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(Circulation. 2001;104:2095.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Ionic Remodeling of Cardiac Purkinje Cells by Congestive Heart Failure

Wei Han, MSc; Denis Chartier, BSc; Danshi Li, MD PhD; Stanley Nattel, MD

From the Department of Medicine, Montreal Heart Institute and University of Montreal (W.H., D.C., D.L., S.N.) and the Department of Pharmacology, McGill University (W.H., S.N.), Montreal, Quebec, Canada.

Correspondence to Stanley Nattel, Montreal Heart Institute Research Center, 5000 Belanger St East, Montreal, Quebec, H1T 1C8, Canada. E-mail nattel{at}icm.umontreal.ca

	Abstract

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Background— Cardiac Purkinje cells (PCs) are important for the generation of triggered arrhythmias, particularly in association with abnormal repolarization. The effects of congestive heart failure (CHF) on the ionic properties of PCs are unknown.

Methods and Results— PCs were isolated from false tendons of control dogs and dogs with ventricular tachypacing-induced CHF. CHF PCs were hypertrophied (capacitance, mean±SEM, 149±4 pF, n=130; versus 128±3 pF, n=150, control; P<0.001). Transient outward current density was reduced in CHF PCs without change in voltage dependence or kinetics. CHF also reduced inward-rectifier current density, with no change in form of the current-voltage relationship. Densities of L- and T-type calcium, rapid and slow delayed rectifier, and Na⁺-Ca²⁺ exchange currents were unaltered by CHF, but L-type calcium current inactivation was slowed at positive potentials. Purkinje fiber action potentials from CHF dogs showed decreased phase 1 amplitudes and elevated plateau voltages and demonstrated twice as much prolongation on exposure to the rapid delayed rectifier blocker E-4031 as control Purkinje fibers.

Conclusions— CHF causes remodeling of important K⁺ and Ca²⁺ currents in cardiac PCs, decreasing repolarization reserve and causing an exaggerated repolarization delay in response to a class III drug. These results have important potential implications regarding ventricular arrhythmogenesis, particularly related to triggered activity in PCs, in patients with CHF.

Key Words: ion channels • remodeling • electrophysiology • antiarrhythmia agents • heart failure

	Introduction

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Congestive heart failure (CHF) predisposes to the generation of ventricular tachyarrhythmias¹ and the occurrence of sudden death.² In addition, CHF promotes drug-induced Torsades de Pointes arrhythmias.³ Abnormal repolarization, related to ion channel remodeling, is important in the arrhythmogenic potential of CHF.^1,4,5

CHF-induced remodeling of ionic currents in ventricular⁵ and atrial⁶ myocytes has been studied in detail. Cardiac Purkinje cells (PCs) are believed to play important roles in the generation of ventricular arrhythmias, particularly those related to triggered activity.^7–9 Ionic currents are altered in subendocardial PCs in regions of myocardial infarction^10–12; however, almost nothing is known about PC ionic remodeling in CHF. The present study was designed to evaluate CHF-induced changes in ionic currents and action potentials (APs) in canine cardiac PCs.

	Methods

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CHF Preparation
CHF was produced as previously described⁶ by pacing the right ventricle at 240 bpm for 3 weeks followed by 2 weeks at 220 bpm. Dogs were then anesthetized with morphine (2 mg/kg SC) and -chloralose (120 mg/kg IV load, 29.25 mg/kg per hour infusion), and a median sternotomy was performed. All animal care and handling procedures followed the guidelines of the Canadian Council on Animal Care. Excised hearts were immersed in Tyrode solution at room temperature. Free-running false tendons were excised into modified Eagle’s MEM (Gibco-BRL; pH 6.8, HEPES-NaOH) containing collagenase (800 to 900 U/mL, Worthington Type-II) and 1% BSA (Sigma), and single PCs were isolated as previously described.^13,14

Solutions
Standard Tyrode solution contained (in mmol/L) NaCl 136, KCl 5.4, MgCl₂ 1, CaCl₂ 1, NaH₂PO₄ 0.33, HEPES 5, and dextrose 10; pH 7.4 (NaOH). High-K⁺ storage solution contained (in mmol/L) KCl 20, KH₂PO₄ 10, dextrose 10, glutamic acid 70, ß-hydroxybutyric acid 10, taurine 10, and EGTA 10; 0.1% BSA; pH 7.4 (KOH). Standard pipette solution contained (in mmol/L) K⁺ aspartate 110, KCl 20, MgCl₂ 1, Mg₂ATP 5, HEPES 10, phosphocreatine 5, GTP 0.1, and EGTA 5; pH 7.2 (KOH). Solutions were equilibrated with 100% O₂.

For K⁺ current measurement, the extracellular solution included 1 µmol/L atropine to eliminate muscarinic K⁺ currents and CdCl₂ (200 µmol/L) or nimodipine (1 µmol/L, for I_K studies) to block Ca²⁺ currents. Na⁺ current contamination was prevented by equimolar substitution of choline for extracellular Na⁺. For currents other than transient outward current (I_to), 1 mmol/L 4-AP was used to block I_to. Rapid delayed rectifier current (I_Kr) was studied as 5 µmol/L E4031-sensitive current and inward rectifier current (I_K1) as 1 mmol/L Ba²⁺-sensitive current. Slow delayed rectifier current (I_Ks) was studied in the presence of 1 µmol/L dofetilide to eliminate I_Kr. For I_Ca recording, the bath solution contained tetraethylammonium chloride, CsCl, and CsOH in place of NaCl, KCl, and NaOH, respectively, and [CaCl₂] was 2 mmol/L. The pipette for I_Ca recording contained (in mmol/L) CsCl 20, Cs-aspartate 110, MgCl₂ 1, EGTA 10, GTP 0.1, ATP-Mg 5, HEPES 10, and Na₂ phosphocreatine 5; pH 7.2 (CsOH). Na⁺-Ca²⁺ exchange (NCX) current (I_NCX) was recorded with ramp pulses and extracellular (in mmol/L, NaCl 140, CaCl₂ 0 or 5, MgCl₂ 1, CsCl 5, HEPES 5, nimodipine 0.001, ouabain 0.01, and ryanodine 0.005; pH 7.2 CsOH) and pipette (in mmol/L, CsCl 90, NaCl 50, MgATP 5, MgCl₂ 3, EGTA 20, CaCl₂ 13, and HEPES 20; pH 7.2, CsOH) solutions designed to suppress K⁺ current, Na⁺-K⁺ ATPase, and sarcoplasmic reticulum Ca²⁺ release.¹⁵

Data Acquisition and Analysis
Whole-cell patch clamp was performed as previously described^13,14 at 36.5°C. Compensated series resistance and capacitive time constants (s) averaged 2.5±0.1 M and 290±10 µs. Leakage compensation was not used. The capacitance of CHF cells was increased (149±4 pF, n=130, versus 128±3 pF in control, n=150; P<0.001), so currents are expressed in terms of density.

Standard microelectrode techniques were used to record action potentials (APs). The Tyrode solution contained (in mmol/L) NaCl 120, KCl 1.5, KH₂PO₄ 1.2, MgSO₄ 0.1, NaHCO₃ 25, CaCl₂ 1.25, and dextrose 5; pH 7.4. Purkinje fiber false tendons were superfused with oxygenated (95% O₂ and 5% CO₂) Tyrode solution at 36°C and impaled with 3 mol/L KCl-filled glass microelectrodes (8 to 20 M) connected to a high input-impedance amplifier.

Nonlinear least-square curve-fitting algorithms were used for curve fitting. Nonpaired t tests were used to compare CHF with control cells. P<0.05 was considered to indicate statistical significance. Group data are expressed as mean±SEM.

	Results

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Changes in Ionic Currents
K⁺ Currents
Figure 1 shows representative I_K1 recordings (panels A and B). CHF significantly reduced I_K1 (C), including the outward component (D), without altering the form of the I_K1 current-voltage relation (Figure 1C, inset).

View larger version (30K): [in this window] [in a new window]   Figure 1. Ba2+-sensitive IK1 in representative control (A) and CHF (B) PCs (voltage protocol in inset delivered at 0.1 Hz). C, Mean±SEM IK1 density in 29 control and 27 CHF cells. *P<0.05, **P<0.01, ***P<0.001 vs control. Inset, Normalized current-voltage relations overlap. D, Currents between -70 and -20 mV on expanded scale. CTL indicates control; TP, test potential.

Figures 2A and 2B illustrate I_to recordings from control and CHF PCs. I_to density was significantly smaller in CHF PCs (Figure 2C); however, there were no differences in the form of the I_to-V relation (Figure 2D). The voltage dependence of I_to inactivation was tested with a 2-pulse protocol as described in Figure 2E. Activation voltage dependence was evaluated from the relation I_TP=a_TPG_max(V_TP-V_R), where I_TP and a_TP are current and activation variable at test potential V_TP, V_R is reversal potential, and G_max is maximal conductance. Half-maximal voltage (V) and activation slope factor (Boltzmann fits) were 8.9±0.7 and 10.3±0.4 mV (control) and 8.9±1.0 and 10.9±1.0 mV (CHF, P=NS). V_R obtained from the reversal of I_to tail currents after 2-ms activating pulses averaged -75.3±2.2 mV. Inactivation V and slope factor were -30±2 and 11±1 mV (control) and -31±2 and 11±1 mV (CHF, P=NS). I_to inactivation kinetics were biexponential, with s unaltered by CHF (Figure 2F). I_to recovery (Figure 2G) was biexponential, with s averaging 42±8 and 1391±67 ms (control) and 52±11 and 1486±108 ms (CHF, n=10 for each, P=NS). I_to frequency dependence was similarly unaffected by CHF (Figure 2H).

View larger version (38K): [in this window] [in a new window]   Figure 2. Ito recordings obtained with 100-ms test pulses at 0.1 Hz from representative control (A) and CHF (B) PCs. C, Mean±SEM Ito density for 37 cells from 13 control dogs and 34 cells from 14 CHF dogs (*P<0.05, **P<0.01, ***P<0.001 vs control at same test potential). D, Mean±SEM Ito-V relations normalized to maximum current in each cell. E, Ito inactivation and activation voltage dependence. Inactivation was evaluated with 1000-ms prepulses followed by a 200-ms test pulse to +50 mV. Activation voltage dependence was analyzed from data obtained with the protocol in panel A according to equation in the text. Data are mean±SEM (n=10 cells/group, inactivation; n=20 cells/group, activation); curves are best-fit Boltzmann relations. F, Mean±SEM inactivation s (n=15 cells/group). G, Ito reactivation time course evaluated by ratio of current (I2) during a 100-ms test pulse (P2, identical to P1) to current (I1) during a conditioning pulse (P1) with varying P1 to P2 interval (protocol delivered at 0.07 Hz). Curves are biexponential fits (n=10 cells/group). H, Ito frequency dependence, determined by ratio of current during the 15th pulse to current during the first pulse of a train of 100-ms depolarizations (n=15 cells/group). CTL indicates control; TP, test potential.

Figure 3 shows results for I_Kr (left) and I_Ks (right). Representative control recordings are shown at the top, with mean step I-V relations, which were unchanged by CHF, in the middle. Activation voltage dependence based on normalized tail currents is shown in bottom panels and was not altered by CHF for either I_Kr or I_Ks.

View larger version (39K): [in this window] [in a new window]   Figure 3. A, Representative E-4031-sensitive IKr recordings. B, Mean±SEM IKr density voltage relations (n=11, control; n=9, CHF). C, Mean±SEM normalized IKr tail currents (n=5 cells/group) and best-fit Boltzmann relations. D, Representative IKs recordings with 4-second depolarizing pulses (0.1 Hz) and 2-second repolarizations to -40 mV to record tail currents. E, Mean±SEM IKs density voltage relations (n=30, control; n=25, CHF). F, Normalized IKs tail I-V relations and best-fit Boltzmann relations. CTL indicates control; TP, test potential.

Ca²⁺ Currents
Representative L-type calcium current (I_Ca.L) recordings are shown in Figures 4A and 4B and point to slowed I_Ca.L decay in CHF. I_Ca.L density was not significantly different between control and CHF cells (Figure 4C). Inactivation s were slowed significantly by CHF at voltages positive to +10 mV (Figure 4D). For example, at +20 mV, _fast increased from 6.0±0.3 to 8.2±0.9 ms (P<0.05) and _slow from 38±2 to 66±8 ms (P<0.01) in CHF PCs. In addition to a slowing of inactivation s, CHF significantly increased the proportion of slow-phase inactivation at positive voltages (Figure 4E). For example, at +30 mV, ₂ accounted for 36±4% of inactivation in control PCs compared with 51±5% in CHF (P<0.05). I_Ca.L inactivation voltage dependence was assessed with a 2-pulse protocol (Figure 4F). Inactivation V and slope factor were -25±2 and -6±1 mV (control) and -26±1 and -7±1 mV (CHF, n=10 cells/group, P=NS). Activation voltage dependence was assessed according to the relation I_TP=a_TPG_max (V_TP-V_R), with V_R obtained from a linear fit to the ascending portion of the I-V relation. Resulting mean V and slope factors were 2.0±1.4 and 6.6±0.2 mV (control) and 0.4±1.4 and 6.5±0.1 mV (CHF, n=10/group, P=NS). Reactivation kinetics at holding potential (HP) (-80 mV) close to the PC resting potential are shown in Figure 4G. Reactivation was monoexponential, with similar s in control (44±7 ms) and CHF PCs (53±6 ms, n=10/group, P=NS). I_Ca.L showed little frequency dependence between 1 and 5 Hz at a HP of -80 mV, and there were no significant CHF-related differences in I_Ca.L frequency dependence (Figure 4H).

View larger version (34K): [in this window] [in a new window]   Figure 4. Representative ICa.L recordings obtained with 250-ms pulses (0.1 Hz) in a control (A) and CHF (B) cell. C, Mean±SEM ICa.L density obtained with the protocol shown in panel A (n=26 and 24 cells in control and CHF, respectively). D, Mean±SEM ICa.L inactivation time constants (n=12 cells, control; n=14, CHF) obtained from recordings with the protocol in panel A. E, Mean±SEM percentage of overall inactivation proceeding by the slower time constant at each test potential. *P<0.05 and **P<0.01 versus control. F, Voltage dependence of ICa.L inactivation (Inact.) and activation (Act.). Steady-state inactivation was assessed with 1-second conditioning pulses followed by a 300-ms test pulse to +10 mV (0.1 Hz). Activation was assessed from data obtained with the protocol shown in panel A, according to the equation in the text. Data are mean±SEM from 10 control and 10 CHF cells; curves are best-fit Bolzmann relations. G, ICa.L reactivation time course, studied with paired 100-ms pulses delivered with varying interpulse intervals at 0.1 Hz. Curves are monoexponential fits (n=10 cells/group). H, ICa.L frequency dependence, determined from the ratio of current during the 15th pulse to current during the first pulse of a train of 100-ms depolarizations from -80 mV to +10 mV at frequencies indicated (n=10 cells/group). CTL indicates control; TP, test potential.

All PCs possessed a relatively large T-type calcium current (I_Ca.T) (Figures 5A and B). I_Ca.T was obtained as previously described^16,17 by digital subtraction of I_Ca elicited at HP -50 mV from I_Ca at a HP of -90 mV. The mean I_Ca.T density voltage relation was not altered by CHF (Figure 5C). I_Ca.T inactivation V and slope factors averaged -61±2 and 4.1±0.4 mV (control) and -63±2 and 4.9±0.5 mV (CHF, n=10/group, P=NS) (Figure 5D). Activation V and slope factors were -32±1 and 10±1 mV (control) and -30±2 and 12±1 mV (CHF), respectively (n=10/group, P=NS). The time course of I_Ca.T inactivation was monoexponential, and there were no significant differences between groups; for example, at -20 mV (voltage of maximum I_Ca.T), s were 4.5±0.2 ms (control, n=10) versus 4.6±0.3 ms (CHF, n=10, P=NS). I_Ca.T reactivation was assessed (Figure 5E) at a test potential (-30 mV) at which no I_Ca.L is activated (Figure 4C). I_Ca.T reactivation s averaged 51±3 ms (control) versus 49±4 ms (CHF, n=10 cells/group, P=NS). The frequency dependence of I_Ca.T (Figure 4F) was greater than that of I_Ca.L but did not differ between groups.

View larger version (31K): [in this window] [in a new window]   Figure 5. ICa.T was obtained by subtracting currents recorded during 250-ms depolarizations (0.1 Hz) from a HP of -50 mV from those with the same depolarization and a HP of -90 mV. Examples are shown for control (A) and CHF (B) cells. C, Mean±SEM ICa.T density voltage relation (n=16 cells/group). D, Voltage dependence of inactivation (Inact.) and activation (Act.) of ICa.T. Steady-state inactivation was assessed with 1-second prepulses from a HP of -90 mV, followed by a 300-ms test pulse to -30 mV (0.1 Hz). Activation was assessed from currents recorded on 250-ms depolarizations, according to the relation ITP=aTPGmax(VTP-VR), with VR obtained from a linear fit to the ascending portion of the I-V relation. Data are mean±SEM for 10 cells/group; curves are best-fit Boltzmann relations. E, ICa.T reactivation time course, studied with paired 100-ms pulses with varying interpulse intervals delivered at 0.1 Hz. Data are mean±SEM (n=10/group); curves are best-fit monoexponentials. F, Frequency dependence of ICa.T, obtained with 15 100-ms pulses from -90 to -30 mV. Current during the 15th pulse was normalized to current during the first pulse (n=10 cells/group). CTL indicates control; TP, test potential.

NCX
Figure 6A shows I_NCX as determined from current during ramp depolarizations from -60 to +50 mV in the presence of 5 and 0 mmol/L [Ca²⁺]_o. Reverse-mode I_NCX is substantial in the presence of 5 mmol/L Ca²⁺ and is absent in the presence of 0 mmol/L Ca²⁺, allowing I_NCX to be calculated from the difference between the two current recordings.¹⁵ The results in Figure 6B show that I_NCX amplitude was larger in CHF cells but that after correction for cell size (capacitance normalization), there were no significant differences.

View larger version (20K): [in this window] [in a new window]   Figure 6. A, Currents recorded during 164-ms ramps from -60 to +50 mV in the presence of 0 and 5 mmol/L [Ca2+]o. B, Mean±SEM (24 cells/group) INCX amplitude (left) and density (right). CTL indicates control; RP, ramp potential.

Changes in APs
PC AP characteristics were first recorded at a total of 42 sites from 11 control dogs and 37 sites in 8 CHF dogs at 1 Hz. Phase 1 repolarization was less marked in CHF, and the plateau voltage was higher (Figure 7, top). There were no significant differences in resting potential, AP amplitude, or AP duration (APD) between control and CHF cells, but plateau-voltage was significantly more positive and phase 1 amplitude significantly smaller in CHF PCs (Table).

View larger version (24K): [in this window] [in a new window]   Figure 7. AP results for control (left) and CHF (right) Purkinje fiber preparations. A and B, Typical AP recordings. Vertical line to the left of each AP shows phase-1 amplitude. P.V. indicates the point used to measure voltage at the onset of the plateau. C and D, Effects of E-4031 on APs at 1 Hz in control (C) and CHF (D) PCs. E and F, Effects of E-4031 on mean±SEM APD in 20 control (E) and 26 CHF (F) PCs. *P<0.05, **P<0.01, ***P<0.001 for difference between CHF and control PCs under corresponding conditions with respect to frequency and drug. CTL indicates control.

View this table: [in this window] [in a new window]   Table 1. Comparison of AP Properties in Control and CHF PCs at 1 Hz

To evaluate the possibility that the response to an I_Kr-blocking class III drug may be altered in CHF PCs, APs were recorded from control and CHF PCs in free-running false tendons with standard microelectrodes before and after exposure to E-4031 (1 µmol/L). The results are illustrated in Figures 7C and 7D, and mean data are provided in Figures 7E and 7F. As in the first series of experiments, predrug APDs were not significantly different between control and CHF PCs. However, E-4031 had substantially larger effects on APD in CHF PCs, so that APD₉₅ after the drug was significantly greater in CHF cells at all frequencies (P<0.001 for each). For example, E-4031-induced APD₉₅ increases averaged 103±45 ms (25±10%) at 1 Hz in control preparations compared with 231±40 ms (54±10%) in CHF preparations.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We have evaluated the effects of CHF on K⁺ currents, Ca²⁺ currents, NCX, and AP properties of PCs from free-running ventricular false tendons. CHF decreased PC I_to and I_K1 density and slowed the inactivation of I_Ca.L without altering its density. I_Ks, I_Kr, I_NCX, and I_Ca.T were unaffected. CHF reduced the amplitude of phase 1 repolarization, increased plateau voltage, and enhanced the APD-prolonging effect of E-4031.

Comparison with Previous Studies of CHF-Induced Ionic Remodeling
There have been extensive studies of the ionic remodeling of K⁺ and Ca²⁺ channels in ventricular myocytes of patients and experimental animals with CHF.^5,18,19 I_to is quite consistently reduced^5,20–22 by an average of 35% in ventricular myocytes of patients with CHF.¹⁸ I_K1 also tends to be decreased by 25%.¹⁸ Although there is some variability in the results for I_Ca.L,^5,23 overall there seems to be no change in I_Ca.L density or kinetics. Recent studies point to downregulation of I_Ks and possibly I_Kr in ventricular myocytes of rabbits with tachypacing-induced CHF.²⁴ Atrial ionic remodeling in CHF has been studied to a lesser extent, but recent studies suggest that I_to, I_Ks, and I_Ca are all decreased and I_NCX is increased, with no change in kinetics or voltage dependence and no change in I_K1 or I_Kr.⁶

Several aspects of PC ionic remodeling resemble previously reported findings in ventricular myocytes: I_to and I_K1 were reduced and I_Ca.L density was unchanged. These observations suggest that the mechanisms leading to CHF-induced I_to and I_K1 downregulation at the ventricular level also likely operate on free-running Purkinje fiber false tendons. The CHF-induced slowing of I_Ca inactivation that we observed has not, to our knowledge, been reported in ventricular myocytes. We did not observe changes in I_Kr or I_Ks, suggesting that PCs may be spared from the I_K downregulation occurring with CHF in ventricular myocytes²⁴ and possibly explaining why overall APD was not prolonged in CHF PCs. Unlike typical findings in ventricular⁵ and atrial⁶ myocytes, I_NCX density was not increased by CHF in PCs.

Relationship to Other Studies in PCs
I_to, I_Ca, I_K1, and E-4031-sensitive current have been studied in PCs from the subendocardial Purkinje fiber network overlying 24- to 48-hour-old myocardial infarctions.^10–12 I_to density was reduced by >50% in the infarct zone, with no change in voltage dependence but a slowing in reactivation.¹⁰ PCs from free-running false tendons had normal I_to properties. Both I_Ca.L and I_Ca.T were reduced in subendocardial PCs from the infarct zone, with no changes in voltage dependence or inactivation kinetics.¹¹ I_K1 was reduced in PCs from the infarct zone, and a very rapidly activating E-4031-sensitive current was increased, with no classical I_K noted in either normal or infarct zone PCs.¹² In general, the abnormalities noted in infarct zone PCs were more severe than those we observed. There are, however, some qualitative similarities in terms of decreases in I_K1 and I_to. In contrast to our findings, myocardial infarction did not affect the electrophysiology in PCs from free-running false tendons, suggesting that myocardial infarction produces severe but localized ionic remodeling in infarct zone PCs whereas CHF produces more generalized but less severe remodeling.

Potential Significance
The present study constitutes the first detailed analysis of CHF-induced ionic remodeling in PCs. PCs are believed to play an important role in ventricular arrhythmogenesis, particularly in the generation of early afterdepolarization-induced arrhythmias.^7,8 The CHF-induced ionic abnormalities we noted in PCs may contribute to promoting the occurrence of arrhythmogenic afterdepolarizations in patients with CHF, particularly in response to interventions such as I_Kr-blocking antiarrhythmic drugs and hypokalemia that prolong APD. Downregulation of I_to, along with slowed inactivation of I_Ca, is likely responsible for the positive shift in the plateau voltage of PCs. The positive shift in plateau voltage and slowed I_Ca.L inactivation at positive voltages would act to promote the occurrence of I_Ca.L-dependent early afterdepolarizations under conditions that delay repolarization.²⁵

The CHF-induced decreases in I_to and I_K1 are likely to reduce the repolarization reserve, the ability of myocardial cells to repolarize when normal repolarizing currents are reduced by drugs, metabolic or electrolyte imbalances, or intercurrent diseases. Consistent with this notion, PCs from dogs with CHF showed twice as great APD prolongation in response to I_Kr blockade with E-4031 compared with control PCs. CHF significantly increases the risk of drug-induced Torsades de Pointes arrhythmias.³ The CHF-induced ionic remodeling that we observed in PCs could clearly play an important role in this clinically important phenomenon.

Potential Limitations
The isolation of PCs from free-running Purkinje fibers is technically challenging, requiring prolonged periods of bath exposure to cell-dissociating enzymes (chunk method). This likely explains the absence of studies of PC remodeling in CHF, despite the large number of studies that have evaluated ventricular myocyte remodeling. I_K is particularly sensitive to isolation technique²⁶ and has been difficult to record in isolated PCs.^12,27 We were able to record robust I_Ks in isolated PCs, but I_Kr was smaller and more difficult to record; therefore, our results regarding I_Kr should be interpreted with caution. The properties of the currents we recorded from normal PCs were generally similar to those reported for studies with other preparations; however, we cannot exclude the possibility that cell isolation might have affected the currents we evaluated. Isolation of PCs uncouples them from other PCs and from ventricular myocytes, removing an important modulator of electrophysiologic function present in vivo. This consideration does not apply to the multicellular preparations we studied, which maintain attachments to both false tendon PCs and underlying muscle intact. We measured currents in the presence of heavily buffered [Ca²⁺]_i. [Ca²⁺]_i buffering was necessary to preserve cell viability under our recording conditions; however, CHF-induced changes in [Ca²⁺]_i could importantly affect currents like the NCX. Such changes would not have been detected in the present study.

	Acknowledgments

 
This work was supported by operating grants from the Canadian Institutes for Health Research and the Quebec Heart and Stroke Foundation. Dr Li was a Heart and Stroke Corporation of Canada/AstraZeneca Fellow. The authors thank Essai and Pfizer Pharmaceuticals for providing E-4031 and dofetilide, Chantal St-Cyr and Chantal Maltais for technical assistance, and Annie Laprade for secretarial help.

Received May 17, 2001; revision received July 20, 2001; accepted July 25, 2001.

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