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Am J Physiol Heart Circ Physiol 283: H2495-H2503, 2002. First published August 15, 2002; doi:10.1152/ajpheart.00389.2002
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Vol. 283, Issue 6, H2495-H2503, December 2002

Properties of potassium currents in Purkinje cells of failing human hearts

Wei Han1,3, Liming Zhang1, Gernot Schram1,2, and Stanley Nattel1,2,3

1 Research Center, Montreal Heart Institute, Montreal, Quebec H1T 1C8; 2 Department of Medicine, University of Montreal, Montreal, Quebec H3C 3J7; and 3 Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec H3G 1Y6, Canada


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cardiac Purkinje fibers play an important role in cardiac arrhythmias, but no information is available about ionic currents in human cardiac Purkinje cells (PCs). PCs and midmyocardial ventricular myocytes (VMs) were isolated from explanted human hearts. K+ currents were evaluated at 37°C with whole cell patch clamp. PCs had clear inward rectifier K+ current (IK1), with a density not significantly different from VMs between -110 and -20 mV. A Cs+-sensitive, time-dependent hyperpolarization-activated current was measurable negative to -60 mV. Transient outward current (Ito) density was smaller, but end pulse sustained current (Isus) was larger, in PCs vs. VMs. Ito recovery was substantially slower in PCs, leading to strong frequency dependence. Unlike VM Ito, which was unaffected by 10 mM tetraethylammonium, Purkinje Ito was strongly inhibited by tetraethylammonium, and Purkinje Ito was 10-fold more sensitive to 4-aminopyridine than VM. PC Isus was also reduced strongly by 10 mM tetraethylammonium. In conclusion, human PCs demonstrate a prominent IK1, a time-dependent hyperpolarization-activated current, and an Ito with pharmacological sensitivity and recovery kinetics different from those in the atrium or ventricle and compatible with a different molecular basis.

ion currents; cardiac Purkinje cells; potassium channel blockers


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CARDIAC PURKINJE FIBERS PLAY a key role in conduction and arrhythmogenesis. As the prime cellular component of the cardiac conducting system, they are critical for assuring appropriate timing and sequence of ventricular contraction. They appear to play a particularly important role as a generator of early afterdepolarizations and an initiator of transmural reentry in torsades de pointes arrhythmias associated with long QT syndromes (2, 3, 10, 23). In addition, there is evidence for significant participation of Purkinje cells (PCs) in ventricular tachyarrhythmias due to delayed afterdepolarizations (35), intraventricular reentry (26), and ventricular fibrillation (7).

K+ currents are a key determinant of cellular repolarization and, consequently, of the occurrence of cardiac arrhythmias. Recent work has shown that the properties of K+ currents in canine PCs differ from those in ventricular myocytes (14). Furthermore, the properties and molecular basis of specific K+ currents in human cardiac cells may be chamber specific and distinct from those of corresponding regions in hearts of other animals (11, 19, 41, 43). We were unable to identify any voltage-clamp studies of human cardiac PCs in the literature. The objectives of the present study were to isolate PCs from free-running false tendons of human hearts explanted at the time of cardiac transplantation and to characterize a variety of ionic currents by voltage-clamp recording.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Isolation

Free-running false tendons and left ventricular free wall midmyocardium were obtained from nine explanted failing human hearts removed at the time of cardiac transplantation. Midmyocardial myocytes were used because they share some repolarization and phase 0 upstroke properties with Purkinje tissue (32). Table 1 lists the clinical characteristics of the patient population. Procedures for obtaining the tissues were approved by the Research Ethics Committee of the Montreal Heart Institute.

                              
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  		Table 1.   Patient characteristics

Free-running endocardial false tendons were excised quickly from both ventricles into modified Eagle's minimum essential medium (pH 7.0 with HEPES-NaOH; GIBCO-BRL), followed by incubation with the same solution containing collagenase (1,500 U/ml; Worthington type II) and 1% bovine serum albumin (Sigma). False tendons were distinguished from trabeculae in that they were pale in color and thinner and were removed from either ventricle by cutting with fine scissors. The fibers were agitated by continuous bubbling with 100% O2 in a 37°C shaker bath for 4-6 h. After the endothelial sheath had been digested, revealing single cells and/or cell columns under light microscope, the digested fibers were washed twice with the high-K+ storage solution and were incubated for an additional 10 min. Individual cells were dispersed by gentle hand pipetting, harvested by centrifugation, and kept in a high-K+ storage solution. Human ventricular myocytes were isolated with the use of coronary artery perfusion methods described previously in detail (19). Both PCs and ventricular myocytes were Ca2+ tolerant and were studied concurrently within 18 h of isolation. PCs were characterized by a typical spindle-shaped, narrower, and longer morphology with much less prominent cross-striations (Fig. 1) compared with ventricular myocytes, from which they looked clearly different under light microscopy.


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  		Fig. 1.   Photomicrographs of two human cardiac Purkinje cells (PCs) (left) and ventricular myocytes (VMs) (right) at comparable magnifications. The horizontal scales indicate 20 µm. PCs were typically longer and thinner than VMs and had less prominent cross-striations.

Solutions

Standard Tyrode solution for cell isolation and patch-clamp studies contained (in mM) 136 NaCl, 5.4 KCl, 1.0 MgCl2, 1.0 CaCl2, 0.33 NaH2PO4, 5.0 HEPES, and 10 dextrose, pH 7.4 (adjusted with NaOH). High-K+ storage solution contained (in mM) 20 KCl, 10 KH2PO4, 10 dextrose, 70 glutamic acid, 10 beta -hydroxybutyric acid, 10 taurine, 10 EGTA, and 0.1% albumin, pH 7.4 (adjusted with KOH). The pipette solution contained (in mM) 110 K+-aspartate, 20 KCl, 1 MgCl2, 5 Mg2ATP, 10 HEPES, 5 phosphocreatine, 0.1 GTP, and 5 EGTA, pH 7.2 (adjusted with KOH).

Atropine (1 µM) was included in the extracellular solution to eliminate any basal activity of acetylcholine-activated current (16) and 4-aminopyridine (4-AP)-dependent K+ currents (30). CdCl2 (200 µM) was added to block L-type Ca2+ current (ICa) and Ca2+-activated Cl- current. T-type Ca2+ current was suppressed by holding at -50 mV and in one protocol in which a more negative holding potential (HP) (-80 mV) was used by pulsing to a voltage (+50 mV) at which T-type ICa in PCs is minimal (30). Contamination by Na+ current was prevented with an HP of -50 mV or, when a more negative HP was necessary, with Tris · Cl substitution for extracellular NaCl.

Data Acquisition and Analysis

The whole cell patch-clamp technique was used to record currents at 37°C as previously described in detail (11, 41). The capacitance of PCs averaged 199 ± 17 pF (n = 20) and the capacitance of midmyocardial myocytes was 237 ± 23 pF (n = 18). Compensated series resistances and capacitive time constants averaged 2.7 ± 0.2 MOmega and 0.40 ± 0.02 ms for PCs and 1.7 ± 0.4 MOmega and 0.40 ± 0.05 ms for ventricular myocytes, respectively. Maximum mean voltage drop across the series resistance was 6 mV. Junction potentials between bath and pipette solutions averaged 10 mV and were not corrected. Drug effects were studied at steady state, after 5 min of exposure at each concentration.

Group data are presented as means ± SE. Nonlinear curve fitting was performed with Clampfit in pCLAMP 6. Student's t-tests were used for statistical comparisons. A two-tailed P < 0.05 indicated statistical significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Inward-Rectifier Current

The inward rectifier K+ current (IK1) was recorded as current sensitive to 1 mM Ba2+. Figure 2, A and B, shows typical recordings obtained from a PC and a ventricular myocyte, respectively, on voltage steps from -40 mV before and after the addition of 1 mM Ba2+ to the extracellular solution. In these cells, virtually all of the current elicited on voltage steps from -40 mV was suppressed by 1 mM Ba2+. Ba2+-sensitive currents were obtained by digital subtraction of recordings before and after Ba2+, as illustrated in Fig. 2, A and B. Figure 2C shows means ± SE of IK1 density in five PCs and nine ventricular myocytes as a function of test potential. Currents in both cell types reversed at approximately -70 mV, which, when corrected for the junction potential, provide an estimated reversal potential of approximately -80 mV. IK1 in both cell types showed strong inward rectification with a small but distinct outward component between approximately -70 and -20 mV, as shown on an expanded scale in Fig. 2D. No statistically significant differences in IK1 density were observed over the voltage range between -110 and -20 mV; however, IK1 density was significantly greater in ventricular myocytes than in PCs at -120 mV.


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  		Fig. 2.   A and B: inward rectifier K+ current (IK1) before and after exposure to 1 mM Ba2+ and Ba2+-sensitive IK1 (right) obtained by digital subtraction of Ba2+ resistant (middle) from pre-Ba2+ current (left) in a PC (A) and a VM (B). C: mean ± SE IK1 density-voltage relationship in PCs and VMs. * P < 0.05, PC vs. VM. D: IK1 over the range of -80 to -20 mV on an enlarged scale. Note that junction potentials were not corrected, so true voltages are ~10 mV negative to those shown. TP, test potential.

Hyperpolarization-Activated Current

In 5 of 11 PCs subjected to hyperpolarizing steps, clear time-dependent currents were seen on hyperpolarization. Figure 3A shows time-dependent hyperpolarization-activated current (IH) elicited on 3,910-ms voltage steps from an HP of -50 mV. Such currents were strongly suppressed by 2 mM Cs+, as shown in Fig. 3B. Figure 3C shows Cs+-sensitive current obtained by digital subtraction. Figure 3D shows mean IH density-voltage relations in five PCs. IH was relatively large (e.g., mean density -4.5 ± 1.0 pA/pF at -120 mV). Measurable time-dependent inward currents were detectable at voltages as positive as -60 mV. Current activation kinetics were biexponential, with mean time constants (tau ) in five PCs as shown in Fig. 3E. The proportion of activation in the faster phase decreased progressively at more positive voltages (Fig. 3F). Current activation became slower at more positive potentials, both because of significant voltage-dependent slowing of the rapid phase time constant (tau fast) and a decreasing proportion of fast phase activation at more positive voltages. No time-dependent activating current was observed on hyperpolarization of ventricular myocytes (12 cells from 4 hearts).


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  		Fig. 3.   A and B: representative hyperpolarization-activated current (IH) recordings in the absence (A) and presence (B) of 2 mM Cs+ in a PC obtained with the protocol shown in B, inset. C: Cs+ -sensitive current obtained by digital subtraction of currents in B from those in A. D: mean ± SE density-voltage relationship of time-dependent current activated by hyperpolarizing steps from -50 mV to various TP values. E: voltage-dependent IH activation fast (tau fast) and slow (tau slow) time constants obtained from biexponential fits of time-dependent current. F: portion of activation in fast component as a function of TP. Af and As, amplitude of the fast and slow phase, respectively.

Transient Outward Current

Voltage- and time-dependent properties. Figure 4, A and B, shows typical recordings of transient outward current (Ito) from a PC and a ventricular myocyte. Because of slower inactivation in PCs, longer pulses (300 ms) were used for PCs to permit steady-state inactivation (note the difference in time scale for Fig. 4, A vs. B). Ito activated and inactivated rapidly in both cell types, but the sustained "pedestal" component was larger in PCs. Inactivation was best fitted by biexponential functions. Figure 4C shows mean inactivation time constants in eight PCs and seven ventricular myocytes. Both kinetic components were slower in PCs than in ventricular muscle. Figure 4D shows current density-voltage relationships for Ito in eight PCs and seven ventricular myocytes. Ito density (measured from peak current to end-pulse steady state) was significantly smaller in PCs between +20 and +60 mV. However, sustained current (Isus; measured from the end-pulse level to the 0-current level) density in the same cells was significantly larger between -10 and +60 mV in PCs (Fig. 4E). Figure 4F shows mean data for Ito activation and inactivation voltage dependence based on recordings in five PCs and five ventricular myocytes. Inactivation voltage dependence was determined with the use of 1,000-ms conditioning pulses followed by 200-ms test pulses to +50 mV. Activation voltage dependence was determined from the relation IV = Imax(GV/Gmax)(V - Vrev), where IV and Imax are currents at voltage (V) and maximum current (at the most positive test potential), respectively; GV and Gmax are conductances at voltage and maximum conductance, respectively; and Vrev is reversal potential. Data were fitted by Boltzmann relations, as illustrated in Fig. 4. The half-maximal inactivation voltage averaged -27 ± 2 mV in PCs and -21 ± 2 mV in ventricular myocytes (P = not significant). The inactivation slope factor in PCs was -13 ± 2 mV, significantly larger than in ventricular myocytes (-5 ± 1 mV, P < 0.01). Half-maximal activation occurred at +20 ± 9 and +17 ± 6 mV in PCs and ventricular myocytes, respectively (P = not significant), and the activation slope factor averaged 13 ± 5 and 14 ± 5 mV in PCs and ventricular myocytes (P = not significant), respectively.


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  		Fig. 4.   A and B: transient outward current (Ito) recorded with the voltage protocol in inset at 0.1 Hz in a PC and a VM, respectively. C: time constants of Ito inactivation. D: mean ± SE Ito density (measured from peak to end-pulse steady-state current) as a function of TP. E: sustained currents (Isus; measured from end-pulse to 0-current level) as a function of TP in PCs and VMs. F: voltage dependence of inactivation and activation in PCs and VMs. Curves are best-fit Bolzmann relations. * P < 0.05, ** P < 0.01, and *** P < 0.001, PCs vs. VMs in C-E.

An analysis of Ito recovery kinetics is shown in Fig. 5. Figure 5A shows Ito recordings during paired pulses from a PC and a ventricular myocyte during 100-ms conditioning pulses (P1) from a HP of -80 mV to +50 mV, followed by currents during test pulses (P2) with the same duration and voltage at varying P1-P2 intervals (paired pulse protocol delivered at 0.1 Hz in ventricular myocytes and 0.066 Hz in PCs). PC Ito recovery was extremely slow, whereas Ito recovery was much faster in the ventricular myocyte. Figure 5, B and C, shows means ± SE data for the time dependence of Ito reactivation in PCs (n = 4) and ventricular myocytes (n = 6), respectively. The ratio of Ito during P2 to that in P1 (I2/I1 ratio) was plotted as a function of the P1-P2 interval and fit with a biexponential function. Ventricular muscle Ito had a larger portion (69 ± 5%) of reactivation in the rapid phase with a time constant (tau 1) of 12 ± 1 ms, whereas PC Ito had a smaller rapid phase (38 ± 2% of total, P < 0.01) with a time constant of 31 ± 7 ms. The majority of Purkinje Ito recovery (62 ± 1%) proceeded very slowly, with a slow phase time constant (tau 2) of 1,575 ± 189 ms (n = 4). Ventricular Ito slow phase tau 2 averaged 217 ± 55 ms (n = 6, P < 0.001 vs. PC). Correspondingly, Purkinje Ito was much more frequency dependent than ventricular Ito (Fig. 5D).


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  		Fig. 5.   A: Ito recorded during a 100-ms pulse to +50 mV (P1) and during an identical subsequent pulse (P2) at varying P1-P2 intervals in a PC (left) and a VM (right). B: mean ± SE Ito recorded during P2 pulses (I2) normalized to current during the P1 pulse (I1) as a function of the P1-P2 interval in PCs. The recovery time course was fitted by a biexponential function as shown. Values provided for tau 1 and tau 2 are means for biexponential fits in four cells. C: data obtained from 6 VMs with the protocol illustrated in A and analyzed as shown in B. D: Ito frequency dependence as determined by %decrease of current during the 10th pulse relative to current during the 1st pulse of a train of 100-ms depolarizations to +50 mV at the frequencies shown. ** P < 0.01 and *** P < 0.001 for value in PCs vs. VMs at the same frequency.

Sensitivity to K+ channel blockers. Sensitivities to the K+ channel blockers tetraethylammonium (TEA) and 4-AP are characteristic properties of K+ currents (24, 25). Figure 6, A and B, shows recordings of Ito from a representative PC and a ventricular myocyte obtained on depolarization to +50 mV before and after exposure to a series of 4-AP concentrations. Whereas 500 µM 4-AP almost completely suppressed Ito in the PC, it caused <50% Ito inhibition in the ventricular myocyte. Figure 6C shows mean concentration-response data, along with best fit Hill equations. The 50% blocking concentration (IC50) for 4-AP at +50 mV was 56 ± 7 µM for Purkinje Ito (n = 4), significantly less than the IC50 of 511 ± 110 µM (n = 5, P < 0.01) for ventricular Ito. Hill coefficients averaged 1.9 ± 0.6 and 1.8 ± 0.6 (P = not significant), respectively.


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  		Fig. 6.   Recordings of PC (A) and ventricular muscle (B) Ito on depolarization to +50 mV before (CTL) and during exposure to a series of 4-aminopyridine (4-AP) concentrations. C: concentration-response curves for Ito inhibition by 4-AP, along with Hill equation fits (n = 4 PCs, 5 VMs). D: Ito recordings from a VM before and after exposure to 10 mM tetraethylammonium (TEA), respectively. E: Ito in the absence and presence of 10 mM TEA in a PC. E, inset: PC Ito density (n = 5) in the absence and presence of 10 mM TEA. * P < 0.05 vs. control. All drug effects were evaluated after 5 min of exposure at each concentration.

Figure 6, D and E, illustrates the response of a ventricular myocyte and a PC to 10 mM TEA. Ito in the ventricular myocyte was not appreciably different before compared with after TEA exposure (Fig. 6D). In four ventricular myocytes, TEA did not significantly affect Ito, with a density averaging 9.3 ± 1.3 and 9.1 ± 1.4 pA/pF (P not significant) at +50 mV, respectively, before and after TEA. In contrast, the same concentration of TEA strongly inhibited Purkinje Ito, as illustrated by currents from one PC recorded before and after TEA (Fig. 6E). Figure 6E, inset, shows mean Ito in five PCs before and after TEA, which caused a 67 ± 9% (P < 0.05) current reduction.

Sustained component. As mentioned in Transient Outward Current, we found a substantially larger sustained end-pulse current after Ito inactivation in human PCs than in ventricular myocytes. The end-pulse current in PCs was inhibited by 4-AP, with an IC50 of 54 ± 15 µM at +50 mV. For ventricular myocytes, 4-AP had no significant effect on end-pulse current, with a mean 4.6 ± 2.1% reduction at 1 mM. The end-pulse sustained component in PCs was also sensitive to TEA, which decreased Isus by 46 ± 5% (n = 5, P < 0.01) at 10 mM, in contrast to a 4.3 ± 1.7% decrease (P = not significant) in ventricular myocytes at the same concentration. In an attempt to dissociate Isus from Ito, we recorded Isus in additional cells with the use of 100-ms prepulses from -50 to +50 mV 10 ms before test depolarizations to suppress Ito, as previously used to study ultrarapid delayed rectifier current (IKur) in human atrial myocytes (11, 19, 37), followed by a repolarizing step to -40 mV to observe tail currents. Isus either activated very rapidly or was instantaneous (activation kinetics could not be resolved) and showed slow inactivation (Fig. 7, A and C) with no tail currents. 4-AP (50 µM) decreased the current modestly by 33 ± 8% (P = not significant) in five cells (Fig. 7B). TEA (10 mM) reduced the current (Fig. 7D) by 42 ± 9% (P < 0.05, n = 5).


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  		Fig. 7.   A and B: Purkinje Isus elicited by the protocol shown in A, inset, before and 5 min after exposure to 50 µM 4-AP. C and D: Purkinje Isus before and 5 min after 10 mM TEA. B and D, insets: mean data. NS, not significant.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have succeeded in isolating human cardiac PCs, in which we have characterized a variety of ionic currents, including IK1, IH, Ito, and Isus. These currents were compared with corresponding currents in human ventricular myocytes with some potentially important differences observed, particularly for Ito and Isus.

Comparison with Previous Studies of Corresponding Currents in PCs

Although the free-running Purkinje fiber false tendon preparation was widely used for classical voltage- clamp studies in multicellular preparations, much less work has been done in isolated PCs, largely because of the difficulty of isolating single PCs. Cordeiro et al. (6) found IK1 to be much smaller in rabbit PCs than in ventricular myocytes. In the present study, we found human PC IK1 to be substantial. PC IK1 density in our study was significantly smaller than that in ventricular myocytes at -120 mV, but was not significantly different from ventricular muscle IK1 density between -110 and 0 mV. Cordeiro et al. (6) found Ito to be inhibited by 4-AP, but the concentration dependence of inhibition was not characterized. We (14) previously evaluated in detail the properties of canine PC Ito. Like human PC Ito in the present study, canine PC Ito recovered more slowly than ventricular Ito, with a large, slowly recovering component. Canine PC Ito had time constants averaging 35 and 1,427 ms, quite similar to the values of 31 and 1,575 ms for human PC Ito in the present study. The 4-AP sensitivities of canine PC and ventricular Ito were also quite similar to those of human PC and ventricular Ito in the present study (14). Additional findings in canine hearts similar to those in the present study were the TEA sensitivity of canine PC Ito and the TEA insensitivity of canine ventricular Ito (14).

Callewaert et al. (4) studied pacemaker current in single sheep PCs and found that activation accelerated at more negative potentials. Properties of the IH we recorded in human PCs are similar to those described by Cellewaert et al. (4). Human PC IH had a rapid kinetic component that activated more rapidly and constituted a larger proportion of current activation at more negative potentials. We found human PC IH activation kinetics to be biexponential, whereas Callewaert (4) used a single time constant to characterize pacemaker current activation. The voltage dependence and time dependence of IH in human PCs would be consistent with a role in pacemaker function, although any such inferences must be very cautious in view of our inability to record action potentials from human PCs.

Comparison with Other Studies of K+ Currents in the Human Heart

We were unable to identify previous studies of ionic currents in human cardiac PCs with which to compare our results. Several studies have evaluated human ventricular myocyte Ito. Wettwer et al. (40) found Ito activation and inactivation voltage dependence in human ventricular muscle cells that was similar to what we observed. They obtained only a single recovery time constant (~20 ms), similar to the rapid phase time constant in our ventricular myocytes. Näbauer et al. (22) observed regional differences in human ventricular Ito, with Ito being smaller and recovering more slowly in subendocardial compared with subepicardial tissue. Time constants of Ito recovery were of the order of 10 and 900 ms for both tissue types, with the fast phase constituting 89% of recovery in subepicardium vs. 4% of subendocardium. Li et al. (18) characterized Ito in epicardial, midmyocardial, and endocardial regions of the human heart, reporting biexponential recovery with time constants averaging 12, 20, and 34 ms for fast phase recovery in epicardium, midmyocardium, and endocardium, respectively, and slow phase time constants of 229, 254, and 490 ms. Recovery was significantly slower in endocardium than in epicardium or midmyocardium, and midmyocardial values were of the same order as our observations for midmyocardial ventricular myocytes. Amos et al. (1) studied Ito in human atrial and ventricular subepicardial cells. They noted a recovery time constant of 24 ms for ventricular Ito. Neither atrial nor ventricular Ito were affected by 20 mM TEA (1). Cerbai et al. (5) recorded hyperpolarization-activated current (If) in ventricular myocytes from failing human hearts. They noted a density of ~2 pA/pF at -120 mV, about half of the value for IH in human PCs in the present study. If activation was found to be monoexponential, with a time constant that increased at more positive voltages, consistent with the voltage dependence of IH activation kinetics that we observed.

In the present study, we noted a larger Isus in human PCs compared with ventricular myocytes. Human atrial myocytes also have a larger Isus compared with ventricular myocytes (11). Several observations suggest that PC Isus has a different basis compared with atrial. Human atrial Isus shows tail currents, even at 37°C (12); we did not observe Isus tail currents in human PCs at either room temperature or 37°C. Human atrial Isus is insensitive to TEA (37), whereas Purkinje Isus was substantially inhibited by TEA. In fact, the response of PC end pulse current to 4-AP (IC50 54 ± 15 µM) and 10 mM TEA (46 ± 5% reduction) was quite similar to the effects on PC Ito of 4-AP (IC50 56 ± 7 µM) and 10 mM TEA (67 ± 9%), suggesting that PC Isus is a non- or slowly inactivating component of PC Ito rather than a distinct TEA-insensitive current like the IKur that underlies most of human atrial Isus (11, 19).

Potential Significance

PCs play an important role in a variety of cardiac arrhythmia mechanisms (2, 3, 7, 10, 23, 26, 35). They seem to be particularly significant in the generation of torsades de pointes arrhythmias associated with the long QT syndrome (2, 3, 10, 23), in which abnormalities in repolarization are central. It is therefore important to understand the properties of K+ currents that govern PC repolarization. The present study is, to our knowledge, the first voltage-clamp study of K+ currents in human cardiac PCs. We previously showed that canine cardiac PCs have Ito properties different from those of ventricular and atrial Ito (14). In the present study, we found that human PC Ito has very similar voltage-dependent, kinetic, and pharmacological sensitivity patterns to canine PC Ito, with clear differences from human ventricular Ito. Furthermore, the sensitivity of human PC Ito to TEA contrasts with the TEA insensitivity to concentrations as high as 100 mM (9, 29, 33) of the alpha -subunits (Kv1.4, Kv4.2, and Kv4.3) believed to underlie mammalian atrial and ventricular Ito (25, 33, 38). These results are compatible with a role for alpha -subunits other than Kv1.4 and Kv4.x in human PC Ito, such as TEA-sensitive Kv3-related subunits (14), which have been found to be expressed at the mRNA (15) and protein (unpublished data) levels in canine PCs.

If is believed to play an important role in pacemaker function of automatic cardiac tissues, like Purkinje fibers (8, 31). We found a robust current compatible with If in human cardiac PCs, consistent with PC function as cardiac escape pacemakers. We have referred to the current as IH rather than If, because due to the limited number of cells available we could not characterize the current in biophysical detail. Substantial IH was present at -70 mV, and measurable current was still present at -60 mV, compatible with the voltage range of pacemaker activity in PCs of other species. Shi et al. (31) have shown that rabbit Purkinje fibers are rich in mRNA corresponding to hyperpolarization-activated cation channel (HCN)1 and HCN4, which encode cyclic nucleotide-sensitive cation channels with distinctive properties that strongly resemble If (21). Because expression studies of HCN channels have generally been performed at room temperature (21), it is difficult to compare directly the kinetics and voltage dependence of human PC IH components with those of cloned channels. Further work in this area is of potentially great interest.

Potential Limitations

Isolation of PCs from human cardiac false tendons was very difficult, with a very small number of Ca2+-tolerant PCs available from each isolation. Action potentials could be recorded only rarely and had an abnormal, triangularized morphology. We were unable to record delayed rectifier K+ currents, known to be particularly sensitive to isolation technique (42). Of note, PCs are notoriously difficult to isolate even from normal animal hearts, and several studies have been unable to record significant IK from PCs (6, 27) isolated from the hearts of animals in which IK blockers are known to prolong Purkinje fiber action potential duration (20, 34). The currents we selected for study were large and had reproducible properties across cells. Ito, in particular, is known to be resistant to cell isolation (42).

Hearts from which PCs were isolated were clearly diseased and patients were taking a variety of medications that could have affected the results. These are well-recognized limitations of virtually all voltage- clamp studies of human ventricular myocytes in the literature, which have almost invariably been obtained from explanted recipient heart tissue obtained at the time of heart transplantation. The only exceptions are studies from countries in which explanted donor hearts are used for valve transplantation and cardiac tissue may be available for electrophysiological study (36), and studies of small (~5 mg) tissue cores obtained by myocardial biopsy (17, 39). Myocardial biopsy specimens are not useful for PC isolation. Despite the fact that the PCs we obtained were from diseased hearts, we were able to record large currents with analyzable properties compatible with those of corresponding currents previously studied in animal models. PC ionic currents are remodeled by congestive heart failure (13), with downregulation of both IK1 and Ito densities but no change in their other properties. We were careful to compare currents in PCs with those of ventricular myocytes from the same hearts; because comparable downregulation occurs in ventricular myocytes (28) and PCs (13), the comparisons should have at least qualitative validity. Nevertheless, our results must be considered in the context of the potential role of cardiac disease and cell isolation effects.

We identified PCs on the basis of their characteristic appearance and the fact that they were isolated from free-running false tendons. Previous studies (4, 6, 27) have used similar approaches. Unfortunately, there is no independent marker that typifies PCs with absolute certainty, requiring us (as well as previous investigators) to rely on these criteria. The characteristic features of human PC Ito we observed that are different from those of human ventricular Ito in the present study and from mammalian atrial or ventricular Ito in numerous previous studies make it unlikely that our PC population was contaminated by ventricular myocytes.

Unlike Cerbai et al. (5), we were unable to record If from ventricular cardiomyocytes of failing hearts. We are unable to account for this discrepancy in a direct way, although it may be related to differences in patient population and/or recording methods. We used Cd2+ to inhibit ICa in studies of Ito and Isus. Divalent cations like Cd2+ can produce shifts in current voltage dependence by neutralizing fixed negative surface charges, a phenomenon that must be kept in mind when considering our results.


    ACKNOWLEDGEMENTS

We thank Evelyn Landry for expert technical assistance and France Thériault and Annie Laprade for secretarial help with the manuscript.


    FOOTNOTES

This study was funded, in part, by the Canadian Institutes of Health Research and the Quebec Heart Foundation. W. Han was supported by a Heart and Stroke Foundation of Canada studentship and G. Schram is the recipient of a Canada Institutes of Health Research/Aventis fellowship.

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

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.

August 15, 2002;10.1152/ajpheart.00389.2002

Received 6 May 2002; accepted in final form 29 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 283(6):H2495-H2503
0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society


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