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Gene Transfer of a Synthetic Pacemaker Channel Into the Heart
A Novel Strategy for Biological Pacing
Yuji Kashiwakura, MD, PhD; Hee Cheol Cho, PhD; Andreas S. Barth, MD;
Ezana Azene, PhD; Eduardo Marbán, MD, PhD
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Background—One key element of natural pacemakers is the pacemaker current encoded by the hyperpolarization-activated
nucleotide-gated channel (HCN) gene family. Although HCN gene transfer has been used to engineer biological
pacemakers, this strategy may be confounded by unpredictable consequences of heteromultimerization with endogenous
HCN family members and limited flexibility with regard to frequency tuning of the engineered pacemaker.
Methods and Results—To circumvent these limitations, we converted a depolarization-activated potassium-selective
channel, Kv1.4, into a hyperpolarization-activated nonselective channel by site-directed mutagenesis (R447N, L448A,
and R453I in S4 and G528S in the pore). Gene transfer into ventricular myocardium demonstrated the ability of this
construct to induce pacemaker activity with spontaneous action potential oscillations in adult ventricular myocytes and
idioventricular rhythms by in vivo electrocardiography.
Conclusions—Given the sparse expression of Kv1 family channels in the human ventricle, gene transfer of a synthetic
pacemaker channel based on the Kv1 family has novel therapeutic potential as a biological alternative to electronic
pacemakers. (Circulation. 2006;114:1682-1686.)
Key Words: gene therapy 䡲 ion channels 䡲 pacing
I
n the sinoatrial node, pacemaker activity is generated by
a balance of depolarizing and repolarizing currents
whose gating and permeation properties, in ensemble,
create a stable oscillator.1 Hyperpolarization-activated nucleotide-gated channel (HCN) family genes figure prominently in physiological automaticity, and transfer of such
genes into quiescent heart tissue has been explored as
1 way of creating a biopacemaker.2– 4 However, use of
HCN genes may be confounded by unpredictable consequences of heteromultimerization with multiple endogenous HCN family members in the target cell.5,6 Because
HCN
is
Methods
Plasmid Construction, Adenovirus Preparation,
and Mutation
Human Kv1.4 cDNA was subcloned from XL-4 (Origene Technologies, Inc, Rockville, Md) to pTracerCMV2 (Invitrogen, Carlsbad,
Calif) by EcoRI and NotI sites. The bicistronic adenovirus shuttle
vector pAdCIG was used for generation of adeno/SPC-IRES green
fluorescent protein (GFP). Adenovirus was produced as previously
described.9 Oligonucleotide mutagenesis was performed with a
site-direct mutagenesis kit (Stratagene, La Jolla, Calif).
Transient Transfections
Clinical Perspective p 1686
Twenty-four hours before transfection, HEK293 cells (ATCC,
American Type Culture Collection, Manassas, Va) were seeded at a
density of 2.0⫻105 per 35 mm. Cells were transfected with 2 ␮g per
well plasmid DNA with Lipofectamine 2000 (Invitrogen. After 4
hours, transfection media were replaced with normal growth media.
expressed in ventricular myocytes and may contribute to
arrhythmogenesis,7,8 HCN gene transfer in vivo may have
unpredictable consequences. Moreover, the use of wild-type
channels offers little flexibility with regard to frequency
tuning of the engineered pacemaker. To avoid these limitations with HCN gene transfer in biopacemakers, we created a
synthetic pacemaker channel (SPC). By targeted mutagenesis
involving ⬍2% of the sequence, we converted the human
Kv1.4 depolarization-activated potassium-selective channel
into a hyperpolarization-activated nonselective channel suitable for biopacing applications.
Electrophysiology
Experiments were performed with the use of the whole-cell patchclamp technique10 at 37°C with an Axopatch 200B amplifier (Axon
Instruments Inc, Foster City, Calif) while sampling at 10 kHz for
voltage-clamp or 2 kHz for current-clamp recordings filtered at 2
kHz. Pipettes had tip resistances of 2 to 4 mol/L⍀ when filled with
the internal recording solution. Because we had demonstrated that
Received April 19, 2006; revision received July 15, 2006; accepted August 7, 2006.
From the Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Md.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.106.634865/DC1.
Correspondence to Eduardo Marbán, MD, PhD, Division of Cardiology, Johns Hopkins University School of Medicine, 858 Ross Bldg, 720 Rutland
Ave, Baltimore, MD 21205. E-mail [email protected]
© 2006 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/CIRCULATIONAHA.106.634865
1682
Kashiwakura et al
adenovirus infection itself did not modify the electrophysiology of
guinea pig myocytes,11 control patch-clamp experiments was performed on uninfected (nongreen) left ventricular myocytes isolated
from SPC adenovirus (AdSPC)–injected guinea pig.
Cells were superfused with a physiological saline (Tyrode’s)
solution containing 135 mmol/L NaCl, 5 mmol/L KCl, 1.8 mmol/L
CaCl2, 10 mmol/L glucose, 1 mmol/L MgCl2, and 10 mmol/L
HEPES; pH was adjusted to 7.4 with NaOH. The pipette solution
was composed of 130 mmol/L K-glutamate, 10 mmol/L KCl,
10 mmol/L Na-HEPES, 2 mmol/L ethyleneglycoltetraacetic acid,
5 mmol/L Mg–adenosine triphosphate, and 1 mmol/L MgCl2; pH
was adjusted to 7.3 with KOH. Action potential (AP) oscillations
were initiated by brief depolarizing current pulses (2 ms, 300 to 700
pA, 110% threshold) at 0.33 Hz at 32°C. When we measured SPC
current in adult myocytes, 5 ␮mol/L BaCl2 was added in bath
solution. Data are mean⫾SEM.
Animal Procedure and Myocyte Isolation
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Adenoviruses were injected into the left ventricular free wall of
guinea pigs. Adult female guinea pigs (weight, 250 to 300 g; Hilltop
Lab Animals, Inc, Scottdale, Pa) were anesthetized with 4% isoflurane, intubated, and placed on a ventilator with a vaporizer supplying
1.5% to 2% isoflurane. After lateral thoracotomy, a 30-gauge needle
was inserted at the free wall of the left ventricle. An adenovirus of
3⫻1010 plaque-forming units AdSPC or 3⫻1010 plaque-forming
units GFP (control group) was injected into the left ventricle.
Forty-eight to 72 hours after injections were performed, free wall
myocytes of the left ventricle were isolated by standard techniques.12
The yield of transduced myocytes, identifiable by their vivid green
fluorescence by epifluorescence imaging, was ⬇3% to 5% as judged
by visual assessments when cells were dispersed into the electrophysiology recording chamber. The work presented was performed
in accordance with National Institutes of Health guidelines for the
care and use of laboratory animals and was performed in accordance
with the guidelines of the Animal Care and Use Committee of Johns
Hopkins University.
Electrocardiograms
Surface ECGs (MP100; BIOPAC Systems, Inc, Goleta Calif) were
recorded 72 hours after adenoviral injection as previously described.13 Guinea pigs were lightly sedated with isoflurane, and
needle electrodes were placed under the skin. Electrode positions
were optimized to obtain maximal-amplitude recordings. ECGs were
simultaneously recorded from standard limb leads I, II, and III. To
detect ventricular beats effectively, we used methacholine (0.1 to 0.5
mg/g; Sigma Chemical Co, St. Louis, Mo) by intraperitoneal
injection to induce bradycardia. We confirmed the origin of ventricular beats by mapping the left ventricular free wall with a hand-held
electrode.
The authors had full access to the data and take full responsibility
for its integrity. All authors have read and agree to the manuscript as
written.
Results
Creation of SPC
In the Kv1.4 backbone, we introduced 3 point mutations
(R447N, L448A, and R453I) in the S4 segment and a single
mutation (G528S) in the pore (Figure 1).14,15 When expressed
in HEK293 cells, wild-type human Kv1.4 showed the expected depolarization-activated outward current (Figure 2A).
The S4 triple mutant changed the gating to hyperpolarizationactivated from depolarization-activated (Figure 2B, panel a;
current record in high-potassium external solution) but did
not alter the K⫹-selective reversal potential. As shown in
Figure 2B, panel b, the reversal potentials of this mutant in
normal Tyrode’s or high potassium external solution were
⫺79.1⫾2.0 or 0.8⫾0.5 mV, respectively (n⫽5). To create a
Creation of Synthetic Pacemaker Channel
1683
Figure 1. Design of synthetic pacemaker channel. To convert
human Kv1.4 channel into a pacemaker channel, we mutated
residues in the voltage-sensor segment S415 and the selectivity
filter part of the pore.14 S4 triple mutations (R447N, L448A, and
R453I) altered the gating from depolarization-activated to hyperpolarization-activated, and the pore mutation (G528S) renders
the K⫹-selective channel to a channel selective for both Na⫹
and K⫹, resulting in the positive shift of voltage activation.
repolarizing current at negative potentials, we further mutated
the pore (G528S) so as to render the channel nonselective for
Na⫹ versus K⫹. The pore mutant alone expressed depolarizationactivated outward current with little inward current (Figure 2C).
Combining the S4 and pore mutations resulted in SPC that
showed hyperpolarization-activated inward current under physiological conditions (Figure 2D). The mean current density of
SPC at ⫺130 mV was ⫺30.3⫾4.4 pA/pF (n⫽10). Tail current
analysis revealed a reversal potential of ⫺10.5⫾1.7 mV (n⫽5)
and no significant deactivation in a physiological voltage range
(Figure 2E). We detected little depolarization-activated current
in SPC by another protocol (data not shown). With the use of
normal Tyrode’s external solutions, the sodium-to-potassium
permeability ratio (PNa/PK) was calculated to be 0.66 by the
Goldman-Hodgkin-Katz formula. CsCl (2 mmol/L) did not
affect SPC current (data not shown), whereas it completely
blocked currents encoded by the HCN gene family.16
No Heteromultimerization of SPC With HCN
Gene Family
Wild-type Kv1.4 has been reported previously not to multimerize with the HCN gene family.17 Before in vivo use of
SPC, we verified that SPC was unable to multimerize with
HCN1 by cotransfection into human embryonic kidney cells
and by analyzing reversal potentials. Wild-type HCN1 (Figure 3A, panel a, left), when expressed alone, had a reversal
potential of ⫺36.1⫾1.4 mV, whereas HCN cotransfected
with SPC exhibited a reversal potential of ⫺22.0⫾8.0 mV
(n⫽5 for each; tail currents not shown). Superfusion with
2 mmol/L CsCl to block HCN1 homomultimers left behind a
current that reversed at ⫺11.1⫾2.3 mV, which is indistinguishable from the reversal potential of SPC alone (Figure
3A, panel c, right). The clean pharmacological separation
suggests the absence of any functional SPC-HCN heteromultimers. We also excluded the possibility that SPC expression
might affect native sodium, potassium, or calcium currents in
adult guinea pig myocytes (data not shown).
Pacemaker Abilities of SPC In Vivo
Next, to test its pacemaker ability in the adult ventricle, we
made bicistronic (GFP-tagged) AdSPC and injected it into
guinea pig heart. Seventy-two hours after virus injection,
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October 17, 2006
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Figure 2. A, Current records of human wild-type Kv1.4. B, panel a, Currents of S4 triple mutations in high-potassium external solution
(140 mmol/L); panel b, I-V relationship of instantaneous currents at various test potentials (20 to ⫺140 mV) measured 4 to 8 ms after a
1.7-ms prepulse to ⫺120 mV. HiK indicates high-potassium external solution; NT, normal Tyrode’s external solution. I-V relationship
was fitted by linear regression (R⫽0.985 for NT, R⫽0.979 for HiK). C, Currents through the pore mutant. Wild-type Kv1.4 showed typical transient outward current on depolarization. In pore mutant, current magnitude was reduced compared with wild-type, and the
reversal potential was changed to ⫺10 mV from ⫺80 mV. D, Currents through a channel carrying S4 triple and pore mutations (SPC)
and I-V relationship from mean current density. By combining S4 and pore mutations, we created SPC showing hyperpolarization-activated measurable inward current in physiological voltage range. E, Tail currents of SPC and I-V relationship of instantaneous currents
at various test potential (20 to ⫺100 mV) between 4 and 8 ms after 1.7-ms prepulse to ⫺120 mV. I-V relationship was fitted by linear
regression (R⫽0.981). Reversal potential in normal Tyrode’s external solution was ⫺10.5⫾1.7 mV. There was little significant deactivation in a physiological voltage range. We recorded all currents in normal Tyrode’s as an external solution. For S4 triple mutant, we also
used high-potassium external solution to highlight hyperpolarized-activated inward current.
isolated ventricular myocytes transduced with AdSPC were
examined by whole-cell voltage clamp. There was little
measurable pacemaker current in control cells from injected
animals (data not shown). In contrast, we detected hyperpolarization-activated inward current in AdSPC-transduced
myocytes (Figure 3B, panel a). Mean current densities at ⫺80
or ⫺160 mV equalled ⫺7.2⫾1.3 or ⫺59.7⫾5.5 pA/pF,
respectively (n⫽5 each; Figure 3B, panel b). We also
examined APs in control (n⫽13) and SPC-transduced cells
(n⫽14). Control cells never exhibited spontaneous AP oscillations, whereas half of SPC-transduced cells (7 of 14)
showed spontaneous AP oscillations. In the experiment
Figure 3. A, Currents and I-V relationship of
instantaneous currents at various test potentials (20 to ⫺140 mV) measured 4 to 8 ms
after 1.7 ms prepulse to ⫺120 mV. A, panel
a, Wild-type mouse HCN1. Reversal potential was ⫺36.1⫾1.4 mV. I-V relationship was
fitted by second-order polynomial function.
A, panel b, Cotransfection of wild-type
mHCN1 and SPC. I-V relationship was fitted
by second-order polynomial function. A,
panel c, Cotransfection after addition of
2 mmol/L CsCl in the external solution of A,
panel b. I-V relationship was fitted by linear
regression (R⫽0.998). B, panel a, Currents in
AdSPC-transduced myocyte with the use of
normal Tyrode’s without potassium in the
external solution in order to suppress IK1. We
did not use barium to prevent IK1 because
barium affected SPC current (data not
shown). B, panel b, I-V relationship from
mean current density of B, panel a. In this
condition, mean current density was
⫺7.2⫾1.3 pA/pF at ⫺80 mV. C, Spontaneous AP oscillation after triggered AP by brief
depolarizing current pulses in AdSPCtransduced myocyte.
Kashiwakura et al
Creation of Synthetic Pacemaker Channel
1685
Figure 4. In vivo ECG demonstrated
induced idioventricular rhythms. A, ECG
leads II, I, III. Overview of idioventricular
rhythms is shown. Arrows indicate start
of idioventricular rhythms (150 bpm). B,
panel a, Junctional beats as intrinsic
rhythms of guinea pig after methacholine
injection; panel b, High-magnitude ECG
of idioventricular rhythms of dashed-line
square of ECG (A); panel c, Pace mapping of left ventricular free wall with
hand-held electrode. Arrows indicate
artifact of pacing (150 bpm). Note that
idioventricular rhythms were identical in
polarity and similar in morphology to
pacing beats, indicating that idioventricular rhythms originated from left ventricular free wall. In control group (GFP adenovirus injection), no ventricular ectopic
beats were observed (data not shown).
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shown here, we could detect fast spontaneous AP oscillations
(mean rate ⬎200 bpm; Figure 3C), with maximal diastolic
potential and phase-4 slope of 53.6⫾2.5 mV and 10.4 mV/s,
respectively. There was no significant difference in evoked
AP durations (306.2⫾12.5 ms in control versus 303.2⫾10.9
ms in AdSPC-transduced cells). Given these results, we
concluded that SPC can induce pacemaker activity in guinea
pig myocytes.
When the mechanism of spontaneous AP oscillation is
considered, the combination of a positive shift of resting
membrane potential and generation of hyperpolarizationactivated inward current is key. As membrane potential shifts
positively, membrane resistance becomes lower, such that
even small currents can produce relatively large changes of
membrane potential. Neonatal myocytes exhibit spontaneous
AP oscillations partially because their resting membrane
potential is depolarized relative to that of adult myocytes, in
addition to native If. In our case, a small current could be
produced by SPC at the level of maximal diastolic potential
(from ⫺55 to ⬇⫺40 mV). Furthermore, transduction of SPC
whose reversal potential is ⫺10 mV shifted the membrane
potential positively. Taken together, SPC transduction resulted in a positive shift of membrane potential in adult
myocytes, in which even small hyperpolarization-activated
inward current produced by SPC could contribute to spontaneous AP oscillation.
To confirm the ability of SPC to induce pacemaker activity
in vivo, ECGs were performed 72 hours after AdSPC injection. During ECG recording, methacholine (0.1 to 0.5 mg/g)
was administered by intraperitoneal injection to induce bradycardia. Control animals (GFP adenovirus; n⫽6) showed no
ectopic ventricular beats, whereas frequent monomorphic
idioventricular beats could be detected in animals injected
with AdSPC (n⫽6). In representative experiments (Figure 4),
ECG with pace mapping demonstrated idioventricular
rhythms (150 bpm) originating from the injection site (left
ventricular free wall). These results demonstrated directly
that SPC worked as a pacemaker in vivo.
Discussion
The number of SPC-transduced cells that are minimally
necessary for pacemaker activity is an important and as-yetunanswered issue. SPC was delivered by focal intramuscular
injection to the apex of the heart. The 2% to 3% of
transduction rate thus reflects the very limited site of virus
injection, thereby creating a concentrated number of transduced cells in a small area (yielding a small percentage of
transduced cells among all ventricular cells). Because induced ventricular pacemakers do not enjoy the impedance
mismatch of the sinoatrial node, it is likely that more
ventricular cells will have to be “pacemakers” for the
biopacemaker to function in vivo (relative to the number of
cells in the sinoatrial node).
Flexibility for Frequency Tuning of SPC
Unlike previous studies with adenoviral HCN2 delivered into
other regions of the heart,2,3 we induced biopacemaker
activity with SPC in ventricular myocardium. An alternative
approach has been to use mesenchymal stem cells as a
platform for gene delivery to the ventricle.4 Such cells do not
fully differentiate into heart cells (although they can differentiate into bone, cartilage, or adipose tissue18), and their
persistence over time has not been demonstrated. Direct gene
transfer of SPC avoids many of these potential complications
and uncertainties (while admittedly introducing others). Another potential advantage of SPC is its flexibility for frequency tuning of synthetic pacemaker strategy. We investigated 3 sets of S4 mutations and 5 different pore mutations,
yielding a total of possible 15 combinations of S4 and pore
mutations. Some of these other mutants also expressed
hyperpolarization-activated inward current in physiological
conditions. For example, combining the S4 triple mutation
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October 17, 2006
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with another pore mutation (V525S, VGYG3 SGYG) displayed a current density of ⫺6.1 pA/pF at ⫺100 mV with a
reversal potential of ⫺25 mV in human embryonic kidney
cells (Figure I in the online-only Data Supplement). When
expressed in vivo, this V525S pore mutant combined with the
S4 mutations also showed slow idioventricular rhythms (55
bpm) for short periods (Figure II in the online-only Data
Supplement). These results indicate that specific mutations
could favor specific heart rates that can be achieved in vivo
by combining the S4 mutations with different pore mutants.
Thus, by combining various S4 mutations with pore mutations, we can prepare a broad range of candidates for
synthetic pacemakers and choose the one best suited to
accomplish a therapeutic goal, namely, pacing at any given
desired basal heart rate.
In summary, by selective mutagenesis of S4 and the pore in
the human Kv1.4 channel, we succeeded in creating a novel
pacemaker channel. This channel showed hyperpolarizationactivated inward currents with steady activation under physiological conditions. Gene transfer of SPC induced pacemaker activity in guinea pig adult ventricular myocardium
and produced idioventricular rhythms on ECG. Given the
sparse expression of Kv1 family channels in the human
ventricle19,20 and the capability of tuning the frequency of
oscillation to any given desired rate range, SPCs based on the
Kv1 family have the potential to be novel therapeutic tools
for the creation of biopacemakers.
Acknowledgments
We thank Peihong Dong for technical support in plasmid constructions and Michelle K. Leppo for animal procedures.
Sources of Funding
This study was supported by the Donald W. Reynolds Foundation.
Dr Marbán holds the Michel Mirowski, MD, Professorship of Johns
Hopkins University.
Disclosures
Excigen, Inc has licensed intellectual property related to biological
pacemakers from Johns Hopkins University. Dr Marbán is a founder
of, stockholder of, and consultant to Excigen. No research funding
was provided by Excigen. The other authors report no conflicts.
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CLINICAL PERSPECTIVE
Electronic pacemakers are effective but have limitations, including the risks of hardware implantation, limited battery life,
and insufficient rate response to physiological stimuli. Engineered biological pacemakers promise to overcome these
limitations. With the use of simple gene delivery methods, heart cells that are normally not pacemakers can be converted
into automatically firing cells. This form of gene therapy is focal and reversible by conventional electrophysiological
approaches. In the present study, we show that a pacemaker channel gene can be engineered from scratch to create a
“synthetic pacemaker channel.” The use of this man-made gene enables the selective modulation of pacemaker properties,
such as tailoring the rate to meet an individual patient’s needs. Important issues, including duration of transgene expression
and long-term safety of the gene therapy approach, need to be addressed before clinical studies can be initiated.
Gene Transfer of a Synthetic Pacemaker Channel Into the Heart: A Novel Strategy for
Biological Pacing
Yuji Kashiwakura, Hee Cheol Cho, Andreas S. Barth, Ezana Azene and Eduardo Marbán
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Circulation. 2006;114:1682-1686; originally published online October 9, 2006;
doi: 10.1161/CIRCULATIONAHA.106.634865
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2006 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7322. Online ISSN: 1524-4539
The online version of this article, along with updated information and services, is located on the
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http://circ.ahajournals.org/content/114/16/1682
Data Supplement (unedited) at:
http://circ.ahajournals.org/content/suppl/2006/10/16/CIRCULATIONAHA.106.634865.DC1
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