped myocytes was used to elicit Ca2+ transients. Electrical pacing was produced by 10-ms pulses at a voltage 1.5 times threshold using two platinum electrodes separated by 2 cm in the experimental chamber.


Figure 1
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  		Fig. 1. Ca2+-transient frequency response at 35°C. A: representative recordings of a pulmonary vein (PV) cardiomyocyte frequency response (0.1, 0.2, 0.5, 1.0, 2.0, 3.0, and 4.0 Hz). The Ca2+ concentrations were measured with the Ca2+ indicator indo-1 AM. The electronically controlled shutter was closed during the initial pacing period at each frequency to minimize photobleaching. Insets show that at low frequencies the response was regular, whereas at higher frequencies (≥2 Hz), the response was often irregular and sometimes showed alternans. [Ca2+]i, intracellular Ca2+ concentration. B and C: representative time-averaged Ca2+ fluorescence recordings for PV and left atrium (LA) cardiomyocytes when paced at 0.1 Hz (B) and 1.0 Hz (C). {Delta}[Ca2+]i indicates change in Ca2+ concentration from diastolic level. The measurement of amplitude, peak time (Tpeak), time from stimulus to half-decay (TS1/2D), and the time constant (tau) are illustrated in C.

 

Figure 8
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  		Fig. 8. Summary of Ca2+-transient frequency-response measurements recorded at room temperature in cells from normal (Ctrl) dogs and dogs subjected to 7 days of rapid atrial pacing (RAP). A and B: responses at 0.5 and 1.0 Hz for the amplitude of the Ca2+-fluorescence transients (A) and the time from stimulus to half-decay (B). Values are means ± SE; n = 14 cells/measurement. *P < 0.05 for RAP vs. corresponding Ctrl value. No significant differences between LA and PV.

 
For the field-stimulated myocyte experiments, three protocols were used, one for basic frequency-response recordings and alternans measurements, one for spontaneous Ca2+-transient observations, and another to study caffeine-induced Ca2+ transients. To establish frequency-response properties, the pacing frequency (F) was gradually increased (F = 0.1, 0.2 or 0.25, 0.5, 1.0, 2.0, 3.0, and 4.0 Hz), allowing at least 60 s (F = 0.1–0.5 Hz) or at least 40 stimuli (F = 1.0–4.0 Hz) to reach steady state. The illumination shutter was closed for the first half of each pacing period to minimize photobleaching. NT (1.8 mM Ca2+) was the only extracellular solution used with the first protocol. The protocol to study spontaneous Ca2+ transients involved 1 min pacing at 0.4 Hz (baseline solution), 1.5 min rest (test solution; incubation period), 1 min pacing at 0.4 Hz (test solution), 0.5 min pacing at 2 Hz (test solution), and 1 min rest (test solution; observation period). NT (1.8 mM Ca2+) was used as a baseline solution, and test solutions consisted of only NT containing 1.8 mM Ca2+ (control) or NT (1.8 mM Ca2+) plus 0.5 µM ryanodine, 2.0 µM ryanodine, or 1.0 µM isoproterenol. To study caffeine-induced Ca2+ transients, nonvoltage-clamped cells were paced at 1 Hz for 60 s, and 10 mM caffeine (in NT without Ca2+) was applied with a rapid perfusion system in direct vicinity of the myocyte, producing the desired caffeine exposure within 2 s after the last electrical stimulus.

In the last set of experiments, Ca2+ fluorescence and L-type Ca2+ current (ICaL) were simultaneously measured using the patch-clamp technique. The cells were superfused at 35°C with 1.8 mM Ca2+-NT solution plus 5 mM 4-aminopyridine [to block transient outward current (Ito)] and 50 µM niflumic acid [to block Ca2+-dependent Cl– current (ICl.Ca)]. The internal solution was composed of (in mM) 110 potassium aspartate, 20 KCl, 1 MgCl2, 5 MgATP, 0.1 GTP (lithium salt), 10 HEPES, 5 sodium phosphocreatine, and 0.005 indo-1 potassium salt (pH 7.3, KOH). Fluorescence background, as measured directly on the cell before indo-1 loading, was removed, and transients were measured at the center of the myocyte as described above. The cells were held at –50 mV and depolarized to 0 mV for 300 ms at 0.5 Hz for 120 pulses, and the last 20 pulses were time averaged and kept for analysis. A current-voltage analysis protocol was also performed with 300-ms depolarizing pulses to voltages from –40 to 30 mV at 0.1 Hz.

Ratiometric intracellular Ca2+ measurements were converted into intracellular Ca2+ concentration ([Ca2+]i) with the formula [Ca2+]i = KD x beta x (R – Rmin)/(Rmax – R) (11), where KD is the dissociation constant, R is the ratiometric Ca2+ measurement (emission at 400nm/emission at 500 nm), Rmin is the minimum value of R, and Rmax is the maximum value of R. Calibration was performed with the method described in Ref. 26 with a KD of 844 nM (2). Briefly, for calibration with the AM form of indo, the following NT solutions (with NaCl replaced by Tris·Cl) were used: solution A, Ca2+ free, 5 mM EGTA free, 10 µM ionomycin, 10 mM 2-deoxyglucose (to determine Rmin) and solution B, EGTA free, 5 mM Ca2+, 10 µM ionomycin, 10 mM 2-deoxyglucose (to determine Rmax). beta was taken as the ratio of fluorescence at 500 nm in solution A over the value in solution B. In some experiments, a glass pipette was used to rupture the membrane for Rmax measurements (Rmin = 0.43, Rmax = 2.34, and beta = 2.32; n = 4–5). For calibration with the non-AM form of indo, the same external solutions were used with the omission of ionomycin. The internal solutions were the same as for the patch-clamp experiments with the addition of 5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) for Rmin measurements. Because different internal solutions were required for Rmin and Rmax, we used the beta value obtained above (Rmin = 0.51 and Rmax = 2.39; n = 6).

Data analysis. All data processing was performed with Igor Pro software. For frequency-response, a minimum of 4 transients (at 0.1 Hz) or a maximum of 20 transients (>1.0 Hz) were time-averaged and then filtered with a smoothing function of width 9. The baseline, amplitude, time to peak, and time from stimulus to half-decay of the Ca2+ transient were measured (Fig. 1C). A monoexponential fit was applied from 5% of the decay to the end of the cycle or to 2,000 ms after the peak for frequencies <1 Hz, and the time constant (tau) was determined. To quantify alternans and beat-to-beat regularity of Ca2+ transients, a cyclic autocorrelation was performed on a minimum of 8 beats (at 0.2 Hz) and a maximum of 24 beats (>1.0 Hz) after baseline removal. The autocorrelation was then normalized to one. The amplitude of the correlation peaks was then measured, and statistical analysis was applied. The irregularity index (IRI) was defined as IRI = (SD of the peaks)/(mean value of the peaks) x 100%. Signals were defined to be irregular if IRI was >2%. If the IRI defined the response as irregular, but sorting beats separated by 1, 2, or 3 beats produced individual groups that all became regular (IRI <2%), the signal was said to show alternans. This method of analysis proved to be more reproducible than amplitude detection (too sensitive to noise), and to calculation of the area under each transient (too sensitive to baseline setting and drift). Finally, spontaneous Ca2+ transients were observed during the first minute after Ca2+ loading by pacing at 2.0 Hz. A successful event was defined as an unstimulated Ca2+ transient (accompanied by cellular contraction) that was at least 50% of the steady-state signal at 0.4 Hz.

All statistics were performed in Prism (GraphPad). Frequency-response parameters were analyzed for LA-PV differences with two-way ANOVA. If a statistically significant difference was found, a Bonferroni-adjusted t-test was used to define individual frequencies at which significant differences occurred. For strict two-sample comparisons, paired or unpaired t-tests were used as appropriate. Values are expressed as means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Figure 1A illustrates the results of a representative Ca2+-transient measurement of the frequency response in a PV cardiomyocyte at 35°C. In general, PV cells followed a regular 1:1 response-stimulus pattern at low pacing frequencies while exhibiting either irregular activity, alternans, or block at higher frequencies (≥2 Hz). The same tendency was also observed in LA cardiomyocytes. Figure 1, B and C, shows representative time-averaged traces for typical PV and LA cells at 0.1 and 1.0 Hz, respectively. At both frequencies, the PV and LA Ca2+ transients were quite similar.

Mean results for the Ca2+-transient amplitude, diastolic level, time to peak, and decay time constant (Tau) are shown in Fig. 2, A–D, for 12 cells in each group at 35°C. There were no significant differences between LA and PV cardiomyocytes. A subportion of the cell population (n = 5 LA and n = 7 PV) had a 1:1 response up to 3.0 Hz, as displayed in the inset of Fig. 2A. Interestingly, both LA and PV myocytes had a negative frequency response at low frequencies followed by flat responses from intermediate to higher frequencies, which is in line with previous observations in isolated canine LA myocytes (36). Again, there were no significant differences between LA and PV cells. Because lowering temperature decreases metabolic requirements and produces more stable and robust Ca2+ transients, we repeated the protocol at room temperature. The results at room temperature are summarized in Fig. 3. There were no significant differences at any frequencies in Ca2+-transient amplitude (A), diastolic level (B), time to peak (C), or decay time constant (D).


Figure 2
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  		Fig. 2. Summary of Ca2+ fluorescence frequency-response measurements at 35°C. The frequency response (0.1–1.0 Hz) of the amplitude of the Ca2+ transients (A); the Ca2+ diastolic level (B); the time from stimulus to peak (C); and tau, the time constant of the monoexponential fit to the Ca2+-transient decay curve (D). Inset in A shows data for a reduced number of samples that had a 1:1 response up to 3 Hz. Values are means ± SE; n = 12 cells studied at all frequencies, except for inset for which n = 5–7 cells; no significant differences between PV and LA cells.

 

Figure 3
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  		Fig. 3. Summary of Ca2+-transient frequency-response measurements at room temperature. The frequency response (0.1–1.0 Hz) of the amplitude of Ca2+ transients (A); the Ca2+ diastolic level (B); the time from stimulus to peak (C); and tau (D). Values are means ± SE; n = 13 and 12 cells for LA and PV, respectively; Bonferroni test showed no significant differences at any frequencies.

 
Figure 4 illustrates various Ca2+-transient response patterns and their analysis. Figure 4A depicts the three different general Ca2+-response patterns that we observed. As described in METHODS, the baseline-removed fluorescence signals (Fig. 4A, top) were autocorrelated and normalized to unity, providing the signals shown in Fig. 4A, bottom. Autocorrelation peaks were then measured, and the IRI was evaluated as described in METHODS. For the three examples in Fig. 4A, IRI values were 0.94% (left), 3.97% (middle), and 6.93% (right), respectively. However, for Fig. 4A, right, when the peaks were divided into two groups (even and odd peaks), the IRI of each group, respectively, became 0.10 and 0.17%, indicating alternans. Figure 4B shows mean IRI values obtained at room temperature. As the pacing frequency increased, the signal became more and more irregular (increasing IRI). No statistically significant IRI differences were found between PV and LA myocytes studied at room temperature. Figure 4C shows IRI data at 35°C, which indicate significantly greater irregularity in the response of LA myocytes at 4.0 Hz. Figure 4D shows the percentage of cells displaying each type of behavior at 35°C and various frequencies. Although LA cells showed a tendency to more frequent alternans, the prevalence of regular responses was similar in LA vs. PV cells, and regular responses were seen at least as often in PV compared with LA cardiomyocytes.


Figure 4
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  		Fig. 4. Irregularity and alternans in Ca2+-transient behavior. A: examples of regular signals, irregular signals, and signals with alternans, all from data in atrial myocytes at room temperature. Top: original Ca2+-transient recordings at 1.0 Hz (baseline removed); bottom: corresponding cyclic autocorrelation signals used in the calculation of the irregularity index (see text). B and C: index of irregularity at room temperature (B) and at 35°C (C). D: proportion of cells with a regular signal, an irregular signal, an irregular signal with alternans, and without measurements (cells that did not respond at higher frequencies) at 35°C (see text for classification criteria). Values in B and C are means ± SE. *Bonferroni posttest showed significant difference between LA and PV at 4.0 Hz in C; for B–D, n = 12–13 cells for each measurement. Irregularity index analysis was performed directly on ratiometric raw data.

 
In the next set of experiments, spontaneous Ca2+-transient events were elicited by rate-related Ca2+ loading, under control conditions and then in the presence of manipulations (0.5–2 µM ryanodine, 1 µM isoproterenol) previously reported to preferentially induce triggered activity in PV cardiomyocytes (16). Under control conditions, small numbers of Ca2+-transient events were seen during the no-pacing interval after 2 Hz pacing (Fig. 5A, top). Infusion of isoproterenol enhanced Ca2+-transient amplitude and increased the number of spontaneous Ca2+ transients after 2 Hz pacing (Fig. 5A, bottom). Figure 5B shows the percentage of cells showing spontaneous Ca2+ transients. In control NT, more than one-half of the cells had at least one spontaneous transient in both PV and atrial groups (Fig. 5B), although the number of events per cell was relatively small (Fig. 5C). The application of ryanodine reduced the occurrence of these spontaneous Ca2+-transient events, whereas the application of isoproterenol had the opposite effect, with some cells exhibiting up to 20 spontaneous Ca2+ transients during the 1-min post-2-Hz pacing observation period in the presence of isoproterenol. However, neither the percentage of cells showing spontaneous Ca2+ transients nor the number of events per cell were significantly different between PV and LA cardiomyocytes. We then analyzed the response to isoproterenol to determine whether there were differences between LA and PV cardiomyocytes. Although isoproterenol significantly increased Ca2+-transient amplitude (Fig. 6A) and accelerated decay kinetics (Fig. 6B), there were no significant differences in response between LA and PV cardiomyocytes.


Figure 5
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  		Fig. 5. Analysis of spontaneous Ca2+ transients after rapid pacing at 35°C. A: protocol is shown schematically at top. The shutter was closed to minimize photobleaching, except when measurements were required. Ca2+-transient recordings on top represent a control experiment in normal Tyrode (NT) solution, whereas recordings on bottom represent an experiment in the presence of isoproterenol (Iso). The analysis of spontaneous Ca2+ transients was obtained during a 1-min period without pacing after 30 s of 2 Hz stimulation, as indicated by the gray bar. Inset shows the transition between the end of fast pacing (first 9 beats) and spontaneous Ca2+-transient events (last 5 beats). B and C: quantification of spontaneous Ca2+-transient events, with the percentage of cells with spontaneous release (B) and the number of spontaneous transients events/cell (C) in control situations or in the presence of 0.5 µM ryanodine (Ry), 2.0 µM Ry, or 1.0 µM isoproterenol. Values in C are means ± SE; n = 24 cells for control, n = 8 each for 0.5 µM Ry, 2.0 µM Ry, and 1.0 µM isoproterenol. No significant differences between LA and PV myocytes in any condition, t-test; P > 0.05.

 

Figure 6
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  		Fig. 6. Response to isoproterenol at 35°C. A and B: relative response at low pacing frequencies (0.4 Hz), using data from the protocol presented in Fig. 5A (end of each 0.4 Hz pacing period) for Ca2+-transient amplitude (A) and fitted decay time constants (B). Values are means ± SE; n = 8 cells for each measurement. *P < 0.05 for results in the presence of isoproterenol vs. control (Ctrl). There were no significant differences between PV and LA cells.

 
The Ca2+-transient properties of a cardiomyocyte rely on the ability of the cell to store and release Ca2+ in the sarcoplasmic reticulum (SR). To assess the relative amount of Ca2+ present in the SR of PV vs. LA cardiomyocytes after 1 min of pacing at 1.0 Hz, we used the local application of 10 mM caffeine (36). Caffeine directly releases Ca2+ from SR stores, and the size of the Ca2+-induced transient is an index of stored Ca2+. Figure 7A shows a representative experiment in which the caffeine-induced Ca2+ transient is approximately two to four times larger than those resulting from electrical pacing, and the relaxation rate is roughly approximately seven times slower. Mean amplitude (Fig. 7B) and decay kinetics (Fig. 7C) for PV and LA cardiomyocytes are comparable, indicating similar releasable Ca2+ stores in both types of cells.


Figure 7
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  		Fig. 7. Response to 10 mM caffeine at 35°C. A: representative experiment in which a PV cardiomyocyte was paced at 1 Hz for 60 s, after which 10 mM caffeine was applied locally via a rapid perfusion system to create a caffeine-induced transient. B and C: summary of the Ca2+-transient amplitude (B) and decay time constant (C) of the caffeine-induced transient. Values are means ± SE; n = 5–6 cells at each point. There were no significant differences between LA and PV myocytes. Open bar denotes LA; solid bar denotes PV.

 
We also studied Ca2+ transients in cells isolated from dogs subjected to 1 wk of atrial tachypacing, known to enhance the inducibility of atrial tachyarrhythmias and AF (3, 35). We measured Ca2+ transients at room temperature at two different pacing frequencies (0.5 and 1.0 Hz). There were no significant differences in Ca2+-transient amplitude (Fig. 8A) or kinetics (Fig. 8B) between PV and LA cardiomyocytes from dogs subjected to atrial tachypacing.

It is possible that the differences in Ca2+ handling between PV and LA myocytes could be masked by differences in AP properties. To compare LA and PV Ca2+ transients under voltage-controlled conditions, we measured Ca2+ transients under voltage clamp with the non-AM form of indo-1 in the internal solution. The cells were held at –50 mV and depolarized to 0 mV for 300 ms at a frequency of 0.5 Hz. The first 100 pulses were used to load the cells with Ca2+, and the last 20 pulses were used to simultaneously measure ICaL and Ca2+ transients (Fig. 9, A–C). The average capacitance was similar for both cell groups [83.3 ± 7.5 vs. 88.2 ± 8.6 pF; PV vs. LA, P = not significant (NS)]. Simultaneously measured current and Ca2+-transient recordings from a PV and a LA myocyte are shown in Fig. 9A. There was considerable variability in Ca2+-transient amplitudes recorded from different cells (Fig. 9B), but the distributions of individual values were quite similar for cells from either region. The overall average Ca2+-transient amplitudes (123 ± 26 nM in LA vs. 100 ± 19 nM in PV; P = NS; n = 10/group) were not significantly different between regions. Mean ICaL densities were significantly less (P < 0.05, ANOVA) in PV cells (Fig. 9C), but the differences were of the order of 10%, smaller than we noted under different conditions (strongly buffered intracellular Ca2+, no indo-1) in a previous study (9).


Figure 9
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  		Fig. 9. Simultaneous L-type Ca2+ current (ICaL) and Ca2+-transient recordings. A: average of last 20 of 120 pulses at 0.5 Hz with protocol shown in the inset of A. Representative ICaL (top) and Ca2+-transient (bottom) recordings from a PV (left) and an LA (right) myocyte. B: distribution of Ca2+-transient amplitudes. Each point represents the mean value from one cell (n = 10). C: ICaL current-voltage relation as measured at 0.1 Hz using the protocol shown in the inset. *P < 0.05 using 2-way ANOVA; n = 9–10 for each data point.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we investigated the Ca2+-handling properties of isolated PV and LA cardiomyocytes, including the Ca2+ fluorescence frequency response, Ca2+ transients under voltage-clamped conditions, beat-to-beat regularity, alternans behavior, spontaneous Ca2+ transients after rapid pacing, the response to beta-adrenergic stimulation, and caffeine-induced transients. The results show that the Ca2+-handling properties of canine PV cardiomyocytes are similar to those found in LA cells and thus suggest that intrinsically distinct PV Ca2+-handling characteristics are not likely to be the basis for the reported PV propensity for ectopic activity.

Similarities and differences between basic properties of PV and atrial cardiomyocytes. Atrial and PV cardiomyocytes are morphologically similar (7, 37). Evidence for specialized conduction cells has been reported in PVs from patients with AF (29). Although distal PV cardiomyocytes run longitudinally, proximal PV sleeves contain cells that run both perpendicular and parallel to the vessels, sometimes with abrupt changes in cardiomyocyte orientation (14, 15, 37). These zones of abrupt orientation change have been associated with block or slow conduction (1, 14, 15). In addition, PV cardiomyocytes express similar connexin 40, but less connexin 43, compared with atria (37).

PV cardiomyocytes have more positive resting potentials, shorter AP duration, and smaller AP amplitude than atrial cardiomyocytes (9). In humans, the PV effective refractory period is shorter in the distal PV (4, 20), and one study reported shorter refractory periods in PV vs. atrium in AF patients (18). Canine PV cardiomyocytes exhibit smaller inward rectifier K+ current (IK1), Ito, and ICaL, and larger rapid delayed rectifier K+ current (IKr) and slow delayed rectifier K+ current (IKs), densities than in atria (9).

Evidence for a role of Ca2+-handling mechanisms in ectopic activity of PVs. Although the importance of PV activity in clinical AF is well recognized, underlying mechanisms are poorly understood (24). Several reports described indirect findings suggesting the possibility that intrinsically abnormal PV Ca2+ handling predisposes them to triggered arrhythmias. Honjo et al. (16) reported that spontaneous firing in normal canine PVs could be induced by rapid stimulation in the presence of interventions (low-dose ryanodine, isoproterenol) that enhance cellular Ca2+ loading and could be suppressed by depleting SR Ca2+ stores with cyclopenzanoic acid or thapsigargin. Chen et al. (6) showed a propensity for DAD formation in PV cardiomyocytes, and Ca2+-handling is known to be crucial to the generation of DADs (34). Patterson et al. (28) recently described rapid firing in PV myocardial sleeves in response to stimulation of local vagal and sympathetic nerves. Ca2+-transient inhibition with ryanodine suppressed rapid firing, as did removal of extracellular Ca2+ and beta-adrenoceptor blockade (28). Despite the above observations consistent with PV Ca2+-handling abnormalities, our careful analyses of PV and LA Ca2+-handling properties revealed no differences, suggesting that other mechanisms must have been responsible for the arrhythmogenic PV responses described in the literature.

Potential significance of our findings. To our knowledge, the present study is the first to examine directly the Ca2+-handling properties of PV cardiomyocytes and to compare them with those of LA cells. AP-induced Ca2+ entry via L-type channels (ICaL) opens ryanodine receptors and triggers Ca2+-induced Ca2+ release from the SR. After repolarization, Ca2+ is removed by the SR Ca2+-ATPase pump and the NCX (34). In our study, LA and PV cardiomyocytes had indistinguishable Ca2+-transient amplitudes and kinetics at different frequencies and temperatures, suggesting similarity in their Ca2+-handling machinery. The similarity in NCX function inferred from our caffeine-induced Ca2+-transient decay data agrees with direct patch-clamp measurements of NCX current (9) and with Western blot and immunohistochemical analyses of NCX protein expression (21) in PV vs. LA cardiomyocytes.

Conditions increasing Ca2+ loading can cause spontaneous Ca2+ release leading to DADs and triggered activity (31, 34). A couple of studies point to PV susceptibility to triggered activity under conditions that increase Ca2+ loading (16, 28). For that reason, we compared PV and LA cardiomyocyte responses with Ca2+ loading induced by rapid pacing (Figs. 4 and 5), ryanodine or isoproterenol administration (Fig. 5), and atrial tachycardia-induced remodeling (Fig. 8). Rapid pacing led to alternans and irregular Ca2+-transient responses, but with no greater predilection in PVs than LA. Isoproterenol enhanced Ca2+ transients and increased the number of spontaneous Ca2+ transients after rapid pacing, but no more so in PVs than LA. Finally, there were no clear differences in Ca2+ transients from PVs vs. LA in cells from atrial-tachypaced dogs.

The lack of abnormal behavior of AT-remodeled PV cardiomyocytes that we observed differs from the observations of Chen et al. (6) in isolated cardiomyocytes but agrees with more recent studies by Cha et al. (3). Our results also differ from studies reporting that 0.5–2 µM ryanodine led to pacing-induced triggered activity in PVs (16). When ryanodine is present at moderate concentrations (0.1–10 µM), the SR Ca2+-release channels (ryanodine receptors) enter a subconductance state and can show greater Ca2+-induced/Ca2+-release gain (33). With continued exposure, ryanodine progressively empties the SR by enhancing Ca2+ leak through the subconductance ryanodine receptor state, substantially decreasing steady-state Ca2+-transient amplitude. This reduction in SR Ca2+ content is likely responsible for the decrease in spontaneous activity we saw with ryanodine.

The present study makes it unlikely that abnormalities in Ca2+-handling properties intrinsic to normal PV cardiomyocytes produce a predisposition to the generation of ectopic activity. We were disappointed with these findings, in light of indirect evidence in the literature pointing to the possibility that peculiarities in PV Ca2+ handling are responsible for the arrhythmogenic behavior of PVs. In addition, we have previously shown different AP and ICaL properties between PV and LA cardiomyocytes (9). Presumably, the net effect of these differences is insufficient to produce detectable Ca2+-handling alterations under the conditions of the present study. Despite careful and rigorous analysis of PV vs. LA Ca2+ transients under a variety of conditions, we were unable to uncover any significant differences that would account for differential PV susceptibility to arrhythmogenic influences. It is possible that, despite these similar cellular Ca2+-handling properties, PV cardiomyocytes may be predisposed to Ca2+ release-induced spontaneous activity because of other factors. For example, the thin (and in some areas poorly coupled) PV cardiomyocyte layer may impose a reduced electrotonic sink on cells with spontaneous Ca2+ release events. This could favor spontaneous local activity, just as limited coupling to adjacent atrium contributes to the pacemaker function of sinus node cells. In addition, it is possible that human PV Ca2+-handling properties are different from those in dogs and/or that AF-promoting conditions differentially affect Ca2+ handling in PVs vs. other parts of the atria.

Potential limitations. The present findings in normal experimental animals cannot exclude the possibility that arrhythmogenic PVs in paroxysmal AF patients have enhanced susceptibility to triggered activity or abnormal Ca2+ handling. Arrhythmogenic PVs in paroxysmal AF patients have altered structural and histopathological properties (12, 29). Animal models that mimic the pathophysiology of such patients do not presently exist. A variety of intrinsic PV features in normal experimental animals have been suggested to be related to the role of PVs in clinical AF (1, 5–9, 15, 16, 19, 22). The present study shows that intrinsic atrial-PV differences in Ca2+ handling (defined as differences inherent to the properties of normal atrial and PV cardiomyocytes), although an attractive candidate mechanism, do not exist in normal dogs.

Another limitation relates to the use of random-source mongrel dogs, for which the age is unknown. Gender- and age-related differences in Ca2+-handling properties might have been present for the myocytes used in this study, but gender/age bias was minimized by using corresponding numbers of PV and LA myocytes for each animal.

We have expressed [Ca2+]i in terms of concentrations, rather than the ratio of fluorescence at 400 to 500 nm. In vivo Ca2+ indicator calibration involves a number of uncertainties (2). It is possible, by applying a nonlinear transformation involved in the conversion to concentrations, to artificially introduce errors into the results. However, we performed the same analyses on the untransformed ratiometric data and obtained the same conclusions, making important distortion by transformation unlikely.

Our comparisons between normal and rapidly paced dogs were obtained at only two frequencies (0.5 and 1 Hz) because 1:1 response could not be obtained in all cells at >1 Hz, and we wanted to obtain data in as many cells as possible at consistent frequencies from each dog. Thus we cannot exclude Ca2+-transient differences between LA and PV at higher frequencies in rapidly paced dogs. We have shown previously that removing the PVs does not alter arrhythmia inducibility in PV-LA preparations from dogs subjected to 7-day atrial tachypacing (3). The dogs we used to study rapid-pacing effects were submitted to 7-day atrial tachypacing, which produces clear AF promotion but less sustained AF than longer periods of atrial tachycardia (10). We cannot exclude the possibility that longer periods of atrial tachycardia remodeling and/or other forms of more severe AF-promoting pathology could differentially affect PV Ca2+ handling.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Canadian Institutes of Health Research, the Quebec Heart and Stroke Foundation, and the Mathematics of Information Technology and Complex Systems Network of Centers of Excellence. P. Coutu was supported by a National Science and Engineering Council Fellowship.


   ACKNOWLEDGMENTS
 
We thank Evelyn Landry, Chantal Maltais, Nathalie L'Heureux, Richard Yeh, Xiao Yan Qi, and Chantal St-Cyr for technical support and France Thériault for secretarial help with the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Nattel, Research Center, Montreal Heart Institute, 5000 Belanger St. East, Montreal, Quebec, Canada H1T 1C8 (e-mail: stanley.nattel{at}icm-mhi.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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