Adrenergic regulation of a key cardiac ... - Wiley Online Library

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J Physiol 586.2 (2008) pp 627–637

Adrenergic regulation of a key cardiac potassium channel can contribute to atrial fibrillation: evidence from an IKs transgenic mouse Kevin J. Sampson1 , Cecile Terrenoire1 , Daniel O. Cervantes1 , Riyaz A. Kaba2 , Nicholas S. Peters2 and Robert S. Kass1 1 2

Department of Pharmacology, Columbia University Medical Center, New York, NY, USA Department of Cardiac Electrophysiology, Imperial College, St Mary’s Hospital, London, UK

Inherited gain-of-function mutations of genes coding for subunits of the heart slow potassium (IKs ) channel can cause familial atrial fibrillation (AF). Here we consider a potentially more prevalent mechanism and hypothesize that β-adrenergic receptor (β-AR)-mediated regulation of the IKs channel, a natural gain-of-function pathway, can also lead to AF. Using a transgenic IKs channel mouse model, we studied the role of the channel and its regulation by β-AR stimulation on atrial arrhythmias. In vivo administration of isoprenaline (isoproterenol) predisposes IKs channel transgenic mice but not wild-type (WT) littermates that lack IKs to prolonged atrial arrhythmias. Patch-clamp analysis demonstrated expression and isoprenaline-mediated regulation of IKs in atrial myocytes from transgenic but not WT littermates. Furthermore, computational modelling revealed that β-AR stimulation-dependent accumulation of open IKs channels accounts for the pro-arrhythmic substrate. Our results provide evidence that β-AR-regulated IKs channels can play a role in AF and imply that specific IKs deregulation, perhaps through disruption of the IKs macromolecular complex necessary for β-AR-mediated IKs channel regulation, may be a novel therapeutic strategy for treating this most common arrhythmia. (Received 24 July 2007; accepted after revision 12 November 2007; first published online 15 November 2007) Corresponding author R. S. Kass: Department of Pharmacology, Columbia University Medical Center, 630 W 168th St, New York, NY 10032, USA. Email: [email protected]

Approximately 2.5 million Americans now suffer from atrial fibrillation (AF), a number that is expected to exceed 5 million by 2050 (Waldo, 2006). AF is associated with increased risk of stroke, heart failure and death. The incidence of AF increases in the presence of structural heart disease and with age (Heist & Ruskin, 2006) and can also occur in association with other cardiovascular conditions including hypertension and heart failure (Camm, 2006; Prystowsky, 2006). At the cellular and molecular level, multiple ion channel changes have been shown to accompany and underlie AF in a number of experimental models (Heist & Ruskin, 2006). A common mechanism has been inferred in which changes in these ionic pathways contribute to decreased atrial action potential duration (APD) and subsequent atrial refractory periods (Heist & Ruskin, 2006). Multiple approaches have been taken in the management of AF including tissue ablation,

K. J. Sampson and C. Terrenoire contributed equally to this work. This paper has online supplemental material. C 2008 The Authors. Journal compilation C 2008 The Physiological Society

anticoagulant prophylaxis, and targeted block of selected ion channels (Camm, 2006; Prystowsky, 2006). However, despite dramatic advances in the therapy of some arrhythmias, contemporary treatment approaches to AF are still seriously limited. In particular, ion channel blocking anti-arrhythmic agents have not reduced mortality, and pro-arrhythmia with these agents is an increasing problem clinically and in drug development (Waldo, 2006), suggesting that an alternative method to regulate specific contributions to the arrhythmic substrate in AF is highly desirable. Among the multiple ion channels known to contribute to AF, several recent studies have refocused attention on possible roles of the slowly activating IKs potassium channel, formed by assembly of KCNQ1α and KCNE1β subunits, as well as the adaptor protein Yotiao (Marx et al. 2002). The discovery of inherited gain-of-function mutations in KCNQ1 which cause both AF and short QT syndrome (Chen et al. 2003; Wolpert et al. 2005) have provided a clear link between increased IKs activity and risk of AF in humans. Of note, both of the reported congenital AF mutations require co-assembly of the channel α and β DOI: 10.1113/jphysiol.2007.141333

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subunits to cause the important functional consequence: markedly increased activation of the IKs channel during repetitive stimulation. It is interesting that subsequent reports have revealed that mutations in auxiliary subunits (KCNE2), which also increase IKs , are also associated with familial forms of AF. Thus, this channel and particularly conditions which promote its activation during repetitive activity, can clearly contribute to the substrate favouring AF. This evidence based on molecular genetics is consistent with pharmacological data which indicate that the use of azimilide, amiodarone and dronedarone, which affect multiple targets including IKs , are better than more specific rapidly activating delayed rectifier (IKr ) blockers in the treatment of AF (Kerr et al. 2006). Finally, heart failure (HF) predisposes to AF and is known to be accompanied by an increase in basal catecholamine levels (Brodde et al. 2006). In addition, the IKs channel is regulated by catecholamines in a manner that promotes increased IKs activity during repetitive stimulation (Marx et al. 2002; Terrenoire et al. 2005). However, in pacing-induced HF in rabbits, it has been reported that IKs is downregulated indicating that its role may be diminished in failing hearts (Tsuji et al. 2006). Here we have investigated a possible role of IKs and its regulation by the sympathetic nervous system (Terrenoire et al. 2005) in AF to test the hypothesis that protein kinase A (PKA)-phosphorylated IKs channels (Kurokawa et al. 2003; Yang et al. 2003) contribute to susceptibility to, and maintenance of, atrial tachyarrhythmias. Our approach was to combine whole animal in vivo studies with cellular in vitro and computational studies to explore a possible role of β-AR-modulated IKs in AF. Methods Transgenic mice and isolation of cardiac atrial myocytes

Transgenic mice expressing hKCNE1–hKCNQ1 fusion protein in the heart have been previously described (Marx et al. 2002; Chiello Tracy et al. 2003). Mice were killed by intraperitoneal injection of a lethal dose of pentobarbital (100 mg kg−1 ) and atrial myocytes were isolated using the methodology of Mitra & Morad (1985) following protocols previously described (Dilly et al. 2004) and approved by the Institutional Animal Care and Use Committee at Columbia University. Single-cell electrophysiology

Cells were plated in culture dishes which were then placed on the stage of an inverted microscope (Nikon Diaphot 200, Nikon Instruments Inc., Melville, NY, USA). IKs was recorded at room temperature (22◦ C)in atrial myocytes from both 3-month-old and 1-year-old transgenic mice. The whole-cell configuration of the

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patch-clamp technique (series resistance, 2–3 M) was carried out with an Axopatch 200B amplifier (Axon Instruments, Union City, CA, USA) using voltage-clamp protocols that have been previously described (Terrenoire et al. 2005). External solutions contained (mm): NaCl 132, KCl 4.8, CaCl2 2, MgCl2 1.2, Hepes 10 and glucose 5; pH 7.4. E-4031 (5 μm, Wako, Osaka, Japan) and nisoldipine (1 μm, gift from Bayer AG, Leverkusen, Germany) were used to block IKr and ICa , respectively. Na+ channels were inactivation eliminated in these experiments by preceding IKS activation pulses with prepulses to −40 mV for 20 ms. The internal solution contained (mm): potassium aspartate 110, ATP-K2 5, EGTA 11, Hepes 10, CaCl2 5.5 (free [Ca2+ ]i was 100 nm) and MgCl2 1; pH 7.3. In some experiments, 1 μm isoprenaline (isoproterenol) (Sigma-Aldrich Corp., St Louis, MO, USA) was combined with 1 μm okadaic acid (EMD Chemicals Inc., La Jolla, CA, USA) in external solutions to stimulate I Ks current. Animals and pre-operative preparation

Male and female wild-type (WT) (IKs −/− ) and transgenic IKs +/+ 3- to 4-month-old (23.0–38.0 g) and 12- to 17-month-old mice (30.0–49.7 g) were anaesthetized by intraperitoneal injection of pentobarbital (0.05–0.07 mg (g body weight)−1 ). Body temperature was monitored during the whole experiment using a rectal probe and was maintained between 34◦ C and 37◦ C with a warming light. Intubation was performed by direct laryngoscopy (Berul et al. 1996) with a catheter connected to a rodent ventilator for continuous mechanical ventilation (respiratory rate, 120 min−1 ). Before surgery, a six-lead ECG (I, II, III, aVR, aVL and aVF) was recorded from the anaesthetized mice by placement of subcutaneous 22-gauge needle electrodes in each limb. Then a 1.1 F eight-electrode catheter (Model ERP-800, Millar Instruments Inc., Houston, TX, USA) was inserted via the jugular vein (Berul et al. 1998) and the tip advanced into the right ventricle, with the more proximal electrodes therefore extending into the right atrium, for recording intracardiac bipolar electrograms as well as for pacing the heart using consecutive electrode pairs. After insertion of the eight-electrode catheter, two ECG leads along with intracardiac signals were recorded throughout the procedure and were pre-amplified, filtered and sampled. All data were acquired, digitized and stored using a waveform recording system (DI-720, DATAQ Instruments, Akron, OH, USA) and analysed on a windaq waveform browser (DATAQ Instruments). Programmed electrical stimulation protocol

Bipolar pacing delivered between adjacent electrodes was performed using 1 ms current pulses with amplitude C 2008 The Authors. Journal compilation C 2008 The Physiological Society

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Adrenergic K+ channel regulation and AF

of two times diastolic capture threshold. A 2 s burst-pacing protocol was applied in an attempt to induce potential atrial arrhythmias (AAs) (Verheule et al. 2004). Burst-pacing protocols commenced at a cycle length (CL) of 40 ms and decreased by 2 ms in each successive burst down to a CL of 20 ms with 3 s intervals between bursts. This burst series was repeated after an interval of 2 min. Reproducibly induced AA is defined in this study as an irregular atrial rhythm lasting for at least 1 s and induced more than once. The identical burst-pacing and stimulation protocols were repeated after peritoneal administration of isoprenaline solution (0.5 mg kg−1 ) prepared in saline. All ECG parameters and analysed intracardiac signals (online supplemental material) and AA inducibility were determined for control and after administration of isoprenaline. The dose of isoprenaline used in these experiments (0.5 mg kg−1 i.p.) produced more than 20% increase in basal heart rate in all groups tested. Western blot experiments

Mouse atrial homogenates (obtained after completion of in vivo experiments) and Chinese Hamster Ovary (CHO) cell lysates (prepared from a cell line stably expressing hKCNE1–hKCNQ1 channels), were size-fractionated on 7.5% SDS PAGE. Immunoblots were developed using a commercial KCNQ1 antibody to detect total KCNQ1 (C20, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Data analysis

Patch-clamp data, shown as mean ± s.e.m., were acquired using pCLAMP 8.0 (Axon Instruments, Union City, CA, USA) and analysed with Origin 7.0 (OriginLab Corp., Northampton, MA, USA) and Clampfit 8.2 (Axon Instruments). Statistical data analysis was assessed with Student’s t test for simple comparisons: differences at P < 0.05 were considered to be significant. Computational methods

Simulation of IKs and its response to adrenergic stimulation was performed using the model formulation previously published (Terrenoire et al. 2005). For whole-cell simulation of atrial action potentials, a revised version of the membrane model of Courtemanche et al. (1998) was used. Our modifications replaced the IKs channel, L-type Ca2+ channel and sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump formulation with that from our previous work allowing these pathways to respond to adrenergic stimulation. The equations governing these channels are reproduced in supplemental materials. All simulations were run in Matlab (Mathworks Co., Natick, C 2008 The Authors. Journal compilation C 2008 The Physiological Society

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MA, USA) and run on a dual processor power Mac computer (Apple Computers, CA, USA). Results Electrophysiological characterization of IKs mice

In order to explore the role of IKs in AAs, we used a transgenic mouse model previously developed and described by us (Marx et al. 2002; Chiello Tracy et al. 2003). This mouse expresses an hKCNE1–hKCNQ1 fusion protein in a cardiac directed manner, reconstituting cardiac IKs channels which are not expressed in WT mice. Here we sought first to determine whether the channel was expressed in atrial myocytes and also whether channel kinetics were sensitive to adrenergic stimulation as is the case in ventricular cells (Terrenoire et al. 2005). Western blotting of atrial tissue was performed in the transgenic mice (IKs +/+ mice) and compared with results from their WT littermates as well as controls of hKCNE1–hKCNQ1 stably expressed in CHO cells. The results indicate expression of the fusion protein in atria of IKs but not WT mice (Fig. 1A). Whole-cell patch-clamp recordings confirmed IKs expression in atrial myocytes isolated from transgenic but not WT mice (Fig. 1B and D). We found that atrial IKs was responsive to isoprenaline with both the characteristic increase in current density (Fig. 1C) as well as significant slowing of deactivation (Fig. 1E), this latter effect favouring beat-dependent accumulation of IKs channels and shortening of APD during repetitive stimulation. We also carried out experiments in WT and IKs +/+ mice to test for compensatory changes in transient outward currents, the major mouse potassium channel currents (Nerbonne & Kass, 2005), and found no difference in the two groups. We further tested for effects of isoprenaline on potassium currents in WT atrial myocytes and found no effect. Finally, we blocked IKS with chromanol 293B (50–100 μm) in atrial myocytes isolated from IKs +/+ mice, and found no change in the effects of isoprenaline on the remaining potassium currents. These control data are summarized as online supplemental material. Susceptibility of IKs mice to AAs

Following the cellular electrophysiological characterization of the mice, we next sought to test susceptibility to AAs using a burst pacing protocol (see Methods) that was repeated before and after application of isoprenaline in whole-animal experiments. Two age groups were studied (3–4 months old and 12–17 months old) to examine the possibility that arrhythmia susceptibility may be affected by age in our transgenic mice. In both WT and IKs +/+ mice, no arrhythmias were seen in control conditions but application of isoprenaline

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made the IKs +/+ mice susceptible to arrhythmia following burst stimulation. After application of isoprenaline, 6 out of 10 IKs +/+ 12- to 17-month-old mice and 3 out of 7 IKs +/+ 3- to 4-month-old mice had sustained reproducible AAs as summarized in Table 1. Independent of age, reproducible AAs were only observed in transgenic mice indicating a contribution of the presence of the IKs channel to induction of AAs in IKs +/+ mice. The strict definition of AAs used above (Methods) leaves out some important information about the nature of all induced arrhythmic events. WT mice in fact experienced arrhythmias after isoprenaline challenge but infrequently and of very short duration. In order to illustrate the IKs channel-dependent prolongation of the observed arrhythmias, we generated histograms (Fig. 2) of the duration and incidence of atrial events for the young and old mice, in the presence and absence of the IKs transgene (WT versus IKs +/+ ). Figure 2 clearly shows the dramatic increase in both the incidence and the duration of atrial tachyarrhythmias seen in the presence of the transgene and β-adrenergic stimulation. Figure 3 shows one example of the short-lived atrial events following burst stimulation in a young WT mouse. Figure 3A shows the electrograms following burst stimulation in control conditions and Fig. 3B shows them following adrenergic challenge and burst stimulation. The data from the lower

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Table 1. Impact of IKs expression on induction of atrial arrhythmias in mice 3- to 4-month-old mice 12- to 17-month-old mice

Baseline Iso

WT (n = 7)

IKs +/+ (n = 7)

WT (n = 6)

IKs +/+ (n = 10)

0/7 0/7

0/7 3/7

0/6 0/6

0/10 6/10

Incidence of atrial arrhythmias following burst pacing in control and following isoprenaline (Iso) application. Atrial arrhythmias are only included here if they lasted longer than 1 s and were reproducible.

right atrial lead in Fig. 3 show abnormal atrial excitation and taken together with the data of lead 1 illustrate an uncoupling of atrial excitation from ventricular excitation. By contrast, the presence of IKs in the transgenic mice caused drastically prolonged events as seen in Fig. 4. Again complex atrial signals and irregular ventricular excitation are present but in this case they last for over 100 s in a self-sustained manner. Taken together, the results (summarized in Fig. 2) suggest that the modulated IKs channel plays a large role in the duration and frequency of the observed arrhythmia events.

Figure 1. Expression and β-AR regulation of IKs in atria of transgenic mice A, immunoblots using anti-KCNQ1 antibodies to detect the fusion protein hKCNE1–hKCNQ1. From left to right: first lane, CHO cells stably expressing hKCNE1-hKCNQ1; second and third lanes, atria from 3-month-old and 1-year-old WT mice, respectively; fourth and fifth lanes, atria from 3-month-old and 1-year-old IKs +/+ mice, respectively. B, typical current recorded in WT atrial myocytes using the stimulation protocol shown above the trace (holding potential, −75 mV; stimulation frequency, 0.06 Hz). C, typical current recorded in an IKs +/+ atrial myocyte using the stimulation protocol shown in B. IKs , first recorded in control (ctrl) conditions was increased by external application of 1 μM isoprenaline with 1 μM okadaic acid (iso/OA). For B and C, dashed line indicates the level of zero current. D, tail current amplitude measured at −40 mV in control conditions in WT (grey column, n = 7) and in IKs +/+ (IKs , black column, n = 7) atrial myocytes. E, IKs deactivation rate measured at −40 mV in IKs +/+ atrial myocytes in control conditions (white column, n = 3) and in the presence of 1 μM isoprenaline with 1 μM okadaic acid (iso/OA, grey column, n = 3). For D and E, asterisks indicate a statistically significant difference (P < 0.05) as assessed with Student’s t test. C 2008 The Authors. Journal compilation C 2008 The Physiological Society

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Simulated channel response to adrenergic challenge: a computational analysis

In order to better understand a possible role of isoprenaline-mediated IKs kinetics in the ability of the mice to sustain an arrhythmic event, we next examined simulated channel activity in a voltage-clamp setting. In so doing, the contribution of this slowly activating channel can be dissected for the extremely fast rates associated with the mouse arrhythmias. As in previous simulations of cardiac ventricular myocyte β-AR signalling, the channel was examined in its unphosphorylated (control) and completely PKA-phosphorylated (maximal isoprenaline exposure) state for a series of square-pulse, voltage-clamp trains. Figure 5A shows the results for a 15 ms square pulse (similar in duration to murine atrial action potentials) driven at 30 Hz. In this figure the continuous lines illustrate the response of IKs channels to a constant depolarizing pulse and are shown for comparison with the steady-state response of the high-frequency pulse trains. Not unexpectedly, the channel accumulates in the open state at this high frequency and despite the fact that only 3–4% of all channels open per pulse, an instantaneous outward K+ current develops over time marking a drastic increase in the contribution of this slowly activating channel to fast, brief depolarization. Figure 5 also shows that the unphosphorylated (control) channel deactivates faster and hence more completely between pulses, and consequently contributes much less current than its phosphorylated counterpart. Figure 5B and C show the effect of altering the pulse duration and frequency on channel accumulation in both states: the kinetic differences between phosphorylated (regulated) states causes the stimulated channel to accumulate more over all rates and durations. For the 40 Hz stimulation rate used in the mouse studies to induce arrhythmias, even a very brief depolarization of 5 ms can result in the accumulation of 11% of IKs channels in the open state thereby contributing a constant outward current throughout the action potential. As action potential morphology can vary in both mouse and human atria, we also repeated these computations using a different plateau potential and with triangular waveforms, and found similar results (see online supplemental material). Thus the modelling indicates that during even very brief murine atrial action potentials, accumulation of IKs will contribute to action potential shortening. In an effort to understand whether IKs channels and isoprenaline-mediated channel kinetics may play a role in human atrial tachyarrhythmias, the voltage-clamp simulations were repeated for 150 ms duration pulses, of the order of human atrial APD, at fast rates. Figure 6A shows the results for a 150 ms voltage pulse applied at 3 Hz where there is very little accumulation of the channel in the basal state and a large accumulation in the β-AR-regulated C 2008 The Authors. Journal compilation C 2008 The Physiological Society

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state. Both the increasing speed of activation and the slowing of deactivation play a role in this accumulation when the diastolic interval is sufficiently short to prevent complete deactivation of the channels between stimuli. Figure 6B, which summarizes results for a wide range of frequencies, clearly indicates that increased adrenergic tone would result in a larger repolarizing current from IKs in all conditions. In a previous paper (Terrenoire et al. 2005), we showed that β-AR stimulation of this channel in concert with β-AR-mediated regulation of L-type Ca2+ channels and the SERCA pump produce ventricular AP shortening at all rates of stimulation investigated. In this study we incorporated our previous kinetic models of these three phosphorylation targets within a human atrial action potential model (Courtemanche et al. 1998) (see online supplemental material). It is not surprising, as seen in Fig. 7, that increased adrenergic tone results in a shortening of the APD as well as changes in AP morphology due to the increases in L-type Ca2+ current. In the steady state, phosphorylation results in a decrease in APD at 90% repolarization (APD90 ) from 246 to 198 ms. This nearly 50 ms decrease is seen at all rates. At 2 Hz, APD90 decreases from 227 ms in the unstimulated myocyte to 174 ms following adrenergic challenge. Similarly at a steady pacing rate of 4 Hz, the decrease in APD90 is from 207 to 144 ms. The shortening of APD plays both a protective role, maximizing diastolic time in the face of adrenergic challenge and concomitant rate increase, and a detrimental

Figure 2. Effect of age and the presence of the IKs transgene on atrial arrhythmia duration in the presence of isoprenaline A and B, WT mice show no sustained events > 1 s regardless of age. C and D, mice homozygous for the hKCNE1–hKCNQ1 channel experience a higher frequency of events with a markedly prolonged duration in both age groups.

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one, shortening APD and thus path length allowing for more stability of arrhythmic events. Discussion The work in this study was largely motivated by recent molecular genetic studies that have refocused interest on possible contributions of the slowly activating delayed rectifier current I Ks to arrhythmia susceptibility in human atria. The discovery of mutations in the IKs α subunit KCNQ1 that cause congenital atrial fibrillation which, at least in some cases, is accompanied by short QT syndrome, unquestionably indicates the potential contribution to AA susceptibility of activated IKs channels (Chen et al. 2003; Hong et al. 2005). Of importance, in both cases the functional consequences of the KCNQ1 mutations

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require coexpression of KCNE1 (the critical IKs auxiliary subunit) and both promote and stabilize IKs channels in the open state. The result is excessive outward current which can dramatically shorten APD in tissue expressing the mutant channels and thus, by implication, both KCNE1 and KCNQ1 clearly contribute to important repolarizing currents in human atria. Subsequent studies revealing contributions of mutations in other KCNE1 family members to inherited atrial fibrillation (Yang et al. 2004), as well as studies in KCNE1-null mice (Temple et al. 2005), reinforce potentially important roles of KCNQ1 and its regulatory subunits in atrial electrical activity. It is interesting that the functional consequences of the mutations identified in congenital atrial fibrillation resemble, to a certain extent, extreme regulatory responses of the IKs channel to β-AR stimulation: a requirement of

Figure 3. Non-sustained atrial arrhythmia (AA) in 3- to 4-month-old wild-type mouse ECG and intracardiac electrograms during right atrial burst pacing at cycle length of 24 ms at baseline (A) and following isoprenaline injection (0.5 mg kg−1 ) (B). AA with irregular atrial electrical activity and variable ventricular response was induced after isoprenaline administration. The termination of this arrhythmia occurred spontaneously 0.86 s after the initiation of arrhythmia. I, ECG lead I; LRA, low right atrial electrogram; STIM, stimulus channel showing the last five pulses in the 2 s train (see Methods). C 2008 The Authors. Journal compilation C 2008 The Physiological Society

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coassembly of KCNQ1 and KCNE1 and beat-dependent increased outward current due, in part, to altered channel deactivation kinetics (Kurokawa et al. 2003; Terrenoire et al. 2005). Here we have examined the role of the incompletely deactivated IKs channel in generating a substrate conducive to atrial arrhythmogenesis, and investigated whether there is a link between the β-AR regulation of the IKs channel and tachyarrhythmia stability in a mammalian whole-animal model. Our results show that the IKs channel is regulated in response to β-AR stimulation in atrial tissue from a transgenic hKCNE1–hKCNQ1 mouse. This regulation results in faster activation and slower deactivation, probably resulting in an increased stabilization of reentrant AAs in mice. Computational analysis explains how the slow-activating channel can play

a role at fast rates even when APD is exceptionally brief, as in the mouse atria, and how the IKs channel regulation (PKA-dependent phosphorylation) may be important in setting the substrate for human tachyarrhythmias. Taken together, this puts a renewed emphasis on a role of the IKs channels in disease states where increased adrenergic tone is expected. IKs phosphorylation and AA induction in mice

Using our previously developed mouse model with the hKCNE1–hKCNQ1 tandem multimer transgene (Marx et al. 2002), we explored the baseline cardiac electrophysiology of these mice and their response to isoprenaline with an emphasis on atrial activity. Use of a transgenic mouse model is advantageous as WT mice have no

Figure 4. Induction of sustained atrial arrhythmia (AA) in 12- to 17-month-old IKs +/+ mouse ECG and intracardiac electrograms during right atrial burst pacing at cycle length of 24 ms at baseline (A) and following isoprenaline injection (0.5 mg kg−1 ) (B). AA, with irregular atrial electrical activity and variable ventricular response was induced after isoprenaline administration. The termination of this arrhythmia occurred spontaneously 109 s after the initiation of arrhythmia. I, ECG lead I; LRA, low right atrial electrogram; STIM, stimulus channel showing the last four pulses in the 2 s train (see Methods). C 2008 The Authors. Journal compilation C 2008 The Physiological Society

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substantial native I Ks current (Harrell et al. 2007). Expression of the channel alone had no substantial effect on atrial effective refractory period (at a pacing CL of 80 ms) or conduction times although it did result in a modest slowing of sinus rate (see Table S-2 in online supplemental material). We next tested the inducibility of AAs in both WT and transgenic mice. For all the mice, no AAs were seen in baseline conditions. However, application of isoprenaline in conjunction with the burst pacing protocol produced atrial arrhythmic events in all groups of mice. In contrast to the baseline electrophysiological measurements, the presence of the channel greatly changed the nature of inducible AAs seen in the mice. In WT mice, no reproducible sustained (> 1 s) events could be observed, whereas prolonged reproducible events were observed in transgenic mice. Because age is a strong risk factor for AAs, we examined two populations of mice both young (3–4 months) and old (12–17months) (Feinberg et al. 1995). As seen in Fig. 2, the IKs +/+ mice, in both age groups, had both a prolongation of the duration of arrhythmic events and a marked increase in the number of observed arrhythmias. These data suggest that the phosphorylated potassium channel participates in the stabilization of very high-frequency arrhythmias which might be due to reentry. The participation of this channel in very brief APs at very high frequency is

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contrary to its well known slow activation process. To better understand the operation of the channels at these rates, simulations were performed for brief square-wave pulses at high frequencies similar to the AA inducing protocols.

Slowed deactivation and channel accumulation

As reported before (Terrenoire et al. 2005), the slow deactivation process of the IKs channel can lead to an accumulation of channels in the open state at fast rates. Using our previously developed channel model that is responsive to β-AR stimulation, we are able to observe how the channel behaves at the extremely fast rates associated with the mouse study. In Fig. 5C, we can see that for a 15 ms depolarization, which is of the order of a mouse APD, channel accumulation is negligible at normal mouse rates (< 10 Hz). This is consistent with the electrophysiological data showing little or no effect of the channel on baseline properties of the myocytes (see online supplemental material, Table S-2). However, as rates escalate due to burst pacing or reentrant tachyarrhythmia, channels accumulate and at all rates the phosphorylated channel accumulates significantly more than the stimulated channel. This behaviour of the IKs channel is consistent with the

Figure 5. Computational modelling of the IKs channel for the stimulus protocol used in the mouse studies Trains of square pulses to +10 mV were applied to the channel for different pulse durations and interpulse (stimulus) rates. A, an example for a 40 Hz stimulus train and a 15 ms pulse; blue lines for the basal channel and red lines for the phosphorylated channel. Monotonically increasing lines show the channel response to a sustained depolarization as a control. The unstimulated channel shows modest accumulation at the end of the train while the phosphorylated channel deactivates more slowly resulting in a larger instantaneous current relative to the total channel density. B and C, summary of the relative accumulation of the basal (blue) and stimulated (red) channel for different pulse durations or rates. C 2008 The Authors. Journal compilation C 2008 The Physiological Society

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marked effect on AAs stability without large changes in baseline Atrial Effective Refractory Period (AERP) or conduction times as seen in our in vivo experiments. These computational results predict that APD would be shortened in our transgenic mice especially at fast rates as reported previously by us in our IKs mouse (Chiello Tracy et al. 2003) and by others in rat preparations (Munoz et al. 2007). Recent work in rat monolayers also has suggested a role for accumulated IKs in altering post-repolarization refractoriness, further altering the arrhythmogenic substrate (Munoz et al. 2007). Extending this computational analysis to examine the effect of channel accumulation with and without adrenergic challenge at human atrial rates and APD, Fig. 6 shows that the phosphorylated channel is predicted to play an increasingly large role, as a function of rate, in the repolarization of atrial myocytes. The slow deactivation makes the function of the channel at fast

Figure 6. Accumulation of current in simulations of IKs for pulse durations consistent with human atrial activity Here we repeated the voltage-clamp simulations as shown in Fig. 5 for 150 ms pulses at different rates. A, the basal (blue) versus stimulated (red) activity for a 150 ms pulse at 3 Hz. Again, the phosphorylated channel results in a significantly increased instantaneous current after pacing at high rates. B, the normalized current (relative to the maximum current density) at the end of the train as a function of stimulus rate. C 2008 The Authors. Journal compilation C 2008 The Physiological Society

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rates significantly different than the characteristic slowly activating channel as a majority of the channels reside in the open state at the beginning of an AP resulting in a large I Ks current throughout the AP. The subsequent APD shortening, reduces reentrant path length thus rendering the atria more capable of supporting self-sustained tachyarrhythmic activity. The increased duration of the atrial events in the IKs transgenic mice is consistent with the idea of the activity of the channel promoting a substrate favourable to stable reentry.

Figure 7. Effect of β-AR-mediated IKs regulation on action potential morphology and duration in a human atrial action potential model A, at 1 Hz, change in β-AR-stimulated regulation of IKs , ICaL and Ca2+ ATPase (SERCA) from their basal levels (continuous line) to stimulated levels (dashed line) results in an increased plateau voltage and decreased APD at 90% repolarization (APD90 ). B, the APD at basal phosphorylation levels (blue) and following stimulation (red) are shown for a series of stimulation rates. At all rates, a nearly 50 ms decrease in APD90 is seen and the unstimulated channel is incapable of pacing one to one beyond 4 Hz whereas the phosphorylated channel continues to adapt.

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Implications for human arrhythmias

Recent clinical trials have had mixed results on the efficacy of class III anti-arrhythmic agents (Scholz, 1994) on AF (Camm, 2006). One drug that is effective in the treatment of AF is amiodarone. This drug has a combination of effects including β-blocker activity as well as classic class III actions. The utility of such a drug is consistent with the mouse model of AAs presented here in that IKs and, in particular, its phosphorylated form may have a large role in setting the substrate for prolonged atrial reentry. We have previously shown that PKA-dependent functional regulation of the IKs channel requires assembly of a macromolecular signalling complex coordinated by binding of the A-kinase anchoring protein Yotiao to the KCNQ1 carboxy terminus (Marx et al. 2002). Our present results raise the possibility that drugs capable of specifically reducing IKs channel phosphorylation and/or the functional consequences of phosphorylation by disruption of the IKs macromolecular complex which would leave β-AR regulation of other downstream targets intact, might prove to be a novel, specific and even more effective approach to management of AF in the future. References Berul CI, Aronovitz MJ, Wang PJ & Mendelsohn ME (1996). In vivo cardiac electrophysiology studies in the mouse. Circulation 94, 2641–2648. Berul CI, Christe ME, Aronovitz MJ, Maguire CT, Seidman CE, Seidman JG & Mendelsohn ME (1998). Familial hypertrophic cardiomyopathy mice display gender differences in electrophysiological abnormalities. J Interv Card Electrophysiol 2, 7–14. Brodde OE, Bruck H & Leineweber K (2006). Cardiac adrenoceptors: physiological and pathophysiological relevance. J Pharmacol Sci 100, 323–337. Camm J (2006). Medical management of atrial fibrillation: state of the art. J Cardiovasc Electrophysiol 17 (Suppl. 2), S2–S6. Chen YH, Xu SJ, Bendahhou S, Wang XL, Wang Y, Xu WY et al. (2003). KCNQ1 gain-of-function mutation in familial atrial fibrillation. Science 299, 251–254. Chiello Tracy C, Cabo C, Coromilas J, Kurokawa J, Kass RS & Wit AL (2003). Electrophysiological consequences of human IKs channel expression in adult murine heart. Am J Physiol Heart Circ Physiol 284, H168–H175. Courtemanche M, Ramirez RJ & Nattel S (1998). Ionic mechanisms underlying human atrial action potential properties: insights from a mathematical model. Am J Physiol Heart Circ Physiol 275, H301–H321. Dilly KW, Kurokawa J, Terrenoire C, Reiken S, Lederer WJ, Marks AR & Kass RS (2004). Overexpression of β2-adrenergic receptors cAMP-dependent protein kinase phosphorylates and modulates slow delayed rectifier potassium channels expressed in murine heart: evidence for receptor/channel co-localization. J Biol Chem 279, 40778–40787.

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Acknowledgements This work was funded by National Institutes of Health grant 1R01 HL-44365-15 awarded to R.S.K.

Supplemental material Online supplemental material for this paper can be accessed at: http://jp.physoc.org/cgi/content/full/jphysiol.2007.141333/DC1 and http://www.blackwell-synergy.com/doi/suppl/10.1113/jphysiol. 2007.141333