Characterization of a novel multifunctional resveratrol derivative for the treatment of atrial fibrillation

Article abstract of DOI:10.1111/bph.12409

Background and Purpose Atrial fibrillation (AF) is the most common cardiac arrhythmia and is associated with an increased risk for stroke, heart failure and cardiovascular-related mortality. Candidate targets for anti-AF drugs include a potassium channel K_v1.5, and the ionic currents I _KACh and late I_Na, along with increased oxidative stress and activation of NFAT-mediated gene transcription. As pharmacological management of AF is currently suboptimal, we have designed and characterized a multifunctional small molecule, compound 1 (C1), to target these ion channels and pathways. Experimental Approach We made whole-cell patch-clamp recordings of recombinant ion channels, human atrial I_Kur, rat atrial I _KACh, cellular recordings of contractility and calcium transient measurements in tsA201 cells, human atrial samples and rat myocytes. We also used a model of inducible AF in dogs. Key Results C1 inhibited human peak and late K_v1.5 currents, frequency-dependently, with IC₅₀ of 0.36 and 0.11 μmol·L^-1 respectively. C1 inhibited I _KACh (IC₅₀ of 1.9 μmol·L^-1) and the Na_v1.5 sodium channel current (IC₅₀s of 3 and 1 μmol·L^-1 for peak and late components respectively). C1 (1 μmol·L^-1) significantly delayed contractile and calcium dysfunction in rat ventricular myocytes treated with 3 nmol·L ^-1 sea anemone toxin (ATX-II). C1 weakly inhibited the hERG channel and maintained antioxidant and NFAT-inhibitory properties comparable to the parent molecule, resveratrol. In a model of inducible AF in conscious dogs, C1 (1 mg·kg^-1) reduced the average and total AF duration. Conclusion and Implications C1 behaved as a promising multifunctional small molecule targeting a number of key pathways involved in AF.

Full text of DOI:10.1111/bph.12409

Characterization of a novel multi-functional resveratrol
derivative for the treatment of atrial fibrillation.1
Istvan Baczko, MD, PhD*1; David Liknes*2; Wei Yang2, PhD; Kevin C Hamming2, PhD; Gavin
Searle2, PhD; Kristian Jaeger, MD2; Zoltan Husti1; Viktor Juhasz1; Gergely Klausz3; Robert
Pap3; Laszlo Saghy3; Andras Varro, MD, PhD, DSc1,4; Vernon Dolinsky5, PhD; Shaohua Wang6,
MD; Vivek Rauniyar7, PhD; Dennis Hall7, PhD; Jason R. Dyck, PhD8; Peter E. Light, PhD2#.
1. Department of Pharmacology and Pharmacotherapy, University of Szeged, Dom ter 12. 6720,
Szeged, Hungary.
2. Department of Pharmacology, Cardiovascular Research Centre and the Alberta Diabetes
Institute, Li Ka Shing Research Centre, University of Alberta, Edmonton Alberta, Canada.
3. 2nd Department of Medicine and Cardiology Centre, University of Szeged, Koranyi fasor 6.
6720, Szeged, Hungary.
4. Division of Cardiovascular Pharmacology, Hungarian Academy of Sciences, Szeged,
Hungary.
5. Department of Pharmacology and Therapeutics, Manitoba Institute of Child Health, University
of Manitoba, Winnipeg, Manitoba, Canada.
6. Division of Cardiac Surgery, Faculty of Medicine and Dentistry, University of Alberta,
Edmonton Alberta, Canada.
7. Department of Chemistry, University of Alberta, Edmonton Alberta, Canada.
8. Department of Pediatrics, Cardiovascular Research Centre and the Alberta Diabetes Institute,
University of Alberta, Edmonton Alberta, Canada.
* NOTE: These two authors contributed equally to the work.
# Corresponding author.
Abstract word count: 248
Text body word count: 4,762 (excluding abstract, references and figure legends).
Keywords: atrial fibrillation; pharmacology; Kv1.5; resveratrol; electrophysiology; ion
channels.
This article has been accepted for publication and undergone full peer review but has not been through the
copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version
and the Version of Record. Please cite this article as doi: 10.1111/bph.12409
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
Abstract.
Background and Purpose. Atrial fibrillation (AF) is the most common cardiac arrhythmia and is
associated with an increased risk for stroke, heart failure and cardiovascular-related mortality.
Candidate targets for anti-AF drugs include the ion channels Kv1.5, IKACh and late INa. AF is also
associated with oxidative stress and activation of NFAT-mediated gene-transcriptional pathways.
Since current pharmacological management of AF is sub-optimal, we designed and characterized
a multi-functional small molecule, compound 1 (C1), to target these ion channels and pathways.
Experimental approach. To assess the characteristics and efficacy of C1, we employed whole
cell patch-clamp recordings of recombinant ion channels, human atrial IKur, rat atrial IKACh
,
cellular recordings of contractility, calcium transient measurements and an in vivo large animal
model of inducible AF.
Key Results. C1 inhibited human peak and late Kv1.5 currents in a frequency-dependent manner
with an IC50 of 0.36 and 0.11 mol/L respectively. C1 inhibited IKACh (IC50 of 1.9 mol/L) and
the Nav1.5 sodium channel current (IC50s of 3 and 1 mol/L for peak and late components
respectively. C1 (1 mol/L) significantly delayed contractile and calcium dysfunction in rat
ventricular myocytes treated with 3 nmol/L ATX-II. C1 weakly inhibited the hERG channel and
maintained similar anti-oxidant and NFAT-inhibitory properties as the parent molecule
resveratrol. In a conscious dog model of inducible AF, C1 (1mg/kg) significantly reduced the
average and total AF duration.
Conclusion and Implications. These findings indicate that C1 is a promising multi-functional
small molecule that targets a number of key pathways involved in AF.
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
Introduction
AF is the most common type of cardiac arrhythmia and the incidence of AF is increasing
with an aging population (McManus et al., 2012; Naccarelli et al., 2009). While AF does not
generally induce sudden cardiac death, AF increases the risk of stroke 5-fold (McManus et al.,
2012; Wolf et al., 1991; Hart et al., 2001), can cause tachycardia-induced cardiomyopathy
(Nerheim et al., 2004) and adverse electrical and structural remodeling of the heart (Franz et al.,
1997; Anter et al., 2009). Furthermore, ~30% of patients undergoing coronary artery by-pass
graft (CABG) surgery develop transient post-operative AF (Mathew et al., 2004; Andrews et al.,
1991). Current pharmacotherapy of AF is still sub-optimal, since some of the drugs used for
rhythm control also display significant risk for acquired LQT and Torsades des Pointes
arrhythmias (Taira et al., 2010) and may increase the incidence of vascular events (Connolly et
al., 2011). Therefore, there is a significant need for the development of improved therapeutics for
AF.
Over the last decade, studies on the cellular pathways in AF have revealed potential
therapeutic targets to develop new anti-AF drugs. One important target is the ultra rapid
potassium channel (IKur/ Kv1.5) preferentially expressed in the atria, but not the ventricle
(Ravens et al., 2011; Gaborit et al., 2007; Wang et al., 1993; Tamargo et al., 2009). In addition
to Kv1.5, IKACh (Ehrlich and Nattel, 2009), voltage-gated sodium channels (Burashnikov et al.,
2012; Burashnikov et al., 2007), oxidative stress (Carnes et al., 2001; Mihm et al., 2001) and
activation of the nuclear factor of activated T-cells (NFAT) (Lin et al., 2004) have all been
implicated in AF development. Given the complex etiology of AF, it has been suggested that
drugs targeting multiple pathways involved in AF development may be more effective (Dobrev
et al., 2010; Dobrev et al., 2012). To date, AF drugs have been exclusively targeted towards ion
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
channels. However, non-ionic remodeling events also likely contribute to the initiation and
maintenance of AF as well. It has been further suggested that targeting maladaptive remodeling
events in AF may also be required for effective control of AF (Burashnikov et al., 2011).
Therefore, based on these criteria, a multi-functional small molecule drug for AF should ideally
possess the following properties; 1) frequency dependent Kv1.5 inhibition, 2) IKACh inhibition 3)
late sodium current inhibition 4) lack of hERG channel inhibition 5) display atrial specificity i.e.
no effect on repolarization and excitation-contraction coupling in ventricular tissue 6) anti-
oxidant properties 7) NFAT inhibition and 8) functional efficacy in a large animal model of
inducible AF.
Resveratrol is a bioactive polyphenol found in red grape products that has been
investigated intensively for its cardio-protective and anti-aging properties (Dolinsky et al., 2011;
Brisdelli et al., 2009). We have previously shown that resveratrol 1) inhibits NFAT activation
and hypertrophic remodeling in cardiac tissue (Chan et al., 2008; Dolinsky et al., 2009) and 2)
inhibits the late INa current preferentially over peak current (Wallace et al., 2006). Based on these
multi-functional properties, the aim of the present study was to synthesize a novel small
molecule using resveratrol as the parent molecule, termed compound 1 (C1), and characterize its
anti-oxidant, NFAT-inhibitory effects and electrophysiological profile with respect to Kv1.5,
IKACh, late INa and hERG, and its in vivo efficacy in a large animal model of inducible AF.
Methods
Chemical synthesis of compounds
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
Details of the chemical synthesis procedures used to generate four unique resveratrol
hybrid bis-ortho-substituted biphenyl diamide derivatives are provided in the online
supplementary materials section.
Cell transfection
The human Nav1.5 and hERG clones were generously provided by Dr. Arthur Brown
(Case Western Reserve University, USA) and Dr. Henry Duff (University of Calgary, Canada)
respectively. Expression vectors were co-transfected into the tsA201 cell line with a GFP
expression vector using the calcium phosphate precipitation technique. TsA201 cells are a
transformed HEK293 cell line expressing the temperature-sensitive antigen to increase
expression levels of recombinant proteins).
Transfected cells were then used within 48 hrs. All in vitro experiments were performed
at room temperature (20-22oC).
Kv1.5 currents
Recordings were made from single tsA201 cells stabling expressing the human heart
Kv1.5 gene. Pipettes were pulled from borosilicate glass capillary tubing (Warner instruments,
Hamden, CT USA) and tips were fire-polished, producing resistances of 1–3 MΩ. The pipette
solution contained (in mmol/L): 140 KCl, 1 CaCl2, 1 MgCl2 , 10 2-[4-(2-hydroxyethyl)-1-
piperazinyl]ethanesulphonic acid (HEPES) and 10 ethylene glycol-bis-(2-aminoethyl ether)-
N,N,N′,N′-tetraacetic acid (EGTA). The pH was adjusted to 7.3 with KOH. 2 mmol/L MgATP
was added immediately before use. Cells were bathed in extracellular solution contained (in
mmol/L): 135 NaCl, 5.4 KCl, 1 CaCl2, 1.2 MgCl2, 10 HEPES and 5 Glucose (pH adjusted to 7.4
with NaOH). Solutions were applied to cells using a multi-input perfusion pipette (switch time <
2 s). Once a giga-seal was formed, the patch was ruptured and the whole-cell voltage-clamp
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
technique was used to record Kv1.5 currents. Currents were elicited by 120 ms duration test
pulses to +40 mV from a holding potential of -100 mV with a cycle length of 0.3 Hz, 1 Hz or 3
Hz respectively. Data were acquired using an Axopatch 200B patch-clamp amplifier and
Clampex 8.2 software (Axon Instruments, Foster City, CA, USA).
Nav1.5 currents
Single tsA201 cells transfected with mammalian expression vectors encoding the human
heart NaV1.5 were used for recording of whole-cell Nav1.5 currents as described previously
(Wallace et al., 2006). Voltage steps from a holding potential of -120 mV to 40 mV every 10mV
was used to generate the current-voltage (I –V) relationship for peak INa.
hERG currents
TsA201 cells were transfected with a mammalian expression vector encoding the hERG
channel clone (Kv11.2) as described for Nav1.5 above and hERG currents were recorded as
described previously (Kikuchi et al., 2005).
Human atrial IKur currents
For experiments on human atrial IKur (Kv1.5), atrial appendage samples were collected
during CABG surgery (University Hospital, University of Alberta, Canada) and enzymatically
dissociated into single atrial myocytes. IKur currents were then measured using a modification of
the whole-cell recording protocol to inactivate Ito currents as described previously (Cai et al.,
2007). Atrial tissue was collected from 7 patients with 1) prior ethical approval from the
University of Alberta human ethics board and 2) informed patient consent.
Rat atrial IKACh and recombinant IK1 currents.
Rat atrial myocytes were isolated as described previously (Komukai et al., 2002). Whole-
cell recordings of carbachol-induced IKACh currents were made using the same solutions used for
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
Kv1.5 and a voltage step protocol from -110 to +50 mV with a holding potential of -80 mV. IK1
whole-cell currents were measured in tsA201 cells transiently transfected with the Kir2.1 clone
using the same solutions and voltage protocols used for IKACh currents.
Cell shortening and calcium transient recordings
These were measured in rat ventricular myocytes using standard procedures previously
published by our laboratory (Wallace et al., 2006; Baczko et al., 2005).
NFAT Reporter Assay
Neonatal rat ventricular myocytes were cultured as described previously(Chan et al.,
2008), and infected with Ad.GFP or Ad.NFAT-Luc-Promoter adenovirus (Seven Hills
Bioreagents) at the multiplicity of infection (MOI) of 10. Cells were treated 24 h post-infection
with vehicle or varying concentrations of C1 or resveratrol and/or angiotensin II (1 mol/L for
24 hours). Cells were harvested with the reporter lysis buffer supplied in the luciferase assay
system kit (Promega) and luminescence measured as per the manufacturer's instructions.
Antioxidant assay
A Diphenylpicrylhydrazyl (DPPH) assay was used to determine the anti-oxidant capacity
of C1 and resveratrol. A solution of H2O2 at a concentration of 200 nmol/L was mixed with
DPPH alone or DPPH with either resveratrol or C1 at various concentrations. The absorbance of
these solutions was measured at 517 nm, with a lower absorbance representing an increase in
anti-oxidant capacity.
In vivo electrophysiology
All experiments were carried out in compliance with the Guide for the Care and Use of
Laboratory Animals (USA NIH publication No 85–23, revised 1996) and approved by the
Department of Animal Health and Food Control of the Ministry of Agriculture and Rural
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
Development, Hungary (XIII/01031/000/2008). Full experimental details are provided in the
online supplementary materials section.
Statistics
Data were expressed as mean ± S.E.M. In whole animal experiments, each animal served
as its own control, after one-way analysis of variance (ANOVA), the groups were compared in
pairs by means of Student’s t-test. In cell-based experiments one-way ANOVAs or paired
Student’s t-tests were used as appropriate. A level of p<0.05 was considered to be significant.
Ion channel nomenclature conforms to the British Journal of Pharmaology Guide to
Receptors and Channels (Alexander, Mathie and Peters, 2011.).
Results
Effects of C1 on Kv1.5 currents
The Kv1.5 inhibitory effects of four novel resveratrol derivatives (Figure 1A) were
compared to the parent molecule resveratrol. Resveratrol is a weak inhibitor of Kv1.5 (IC50 = 66
mol/L, Figure 1B). Of the four derivatives synthesized, compound 1 (C1) was found to be the
most potent Kv1.5 inhibitor (IC50s = 0.36 mol/L and 0.11 mol/L for peak and late current
inhibition respectively, Figure 1B-E). Compounds 2-4 displayed intermediate Kv1.5 peak current
inhibitory potencies of 8.3, 10.9 and 11.2 mol/L respectively (Figure 1B). Therefore, only C1
was selected for further characterization in this study.
Frequency-dependent effects of C1 on Kv1.5 currents
The frequency-dependent effects of C1 were tested on whole-cell Kv1.5 currents in the
Kv1.5 stably expressing cell line. At 1 Hz, 0.3 mol/L C1 displayed a time-dependent inhibition
of Kv1.5 currents, reaching a steady-state after 15 sec (Itest/Icontrol = 0.74  0.02). In contrast, at a
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
higher stimulation frequency of 3 Hz, the observed C1 inhibition was increased, reaching a
steady-state level of 0.50  0.03 when compared to control (n= 5 cells, Figure 2A, B).
Effects of C1 on human atrial IKur currents.
Single atrial myocytes were isolated from human atrial appendage samples obtained
during CABG surgery and whole-cell IKur currents measured. Application of C1 (0.3 mol/L)
reversibly inhibited IKur to 31.0  1.8% of control values (n = 3 cells, Figure 2C, D), a value
similar to that observed on recombinant Kv1.5 (Figures 1D and 2A, B).
Effects of C1 on rat atrial IKACh and recombinant IK1 currents.
Single atrial cells were isolated from rat hearts and IKACh currents were elicited by
application of carbachol. C1 significantly inhibited carbachol-induced rat atrial IKACh currents
with an IC50 of 1.9 mol/L (Figure 3.). In contrast, 3 mol/L C1 had no significant inhibitory
effect on recombinant IK1 (Kir2.1) whole-cell currents transiently expressed in tsA201 cells (see
supplementary material for results).
Effects of C1 on human Nav1.5 currents
The effects of C1 were tested on peak and late recombinant Nav1.5 whole-cell currents.
Application of 3 mol/L C1 resulted in a 50% inhibition of peak current (Figure 4A, B). To
establish the concentration-response curves for peak and late Nav1.5 currents, cells were treated
with the sea anemone toxin ATX-II (3 nmol/L) to induce the late current component and the
inhibitory effects of C1 at differing concentrations tested. C1 preferentially inhibits the late
current when compared to peak current (IC50s = 1.1 mol/L vs 3.2 mol/L respectively, Figure
4C, D).
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
Effects of C1 on hERG currents
Inhibition of the hERG (IKr, Kv11.2) is highly undesirable and may result in
arrhythmogenic QT prolongation (Sanguinetti et al., 1996; Sanguinetti et al., 2006; Taira et al.,
2010). Therefore, we tested the effects of C1 on recombinant whole-cell hERG channel currents.
hERG currents were positively identified by their characteristic inactivation at positive holding
potentials, large tail currents and inhibition by 1 mol/L E-4031 (Figure 5A). Construction of
concentration-inhibition curves revealed that C1 is a weak inhibitor of peak and tail hERG
currents (Figure 5B, C), with IC50s of 30 and 25 mol/L respectively, values that are greater than
100-fold higher than peak and late Kv1.5 inhibition.
C1 Anti-oxidant capacity and NFAT activation
Anti-oxidant therapy has been shown to reduce the incidence of post-operative AF in
humans (Carnes et al., 2001) and reactive oxygen species also directly activate Kv1.5 channels
(Caouette et al., 2003). As resveratrol is a known anti-oxidant, we compared the anti-oxidant
properties of C1 with resveratrol. At 10 mol/L, resveratrol and C1 displayed significant
antioxidant capacities (0.59  0.04 vs 0.77  0.02 of maximal DPPH 517 nm absorbance signal,
Figure 6A). At 100 mol/L, these values were 0.09  0.02 and 0.19  0.02 for resveratrol and C1
respectively.
We have previously shown that resveratrol inhibits NFAT activation induced by
phenylephrine contributing to a reduction in maladaptive hypertrophy in neonatal rat ventricular
myocytes (NRVMs)(Chan et al., 2008). Accordingly, we tested the effects of C1 on NFAT
activation in NRVMs. Treatment of NRVMs with 0.01-25 mol/L of C1 resulted in a significant
reduction in NFAT activity when compared to no treatment (Figure 6B). These experiments
revealed that C1 significantly reduced NFAT activity at concentrations of 0.1 mol/L and higher,
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
with 1 mol/L of C1 exhibiting half-maximal inhibition (Figure 6B). Since 1 mol/L of C1
inhibited NFAT activation by 50%, we used this concentration to determine whether C1 was able
to inhibit NFAT activation in response to a pro-hypertrophic stimuli. NRVMs were infected as
above and treated with vehicle, AngII (1 mol/L), C1 (1 mol/L) or AngII + C1 (1 mol/L) for
24 hours. As expected, treatment of NRVM with 1 mol/L of AngII significantly increased
NFAT activation by 504  73%, whereas 1 mol/L of C1 inhibited NFAT activation by
approximately 50%. C1 also significantly reduced AngII-induced activation of NFAT to 160 
23% of control values (Figure 6C.).
Effects of C1 on ventricular myocyte excitation-contraction coupling
A desirable property of potential compounds for AF is a minimal effect on excitation-
contraction coupling in ventricular myocytes. Single rat ventricular myocytes were field
stimulated at 1 Hz; contractility and calcium transients were then measured by edge-detection
and calcium-imaging. Application of 3 mol/L C1 had no significant effect on 1) diastolic
resting cell length or calcium levels or 2) peak systolic contractility or calcium transient
amplitude (Figure 7A, B).
The results presented in Figure 3 indicate that C1 inhibits the late component of the
cardiac voltage-gated sodium channel current compared to peak current. Therefore, the effects of
C1 were tested in a single ventricular myocyte model of late sodium current-induced
disturbances in calcium homeostasis by pretreatment of myocytes with 3 nmol/L ATX-II. ATX-
II pretreatment results in markedly elevated diastolic calcium levels and the loss of uniform
calcium transients (Figure 7C, E, F). In contrast, application of C1 (1 mol/L), significantly
reduced the magnitude of ATX-II-induced diastolic calcium elevations (C1 = 5.9  2.9 % vs
control = 57.8  5.9% of maximal transient amplitude, p<0.01, n=5 cells,) and significantly
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
delayed the onset of calcium transient dysfunction (C1 = 5.5  0.8 min vs 3.1  0.4 min for
control, p<0.01, n = 10 cells, Figure 7D, E, F).
Effects of C1 in a large animal model of inducible AF
Right atrial effective refractory period measurements before the commencement of rapid
atrial pacing at 400 beats/min yielded 118 ± 3.7 and 130 ± 3.2 ms in conscious dogs (n = 5; at
basic cycle lengths of 150 and 300 ms, respectively). Rapid right atrial pacing for 6 to 7 weeks
resulted in a significant decrease of right AERP, since AERP decreased below 80 ms in all five
animals, and were defined as 79 ms for the following reason: the lower adjustable limit for S1-S2
intervals in the pacemakers available for this study was 80 ms, therefore the exact AERP after 7
weeks of rapid atrial pacing and immediately before C1 administration could not be determined
(measured AERP values were less than 80 ms in all dogs at both basic cycle lengths since an S1-
S2 interval of 80 ms still evoked a P wave), although statistical comparison of AERP values was
not possible. AERP measurements following C1 administration yielded 87.5 ± 2.50 ms at 150 ms
BCL in 4 animals (in one animal AERP was less than 80 ms), 90 ± 3.16 ms at 300 ms BCL,
respectively, following 0.3 mg/kg C1; and 90.0 ± 3.16 ms at 150 ms BCL, 100 ± 3.16 ms at 300
ms BCL, respectively, following 1 mg/kg C1 (n = 5; with the exception of AERP measurement
at 150 ms BCL following 0.3 mg/kg C1).
The incidence of AF was not influenced by administration of C1 (86.4 ± 6.4 % in control vs.
71.2 ± 15.7 % and 66.4 ± 15.7 % following 0.3 and 1 mg/kg C1, respectively, both p>0.05).
However, as typical ECG recordings and grouped data show, the total duration of AF was
significantly reduced by 1 mg/kg C1 administration (Figure 8 B-D), and the average duration of
AF episodes was also significantly decreased by 1 mg/kg C1 (Figure 8 A-D). These results
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
clearly demonstrate in vivo efficacy of C1 against atrial fibrillation in conscious dogs. HPLC
analysis of blood plasma collected within 5 min of IV bolus injection showed that we obtained
concentration ranges of 0.32 to 0.79 mol/L and 0.7 to 3.0 mol/L for the 0.3mg/kg and 1mg/kg
dosing respectively.
None of the investigated doses exhibited any QT interval prolonging effect in five
conscious dogs, yielding 261.7 ± 9.37 ms in control vs. 260.9 ± 8.77 ms (p = 0.15) following 0.3
mg/kg C1 and 257.3 ± 9.05 ms (p= 0.55) following 1 mg/kg C1.
Discussion
Ionic effects of C1
As decreased atrial action potential refractory period contributes to the development of
AF, the design of atrial specific repolarization delaying drugs has received much recent attention
(Dobrev et al., 2010; Tamargo et al., 2009). Specifically, inhibition of the IKur/Kv1.5 repolarizing
potassium channel that is predominately expressed in atria rather than ventricle (Gaborit et al.,
2007; Tamargo et al., 2009; Wang et al., 1993) has emerged as an attractive therapeutic target
(Tamargo et al., 2009). Results generated in this study on recombinant Kv1.5 and human atrial
myocyte IKur currents indicate that C1 is an effective inhibitor of this atrial-specific ion channel.
The calculated IC50s for Kv1.5 are in the 0.11-0.36 mol/L range for late and peak Kv1.5 current
inhibition, which are 180-600-fold lower than observed for the parent resveratrol molecule (66
mol/L). Furthermore, these IC50 values are 10-30-fold lower than those reported for the recently
developed AF drug vernakalant that is in clinical use in Europe (Camm et al., 2012). Further
analysis revealed that C1 is a more effective Kv1.5 inhibitor at a higher stimulatory frequency of
3 Hz compared to 1 Hz indicating that C1 may display increased inhibitory efficacy in
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
fibrillatory tissue. Therefore, our results indicate that C1 has a favorable pharmacological and
frequency-dependent inhibitory profile for Kv1.5 that are both desirable properties for novel AF
therapeutics.
Recent research has suggested that inhibition of peak and late atrial sodium currents may
also be an attractive strategy to suppress AF (Burashnikov et al., 2012; Burashnikov et al.,
2007). Inhibition of peak sodium current in a frequency-dependent manner may reduce the
occurrence of premature action potentials and reduce the incidence of AF. Whereas the induction
of the Nav1.5 late current may not only increase the risk of early after depolarization (EAD)-
induced arrhythmias but also lead to excessive sodium loading within cells that may contribute to
chronic calcium loading that is considered to be a primary trigger for EADs and calcineurin-
mediated activation of the NFAT gene transcriptional pathway that contributes to maladaptive
hypertrophic remodeling observed in chronic AF (Lin et al., 2004). Previous research from our
group reported that the C1 parent molecule resveratrol preferentially inhibited late over peak
Nav1.5 currents (Wallace et al., 2006, 26 vs 77 mol/L respectively). Our results indicate that
C1 inhibits the peak and late components of recombinant human heart Nav1.5 currents more
potently than resveratrol with IC50s of 3.2 and 1.1 mol/L respectively. While we did not directly
compare atrial and ventricular sodium currents, these findings warrant further investigation with
respect to atrial-specific sodium channel inhibition by C1. However, results from ventricular
myocytes indicate that C1 does not affect excitation/contraction-coupling at concentrations in the
low micromolar range. In contrast, C1 was effective at reducing calcium transient abnormalities
caused by late sodium current induction with ATX-II. These findings suggest C1 will not
interfere with ventricular EC-coupling at concentrations effective at inhibiting atrial Kv1.5 and
peak/late sodium channel currents.
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
Inhibition of the hERG (Kv11.2) potassium channel is a highly undesirable property of
any new drug as significant hERG inhibition can lead to excessive action potential prolongation
and life-threatening Torsade de Pointes arrhythmias (Sanguinetti et al., 2006). Accordingly, we
tested the effects of C1 on recombinant hERG channel whole-cell currents and observed that C1
is a weak inhibitor of peak and tail hERG currents with IC50 values of 30 and 25 mol/L
respectively. These results indicate that C1 is unlikely to exhibit any QT prolongation via hERG
channel inhibition at concentrations shown to be effective at inhibiting Kv1.5, Nav1.5 and IKACh
channels.
With respect to the overall ion channel inhibitory profile tested in this study, C1 displays
marked selectivity in the following order of IC50 values Kv1.5 (late) <Kv1.5 (peak) <Nav.1.5
(late) <IKACh <Nav1.5 (peak) <<hERG (tail) <hERG (peak). These results are summarized in
Figure 8 and indicate that C1 displays a favorable ion channel inhibitory profile that merits
further characterization as a potential AF therapeutic.
Non-ionic effects of C1
The generation of reactive oxygen species (ROS) in the fibrillating atria likely contributes
to the disturbances in the ionic and non-ionic milieu observed in AF (Carnes et al., 2001; Mihm
et al., 2001). Furthermore, it has been shown that Kv1.5 activity is increased in the presence of
the ROS H2O2 (Caouette et al., 2003). Resveratrol possesses a widely described anti-oxidant
capacity that likely contributes to its well-documented biological effects (Brisdelli et al., 2009;
Chung et al., 2012; Dolinsky et al., 2011; Wu et al., 2011). Our results show that C1 possesses
similar anti-oxidant capacity to resveratrol. While the concentrations of C1 required to yield
significant hydrogen peroxide quenching are higher than its Kv1.5 and Nav1.5 inhibitory effects,
C1 levels may accumulate in the plasma-membrane as resveratrol is known to partition into lipid
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
membranes leading to significant tissue accumulation (Sale et al., 2004). Furthermore, binding of
C1 to Kv1.5 may additionally contribute to inhibition of Kv1.5 in the presence of ROS by
decreasing the ROS-induced activation (Caouette et al., 2003).
Activation of the NFAT transcription pathway is an important signalling pathway that
contributes to the initiation of maladaptive hypertrophy and chronic AF (Cha et al., 2004;
Wilkins et al., 2004). It has been shown that activation and nuclear translocation of NFAT is
increased in AF (Lin et al., 2004). Our previous research indicates that resveratrol inhibits NFAT
nuclear translocation and reduces phenylephrine-induced hypertrophy in cardiac myocytes (Chan
et al., 2008). Interestingly, our results also indicate that C1 inhibits basal and angiotensin-II
induced NFAT activation at similar concentrations (IC50 ~ 1 mol/L) to resveratrol. While this
effect may not play a major role in the reduction of AF in the acute setting, it may become more
important in the prevention of transient AF conversion into chronic AF as the atria becomes
remodeled.
In vivo efficacy
To test the in vivo efficacy of C1, we used a chronically instrumented conscious dog
model, where electrophysiological changes favoring the development of AF were elicited by
chronic rapid right atrial pacing (Figure 8A). Atrial tachypacing in dogs is an established AF
model leading to electrical and structural remodeling (Morillo et al., 1995; Gaspo et al., 1997).
Atrial tachycardia and flutter have also been shown to lead to similar atrial remodeling in
humans (Franz et al., 1997). Therefore, the significantly decreased duration of AF (Figure 8B, C,
D) and possibly increased AERP following C1 administration, provides in vivo evidence that C1
may be beneficial in the treatment of certain forms of atrial fibrillation in patients.
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15

Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
Interestingly, treatment with C1 (0.3 and 1 mg/kg, i.v.) did not reduce the incidence of
burst induced AF in this study, however, it significantly reduced the duration of AF episodes.
Selective and non-selective IKur blocking compounds have been shown to reduce both the
incidence and duration of AF (Ford and Milnes, 2008). However, one must consider the
electrical and structural remodeling of the atria following chronic atrial fibrillation in patients
and following chronic rapid atrial pacing in animal models (Nattel et al., 2007). Remodeling can
alter the efficacy and effective doses of compounds developed for the treatment of AF. Although
no change in IKur density was previously observed in a tachypaced large dog AF model (Yue et
al., 1997), the down-regulation of IKur has been demonstrated in humans with chronic AF (Van
Wagoner et al., 1997; Bosch et al., 1999; Caballero et al., 2010). Such down-regulation of Kv1.5
in this model, if present, might have influenced the in vivo effect of C1, making the IKur current
blocking effect less important in this particular model. Other compounds blocking IKur that we
recently investigated in our laboratory (unpublished data) in the same model (with similar IC50
values for IKur) significantly decreased the incidence of burst-induced AF in 3 mg/kg i.v. dose or
higher. A recent review summarizing data on the cardiac electrophysiological effects of novel
IKur blockers also found that they were effective against AF induction in i.v. doses of 1-3 mg/kg
and higher (Ford and Milnes, 2008). In the present study, due to limits in the synthesized amount
of C1 (required for several parallel in vitro studies), it was not feasible to investigate C1 in vivo
at a dose of 3 mg/kg.
Importantly, C1 did not prolong the QT interval in conscious dogs in the present study
suggesting that C1 is unlikely to provoke ventricular arrhythmias based on repolarization
disturbances. These latter findings support our results on weak C1 hERG channel inhibition and
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16

Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
a lack of effect of C1 on EC-coupling in single ventricular myocytes. As this animal model
represents AF in the setting of previously remodeled atria leading to an increased induction of
AF, the pronounced acute effects observed with C1 are likely mediated by its ion channel
inhibitory and anti-oxidant properties, rather than inhibition of NFAT activation. Whether
chronic treatment with C1 reduces maladaptive remodeling remains to be determined by future
long-term studies.
Summary
The etiology of AF is complex and a number of ionic and non-ionic pathways contribute
the initiation and maintenance of AF (Lin et al., 2004; Franz et al., 1997). Therefore, inhibition
of more than one of these pathways is likely to provide greater anti-fibrillatory efficacy than one
pathway alone (Dobrev et al., 2010; Ehrlich et al., 2009; Dobrev et al., 2012). Our results
indicate that the resveratrol derivative C1 is an effective inhibitor of several potential targets
involved in AF development and confirmed that C1 is also effective at reducing the duration of
AF episodes in a large animal model of inducible AF.
Limitations of the study
While the effects of C1 on human atrial cell IKur were measured, the effects of C1 on
human atrial cell action potentials were not studied due to the limited supply of atrial appendage
tissue and the limited number of viable cells following enzymatic digestion. For practical
reasons, all ionic currents were measured at room temperature (20-22°C) rather than at 37°C.
The authors acknowledge that cardiac rhythm status of the donor could influence the
electrophysiological response to C1 in human atrial cells, however no information was available
on the cardiac rhythm status of the patients whose atrial tissue samples were used in this study.
In the conscious dog model of AF, the AERP could not be measured below 80 ms, since this is
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17

Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
the shortest S1-S2 duration that can be set in the human pacemakers used in this study. The
conscious animal experimental setup did not allow us to directly measure AERP since it would
have required anaesthesia and thoracotomia. Therefore, the exact statistical evaluation of the
possible atrial ERP prolonging effect of C1 was not performed in this study.
Acknowledgements
We would like to acknowledge the expert technical assistance of Beth Hunter and Lynn
Jones for the isolation of human and rat cardiac myocytes. We would also like to thank Irina
Manisali and the Centre for Drug Research and Development (Vancouver, Canada) for their help
in obtaining the C1 HPLC data from dog plasma.
Funding sources
Funding was provided to P.E.L. and J.R.B.D. by the Canadian Institutes of Health
Research and Technology Entrepreneurs and Companies (TEC) Edmonton. P.E.L. is holder of
the Charles A. Allard Chair in Diabetes Research. J.R.B.D is a Senior Scholar of Alberta
Innovates Health Solutions. This work was also funded by grants from the Hungarian National
Office for Research and Technology, Ányos Jedlik Programs ((NKFP_07_01-RYT07_AF), the
Hungarian National Development Agency co-financed by the European Regional Fund
(TÁMOP-4.2.2/B-10/1-2010-0012) and the Hungarian Academy of Sciences and the Hungarian
Scientific Research Fund (OTKA NK-104331).
Disclosures
None.
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
Figure Legends:
Figure 1. Effects of Resveratrol and its derivatives on recombinant Kv1.5 current inhibition. A,
Chemical structures of resveratrol and the four resveratrol derivatives; C1-C4. B, Concentration-
inhibition curves of the effect of resveratrol and the four resveratrol derivatives (C1-C4) on
Kv1.5 peak current amplitude (IC50s = 66.0, 0.36, 8.3, 10.9 and 11.2 mol/L respectively, n = 4-7
cells for each concentration). C, Representative traces before and after application of 0.3mol/L
C1. D, Current-voltage curves of Kv1.5 peak and late currents before and after application of 0.3
mol/L C1 compared to control. E, Concentration-inhibition curves of the effect of C1 on peak
and late Kv1.5 current compared to control (peak current IC50 = 0.36 mol/L, late current IC50 =
0.11 mol/L, n = 4-6 cells for each concentration).
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
Figure 2. Effects of C1 on human atrial IKur. A, Representative traces of frequency-dependent
effect on steady-state current by application of 0.3 mol/L C1 compared to controls (n=5). Top:
Effect on steady state current at 1Hz stimulation (Itest/Icontrol = 0.74  0.02). Bottom: Effect at 3
Hz stimulation (Itest/Icontrol = 0.50  0.03). B: Use-dependent effects of 0.3 mol/L C1 on IKur at 1
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25

Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
and 3 Hz stimulation. C, Representative traces of effects of 0.3 mol/L C1 on IKur before and
after application, and recovery after washout. D, Current-voltage curve of the effect of the
application of 0.3 mol/L C1 on IKur current, and after washout, compared to control, n = 5
cells).
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
Figure 3. Effect of C1 on rat atrial IKACh currents. A, Representative traces of the effects of
3mol/L C1 on IKACh whole-cell currents induced by 10 mol/L carbachol. B, Grouped data
current-voltage curves of control, carbachol and carbachol + 3 mol/L C1 (n = 5 cells, currents
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27

Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
normalized to control values). ＊, # denote P<0.05, vs. control and carbachol, respectively. C,
Concentration-inhibition curve of the effect of C1 on IKACh current (n = 4-6 cells for each
concentration).
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Multi-functional small molecule for atrial fibrillation Baczko and Liknes et al 2013
Figure 4. Effect of C1 on Nav1.5 currents. 3 nmol/L ATX-II was added to induce the late
sodium current component where late current recordings are indicated. A, Representative traces
of effects on Nav1.5 current with control, on application of 3 mol/L C1, and after washout. B,
Current-voltage curves on application of 3 mol/L C1 and after washout, normalized to control
values. C, Representative traces of control and the effect of application of 0.3, 1, and 3 mol/L
C1. D, Concentration-inhibition curve of the effect of C1 on peak and late Nav1.5 current (late
current IC50 = 1.1 mol/L, peak current IC50 = 3.2 mol/L, n = 4-7 cells for each concentration).
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29