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Cardiovascular Research 2003 59(3):620-627; doi:10.1016/S0008-6363(03)00507-8
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Copyright © 2003, European Society of Cardiology

Spontaneous activity induced in rabbit Purkinje myocytes during coupling to a depolarized model cell

Delilah J. Huelsinga, Kenneth W. Spitzerb and Andrew E. Pollarda,*

aCardiac Rhythm Management Lab, Department of Biomedical Engineering, University of Alabama-Birmingham, Volker Hall B140, 1670 University Blvd., Birmingham, AL 35294, USA
bNora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah School of Medicine, Salt Lake City, UT 84112, USA

pollard{at}crml.uab.edu

* Corresponding author. Tel.: +1-205-975-4710; fax: +1-205-975-4720.

Received 20 December 2002; accepted 20 June 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The development of an "injury current" secondary to heterogeneous ion accumulation and cellular uncoupling across the ischemic border zone has been implicated as a trigger for arrhythmias arising during acute ischemia. The purpose of the present study was to determine the effects of injury current across the Purkinje–ventricular interface in the development of abnormal automaticity. Methods: Patch clamp and electronic cell coupling techniques were used to record action potentials from and to apply injury current to isolated rabbit Purkinje myocytes. Injury current was dependent upon: (1) a coupling resistance, which was varied to simulate different degrees of cellular uncoupling, and (2) the difference in Purkinje membrane potential and depolarized ischemic myocardium, which was represented by a passive resistor–capacitor circuit with initial voltages of –70, –60, or –50 mV. Results: During coupling to the moderately depolarized cell (–60 or –50 mV), Purkinje myocytes developed repetitive, spontaneous activity within a window of coupling resistances. This abnormal automaticity was dependent upon L-type calcium current, as cadmium or nifedipine completely suppressed coupling-induced spontaneous activity. Conclusions: Our results demonstrate that injury current alone can induce spontaneous activity in normal Purkinje myocytes. The level of myocardial depolarization and the degree of cellular uncoupling required to induce this activity suggest spontaneous Purkinje activity induced by injury current as a potent trigger for acute ischemic arrhythmias.

KEYWORDS Arrhythmia (mechanisms); Calcium current; Cell communication; Gap junctions; Ischemia


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Acute myocardial ischemia is accompanied by a myriad of changes in ionic and metabolic components that, in turn, alter the action potential [1,2]. Such alterations include depolarization of the rest potential (Vrest), altered refractoriness, and increased gap junctional resistance (Rj). These electrophysiologic changes evolve both temporally and spatially such that a border zone develops between ischemic and nonischemic myocytes within minutes, providing the anatomic substrate and the electrophysiologic trigger for arrhythmias. Importantly, the heterogeneous nature of these changes is critical to the development of "injury current" flow from ischemic to nonischemic myocytes, which has been suggested as a mechanism for acute ischemic arrhythmias [3–5].

One useful methodology for studying the consequences of cellular uncoupling and heterogeneous electrophysiologic changes across the ischemic border is the cell coupling approach. Using this paradigm, isolated myocytes can be electrically connected with a variable Rj to model cells, whose "membrane properties" and "rest potential" can be altered to model various stages of injury. Previous investigators have electronically coupled real myocytes to depolarized, passive model cells to demonstrate arrhythmogenic activity at the cellular level. This previous work has primarily focused on the initiation or suppression of afterdepolarizations by injury current in myocytes exposed to catecholamines [6–8]. In most cases, relatively large magnitudes of injury current, attained by coupling the myocyte to the model cell with Vrest set to values of –30 mV and above, were required to induce afterdepolarizations.

While similar in methodology, the present study distinguishes itself in focus. We hypothesized that injury current arising at the ischemic border from moderately depolarized, passive cells could induce spontaneous activity in addition to afterdepolarizations. To examine the conditions for the genesis of spontaneous activity at the ischemic border, we used the cell coupling approach to represent the subendocardial border zone, in which nonischemic Purkinje myocytes are weakly coupled to ischemic, depolarized ventricular myocytes.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Myocyte isolation
Single Purkinje myocytes and Purkinje aggregates were isolated from the left ventricle of adult rabbit hearts, as previously described [7]. Animal care and treatment conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Isolated myocytes were stored until use later that day in a high potassium storage solution that contained (in mM): potassium glutamate 100, potassium aspartate 10, KCl 25, KH2PO4 10, MgSO4 2, taurine 20, creatine 5, EGTA 0.5, glucose 20, HEPES 5, and BSA 2%.

2.2 Electrical recordings
Myocytes were placed in a glass-bottom, temperature-controlled bath (36°C) and continuously superfused with normal bathing solution that contained (in mM): NaCl 126, KCl 4.4, MgCl2 1.0, CaCl2 1.0, glucose 11, and HEPES 24, titrated with 13.0 mM NaOH (pH 7.4). Action potentials were recorded using suction pipettes that had resistances of 2–5 M{Omega} when filled with solution that contained (in mM): NaCl 10, KCl 113, MgCl2 0.5, HEPES 10, K2ATP 5.0, dextrose 5.5, pH adjusted to 7.1 with 11 mM KOH. Transmembrane potentials (Vm,p) were recorded with an Axoclamp 2B amplifier system (Axon Instruments, Inc., Foster City, CA). Pipette series resistance was compensated before attachment, and pipette capacitance was minimized by maintaining a low level of solution in the bath. Vm,p was digitized with a 12-bit A/D converter (Digidata 1320, Axon Instruments) and recorded and analyzed using PCLAMP 8 software (Axon Instruments).

We used an electronic circuit to couple Purkinje myocytes to a passive, resistor–capacitor model of a rabbit ventricular myocyte (hereafter referred to as "V cell"). The V cell had an input resistance of 30 M{Omega} and a capacitance of 90 pF, which are typical values of input resistance and capacitance for rabbit ventricular myocytes [9]. Additionally, the V cell had a variable set potential (Vrest,v) that we varied in 10 mV increments from –70 to –40 mV to mimic different levels of ischemic ventricular depolarization. As previously described [7], the coupling circuit included two amplifiers with variable gain to compute the voltage differences between the Purkinje myocyte and the V cell. That output was sent to voltage-to-current convertors with fixed gain to supply equal and opposite coupling currents of ±(Vm,p–Vm,v)/Rj simultaneously to the myocyte and V cell. These coupling currents were supplied via the suction pipette and, therefore, were intracellular currents. Rj represented a gap junctional resistance that could be varied from 0 to 2000 M{Omega}.

2.3 Coupling protocol
To determine if coupling to a depolarized V cell initiated spontaneous activity in Purkinje myocytes, we recorded Vm,p and the coupling current (shown as stimulus+"injury" current) during a standard coupling protocol, outlined as follows. First, a train of intrinsic, uncoupled Purkinje action potentials paced at a basic cycle length of 2 s was recorded. Next, the Purkinje myocyte was coupled during diastole to the V cell with Vrest,v=–70 mV and Rj=1000 M{Omega} initially. Pacing was discontinued, and Vm,p and the coupling current were recorded for the next 10–60 s. Finally, the Purkinje myocyte and V cell were uncoupled, pacing was resumed, and another train of action potentials was recorded to establish that stable activity at the basic cycle length resumed. This procedure was repeated for several values of Rj (typically between 200 and 2000 M{Omega}) and Vrest,v to determine the conditions under which spontaneous activity in the Purkinje myocytes was induced. In all coupling current traces, positive current indicates depolarizing current supplied when Vrest,v acted as a source, while negative current indicates repolarizing current supplied when Vrest,v acted as a sink.

2.4 Drugs
In a subset of experiments, one of four pharmacologic interventions was used to determine the ionic basis for coupling-induced spontaneous activity: (1) equimolar Li+ substitution for Na+ in the normal bathing solution; (2) 50 µM NiCl2; (3) 100 µM CdCl2; or (4) 10 µM nifedipine+10 µM CdCl2. Nifedipine (Sigma) was prepared as a 10 mM stock solution and stored at –20°C until use, when it was diluted in normal bathing solution.

2.5 Data analysis
All averaged data are presented as mean±S.E. Uncoupled action potentials were characterized by measures of Vrest, action potential amplitude (APA), and action potential duration (APD) at –60 mV. Statistical differences between averages measured in different groups of myocytes were established using Student's t-test. A value of P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Coupling to Vrest,v≤–70 mV
Purkinje myocytes coupled to mildly depolarized Vrest,v demonstrated some depolarization of the rest potential but no spontaneous activity. Fig. 1 shows the coupling current (A) and Vm,p (B) from one experiment. A train of paced responses in the uncoupled Purkinje myocyte was followed by coupling (shaded bar) at Rj=300 M{Omega} for 10 s. Pacing was discontinued during coupling and resumed at the end of the coupling period. Although diastolic injury current depolarized resting Vm,p from –77 to –75 mV, no spontaneous activity occurred. These results were consistently observed in all experiments (n=19).


Figure 1
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  		Fig. 1 Injury current and membrane potential recorded during coupling to mildly depolarized model cell. During coupling, Vm,p was electronically altered by an injury current that represented the current that would have flowed from a depolarized (Vrest,v=–70 mV) cell characterized by a "membrane resistance" of 30 M{Omega} and a "membrane capacitance" of 90 pF. Panel A shows the current (stimulus+injury) applied intracellularly to the Purkinje myocyte. Panel B shows Vm,p. The coupling protocol consisted of a train of basic stimuli while the cells were uncoupled, followed by a period of coupling during which no pacing stimuli were given, and ending with another train of basic stimuli after the cells were uncoupled.

 
3.2 Coupling to Vrest,v≥–60 mV
We were particularly interested in the effects of Purkinje–ventricular coupling when there was moderate depolarization of Vrest,v to values between –60 and –50 mV since that level of depolarization corresponds to the measured rise in rest potential after 15 min of experimental ischemia [10]. Coupling to moderately depolarized Vrest,v induced spontaneous activity in Purkinje myocytes at intermediate Rj values. Fig. 2 shows the coupling current and Vm,p from an experiment in which Vrest,v was set to –50 mV and Rj was varied. At Rj=470 M{Omega}, the injury current induced a slight depolarization of Vm,p but no spontaneous activity (Fig. 2A). During coupling with an intermediate value of 400 M{Omega}, the larger injury current induced a sustained train of spontaneous action potentials that stopped only when coupling was discontinued (Fig. 2B). With stronger coupling of Rj=140 M{Omega}, a single Purkinje action potential was elicited, but subsequent responses were largely suppressed by coupling (Fig. 2C). Thus, spontaneous activity occurred within an "Rj window" during coupling to depolarized Vrest,v. For Rj values above the window, Vm,p was not sufficiently depolarized to reach threshold. For Rj values below the window, too much injury current effectively voltage-clamped the Purkinje myocyte.


Figure 2
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  		Fig. 2 Coupling current and membrane voltages recorded with Vrest,v=–50 mV and Rj=470 M{Omega} (panel A), Rj=400 M{Omega} (panel B), and Rj=140 M{Omega} (panel C). The insets below each panel highlight the first 1.35 s of coupling on an expanded time scale. The dashed traces included in the Vm insets show the changes in Vm,v associated with the coupling current. The open and filled arrows indicate the times at which Vm,p crossed over Vm,v and caused reversal of coupling current.

 
The injury current reversed direction as Vrest,v acted first as an electrical source then as an electrical load during each spontaneous action potential. For example, the inset of Fig. 2B shows that during diastole, the injury current was positive and supplied depolarizing current to the Purkinje myocyte. During the upstroke, the injury current reversed direction when Vm,p exceeded Vm,v (open arrow), supplying repolarizing current to the Purkinje myocyte and depolarizing current to the V cell. Likewise, during repolarization when Vm,p<Vm,v (filled arrow), the current again reversed direction to promote "diastolic depolarization" of the Purkinje myocyte. This pattern of injury current reversal repeated throughout the coupling interval to promote spontaneous activity.

We found that in eight of 15 single Purkinje myocytes, spontaneous activity was induced by coupling to Vrest,v=–60 mV with a combined mean Rj of 830±113 M{Omega}. The mean Rj value at which spontaneous activity was observed in this group of myocytes ranged from 350 to 1200 M{Omega}. These myocytes additionally displayed spontaneous activity during coupling to more depolarized Vrest,v. However, the Rj window for spontaneous activity was shifted to higher Rj values to offset the increase in injury current caused by the larger voltage gradient. In the other seven myocytes, spontaneous activity was not observed at Vrest,v=–60 mV at any value of Rj. However, this second group of myocytes did display spontaneous activity during coupling to Vrest,v=–50 mV with a combined mean Rj of 1190±155 M{Omega}. The mean Rj value at which spontaneous activity was observed in this group of myocytes ranged from 600 to 2000 M{Omega}. Though the second group of myocytes tended to require weaker coupling at more depolarized Vrest,v, the difference in the mean Rj values at which spontaneous activity was observed in these groups was not statistically significant (P=0.09).

To understand why this bimodal distribution existed, we compared the action potential characteristics between these groups. In the group of myocytes that demonstrated spontaneous activity during coupling to Vrest,v=–60 mV, mean Vrest measured –78.3±0.7 mV; mean APA measured 119.8±1.5 mV; and APD measured 456.9±74.2 mV. In the other group of myocytes, mean Vrest measured –77.2±0.7 mV; mean APA measured 119.3±1.7 mV; and APD measured 467.9±114.1 mV. These differences were not statistically different between the two groups, suggesting that these parameters did not influence the level of ventricular depolarization required for the development of spontaneous activity. However, the average diastolic membrane resistance of the myocytes that fired spontaneously during coupling to Vrest,v=–60 mV was significantly higher (237±32 M{Omega}) than that of the myocytes that required Vrest,v=–50 mV (89±6 M{Omega}). Thus, the injury current required to induce spontaneous activity was dependent upon the membrane resistance of the myocytes.

We additionally observed spontaneous activity in four Purkinje aggregates coupled to depolarized Vrest,v. One Purkinje aggregate fired spontaneously during coupling to Vrest,v=–60 mV at Rj values less than or equal to 300 M{Omega}. Two other aggregates fired spontaneously during coupling to Vrest,v=–50 mV at Rj values less than or equal to 400 M{Omega}. The other aggregate required Vrest,v≥–40 mV to demonstrate spontaneous activity during coupling at Rj≤250 M{Omega}. The typical Rj window for spontaneous activity in Purkinje aggregates coupled to depolarized Vrest,v was much lower than in single Purkinje myocytes at each Vrest,v value because the input resistance of the aggregates was lower (55±9 M{Omega}). Since injury current was directly related to the voltage difference (Vm,p–Vrest,v) and inversely related to Rj, Purkinje aggregates required more injury current than single myocytes to induce spontaneous activity.

3.3 Cd2+ and nifedipine suppress spontaneous activity
To elucidate the ionic mechanism(s) promoting the development of spontaneous action potentials during coupling to the depolarized V cell, we performed the coupling protocol before and during superfusion of 50 µM NiCl2 to inhibit T-type calcium current (ICa,T), 100 µM CdCl2 or 10 µM Cd2++10 µM nifedipine to inhibit ICa,L, and equimolar substitution of LiCl2 for NaCl to inhibit sodium–calcium exchange. Neither inhibition of T-type calcium current (n=3) nor inhibition of sodium–calcium exchange (n=3) suppressed spontaneous activity (not shown). In contrast, inhibition of L-type calcium current with 100 µM Cd2+ (n=2) or 10 µM Cd2++10 µM nifedipine (n=2) did suppress coupling-induced spontaneous activity. Fig. 3 shows action potentials recorded before (A) and during (B) superfusion of 10 µM Cd2++10 µM nifedipine. In normal bathing solution, coupling to Vrest,v=–60 mV induced a train of spontaneous action potentials (Fig. 3A), as previously described. While the pipette attachment was maintained, the superfusate was switched to the Cd2++nifedipine mixture and action potentials before, during, and after coupling to the V cell were again recorded over a range of Rj and Vrest,v values. ICa,L inhibition prevented spontaneous activity during coupling (Fig. 3B). Similar results were observed during superfusion of 100 µM CdCl2.


Figure 3
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  		Fig. 3 Vm,p recorded before, during, and after coupling. In panels A and B, Vrest,v=–60 mV and Rj=1200 M{Omega}. (A) Action potentials recorded in normal Tyrode's solution. (B) Action potentials recorded during superfusion of 10 µM Cd2++10 µM nifedipine. The inset illustrates the APD shortening and lowering of the AP plateau that accompanied Cd2++nifedipine superfusion.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Previously, we reported the development of spontaneous activity in isoproterenol-superfused Purkinje myocytes upon coupling to a depolarized, model ventricular cell [7]. In the present study, we showed that coupling-induced spontaneous activity did not require exposure to catecholamines. Instead, weak coupling to moderately depolarized Vrest,v alone was sufficient to induce spontaneous activity. Further, our results suggest that these spontaneous action potentials are mediated by L-type calcium current.

4.1 ICa,L as a mechanism for spontaneous activity
In pacemaker cells, a number of inward ionic currents are active during diastolic depolarization, including the hyperpolarization-activated current (If), a sustained inward sodium current (Ist), and the two calcium currents ICa,T and ICa,L [11]. Typically, the ionic current governing early diastolic depolarization (Vm=–60 to –40 mV) has been identified as either If or ICa,T because it has been widely accepted that the activation threshold for ICa,L is positive to –40 mV. Verheijck et al. [12] recently demonstrated, however, that ICa,L is activated and contributes to both early diastolic depolarization and the upstroke in rabbit sinoatrial nodal action potentials. In that study, 5 µM nifedipine selectively inhibited ICa,L and abolished spontaneous activity.

To test the hypothesis that ICa,L inhibition would also suppress the coupling-induced depolarization observed in the present study, we used either 100 µM CdCl2 or a combination of 10 µM CdCl2+10 µM nifedipine to inhibit ICa,L in rabbit Purkinje myocytes. While 100–500 µM CdCl2 is routinely used to suppress L-type calcium current, it has previously been shown that this level of cadmium concentration may also partially inhibit sodium–calcium exchange [13] and T-type calcium current [14]. Low concentrations of cadmium and nifedipine nearly completely inhibit L-type calcium current while having minimal effects on sodium–calcium exchange [13] or T-type calcium current [14]. Thus, we used both an intermediate concentration of cadmium alone and a combination of low concentrations of cadmium and nifedipine, and we grouped these results to demonstrate that ICa,L inhibition completely suppressed the spontaneous activity that occurred during coupling to depolarized Vrest,v (n=4). In contrast, inhibition of ICa,T (n=3) or sodium–calcium exchange (n=3), which are expected to be active, inward currents in the relevant voltage range, did not suppress coupling-induced spontaneous activity.

4.2 Depolarization of Vrest,v during ischemia
The finding that Vrest,v depolarization between –60 and –50 mV triggered spontaneous Purkinje activity is important because this moderate degree of Vrest depolarization is consistent with the onset of ischemia in a number of experimental preparations [10,15]. For example, Kleber measured epicardial Vrest in guinea pig hearts subjected to aortic occlusion. From a control value of –80 mV, Vrest increased to –70 mV within 3 min of occlusion, to –60 mV within 7 min of occlusion, and to –50 mV within 15 min of occlusion [10]. This depolarization depends primarily upon Ko+ accumulation. As shown by Hill and Gettes, that accumulation follows a triphasic time course in which an immediate, rapid rise in [K+]o is followed 5–10 min later by a plateau phase, which is followed by a final phase with steady [K+]o increases 25 min after occlusion [16]. Severe Vrest depolarization and myocardial inexcitability are primarily associated with this later phase.

In this regard, we distinguish our findings from previous studies that used a similar cell coupling approach to apply injury current. Using guinea pig ventricular myocytes, Kumar and Joyner showed that induction of early afterdepolarizations required isoproterenol, forskolin, or Bay K8644 and injury current arising from coupling to Vrest,v=0 mV [6]. Similarly, Verkerk et al. showed that sheep myocytes exposed to norepinephrine developed delayed afterdepolarizations and triggered activity with Vrest,v≥–33 mV [17]. In contrast, our results demonstrate that rabbit Purkinje myocytes demonstrate coupling-induced spontaneous activity at moderate levels of ventricular depolarization that corresponded to values measured during early ischemia. Additionally, while previous studies have focused on the development of afterdepolarizations and triggered activity during injury current and exposure to catecholamines [6–8], the focus for the present study was on the development of spontaneous activity rather than afterdepolarizations with injury current alone. Our results demonstrated that spontaneous activity developed during injury current alone and did not require the presence of catecholamines.

4.3 Membrane resistance
Spontaneous activity likely developed with smaller injury currents in the present study because the membrane resistance of rabbit Purkinje myocytes is relatively high compared with ventricular myocytes used in previous studies. Membrane resistance is inversely related to density of inward rectifier current (IK1). As a result, myocytes with high membrane resistance, such as Purkinje and pacemaker cells, require very little depolarizing current for action potential initiation. In a recent report, ventricular myocytes were converted to pacemaker cells by genetic suppression of IK1 [18]. Thus, increased membrane resistance alone may be an important mechanism for focal arrhythmias.

Additionally, the mismatch of input resistances between neighboring tissues may present a mechanism for injury current-induced arrhythmias. We have previously demonstrated that early after depolarizations were preferentially suppressed in single Purkinje myocytes (high membrane resistance) compared with aggregates (low input resistance) during coupling to a well-polarized V cell [7]. Similarly, in the present study, membrane resistance determined the injury current required to induce spontaneous activity. Myocytes with high membrane resistance represented a smaller electrical load and demonstrated spontaneous activity at less depolarized Vrest,v and/or weaker coupling than myocytes with low membrane resistance. A better survival of the culturing process by either group of single Purkinje myocytes can be excluded as the difference in their response to coupling to Vrest,v=–60 mV since none of the other action potential parameters measured (Vrest, APD, APA) were significantly different between the groups. Thus, membrane resistance was the determining factor of the quantity of injury current required to induce spontaneous activity.

4.4 Cellular uncoupling during ischemia
Cell coupling at the Purkinje–ventricular junction is decidedly weak under normal conditions [19], while simulated ischemia preferentially inhibits conduction across the Purkinje–ventricular junction [20], suggesting critical increases in Rj at the Purkinje–ventricular interface during ischemia. Such cellular uncoupling is correlated with the secondary rise in Vrest during acute ischemia [21]. Rising [H+]i and [Ca2+]i and depletion of ATP during this period cause gap junctions to close [22] in an attempt to protect surviving myocytes from reperfusion injury [23]. However, several investigators have clearly demonstrated the onset of phase 1b arrhythmias with cellular uncoupling [21,24]. In the present study, we have demonstrated an Rj window of vulnerability to spontaneous activity during ischemic coupling. Stronger coupling (low Rj) between the cells suppressed spontaneous activity by clamping Purkinje myocytes to a depolarized potential near Vrest,v. In contrast, weak coupling associated with high Rj is required to induce spontaneous activity as Vrest,v depolarizes to modest values between –60 and –50 mV. Our results are consistent with previous reports that indicate that a degree of cellular uncoupling is required for the development of injury current-induced arrhythmias [24].

4.5 Limitations
We modeled the subendocardial border zone simply by assuming a depolarized, inexcitable ventricular cell was electrically coupled to a normal Purkinje myocyte. This approach was limited in that we did not attempt to model changes in pH, [Ca2+]i, or metabolic factors that occur secondary to ischemia. However, we were primarily interested in the effects of injury current, which required only a difference in membrane potentials between coupled cells. Further, by utilizing this cell coupling approach, we were able to alter Vrest,v and Rj independent of any other electrophysiologic change. Another limitation of the study was the all-or-nothing application of the injury current. Because electrophysiologic changes secondary to ischemia occur gradually, an interesting follow-up to this study would include a ramp protocol to represent gradual depolarization and uncoupling.

Our defined level of "mild depolarization" was set at –70 mV. Though we used a passive cell incapable of generating an action potential, ventricular myocytes depolarized to this level may still respond actively to electrical stimuli. Purkinje–ventricular coupling under those conditions would likely increase the excitability of both cells and generate some interesting dynamics. The focus for this manuscript, however, was strictly on the development of spontaneous activity in Purkinje myocytes due to ventricular depolarization.

Finally, our results do not eliminate If or Ist as mechanisms of coupling-induced automaticity. However, these currents have not been identified in isolated rabbit Purkinje myocytes, which do not demonstrate automaticity normally [25], suggesting that the combined magnitude of these currents is quite small if they are present at all. Taken together, these results collectively suggest ICa,L as a mechanism for the development of abnormal automaticity in Purkinje myocytes weakly coupled to ischemic myocardium.

4.6 Implications
Alterations in [K+]o and Vrest in the myocardium occur heterogeneously, with gradients appearing across the ischemic border. A voltage gradient at the border zone sets up a driving force for injury current, and even small injury currents that slightly depolarize normal myocytes may facilitate acute ischemic arrhythmias by increasing excitability of tissue on the normal side of the ischemic border. Coronel et al. showed during LAD occlusion in pig hearts that diastolic injury current across the border zone depolarized normal tissue and reduced the stimulation threshold [4]. Larger diastolic injury currents may depolarize myocytes on the normal side of the ischemic border sufficiently to initiate ectopic beats. Janse et al. quantified injury current magnitudes during acute ischemic arrhythmias in pig and dog hearts, and they reported that the earliest activity of these arrhythmias always occurred on the normal side of the ischemic border at sites where injury current was significant [3]. At the subendocardial border zone, large injury currents can develop between Purkinje and ventricular myocytes because Purkinje fibers maintain relatively normal electrophysiological properties after occlusion while neighboring ventricular myocytes demonstrate depolarized Vrest [26]. Our results show that such injury currents can induce spontaneous activity in normal Purkinje myocytes, which may provide the trigger for acute ischemic arrhythmias that are then maintained by reentrant mechanisms.

Time for primary review 61 days.


   Acknowledgements
 
The authors thank Melody Kingsley for her technical assistance. This work was supported by grants from the Whitaker Foundation (D.J. Huelsing), American Heart Association 0030074N (D.J. Huelsing) and 0051196B (A.E. Pollard), National Science Foundation BES-9903466 (A.E. Pollard), and National Heart, Lung and Blood Institute Awards HL67728 (A.E. Pollard), HL67961 (A.E. Pollard), and HL52338 (K.W. Spitzer).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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