Muscarinic stimulation and pinacidil produce similar facilitation of tachyarrhythmia induction in rat isolated atria

Journal of Molecular and Cellular Cardiology 65 (2013) 120–126

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Original article

Muscarinic stimulation and pinacidil produce similar facilitation of tachyarrhythmia induction in rat isolated atria Nivaldo Zafalon Jr. a b

, Natália F. Oshiyama a, José W.M. Bassani a,b, Rosana A. Bassani b,⁎

a,1

Department of Biomedical Engineering/FEEC, University of Campinas, Caixa Postal 6040, 13084-971 Campinas, SP, Brazil Center for Biomedical Engineering, University of Campinas, Caixa Postal 6040, 13084-971 Campinas, SP, Brazil

a r t i c l e

i n f o

Article history: Received 13 September 2013 Accepted 9 October 2013 Available online 17 October 2013 Keywords: Atrial tachyarrhythmia Electrical propagation Inward rectifier potassium currents KATP channel Action potential

a b s t r a c t Atrial tachyarrhythmias, the most common type of cardiac arrhythmias, are associated with greater stroke risk. Muscarinic cholinergic agonists have been shown to facilitate atrial tachyarrhythmia maintenance in the absence of cardiac disease. This has been attributed to action potential shortening, which enhances myocardial electrical anisotropy, and thus creates a substrate for reentrant excitation. In this study, we describe a similar effect of the ATP-sensitive K+ channel (KATP) opener pinacidil on tachyarrhythmia induction in isolated rat atria. Pinacidil, which activates a weakly inwardly-rectifying current in isolated atrial myocytes, enhanced arrhythmia induction in the right and left atria. This effect was abolished by the KATP blocker glibenclamide, but not by atropine, which rules out a possible indirect effect due to stimulation of acetylcholine release. However, pinacidil attenuated carbachol-induced tachyarrhythmia facilitation, which may indicate that the action of these agonists converges to a common cellular mechanism. Both agonists caused marked action potential shortening in isolated atrial myocytes. Moreover, during arrhythmia in the presence of pinacidil and carbachol, the atrial vectorelectrographic patterns were similar and consistent with reentrant propagation of the electrical activity. From these results, we conclude that the KATP channel opening is pro-arrhythmic in atrial tissue, which may pose as an additional risk in the scenario of myocardial hypoxia. Moreover, the similarity of the electrophysiological effects of pinacidil and carbachol is suggestive that the sole increase in background K+ conductance is sufficient for atrial tachyarrhythmia facilitation. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Atrial tachyarrhythmias (AT), the most frequent type of sustained arrhythmias, are associated with significant morbidity and mortality, and considered as a risk factor for stroke, cardiomyopathy and peripheral embolism (e.g., [1–4]). Because AT prevalence increases with age [1,5], it is expected that a considerable part of the population worldwide should be afflicted by it in the next decades, in face of the progressive rise in life expectancy in most countries. Thus, it is not surprising that much effort has been expended for better understanding of the mechanisms Abbreviations: ACh, acetylcholine; AP, action potential; APD, action potential duration; AR, right atrial spontaneous rate; AT, atrial tachyarrhythmia; cAMP, 3′-5′ cyclic adenosine monophosphate; CCh, carbachol; High, high-inducibility stimulation protocol; IK(ACh), acetylcholine-regulated K+ current; IK(ATP), ATP-sensitive K+ current; KATP channel, ATPsensitive K+ channel; Low, low-inducibility stimulation protocol; SUR, sulfonylurea receptor; TI, tachyarrhythmia induction index; Vm, membrane potential. ⁎ Corresponding author at: Centro de Engenharia Biomédica, Universidade Estadual de Campinas, R. Alexander Fleming 163, 13083-881 Campinas, SP, Brazil. Tel.: +55 19 3521 9289, +55 19 3521 9261; fax: +55 19 3289 3346. E-mail addresses: profnivaldo@fiap.com.br (N. Zafalon), [email protected] (R.A. Bassani). 1 Present address: Department of Microcontrolled Systems, Faculdade de Informática e Administração Paulista (FIAP), Av. Lins de Vasconcelos 1222, 01538-001 São Paulo, SP, Brazil. 0022-2828/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.yjmcc.2013.10.004

underlying this type of arrhythmia, as well as for improvement of the therapeutic and preventive approaches [3]. It is well known that stimulation of muscarinic cholinergic receptors can facilitate AT occurrence and maintenance [6–9], a mechanism likely to underlie the vagally-mediated form of atrial fibrillation [10–12]. This effect has been largely attributed to the establishment of a functional substrate for reentrant propagation of the electrical activity by abbreviation of the action potential (AP) and thus of the refractory period of atrial myocytes [6,7,9,13,14]. In the adult heart, the predominant muscarinic receptors are of the m2 subtype, which are coupled to a pertussis toxin-sensitive G protein (Gi/Go). Classically, muscarinic stimulation produces functional antagonism of the β-adrenergic pathway. Most of this antagonism is exerted on the cAMP synthesis, as the α subunit of the G protein coupled to m2 receptors inhibits adenylate cyclase activity [15]. In the atrial tissue, m2 receptor stimulation recruits an additional mechanism: the activation of inward rectifier K+ channels by the βγ subunits of the G protein, resulting in the activation of a ligand-regulated, background hyperpolarizing current (IK(ACh)), to which is largely attributed the negative chronotropic effect, as well as the AP shortening effect of muscarinic agonists [15–19]. Using an in vitro rat model of muscarinic AT [20], Zafalon et al. [8] observed that arrhythmia induction and maintenance could be suppressed by agents that activate the cAMP–protein kinase A signaling

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pathway, such as β-adrenoceptor agonists, as well as phosphodiesterase and protein phosphatase inhibitors. However, amiodarone, which blocks IK(ACh) and prevents AP shortening by muscarinic agonists [21,22], was also able to antagonize the proarrhythmic effects of muscarinic stimulation [8]. Thus, although both mechanisms seem to be involved in the muscarinic tachyarrhythmia facilitation, it was not clear if this effect could be produced by recruitment of only one of them. It has been shown that IK(ACh) activation is necessary for the manifestation of muscarinic proarrhythmogenesis [23]. However, it still remained to be demonstrated whether the increase in background K+ conductance alone is sufficient for AT facilitation. The goal of the present study was to investigate the ability of the induction of the ATP-sensitive K+ current (IK(ATP)), a muscarinicindependent, background K+ current, to reproduce the proarrhythmic effect of muscarinic stimulation. The pore-forming subunits of the channels that conduct IK(ACh) and IK(ATP) belong to the family of inward rectifier K+ channels (Kir family: Kir3.x and Kir6.x, respectively), and are well expressed in atrial cells [19,24]. The ATP-sensitive K+ (KATP) channel has constitutive activity and is negatively regulated by intracellular adenine nucleotides: IK(ATP) may be induced by ATP dissociation from its binding sites at the channel or by pharmacological agents that cause the channel to open by binding to the sulfonylurea receptor (SUR), a regulatory subunit [19]. In this study, we compared the effects of the KATP channel opener pinacidil [25] and of the muscarinic cholinergic agonist carbachol (CCh) on the electrical behavior of both unicellular and multicellular atrial preparations, to explore the hypothesis that the increase in sarcolemmal background conductance to K+ is sufficient to promote AT facilitation. 2. Materials and methods

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while the high inducibility protocol (High) consisted of a triple train at 67 Hz, with 3 blocks of 20 pulses separated by 1.3 s intervals. Only one induction protocol was used in each preparation, i.e., before and during exposure to drugs. A trial was considered as the single application of a given induction protocol, whereas an episode was the succession of 8–10 trials applied at 1 min intervals. The tachyarrhythmia induction index (TI) was the fraction of trials in an episode that were successful at evoking AT, which was identified as high rate (N 10 Hz) oscillations of the electrographic signal after the electrical stimulation was interrupted, with duration greater than 10 s (Fig. 1). If the AT lasted longer than 2 min, it was reverted with the same stimulation protocol used for its induction. The interval between episodes was at least 15 min. 2.3. Experimental protocol In the right atria, the stimulation protocol for arrhythmia induction was applied in the absence (control) and presence of increasing concentrations of pinacidil or CCh. In some cases, CCh was added to enhance TI prior to pinacidil exposure. Some experiments were performed in the presence of 1μM atropine and/or 30μM glibenclamide, to which atria were exposed for 30 min prior to TI determination. Pinacidil and CCh effects were assessed after 5–7 min exposure. The total duration of the experiments (including the stabilization period) did not exceed 3.5 h, during which AT induction was previously shown to be reproducible [20]. Solutions were prepared with salts of analytical grade. All drugs were from Sigma Chem Co. Stock solutions were kept at −20° and diluted immediately before use.

2.1. Isolated atrial preparations

2.4. Atrial vectorelectrogram

The right and left atria (weighing 42.4 ± 5.2 mg, N = 22; and 34.4 ± 1.9 mg, N = 6, respectively) were isolated from male adult Wistar rats (6–8 month-old) after euthanasia by exsanguination following cerebral concussion. The protocol for animal care and use was in accordance to the Brazilian laws regarding the use of experimental animals and was approved by the institutional Committee for Ethics in Animal Use (CEEA-CEUA/IB/UNICAMP, doc. number 773-1 and 2587-1C). Atria were mounted horizontally (dorsal surface up) under 5 mN preload in a circular chamber containing modified Krebs–Henseleit solution (mM composition: 115 NaCl, 4.5 KCl, 1.2 KH2PO4, 1.5 MgSO4, 25 NaHCO3, 2.5 CaCl2, 11 glucose, pH 7.4) at 36.5 °C, gassed with 95% O2/5% CO2 [8], to which 0.1 μM propranolol was added to block βadrenoceptors. Experiments started after a 45 min stabilization period. Atrial electrograms were recorded with coated Ag–AgCl wire electrodes and atria were electrically stimulated with a pair of platinum wire electrodes, as described elsewhere [26]. For atrial myocyte isolation, the hearts were perfused via the aorta with Ca2+-free Krebs–Henseleit solution at 37°C containing collagenase I (0.6 mg/ml; Worthington Biochem, Lakewood, NJ, USA) for 15 min. Atria were then removed and incubated with the collagenase solution at 37 °C for additional 15 min. The tissue was rinsed and triturated in a cardioplegic solution [27]. Cells were stored at 4 °C and used within 4 h after isolation.

The atrial vectorelectrogram was determined according to Zafalon et al. [26] from electrograms recorded simultaneously (1kHz acquisition rate) at two leads oriented at 60° (equivalent to the D1 and D2 leads in the Einthoven's triangle). After signal filtering (100 Hz low-pass and

2.2. Tachyarrhythmia induction Atria were field-stimulated with trains of biphasic rectangular voltage pulses with 1 ms total duration, and amplitude 20% higher than the threshold for arrhythmia induction, determined previously in each preparation. Electrical stimulation was applied only during the arrhythmia induction trials. Two stimulation protocols were used, with different effectiveness at tachyarrhythmia induction [8]: the low inducibility protocol (Low) was a single train of 50 pulses at 30 Hz,

Fig. 1. Electrogram traces recorded from an isolated rat right atrium exposed to 1 μM atropine, in the absence (A) and in the presence of 20 μM pinacidil (B), using the high inducibility stimulation protocol. The black bars indicate the application of the last block of the stimulus train (the previous 2 blocks did not evoke arrhythmia, and were omitted for clarity).

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60 Hz notch filters), one of the components was transformed for conversion to the orthogonal coordinate system. Vectorelectrographic loops, which describe the trajectory of the mean electrical vector during the propagation of the electrical activity throughout the atrial tissue, were obtained by plotting the transformed component against the other component.

2.6. Statistical analysis

2.5. Action potential and IK(ATP) recording

3. Results

Membrane potential (Vm) was recorded in isolated atrial myocytes under whole-cell current clamp (ruptured patch) using micropipettes with 5–8 MΩ resistance when filled with the internal solution (mM composition: 130 potassium glutamate, 7 NaCl, 10 HEPES, 0.5 EGTA; pH 7.2). Cells were perfused with modified Tyrode's solution (mM composition: 140 NaCl, 6 KCl, 1.5 MgCl2, 5 HEPES, 11 glucose, 1 CaCl2; pH 7.4) at 23 °C. Action potentials were triggered at 0.5 Hz by 2 ms-long, incremental square current pulses (0.3 nA steps) before and 5 min after addition of 0.6 μM CCh or 20 μM pinacidil. Membrane currents were recorded under voltage-clamp (3–5 MΩ pipette resistance) in response to a voltage ramp from −120 to 60 mV (holding potential = −90 mV) with 500 ms duration. The solutions used were the same for Vm recording, except that the bath CaCl2 concentration was 0.5 mM. The IK(ATP) was considered as the difference of the current recorded in the presence and absence of 20 μM pinacidil. The signals were recorded with an Axopatch 200B amplifier (Axon Instruments, Inc., Union City, CA, USA), acquired at 10 kHz, filtered at 5 kHz, and corrected for the liquid junction potential. Action potential duration was measured at 30%, 50% and 90% repolarization (APD30, APD50 and APD90, respectively).

Under basal conditions, application of the Low stimulation protocol resulted in poor tachyarrhythmia induction in either the right or left atria. Pinacidil treatment did not affect significantly the spontaneous beating rate (AR) in the right atria (3.77 ± 0.14 and 3.65 ± 0.14 Hz in the absence and presence of 20 μM pinacidil, respectively; N = 4; p N 0.62, t test), but it markedly facilitated tachyarrhythmia induction in a concentration-dependent manner in both the right and left atria (p b 0.02, analysis of variance; Figs. 1 and 2A, B). Whereas AT was induced in less than 10% of the trials under control conditions, induction was successful in more than 65% of the trials in the presence of 20 μM pinacidil. This effect was completely reversible by pinacidil washout (Figs. 2A, B). Similar facilitation of AT induction was achieved by exposure to CCh (p b 0.001; Fig. 2C), as previously reported [8,20]. It should be observed that a significant positive relationship between AT rate and agonist concentration was seen for CCh (p b 0.001), but not for pinacidil (p N 0.98), which did not enhance the AT rate above the values seen in the drug-free condition (Fig. 2D). In this in vitro right atrium model, tachyarrhythmia induction by electric stimulation in the absence of drugs seems to completely rely on the release of endogenous acetylcholine (ACh) stores [8,20]. To ascertain whether pinacidil exerted its effects via occupation of muscarinic

Data are presented for paired samples or measures followed by statistical comparison. occur when p ≤ 0.05.

as mean ± standard error. Student's t test one-way analysis of variance for repeated the post-hoc Dunnett's test was used for Statistical significance was considered to

Fig. 2. Pinacidil increased the tachyarrhythmia induction index (TI) in a concentration-dependent manner in isolated rat right (A) and left atria (B). A similar effect was observed during exposure to carbachol (CCh, panel C) in the right atria. D: the mean AT rate in the right atria in the absence and presence of increasing CCh and pinacidil concentrations. The low inducibility stimulation protocol was used in these experiments. Data are means ± SEM; N was 4 for data in panels A and B, and 5 for panels C and D. *p b 0.05 vs. in the absence of drugs (Dunnett's test).

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receptors or by promoting ACh release from cholinergic nerve terminals, atria were treated with 1 μM atropine before and during pinacidil incubation. To maximize AT induction under these conditions, we used the High stimulation protocol. As shown in Fig. 3A, this protocol induced tachyarrhythmia in more than 70% of the trials in the absence of drugs, but it was totally ineffective in the presence of atropine, as observed previously [8,20]. Nevertheless, atropine did not prevent tachyarrhythmia facilitation by pinacidil (p b 0.001, analysis of variance), which, at 20 μM, was able to restore TI to the pre-atropine value, and to elevate TI above 0.9 at 50 μM. Fig. 3B shows that, although pinacidil could increase TI in the presence of atropine, its effect was abolished by the addition of the KATP channel antagonist glibenclamide. Neither atropine, nor glibenclamide significantly affected AR (3.70 ± 0.10 and 3.70 ± 0.11 Hz before and after atropine addition, respectively, p N 0.39; 3.05 ± 0.17 and 3.10 ± 0.08 Hz before and after glibenclamide addition, respectively, p N 0.84; N = 4). We have previously shown that exogenous muscarinic cholinergic agonists are able to produce a concentration-dependent increase in TI in atria stimulated with the Low protocol [8]. Aiming at investigating whether pinacidil and muscarinic stimulation produced interacting effects, the former was tested in the right atria in which tachyarrhythmia induction by the Low protocol was previously enhanced by the addition of CCh. The muscarinic agonist produced a significant, concentrationdependent negative chronotropic effect (p b 0.02) and greatly enhanced TI (p b 0.001; Table 1). Surprisingly, the addition of pinacidil, although not affecting AR (p N 0.99), depressed TI (p b 0.001), thus reverting the CCh effect (Table 1). Membrane current was measured in isolated atrial myocytes to confirm the activation of IK(ATP) by pinacidil in this preparation. As seen in Fig. 4, 20 μM pinacidil induced a current with reversal potential of approximately −77 mV, and weak inward rectification at Vm values

Fig. 3. Tachyarrhythmia induction index (TI) determined in isolated rat right atria using the high inducibility stimulation protocol. The increase in TI by pinacidil occurred in the presence of atropine (A, B), but was abolished by glibenclamide (B). Data are means ± SEM (N = 4). A: #p b 0.01 vs. in the presence of atropine; *p b 0.01 vs. in the absence of pinacidil. B: *p b 0.01 vs. atropine + pinacidil (Dunnett's test).

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Table 1 Spontaneous atrial rate at sinus rhythm (AR) and arrhythmia induction index (TI) in isolated rat right atria in the absence and presence of carbachol (CCh) and pinacidil, using the low inducibility stimulation protocol. Pinacidil was added during exposure to 0.6 μM CCh. Data are mean ± SEM (N = 4). CCh

Pinacidil

AR (Hz)

TI

– 0.3 μM 0.6 μM 0.6 μM 0.6 μM 0.6 μM

– – – 5 μM 10 μM 20 μM

3.72 ± 0.05 1.97 ± 0.66⁎ 1.27 ± 0.44⁎ 1.37 ± 0.47⁎ 1.42 ± 0.50⁎ 1.42 ± 0.49⁎

0.06 ± 0.06 0.28 ± 0.06 0.78 ± 0.08⁎ 0.31 ± 0.08# 0.22 ± 0.15# 0.12 ± 0.05#

⁎ p b 0.05 vs. in the absence of drugs. # p b 0.01 vs. 0.6 μM CCh alone (Dunnett's test).

negative to 60 mV, which is characteristic of IK(ATP) and contrasts with the strong rectification shown by IK(ACh) (e.g., [19,24,28,29]). Because a substantial, nearly linear outward current was present at positive Vm values within the range of the AP peak, one might predict that pinacidil might, in addition to abbreviating the AP, depress its peak voltage. In isolated atrial myocytes, treatment with 0.6 μM CCh caused significant diastolic hyperpolarization (Vm changed from −74.5 ± 0.7 to −93.5±1.1mV; N=5; p=0.001, Student's t test for paired samples) without significant change in the AP peak (41.3 ± 9.2 and 30.8 ± 11.1 mV in the presence and absence of CCh, respectively; p N 0.44). As expected, marked acceleration of the AP repolarization was observed (p b 0.01; Figs. 5A and B). The effects of 20 μM pinacidil were qualitatively similar to those of CCh. Diastolic hyperpolarization was considerably smaller (diastolic Vm: −79.4 ± 5.5 vs. −75.4 ± 4.6 to mV; N = 6; p = 0.054), and in some cells pinacidil depressed the AP peak, as predicted from the IK(ATP) current–voltage relationship (Fig. 5C), although this effect was not significant in the pooled cell population (6.7 ± 12.4 vs. 30.9 ± 13.0 mV; p N 0.12). As CCh, pinacidil decreased APD at all levels of repolarization (p b 0.01). Similarly as observed for arrhythmia induction, the effects of pinacidil on the atrial AP, but not those of CCh, were reverted after washout. It is interesting to observe the association between the effects of the agonists on the diastolic Vm and the AR: while CCh caused strong diastolic hyperpolarization in isolated myocytes and depressed right atrial chronotropic activity, none of these effects were significant for pinacidil. Vectorelectrograms were determined in the right atria with the aim of gaining insight into the pattern of electrical propagation during the tachyarrhythmia evoked in the presence of CCh and pinacidil. Fig. 6A shows that at sinus rhythm the trajectory of the mean electrical vector crossed the origin at each activity cycle, indicating the existence of electric rest over all the tissue, i.e., electrical diastole [26]. However, over successive excitation cycles during muscarinic AT, an elliptical, nearly stable trajectory was observed, which never crossed the origin (Fig. 6B). The same kind of trajectory was observed during the AT

Fig. 4. Current density × voltage relation determined in isolated rat atrial myocytes during the application of a 500 ms-long voltage ramp from −120 to 60 mV. The ATP-dependent K+ current (IK(ATP)) was considered as the difference of the membrane current measured in the presence and in the absence of 20 μM pinacidil. Points are means and SEM (N = 4).

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Fig. 5. Action potentials recorded from isolated rat atrial myocytes before (control) and during exposure to 0.6 μM carbachol (CCh, A) or 20 μM pinacidil (C). Mean and SEM values of action potential duration at 30%, 50% and 90% repolarization (APD30, APD50 and APD90, respectively) before and during exposure to CCh (B) or pinacidil (D). *p b 0.01 vs. control (Student's t test for paired samples).

facilitated by pinacidil in the presence of atropine (Fig. 6C), which indicates that the overall pattern of electrical propagation was similar in both cases, and consistent with reentrant activity (i.e., stable trajectory and absence of electrical diastole). 4. Discussion The present results indicate the marked parallelism of the effects of the muscarinic agonist CCh and the KATP channel opener pinacidil at enhancing tachyarrhythmia induction in rat right and left atria, despite the lack of similarity of their chronotropic effects. Both muscarinic stimulation and pinacidil, which induce an increase of background membrane K+ conductance via different inward rectifier K+ channels, greatly accelerated repolarization in atrial myocytes and produced a comparable alteration in the pattern of atrial electrical propagation, which is suggestive of reentry. Although it has previously been proposed that IK(ACh) is necessary for muscarinic AT induction, as the latter is abolished in IK(ACh)-deficient mice [23], the similarity between the effects of CCh and pinacidil in the present experiments indicates that the induction of a background K+ current per se increases the myocardial susceptibility to AT. In this study, CCh and pinacidil, in addition to producing comparable AP shortening, facilitated AT induction in isolated atria, with an electrical propagation pattern indicative of reentry. The creation of a substrate for reentrant propagation by refractoriness abbreviation (and consequently decrease in the wavelength of the electrical impulse), and enhancing dispersion of repolarization, as well as rotor stabilization, is considered to underlie the pro-arrhythmic effect of both muscarinic agonists or KATP channel openers [6,13,30–33]. In a previous study [8], we observed that muscarinic AT facilitation could be counteracted by either amiodarone (which inhibits IK(ACh) [21,22]) or agents that increase signaling via the canonical β-adrenergic pathway. Although the latter agents may exert their effects by affecting several cellular targets (e.g., ion currents, proteins involved in Ca2 + handling, myofilaments), the data in the present report support the hypothesis that the sole stimulation of a K+ background current seems to suffice

for AT facilitation. A likely possibility to explain the previously observed β-adrenergic anti-arrhythmic influence in this atrial model might be the prevention of IK(ACh)-dependent AP abbreviation, as Sosunov et al. [34] reported that β-adrenergic agonists, although decreasing APD when applied alone, can prolong the atrial AP previously shortened by ACh. We observed that raising CCh concentration led not only to enhanced AT induction, but also to increase in the AT rate, whereas the latter effect was absent for pinacidil. This difference might be related to the involvement of multiple signaling pathways coupled to muscarinic receptors. Alternatively, it is possible that further decrease in impulse wavelength might ensue due to a decrease in the electrical conduction velocity under strong muscarinic stimulation, an effect that is negligible in sub-micromolar agonist concentrations [13,35; Marques JLB, Bassani RA, Bassani JWM, unpublished results]. As a result, the tissue area required for the establishment of reentrant circuits would be reduced, which would possibly give rise to more reentrant wavelets [36]. Pinacidil, on the other hand, although causing AP shortening, was reported not to affect myocardial electrical conduction [37]. Because pinacidil was shown not to affect the atrial APD in the perfused mouse hearts [38], but still could markedly facilitate AT induction and maintenance in the present isolated rat atria model, one might suppose that the latter effect could be attained via an indirect mechanism, namely stimulation of neurotransmitter release from nerve terminals in the atrial tissue [39,40]. However, the proarrhythmic effect of pinacidil was maintained in the presence of not only the β-adrenoceptor blocker propranolol, but also atropine, which consistently suppresses AT induction dependent on ACh release or exogenous muscarinic stimulation [8,20]. This observation, in association with the suppression of pinacidil proarrhythmic effect by the KATP channel blocker glibenclamide [22] and the electrophysiologic effects of pinacidil on atrial myocytes (namely, the activation of an outward current and marked AP shortening), provides evidence of a direct effect of pinacidil on KATP channels in the rat atrial tissue. It should be noted that pinacidil exposure subsequently to AT enhancement by muscarinic stimulation caused an apparently

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atrial tissues, including arrhythmia facilitation [41,42], stresses the translational relevance of the present in vitro rat AT model for the study of KATP channel-related arrhythmogenesis and test of antiarrhythmic therapeutic approaches. The role of IK(ATP) in the susceptibility to arrhythmias has been a controversial issue [32,43]. While the activation of this current may exert a protective influence against certain types of arrhythmias, particularly those involving Ca2 + overload and occurrence of afterdepolarizations [44], in which AP shortening is beneficial, it should also be considered that refractoriness shortening may be associated to the creation of a functional substrate for reentrant propagation, which may be especially significant when in conjunction with generation of ectopic excitation. Studies have shown an increased incidence of arrhythmia upon ischemia and metabolic stress, which can be attributed to enhanced activity of KATP channels [45,46]. Moreover, greater propensity to atrial fibrillation was reported in carriers of a gain-of-function Kir6.1 mutation [47]. The present findings, which show that activation of either muscarinic receptor-dependent or KATP channel-mediated current is sufficient to promote reentrant AT emphasize the potential of both types of channel as promising targets for antiarrhythmic therapy. This is especially relevant in view of recent reports that atrial remodeling by chronic AT and cardiomyopathy leads to enhanced molecular and functional expression of these channels [18,32,42,45]. Moreover, it is possible that muscarinic receptorcoupled channels and KATP channels are not the only ones that might be involved in arrhythmia-promoting an increase in K+ efflux, as a recent study described AP shortening and enhanced arrhythmia susceptibility in post-myocardial infarction ventricular myocytes due to activation of small-conductance Ca2+ activated K+ channels [48]. 5. Conclusions The present data support the hypothesis that the sole increase in background K+ conductance is sufficient for facilitation of AT induction and maintenance. Disclosures None declared. Fig. 6. Vectorelectrograms determined in isolated rat right atria beating at sinus rhythm (A; points are mean ± SEM for 7 successive cycles), and during tachyarrhythmia evoked in the presence of 0.6 μM carbachol (B; 14 successive cycles) and 10 μM pinacidil + 1 μM atropine (C; 37 successive cycles). The arrows indicate the direction of the mean electric vector trajectory.

paradoxical inhibition of AT facilitation, although both pinacidil and CCh isolatedly exerted similar proarrhythmic effects. While this indicates that their effects are non-additive, it also may be interpreted as the existence of a common mechanism by which both drugs facilitate arrhythmia, namely the increase in background K+ efflux. The present findings are in agreement with the mutual attenuation of atrial inwardly rectifier K+ currents (IK(ATP), IK(ACh) and IK1) described by WellnerKienitz et al. [29], who attributed this interference to changes in the driving force for the transmembrane K+ flux, such as subsarcolemmal depletion of the ion by the concurrent stimulation of another inwardly rectifier K+ current. It is also important to point out the contrast between the pharmacological profile of KATP channels in the atria of two common rodent models: rat and mouse. While the latter is unresponsive to pinacidil, which is suggestive of a low level of expression of the SUR2A subunit via which pinacidil promotes channel opening [38], in rat atrial myocardium, where SUR2A is well expressed [28], pinacidil induces a robust IK(ATP) and causes marked AP shortening [28,29; present results]. The resemblance of pinacidil effects in rat and human

Acknowledgments We are indebted to Ms. Elizângela S. Oliveira and Mr. Sérgio P. Moura for the technical support. This study was partially supported by CNPq (N. 300632/2005-3, 302996/2011-7 and 141175/2002-8) and FAPESP (2011/19805-3). References [1] Benjamin EJ, Levy D, Vaziri SM, D'Agostino RB, Belanger AJ, Wolf PA. Independent risk factors for atrial fibrillation in a population-based cohort: the Framingham Heart Study. JAMA 1994;271:840–4. [2] Cha YM, Redfield MM, Shen WK, Gersh BJ. Atrial fibrillation and ventricular dysfunction: a vicious electromechanical cycle. Circulation 2004;15:2839–43. [3] Prystowsky EN. The history of atrial fibrillation: the last 100 years. J Cardiovasc Electrophysiol 2008;19:575–82. [4] Lin C, Edwards C, Armstrong GP, Scott A, Patel H, Hart H, et al. Prevalence and prognostic significance of left ventricular dysfunction in patients presenting acutely with atrial fibrillation. Clin Med Insights 2010;4:23–9. [5] Feinberg WM, Blackshear JL, Laupacis A, Kronmal R, Hart RG. Prevalence, age distribution and gender of patients with atrial fibrillation: analysis and implications. Arch Intern Med 1995;155:469–73. [6] Allessie MA, Lammers WJEP, Bonke IM, Hollen J. Intra-atrial reentry as a mechanism for atrial flutter induced by acetylcholine and rapid pacing in the dog. Circulation 1984;70:123–35. [7] Schuessler RB, Grayson TM, Bromberg BI, Cox JL, Boineau JP. Cholinergicallymediated tachyarrhythmias induced by a single stimulus in the isolated canine right atrium. Circ Res 1992;71:1254–67. [8] Zafalon Jr N, Bassani JWM, Bassani RA. Cholinergic–adrenergic antagonism in the induction of tachycardia by electrical stimulation in isolated rat atria. J Mol Cell Cardiol 2004;37:127–35.

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