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J Physiol (2003), 551.3, pp. 801-813
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.046417

Cellular electrophysiology of canine pulmonary vein cardiomyocytes: action potential and ionic current properties

Joachim R. Ehrlich*, Tae-Joon Cha*, Liming Zhang*, Denis Chartier*, Peter Melnyk*‡, Stefan H. Hohnloser§ and Stanley Nattel*†

	ABSTRACT

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Pulmonary vein (PV) cardiomyocytes play an important role in atrial fibrillation; however, little is known about their specific cellular electrophysiological properties. We applied standard microelectrode recording and whole-cell patch-clamp to evaluate action potentials and ionic currents in canine PVs and left atrium (LA) free wall. Resting membrane potential (RMP) averaged -66 ± 1 mV in PVs and -74 ± 1 mV in LA (P < 0.0001) and action potential amplitude averaged 76 ± 2 mV in PVs vs. 95 ± 2 mV in LA (P < 0.0001). PVs had smaller maximum phase 0 upstroke velocity (V_max: 98 ± 9 vs. 259 ± 16 V s^-1, P < 0.0001) and action potential duration (APD): e.g. at 2 Hz, APD to 90 % repolarization in PVs was 84 % of LA (P < 0.05). Na⁺ current density under voltage-clamp conditions was similar in PV and LA, suggesting that smaller V_max in PVs was due to reduced RMP. Inward rectifier current density in the PV cardiomyocytes was ~58 % that in the LA, potentially accounting for the less negative RMP in PVs. Slow and rapid delayed rectifier currents were greater in the PV (by ~60 and ~50 %, respectively), whereas transient outward K⁺ current and L-type Ca²⁺ current were significantly smaller (by ~25 and ~30 %, respectively). Na⁺-Ca²⁺-exchange (NCX) current and T-type Ca²⁺ current were not significantly different. In conclusion, PV cardiomyocytes have a discrete distribution of transmembrane ion currents associated with specific action potential properties, with potential implications for understanding PV electrical activity in cardiac arrhythmias.

(Received 5 May 2003; accepted after revision 7 July 2003; first published online 7 July 2003)
Corresponding author S. Nattel: Research Center, Montreal Heart Institute, 5000 Belanger St E, Montreal, Quebec, Canada, H1T 1C8. Email: nattel{at}icm.umontreal.ca

	INTRODUCTION

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Spontaneous activity of the pulmonary veins (PVs) was first recognized in the 1800s (Brunton & Fayrer, 1876), and the ability of the pulmonary veins to conduct electrical activation from the atria was noted in the 1970s (Spach et al. 1972). Cheung first recorded cardiomyocyte action potentials in guinea-pig PVs, and showed that they played a role in digitalis-toxic arrhythmias (Cheung, 1980, 1981). These studies demonstrated that distal PV cardiomyocytes (with shorter action potential duration (APD) and smaller action potential (AP) amplitudes than proximal cells) were capable of pacemaking but were usually dominated by sinus node activity. Electrical activity appeared to be a characteristic of cells from the cardiomyocyte sleeve, as smooth muscle cells were unable to generate APs (Cheung, 1980).

It has recently been demonstrated that electrical activity in pulmonary vein tissue plays an important role in atrial fibrillation (AF) in man (Haissaguerre et al. 1998). AF is the most common cardiac arrhythmia and is associated with considerable morbidity and mortality (Benjamin et al. 1998), and the discovery of the role of the PVs was an important advance in understanding and treating the arrhythmia (Allessie et al. 2001; Nattel, 2002). Although initial work emphasized PVs as a trigger for AF, recent studies suggest that the PVs may also be important in AF maintenance (Wu et al. 2001).

Relatively little is known about the cellular electrophysiology of the PV myocardial sleeve believed to be important in arrhythmogenesis. Studies by Chen et al. (2000, 2001, 2002) have described various arrhythmogenic cellular properties of PV myocytes, but the authors did not record APs at physiological temperature or compare PV cardiomyocyte properties to those of a reference atrial region. The anatomical arrangement of PV fibres results in prominent conduction delays, particularly close to the refractory period (Hocini et al. 2002; Verheule et al. 2002), and recent optical mapping studies show that atrial premature beats preferentially induce re-entry in PVs (Arora et al. 2003). The latter authors noted a potentially important role of a smaller PV phase 0 upstroke velocity (V_max) relative to the left atrium (LA) in favouring re-entry, and speculated that either reduced sodium current (I_Na) or I_Na inactivation by lower resting potentials could be involved.

The present study examined the hypothesis that PV cardiomyocytes have distinct AP characteristics, based on a discrete distribution of ionic currents. We therefore compared AP and ionic-current properties of PV cardiomyocytes with those of LA cardiomyocytes under as physiological conditions as possible.

	METHODS

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Animal and tissue handling

Adult mongrel dogs of either sex weighing 23-38 kg were anaesthetized with pentobarbital (30 mg kg^-1 I.V.) and artificially ventilated. Hearts and adjacent lung tissue were excised via a left thoracotomy and immersed in oxygenated Tyrode solution. Removal of the heart and lungs produced circulatory arrest, resulting in effective and humane killing. Animal care procedures followed Canadian Council on Animal Care guidelines and were approved by the Animal Research Ethics Committee of the Montreal Heart Institute.

For cell isolation, the proximal circumflex coronary artery was cannulated and distal ends of PV myocardial sleeves were marked with silk thread prior to enzyme perfusion with collagenase (100 U ml^-1, Worthington, type II), to facilitate identification after enzymatic digestion. Cell isolation was performed as previously described (Li et al. 2001). PVs were well perfused and single myocytes could be isolated from all PVs. There were no appreciable differences in the results from different PVs, and therefore the results of cells from all PVs were pooled for analysis. PV cardiomyocytes were rod-shaped with cross-striations and in overall shape similar to LA free-wall cardiomyocytes. After isolation, cells were stored at 4 °C and studied within 12 h.

Figure 1A shows a typical LA preparation with PVs attached, with locations of the PV myocardial sleeve and LA regions for cell isolation indicated. Figure 1B and C illustrate regions from which cardiomyocytes were isolated in PV sleeves. PV cells were isolated as far distally from the LA-PV junction as possible, generally 4-10 mm from the junction. Figure 1D is a photomicrograph of a longitudinal PV section, with the distal end of the myocardial sleeve at the bottom. As shown by progressively higher-powered sections in Fig. 1E and F, the cardiomyocyte-containing sleeve was separated from smooth muscle cells by connective tissue, consistent with the electrical independence of PV cardiomyocytes and smooth muscle cells previously reported (Cheung, 1980, 1981).

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Figure 1. Canine atrial preparation with adjacent PVs A, photograph of a typical preparation indicating locations at which LA and PV cells were obtained (dashed lines) and APs were recorded with standard microelectrode technique. B and C, photomicrographs of PV sections (Masson trichrome staining) under low and high magnification, showing the myocardial sleeve that extends from the LA onto PVs. D-F, longitudinal Gomori-stained PV sections at progressively higher magnification (squares highlight areas magnified in subsequent panels), showing connective tissue (green) and myocytes (pink). RSPV: right superior PV; LSPV: left superior PV; RIPV: right inferior PV; LIPV: left inferior PV; S: PV myocardial sleeve; PW: posterior wall.

For standard microelectrode experiments, intact tissue preparations including the LA and adjacent PVs (as in Fig. 1A) were mounted in a chamber and perfused via the left circumflex coronary artery with oxygenated Krebs solution at 36 ± 0.5 °C. Fine-tipped microelectrodes (resistance 15-20 M when filled with 3 M KCl) coupled to a high-input impedance amplifier were used to record APs as previously described (Kneller et al. 2002).

Solutions

The solution for cell storage contained (mM): KCl 20, KH₂PO₄ 10, dextrose 10, mannitol 40, L-glutamic acid 70, -OH-butyric acid 10, taurine 20, EGTA 10 and 0.1 % BSA (pH 7.3, KOH). Tyrode (extracellular) solution contained (mM): NaCl 136, KCl 5.4, MgCl₂ 1, CaCl₂ 1, NaH₂PO₄ 0.33, Hepes 5 and dextrose 10 (pH 7.35, NaOH). For recording of delayed-rectifier current (I_K), nifedipine (5 µM), 4-aminopyridine (2 mM) and atropine (200 nM) were added to suppress Ca²⁺ current (I_Ca), transient outward current (I_to) and 4-aminopyridine (4AP)-dependent muscarinic K⁺ currents (Yue et al. 1997). Nifedipine was used rather than inorganic Ca²⁺ channel blockers because of the latter's effects on I_K (Yue et al. 1997; Virag et al. 2001). Dofetilide (1 µM) was added for slow I_K (I_Ks) recordings and rapid I_K (I_Kr) was recorded as chromanol 293B (50 µM)-resistant current (Bosch et al. 1998). For I_to and inward-rectifier K⁺ current (I_K1) recording, nifedipine was replaced by CdCl₂ (200 µM). I_K1 was recorded as 1 mM barium-sensitive current upon stepping from -40 mV to voltages between -120 and -10 mV. Na⁺ current (I_Na) contamination was avoided by using a holding potential (HP) of -50 mV or by substitution of equimolar Tris-HCl for NaCl. The internal solution for K⁺ current recording contained (mM): potassium aspartate 110, KCl 20, MgCl₂ 1, MgATP 5, GTP (lithium salt) 0.1, Hepes 10, sodium phosphocreatine 5 and EGTA 5.0 (pH 7.3, KOH).

The external solution for I_Ca recording contained (mM): tetraethylammonium (TEA)-Cl 136, CsCl 5.4, CaCl₂ 2, MgCl₂ 0.8, Hepes 10 and dextrose 10 (pH 7.4, CsOH). Niflumic acid (50 µM) was added to inhibit Ca²⁺-dependent Cl^- current (I_Cl,Ca) (Schlotthauer & Bers, 2000). The internal solution for I_Ca recording contained (mM): CsCl 120, TEA-Cl 20, MgCl₂ 1, MgATP 5, GTP (lithium salt) 0.1, EGTA 10 and Hepes 10 (pH 7.3, CsOH). Na⁺-Ca²⁺-exchange current (I_NCX) was recorded with an extracellular solution containing (mM): NaCl 140, CaCl₂ 2, MgCl₂ 1, dextrose 10, Hepes 10, niflumic acid 0.1 and nifedipine 5 µM. The internal solution contained (mM): CsCl 130, NaCl 5, MgATP 4, Hepes 10 (pH 7.3, CsOH). For I_Na recording, the external solution contained (mM): NaCl 5, CsCl 132.5, MgCl₂ 1.0, CaCl₂ 1.0, Hepes 20, glucose 11 (pH 7.35, CsOH), CdCl₂ 0.1 (to block I_Ca). The internal solution was NaCl 5, CsF 135, Hepes 5, EGTA 10, MgATP 5 (pH 7.2, CsOH). For standard microelectrode experiments, a solution containing (mM): NaCl 120, KCl 4, KH₂PO₄ 1.2, MgSO₄ 1.2, NaHCO₃ 25, CaCl₂ 1.25 and dextrose 5 (95 % O₂-5 % CO₂, pH 7.4), was used to perfuse the tissue.

Data acquisition and analysis

Currents were recorded with whole-cell patch-clamp at 36 ± 0.5 °C as previously described (Yue et al. 1996), except for I_Na which was recorded at room temperature. Borosilicate glass electrodes had tip resistances between 1.5 and 3.0 M when filled. Compensated series resistance and capacitive time constants averaged 3.6 ± 0.2 M and 287 ± 2 µs. Maximum voltage drop across the series resistance averaged ~8 mV for I_Na and < 5 mV for all other currents. The sampling frequency was 40 kHz for I_Na, 10 kHz for I_Ca, I_to and I_NCX and 1 kHz for I_Kr, I_Kg, and I_Kl, and filtering was performed at half the sampling frequency. Cell capacitance averaged 87 ± 3 pF for PV and 91 ± 3 pF for LA myocytes (n = 150 per group, P = n.s.). To control for cell size variability, currents are expressed as densities (pA pF^-1). Junction potentials between bath and pipette solution averaged 11.8 ± 0.9 mV and were not compensated. V_max was determined by electronic differentiation.

Non-linear algorithms were used for curve fitting. Unpaired t tests were used to compare individual non-repeated measures results from LA vs. PV. Analysis of variance (ANOVA) was used to compare repeated measures data (such as current densities, which were evaluated with repeated voltage steps over a wide range of voltages for each individual cell). P < 0.05 was considered statistically significant. Data are expressed as means ± S.E.M.

	RESULTS

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AP recordings

The mean resting membrane potential (RMP) of APs in PVs from multicellular LA-PV preparations, as recorded with standard fine-tipped microelectrodes, was -65.7 ± 0.9 mV, compared to -74.2 ± 1.1 mV in LA (n = 68, 33 cells, respectively, P < 0.0001). No spontaneous phase 4 depolarization or afterdepolarizations were observed. PV APs demonstrated a smaller phase 1 repolarization slope and a shorter plateau (Fig. 2A and B). Recordings obtained by impaling deeper than the layer in which APs could be recorded (about 5-10 cells deep) showed only a stable RMP of ~-40 mV, presumably from the smooth muscle cell layer, without electrotonic influence from cardiomyocyte activity (Fig. 2C). V_max was greater in the LA (Fig. 2D) than PV (Fig. 2E), averaging 98 ± 9 V s^-1 in PV cells (n = 83) vs. 259 ± 16 V s^-1 in LA (n = 54, P < 0.0001). Mean RMP, overshoot (OS) and AP duration (APD) at 1 Hz were smaller in PV than LA (Fig. 2F). APD was less in PVs over a broad range of frequencies (0.5-2 Hz).

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Figure 2. Action potential properties of LA and PV cardiomyocytes Recordings of action potentials from the LA (A) and PV (B) and a transmembrane potential in the smooth muscle cell layer (SMC) (C). No spontaneous diastolic depolarizations were observed. D and E, left, typical action potential recordings from LA and PV; right, phase 0 upstrokes of the same APs on an expanded time scale. F, mean (± S.E.M.) action potential properties of LA and PV cardiomyocytes. APA, action potential amplitude; APD90 and APD50, APD at 90 or 50 % repolarization, respectively; RMP, resting membrane potential; OS, overshoot; Vmax, maximum phase 0 upstroke velocity. ** P < 0.005, *** P < 0.0001 for LA vs. PV.

Sodium current (I_Na)

I_Na recordings are illustrated in Fig. 3A and B. Mean I_Na densities (Fig. 3C) were not different between the PV and LA. Peak I_Na averaged -16.1 ± 2.6 pA pF^-1 in LA and -17.7 ± 2.3 pA pF^-1 (P = n.s., ANOVA) in PV cardiomyocytes. Half-maximal inactivation voltages based on current densities following 1000 ms depolarizing prepulses (Fig. 3D) were -104.3 ± 2.0 for LA (n = 4) and -104.1 ± 2.8 mV for PV (n = 6) cardiomyocytes (P = n.s.). Half-maximal activation voltages (based on the relationship I_v = I_max(V - V_r)(G_v/G_max), where I_v and G_v represent current and conductance at voltage V; V_r represents reversal potential (0 mV); and I_max and G_max represent current and conductance at the most positive test potential, respectively; Fig. 3E), were -49.9 ± 3.6 mV for LA and -48.2 ± 4.3 mV for PV (n = 5 cells per group, P = n.s.). Activation and inactivation time constants (; mono-exponential curve fits) were similar (e.g. at -35 mV, activation was 0.20 ± 0.05 for LA and 0.24 ± 0.03 ms for PV, P = n.s.; inactivation was 0.81 ± 0.1 vs. 0.90 ± 0.15 ms, respectively, n = 5 per measurement, P = n.s.; Fig. 3F).

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Figure 3. Sodium current (INa) A and B, representative INa recordings (obtained with the protocol shown in inset to A, delivered at 0.1 Hz) from LA and PV cardiomyocytes. C, INa density-voltage relations (n = 10 cells per data point). Mean LA and PV current densities were not different (P = n.s., ANOVA). D, voltage dependence of INa inactivation, evaluated with 1000 ms prepulses to various voltages followed by a 40 ms test pulse to -40 mV, was not different between cardiomyocytes from LA and PV (V1/2 = -104.3 ± 2.0 vs. -104.1 ± 2.8 mV, respectively, n = 4 and 6 cells, P = n.s.). E, similarly, V1/2 of activation was not different (-49.9 ± 3.5 mV vs. -48.2 ± 4.3 mV, respectively, n = 5 cells each, P = n.s.). Time constants for current activation and inactivation (F) were similar between the two tissues (e.g. at -35 mV activation was 0.20 ± 0.05 for LA and 0.24 ± 0.03 ms for PV, P = n.s. and inactivation was 0.81 ± 0.1 vs. 0.90 ± 0.15 ms, respectively, P = n.s.). Open symbols represent PV, filled symbols represent LA. TP, test potential.

Inward rectifier current (I_K1)

Representative I_K1 recordings are illustrated in Fig. 4A and B. I_K1 density (end-pulse) was smaller in PV cardiomyocytes (Fig. 4C), including the outward-current portion shown on an expanded scale in the inset. Over all voltages, PV I_K1 density averaged ~58 % of that in LA and was significantly smaller (P = 0.002, ANOVA). I_K1 reversed at ~-70 mV: when corrected for junction potential, V_r was ~-80 mV. No time-dependent currents were observed in the presence of Ba²⁺ upon hyperpolarization of PV or LA cardiomyocytes, excluding the presence of the non-selective cation pacemaker current I_f.

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Figure 4. Inward rectifier current (IK1) A and B, representative 1 mM Ba2+-sensitive IK1 recordings obtained with the voltage protocol shown in the inset (0.1 Hz) in a LA and a PV cardiomyocyte. C, mean ± S.E.M. IK1 density-voltage relationship (8 cells each for LA and PV), with magnified outward component of the current in the inset. Mean LA currents were significantly greater than PV current as determined by ANOVA analysis (P = 0.002). TP, test potential.

Delayed rectifier current (I_K)

I_Ks recordings from each tissue are illustrated in Fig. 5A and B. The density of time-dependent I_Ks during a depolarizing voltage step (Fig. 5C) and following repolarization (Fig. 5D) was significantly greater in PV cardiomyocytes (P < 0.0001 for each, ANOVA), by ~50-70 %. Activation voltage dependence was assessed by normalizing tail current amplitudes to maximum current and was comparable in both cell types (mean V_1/2 = +20.2 ± 3.2 mV for LA vs. +21.9 ± 1.6 mV for PV, P = n.s.).

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Figure 5. Delayed rectifier potassium current components (IKs and IKr) A and B, representative IKs recordings obtained with 4 s depolarizations (0.1 Hz) followed by 2 s repolarizations to -30 mV in the presence of 1 µM dofetilide from a LA (A) and a PV (B) cardiomyocyte. C, mean ± S.E.M. IKs density-voltage relations from 12 cells. IKs denisty was greater in PV than in LA cardiomyocytes (P < 0.0001, ANOVA). D, mean ± S.E.M. IKs tail density-voltage relations (n = 12 per group). E, representative recordings of chromanol 293B (50 µM)-resistant IKr tail currents before and after application of 1 µM dofetilide obtained with the protocol in A (0.1 Hz). F, mean ± S.E.M. IKr tail current-voltage relationships and Boltzmann fits to mean data. IKr densities were significantly larger in the PV (P = 0.01, ANOVA). TP, test potential.

I_Ks activation kinetics at +40 mV were bi-exponential, with a significantly smaller slow-phase time constant (_s) for PV cardiomyocytes (1776 ± 210 ms vs. 3084 ± 32 ms in LA, P < 0.05), whereas the fast-phase time constant (_f) was not significantly different (333 ± 78 ms vs. 394 ± 67 ms). PV tail-current deactivation was also faster ( = 89 ± 10 vs. 155 ± 27 ms, P < 0.05).

The rapid component of the delayed rectifier current (I_Kr) is very sensitive to isolation technique (Yue et al. 1996), and was relatively small in the cells obtained in the present study (Fig. 5E). I_Kr tails were clearly dofetilide sensitive (Fig. 5E), and were significantly larger in PV than in LA cardiomyocytes (Fig. 5F; P = 0.01, ANOVA). For example, mean I_Kr tail densities at +20 mV were 0.16 ± 0.02 pA pF^-1 for LA and 0.24 ± 0.03 pA pF^-1 for PV myocytes (n = 10 cells per group). Activation voltage dependence was similar in both regions: V_1/2 was -15.2 ± 1.9 for LA and -15.9 ± 0.8 mV for PV myocytes.

Transient outward current (I_to)

I_to in LA (Fig. 6A) and PV (Fig. 6B) cells had similar morphology, but mean I_to density was significantly larger in LA cells (Fig. 6C; P = 0.0001, ANOVA). Inactivation kinetics were well fitted by a mono-exponential relationship, and inactivation time constants were similar in LA and PV (Fig. 6D). Time to peak current, an index of current activation speed, was also similar for LA and PV.

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Figure 6. Transient outward current (Ito) A and B, typical Ito recordings obtained with 100 ms depolarizing pulses from -50 mV. C, mean ± S.E.M. Ito density-voltage relations for 43 LA and 37 PV cells. Ito density was greater in LA than in PV cells (P = 0.0001, ANOVA); inset: Ito-V relations of currents normalized to values at +50 mV. D, time to peak Ito and Ito inactivation time constants (inact.; n = 10 cells per group). E, mean ± S.E.M. data for voltage dependence of Ito inactivation (Inact.) and activation (Act.; n = 10 cells per observation). F, mean ± S.E.M. current during P2 normalized to current during P1, as a function of P1-P2 interval (protocol in inset). Curves are mono-exponential fits to mean ± S.E.M. data (n = 9 cells each). All protocols were at 0.1 Hz. TP, test potential.

Activation voltage dependence of I_to was assessed from data obtained with the protocol shown in Fig. 6A, based on the relationship I_v = I_max(V - V_r)(G_v/G_max). V_r, as evaluated by tail currents following 2.2 ms depolarizations to +50 mV, averaged -70 ± 4 mV and -68 ± 3 mV (without correction for junction potential) in LA and PV cells, respectively (6 cells per group, P = n.s.). V_1/2 averaged +11.8 ± 1.5 mV (LA) vs. +12.5 ± 1.6 mV (PV) (10 cells each, P = n.s.) and corresponding slope factors were 11.9 ± 0.5 and 11.0 ± 0.8 mV (P = n.s.). Inactivation voltage dependence was studied with 1000 ms prepulses followed by 200 ms test pulses to +50 mV. Currents were normalized to values at +50 mV and fitted with Boltzmann functions. Inactivation V_1/2 averaged -27.4 ± 2.0 and -29.9 ± 1.8 mV (P = n.s.) in LA and PV, and corresponding slope factors were -5.9 ± 0.3 and -7.8 ± 1.1 mV (n = 10 cells per group, P = n.s., Fig. 6E). A paired-pulse protocol, with identical 150 ms depolarizations (P1 and P2) from -70 to +50 mV with varying P1-P2 intervals, was used to test recovery kinetics. Current during P2 normalized to current during P1 was a mono-exponential function of P1-P2 interval (Fig. 6F). Recovery time constants averaged 29.3 ± 3.8 ms for LA vs. 28.7 ± 2.8 ms for PV cardiomyocytes (n = 9 cells per group, P = n.s.).

L-type (I_Ca,L) and T-type (I_Ca,T) calcium current

Representative I_Ca,L recordings are shown in Fig. 7A and B. I_Ca,L was smaller in PV cardiomyocytes (Fig. 7C; P < 0.001, ANOVA). For example, at +10 mV I_Ca,L density averaged -11.1 ± 1.3 in LA and -7.1 ± 1.0 pA pF^-1 in PV cells respectively (n = 10 cells per group). No difference between the two current-voltage (I-V) relationships was apparent after normalization to maximal current at +10 mV (Fig. 7D). The voltage dependence of I_Ca,L activation and inactivation was evaluated in the same overall fashion as for I_Na. V_r determined by linear extrapolation of the ascending limb of the I-V curve to the voltage axis was similar for PV and LA cardiomyocytes (+66.0 ± 1.8 vs. +65.8 ± 2.8 mV, n = 10 cells each, P = n.s.). Activation V_1/2 averaged -1.8 ± 1.1 for LA vs. -2.6 ± 1.8 mV for PV (P = n.s.) and slope factors were 4.8 ± 0.3 and 5.2 ± 0.4 mV (P = n.s.; Fig. 7E). Inactivation voltage dependence was assessed with 1000 ms prepulses followed by 300 ms test pulses to +10 mV. Inactivation V_1/2 averaged -34.0 ± 1.7 in LA vs. -35.1 ± 1.2 mV in PV myocytes; slope factors were -6.1 ± 0.2 vs. -6.6 ± 1.3 mV (n = 9 cells per group, P = n.s.; Fig. 7E). Inactivation of L-type current was bi-exponential, with no differences in time constants (Fig. 7F). Recovery time constants for LA and PV were not different, averaging 35.9 ± 4.0 and 42.4 ± 5.4 ms, respectively (n = 8 cells per group, P = n.s.; Fig. 7G). I_Ca,L frequency dependence was also similar for both regions (Fig. 7H).

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Figure 7. L-type calcium current (ICa,L) ICa,L recordings during 240 ms depolarizing steps from a HP of -50 mV in LA (A) and PV (B) cardiomyocytes (0.1 Hz). C, mean ± S.E.M. ICa,L density-voltage relations, (n = 10 cells per group). Mean PV ICa,L density was smaller than LA current density (P < 0.001, ANOVA). D, normalized ICa,L -voltage relationships for both tissues superimposed. E, voltage dependence of ICa,L inactivation and activation. Curves are Boltzmann fits to mean data (n = 10 cells per group). F, inactivation time constants (slow and fast) obtained by bi-exponential curve fitting (n = 10 cells each). G, ICa,L recovery kinetics studied with paired 300 ms pulses to +10 mV (0.1 Hz). Mono-exponential fits to mean data are shown (n = 8 cells per group). H, ICa,L frequency dependence for 200 ms pulses from -80 mV to +10 mV, with current at steady state normalized to current during first pulse of train (n = 6 cells per group). Open symbols represent PV, closed symbols LA. TP, test potential; act., activation; inact., inactivation.

Representative recordings of I_Ca,T are shown in Fig. 8A and B. T-type current was obtained by subtracting currents recorded with a holding potential of -50 mV from current recorded with a holding potential of -90 mV (top of each panel), as previously described (Tseng & Boyden, 1989; Hirano et al. 1989). At the bottom of panels A and B is shown subtracted current, representing I_Ca,T. Mean I_Ca,T-voltage relationships are shown in Fig. 8C and were not different between the two tissues (e.g. at -20 mV: -1.5 ± 0.2 for LA vs. -1.5 ± 0.4 pA pF^-1 for PV, n = 7 and 8 cells, respectively; P = n.s., ANOVA).

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Figure 8. T-type calcium current (ICa,T) and Na+,Ca2+-exchange current (INCX) A and B, representative recordings of calcium current from LA and PV cardiomyocytes respectively. The top of each panel shows current recorded with holding potentials of -90 mV (open circles) and -50 mV (filled circles). The bottom shows subtracted current, representing ICa,T. C, mean ICa,T density-voltage relationship (n = 7 and 8 cells for LA and PV, respectively, P = n.s., ANOVA). D and E, representative recordings obtained with the protocol used to evaluate INCX from LA and PV cardiomyocytes, respectively at 0.1 Hz. Recordings before (filled squares) and after (open squares) removal of external sodium are shown. F, mean ± S.E.M. INCX density-voltage relationship (8 cells each, P = n.s., ANOVA). TP, test potential.

Na⁺-Ca²⁺-exchange current (I_NCX)

After a brief (5 ms) depolarization from an HP of -70 mV, I_NCX was recorded during 100 ms hyperpolarizing pulses, as previously described (Yue et al. 1997; Zygmunt et al. 2000). External NaCl was then rapidly replaced with LiCl to suppress I_NCX, and the current was re-recorded. Figure 8D and E shows currents before and after removal of external Na⁺ in the two regions. Mean current densities (Fig. 8F) were not different (e.g. at -120 mV: -1.7 ± 0.4 (LA) and -1.8 ± 0.6 (PV) pA pF^-1, n = 8 cells each, P = n.s., ANOVA).

	DISCUSSION

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We have shown that PV cardiomyocytes have distinct cellular electrophysiological properties compared to LA free-wall cells. These properties include reduced V_max, decreased RMP associated with reduced I_K1 density and decreased APD associated with increased I_Kr and I_Ks and reduced I_Ca,L.

AP characteristics of PV cardiomyocytes

Since technical factors (types of preparation, recording methods, solutions, etc.) can strongly influence AP characteristics, it is important that PV properties be compared directly with those of a reference region measured using the same methods in the same hearts in order to understand how PV APs compare to APs in other atrial tissues. Our study points to important physiological differences between APs in PV and LA. The PV RMPs in our study are compatible with those in previous studies (Cheung, 1980; Chen et al. 2000). The APD properties of PVs in our study are consistent with measurements of effective refractory periods in human PVs, which indicate briefer PV refractoriness compared to LA (Chen et al. 1999).

We did not observe pacemaker activity in PVs, but cannot totally exclude intrinsic pacemaker activity as our preparations were generally electrically paced. However, we can exclude pacemaker or spontaneous arrhythmic activity at frequencies above 0.5 Hz (rate = 30 min^-1), since we were always able to capture LA and/or PV preparations at this frequency and we did not see significant spontaneous phase 4 diastolic depolarization. In this respect, our results differ from those of Chen et al. (2000, 2001) who observed important pacemaker and arrhythmic triggered activity in cardiomyocytes isolated from the myocardial sleeves around normal PVs. However, our results are consistent with findings from several other laboratories. For example, in the initial study of electrical activity in PV tissue, Cheung (1980) noted spontaneous activity in only 7 of 17 guinea-pig PV preparations. When spontaneous activity was present, it was extremely slow, below a frequency of 0.5 Hz. More recently, Hocini et al. (2002) observed no spontaneous canine PV activity. These two studies, as well as ours, were performed with fine-tipped standard microelectrodes in multicellular preparations, unlike the studies by Chen et al. (2001, 2002) that used whole-cell tight-seal patch-clamp methods on isolated myocytes, with the attendant potential effects of cell isolation on ionic currents. While we cannot exclude spontaneous pacemaker activity at a frequency below 0.5 Hz (rate = 30 min^-1), it cannot be assumed that such slow pacemaker activity would play an important role in cardiac arrhythmogenesis.

Relationship between our findings and those of previous studies of PV ion currents

Although a variety of ionic currents have been characterized in PV smooth muscle cells (Weir et al. 1998), these cells are not known to be capable of initiating cardiac electrical activity (Cheung, 1980, 1981). Michelakis et al. (2001) described Ba²⁺-sensitive inward currents compatible with I_K1 and 4AP-sensitive outward currents compatible with I_to in PV cardiomyocytes. They also demonstrated the presence of a broad range of Kir and Kv subunits on immunoblots of PV tissue and showed by immunohistochemistry that Kir1.1, 2.1 and 3.2 subunits differentially localize to cardiomyocytes vs. smooth muscle cells, consistent with a 'sphincter-like' action of PV cardiomyocytes in response to Ba²⁺ (Michelakis et al. 2001).

In order to understand the specific ionic electrophysiology of PVs, we analysed PV cardiomyocyte ionic current properties in relation to those of a reference atrial region. Two papers by Chen et al. (2001, 2002) described ionic currents in PV cardiomyocytes, but neither study compared PV currents with currents in cells from another atrial region. PV currents in our study were qualitatively very similar to those previously reported from canine atrial myocytes (Yue et al. 1996, 1997; Li et al. 2001). On the other hand, the properties of PV currents reported by Chen et al. (2001, 2002) showed some discrepancies from corresponding currents previously characterized in other systems. For example, the I_K1 current-voltage relations in the studies by Chen et al. (2001, 2002) reversed between -30 and -40 mV, difficult to reconcile with the RMPs they recorded of -65 mV. I_to activated at ~+20 mV, much more positive than the typical I_to activation threshold of ~-10 mV (Li et al. 1998). In some cases, current-voltage relations for I_K1, I_K and I_to had unusual forms (Chen et al. 2002). Some of the discrepancies may be due to the recording of all currents with the same external and internal solutions, resulting in substantial current overlap. Others may be due to the extremely small current amplitudes, perhaps related to the unusual method of cell isolation involving retrograde perfusion through the distal lumen of the vein (Chen et al. 2002).

Potential relationship to atrial arrhythmias

The PVs are known to be highly important in clinical AF (Cheung, 1980, 1981; Haissaguerre et al. 1998; Chen et al. 1999, 2000, 2001, 2002; Wu et al. 2001). In clinical medicine, segmental isolation of PVs with radiofrequency catheter-ablation techniques has efficacy in treatment of persistent AF (Pappone et al. 2000; Oral et al. 2002) and the most rapid activity during experimental AF is in the PV region (Morillo et al. 1995; Wu et al. 2001). Thus, PVs appear to be involved not only in initiating AF but also in its maintenance. The specific cellular electrophysiological properties of PV cardiomycoytes establish the cellular electrophysiological milieu for their electrical activity.

Although triggered activity from the PVs may underlie spontaneous ectopic activity (Chen et al. 2000, 2001), evidence has also been obtained to suggest that the PVs may be a privileged site for atrial reentry (Arora et al. 2003). Slowed conduction related to reduced phase 0 upstroke properties may play a role in PV re-entry (Arora et al. 2003). Verheule et al. (2002) have noted that PVs possess comparable connexin 43 but reduced connexin 40 expression compared to atrial myocardium, and have a circumferential fibre orientation that the authors suggest may favour rapid circular reentry. Arora et al. (2003) noted a potentially important role of reduced V_max in PVs compared to LA in favouring PV re-entry, and speculated that reduced I_Na or I_Na inactivation by reduced RMP could be involved. Our studies show that PV V_max is indeed less than that in LA, but that I_Na is comparable in both tissues, suggesting that I_Na inactivation by reduced RMP due to decreased I_K1 is probably the underlying mechanism. Furthermore, we found that larger densities of important repolarizing outward currents (I_Kr and I_Ks) and smaller plateau inward current (I_Ca,L) are associated with shorter PV APDs. Faster repolarization should lead to an ability to fire at more rapid rates, promoting re-entry and leading to AF, as postulated in a recent report of familial AF due to a gain-of-function mutation in KvLQT1/I_Ks (Chen et al. 2003). In addition to having distinct AP properties that may contribute to AF promotion, the complex architecture of PVs also contributes importantly to conduction delays and the promotion of re-entry in PVs (Hocini et al. 2002).

Honjo et al. (2003) recently showed that repetitive spontaneous excitation can be induced by rapid pacing in the presence of ryanodine to alter Ca²⁺ release channel function. Reduced I_K1 has been shown to promote the development of delayed afterdepolarizations (Pogwizd et al. 2001), and therefore our observation that PVs have a smaller I_K1 compared to LA free wall may help to explain the PV arrhythmogenesis observed by Honjo et al. (2003).

Potential limitations

Ion currents are sensitive to isolation technique. Great care was therefore taken to record the same ionic currents from similar numbers of PV and LA cells from each dog, so any influence of the isolation procedure would be equally distributed. When recordings could be obtained from cells of only one tissue, they were rejected for analysis. Furthermore, ion current recordings can change over time due to rundown. Therefore, all currents were recorded after the same time intervals and with protocols applied in the same order for both cell types. LA I_Kr recordings showed a smaller current density than previously reported from our laboratory (Li et al. 2001), reflecting the sensitivity of I_K to cell isolation (Yue et al. 1996) and differences in the collagenase lots presently available compared to the ones we used at the time of the previous study. This highlights the importance of simultaneously comparing currents of interest in a region with those in a reference cell type, to control for isolation-related factors. Another limitation is that we were unable to record the ultrarapid delayed rectifier (I_Kur.d), another current that is very sensitive to isolation, in cells isolated over the course of the present study. We have applied immunohistochemistry to compare ion channel subunit distribution between LA and PV cardiomyocytes (Melnyk et al. 2002) and found no LA-PV distribution differences in the ion channel subunit, Kv3.1, underlying I_Kur.d (Yue et al. 2000). In contrast to the similar expression of Kv3.1 in PV and LA, ERG and KvLQT1 subunits were found to be more strongly expressed in PV, and Kir2.3 subunits less strongly expressed in PV, compared to LA cardiomyocytes (Melnyk et al. 2002), providing potential molecular bases for the differences in I_Kr, I_Ks and I_K1 reported in the present paper.

Atrial cellular properties are spatially heterogeneous and we compared PV properties to those of only one other region, the LA free-wall. However, LA free-wall cells have shorter APDs and similar RMPs to right atrial cells (Li et al. 2001), therefore, the shorter PV APs and smaller RMPs that we observed vs. LA should also hold relative to right atrial (RA) regions.

We took precautions to ensure good voltage control during I_Na recording, including the use of low [Na⁺]_o, the use of small cells and recording at reduced temperature compared to the other currents. Signs of loss of voltage control were not observed and the I_Na density-voltage relation was quite symmetrical; however, the peak voltage drop across series resistance was larger for I_Na recordings compared to the other currents. This may have led to some loss of precision in relating large I_Na recordings to the applied voltages, which must be considered in the interpretation of our data. On the other hand, the currents were very similar between LA and PV and any error should have applied equally to cells from both regions, so that this limitation should not affect our conclusion that LA and PV I_Na values are similar. Similar considerations apply to the inactivation voltage dependence of I_Na. I_Na inactivation voltage dependence is known to shift rapidly in the hyperpolarizing direction upon establishment of the whole-cell patch-clamp mode, resulting in half-inactivation voltages negative to -100 mV (Barber et al. 1991; Zhang & Siegelbaum, 1992). After the initial inactivation voltage shift, voltage dependence stabilizes and we were therefore careful to measure I_Na in both LA and PV at similar times, following stabilization of inactivation voltage dependence. A hyperpolarizing pulse to -140 mV was used to remove inactivation before I_Na measurement at each test potential, preventing the hyperpolarizing shift in inactivation voltage from affecting the currents measured.

We measured I_NCX with intracellular contents fixed by cell dialysis. Although this approach provides a good estimate of NCX ion transport under one set of conditions, it does not indicate NCX function under dynamic physiological conditions in which NCX activity varies with activation rate, action potential changes, varying Ca²⁺ loads and buffering, Na⁺ movement, etc. We can only state, therefore, that there appears to be no intrinsic difference in NCX function between LA and PV cardiomyocytes, which is supported by the equal expression levels of NCX protein found upon immunohistochemical analysis (Melnyk et al. 2002). A more detailed analysis of NCX function, as well as that of other components of the Ca²⁺-handling system, would be very relevant and appropriate but is beyond the scope of the present study.

As cell isolation can have important effects on APs, our AP recordings were performed not on isolated cells, but rather with the use of standard microelectrodes in intact multicellular atrial preparations. Intact intercellular coupling in multicellular preparations may affect cellular properties such as APD by virtue of a 'smoothing effect' over cell populations. This could account for some of the differences between our findings and previous studies recording APs from single atrial myocytes. However, since cell coupling is intact in the in vivo setting, and since cell isolation can alter AP properties by suppressing membrane currents, we believe our approach to be physiologically justified. We compared PV ionic current and AP properties to those in the LA of normal atrial preparations, as we feel that this is an essential starting point before properties can be compared in diseased tissues. Major changes in ionic properties and in tissue coupling can occur in pathological models susceptible to atrial arrhythmias (Yue et al. 1997; Li et al. 1999, 2000), and further comparisons of PV and LA properties in such models is warranted.

Conclusions

PV myocytes exhibit characteristic AP features, with a less negative RMP, a smaller V_max and a shorter APD relative to LA free-wall cells. These AP properties are due to smaller PV I_K1 and I_Ca, as well as larger I_Kr and I_Ks, compared to the LA. These distinct electrophysiological properties are important for understanding the cellular determinants of PV electrical activity and may contribute to the favourable PV substrate for re-entry.

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Acknowledgments

This study was supported by the Canadian Institutes of Health Research, the Quebec Heart and Stroke Foundation and the Mathematics of Information Technology and Complex Systems (MITACS) Network of Centers of Excellence. The authors thank Evelyn Landry, Nathalie L'Heureux, and Chantal Maltais for technical assistance and France Thériault for secretarial help. J.R.E. is a Heart and Stroke Foundation of Canada (HSFC) Research Fellow and P.M. is supported by a HSFC studentship.

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