INTRODUCTION

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PURKINJE (P)-to-ventricular (V) conduction is an essential step in the cardiac excitation sequence. The P network is a thin layer of tissue that is coupled to the much thicker ventricular myocardium at numerous, discrete sites in the subendocardium that are termed Purkinje-ventricular junctions (PVJs) (28, 30, 39). Electrotonic interactions within this multidimensional syncytium contribute to asymmetric (19) and discontinuous (45) conduction at these PVJs. Asymmetric conduction refers to the directional difference in safety factor for propagation at the PVJ; this is manifested in unidirectional (P to V) block under both pathophysiological (12, 45) and physiological (26, 28) conditions. Unidirectional block is thought to occur because the large, less excitable V mass imposes an electrical load on the smaller, more excitable P layer (30). Purkinje-ventricular (PV) conduction is described as discontinuous because the conduction delay between the two regions is quite large (3-6 ms) (39, 41, 45), considering the relatively short distance (0.1-1.0 mm) traveled by the impulse (39). Sparse intercellular connections between the P and V regions form a high-resistance barrier (29, 39) that contributes to the discontinuity in PV conduction (19). Although this barrier may benefit P-to-V conduction by shielding the P layer from the electrical load imposed by the V mass (17, 19, 30), ischemic conditions increase the resistance of the barrier, thereby uncoupling the P and V regions (41). This may produce triggered arrhythmias secondary to prolonged P action potentials (22) or reentry due to the combined presence of unidirectional block and slow conduction (13, 32).

Although unidirectional block at the PVJ has traditionally been attributed to these electrotonic interactions, intrinsic differences between P and V myocytes may also underlie directional differences in conduction at the PVJ. For example, P myocytes are typically larger than V myocytes (8). Joyner et al. (18) showed that conduction from a large V cell to a small V cell succeeded at higher values of junctional resistance (R_j) than conduction from a small V cell to a large V cell. Early partial repolarization of the stimulated, or source, cell limited the R_j that could be imposed before conduction failed, i.e., the critical R_j. This repolarization, termed source loading, was due to the electrical load imposed by the nonstimulated, or sink, cell. Ionic mechanisms that may induce directional differences in conduction include P and V differences in the inward rectifier current (I_K1), Na⁺ channel density, the L-type calcium current (I_Ca), and the transient outward current (I_to). I_K1 is smaller in P myocytes than in V myocytes, resulting in a larger diastolic membrane resistance (R_m) in P myocytes (7). This larger R_m should yield a smaller current threshold for P myocytes. Additionally, P myocytes have a greater density of Na⁺ channels (10) and, therefore, a faster maximum upstroke velocity (_max) than V myocytes (8, 44). In computer simulations of conduction between regions of differing excitability separated by a resistive barrier, unidirectional block occurred in the direction of high to low excitability (19). Intrinsic P and V differences during early repolarization may also influence the success of PV conduction. Blocking I_Ca decreased the critical R_j for conduction between electrically coupled V myocytes by reducing the source available for conduction (37). Additionally, in canine papillary muscle preparations that included PVJs, blocking I_Ca increased P-to-V conduction delay, whereas enhancing I_Ca decreased P-to-V delay (45). I_to is responsible for the early, rapid repolarization during phase 1 of the action potential. Because cellular differences in I_to cause greater phase 1 repolarization in P than in V myocytes (1, 6, 8, 9), P-to-V and V-to-P conduction may be differentially modulated by I_to.

Because electrotonic interactions within the multidimensional syncytium make it difficult to assess how these intrinsic cellular properties influence PV conduction in intact preparations, we used a two-myocyte experimental system (38). Because this system does not require functional gap junctions, it was straightforward to systematically vary R_j between the cells and measure conduction delay and early partial repolarization during P-to-V and V-to-P conduction. Companion numerical simulations provided insight into how differences in intrinsic phase 1 repolarization influenced P-to-V conduction. Our results demonstrate that, even in a two-myocyte system, V-to-P conduction is highly favored over P-to-V conduction. The directional difference in critical R_j likely resulted from differences in diastolic R_m and intrinsic phase 1 repolarization.

	MATERIALS AND METHODS

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Cell isolation. Single P and V myocytes were isolated from rabbit hearts using previously published techniques (7, 33). Adult, male rabbits weighing 2.0-3.0 kg were anesthetized with 1 ml/kg pentobarbital sodium and 0.5 ml heparin to prevent clotting. After rapid isolation of the heart, the aorta was cannulated for Langendorff perfusion. The heart was then perfused for 6-8 min with nominally Ca²⁺-free Tyrode solution, followed by 18 min with enzyme solution containing 0.1 mM Ca²⁺, and 5 min of enzyme washout with 0.1 mM Ca²⁺ Tyrode solution containing no enzymes.

Free-running P fibers were dissected from both ventricles, put into a small bath containing enzyme solution, and agitated with a stream of 100% O₂. The temperature was maintained at 37°C. Single P myocytes were periodically removed from the bath and stored in 0.1 mM Ca²⁺ solution, and enzyme solution was added to the remaining P fibers in the bath to maintain a 2-ml volume. Cell dissociation required 15-60 min under these conditions. After the P fibers were dissected from both ventricles, the left ventricle was minced and gently agitated for 6-10 min in 0.1 mM Ca²⁺ Tyrode solution. The isolated cells were stored at room temperature in 0.1 mM Ca²⁺ Tyrode solution until use.

Solutions. Nominally Ca²⁺-free Tyrode solution contained (in mM) 126 NaCl, 5.4 KCl, 5.0 MgCl₂, 22 glucose, 1.0 NaH₂PO₄, 20 taurine, 5 creatine, 5 sodium pyruvate, and 24 N-2-hydroxyethylpiperazine-N '-2-ethanesulfonic acid (HEPES), with pH adjusted to 7.4 with NaOH. The enzyme solution had the same composition, except it also contained 1 mg/ml collagenase (type II, Worthington Biochemical, Freehold, NJ), 0.1 mg/ml protease (type XIV, Sigma Chemical, St. Louis, MO), and 0.1 mM CaCl₂.

The normal bathing solution during the experiments contained (in mM) 126 NaCl, 5.4 KCl, 1.0 MgCl₂, 1.0 CaCl₂, 11 glucose, and 24 HEPES, titrated with 13.0 mM NaOH (pH 7.4). Two pipette filling solutions were used: the ethylene glycol-bis(-aminoethyl ether)-N,N,N ',N '-tetraacetic acid (EGTA) filling solution and the normal filling solution. The EGTA filling solution contained (in mM) 15 NaCl, 30 KCl, 1.0 MgCl₂, 5.0 HEPES, 10 EGTA, 5.0 K₂ATP, and 90 potassium aspartate, with pH adjusted to 7.2 with KOH. The potassium aspartate caused a liquid junction potential (10 mV) (9), and results obtained using the EGTA filling solution were corrected by this amount. The normal filling solution contained (in mM) 113 KCl, 10 NaCl, 5.5 glucose, 5.0 K₂ATP, 0.5 MgCl₂, 10 HEPES, and 11 KOH (pH 7.2).

Electrical recordings. P and V myocytes were placed in a glass-bottom, temperature-controlled bath (36°C) and continuously bathed with the normal solution at a rate of 1-2 ml/min. Only quiescent, rod-shaped cells were studied. Transmembrane potentials were recorded with an Axoclamp 2B amplifier system (Axon Instruments, Foster City, CA). Suction pipettes were made from borosilicate glass (no. 7052, OD 1.65 mm, ID 1.20 mm, A-M Systems, Everett, WA), and after they were fire polished and filled, they had resistances of 3-6 M. Pipette series resistance was carefully compensated before cell attachment. Pipette capacitance was minimized by maintaining a low level (1 mm) of solution in the bath. Action potentials were initiated with intracellular current injection (cycle length = 1 or 2 s). The stimulus duration was 3 ms, and the current stimulus magnitude (I_stim) was just sufficient to establish 1:1 pacing during the coupling experiments. The P transmembrane voltage (V_m,p) and the V transmembrane voltage (V_m,v) were filtered at 1 kHz and digitized at 4 kHz with a 12-bit analog-to-digital converter (Digidata 1200A, Axon Instruments) and recorded with a computer using pCLAMP 6 software (Axon Instruments) for the coupling experiments. In additional experiments, current thresholds were measured in single P and V myocytes with 3-ms (I_th,3) and 30-ms (I_th,30) pulses to compare how P and V current thresholds changed with pulse duration. Current threshold was defined as the smallest current needed to initiate an action potential and, therefore, represented a measure of excitability in the uncoupled cell. These action potentials were filtered at 5 kHz and digitized at 10 kHz to examine the effect of sampling rate on the action potential upstroke.

Electrical coupling of the two myocytes was achieved using the electronic circuit first described by Tan and Joyner (38). Briefly, two amplifiers with variable gain computed the membrane voltage differences, (V_m,p  V_m,v) and (V_m,v  V_m,p). That output was sent to voltage-to-current convertors with fixed gain to simultaneously supply a current of (V_m,p  V_m,v)/R_j to the V myocyte and (V_m,v  V_m,p)/R_j to the P myocyte. We defined this coupling current, I_c, as positive when it flowed into the stimulated cell, either via an external stimulus or from the nonstimulated cell, and negative when it flowed from the stimulated cell to the nonstimulated cell. R_j was determined by the gains of the convertors and amplifiers and could be varied from 0 to 1,000 M in our system.

Our procedure for studying conduction between P and V myocytes was to first establish pipette attachments in both cells. Before coupling, both cells were stimulated, and the intrinsic action potentials were recorded for each cell. The cells were then electrically coupled with an R_j of ~50 M. Pacing stimuli were delivered only to the P myocyte to elicit P-to-V conduction. To determine the critical R_j for P-to-V conduction, R_j was stepwise increased until conduction failed. To elicit V-to-P conduction, pacing stimuli were delivered only to the V myocyte. We determined the critical R_j for V-to-P conduction by stepwise increasing R_j until conduction failed. Note that because there was an upper bound on the R_j that could be imposed between the cells, we could not measure critical R_j above 1,000 M. Therefore, for conduction that did not fail before the upper bound was reached, we considered the critical R_j equal to 1,000 M.

Diastolic membrane resistance (R_m, M) and capacitance (C_m, pF) were determined using intracellular injection of small hyperpolarizing, constant-current pulses (I = 0.1-0.5 nA, 100-ms duration) to elicit small changes in the transmembrane potential (V_m = 3-11 mV) of uncoupled cells. R_m was calculated as V_m/I, and C_m was calculated as /R_m, where  was the membrane time constant determined with an exponential fit. Using Clampfit 6 software (Axon Instruments), we differentiated V_m with respect to time and identified the maximum derivative as the maximum upstroke velocity, _max. We defined the time of activation as the time of _max. Reported conduction delays reflected the difference in activation times between coupled cells. All tabulated data are presented as means ± SD. Statistical analysis included analysis of variance with repeated measures using the SPSS package (SPSS, Chicago, IL).

Computer simulations. To model the coupled cells, we used the DiFrancesco-Noble (DN) membrane equations (4) to describe the ionic currents for a single P cell and the Luo-Rudy (LRd) membrane equations (24, 46) to describe the ionic currents for a single V cell. Action potentials were calculated by numerically solving the following equations

(1)

(2)

where R_j was the junctional resistance (M), C_m was the membrane capacitance (µF/cm²), I_ion,p and I_ion,v were total ionic current in the P and the V myocyte, respectively (µA/cm²), and k (1.26 × 10⁴ cm²) depended on the surface-to-volume ratio and the spatial increment. I_c flowing from the model P cell to the model V cell was simply equal to (V_m,v  V_m,p)/R_j from Eq. 1. For V-to-P current flow, I_c was equal to (V_m,p  V_m,v)/R_j from Eq. 2. All simulations were performed on a Sun Microsystems SPARC4 workstation. Solution times were typically 2 min.

Parameter scaling for _max. Because the current that a source cell may provide to a sink cell is related to _max, we wanted to ensure that the ratio of _max measured in the P myocyte to _max measured in the V myocyte (_max,p/_max,v) was maintained in the simulations. Therefore, we used the well-known relationship between the maximum sodium conductance (_Na) and _max, and we increased _Na in the DN model from 11.2 to 12.7 mS/µF to obtain a concomitant increase in _max from 177 to 200 V/s. In the LRd model, we decreased _Na from 16.0 to 6.35 mS/µF, which reduced _max from 365 to 135 V/s. We chose these values to reflect average _max values measured at the 4-kHz sampling rate in the uncoupled P and V myocyte pairs.

Parameter scaling for I_to and I_Ca. Both the DN and LRd models include formulations of I_Ca, but only the DN model has a formulation of I_to. To achieve different plateau potentials for the model P cell, we varied either I_to or I_Ca of the model P cell. This allowed us to determine relative effects on critical R_j, early partial repolarization, and conduction delay during P-to-V conduction.

	RESULTS

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Cell size and excitability. Conduction was studied in a total of eight cell pairs, four Purkinje-ventricular (PV) myocyte pairs and, for comparison, four ventricular-ventricular (VV') myocyte pairs. Ten additional uncoupled P and V myocytes were used to compare the effect of short and long duration pulses on current threshold and to examine the effect of sampling rate on _max. Table 1 summarizes diastolic membrane resistance, membrane capacitance, and resting potential measured in uncoupled myocytes from the PV and VV' pairs. The mean R_m in the P myocyte (R_m,p) was 3.4 times greater than the mean R_m in the V myocyte (R_m,v) (98.0 vs. 28.9 M, respectively). Because this largely reflects the much smaller I_K1 in rabbit P myocytes (7), we used C_m instead of R_m as a measure of cell size. In two of the four PV myocyte pairs, the P myocyte was larger than the V myocyte. On average, the P myocytes were 16% larger than the V myocytes (67.1 vs. 57.7 pF, respectively). For each VV' myocyte pair, the data presented in Table 1 are ordered by membrane capacitance, such that V denotes the larger myocyte and V' the smaller myocyte. The mean ratio of C_m in the V myocyte to C_m in the V' myocyte (C_m,v/C_m,v') was 1.6 (58.5 pF/36.2 pF). The mean V_rest values were consistent with reported values for resting potential in the literature (5, 8).

Tables 2 and 3 present values of the stimulus current used during conduction in PV and VV' myocyte pairs, current thresholds assessed with 3-ms and 30-ms pulses in uncoupled P and V myocytes, and maximum upstroke velocity measured in uncoupled myocytes at 4- and 10-kHz sampling rates. On average, I_stim in the V myocyte (I_stim,v) required during V-to-P conduction was 60% higher than I_stim in the P myocyte (I_stim,p) during P-to-V conduction (1.6 vs. 1.0 nA, respectively). This was consistent with higher current thresholds in uncoupled V myocytes than in P myocytes. With 3-ms-duration pulses, V myocytes required 2.7 times (0.84 vs. 0.31 nA) more current than P myocytes to initiate an action potential. Long-duration pulses, representative of long conduction delays, revealed an even larger excitability difference, as I_th,30 was 8.75 times (0.35 vs. 0.04 nA, respectively) higher in the V myocytes. _max,p averaged 1.47 times higher than _max,v (200.2 vs. 135.9 V/s, respectively) in the PV myocyte pairs when sampled at 4 kHz. Action potentials digitized at 10 kHz had faster upstrokes, but neither the timing of _max nor the ratio _max,p/_max,v were significantly affected by the sampling rate. In the VV' myocyte pairs, the mean values of I_stim,v and I_stim in the V' myocyte (I_stim,v') were not significantly different. Similarly, there was no significant difference between _max,v and _max,v'.

P-to-V and V-to-P conduction. Figure 1 shows action potentials and coupling currents recorded at R_j = 100 M from a PV myocyte pair (P3-V3) during P-to-V and V-to-P conduction. In Fig. 1, each panel depicts the complete record (either V_m or I_c) in insets at left and the initial portion of the record (encompassing the upstroke and early repolarization) in an expanded timescale at right. During P-to-V conduction (Fig. 1, top left), the P action potential demonstrated a spike-and-dome configuration (25). The P upstroke was followed by a large, early partial repolarization of 71.4 mV, forming the spike (open arrow). On activation of the V myocyte, the large, secondary depolarization of the P myocyte formed the dome (closed arrow). P-to-V conduction delay measured 9.3 ms. During V-to-P conduction (Fig. 1, top right), there was no measurable early partial repolarization of the V myocyte because conduction was completed during the upstroke rather than after the action potential peak. V-to-P delay measured only 1.3 ms. The directional difference in conduction delay was due largely to the difference in diastolic R_m between the P and V myocytes. R_m,p measured 107.0 M, whereas R_m,v measured 41.0 M. As a result, the stimulus current required for activation was smaller for the P myocyte (1.0 nA) than for the V myocyte (1.5 nA). Because the P myocyte reached threshold faster than the V myocyte, V-to-P delay was shorter than P-to-V delay.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Purkinje (P, solid line) and ventricular (V, dashed line) action potentials (top) and coupling currents (bottom) recorded from PV myocyte pair P3-V3 at junctional resistance (Rj) = 100 M. For each panel, insets at left depict complete record, and initial portion of record is shown in expanded timescale at right. Top left: P-to-V conduction after P cell stimulation. Open arrow points to spike; closed arrow points to dome. Top right: V-to-P conduction after V cell stimulation. Bottom left: stimulus + coupling current during P-to-V conduction. For descriptions of a-d, see RESULTS. Bottom right: stimulus + coupling current during V-to-P conduction. For descriptions of a-e, see RESULTS.

The coupling current I_c supplied from the pipettes to the myocytes by the coupling circuit was larger during P-to-V (Fig. 1, bottom left) than V-to-P (Fig. 1, bottom right) conduction. The initial positive deflection, marked a at Fig. 1, bottom left, shows the stimulus applied to the P myocyte. As the V myocyte loaded the P myocyte during P-to-V conduction, I_c rapidly decreased to 1.0 nA (b). This outward current contributed to the large, early partial repolarization of the P action potential. I_c became less negative as V_m,v slowly approached threshold. When the V myocyte fired an action potential and V_m,v rose above V_m,p, the coupling current became positive as the V myocyte supplied current to the P myocyte (c). This formed the dome of the P action potential. As shown in the inset, I_c reversed direction again (d) as V_m,v fell below V_m,p during late repolarization. In Fig. 1, bottom right, the initial positive deflection, marked a, shows the stimulus applied to the V myocyte to initiate V-to-P conduction. The P myocyte exerted a smaller electrical load upon the V myocyte, as I_c fell to 0.7 nA (b). I_c was positive briefly (c) when the P activation caused the notch in the upstroke of the V action potential, then became negative again (d) as V_m,v remained higher than V_m,p during the plateau. As shown in the inset, I_c reversed direction (e) during late repolarization, as V_m,v fell below V_m,p.

Results from computer simulations (not shown) were consistent with the experimental results, with directional differences in the amount of early partial repolarization, the conduction delay, and the magnitude of the coupling current. During P-to-V conduction, there was 40.6 mV of early partial repolarization of the model P cell. By comparison, there was no measurable early partial repolarization of the model V cell during V-to-P conduction. P-to-V conduction delay was 6.9 ms, and V-to-P conduction delay was 3.8 ms. Peak I_c was 1.8 nA during P-to-V conduction and 1.3 nA during V-to-P conduction.

Conduction at the critical R_j. To determine whether conduction in PV myocyte pairs demonstrated unidirectional block, we systematically varied the imposed junctional resistance to identify the critical R_j values for P-to-V and V-to-P conduction. Figure 2 shows action potentials recorded at the critical R_j from myocyte pair P3-V3 and calculated at the critical R_j during simulations. During P-to-V conduction at R_j = 110 M in the myocyte pair (Fig. 2, top left), the P action potential again had a spike-and-dome configuration. However, the amount of early partial repolarization increased to 81.9 mV, and P-to-V conduction delay increased to 15.6 ms, with only a 10-M increase in R_j. V-to-P conduction at R_j = 1,000 M (Fig. 2, top right) was slower than at R_j = 100 M. Thus, the P myocyte imposed a load on the V myocyte after the upstroke, and early partial repolarization of the V action potential measured 8.4 mV. Activation of the P myocyte formed a notch in the V action potential as a slight secondary depolarization interrupted early repolarization. V-to-P conduction delay measured 17.6 ms. Though the P myocyte was approximately twice as large as the V myocyte (76.7 vs. 38.9 pF), the directional differences in early partial repolarization and critical R_j indicated that conduction was favored in the V-to-P direction.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   P and V action potentials recorded from myocyte pair P3-V3 at critical Rj (top) and calculated at critical Rj in simulations for P-to-V and V-to-P conduction (bottom). Top left: P-to-V conduction in myocytes. Cells were coupled at Rj = 110 M. Top right: V-to-P conduction. Cells were coupled at Rj = 1,000 M. Bottom left: P-to-V conduction in simulations. Model cells were coupled at Rj = 142 M. Bottom right: V-to-P conduction in simulations. Model cells were coupled at Rj = 3,590 M.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Conduction in a VV' myocyte pair. Solid line shows V action potential; dashed line shows V' action potential. Top left: V-to-V' conduction after V stimulation. Myocytes were coupled at critical Rj for V-to-V' conduction (250 M). Top right: V'-to-V conduction after V' stimulation. Myocytes were coupled at critical Rj for V'-to-V conduction (110 M). Bottom left: V-to-V' conduction coupled at Rj = 100 M. Bottom right: V'-to-V conduction coupled at Rj = 100 M.

I_to, I_Ca, and source loading. We next considered the contribution of intrinsic phase 1 repolarization to the total early partial repolarization during conduction at the critical R_j. Figure 4 shows action potentials recorded from myocyte pair P3-V3 before and after coupling at the critical R_j. Recall that early partial repolarization was 81.9 mV in the coupled P myocyte during P-to-V conduction (Fig. 2). During this same period, V_m,p fell by 72.0 mV in the uncoupled P myocyte (Fig. 4, left), demonstrating that intrinsic phase 1 repolarization accounted for much of the partial repolarization observed during P-to-V conduction at the critical R_j. By comparison, the uncoupled V action potential (Fig. 4, right) demonstrated much less intrinsic phase 1 repolarization. During the time of conduction, V_m,v fell by only 4.7 mV in the uncoupled myocyte.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Action potentials recorded from myocyte pair P3-V3. Left: action potentials from isolated P myocyte before (P, uncoupled) and after coupling (P, coupled). The P action potential after coupling is same P trace as in Fig. 2, top left. Right: action potentials from isolated V myocyte before (V, uncoupled) and after coupling (V, coupled). The V action potential after coupling is the same V trace as in Fig. 2, top right.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Effect of changing the magnitude of transient outward current (Ito) or calcium current (ICa) on critical Rj for P-to-V conduction in simulations. , Values of critical Rj for different ICa; , values of critical Rj for different Ito. Intersection of Ito and ICa curves marks critical Rj for normal values of Ito and ICa.

	DISCUSSION

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Previous investigators have demonstrated unidirectional block in pairs of isolated V myocytes that were electrically coupled by a variable resistance (18). The critical R_j at which conduction failed in those ventricular cell pairs was a function of the cell sizes and could be modified by altering I_Ca in the source cell (18, 21, 37). Our approach is unique in that we couple P and V myocytes together. Because these cells and their ionic currents differ fundamentally, the unidirectional (P to V) block that occurred over a wide range of R_j values could not be ascribed simply to differences in cell size. In fact, mean C_m,p was not significantly different from mean C_m,v, although the mean values of critical R_j for P-to-V and V-to-P conduction were significantly different. Additionally, we found that directional differences in early partial repolarization and the critical R_j for conduction between a P myocyte and a V myocyte were greater than directional differences between two V myocytes. Our results suggest that this difference is related to cellular differences in I_to, I_Ca, and diastolic R_m.

In general, the success of conduction depends on three factors: 1) the ionic current generated by a source cell, 2) the current required by a sink cell, and 3) the resistive pathway between the source and the sink. During conduction in normal ventricular muscle or within the P network, there is minimal conduction delay between neighboring regions. Therefore, the source is provided by the Na⁺ current (35, 36). However, conduction through the PVJ is discontinuous, and physiological conduction delays are 3-6 ms (39, 41, 45), whereas pathophysiological conditions and cellular uncouplers can extend this delay an additional 5-10 ms (16, 26, 42). Thus the source depends not only on the current generated during the upstroke but also on the current generated during the early plateau (18). Because normal conduction across the PVJ occurs from the P network to the myocardium (P to V), we examined how the dominant early plateau currents of P myocytes, I_Ca and I_to, influenced P-to-V conduction. Although our model results predicted that the critical R_j for P-to-V conduction was more sensitive to changes in I_to than in I_Ca, the relative influences of I_to and I_Ca on unidirectional block at the PVJ will depend on their relative current densities. In canine P myocytes, peak I_to density measured 20 pA/pF (15) and peak I_Ca density measured 6.0 pA/pF (3), whereas in canine V myocytes, peak I_to density measured 4.9 pA/pF (23) and peak I_Ca density measured 15 pA/pF (40). We know of no published data on the density of I_to and I_Ca in isolated rabbit P myocytes; however, in rabbit V myocytes, peak I_to density measured 3.69 pA/pF (9), whereas peak I_Ca density measured 15 pA/pF (14). These intrinsic differences in peak I_to and I_Ca densities likely contribute to unidirectional block at the PVJ by ensuring greater phase 1 repolarization in P myocytes, subsequently inducing large directional differences in early partial repolarization and current source for conduction.

The success of conduction also depends on the excitability of the sink, which is largely determined by diastolic input resistance (R_in). During P-to-V conduction in intact tissue preparations, the input resistance of the sink depends primarily on the size, intercellular coupling, and membrane excitability of the ventricular mass. In isolated P and V myocytes, however, the largest fraction of R_in is provided by the diastolic R_m, which is determined mainly by the conductance of I_K1 (11, 27). I_K1 helps maintain the resting potential, but because of its marked rectification at depolarized potentials, it is small during the action potential. We have recently found that rabbit I_K1 current density is two to three times smaller in P than V myocytes (7). This accounts for the higher R_m in P myocytes (98.0 M) than in V myocytes (28.9 M) and contributes to the lower current threshold for P myocytes (I_th,3 = 0.31 nA) than V myocytes (I_th,3 = 0.84 nA). Furthermore, we assessed the excitability of these myocytes with respect to discontinuous conduction by measuring the current threshold with long-duration pulses. I_th,30 was 8.75 times higher in the V myocytes; this difference was statistically significant (P = 0.002). Thus a V myocyte acted as a larger sink during P-to-V conduction than a P myocyte acted during V-to-P conduction, largely contributing to the nearly 11-fold difference between critical R_j for P-to-V and V-to-P conduction.

By using the coupling-clamp circuit of Tan and Joyner (38), we were able not only to evaluate the source in terms of intrinsic phase 1 repolarization and the sink in terms of excitability, but we were also able to vary the resistive pathway (R_j) between the source and the sink. Low gap junction density at PVJs (29) is consistent with the well-known electrical isolation of P fibers from the underlying V muscle. Thus, we deliberately imposed high values of R_j between the myocytes to yield large conduction delays, thereby modeling discontinuous conduction at the PVJ (25, 26).

It is important to evaluate our findings with respect to certain limitations of the experimental protocol and the modeling study. We used P myocytes isolated from free-running strands as a model for the subendocardial P myocytes that occur at PVJs. Locating PVJs requires extensive mapping of the subendocardium (41), whereas locating the free-running strands required only visual identification. We felt this approach was justified because a previous comparison of the two cell types in dog hearts revealed close similarity in structure, input resistance, and action potential configuration (2).

Another concern was that the high value of critical R_j for V-to-P conduction reflected pacemaker activity in the P myocyte rather than successful V-to-P conduction. Whereas the pattern of ventricular activation during P-to-V conduction at the critical R_j showed a rapid rise of potential followed by a slow rise to threshold, the P myocyte had a very slow, nearly linear rise in potential during V-to-P conduction at the critical R_j (top panels of Fig. 2). However, single P myocytes isolated from a variety of species including dog, sheep, cow, and rabbit generally do not display phase 4 depolarization and automaticity (5, 8, 31, 33, 34). These cells have stable resting potentials between 80 and 90 mV. For isolated cells with resting potentials above 75 mV, infrequent spontaneous activity consisting of transient depolarization occasionally reached threshold (5, 31, 33). In the present study, cell P4 had a resting potential of 70.9 mV (Table 1). However, we did not observe automaticity in this cell or any of the cells included in this study. Moreover, we would expect that if an underlying pacemaker conductance were to artificially raise the critical R_j for V-to-P conduction, we would see the largest critical R_j in cell pair P4-V4 because the elevated resting potential might predispose the P myocyte to automaticity. In fact, this cell pair demonstrated the lowest critical R_j of the four PV cell pairs. Thus P activation at R_j = 1,000 M likely represents V-to-P conduction rather than pacemaker activity. A related limitation of the experimental procedure was that critical R_j measurements were bounded at 1,000 M. In three of the four PV myocyte pairs, V-to-P conduction succeeded at R_j = 1,000 M and may have succeeded at higher R_j values. Thus the directional difference in critical R_j is likely larger than the difference documented here.

The 4-kHz sampling rate used in this study underestimated _max. Under identical conditions except for a 10-kHz sampling rate, mean _max was 2.6 times higher in single P myocytes and 2.9 times higher in single V myocytes. However, the sampling rate affected only the magnitude rather than the timing of _max, so we were able to use _max measured with the 4-kHz sampling rate to determine activation times and, thus, the conduction delay between cells.

In the simulations, we assumed that _max,p/_max,v was equal to that measured in the uncoupled myocytes (1.47) with a 4-kHz sampling rate. Altering _Na and, therefore _max, in the models to reflect _max,p/_max,v measured with the 10-kHz sampling rate (1.32) in the myocytes subsequently changed the critical R_j and early partial repolarization values calculated during conduction. However, because the sampling rate affected the magnitudes rather than the ratio of _max, the directional differences in critical R_j and early partial repolarization were not significantly affected in the simulations. For _max,p/_max,v = 1.47, the directional difference in critical R_j was 25.3 (3,590 M/142 M) and in early partial repolarization was 6.1 (77.1 mV/12.7 mV). For _max,p/_max,v = 1.32, the directional difference in critical R_j was also 25.3 (2,820 M/111 M) and in early partial repolarization was 6.0 (71.4 mV/11.8 mV). Another limitation of the modeling studies included the membrane equations chosen to represent P and V action potentials. Neither was developed from isolated rabbit heart cell data, and this may well explain quantitative differences between experimental recordings and the simulated action potentials. However, our primary objective with the simulations was to represent qualitative differences between P and V cells in the upstroke and the early plateau of the action potentials. The most marked difference was that in the magnitude of I_to and the resulting phase 1 repolarization; the DN membrane equations included a formulation of I_to, whereas the LRd equations did not. The resulting difference in plateau potentials allowed us to relate intrinsic phase 1 repolarization to the critical R_j for P-to-V conduction.

In normal subendocardial preparations, the safety factor for P-to-V conduction varies among the PVJs (26). This may be explained by inherent variation in the structure of each PVJ (26, 39) or in the source current for P-to-V conduction at different PVJs. For example, there are large variations (0.1-1.0 mm) in the distance spanned by PVJs because the strands connecting the P and V regions vary in length (39). Because R_j depends on the intercellular connections within the strands, this may, in part, explain variations in the P-to-V delay at different PVJs in the same preparation. Additionally, Verkerk et al. (43) observed two types of action potentials in single P myocytes, one with large phase 1 repolarization and a relatively negative plateau and another with little phase 1 repolarization and a more positive plateau. These observed differences in action potential configuration were due to differences in I_to and I_Ca density. Results from the present study suggest heterogeneity in intrinsic phase 1 repolarization will cause variation in safety factor for P-to-V conduction. Under ischemic conditions, the number of functional gap junctions is reduced (20, 29), and, therefore, R_j is increased. Because the critical R_j will vary from junction to junction, this increase in R_j will likely induce unidirectional block at some junctions, but not others. If conduction over the return pathway through the myocardium is slow enough to allow recovery at the sites of block, that impulse may excite the P network retrogradely, initiating circus movement reentry.