ION CHANNELS, RECEPTORS AND TRANSPORTERS

Beta-adrenergic stimulation reverses the IKr–IKs dominantpattern during cardiac action potential

Tamas Banyasz & Zhong Jian & Balazs Horvath &

Shaden Khabbaz & Leighton T. Izu & Ye Chen-Izu

Received: 13 August 2013 /Revised: 6 January 2014 /Accepted: 28 January 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract β-Adrenergic stimulation differentially modulatesdifferent K+ channels and thus fine-tunes cardiac action po-tential (AP) repolarization. However, it remains unclear howthe proportion of IKs, IKr, and IK1 currents in the same cellwould be altered by β-adrenergic stimulation, which wouldchange the relative contribution of individual K+ current to thetotal repolarization reserve. In this study, we used an innova-tive AP-clamp sequential dissection technique to directly re-cord the dynamic IKs, IKr, and IK1 currents during the AP inguinea pig ventricular myocytes under physiologically rele-vant conditions. Our data provide quantitative measures of themagnitude and time course of IKs, IKr, and IK1 currents in thesame cell under its own steady-state AP, in a physiologicalmilieu, and with preserved Ca2+ homeostasis. We found thatisoproterenol treatment significantly enhanced IKs, moderatelyincreased IK1, but slightly decreased IKr in a dose-dependentmanner. The dominance pattern of the K+ currents was IKr>IK1>IKs at the control condition, but reversed to IKr<IK1<IKsfollowing β-adrenergic stimulation. We systematically

determined the changes in the relative contribution of IKs,IKr, and IK1 to cardiac repolarization during AP at differentadrenergic states. In conclusion, the β-adrenergic stimulationfine-tunes the cardiac AP morphology by shifting the powerof different K+ currents in a dose-dependent manner. Thisknowledge is important for designing antiarrhythmic drugstrategies to treat hearts exposed to various sympathetic tones.

Keywords Cardiac .Myocyte . Potassium channel .

Beta-adrenergic . Calcium . Action potential

Introduction

Cardiac action potential (AP) is fine-tuned by adrenergic tone.Extensive studies have shown that K+ channels essential for thecardiac AP repolarization are intricately regulated by β-adrenergic stimulation, and different K+ channels—IKs, IKr,and IK1—showdifferent sensitivities toβ-adrenergic stimulation[10, 25, 26, 28]. In all previous studies, however, IKs, IKr, and IK1were each recorded from different cells and using different V-clamp conditions (i.e., voltage protocol, ionic composition, Ca2+

buffering). Thus, it remains unknown how β-adrenergic stimu-lation coordinately regulates all these K+ currents in the samecell during the AP, nor is it clear how variousβ-adrenergic statesmay change the relative contribution of each K+ channel to thetotal repolarization reserve. Yet, such knowledge is essential fordesigning antiarrhythmic strategies using specific K+ channelblockers. Recently, we have developed an innovative AP-clampsequential dissection (called “onion-peeling”) method that givesus unprecedented ability to measure multiple ionic currentsduring the AP in the single myocyte [1, 4]. The onion-peelingdata enable us, for the first time, to analyze the proportion ofdifferent currents flowing in the same cell during the AP underphysiologically relevant conditions. The first goal of this study isto determine the relative contribution of IKs, IKr, and IK1 to the

Tamas Banyasz and Zhong Jian are the first authors with equalcontribution.

T. Banyasz : Z. Jian : B. Horvath : S. Khabbaz : L. T. Izu :Y. Chen-IzuDepartment of Pharmacology, University of California, 2221 TupperHall, 451 Health Science Drive, Davis, CA 95616, USA

T. BanyaszDepartment of Physiology, University of Debrecen, MHSC,Debrecen, Hungary

Y. Chen-IzuDepartment of Biomedical Engineering, University of California,Davis, CA, USA

Y. Chen-Izu (*)Department of Internal Medicine, Division of Cardiology, Universityof California, Davis, CA, USAe-mail: ychenizu@ucdavis.edu

Pflugers Arch - Eur J PhysiolDOI 10.1007/s00424-014-1465-7

AP repolarization in response to various extent of β-adrenergicstimulation; such in-depth knowledge is important for under-standing how cardiac APs are altered under various sympathetictones during exercise, stress, or diseases.

β-Adrenergic stimulation can affect the K+ channels directlyand indirectly. Downstream from β-adrenergic stimulation, ac-tivation of the cyclic AMP-dependent protein kinase A (PKA)causes phosphorylation of many ion channels and Ca2+ han-dling proteins. PKA phosphorylation of K+ channels directlymodifies the magnitude and the kinetics of K+ currents [10].Meanwhile, PKA phosphorylation of Ca2+ handling proteinssuch as the ryanodine receptor, the sarcoplasmic reticulum Ca2+

pump, and the Ca2+-calmodulin-dependent protein kinase II(CaMKII) can alter the Ca2+ homeostasis of cardiac myocytes[9]. Altered Ca2+ homeostasis can exert a secondary effect toalter the K+ currents because K+ channels are sensitive to Ca2+/CaMKII modification. In order to understand the full impact ofβ-adrenergic stimulation onmodulating the K+ currents and APrepolarization, we need to maintain physiologic Ca2+ cyclingduring the AP. However, most of our current knowledge on β-adrenergic modulation of K+ currents is based on the V-clampdata obtained when the intracellular Ca2+ was buffered byexogenous Ca2+ buffers (ethylene glycol tetraacetic acid—EGTA or 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraaceticacid—BAPTA). In 1998, Zaza et al. [30] conducted an elegantstudy to show that Ca2+ can reduce IK1 during the AP inventricular myocytes. Since then, many studies found Ca2+

sensitivity of other K+ currents [8, 10, 22]. The data from thesestudies suggest that the Ca2+ transient during the AP can signif-icantly modify the K+ currents. Nonetheless, the early experi-ments still used 1 mM EGTA in the pipette solution, with theassumption that low EGTA concentration might not interferewith Ca2+ homeostasis [22, 30]. Contrary to this assumption, wefound that EGTA at 1 mM almost eliminated the Ca2+ transientduring AP; hence, the data from the previous studies need to bereinterpreted. The second goal of this study is to determine thefull impact of β-adrenergic stimulation on modulating the K+

currents during the AP with normal cycling Ca2+ under physi-ologically relevant condition. The overarching goal is to sys-tematically determine the changes in the relative contribution ofIKs, IKr, and IK1 to cardiac repolarization during AP at differentadrenergic states under physiologically relevant conditions.

This knowledge is important for designing effective andsafe therapeutic strategies using K channel inhibitors (Singh–Williams class III antiarrhythmic drugs) to treat hearts ex-posed to various sympathetic tones.

Methods

All laboratory procedures in this study conform to the Guidefor the Care and Use of Laboratory Animals published by theUS National Institutes of Health, the Guide for the Care and

Use of Laboratory Animals laid out by Animal Care Commit-tee of the University of California (UC). The animal use wasapproved by the UC Davis Institutional Animal Care and UseCommittee (IACUC, protocol #16914).

Cell isolation

Hartley guinea pigs (male, 3–4 months old, purchased fromCharles River Laboratories USA) were first injected withheparin (800 U, i.p.) and then anesthetized with nembutal(100 mg/kg, i.p.). After achieving deep anesthesia to suppressspinal cord reflexes, a standard enzymatic technique was usedto isolate ventricular myocytes [3].

Electrophysiology

Cells were continuously superfused with a modified Tyrodesolution supplemented with bicarbonate (BTy) containing (inmmol/L) NaCl 120, KCl 5, CaCl2 2, MgCl2 1, HEPES 10,NaHCO3 25, and glucose 10. pH was set to 7.3. BTy was keptin glass flasks with airtight cap and used within 6 h afterpreparation; previous tests confirmed that there was no pH shiftwithin this period. The pipette solution contained (in mmol/L)K-aspartate 115, KCl 45,Mg-ATP 3, HEPES 5, and cAMP 0.1;pH was set to 7.25 using KOH. Borosilicate glass pipettes werefabricated with Sutter (Sutter Instrument Company, NovatoCA, USA) laser puller having resistance of 1.8–2.5 MΩ afterfilling with pipette solution. Experiments were recorded usingAxopatch 200B Amplifier, DigiData 1440A Analog/DigitalConverter, and pClamp10 software (Molecular Devices, Sun-nyvale CA, USA). Series resistance of the pipette and inputresistance of the cell were fully compensated. Cell capacitancecompensation was 80 %. The access resistance was continu-ously monitored during the experiment and only cells havingconstant access resistance (< 5 MOhm) were used for analysis.

The self-AP-clamp sequential dissection (called “onion-peeling” from here on) experiments were conducted as de-scribed in our previous publication [1]. Briefly, after estab-lishing the ruptured patch whole-cell clamp configuration, thecell was paced at 1 Hz frequency under I-clamp mode to reachthe steady-state action potential. The cell’s steady-state APwas recorded. After switching to V-clamp mode, this APwaveform was applied as voltage command onto the samecell at 1 Hz frequency. After recording “zero current,” specificion channel blockers were applied sequentially and compen-sation current recorded. The K+ currents were obtained usingthe following specific inhibitors: 1 μM chromanol 293B wasused to obtain IKs; 1 μM E4031 for IKr; and 50 μM Ba2+ forIK1, respectively. A number of earlier studies have shown thateach blocker is highly specific at the concentrations used. Forstudying the dose-dependent β-adrenergic stimulation effects,the cells were exposed to isoproterenol at the given concen-tration throughout the entire onion-peeling experiments.

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[Ca2+]i measurement

The intracellular Ca2+ concentration ([Ca2+]i) was measuredusing Fura-2 ratiometric method [3]. Briefly, Fura-2 K+ saltwas added into the pipette solution at a concentration of20 μM and diffused into the cytosol through the rupturedpatch during paced cell contraction to reach steady state.The IonOptix system (IonOptix Inc. USA) with dual excita-tion at 340 and 380 nm and single emission >510 nm (throughemission filter 510–645 nm) was used to measure the Fura-2fluorescence ratio. The IonOptix system was synchronizedwith the electrophysiology setup to simultaneously measurethe [Ca2+]i and the electric signals.

Statistical analysis

The numerical values are calculated for the mean value, thestandard deviation (SD), and the standard error of mean(SEM). The mean±SEM values are shown in the bar charts

in the figures. The mean±SD values are reported in the text.The number of cells and the number of animals in eachexperimental group were reported in the legends. Statisticalsignificance of the difference between different groups wasevaluated using Student’s t test and deemed significant ifp<0.05.

Results

Recording of three major K+ currents during the APwith Ca2+

cycling in the single myocyte

We used the onion-peeling technique to record the ion currentsthat are naturally flowing during the AP in the guinea pigventricular myocyte when the cell is undergoing normal exci-tation–contraction coupling under physiological conditionand following β-adrenergic stimulation. Figure 1a demon-strates a typical onion-peeling experiment in which we

A BCTRL ISO

C DCTRL ISO

Fig. 1 β-Adrenergic stimulationeffects on the AP, the three K+

currents, and the Ca2+ transient. a,b AP-clamp sequential dissectionexperiments to directly record thesteady-state AP (upper panel) at1 Hz pacing rate, the three K+

currents (mid panel) in the samecell, and the Ca2+ transients(lower panel) under physiologicalcondition (CTRL) and following30 nM isoproterenol (ISO)treatment. Note that the Ca2+

transient during the AP ispreserved by having theendogenous Ca2+ buffers andwithout adding any exogenousCa2+ buffer (no EGTA) in pipettesolution. c, d Ca2+ transients werelargely eliminated by using 1 mMEGTA in the pipette solutionunder both the control conditionand following ISO treatment

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recorded three major K+ currents—IKs, IKr, and IK1—during theAP in the same cell. The steady-state AP (upper panel) wasrecorded under I-clamp mode, with 1 Hz pacing frequency, atbody temperature (36±0.3 °C). Then this AP waveform wasused as the voltage command under V-clampmode to record theion currents flowing under the AP. The IKs, IKr, and IK1 currents(middle panel) were pharmacologically dissected out one-by-one from the same cell by sequentially adding chromanol 293B1 μM, E4031 1 μM, and Ba2+ 50 μM. The data show that theintracellular Ca2+ transient during the AP cycle (lower panel)was preserved under our experimental conditions.

Next, we studied the effects of β-adrenergic stimulation onfine-tuning the three K+ channels by using isoproterenol at 3,10, and 30 nM concentrations. As shown in Fig. 1b, isoproter-enol shortened the AP duration (upper panel) and differentiallymodified the profiles of the IKs, IKr, and IK1 currents during theAP (middle panel, see below for detailed analysis). Isoprotere-nol also slightly increased the amplitude of the Ca2+ transient(lower panel), which should contribute to the Ca2+-dependentchanges in the K+ currents during the AP with Ca2+ cycling.

Preserving Ca2+ homeostasis during self-AP-clampby eliminating exogenous Ca2+ buffer

In order to understand the full impact of β-adrenergic stimula-tion on altering the K+ currents during AP through PKA phos-phorylation and Ca2+/CaMKII signaling, we designed experi-mental conditions to preserve the Ca2+ transient during the APcycle by eliminating exogenous Ca2+ buffer. In literature,pioneering studies aimed at measuring K+ currents while pre-serving Ca2+ signaling still used 0.5–1.0 mM EGTA in thepipette solution, assuming that low concentrations of EGTAwould not interfere with Ca2+ transients [22, 30]; however, theactual Ca2+ concentration was not measured in those earlyexperiments. We conducted experiments to simultaneously re-cord the Ca2+ transient and ion currents during sAP-clamp. Theresult shows that having 0.5–1 mM EGTA in the pipette solu-tion largely eliminated the Ca2+ transient after pacing at 1 Hz toreach steady state (Fig. 1c); the same result was seen in thepresence of 30 nM isoproterenol (Fig. 1d). We also observedthat the Ca2+ transient during AP was high at the beginning ofpacing, but gradually declined during pacing, and then dimin-ished after reaching steady state (shown in Fig. 1c, d). Further-more, our earlier experiments [1] using 10 mM EGTA in thepipette solution caused the Ca2+ transient to rapidly declineduring pacing. Retrospectively, this is not surprising becauseEGTA should diffuse into the cell and gradually buffer thecytosolic Ca2+ while the cell is being paced; the speed ofCa2+ buffering is slowed with low EGTA concentration, buteliminates the Ca2+ transient at steady state nonetheless. It isnoteworthy that the Ca2+ transient is preserved in our sAP-clamp experiments by eliminating exogenous Ca2+ buffer in thepipette solution. We surmise that since the myocyte cell

membrane (size ~150×40×30 μm) is substantially larger thanthe pipette tip (diameter ~1 μm) and the ion channels andtransporters are functioning normally while being paced underthe cell’s own steady-state AP, the myocyte should maintain itsionic homeostasis in our sAP-clamp experiments. The fact thatthe myocyte experiences its natural state of excitation–contrac-tion coupling (with AP, Ca2+ transient, and contraction) distin-guishes our sAP-clamp experiments from the traditional V-clamp experiments using simplified conditions (i.e., rectangularvoltage waveform, ion substitution, exogenous Ca2+ buffer)that disrupt the ionic homeostasis and Ca2+ transient.

β-Adrenergic stimulation effect on IKr during the APwith Ca2+ cycling

Figure 2a shows the profile of E4031-sensitive IKr currentduring the AP with Ca2+ cycling. The current density (normal-ized to the cell capacitance) of IKr was 0 during diastole,remained small during the AP phases 1 and 2, increased rapidlyduring the AP phase 3, peaked at the end of phase 3, and thendeclined rapidly back to the diastolic level. The effect of β-adrenergic stimulation on the IKr current was subtle and onlyseen at high isoproterenol concentration, albeit a faster timecourse in correspondence to a shorter AP duration (Fig. 2b).Neither the peak current density nor the profile of IKr during APwas altered by isoproterenol at low concentrations of 3–10 nM.Isoproterenol at 30 nM did not significantly alter the IKr currentdensity during the plateau phase at +20 mV, but caused areduction of IKr during the repolarizing phase as seen at 0 and−20 mV membrane potentials (Fig. 2c). Isoproterenol concen-tration higher than 30 nM routinely evoked afterdepolarizationsin the myocytes and therefore was not suitable for conductingAP-clamp experiments. Our data reveal that the IKr currentduring the AP was largely insensitive to isoproterenol at phys-iological concentrations of 3–30 nM. In comparison, manyprevious V-clamp studies used maximal concentrations of iso-proterenol ranging from 100 nM to 10 μM.

Dose-dependent β-adrenergic tuning of IKs during the APwith Ca2+ cycling

In the absence of β-adrenergic stimulation, the chromanol293B-sensitive IKs was seen as a tiny and slow currentthroughout the AP in the guinea pig ventricular myocyte(Fig. 2d). The IKs current was 0 during diastole, built upslowly during the AP phases 1 and 2, reached a peak valueat the end of phase 2 (near 0 mV membrane potential), andthen declined rapidly during phase 3 in correspondence to APrepolarization. Isoproterenol treatment caused significantchanges in IKs throughout the AP (Fig. 2e). The magnitudeof IKs was augmented by isoproterenol in a dose-dependentmanner, with a slight increase at 3 nM isoproterenol, and asubstantial increase from the control value of 0.152±0.027

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A/F to 2.067±0.223 A/F in 30 nM isoproterenol (Fig. 2f).Importantly, the profile of IKs during AP following β-adrenergic stimulation (Fig. 2e) became similar to that of IKr(Fig. 2b) and even surpassed IKr in magnitude. The peak of thecurrent shifted from mid plateau to phase 3 of AP. Thesubstantial alterations in the IKs current magnitude and timecourse indicate a strong β-adrenergic control of this channel.

Dose-dependent β-adrenergic tuning of IK1 during the APwith Ca2+ cycling

The profile of Ba2+-sensitive IK1 current during the AP withCa2+ cycling is shown in Fig. 3a. During diastole, IK1 waspresent as a sustained outward current. At the upstroke of AP,the IK1 had an instant reduction of the current density which ischaracteristic of inward rectification. During the AP phases 1and 2, the IK1 current remained very small, but then shot upsharply during phase 3, reached the peak value at the end ofphase 3, and then declined rapidly to return to the diastolic level.

The isoproterenol effect on the IK1 profile seemed subtle atfirst glance, but quantitative analysis reveal considerable chang-es in several features (Fig. 3b). The average diastolic currentdensity was not changed by isoproterenol, but the inwardrectification became less obvious. Isoproterenol increased theIK1 current during phase 2 in a dose-dependent manner, asdetermined at the membrane potential of +20, 0, and −20 mV(Fig. 3c). Consequently, isoproterenol treatment significantlyincreased the total charge carried by IK1 during the AP (Fig. 3d).

β-Adrenergic stimulation shifts the relative contributionof individual K+ currents to the total repolarization reserve

The onion-peeling method allows recording of all three K+

currents in the same cell. This enables, for the first time,analysis on how each K+ current contribute to the total repo-larization current within a single cell, without the confoundingeffect of cell-to-cell variations. First, we calculated the sum ofall three K+ currents whichmake up the repolarization current,

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Fig. 2 Dose-dependent β-adrenergic tuning of delayedrectifier K+ currents during theAP. a, b IKr recorded during thecell’s own AP before and after β-adrenergic stimulation using30 nM ISO. In each panel, theupper trace shows the AP and thelower trace shows thecorresponding current. ISO had alittle effect on the amplitude of IKrat lower concentrations butslightly reduced IKr at 30 nM (c).d, e AP and IKs current tracesbefore and after 30 nM ISOtreatment, respectively. ISOincreased IKs during AP in a dose-dependent manner (f). Each pointin c and f represents averagedcurrent values and standard errorfrom 7 to 15 cells isolated fromseven hearts. Student’s t testp values: *p<0.05, **p<0.01,***p<0.001

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and then, we calculated the proportion of each individualK+ current to the total repolarization current (or repolari-zation reserve) at different phases of AP. Figure 4a, bshow the analysis result at +20 and −20 mV membranepotentials, respectively. These points were chosen to giverepresentative values for the plateau and the repolarizationphases of AP.

Isoproterenol treatment shifted the relative contribution ofeach K+ current to the repolarization reserve in a dose-dependent manner. The most striking change is a reversal ofthe dominance of IKr and IKs. Under the control condition inthe absence of isoproterenol, IKr presents the most powerfulrepolarizing power, whereas IKs contributes very little, asmeasured at both +20 and −20 mV. With 3 nM isoproterenol,the relative contribution of IKs increases and that of IKr de-clines. The two become equal at 10 nM isoproterenol, andthen, IKs surpassed IKr at 30 nM isoproterenol. The relativecontribution of IK1 to the total repolarizing current did notshow a significant change.

Consistent with the changes in the currents, the totalcharges carried by the K+ currents were also altered by iso-proterenol treatment. As shown in Fig. 4c, the total K+ chargemovement during the AP was increased by isoproterenol in aconcentration-dependent manner. This charge increase result-ed from increased IKs and IK1 while IKr was unaltered.

Consequently, the total outward K+ charge movement go-ing through the three K+ currents was significantly increased

by isoproterenol treatment (Fig. 4c). Importantly, the relativecontributions of IKs, IKr, and IK1 change significantly withincreasing isoproterenol concentration, due to different sensi-tivities of the individual K+ currents to β-adrenergic stimula-tion. This shift of the relative strength between the currents hasprofound implications on how each individual K+ channelmight contribute to arrhythmogenesis at differentβ adrenergicstates and how to design effective antiarrhythmia drug thera-pies for various pathological conditions.

β-Adrenergic stimulation alters the effects of K+ channelblockers (class III antiarrhythmia drugs) on modifyingcardiac AP

Since different K+ channels have different sensitivities toisoproterenol, it is plausible that the β-adrenergic state of theheart may modify the effect of K+ channel blockers on mod-ulating the AP. To test this, we studied the effects of specificK+ channel blockers on modifying the AP duration in theabsence and presence of 30 nM isoproterenol. Figure 5a, bshows that blocking IKs using 1 μMchromanol 293B caused amoderate lengthening of action potential duration (APD) un-der control condition, but drastically lengthened APD in thepresence of isoproterenol. In comparison, blocking IKr using1 μM E4031 also caused a moderate lengthening of APD inthe absence of isoproterenol (Fig. 5c); however, the APDlengthening remained small in the presence of isoproterenol

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Fig. 3 Dose-dependent β-adrenergic tuning of IK1 current.Representative traces wererecorded in the absence (a) andpresence (b) of 30 nM ISO.Dose–response curves for currentvalues at different membranepotentials and total chargemovement during AP are shownin c and d, respectively. ISOincreased IK1 during AP plateau,but left diastolic current valueunaltered. n=7–15 cells fromseven animals

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(Fig. 5d). The above difference in the IKs versus IKr blockereffect on APD is consistent with the differential regulation ofIKs versus IKr by β-adrenergic stimulation.

Discussion

The main goal of this study is to determine the relativecontributions of three major K+ currents—IKs IKr, and IK1—

to the AP repolarization in response to various degrees of β-adrenergic stimulation. By our best knowledge, this is thefirst time these three K+ currents have been measuredfrom the same cell and during the cardiac AP with Ca2+

cycling. Most previous studies used conventional V-clampexperiments to characterize the biophysical properties ofK+ channels under simplified conditions; the data werethen used in mathematical modeling to predict the dynam-ic profile of the current during AP. However, because ofthe simplifications used in experimental conditions andalso in model assumptions, the model predictions mightdeviate from the physiological reality. Therefore, it iscritically important to compare the model predictions withdirect experimental recording of the dynamic ion currentsduring AP. The present study provides such experimentaldata for evaluating model predictions and for improvingthe models.

Furthermore, we systematically characterized theconcentration-dependent effects of isoproterenol on modulat-ing the three major K+ currents during cardiac AP. Our datashow that isoproterenol treatment facilitates IK1 during the APplateau phase, significantly increases the magnitude of IKs, buthas little effect on IKr. Consequently, isoproterenol increasesthe contribution of IKs but decreases the contribution of IKr tothe total repolarization reserve, leading to a reversal of thedominance of IKs versus IKr in repolarizing the AP (Fig. 4).Therefore, the dominant K+ current switches from IKr underthe control condition to IKs under β-adrenergic stimulationwith 30 nM isoproterenol. Such a reversal of dominancepattern has a significant implication for using specific K+

channel blockers, to treat cardiac arrhythmias.

Effects of β-adrenergic stimulation on IKr

The effects of β-adrenergic stimulation on IKr have beencontroversial in literature. Harmati et al. [10] and Heath et al.[11] reported facilitation of IKr by isoproterenol via PKA andPKC pathways in canine and guinea pig ventricular myocytes.Karle et al. [13] reported a reduction of IKr current amplitudefollowing isoproterenol application in guinea pig ventricularmyocytes. Sanguinetti et al. [23] reported no measurableisoproterenol-induced change of IKr. All of these experimentsused standard V-clamp technique to measure the IKr as the tailcurrent elicited with long square pulses in conjunction withblocking the IKs component. In addition, most previous studiesused high isoproterenol concentration (1–10 μM), whereas weused isoproterenol in the range of 3–30 nM (closer to physio-logical β-adrenergic stimulation range) because higher isopro-terenol induced afterdepolarizations. In this study, we directlyrecorded the IKr current during the AP with Ca2+ cycling. TheIKr profile we recorded is largely consistent with the previousmodel simulations of the current under the control condition[20, 31], although some quantitative differences exist. Our data

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Fig. 4 β-Adrenergic stimulation shifts the relative contribution of indi-vidual K+ currents to the total repolarizing reserve. The three K+ currentsmeasured in the same cell were summed, and then each current wasnormalized to this sum. ISO dose–response curves for normalized valuesmeasured at +20 and −20 mVare shown in a and b, respectively. c Totalamount of K+ chargemovement for the individual K+ current and the sumof the currents during the AP. n=7–15 cells from seven animals

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demonstrate that isoproterenol did not significantly alter IKr inthe concentrations below 30 nM and caused only moderatereduction of IKr at 30 nM. Our data provide the first experi-mental measures on the isoproterenol dose–response of IKr inthe presence of cytosolic Ca2+ transient; these data can be usedto fine-tune the quantitative models.

Effects of β-adrenergic stimulation on IKs

IKs is known to be facilitated by β-adrenergic stimulationaccording to previous V-clamp studies [10, 17, 23, 28]. Ourdata largely agree with the previous findings. The novelfindings from our experiments is thatβ-adrenergic stimulationchanges the profile of IKs during the AP (Fig. 2d, e). Under thecontrol condition, the profile of IKs displays a small and flatcurrent throughout the AP (Fig. 2d), similar to that seen byRocchetti et al. [21] in their pioneering AP-clamp study.However, the isoproterenol effect on altering the IKs profileis much greater in our experiments than that seen in Rocchettiet al. [21]. The peak IKs current density wemeasured was 2.15±0.52 A/F, about four times larger than their measured valuebetween 0.5 and 0.7 A/F. This apparent discrepancy may arisefrom methodological difference. One major difference is inthe pipette solution design. Rocchetti et al. used 1 mM EGTAwhich would buffer the intracellular Ca2+, whereas in ouronion-peeling experiments, the Ca2+ transient during APwas preserved. In our earlier work when IKs was recorded inthe presence of 10 mM EGTA, the peak amplitude was foundlower in the range of 0.4–0.6 A/F [1]. Given that IKs issensitive to Ca2+ [2, 19], differences in these experimentaldata would be expected. Another major difference is thatRocchetti et al. used the AP waveform recorded before iso-proterenol application as the voltage command in their AP-clamp experiment, and then, the IKs current was dissected outas the isoproterenol-induced current. In comparison, we used

the AP waveform recorded after the isoproterenol applicationthat resulted in a higher plateau and a steeper phase 3 repolar-ization. This difference in the AP-clamp command voltageshould result in a larger IKs current seen in our data, since IKs ishighly voltage sensitive in the range of the AP plateau [12].Because we used the AP at the new adrenergic state, the IKscurrents recorded with the onion-peeling method provide anaccurate measure of the β-adrenergic stimulatory effect on IKsunder increased sympathetic tone. Interestingly, following β-adrenergic stimulation, the peak of IKs shifted from mid pla-teau to phase 3 of AP, similar to that of IKr, but the magnitudeof IKs even surpassed that of IKr. The observation that IKs isfacilitated by β-adrenergic stimulation to a much larger extentthan IKr was reported earlier [10, 11, 23]. Nevertheless, this isthe first time when changes in the profile of IKs during APfollowing β-adrenergic stimulation were experimentally re-corded and quantitatively measured.

Increased adrenergic tone also increases heart rate; hence,β-adrenergic-related increase of IKs current should help toshorten the AP duration in support of faster heartbeats. How-ever, when the IKs channel is defective in long QT1 syndrome,the lack of a significant adrenergic-related increase of IKscould be a relevant substrate for arrhythmias. An example ofsuch case is seen in a KCNE1 knockout mouse model inwhich tachycardia-induced heterogeneity blunts the QT adap-tation to heart rate variations [5]. Hence, long QT1 patientswith defective IKs have a greater susceptibility to arrhythmias.

Effects of β-adrenergic stimulation on IK1

The profile of IK1 during the AP with Ca2+ cycling shows asustained outward current during diastole. At the AP upstroke,IK1 rapidly decreased due to inward rectification. Duringphases 2 and 3, IK1 remained small, then sharply increasedwith fast repolarization at the end of phase 3, and then rapidly

A

50 m

V

100 ms

0 mV

Chroma

CTRL

0 mV

ISO

ISO+Chroma

B

C

0 mV

E4031

CTRL

D

0 mV

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Fig. 5 β-Adrenergic state altersthe effects of specific K+ channelinhibitors on modifying the AP.The AP lengthening effect ofusing 1 μM chromanol 293B toblock IKs is moderate undercontrol condition (a) but becameprominent after 30 nM ISOtreatment (b). The APlengthening effect of using 1 μME-4031 to block IKr is similarunder control conditions (c) andafter ISO treatment (d)

Pflugers Arch - Eur J Physiol

declined back to the diastolic level. Isoproterenol caused aslight increase of IK1 during the AP but did not change thediastolic IK1 current density (Fig. 3). Previous studies of theβ-adrenergic stimulation effects on IK1 have reported controver-sial results. Facilitation of IK1 by isoproterenol treatment wasreported by Tromba and Cohen [26], Gadsby [7], and Schereret al. [24], whereas reduction of IK1 by isoproterenol wasreported by Koumi et al. [14], Wischmeyer et al. [29], andFauconnier et al. [6]. The above experiments were all con-ducted using the traditional V-clamp technique. Using the AP-clamp method, Zaza et al. [30] provided data to suggest thatisoproterenol might reduce IK1 during the plateau phase. Incontrast, our data show that isoproterenol facilitated the IK1during the AP. This apparent discrepancy may result fromdifferences in the experimental methods used to measureIK1. We used Ba2+ (50 μM)-sensitive current to estimate IK1,whereas Zaza et al. used the I0K current which was dissectedout by removing K+ from the extracellular solution. The I0Kobtained this way is a composite current containing all K+

currents including IK1, IKr, and IKs. The difference between theBa2+-sensitive current and I0K is expected to change with theisoproterenol treatment, since our data show that both IK1 andIKs were significantly increased by isoproterenol. The secondmajor difference is that Zaza et al. used the AP waveformrecorded before isoproterenol application as the AP-clampvoltage command, and then added isoproterenol to obtainthe isoproterenol-sensitive I0K current. In comparison, weused the AP waveform recorded after the isoproterenol appli-cation that resulted in a higher plateau and a steeper repolar-ization. The third major difference is in the Ca2+ bufferingcondition. Zaza et al. [30] used 1 mM EGTA in their pipettesolution which would have buffered the intracellular Ca2+ andeliminate the Ca2+ transient (Fig. 1a, b). Instead, the Ca2+

transient during AP was preserved in our experiments. Giventhe above differences in the experimental methods, it is diffi-cult to compare the data obtained by us with those reported inZaza et al. [30]. Since the Ca2+ transient during AP is pre-served in our experiments, we assume that our data moreclosely reflect the IK1 flowing in the cell in vivo.

Isoproterenol shifts the relative contribution of individual K+

current to the AP repolarization

The onion-peeling recording of three K+ currents from thesame cell enables, for the first time, analysis of the relativecontribution of each K+ current to the repolarization of AP in asingle cardiac myocyte. We found that, under the controlcondition, IKr and IK1 are the major repolarizing currents whilethe contribution of IKs is minor. But isoproterenol treatmentgreatly increased IKs in a dose-dependent manner, ultimatelymaking it to the most powerful repolarizing current. Mean-while, IKr did not change with isoproterenol treatment, so itsrelative contribution to the total K+ current was significantly

reduced. At the same time, IK1 current magnitude was slightlyincreased, but resulted in no change in its relative contribution.With 30 nM isoproterenol treatment, IKs became the largestcontributor to the total K+ current, surpassing the IKr contri-bution by 4–5 folds. This striking reversal of the relativecontribution by IKr and IKs to the AP repolarization has sig-nificant implications.

The contributions of different K+ currents to the AP repo-larization have been a subject of debate. IK1 and IKr aregenerally considered important repolarizing currents, but therole of IKs has been controversial. Some suggested that IKs iscrucial for repolarization [16, 18], others found IKs contributedvery little to normal repolarization [15, 27]. Our data clearlydemonstrate that the relative contributions of IKs, IKr, and IK1should change with different extent of β-adrenergic stimula-tion. This finding helps to resolve the apparent contradictionreported in previous studies. Since catecholamine levels aresubject to changes during daily activity, exercise, stress, ordiseases, our new observations have high clinical relevance.Our results suggest that the efficacy of class III antiarrhythmicdrugs targeting various K+ channels may change according tothe sympathetic tone. This new mechanistic insight is con-firmed by the differential effects of chromanol 293B andE-4031 on lengthening APD in the absence and presence ofisoproterenol (Fig. 5). Therefore, in the design of new thera-peutic strategies targeting specific K+ channels, the reversal ofthe dominance pattern of IKr and IKs with adrenergic stimula-tion must be taken into account. Our data provide accurateexperimental measures of the three major K currents duringthe AP under physiologically relevant conditions, which con-tribute to important quantitative understanding of the adren-ergic effects on AP repolarization and arrhythmogenesis.

Acknowledgments We are grateful to Dr. Donald M. Bers for doing aninternal review and editing of the manuscript. This work was supported bythe National Institute of Health R01 grant (HL90880) to LTI, YC, and TB;the National Institute of Health R03 grant (AG031944) to YC; AmericanHeart Association National Center Scientist Development Award(0335250 N) to YC; European Society of Cardiology Visiting ScientistAward to BH; the Hungarian Research Fund OTKA (K101196) to TB; andthe startup funds from the University of California to LTI and YC.

Conflict of interest None

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