H-00076-2002.R1 1

Increasing IKs corrects abnormal repolarization in rabbit models of

acquired LQT2 and ventricular hypertrophy

Xiaoping Xu,1 Joseph J. Salata,2 Jixin Wang,2 Ying Wu,1 Gan-Xin Yan,1

Tengxian Liu,1 Roger A. Marinchak,1,3 Peter R. Kowey1,3

1Main Line Health Heart Center, Wynnewood, PA 19096

2Department of Pharmacology, Merck Research Laboratories, West Point, PA 19486

3Jefferson Medical College, Philadelphia, PA 19107

Running head: Normalizing repolarization by enhancing IKs

Mailing proofs: Xiaoping Xu, PhDMain Line Health Heart CenterSuite 558, Medical Office Building East100 Lancaster AvenueWynnewood, PA 19096Phone: (610)-645-2687Fax: (610)-896-0643E-mail: XXUJWANG@AOL.COM

Copyright 2002 by the American Physiological Society.

AJP-Heart Articles in PresS. Published on May 2, 2002 as DOI 10.1152/ajpheart.00076.2002

H-00076-2002.R1 2

Excessive action potential (AP) prolongation and early after-depolarizations (EAD) are

triggers of malignant ventricular arrhythmias. Slowly activating delayed rectifier K+

current (IKs) is important for repolarization of ventricular AP. We examined the effects of

IKs activation by a novel benzodiazepine (L3), on AP of control, dofetilide-treated, and

hypertrophied rabbit ventricular myocytes. In both control and hypertrophied myocytes,

L3 activated IKs via a negative shift in the voltage-dependence of activation and a

slowing of deactivation. L3 had no effect on L-type Ca2+ current or other cardiac K+

currents tested. L3 shortened AP of control, dofetilide-treated and hypertrophied

myocytes more at 0.5 Hz than 2 Hz. Selective activation of IKs by L3 attenuates

prolonged AP and eliminated EAD induced by IKr inhibition in control myocytes at 0.5 Hz

and spontaneous EAD in hypertrophied myocytes at 0.2 Hz. Pharmacological activation

of IKs is a promising new strategy to suppress arrhythmias resulting from excessive AP

prolongation in patients with certain forms of long-QT syndrome or cardiac hypertrophy

and failure.

Keywords: action potential; ion channel; early-afterdepolarization; arrhythmia

H-00076-2002.R1 3

Cardiac hypertrophy and failure cause more than 200,000 deaths annually in the

US. As many as 50% are sudden and due to the onset of polymorphic ventricular

tachycardia (VT) or torsades de pointes, associated with Long-QT syndrome (LQTS)

(7). LQTS can be congenital or acquired. Congenital LQTS most commonly develops

from mutations in genes encoding delayed rectifier K+ channels that cause functional

decreases in repolarizing K+ currents (4, 12, 27). Acquired LQTS is associated with

administration of certain antiarrhythmics, antihistamines, antibiotics and other drugs

(http://georgetowncert.org/qtdrugs_torsades.asp). Many of these agents can cause

excessive action potential (AP) prolongation via inhibition of the rapidly activating

delayed rectifier K+ current (IKr), resulting in a specific form of acquired LQTS (LQT2)

(20). Heart failure may also be considered a common acquired form of LQTS (16).

Functional down-regulation of K+ currents is a recurring theme in hypertrophied and

failing ventricular myocardium (26).

At the cellular level, excessive AP prolongation and unstable repolarization is

consistently observed in ventricular myocytes from hypertrophied and failing hearts,

animal models or patients with LQTS (2, 7, 31). Early after-depolarizations (EAD) occur

in the setting of prolonged AP due commonly to diminished K+ currents or in some

cases to slowly- or non-inactivating components of Ca2+ or Na+ currents. EAD have

been identified as the triggering mechanisms for polymorphic VT (2, 4, 35). Ventricular

myocytes from hypertrophied and failing hearts are highly susceptible to EAD (3, 16, 17,

31). No effective or safe drugs are available for treatment of acquired LQT2 or

ventricular arrhythmias associated with cardiac hypertrophy and failure.

We hypothesized that selective activation of the slowly activating delayed rectifier

K+ current (IKs) could provide an effective antiarrhythmic action in certain forms of LQTS

and heart failure. Recently, we identified a novel benzodiazepine (L3), a selective

activator of IKs, which shortened AP of guinea pig ventricular myocytes (22). In this

study of rabbits, we investigated if L3-induced activation of IKs could abbreviate AP and

suppress EAD resulting from IKr inhibition in acquired LQT2 or diminished IKs in left

ventricular hypertrophy (LVH).

H-00076-2002.R1 4

METHODS

Experimental Animals

Male, New Zealand rabbits (1.4-2.0 kg) underwent unilateral nephrectomy and

contralateral renovascular banding to produce LVH as reported previously (19). Banded

rabbits were studied three months after surgery when documented LVH had developed.

Data were collected from 15 control and 9 LVH rabbits.

Myocyte Isolation

Single ventricular myocytes were isolated using a method described previously

(19). After enzyme perfusion, a thin layer (<1.5 mm) of tissue was dissected from

epicardial (Epi) and endocardial (Endo) surface of the left ventricular free wall

(excluding extreme apex and base) and myocytes were dispersed.

Action Potential Recording

AP were recorded from Epi and Endo myocytes using standard microelectrodes

(25-40 MΩ) filled with 3 M KCl. Cells were superfused with a solution containing (mM):

137 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES (pH=7.4) at 36±0.3oC.

AP were recorded at steady state using various stimulus frequencies (0.2, 0.5 and 2

Hz). Action potential duration (APD) was measured at 90% repolarization (APD90).

Because of the beat-to-beat variation of APD, especially at low frequencies, APD90 was

averaged from 10 consecutive AP.

Membrane Current Recording

Membrane ionic currents were recorded at 36±0.5oC using the whole-cell patch-

clamp technique. Solutions (mM): IKs and IKr-Pipette: 119 K-gluconate, 15 KCl, 3.2

MgCl2, 5 HEPES, 5 EGTA, and 5 K2ATP, pH=7.2, (electrode resistance=3-4 MΩ); IKs-

Bath: 132 NaCl, 2 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, 10 glucose, 1 µM dofetilide,

0.6 µM nisoldipine, pH=7.2; IKr-Bath: 132 NaCl, 5 KCl, 1.2 MgCl2, 10 HEPES, 10

glucose, 0.4 µM nisoldipine, 0.1 µM L-768,673 (a specific IKs blocker, 32), pH=7.2; ICa,L-

H-00076-2002.R1 5

Pipette: 151 CsOH, 10 L-aspartic acid, 20 taurine, 20 TEA chloride, 5 glucose, 10

EGTA, pH=7.5 with H3PO4, 5 MgATP and 0.4 Na2GTP were added before use, final

pH=7.3 (electrode resistance=1-2 MΩ); ICa,L-Bath: 140 NaCl, 5 CsCl, 1 MgCl2, 2 CaCl2,

10 glucose, 10 HEPES, pH=7.4. Series resistance was compensated electronically 70-

80%. Liquid junction potential was compensated in the bath but not under whole-cell

conditions.

Epi myocytes were chosen for IKs recording due to their relatively larger IKs

density than in Endo myocytes (32). IKs was recorded with 1-s depolarizing voltage

steps applied at 12-s inter-pulse intervals from a holding potential (Vh) of -40 mV to test

potentials (Vt) of -20 to +60 mV in 20-mV increments (32). Isochronal activation curves

for IKs were determined from peak tail current amplitudes during return to a Vh of -40 mV

following 5-s test pulses. Tail currents (I) were normalized to the maximal tail current

(Imax) obtained following a step to Vt of +70 mV. A Boltzmann function, I/Imax=1/1+exp

[(V0.5-V)/k] (V0.5=half-maximal activation potential, k=slope factor), was fit to the data.

Current-voltage relations were plotted for averaged data normalized to cell capacitance.

A second-order exponential function was fit to the deactivating tail current on return to –

40 mV following a 5-s test pulse to +30 mV to derive the time constants of IKs

deactivation.

IKr was induced by 500-ms pulses to Vt of –10 mV from a Vh of –50 mV. Six

consecutive pulses were delivered at 0.2 Hz and the current traces were averaged. IKr

was quantified as dofetilide sensitive tail current. ICa,L was recorded with 180-ms test

pulses applied at a 5-s interpulse interval from a Vh of -40 mV to Vt of -30 to +50 mV in

10-mV increments.

Data Analysis

Data are expressed as mean ± SEM. Statistical analyses were performed using

GraphPad Prism 2.0 (GraphPad Software, Inc). A paired t-test was used to compare

control and L3 treatment means. Two-way analysis of variance was used to compare

means involving two different categorical variables. P<0.05 was considered statistically

significant.

H-00076-2002.R1 6

Drugs

L3 (R-L3) was synthesized at Merck Research Laboratories, West Point, PA as

previously described (22). Dofetilide and nisoldipine were gifts from Pfizer and Bayer

AG, respectively.

RESULTS

The effects of L3 on IKs in ventricular myocytes of control rabbits are shown in

Fig. 1. L3 throughout these studies was tested at its maximally effective concentration of

1 µM unless otherwise noted (22). Compared to control (Fig. 1A), L3 increased IKs

amplitude and slowed deactivation (Fig. 1B). L3 increased IKs significantly at all Vt (Fig.

1D). At a Vt of +60 mV, L3 increased IKs density from 2.31±0.35 to 3.05±0.46 pA/pF

(n=10). Percentage increases in IKs declined with increasing membrane potential,

286±20% at -20 mV versus 32±2% at +60 mV (n=10, Fig. 1E). L3 shifted the midpoint

(V0.5) of the voltage-dependence of IKs activation by -17.6 mV (Fig. 1F). In control,

deactivation of IKs was best described by a second order exponential function. L3

increased the fast (τf) and slow (τs) time constants of IKs deactivation from 95±7 to

116±14 ms (n=6, n.s.), and 279±25 to 712±127 ms (n=6, P<0.05), respectively.

Fig. 1C shows the ratio of IKs in the presence of L3 to the control (Fig. 1B/1A) for

a test pulse to +60 mV. During the test pulse, L3 increased activating IKs by a constant

ratio of ~1.4 for the entire duration of the pulse, analogous to the increase in current

density measured at the end of the pulse. Upon repolarization to –40 mV, the initial

deactivating tail current is increased to a degree similar to that during the pulse.

Importantly, however, because L3 slowed IKs deactivation, the relative increase in IKs

was greater at later times in the deactivation process, such that at 1 s after

repolarization, the relative current was increased by ~10 fold.

L3 had no effect on ICa,L at all Vt . There was also no evidence for use-dependent

block of ICa,L during repetitive 500-ms pulses from a Vh of -40 mV to a Vt of +20 mV at

0.5 Hz (data not shown). L3 reduced IKr tail current slightly but not significantly from

86±9 to 82±10 pA (n=6, n.s.), and had no significant effect on inward rectifier K+ current

H-00076-2002.R1 7

(IK1) or transient outward K+ current (Ito) (data not shown). Thus, by all these measures,

L3 was a selective activator of IKs.

L3 concentration-dependently shortened AP in rabbit control ventricular

myocytes (Fig. 2). L3 (1 µM) decreased APD90 at 0.5 and 2 Hz by 23±2% and 17±2% in

Epi and by 21±2% and 14±2% in Endo, respectively (n=8). Percentage decreases in

APD90 were similar between Epi and Endo myocytes, but were more pronounced at 0.5

Hz than 2 Hz.

L3 attenuated AP prolongation and eliminated EAD induced by the specific IKr

blocker, dofetilide, in myocytes isolated from control rabbits (Fig. 3). At a stimulus

frequency of 0.5 Hz, 0.1 µM dofetilide induced EAD in 4/11 Endo myocytes. L3

eliminated EAD in all 4 cells in the continuous presence of 0.1 µM dofetilide (Fig. 3A).

The effects of L3 were reversible; e.g., EAD re-appeared during washout of L3 (data not

shown). In the presence of 0.1 µM dofetilide, L3 decreased APD90 from 315±41 to

245±32 ms in Epi myocytes and 627±135 to 388±70 ms in Endo myocytes at 0.5 Hz

(n=8). L3 effects on APD were frequency-dependent. L3 decreased APD90 by 15±1%

and 13±1% at 2 Hz, and 24±2% and 35±3% at 0.5 Hz in Epi and Endo, respectively

(n=8). AP prolonged by 0.1 µM dofetilide in myocytes from both Epi and Endo were

shortened to a lesser extent at 2 Hz than 0.5 Hz by L3. In the presence of dofetilide, L3

decreased APD90 more in Endo (239±69 ms) than Epi myocytes (70±11 ms) at 0.5 Hz

(n=8, P<0.05).

Three months after renal artery banding, rabbits developed LVH with

characteristic changes. LVH significantly increased heart to body weight ratio from

2.10±0.04 (n=15) to 2.53±0.07 g/kg (n=9, P<0.05) and myocyte size, measured as cell

membrane capacitance, from 158±7 (n=20) to 200±12 pF (n=16, P<0.05). Consistent

with our previous findings (32), IKs density of rabbit LVH myocytes was significantly

lower than that of controls, 1.15±0.21 versus 2.31±0.35 pA/pF (n=10, P<0.05). L3 had

similar effects on IKs of myocytes from both control and LVH rabbits (Figs. 1B & 4A). At

a Vt of +60 mV, L3 increased IKs density from 2.31±0.35 to 3.05±0.46 pA/pF, and from

1.15±0.21 to 1.51±0.26 pA/pF, respectively (n=10). L3 increased IKs by 264±9% at -20

mV and 34±4% at +60 mV in myocytes from LVH rabbits (n=10, Fig. 4B). L3 shifted the

H-00076-2002.R1 8

midpoint (V0.5) of the voltage-dependence of IKs activation in LVH rabbits by -19.0 mV

(Fig. 4C).

As observed previously (32), APD90 of Epi and Endo ventricular myocytes in LVH

rabbits was increased by 21% and 22% at 2 Hz, respectively, compared with controls.

At 0.5 Hz, APD90 increased from 175±15 to 212±27 ms by LVH in Epi, and from 231±18

to 296±35 ms in Endo myocytes, respectively (n≥11). Spontaneous EAD were recorded

in 3/13 myocytes from Endo of LVH rabbits at a stimulus frequency of 0.2 Hz. L3

eliminated EAD in all 3 cells (Fig. 5A). In LVH myocytes, 1 µM L3 decreased APD90 by

19±2% and 13±1% in Epi and by 17±2% and 13±1% in Endo at 0.5 and 2 Hz,

respectively (n=8). Percentage decreases of APD90 by L3 were similar between Epi and

Endo at both 0.5 and 2 Hz. However, L3 decreased APD90 more at 0.5 Hz than 2 Hz.

DISCUSSION

This study provides the first direct evidence that IKs activation can be useful in

reversing excessive AP prolongation and suppressing arrhythmogenic EAD in certain

forms of LQTS. We examined this utility in rabbit models of acquired LQTS using the

selective activator of IKs, L3, described previously in detail (22). In a model of LQT2, IKr

inhibition with dofetilide produced typical reverse frequency-dependent increases in

APD and EAD (25). In a model of LVH induced by renovascular hypertension, APD

were prolonged, spontaneous EAD occurred in 23% of Endo myocytes at 0.2 Hz, and

IKs density was significantly reduced, consistent with our previous findings (32). These

profiles of abnormal repolarization reflect deficiencies in two major repolarizing currents,

IKr and IKs, respectively.

Importantly, in control, dofetilide-treated and LVH myocytes, L3 shortened APD

in a concentration-dependent manner and eliminated EAD in the latter two conditions.

AP mapping across the left ventricular free wall using arterially perfused wedge

preparations shows that endocardium has the longest and epicardium the shortest APD

in both control and LVH rabbits (33). Across the left ventricular free wall, there is a

gradual increase in APD from epicardial side to endocardial side. This differs from

canine left ventricle where the longest APD is recorded in subendocardium (M-cells)

H-00076-2002.R1 9

(34). L3 decreased APD to a similar degree in Epi and Endo myocytes in both control

and LVH (Figs. 2B & 5C), indicating that IKs activation by L3 in rabbits should not

increase transmural dispersion of repolarization (TDR). In dofetilide-treated myocytes,

L3 decreased APD more significantly in Endo than Epi at 0.5 Hz, due partly to

elimination of EAD in Endo. Thus, in acquired LQT2 or abnormally prolonged

repolarization due to IKr inhibition, IKs activation should actually decrease TDR.

L3 decreased APD more at 0.5 Hz than 2 Hz in control, dofetilide-treated and

LVH. Because excessive AP prolongation, EAD and torsades de pointes are associated

with bradycardia, this characteristic of L3 could be particularly beneficial. Because of the

complicated interaction of many time and voltage dependent currents in controlling AP

morphology, it is oversimplified to extrapolate changes in a single current to effects on

APD. Nevertheless, the L3 induced slowing of deactivation may provide a basis for the

greater shortening of APD observed at slower rates. Normally, due to its slow

deactivation, IKs can accumulate more at higher frequencies and thereby contribute to

rate dependent shortening of APD (11, 13, 24). L3 slows IKs deactivation and increases

relative deactivating current more at later times or longer intervals (Fig. 1C). The larger

relative increases in deactivating IKs at longer intervals may lead to greater than normal

accumulation at the slower rates and explain the greater decreases in APD by L3 at 0.5

Hz than at 2 Hz. However, the mechanism underlying the frequency dependent effects

of L3 on APD still requires further study.

L3 concentration-dependently activated IKs and shortened APD in guinea pig

ventricular myocytes (22). In this study, L3 effects on rabbit IKs were studied at a

maximally effective concentration of 1 µM as determined previously for guinea pig. L3

increased IKs in rabbit via a negative shift in the voltage-dependence of activation and a

slowing of deactivation. L3 shifted the V0.5 of rabbit IKs activation by -17.6 mV in control

and by -19.0 mV in LVH, comparable to the shift of -24 mV found previously for guinea

pig. This shift accounts for the larger percent increases of IKs at more negative test

potentials (Figs. 1E & 4B). Deactivation of IKs was best described by a second-order

exponential function. L3 increased both τf (22%) and τs (155%) of deactivation of rabbit

IKs, consistent with the changes in guinea pig (70% and 190%, respectively).

H-00076-2002.R1 10

Our experimental results indicate that L3 (up to 1 µM) is a selective activator of

IKs in rabbit ventricular myocytes. L3 also slows deactivation of IKs. These selective

actions on the gating of IKs are likely the predominant if not only mechanisms underlying

the APD shortening and EAD elimination by L3. Both the negative shift in the voltage

dependence of activation and the slowing of deactivation can increase the contribution

of IKs to AP repolarization.

A reduction of repolarizing K+ currents in LQTS and hypertrophied hearts has

been widely observed. Reductions in the density of Ito and IK1 are commonly observed in

cardiac hypertrophy and failure (26). Reductions in IKs and IKr, which play dominant roles

in ventricular AP repolarization of many mammalian species including rabbit (21), are

also common. In cats, delayed rectifier K+ current density was decreased in right

ventricular hypertrophy induced by pressure overload (14), and LVH induced by aortic

stenosis (10). In rabbits, LVH induced by renovascular hypertension significantly

reduced IKs but not IKr density (32), whereas pacing-induced heart failure reduced both

IKs and IKr (28). In dogs with chronic complete atrioventricular block, IKr density of

mid-myocardial cells was decreased in right ventricle and IKs density was decreased in

both ventricles (30). In failing human hearts, IKs density was decreased by 60% (15).

The reduction in the delayed rectifier K+ currents predisposes ventricular myocytes to

potentially arrhythmogenic EAD (9, 17, 32). Computer models of the mammalian

ventricular AP predict that reducing IKs induces EAD in the endocardial cells during

normal cell-to-cell coupling (29).

Other studies aimed at treating arrhythmias resulting from excessive AP

prolongation and LQTS have explored various strategies for augmenting repolarizing K+

currents. These include, increasing IKr by elevating serum K+ concentrations (5, 6),

administration of ATP-sensitive potassium channel openers (PCO) (1, 8, 23), and over-

expression of HERG K+ channels (IKr) (18).

Study limitations: Since excessive APD prolongation and EAD are associated

with bradycardia, the pacing rates used in this study are below the normal physiologic

rates in the rabbit. The rabbit models used in this study only address the down

regulation of IKr amplitude (acquired LQT2) or IKs density (LVH). In patients with

congenital LQTS, not only is current density affected, but also other properties of IKr or

H-00076-2002.R1 11

IKs channels may change. It is uncertain whether IKs activation will have antiarrhythmic

effects in these patients. Use of IKs activators may not be void of adverse effects in

some situations. Shortening APD by IKs activation may facilitate the development of

reentrant arrhythmia under certain circumstances, particularly in myocardial ischemia

and infarction. The effects of L3 on action potentials under ischemic conditions are

unknown, however, we expect the APD shortening by IKs activation would be very

modest and possibly overwhelmed by activation of the large conductance of the KATP

current.

In conclusion, selective activation of IKs by pharmacological agents is a promising

new strategy to suppress arrhythmias resulting from excessive AP prolongation in

patients with certain forms of LQTS or cardiac hypertrophy and failure.

Acknowledgements

This work was supported in part by the Sharpe Foundation and the Fourjay

Foundation.

H-00076-2002.R1 12

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Circulation. 103:1585-1590, 2001.

33. Yan G-X, Rials SJ, Wu Y, Liu T, Xu X, Marinchak RA, and Kowey PR. Ventricular

hypertrophy amplifies transmural dispersion of repolarization and induces phase

2 early afterdepolarization. Am J Physiol. 281:H1968-H1975, 2001.

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H-00076-2002.R1 17

FIGURE LEGENDS

Figure 1. L3 increases IKs in rabbit control ventricular myocytes. IKs traces shown for

control (A) and 1 µM L3 (B) at Vt of -20, 0, +20, +40, and +60 mV. C: Ratio of IKs in the

presence of L3 to the control (B/A) for a test pulse to +60 mV (top traces). D:

Normalized current-voltage (I-V) relation for IKs in control and after 1 µM L3 (n=10,

*P<0.05, L3 versus control). E: Percentage increases of IKs by 1 µM L3 at various Vt. F:

Negative shift of the voltage-dependence of IKs activation by 1 µM L3 (n=6). Control:

V0.5=14.1 mV, k=12.0 mV; L3: V0.5=-3.5 mV, k=13.9 mV.

Figure 2. L3 concentration-dependently shortens AP in rabbit control ventricular

myocytes. A: Superimposed AP traces recorded in Epi (top) and Endo (bottom)

myocytes at stimulus frequencies of 0.5 and 2 Hz. L3 at 0.1 µM (arrows) shortened AP.

B: Concentration-dependent decrease of APD90 in Epi (top) and Endo (bottom)

myocytes by L3 (n=8, *P<0.05, 2 Hz versus 0.5 Hz).

Figure 3. L3 attenuates AP prolongation and eliminates EAD induced by IKr blockade in

rabbit control ventricular myocytes. A: AP recorded in Epi (top) and Endo (bottom)

myocytes at a stimulus frequency of 0.5 Hz for control, 0.1 µM dofetilide; and 0.1 µM

dofetilide + 1 µM L3. Three superimposed traces are shown for each condition. B:

Decreases of APD90 in Epi and Endo myocytes by 1 µM L3 in the presence of 0.1 µM

dofetilide (n=8, *P<0.05 2 Hz versus 0.5 Hz; +P<0.05 Endo versus Epi).

H-00076-2002.R1 18

Figure 4. L3 increases IKs in rabbit LVH ventricular myocytes. A: Normalized I-V relation

for IKs in LVH and after 1 µM L3 (n=10, *P<0.05, L3 versus LVH). B: Percentage

increases of IKs by 1 µM L3 at various Vt. C: Negative shift of the voltage-dependence of

IKs activation by 1 µM L3 (n=6). Control: V0.5=15.1 mV, k=12.9 mV; L3: V0.5=-3.9 mV,

k=13.2 mV.

Figure 5. L3 normalizes APD and eliminates spontaneous EAD in rabbit LVH

ventricular myocytes. A: L3 at 1 µM eliminated spontaneous EAD in a LVH Endo

myocyte at a stimulus frequency of 0.2 Hz. Three superimposed traces are shown for

each condition. B: L3 at 1 µM (arrows) shortened AP of a LVH Endo myocyte at 0.5 and

2 Hz. C: Average percentage decreases of APD90 by 1 µM L3 in LVH Epi and Endo

myocytes (n=8, *P<0.05, 2 Hz versus 0.5 Hz).

H-00076-2002.R1 19

Figure 1

0

200

400

0

200

400

1 µM L3

voltage (mV)

-20 0 20 40 60

I Ks

(pA

/pF

)

0

1

2

3

Control1 µM L3*

*

*

*

*

voltage (mV)

-20 0 20 40 60

% In

cre

ase

of I K

s

0

100

200

300

A

C

B

Control

F

voltage (mV)

-40 -20 0 20 40 60

I / I m

ax

0.0

0.2

0.4

0.6

0.8

1.0

Control1 µM L3

0

4

8

12

16

pA

pA

ratio

500 ms

D

E

H-00076-2002.R1 20

Figure 2

0.03 0.30.1 1

% D

ecre

ase

of A

PD

90

0

5

10

15

20

25Epi

[L3] (µM)

0.03 0.30.1 1

% D

ecre

ase

of A

PD

90

0

5

10

15

20

25

0.5 Hz 2 Hz

Endo

*

*

*

*

*

*

mV

-90

-60

-30

0

30

mV

-90

-60

-30

0

30

50 ms

A B

0.5 Hz

0.5 Hz

2 Hz

2 Hz

Epi

Endo

H-00076-2002.R1 21

Figure 3m

V

-90

-60

-30

0

30

150 ms

mV

-90

-60

-30

0

30 Control Dofetilide Dofetilide+L3A

B

Epi Endo

% D

ecre

ase

of A

PD

90

0

10

20

30

40 0.5 Hz 2 Hz

+Dofetilide

**

+

H-00076-2002.R1 22

Figure 4

voltage (mV)

-40 -20 0 20 40 60

I / I m

ax

0.0

0.2

0.4

0.6

0.8

1.0

voltage (mV)

-20 0 20 40 60

% In

cre

ase

of I

Ks

0

100

200

300

C

BA

voltage (mV)

-20 0 20 40 60

I Ks

(pA

/pF

)

0

1

2

3

LVH1 µM L3

*

*

*

*

*

LVH1 µM L3

H-00076-2002.R1 23

Figure 5m

V

-90

-60

-30

0

30

150 ms

mV

-90

-60

-30

0

30

75 ms

A

C

B

Epi Endo

% D

ecr

ease

of A

PD

90

0

5

10

15

20

0.5 Hz 2 Hz

* *

LVH 1 µM L3

0.2 Hz 0.2 Hz

0.5 Hz 2 Hz