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Recreating the biological pacemaker.

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THE ANATOMICAL RECORD PART A 280A:1046 –1052 (2004)
Recreating the Biological Pacemaker
MICHAEL R. ROSEN,1–3* RICHARD B. ROBINSON,1 PETER BRINK,3
1,3
AND IRA S. COHEN
1
Center for Molecular Therapeutics, Department of Pharmacology, Columbia
University, New York, New York
2
Department of Pediatrics, Columbia University, New York, New York
3
Institute of Molecular Cardiology, Departments of Physiology and Biophysics, State
University of New York, Stony Brook, New York
ABSTRACT
In recent years, several groups have reported a variety of strategies for developing
biological pacemakers whose ultimate function would be to supplement/replace electronic
pacemakers. Strategies have included gene therapy using naked plasmids or viral vectors
and cell therapy for which both adult human mesenchymal stem cells (hMSCs) and human
embryonic stem cells have been employed. This article reviews the various approaches and
summarizes our own research in which the pacemaker gene, HCN2, is administered via viral
vector or in an hMSC platform to produce pacemaker function in the intact canine heart.
©
2004 Wiley-Liss, Inc.
Key words: pacemaker current; HCN channels; heart block; cardiac arrhythmias; sinoatrial node
In a variety of settings of sinoatrial and/or atrioventricular nodal disease, the ideal solution would be to heal or to
recreate the biological pacemaker of the heart, the sinoatrial node, itself. At present, electronic pacemakers represent the state of the art. However, limited battery life,
the need for permanent catheter implantation into the
heart, and lack of response to autonomic neurohumors all
render them less than optimal therapy (Zivin and Bardy,
2001). Several gene therapy approaches have been explored as alternatives to electronic pacemakers, including
␤2-adrenergic receptor overexpression (Edelberg et al.,
1998, 2001), suppression of inward rectifier current when
a dominant negative construct is expressed together with
the wild-type gene Kir2.1 (Miake et al., 2002), and implantation of vectors carrying the pacemaker gene, HCN2, into
the atrium (Qu et al., 2003) or the bundle branch system
(Plotnikov et al., 2004). The ␤2-adrenergic receptor experiments used a naked plasmid as the vector for gene therapy; the others, an adenoviral vector.
A problem inherent in many gene therapy experiments
(Miake et al., 2002; Qu et al., 2003; Plotnikov et al., 2004)
is the use of viruses to deliver the necessary genes. Use of
replication-deficient adenoviruses having little infectious
potential raises the likelihood of only transient improvement in pacemaker function as well as potential inflammatory responses. Retroviruses and other viral vectors
carry the risk of carcinogenicity and infectivity. One way
to avoid the use of viral vectors is to rely on cell therapy
using human embryonic stem cells to create pacemakers.
Such approaches are not only in their infancy but carry
the problems of identifying appropriate cell lineages, the
©
2004 WILEY-LISS, INC.
possibility of stem cell differentiation into lines other than
pacemaker cells, and potential for neoplasia (Gepstein,
2002). Another cell therapy approach for making biological pacemakers would be to use adult mesenchymal stem
cells as platforms for delivering pacemaker genes to the
heart (Potapova et al., 2004). In this setting, the cells
would be thought of as a biologically inert vector that
could deliver molecular/genetic information to adjoining
myocardium.
We review here our use of both viral gene therapy and
adult human mesenchymal stem cell (hMSC) approaches
to test the hypothesis that biological pacemakers may
become feasible alternatives to electrical pacemakers.
These experiments followed our initial demonstration of
proof of concept that an adenoviral vector containing
HCN2 delivered to the canine left atrium could generate a
pacemaker current 100- to 1,000-fold greater than the
Grant sponsor: USPHS-NHLBI; Grant number: HL-28958, HL20558, GM-55263, HL-67101; Grant sponsor: Guidant Corporation.
*Correspondence to: Michael R. Rosen, Center for Molecular
Therapeutics, Departments of Pharmacology and Pediatrics, Columbia University, 630 West 168 Street, PH 7 West-321, New
York, NY 10032. Fax: 212-305-8351. E-mail: mrr1@columbia.edu
Received 11 March 2004; Accepted 8 June 2004
DOI 10.1002/ar.a.20073
Published online 15 September 2004 in Wiley InterScience
(www.interscience.wiley.com).
RECREATING THE BIOLOGICAL PACEMAKER
native atrial If and could initiate atrial beats (Qu et al.,
2003). In the gene therapy experiment to be summarized
here, we implanted an adenoviral construct into the left
bundle branch (LBB) system and determined whether the
rhythms that developed during transient atrioventricular
block were stable and organized and would result from
IHCN2 induction of automaticity in the region injected. In
the stem cell experiment, we tested the hypothesis that
the expressed pacemaker current need not be generated
within a myocyte, but that it would be sufficient if it were
generated within any cell electrotonically coupled to a
myocyte. For this purpose, we investigated whether hMSCs could be transfected by electroporation with HCN2,
could express functional HCN2 channels, and could drive
the canine ventricle, mimicking the HCN2 overexpression
by adenoviral constructs (Qu et al., 2003), but without the
need for in situ viral delivery.
MATERIALS AND METHODS
All studies were performed using protocols approved by
the Columbia University Institutional Animal Care and
Use Committee.
Viral/Genetic Preparation
An adenoviral construct of murine HCN2 (mHCN2;
Genbank AJ225122) driven by the CMV promoter was
prepared (Qu et al., 2003), purified through plaque assay,
amplified to a large stock, and harvested and titrated after
CsCl banding. The identical procedure was used to construct an adenoviral vector of enhanced GFP (AdGFP),
whose sequence was taken from its original vector
pIRES2-EGFP (Clontech, Palo Alto, CA) at BamHI and
NotI sites and subcloned into the shuttle vector pDC516.
Final titers were AdHCN2, 3.4 ⫻ 1011 ffu/mL; AdGFP,
1.4 ⫻ 1012 ffu/mL; 2–3 ⫻ 1010 ffu of each virus was injected per experiment.
Dissociation of Myocytes and Studies of HCN
Current
Purkinje myocytes were dissociated by modifying a previously published procedure (Boyden et al., 1989). Isolated
cells were transferred to a stage-mounted chamber of an
inverted epifluorescence microscope to identify GFP-expressing cells. To measure pacemaker currents, cells were
superfused with 35°C Tyrode solution containing (in
mmol/L): NaCl, 140; NaOH, 2.3; MgCl2, 1; KCl, 5.4; CaCl2,
1.0; MnCl2, 2; BaCl2, 4; HEPES, 5; glucose, 10; pH 7.4.
Pipette solution contained (in mmol/L) aspartic acid, 130;
KOH, 146; NaCl, 10; CaCl2, 2; EGTA-KOH, 5; Mg-ATP, 2;
HEPES-KOH, 10; pH 7.2. To record pacemaker current,
cells were held at ⫺55 mV and stepped to ⫺55 to ⫺125 mV
for 6 sec, followed by an 8-sec step to ⫺115 mV to measure
tail current.
Human Mesenchymal Stem Cell Maintenance
and Transfection
hMSCs (Poietics hMSC, mesenchymal stem cells, human bone marrow) were purchased from Clonetics/BioWhittaker (Walkersville, MD) and cultured in MSC growing medium (Poietics MSCGM; BioWhittaker) at 37°C in a
humidified atmosphere of 5% CO2. Cells were used from
passages 2– 4. A full-length murine HCN2 cDNA was subcloned into a pIRES2-EGFP vector (BD Biosciences Clon-
1047
tech, Palo Alto, CA). Cells were transfected by electroporation using the Amaxa Biosystems Nucleofector (Amaxa,
Cologne, Germany) technology (Hamm et al., 2002).
Transfection efficiency was 30 – 45%. Study of membrane
currents in hMSCs was via the techniques described
above.
Action Potential Recordings in Coculture
hMSCs were plated onto fibronectin-coated coverslips
using a cloning cylinder to restrict initial plating to a 4
mm diameter circular area. The cells expressed EGFP
alone or EGFP ⫹ mHCN2. Four hours later, the cloning
cylinder was removed and neonatal rat ventricular myocytes, prepared as described previously (Protas and Robinson, 1999), were plated over the entire coverslip. Four to
five days later, the coverslips were placed in a superfusion
chamber maintained at 35°C and action potentials recorded from near the center of the coverslip using a perforated patch electrode (Protas and Robinson, 1999) and
normal physiologic solution. Recordings were conducted
with an Axopatch 200 amplifier and PClamp 8 software
(Axon Instruments). The perforated patch technique was
used with amphotericin B (400 ␮g/mL; Sigma) in the pipette solution.
Intact Animal Studies With Viral Injection or
hMSCs
Adult mongrel dogs were anesthetized using Na-thiopental induction (17 mg/kg IV) followed by inhalational
isoflurane (1.5–2.5%). For viral injection, a custom-modified bipolar 8 Fr steerable catheter (Guidant, St. Paul,
MN) was used for subendocardial delivery of the adenoviral constructs or saline. A 29 G needle, which could be
advanced and retracted by 3 mm, was incorporated into
the core lumen of the catheter. The catheter filled with
sterile saline was introduced into the LV under fluoroscopic control via an 8 Fr arterial introducer sheath, a
stable bundle branch potential electrogram recording was
obtained, the needle tip was verified in the LV wall, and
an adenoviral construct (AdGFP ⫹ AdHCN2 or AdGFP
alone) or normal saline solution (0.6 ml) was injected into
the LBB (Plotnikov et al., 2004).
Animals recovered for 4 –7 days, after which a terminal
study was performed in which they were anesthetized as
above and both cervical vagal trunks isolated as described
previously (Plotnikov et al., 2004). Graded right and left
vagal stimulation was performed via bipolar platinum
iridium electrodes (Rosenshtraukh et al., 1994) to suppress sinus rhythm such that escape pacemaker function
might occur. The chest was then opened and LBB excised
from the left ventricular sites that had been injected.
Comparable procedures were performed for AdGFP- or
saline-injected or control dogs. These were used for microelectrode study or study of ion channels. For microelectrode studies, preparations were placed in a 4 ml chamber
perfused with Tyrode’s solution (37°C; pH 7.3–7.4) and
impaled with 3 mol/l KCl-filled glass capillary microelectrodes. Calibration of the system and recording of transmembrane action potential were performed as described
previously (Anyukhovsky et al., 1996).
For hMSC studies, anesthesia was as above, a thoracotomy was performed, and we injected 106 hMSCs containing mHCN2 ⫹ GFP or GFP alone subepicardially in 0.6 ml
of solution into the left ventricular anterior wall, approx-
1048
ROSEN ET AL.
imately 2 mm deep to the epicardium via a 21 gauge
needle. Animals recovered for 4 –10 days and were anesthetized as above. Both cervical vagal trunks were isolated, the chest opened, and ECGs monitored. Graded
right and left vagal stimulation was performed to suppress sinus rhythm such that escape pacemaker function
might occur. Tissues were then removed for histological
study.
Statistical Analysis
Measurements of cycle lengths of cardiac rhythms and
of isolated LBB were made from at least five beats. Fisher’s exact test, Student’s t-test, or one-way ANOVA were
used as appropriate. Data are expressed as mean ⫾ SEM.
P ⬍ 0.05 was considered significant.
RESULTS
Gene Therapy Protocols
We studied seven animals injected with mHCN2 ⫹
AdGFP, three with AdGFP alone, and three with saline.
Seven of the animals were monitored round the clock
following surgery. We noted that for 2 days following the
injection procedure, there was increased ventricular irritability expressed as multiple premature ventricular depolarizations in the saline-injected, AdGFP-injected, and
AdGFP ⫹ AdHCN2-injected animals. This ectopy was attributed to local tissue injury, as was exemplified by hematoma formation at the injection site in all three groups.
Hence, no studies of pacemaker function were done until
day 4 –7.
Figure 1 demonstrates representative experiments
showing records from one dog injected with AdGFP alone
(Fig. 1A) and one with AdGFP ⫹ AdHCN2 (Fig. 1B). Prior
to vagal stimulation, both dogs had comparable sinus
rates. Following vagal stimulation, the rate of the
AdGFP ⫹ AdHCN2-injected heart was over two times
faster than that injected with AdGFP alone. Overall, baseline sinus cycle length did not differ between control and
AdHCN2-injected groups (484 ⫾ 46 and 506 ⫾ 11 msec,
respectively; P ⬎ 0.05). During vagal stimulation, right
ventricular escape rhythms occurred in four of six controls
and three of seven HCN2 dogs (P ⬎ 0.05), while left
ventricular escape rhythms occurred in four of six controls
and six of seven HCN2-injected dogs (P ⬎ 0.05). Whereas
idioventricular escape of right or left ventricular origin
occurred in all animals, the rate generated was significantly more rapid in the hearts of animals that had received a left bundle branch injection of AdHCN2 than was
seen for left ventricular rhythms of control animals and
for right ventricular rhythms of control or AdHCN2-injected animals (P ⬍ 0.05) (Plotnikov et al., 2004).
Pacemaker function was studied in 16 experiments on
bundle branches from animals injected with AdHCN2 ⫹
AdGFP, AdGFP alone, or saline as well as from the same
site in five control dogs that had not been injected at all.
Rate was most rapid (P ⬍ 0.05) in the AdHCN2-injected
regions (95 ⫾ 10 vs. 42 ⫾ 3 bpm at control sites; P ⬍ 0.05)
(Plotnikov et al., 2004). Figure 2 demonstrates the magnitude of IHCN2 in a representative Purkinje myocyte from
an animal injected with AdHCN2 ⫹ AdGFP and from a
control myocyte. Note the greater magnitude of current in
the setting of HCN2 overexpression.
Stem Cell Therapy Protocols
Whereas nontransfected hMSCs demonstrated no significant time-dependent currents during hyperpolarizations,
HCN2-transfected hMSCs expressed a large time-dependent
inward current activating upon hyperpolarization (Fig. 3).
Moreover, the reversal potential (⫺37.5 ⫾ 1.0 mV at [K⫹]o ⫽
5.4 mM) and response to cesium were consistent with the
pacemaker current, If. Given these properties, the current
expressed in hMSCs would be expected to activate in the
physiologic range of diastolic potentials as long as adequate
gap junctional coupling was present between hMSCs and
adjacent myocytes. That such coupling (involving connexins43 and 40 in hMSCs and connexin43 in myocytes) is in
fact a reality among hMSCs and between hMSCs and myocytes was demonstrated by us recently (Potapova et al.,
2004; Valiunas et al., 2004).
Because a potential advantage of biological over electronic pacemakers would be their hormonal regulation, we
examined the effects of ␤-adrenergic and muscarinic agonists on If recorded in the hMSCs. Isoproterenol induced a
significant leftward shift in the activation curve for If,
consistent with the literature on ␤-adrenergic stimulation
in myocytes. Whereas acetylcholine had no direct effect on
If, it did antagonize the effects of isoproterenol. This is
consistent with the accentuated antagonist action of acetylcholine on If in Purkinje fibers (Chang et al., 1990).
Given the expression of the pacemaker gene in hMSCs,
we hypothesized that HCN2-transfected hMSCs could influence excitability of coupled heart cells. Maximum diastolic potential was ⫺74 ⫾ 1 mV (n ⫽ 5) in neonatal rat
ventricular myocytes cocultured with EGFP-expressing
hMSCs and ⫺67 ⫾ 2 mV (n ⫽ 6) in myocytes cocultured
with hMSCs expressing HCN2 (P ⬍ 0.05). Spontaneous
rate was 93 ⫾ 16 bpm in the former group (n ⫽ 5) and
161 ⫾ 4 bpm in the latter (n ⫽ 6; P ⬍ 0.05). The reduced
maximum diastolic potential is consistent with the observed threshold potential of the expressed current in the
HCN2-transfected hMSCs and indicates an effect of this
depolarizing current on the electrically coupled myocytes
(Potapova et al., 2004).
We next injected HCN2-expressing hMSCs into canine
heart in situ to test whether pacemaker function was
demonstrable. During sinus arrest, five of six animals
receiving hMSCs expressing EGFP ⫹ HCN2 developed
rhythms originating from and pace-mapped to the left
ventricle at a site whose origin approximated that of the
hMSC injection (Potapova et al., 2004). Moreover, the
idioventricular rates of these animals was 61 ⫾ 5 vs. 45 ⫾
1 bpm in animals receiving hMSCs expressing EGFP
alone (P ⬍ 0.05). Study of the region of injection via H&E
staining demonstrated basophilic cells that were immunohistochemically positive for vimentin and for the CD44
antigen. Interdigitation between hMSCs and myocardium
was demonstrable (Potapova et al., 2004).
DISCUSSION
Electronic pacemakers having high reliability and low
morbidity are a primary therapy for complete heart block
or sinus node dysfunction (Zivin and Bardy, 2001). Important negative aspects of electronic pacemakers are their
lack of the biological responsiveness required for the autonomic and physiologic demands on the heart, the need
for maintaining catheter electrodes in the heart, and the
requirement for battery replacement. These limitations
RECREATING THE BIOLOGICAL PACEMAKER
Fig. 1. Representative experiments using AdGFP injection (A) and
AdGFP ⫹ AdHCN2 injection (B). Note that at the outset (left side of each
panel), sinus rhythm of comparable rate occurs in both animals. Vagal
stimulation was followed by a slow idioventricular rhythm in the AdGFPinjected dog (A; the interval between left and right traces lasted 22 sec,
near the outset of which vagal stimulation was initiated). In the AdHCN2-
1049
injected dog (B), vagal stimulation (see arrow) was followed by a far more
rapid idioventricular rhythm. The interval between left and right traces
was of 5-sec duration. Insets are magnifications of the lead II and RV
electrogram impulses indicated in the basic traces. Reprinted with permission from Plotnikov et al. (2004).
1050
ROSEN ET AL.
have provided the impetus for developing biological pacemakers. Among the approaches attempted to provide such
pacemaker function have been upregulation of ␤2-adrenergic receptors, downregulation of the background K⫹ current IK1, and overexpression of HCN2 channels, the molecular correlate of the endogenous cardiac pacemaker
current If (Edelberg et al., 1998, 2001; Miake et al., 2002;
Qu et al., 2003; Plotnikov et al., 2004).
All three approaches have initially employed either
plasmid injection into the heart (␤2-adrenergic receptors)
or viral vectors (IK1 downregulation and HCN2 overexpression). One potential flaw in ␤2-adrenergic receptor
overexpression is that endogenous cardiac pacemaker
mechanisms (which may be diseased) are left intact and
the ␤-receptor is used as a nonspecific stimulator of heart
rate: action will be on not only pacemaker current but on
other catecholamine-sensitive currents. With regard to
IK1, downregulation of this current subtracts an important determinant of repolarization, such that cells lacking
this current manifest prolonged repolarization (Miake et
al., 2002). This may set the stage for excess dispersion of
repolarization. Moreover, it is not yet certain what inward
current drives the pacemaker in this setting; existing data
suggest it is a hybrid current rather than the wild-type If
pacemaker.
We have elected to work with the HCN channel family,
as this family encodes the ␣-subunit of the wild-type If
that provides primary pacemaker initiation in the heart
(DiFrancesco, 1981, 1982). Clearly, HCN2 overexpression
locally in left atrium (Qu et al., 2003) or in the proximal
bundle branch system (Plotnikov et al., 2004) induces both
If-like currents and in situ pacemaker function in transfected myocytes. The voltage dependence of the If conductance results in current flow during phase 4 but not during
the action potential plateau limiting the potential arrhythmogenicity resulting from altered action potential
waveforms. Yet our use of an adenoviral construct to deliver the HCN2 gene to the heart (Qu et al., 2003; Plotnikov et al., 2004) is not an optimal approach because adenoviruses are episomal and the nucleic acids they deliver
do not integrate into the genome. Other viral systems that
generate more sustained transgene expression are accompanied by other important drawbacks that hinder their
application in vivo.
The use of stem cell therapy as an alternative to viral
gene transfer is attractive first of all because there is no
need to depend on a virus. Both embryonic stem cells and
mesenchymal stem cells have been tested here. Several
laboratories are exploring the use of embryonic stem cells
that can be differentiated along a cardiac lineage and
might provide a cell-based control of cardiac rhythm.
Among the advantages of these cells is that they make
functional gap junctions and generate spontaneous
rhythms (Gepstein, 2002). There are problems as well,
however, including the immunogenicity of the cells, the
potential for neoplasia, spatial nonuniformity of the implants, and the proper engineering of pure cardiac lineages (Gepstein, 2002).
We found hMSCs to be an attractive platform for delivering genes to the heart for several reasons: they can be
obtained in relatively large numbers through standard
clinical interventions, are easily expanded in culture, are
capable of long-term transgene expression, and their administration can be autologous or via banked stores (as
they are immunoprivileged). While they might in theory
be differentiated in vitro into cardiac-like cells capable of
spontaneous activity, we have taken a genetic engineering
approach that does not depend on differentiation along a
specific lineage. Moreover, the ex vivo transfection method
we used allows evaluation of DNA integration and engineering of the cell carriers with fail-safe death mechanisms.
Both our adenoviral and our genetically engineered hMSCs expressed an If-like current and were capable of functioning as biological pacemakers in intact canine heart.
Control adenoviruses or control hMSCs expressing only
EGFP did not exert these effects. Hence, the electrical
effects of hMSCs transfected with the mHCN2 gene were
similar to the effects of overexpression of the same gene in
the myocytes in vitro and in vivo. These findings suggest
that hMSCs may serve as an alternative approach for the
delivery of pacemaker genes for cardiac implantation.
Yet there are several important caveats with regard to
our results. Some relate to the technique of construct
injection, particularly in experiments in which adenoviral
constructs were injected into the canine left bundle branch
(Plotnikov et al., 2004). This intervention induces hematoma formation that is for a short period arrhythmogenic.
Importantly, hematoma formation occurred regardless of
whether we injected AdHCN2 ⫹ AdGFP, AdGFP alone, or
saline. In other words, it was the result of trauma from the
needle per se. It is likely that this injury contributed to the
tachycardias that occurred in the first 24 – 48 hr after
cardiac catheterization and that were universal across the
three groups of animals. Moreover, the fact that both in
vivo and in vitro accelerated pacemaker function was seen
only in the animals that received HCN2, despite the fact
that all groups underwent injury, suggests that the expression of pacemaker function was not the result of injury.
A major question relates to the duration of efficacy of
these pacemakers. In the present study, we were only
concerned with demonstrating the feasibility of applying
gene therapy via an adenoviral vector or using hMSCs as
a gene delivery system. Since our studies in vivo lasted
less than 2 weeks, transient transfections were sufficient
to demonstrate proof of concept. Before this approach can
be considered clinically relevant, far longer periods of
study will be required. In this regard, our transfected
hMSCs maintain their green fluorescence for at least 3
months, indicating that we have selected for stable clones
expressing HCN2. Hence, it is likely that persistence of
expression will not pose significant difficulties for more
prolonged studies. Nonetheless, it remains to be determined if the differentiation state of the hMSCs is altered
in situ in the long term, or whether such differentiation
would affect HCN2 expression or biophysical properties.
In addition, we used a murine gene, which is quite close
but not completely identical in sequence to the human
gene. It would be most advantageous to use human genes
and to explore various mutations to optimize activation
and recovery characteristics, as well as neurohumoral response. Such approaches are currently being explored.
In comparing results between the two approaches, it is
important to emphasize the conceptual and practical differences that exist in the design of gene therapy and stem
cell therapy. While both have one endpoint in common—
the delivery of a biological pacemaker— gene therapy uses
specific HCN isoforms to engineer a cardiac myocyte into
a pacemaker cell, whereas hMSC therapy uses stem cells
RECREATING THE BIOLOGICAL PACEMAKER
1051
Fig. 2. Patch clamp recording of native If (control dog) and overexpressed IHCN2 in Purkinje cells. Note the different scales in both panels
and the markedly greater current magnitude in the presence of overexpressed HCN2. Reprinted with permission from Plotnikov et al. (2004).
as a platform to carry specific HCN isoforms to a heart
whose myocytes retain their original function. Gene therapy makes use of preexisting homeotypic cell-cell coupling
among myocytes to facilitate propagation of the pacemaker impulses from those myocytes in which pacemaker
current is overexpressed to those that retain their original
function. In contrast, stem cells depend on heterotypic
coupling of cells with somewhat dissimilar populations of
connexins to deliver pacemaker current alone from a stem
cell to a myocyte whose function is left unchanged. Importantly, and unlike sinus node cells, HCN2-transfected hMSCs are not excitable, because they lack the other currents
necessary to generate an action potential. However, when
transfected, these cells generate a depolarizing current,
which spreads to coupled myocytes, driving myocytes to
threshold. In effect, the myocyte acts like a trip wire
whose hyperpolarization turns on pacemaker current in
the stem cell and whose depolarization turns off the current. We believe that as long as the hMSCs contain the
pacemaker gene and couple to cardiac myocytes via gap
junctions, they will function as a cardiac pacemaker in an
analogous manner to the normal primary pacemaker the
sinoatrial node. We must emphasize that a biological pacemaker needs an optimal size (in terms of cell mass) and an
optimal cell-to-cell coupling for long-term normal function.
In a sense, the outcome of the initial experiments was
Fig. 3. Functional expression of If in hMSCs transfected with mHCN2
gene. If was expressed in hMSCs transfected with the mHCN2 gene (B)
but not in nontransfected stem cells (A). C: The fit by the Boltzmann
equation to the normalized tail currents of If gives a midpoint of ⫺91.8 ⫾
0.9 mV and a slope of 8.8 ⫾ 0.5 mV (n ⫽ 9). If was fully activated around
⫺140 mV with an activation threshold of ⫺60 mV. The inset shows
representative tail currents used to construct If activation curves. The
voltage protocol was to hold at ⫺30 mV and hyperpolarize for 1.5 sec to
voltages between ⫺40 and ⫺160 mV in 10 mV increments followed by
a 1.5-sec voltage step to 20 mV to record the tail currents. Reprinted
with permission from Potapova et al. (2004).
fortunate in that the construct used and the number of
cells administered coupled and functioned as well as they
did. We currently are performing experiments to identify
what might be the optimal cell numbers and coupling
ratios needed to optimize function.
Both gene therapy and stem cell approaches currently
have as one of their shortcomings the passage of seconds
between the last normal heart beat and the onset of pacemaker function. Ideally, such an interval should be 1–2
sec in duration, which may require the engineering of
mutant genes. Given the differences in cell-cell coupling in
gene therapy and stem cell therapy, it is highly likely that
the engineering of the mutant genes necessary to function
as the ultimate biological pacemaker gene will differ importantly between the two approaches. It remains to be
seen, with both biological pacemaker approaches, whether
1052
ROSEN ET AL.
the duration of effect and the functional characteristics of
the biological pacemakers will be competitive with electronic units currently in use and in their planning stages.
Finally, the delivery of hMSCs expressing mHCN2 to
the canine heart is not only a demonstration of feasibility
of preparing hMSC-based biological pacemakers, but is
the first concrete example of a general principle; that is,
hMSCs can be used to deliver a variety of genes to influence the function of tissues capable of forming gap junctional connections. Hence, the payload delivered by hMSCs need not be restricted to membrane channels; any
gene product or small molecule that can permeate gap
junctions (MW ⬍ 1,000, minor diameter ⬍ 1.2 nm) can be
incorporated into the hMSCs and delivered to a gap junctional-coupled tissue as its therapeutic target.
ACKNOWLEDGMENTS
The authors thank Nimee Bhat for assistance in performing the studies and Ms. Eileen Franey for her careful attention to the preparation of the manuscript. Work on gene
therapy alone was supported by Guidant Corporation.
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