© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
β1 and β2-adrenergic receptor subtype effects in German shepherd dogs with inherited lethal ventricular arrhythmias
aDepartment of Pharmacology, Columbia University, College of Physicians and Surgeons, 630 West 168 Street, PH7W-321, New York, NY 10032, USA
bDepartment of Pediatrics, Columbia University, College of Physicians and Surgeons, New York, NY, USA
cCornell University, College of Veterinary Medicine, Ithaca, NY, USA
* Corresponding author. Tel.: +1-212-305-8754; fax: +1-212-305-8351 mrr1{at}columbia.edu
Received 8 March 2000; accepted 30 June 2000
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Objective: Delayed afterdepolarization-induced triggered activity originating in ventricular myocardium is a mechanism for some age-dependent, inherited ventricular tachycardias in a colony of German shepherd dogs. Methods: We used standard microelectrode techniques to study β-adrenergic receptor subtype modulation of the triggered activity in anteroseptal left ventricular myocardium from eleven of these dogs and seven unafflicted, age-matched German shepherd controls. Results: During sustained stimulation at cycle lengths of 300–4000 ms, 10–9–10–7 M isoproterenol concentration-dependently shortened action potential duration (APD) to 90% repolarization more in myocardium from afflicted than from unafflicted dogs. This shortening was prevented by a β1-blocker CGP20712A (10–7 M) while a β2-blocker ICI118551 (10–7 M) did not modify the effect of isoproterenol in either group. The β2-agonist zinterol 10–8–10–6 M had no effect on APD. Stimulation at a cycle length of 250 ms in the presence of 10–7 M isoproterenol induced more triggered AP in myocardium from afflicted than unafflicted dogs. β1-Blockade completely eliminated, while β2-blockade facilitated, and the β2-agonist zinterol did not induce triggered activity in the two groups. Conclusion: Isoproterenol effects on APD and triggered activity in the myocardium of dogs with inherited arrhythmias are due primarily to an abnormality of β1-adrenoceptor mediated signaling that is subject to β2-adrenergic modulation.
KEYWORDS Adrenergic (ant)agonists; Arrhythmia (mechanisms); Sudden death; Ventricular arrhythmias; Impulse formation
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Recently we demonstrated that delayed afterdepolarizations and triggered activity originating from the ventricular myocardium may account for a subset of ventricular tachyarrhythmias in German shepherd dogs with inherited sudden cardiac death [1]. The arrhythmias were documented in vivo, and the triggered activity was observed in sections of anteroseptal left ventricular midmyocardium superfused with the β1- and β2-agonist, isoproterenol. This region of myocardium had a β receptor density of
550 fmol/mg protein, as compared to 350 fmol/mg in controls (P<0.05), and a significantly greater response to isoproterenol. It is well known that under certain conditions the distribution of β1- and β2-adrenergic receptors is altered (e.g. in congestive heart failure [2] or after prolonged treatment with β1-selective antagonists [3]). That under these conditions the receptor population is altered, suggests that pathways subserving the receptors, too, may vary, and with this the expression of cardiac rhythm. Therefore, we hypothesized that an altered ratio of signaling via β1- and β2-receptor systems contributes to arrhythmias in afflicted German shepherd dogs. To assess the contribution of β receptor subtypes, we studied action potential repolarization and triggered activity in left ventricular myocardium of normal German shepherd dogs and those with inherited arrhythmias and sudden death. The pharmacological assay employed isoproterenol, the selective β1- and β2-adrenoceptor blockers, CGP20712A and ICI118551, respectively [4,5], and the β2-agonist zinterol [6].
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2 Methods |
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All studies were performed in accordance with Institutional rules for animal experimentation at Cornell and Columbia Universities, and were approved by the Institutional Animal Care and Use Committee. Holter monitoring was used to identify eleven arrhythmic German shepherd dogs aged 22–32 weeks from a colony bred at Cornell University [7]. As previously reported [1] all eleven animals manifested ventricular ectopy and/or tachycardia during the Holter recordings. Seven unrelated control German shepherds of the same age were obtained from a commercial breeder and were determined to have no spontaneous arrhythmias.
Animals were anesthetized with sodium pentobarbital, 30 mg/kg i.v. The hearts were removed and immersed in Tyrode's solution equilibrated with O2–CO2 (95:5%) at 37°C and containing (in mM): NaCl 131, NaHCO3 18, KCl 4, CaCl2 2.7, MgCl2 0.5, NaH2PO4 1.8, EDTA 0.05 and dextrose 5.5. The 0.5-mm thick sections of midmyocardial tissue were filleted parallel to and 5 mm deep to the epicardial surface of the anteroseptal left ventricular free wall. Preparations were stimulated with 2-ms duration, 2 times threshold square-wave pulses at cycle lengths of 300, 500, 1000, 2000 and 4000 ms in steady state conditions. To study delayed afterdepolarizations and triggered action potentials, preparations were stimulated at a cycle length of 250 ms for 5 min and then stimulation was interrupted. When delayed afterdepolarizations reached threshold and induced triggered activity we calculated the incidence of the triggered action potentials.
Standard microelectrode techniques were used to record transmembrane potentials [8]. Graded concentrations of isoproterenol (10–9–10–7 M) (purchased from Sigma, St. Louis, MO, USA) were superfused and 10 min allowed at each concentration of agonist for its effect to develop fully. In experiments on β-receptor subtype blockade, 10–7 M CGP20712A or 10–7 M ICI118,551 (both purchased from RBI, Natick, MA, USA) were superfused for 45 min prior to as well as throughout isoproterenol administration.
These concentrations of β antagonist were selected because they had exerted maximal blocking effects in our previous studies of β1- and β2 adrenergic stimulation of normal young and adult canine tissue [9]. In other experiments we used the β2-adrenergic agonist, zinterol, 10–8–10–6 M (obtained from Bristol-Myers Squibb). All experiments were performed with the room darkened.
Data are expressed as mean±S.E.M. The statistical method used was one- or two-way ANOVA with Bonferroni's test if the f value permitted. Fisher's exact test was used to evaluate the significance of changes in the incidence of premature or triggered action potentials. A P value <0.05 was considered significant.
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3 Results |
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During the predrug control period, action potential duration to 30, 50 and 90% repolarization (APD30, APD50 and APD90, respectively) was longer in myocardium from afflicted as opposed to unafflicted dogs (Fig. 1, Table 1). Other transmembrane potential characteristics did not differ significantly between the two groups.
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Representative recordings of the effects of isoproterenol alone and in the presence of CGP20712A and ICI118551 are shown in Fig. 2. Note that isoproterenol alone elevated the plateau and accelerated repolarization in unafflicted and afflicted animals. Neither CGP20712A, nor ICI118551 alone (i.e. without isoproterenol) induced significant changes in transmembrane potential. In the presence of CGP20712A, isoproterenol had no effect on the plateau and prolonged repolarization in both groups. In the presence of ICI118551, isoproterenol persisted in elevating the plateau and accelerating repolarization.
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Summary data regarding the plateau are provided in Fig. 3. Note that the isoproterenol-induced increase in plateau potential was more pronounced at the longer stimulus cycle lengths. CGP20712A blocked this effect of isoproterenol (Fig. 3, middle panels), but ICI118551 did not (Fig. 3, right panels). Nevertheless, the magnitude of isoproterenol effect on the plateau was smaller in tissues from afflicted as compared to unafflicted dogs in the presence of ICI118551 (Fig. 3, right panel). In unafflicted dogs, isoproterenol prolonged APD30 at CLs of 1000 and 4000 ms and shortened it at 300 ms (Fig. 4, left panel). In contrast, isoproterenol induced no significant changes in APD30 in afflicted animals. CGP20712A blocked the changes in APD30 in unafflicted dogs but had no effect in afflicted dogs (Fig. 4, middle panels). ICI118551 did not modify the isoproterenol effect on APD30 in either group (Fig. 4, right panels).
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Isoproterenol induced a significant shortening of the APD90, more so in afflicted dogs (Fig. 5, left panels). With CGP20712A, the effect of isoproterenol to shorten APD90 was blocked and there appeared a tendency for the action potential to prolong at a CL of 4000 ms in unafflicted and afflicted groups (Fig. 5, middle panels). ICI118551 did not modify isoproterenol effects in either group (Fig. 5, left panels).
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The β2-agonist zinterol had no effect on MDP,
max or action potential amplitude (data not shown). Zinterol also had no significant effect on APD30 or APD90, but at the highest concentration it did significantly increase plateau amplitude (Figs. 2 and 6
).
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During performance of these protocols, no premature depolarizations were seen in tissues from either group, whether superfused with zinterol or isoproterenol alone, or isoproterenol in combination with CGP20712A. However, in the presence of isoproterenol 10–7 M+ICI118551, premature depolarizations having a maximal incidence at CLs of 500 and 1000 ms often occurred in preparations from afflicted dogs (Fig. 7).
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) represents 10–7 M isoproterenol+10–7 M ICI118551 in afflicted animals. The lower curve (
) includes the results from all other groups (afflicted and afflicted+isoproterenol; unafflicted and afflicted+CGP20712A; unafflicted+ICI118551, unafflicted and afflicted+zinterol, 10–6 M). *, P<0.05 compared with predrug control.In the absence of isoproterenol, neither delayed afterdepolarizations nor triggered activity occurred. When sustained stimulation at CLs of 250 ms was abruptly stopped in the presence of 10–7 M isoproterenol, triggered action potentials and delayed afterdepolarizations were noted in both unafflicted and afflicted animals (Fig. 8) with the incidence of triggered action potentials higher in the latter. CGP20712A eliminated the triggered action potentials and delayed afterdepolarizations in both groups. In contrast, ICI118551 increased the incidence of triggered action potentials, with incidence being higher in the afflicted group. With zinterol 10–6 M (the only concentration that had an effect on the action potential), the incidence of triggered activity was significantly lower than with isoproterenol in both groups.
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Limited data are available regarding β1- vs. β2-adrenoceptor contributions to modulation of electrophysiologic properties and arrhythmia mechanisms in the canine heart. In normal dogs, β1-adrenoceptors predominate in modulating action potential repolarization. Moreover, β2-adrenergic stimulation is reported not to affect refractoriness in intact canine hearts [10], and in isolated Purkinje fibers, the β1-component of isoproterenol action predominates in shortening action potential duration (adult dogs) and elevating the plateau (neonates) [9]. The relative contributions of β1- and β2-adrenoceptor subtype-mediated pathways to repolarization or delayed afterdepolarizations in ventricular myocardium were not investigated in these studies.
However, in sheep Purkinje fibers, delayed afterdepolarizations induced by strophanthidin in the presence of isoproterenol are partially antagonized by the β1-blocker CGP20712A but not by the β2-blocker ICI118551 suggesting that β1-adrenoceptors are primarily responsible for the triggered activity [11].
In the present study APD was significantly longer in myocardium of afflicted than unafflicted dogs, particularly, at CLs
1000 ms. We have recently demonstrated that there is also greater spatial inhomogeneity of repolarization among different regions of the left ventricles of afflicted dogs [12]. Hence, the substrate in the afflicted dogs includes both excessively prolonged repolarization and its increased dispersion, a setting that would favor the occurrence of arrhythmias.
We used isoproterenol as a non-selective β1 and β2-antagonist. Isoproterenol's effects in increasing plateau amplitude and abbreviating the total duration of repolarization in canine ventricular myocardium [1,13] occur primarily through an increase in ICa and IK, although all the major ionic currents contributing to repolarization are affected [14]. Isoproterenol also induces delayed afterdepolarizations and triggered activity in canine ventricular cardiomyocytes, as a result of activation of INa/Ca [15]. In our experiments the isoproterenol-induced shortening of APD was greater in afflicted dogs than in controls, while the magnitude of plateau elevation was similar in both groups. Although the plateau effect was induced by the stimulation of the β1 receptor, which was blocked by CGP20712A, there appeared to be β2-adrenergic modulation as well, in that in the afflicted group the plateau elevation was partly suppressed by ICI118551. Zinterol increased plateau amplitude in both groups but this was seen only at 10–6 M, a concentration that has β1-adrenergic effects, as well [6]. Hence, it is unlikely that there is any β2-adrenergic action to modulate the plateau.
In conditions of presumed calcium overload induced with rapid stimulation (CL=250 ms) and 10–7 M isoproterenol the incidence of triggered action potentials was greater in preparations from afflicted than unafflicted dogs (Fig. 8). The phenomenon was clearly mediated by β1-adrenergic receptors as indicated by the response to CGP20712A, an observation consistent with the findings of others for triggered activity [11] in sheep Purkinje fibers. The greater incidence of triggered activity in the afflicted group can be explained based on our previous demonstration of greater β receptor number and adenylyl cyclase response to isoproterenol in the anteroseptal region of the left ventricles of these animals [1]. Moreover, the effect of ICI118551 plus isoproterenol to further increase the incidence of triggered activity and of premature depolarizations at cycle lengths of 1000 and 500 ms (Fig. 7) suggests that the normal function of the β2-adrenergic receptor pathways here may be to antagonize the β1-adrenergic receptor pathway that facilitates Ca overload. This interpretation is supported by our observation that in the presence of zinterol 10–6 M the incidence of triggered activity is markedly lower than that seen with isoproterenol alone or isoproterenol plus ICI118551 (Fig. 8).
Identifying individual steps in β1-adrenergic receptor signal transduction pathways was not the focus of the present study. Interpreting our data in light of current information about these pathways, it is generally accepted that under normal physiologic conditions β1-adrenergic receptors couple to the stimulatory GTP protein Gs, to stimulate adenylyl cyclase which leads to accumulation of cAMP, activation of cAMP-dependent protein kinase A (PKA) and the phosphorylation of key target proteins including the L-type calcium channel, delayed rectifying potassium current, phospholamban, and troponin I (reviewed in [16]). The increase of inward calcium current explains the elevation of the action potential plateau (Fig. 3), while the increase in the repolarizing potassium current can account for the shortening of APD90 (Fig. 5). Our data indicate that these regulatory cascades linked to β1-adrenergic receptor are functional in both unafflicted and afflicted dogs, although β1-adrenergic effects of isoproterenol on repolarization differ to the extent that there is less plateau elevation and more APD90 shortening in afflicted animals.
In physiologic settings, the β2-receptor couples both to Gs and to a pertussis toxin-sensitive inhibitory Gi protein [17] so that functional responses to β2-adrenergic receptor stimulation may occur through a cAMP-PKA independent mechanism. As a result, β2-adrenergic activation also augments L-type calcium current but through different regulatory cascades than β1 [18]. However, an increase in L-type calcium current should induce a plateau elevation that did not occur with isoproterenol plus CGP20712A in our experiments (Fig. 3). It is likely that failure to see changes in the plateau in this and other [19] studies reflects the overall balance attained between effects on the Ca channel and on outward, repolarizing current.
The electrogenic sodium/calcium exchange current and calcium-activated chloride conductance both contribute to delayed afterdepolarizations and triggered activity in conditions of calcium overload [15]. In our experiments, calcium overload was likely achieved through an increase of L-type calcium current during high rates of stimulation. CGP20712A, which blocks isoproterenol-induced increases in calcium current, decreased the incidence of triggered activity to zero (Fig. 8), and blocks the plateau elevation induced by isoproterenol (Fig. 3). This suggests that activation of β2-adrenoceptors may not contribute to intracellular calcium overload here. However, that in the presence of ICI118551, the incidence of triggered activity increases beyond that recorded in the presence of isoproterenol alone (Figs. 7 and 8
) is consistent with β2 receptor-mediated attenuation of the β1 component. Because the high incidence of triggered activity in the presence of ICI118551 is not associated with a change in plateau height, it is difficult to ascribe this arrhythmia to an increase in inward calcium current. Given that isoproterenol influences intracellular calcium balance through the phosphorylation of phospholamban that can activate calcium uptake by sarcoplasmic reticulum [20] the attenuation of triggered activity induced by β2-adrenoceptor stimulation may involve (among others) this regulatory pathway.
The implications of our findings to the arrhythmias that occur in the intact animal are as follows: we have previously shown that triggered activity attributable to delayed afterdepolarizations occurs in about 17% of afflicted animals [1] the remainder of the arrhythmias are pause-dependent and likely associated with early afterdepolarizations [1,21]. The role of β1-adrenergic receptor stimulation to provoke the delayed afterdepolarization-induced arrhythmias is clear in the current experiments and is likely in the intact animals as well, as the clinical arrhythmia appears to be induced by sinus tachycardia [1]. It is possible that the mechanisms involved here also apply to catecholamine- or exercise-induced tachycardias that occur in human subjects.
Time for primary review 21 days.
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Acknowledgements |
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The authors express their gratitude to Dr. Natalia Egorova for her assistance in performing certain of the experiments 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 and by a grant from the Wild Wings Foundation.
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