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Cardiovascular Research 2004 61(4):715-723; doi:10.1016/j.cardiores.2003.12.025
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Copyright © 2004, European Society of Cardiology

Mechanisms of {alpha}-adrenergic potentiation of ventricular arrhythmias in dogs with inherited arrhythmic sudden death

Eugene A. Sosunova, Maria N. Obreztchikovaa, Evgeny P. Anyukhovskya, N.Sydney Moïseb, Peter Danilo, Jr.a, Richard B. Robinsona and Michael R. Rosena,c,*

aDepartment of Pharmacology, Center for Molecular Therapeutics, College of Physicians and Surgeons of Columbia University, New York, NY, USA
bDepartment of Clinical Sciences, College of Veterinary Medicine of Cornell University, Ithaca, NY, USA
cDepartment of Pediatrics, College of Physicians and Surgeons of Columbia University, New York, NY, USA

* Corresponding author. Department of Pharmacology, College of Physicians and Surgeons of Columbia University, 630 West 168 Street, PH 7West-321, New York, NY, 10032, USA. Tel.: +1-212-305-8754; fax: +1-212-305-8351. mrr1{at}columbia.edu

Received 15 October 2003; revised 11 December 2003; accepted 23 December 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: In German shepherd dogs having inherited arrhythmias and sudden death, pause-dependent arrhythmias are triggered by early afterdepolarizations (EADs) originating from left ventricular (LV) Purkinje fibers (PF). Heterogeneity of LV repolarization provides the arrhythmogenic substrate. To elucidate the mechanisms whereby {alpha}-adrenergic stimulation exacerbates these arrhythmias we tested the effects of phenylephrine on both arrhythmogenic trigger and substrate. Methods and results: We used microelectrode techniques to record action potentials from LV and right ventricular (RV) PF and from midmyocardial sections of anteroseptal, anterobasal and posterobasal LV wall of unafflicted and afflicted dogs. EADs occurred spontaneously in 8 of 12 LV PF and in no RV PF from afflicted dogs and in no PF from unafflicted dogs. In LV PF from afflicted dogs, phenylephrine (10–9–10–5 M) concentration-dependently decreased membrane potential, induced abnormal automaticity at membrane potentials from –65 to –45 mV in 6 LV PF and potentiated EADs in another 6. To determine the mechanisms of membrane depolarization we studied phenylephrine effects on IK1 in voltage-clamped single LV and RV PF cells from afflicted dogs. In LV PF, phenylephrine (10–5 M) reduced IK1 over the range of –120 to –40 mV and had no effects on RV PF. Regional heterogeneity of LV repolarization was observed in afflicted dogs only. Phenylephrine had no effects on repolarization in either group. Conclusion(s): {alpha}-adrenergic stimulation exacerbates arrhythmias in afflicted dogs by increasing the arrhythmogenic trigger while leaving the substrate unchanged. Decrease in IK1 contributes importantly to {alpha}-adrenergic effects on LV PF.

KEYWORDS Arrhythmia; Sudden death; Ion channels


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
In a German shepherd model of spontaneous cardiac arrhythmias and sudden death, pause-dependency characterizes >80% of ventricular tachycardias [1–3], and is triggered by early afterdepolarizations (EADs) originating from left ventricular (LV) Purkinje fibers [4,5]. Heterogeneity of LV repolarization provides the substrate contributing to maintenance of the tachycardias [6]. Arrhythmias in this model respond to autonomic modulation [1,7], and {alpha}-adrenergic stimulation with phenylephrine facilitates the development of ventricular arrhythmias in afflicted animals [5,8]. The phenylephrine effect is realized both by induction of reflex bradycardia and direct myocardial action [8].

This study was designed to investigate the mechanisms of the direct myocardial action of phenylephrine. Accordingly, we studied phenylephrine effects on trigger (free-running Purkinje fibers) and substrate (repolarization in multicellular sections from anterobasal, anteroseptal and posterobasal LV midmyocardium) in afflicted and unafflicted dogs. We selected these sites because they represent normally innervated regions as well as regions of decreased sympathetic innervation in afflicted dogs [9]. To elucidate ionic mechanisms of the {alpha}-adrenergic effect, phenylephrine effects on the inward rectifier current (IK1) were examined in Purkinje myocytes from afflicted dogs.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1 Procedures conformed to the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health Publication No 85-23)
Holter monitoring was used to identify 18 arrhythmic (afflicted) German shepherd dogs aged 22–35 weeks from a colony bred at Cornell University [1]. They displayed premature ventricular complexes (singles, couplets and triplets) and/or ventricular tachycardia (four or more ventricular complexes in a row). Fifteen control (unafflicted) German shepherds of the same age were obtained commercially and were determined to have no spontaneous arrhythmias.

In six animals, we calculated an "arrhythmia score" as follows: number of ventricular premature depolarizations in 24 h: score of 1=<20, 2=21–300, 3=301–6000, 4=6001–18,000, 5=>18,000. Number of multiples (couplets or triplets) in 24 h: score of 1=none, 2=1, 3=2–9, 4=10–29, 5=>30.

2.2 Action potentials in multicellular preparations
Animals were anesthetized with sodium pentobarbital, 30 mg/kg i.v. Hearts were removed through a left lateral thoracotomy and immersed in cold Tyrode's solution equilibrated with 95% O2–5% CO2 and containing (mmol/l): NaCl 131, NaHCO3 18, KCl 4, CaCl2 2.7, MgCl2 0.5, NaH2PO4 1.8, dextrose 5.5. We excised LV and right ventricular (RV) free-running Purkinje fibers or midmyocardial sections (0.5–1 mm thick, parallel to the epicardial surface) from anteroseptal, anterobasal and posterobasal LV. Preparations were placed in a 4-ml chamber, superfused with Tyrode's solution (37 °C, pH 7.3–7.4) at 12 ml/min and stimulated via Teflon-coated silver electrodes with 1–2 ms, rectangular, twice-threshold current pulses. Stabilization required 1–2 h (Purkinje fibers) or 3–4 h (myocardial sections) of stimulation at a cycle length (CL)=1000 ms. Conventional microelectrode techniques were used to record transmembrane potentials.

In Purkinje fibers, the effects of phenylephrine (Sigma; 10–9–10–5 mol/l) were studied on stimulated (CL=800 ms) and automatic action potentials. In midmyocardial sections, frequency dependence of phenylephrine effects was investigated at CL=4000, 2000, 1000, 500, and 300 ms. Steady state was achieved by pacing for 3 min at each CL. Preparations were allowed to equilibrate for 10 min at each agonist concentration. To minimize β-adrenergic action of phenylephrine, propranolol (Sigma, 2 x 10–7 mol/l) was included in all solutions.

2.3 IK1 in single Purkinje cells
Cells were obtained from five RV and ten LV free-running Purkinje fiber bundles of five afflicted dogs. Cells from the two LV Purkinje fiber bundles of each dog were disaggregated and pooled. The enzymatic technique used to disaggregate Purkinje myocytes was similar to one described previously [10]. Fibers were placed in standard Hank's balanced salt solution (Gibco, Invitrogen) nominally free of calcium and containing 1000–1500 U/ml collagenase (type II, Worthington), 1% BSA (Sigma), and 5 mM HEPES–NaOH buffer (pH 6.7). Petri dishes with fibers were placed in a gyrator shaker and agitated (2–3 cps) for 30–40 min at 37 °C. Fibers were than washed twice in a high K+-saline solution (in mmol/l: potassium glutamate 160, HEPES–KOH buffer 5, MgSO4 5.4; pH 6.7) and individual cells were dispersed by gentle hand pipetting.

Myocytes were placed in a heated bath and superfused at constant rate with modified Tyrode's solution containing (mmol/l): NaCl 140, KCl 5.4, MgCl2 1, CaCl2 1.8, HEPES 5, glucose 10; pH 7.4 (NaOH) at 35 °C. Nisoldipine (4 x 10–7 mol/l; a gift from Bayer) was always present in the Tyrode's solution to suppress ICa,L. Borosilicate glass pipettes had tip resistances=1–1.5 M{Omega}. The pipette solution contained (mmol/l): KOH 60, KCl 80, aspartate 40, MgCl2 1, CaCl2 0.65, EGTA 10, Mg2ATP 5, Na creatinine phosphate 5, GTP 0.1, HEPES 5; pH 7.2. Ionic currents were recorded via whole cell patch clamp using a PC equipped with pClamp 8 software, DigiData 1200 series interface and Axopatch 1D amplifier (Axon Instruments). Compensated series resistance=2.3±0.3 M{Omega}. Membrane capacitance did not differ between left and right Purkinje cells (189±13 and 198±10 pF, n = 10 and 11, respectively). The holding potential was –70 mV. The pulse protocol consisted of a 50-ms step to –120 mV, followed by depolarizing ramp to +20 mV (4 s), and back to the holding potential. The pulse protocol was elicited once every 30 s. 10 mmol/l Cs was used to separate IK1 from net membrane current [11]. The slope conductance of IK1 (GK1) at its reversal potential (Vrev) was obtained by fitting data points in the vicinity of Vrev with either second- or third-order polynomials and calculating the derivative at Vrev.

2.4 Statistical analysis
Data are expressed as mean±S.E.M. Microelectrode data were analyzed from impalements maintained throughout each experiment. The statistical technique used was one- or two-way ANOVA for multiple groups or for repeated measures, with Bonferroni's test when the F-value permitted this. Significance of differences in incidence of early afterdepolarizations and abnormal automaticity was evaluated with Fisher's exact test. Non-linear least-square algorithms were used for fitting I–V curves. P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Studies of Purkinje fibers
Because a previous study had demonstrated that the incidence of EAD and EAD-induced triggered activity as well as their responses to autonomic neuromodulators did not differ between anterior and posterior LV Purkinje fibers from afflicted dogs [4], we combined both groups into one. Phenylephrine effects were examined in LV and RV Purkinje fibers stimulated at CL=800 ms or beating spontaneously. Fig. 1A shows representative stimulated APs from LV and RV Purkinje fibers from one afflicted dog. Different phenylephrine effects are seen between the two, with prominent depolarization in LV but not RV Purkinje fibers. Phenylephrine effects on membrane potential varied notably among 12 LV fibers from afflicted animals: from no effect to depolarization to –45 mV at 10–5 mol/l. Average values at different phenylephrine concentrations are shown in Fig. 1B. Pre-exposure of LV Purkinje cells to the {alpha}1-antagonist prazosin (10–6 mol/l) blocked the phenylephrine-induced depolarization (data not shown). There were no significant effects of phenylephrine on MDP in RV fibers from afflicted dogs (Fig. 1C) or in fibers from either ventricle of unafflicted dogs (Fig. 1B and C). AP duration was concentration-dependently prolonged by phenylephrine in all groups (Fig. 1D and E).


Figure 1
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  		Fig. 1 (A) Transmembrane potentials recorded from LV (LPF) and RV (RPF) Purkinje fibers of an afflicted dog in control (Con) and 10–5 mol/l phenylephrine (Phe). Concentration-dependent phenylephrine effects on maximum diastolic potential (MDP) in LPF (B) and RPF (C) and on APD to 90% repolarization (APD90) in LPF (D) and RPF (E) of unafflicted and afflicted dogs. n = 12 LPF and n = 6 RPF/group. *P<0.05 vs. respective control. CL=800 ms.

 
Fig. 2 demonstrates typical examples of phenylephrine effects on spontaneously beating LV Purkinje fibers from afflicted dogs. Phenylephrine either induced EADs in fibers having no arrhythmias in control (A) and potentiated existing EADs (B) or depolarized membrane potential and induced abnormal automaticity at low membrane potential (from –65 to –45 mV) (C). Concentration-dependent effects of phenylephrine are shown in Fig. 3. In control, EAD and EAD-induced triggered activity occurred in 8 of 12 LV Purkinje fibers from afflicted dogs and in no fibers from unafflicted dogs. Phenylephrine did not change these incidences significantly (Fig. 3A). Abnormal automaticity was not seen in either group in control and was initiated by phenylephrine in afflicted but not unafflicted dogs (Fig. 3B). As a result, in the presence of 10–5 mol/l phenylephrine, arrhythmias (either triggered activity or abnormal automaticity) occurred in all fibers from afflicted dogs, while only 1 of 12 fibers from unafflicted dogs developed EADs. As regards RV Purkinje fibers, there were no EADs either in control or phenylephrine in both unafflicted and afflicted dogs. The effects of phenylephrine on MDP in LV fibers were similar to those observed during electrical stimulation: concentration-dependent depolarization in afflicted and no effects in unafflicted dogs (Fig. 3C).


Figure 2
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  		Fig. 2 Examples of spontaneous rhythm in LV Purkinje fibers from afflicted dogs in control and 10–5 mol/l phenylephrine.

 

Figure 3
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  		Fig. 3 Concentration-dependent effects of phenylephrine on the incidence of early afterdepolarizations (A), abnormal automaticity at membrane potentials of –65 to –45 mV (B) and maximum diastolic potential (C) in spontaneously beating LV Purkinje fibers. +P<0.05 vs. respective unafflicted. *P<0.05 vs. respective control (n = 12/group).

 
That the depolarization in LV Purkinje fibers may substantively contribute to the expression of arrhythmias is suggested in Fig. 4. Phenylephrine effects were studied in free-running Purkinje fibers from six afflicted animals. A good correlation between the expression of arrhythmias in the intact dogs and phenylephrine-induced depolarization in LV Purkinje fibers in vitro was seen.


Figure 4
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  		Fig. 4 Correlations between phenylephrine induced depolarization in vitro ({Delta}MDP) and expression of arrhythmias in vivo in six afflicted dogs. Vertical axes, maximum depolarization induced by 10–5 mol/l phenylephrine in two LV Purkinje fibers from each dog. Horizontal axes, score for number of premature ventricular complexes (PVC) (A) or multiples (B). Linear regression lines, correlation coefficients and significance values are shown for both arrhythmia scores.

 
3.2 Voltage clamp experiments
We tested the hypothesis that phenylephrine-induced depolarization of LV Purkinje fibers from afflicted dogs resulted from inhibition of IK1 which is a major determinant of maximum diastolic potential. Because phenylephrine depolarized LV but not RV Purkinje cells from afflicted dogs, both cell types were included in the experiments. IK1 was considered as a Cs-sensitive time-independent current [11]. It has been shown for other species and tissues that {alpha}1-adrenoreceptor stimulation not only inhibits IK1 [12,13] but affects other time-independent currents in myocytes [14], including Na+–K+ pump [15], Na+/Ca2+ exchange [16], and muscarinic receptor-activated K+ current [13]. Therefore, in initiating the voltage clamp study we first determined whether phenylephine might affect Cs-insensitive currents. An example of the three experiments performed is shown in Fig. 5A. Phenylephrine induced a reduction of total membrane current and was then washed out. CsCl was added to the bath after the current had recovered completely. Phenylephrine was added to Cs+ and did not affect membrane current. The same result was observed with the other two myocytes. These data demonstrate that phenylephrine had no effects on Cs-insensitive currents and decreased total membrane current by inhibiting IK1.


Figure 5
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  		Fig. 5 (A) Phenylephrine effects on Cs-insensitive current: original tracings from an LV Purkinje cell of an afflicted dog. Sequence of recordings: control (C), phenylephrine, 10–5 mol/l (P), washout (W), CsCl, 10 mmol/l (Cs), phenylephrine in the presence of Cs (Cs+P). Inset: clamp protocol. (B) Phenylephrine effects on Cs-sensitive current. Original tracings of membrane current during ramp pulse are shown for one LPF and one RPF from an afflicted dog. The sequence of recordings was: control (C), phenylephrine, 10–5 mol/l (P), CsCl, 10 mmol/l in the presence of phenylephrine (P+Cs), washout (W). (C) Cs-sensitive current components were obtained by digital subtraction from currents recorded in control and in the presence of phenylephrine of currents recorded under combined presence of phenylephrine and cesium (P+Cs).

 
Because our initial experiments demonstrated that phenylephrine does not influence the Cs-insensitive current, we assumed that any current recorded in the presence of both phenylephrine and Cs would equal the current in Cs alone. Based on this assumption we designed the experiments in Fig. 5B. In control, a typical inwardly rectifying K+ current was present in both cells [17]. Upon phenylephrine exposure, inward and outward current was reduced in LV but not RV Purkinje cells. This reduction was almost completely reversible upon washout of the drug. Then CsCl was added to block IK1 [17]. In both LV and RV Purkinje cells, the I–V relation did not show inward rectification in the presence of CsCl and intersected the other three curves at about –86 mV (IK1 reversal potential, Vrev). In Fig. 5C, the difference I–V relations were plotted (note the different scales for the vertical axes). The two curves in each panel represent the I–V relation for IK1 in control and during exposure to phenylephrine and illustrate the reduction of the current induced by {alpha}-agonist in LV but not RV Purkinje myocytes.

Table 1 summarizes phenylephrine effects on IK1. In right and left Purkinje myocytes, phenylephrine did not change the reversal potential. In LV, phenylephrine significantly reduced IK1 at –70 and –120 mV with respect to Vrev. There were no effects in RV. The slope conductance at Vrev (Grev) that reflects the ability of IK1 to stabilize resting potential was reduced by phenylephrine in LV but not RV Purkinje myocytes.


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  		Table 1 Parameters of cesium-sensitive inward rectifier current in myocytes isolated from free running Purkinje fibers of afflicted German shepherd dogs in control and in the presence of 10–5 mol/l phenylephrine

 
In some of our experiments, phenylephrine was applied twice. Therefore, the lack of effect on second exposure could result from desensitization. To test this possibility, we performed the experiments in Fig. 6. In four intact Purkinje fiber bundles in which phenylephrine had induced significant membrane depolarization on the first exposure, drug was washed out and applied again. Phenylephrine again produced significant depolarization and its effect on second exposure was not different from the initial effect (Fig. 6A). The experiment in Fig. 6B demonstrates that there is no desensitization of phenylephrine effect on IK1. After the control recording, CsCl was added to the bath. Then phenylephrine was applied in addition to Cs and had no effect on the current. After washing out phenylephrine and Cs, phenylephrine alone was applied again and produced a reduction of membrane current similar to that in the absence of Cs in Fig. 4A and B and Table 1.


Figure 6
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  		Fig. 6 Effects of repetitive phenylephrine application. (A) Phenylephrine effects on MDP in multicellular LV Purkinje fibers from afflicted dogs. Phenylephrine (P) was applied twice: after control (Con) and washout (W) recordings. *P<0.05 vs respective predrug value (n = 4). (B) Original tracings were obtained from an LV Purkinje cell of an afflicted dog. The sequence of recordings was: control (C), cesium chloride, 10 mmol/l (Cs), phenylephrine,10–5 mol/l, in the presence of cesium (Cs+P), washout (W), phenylephrine (P).

 
3.3 Studies of LV midmyocardial sections
In a previous study, we demonstrated significant spatial heterogeneity of midmyocardial repolarization along the LV wall of afflicted but not unafflicted dogs, whereas transmural dispersion of repolarization across the ventricular wall did not differ between the two groups [6]. Therefore, in the present study, we focused on action potential duration and phenylephrine responsiveness in sections from different regions of LV midmyocardium of hearts from afflicted and unafflicted dogs. Fig. 7A shows representative traces illustrating phenylephrine effects on transmembrane potentials in three LV midmyocardial regions of one unafflicted and one afflicted dog at CL=4000 ms. In accordance with our previous findings [6], in control Tyrode's solution a marked difference between unafflicted and afflicted groups is evident with prominent heterogeneity of repolarization along the midmyocardium in the afflicted and minimum heterogeneity in the unafflicted (Fig. 7A, upper traces). In unafflicted dogs, APD90 was not significantly different among LV regions at all CLs, whereas in afflicted dogs, and particularly at long CLs, APD90 was the longest in anterobasal and shortest in anteroseptal regions (Fig. 7B, upper panels). Phenylephrine (10–5 mol/l) did not affect repolarization in both unafflicted and afflicted dogs (Fig. 7A, lower traces). As a result, dispersion of APD90 was prominent before and remained prominent after phenylephrine in afflicted animals and was minimal before and after phenylephrine in unafflicted animals (Fig. 7B, lower panels).


Figure 7
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  		Fig. 7 (A) Representative transmembrane potentials from midmyocardial sections of anteroseptal (AS), anterobasal (AB) and posterobasal (PB) LV wall of one unafflicted and one afflicted dog in control and in phenylephrine (10–5 mol/l) at CL=4000 ms. (B) Steady-state dependence of APD90 on CL in midmyocardium from LV of unafflicted and afflicted dogs in control and in phenylephrine (10–5 mol/l) (n = 9–14 from 8 unafflicted and 6 afflicted dogs).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
It was shown previously that LV Purkinje fibers from afflicted animals generated EADs and triggered activity potentiated by {alpha}1-adrenergic stimulation with phenylephrine [4,5]. This {alpha}-adrenergic effect was suggested as a mechanism for phenylephrine-induced exacerbation of arrhythmia in afflicted animals although the mechanism was not fully elucidated. Moreover, concentration-dependent effects of {alpha}-agonists on stimulated and spontaneously beating Purkinje fibers from afflicted dogs have not been studied. The major new finding in the present study is that phenylephrine produces concentration-dependent depolarization of LV Purkinje fibers from afflicted dogs but not of RV fibers or of fibers from either ventricle of unafflicted dogs. This effect is attributable to phenylephrine-induced reduction of IK1 that is observed in LV but not RV Purkinje myocytes from afflicted animals. While IK1 appears to be an important contributor to the arrhythmogenic substrate in the animals it is likely that other determinants contribute here as well. As for the basis for the abnormality of IK1 described in LV but not RV Purkinje fibers, the experimental result suggests a link between sympathetic innervation and expression of the current that remains to be identified.

In voltage clamp experiments, we deliberately used RV Purkinje myocytes from afflicted dogs as a control because our microelectrode studies showed that their electrophysiology is identical to that of LV or RV Purkinje fibers from unafflicted dogs. The important consequence of the membrane depolarization is that all fibers that depolarize to potentials from –65 to –45 mV develop abnormal automaticity. Thus, phenylephrine stimulates abnormal impulse initiation in LV Purkinje fibers not only by potentiating EADs but by inducing abnormal automaticity at low membrane potentials. With regard to the arrhythmogenic substrate, phenylephrine did not change the regional heterogeneity of LV repolarization that has been considered a substrate for pause-dependent arrhythmias in afflicted dogs [6]. These data suggest that {alpha}1-adrenergic stimulation exacerbates pause-dependent arrhythmias by increasing the arrhythmogenic trigger while leaving the substrate unchanged.

Moreover, as shown in Fig. 4, there is a good relationship between the extent of MDP depolarization induced by {alpha}-adrenergic stimulation and the severity of the arrhythmia. We emphasize that ventricular arrhythmias were analyzed in these animals using 24-h ECG monitoring in the absence of any pharmacological interventions. Therefore, our results imply that baseline plasma levels of neurotransmitters and sympathetic tone in these animals were sufficient to influence abnormal impulse initiation in LV Purkinje fibers.

In accordance with previous findings [18,19], concentration-dependent APD lengthening was produced by phenylephrine. This effect is unlikely associated with changes in IK1: first, in Purkinje myocytes, IK1 is active predominantly in the diastolic potential range and does not significantly control APD [17,20]; second, phenylephrine prolonged APD at CL=800 ms to a similar extent in all four groups studied whereas IK1 inhibition was seen only in LV Purkinje fibers from afflicted dogs. It has been shown that reduction of the slow component of delayed rectifier current (IKs) contributes to phenylephrine action on APD in canine Purkinje myocytes [21]. We have previously shown that at long CLs, at which the IKs contribution to repolarization increases, phenylephrine prolongs APD more in Purkinje fibers of afflicted than unafflicted dogs suggesting greater IKs inhibition in afflicted animals. APD prolongation predisposes to the development of EADs [22]. In our experiments, the extent of APD prolongation in the presence of phenylephrine was associated with EADs only in LV Purkinje fibers of afflicted dogs.

A tendency for EADs and triggered activity to occur in LV Purkinje fibers of afflicted dogs and their atypical response to {alpha}-adrenergic stimulation may derive from a regional delay in development of sympathetic innervation to the LV [9]. Reduced sympathetic innervation can inhibit the functional expression of repolarizing currents and affect all components of the adrenergic receptor signaling cascade [23,24]. The present study shows that baseline function of IK1 was unaltered in LV Purkinje myocytes of afflicted dogs while {alpha}-adrenergic effects were significantly modified (Table 1). This is consistent with earlier observations that in regions of reduced sympathetic innervation an altered responsiveness to both β-adrenergic and {alpha}-adrenergic stimulation occurs [4–6]. Another potential contributor to the occurrence of EADs in our experiments may be the Na/Ca exchange current [25]. This can be upregulated by {alpha} agonist [16], providing a potential mechanism for arrhythmogenicity in this model.

Clinical ventricular arrhythmias in afflicted dogs are influenced by perturbations in the autonomic nervous system [1,7,8]. Because stimulation of {alpha}- and {alpha}-adrenergic receptor types has complex and different effects on electrophysiological properties of Purkinje fibers and ventricular myocardium in afflicted dogs, a net effect of increased sympathetic tone is difficult to predict. Our study shows that {alpha}-adrenergic stimulation facilitates abnormal impulse initiation in LV Purkinje fibers while not affecting heterogeneity of LV repolarization. In contrast, stimulation of β-adrenergic receptors suppresses EADs in Purkinje fibers [4] and equalizes LV repolarization [6]. Such stimulation can also induce delayed afterdepolarizations (DAD) and DAD-induced triggered activity in LV regions lacking normal sympathetic innervation [5]. The extent to which {alpha}-adrenergic and β-adrenergic receptor signaling cascades are modified by chronically reduced sympathetic input in Purkinje fibers and ventricular myocardium would appear to be the determinants of proarrhythmia induced by sympathetic stimulation in afflicted dogs.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
The authors express their gratitude to Nimee Bhat for assistance in performing the studies and to Ms. Eileen Franey for her careful attention to the preparation of the manuscript.

These studies were supported by USPHS-NHLBI grant HL-28958.


   Notes
 
Time for primary review 20 days


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

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