Heart Failure Clin 1 (2005) 287 301

Targeting Genes and Cells in the Progression to

Heart Failure
Motoki Sato, MDa,*, Stephen J. Fuller, PhDa, Roger J. Hajjar, MDb,
Sian E. Harding, PhDa
a

National Heart and Lung Institute, London, UK

Massachusetts General Hospital East, Charlestown, MA, USA

In cell electrophysiologic studies, it is known that

there is no remarkable difference in the contractile
force generated between the nonfailing and the failing
heart at low stimulation rates. As beating frequency
accelerates, however, failing heart muscles show a
decrease in the force of contraction, whereas the force
of contraction in normal myocytes increases. These
phenomena are called the negative force frequency
relationship and the positive force frequency relationship, respectively. Despite advances in the
treatment of cardiovascular diseases including hypertension, ischemic heart disease, and valvular heart
disease, they have not resulted in substantial improvements in survival after CHF ensues. According
to a survey done by the American Heart Association
[1], approximately 4.9 million Americans have CHF
and there are about 550,000 new cases per year.
Half the patients will be dead within 5 years after
being diagnosed with CHF. The outlook is not much
better than for most forms of cancer, and an estimated $24.3 billion was spent for the care of CHF
patients. However, the recent increasing knowledge
of the etiologic basis of CHF and the molecular
mechanism causing it is starting to encourage different therapeutic approaches including gene therapy
and cell transplantation for CHF. In this review,

* Corresponding author. Department of Cardiac Medicine, National Heart and Lung Institute, Faculty of
Medicine, Imperial College of London, Guy Scadding
Building, Dovehouse Street, London SW3 6LY, UK.
E-mail address: m.sato@imperial.ac.uk (M. Sato).

the authors highlight two new strategies for the

treatment of CHF: gene transfer to modulate cardiac function and cell transplantation to repair damaged myocardium.

Targets for intervention

The number of molecular targets for each pathophysiology is likely to increase with advances in the
knowledge of the molecular basis of cardiovascular
disease. With this in mind, the reader should be aware
that the following list is not meant to be exhaustive
but, rather, representative of the molecular targets
that have been pursued by various investigators. In
the following section, a systematic overview of the
more promising molecular targets is provided.

Gene therapy in heart failure

Gene therapy is a technology by which genes are
delivered to human cells, tissues, or organs to correct
a genetic defect or to provide new therapeutic functions for the ultimate purpose of preventing or
treating diseases. Hence, unlike genetically modified
organisms, gene therapy aims at inducing a modification of the genetic information of specific somatic
cells, not the permanent modification of the inheritable genome of a patient. In addition, compared with
conventional drug therapy, gene-based therapy shows
a number of potential advantages including safety,
efficacy, faster development, and modest impact on

1551-7136/05/$ see front matter D 2005 Elsevier Inc. All rights reserved.
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the environment. Recently, many considerations have

made heart failure an appealing clinical candidate
for gene therapy, with progressive understanding of
the molecular mechanism of heart failure. Theoretically, there appears to be three major types of gene
transfer to enhance cardiac function: (1) modulating
calcium homeostasis (2) manipulating b-adrenergic
receptor (b-AR) signaling, and (3) augmenting cardiomyocyte resistance to ischemia and apoptosis.
Modulating calcium homeostasis
The control of intracellular calcium (Ca2+) in cardiomyocytes is central to regulating heart function.
Contraction is initiated when Ca2+ enters the cytoplasm through voltage-dependent Ca2+ channels
(L-type Ca2+ channels) in the sarcolemma. The initiation triggers the release of far larger amounts of
Ca2+ from the sarcoplasmic reticulum (SR) by way
of the SR Ca2+ release channel Ryanodine receptor
(RyR). The free Ca2+ in myocytes interacts with
troponin C, producing disinhibition of the actomyosin
complex and eventually inducing contraction. Alternately, relaxation is induced by removal of free
Ca2+ from the cytosol. There are two main mechanisms for Ca2+ removal: reuptake of Ca2+ into the SR
by way of the energy-dependent Ca2+ pump called
the SR Ca2+-ATPase (SERCA2a) and extrusion of
Ca2+ to the extracellular space by way of the sarcolemmal sodium calcium exchanger (NCX). Intracellular Ca2+ homeostasis, therefore, is maintained
by these Ca2+-handling proteins, and the abnormality
of failing heart can be reflected in alterations in protein level or activity. Although the precise role of
these proteins in heart failure still is controversial, in
the failing heart, NCX often is found upregulated [2],
whereas SERCA2a activity is reduced [3,4]. In addition, phospholamban (PLB), which is a SERCA2a
inhibitory molecule, also is upregulated or PLB phosphorylation is decreased. Hence, in the presence of
upregulation of NCX and impaired SERCA2a, the
Ca2+ store in the SR is reduced. These alterations
partly can explain why the smaller SR Ca2+ release
and contractile dysfunction occur in heart failure. In
this regard, gene transfer to modulate calcium homeostasis (eg, restoration of SERCA2a protein level or
activity and reduction of NCX protein level) might
be predicted to rectify the cardiac dysfunction. Although the alterations in SERCA2a function or
protein levels in human heart failure have remained
controversial [3 6], it has been widely accepted that
SERCA2a regulates Ca2+ handling in cardiac cells
and plays a crucial role in cardiac contractility. Therefore, SERCA2a has been a main target to address in

human heart failure. In contrast, there have been no

in vivo studies with NCX as a potential gene therapeutic target.
Restoration of sarcoplasmic reticulum Ca2+-ATPase
activity (overexpression of sarcoplasmic reticulum
Ca2+-ATPase)
Hajjar et al [7] and Giordano et al [8] initially
demonstrated that the overexpression of SERCA2a
in isolated myocytes using adenoviral gene transfer
resulted in increased contractility and a faster decay
of the cytosolic Ca2+ transient. A similar result also
has been confirmed in human ventricular myocytes
from patients with end-stage heart failure [9,10].
These results show the potential of gene therapy for
heart failure but, at the same time, raise some concerns connected with these inotropic interventions,
such as arrhythmogenicity. It now is widely recognized that inotropic interventions that raise cyclic
AMP (cAMP) by way of stimulating b-ARs or inhibiting phosphodiesterase activity will increase
arrhythmogenicity (eg, the significant increase in
arrhythmia-related mortality in the clinical trial of
milrinone [11]. A strong possibility remains, therefore, that overexpressing SERCA2a can trigger
arrhythmias. Some recent in vivo studies, however,
have answered this critical question directly and indirectly. Ito et al [12] reported that transgenic expression of SERCA2a offsets the influence of pressure
overload by ascending aortic stenosis, which reduced
SERCA2a levels in control mice. This study also
showed better contractile function, higher SR Ca2+
stores, and lower mortality 7 weeks after the stenosis.
These results suggested that rectifying depressed
SERCA2a activity may prevent the transition from
compensatory hypertrophy to early heart failure.
In addition, it is noteworthy that overexpression of
SERCA2a has a chronic protective effect on the
development of heart failure by pressure overload.
In vivo gene transfer to rat hearts using adenoviral
SERCA2a-mediated vectors also showed similar
results [10] and a more direct answer to the concern
of arrhythmogenicity [13]. In rat heart failure models
created by ascending aortic banding, gene transfer of
SERCA2a improved survival rate at 4 weeks after
gene transfer (63% versus 9% in controls) and cardiac
function. Overexpression of SERCA2a induced the
normalization of creatine phosphate and ATP, which
means normal energy reserve. This study indicates
that unlike ordinary pharmacologic inotropic intervention, which increases intracellular Ca2+ concentration due to a rise in cAMP, overexpression of
SERCA2a leads to a decrease in diastolic Ca2+
concentration in cardiomyocytes, underlying the

hf progression: targeting genes

antiapoptotic and antiarrhythmogenic effect. These

experimental studies consistently showed that restoration of SERCA2a activity improved systolic and
diastolic function. Importantly, the results of in vivo
SERCA2a overexpression using adenovirus indicate
the feasibility of cardiac gene transfer into failing
hearts as a therapeutic inotropic intervention without
danger of arrhythmia.
Reduction of phospholamban protein levels
In experimental studies, the ablation of PLB,
which theoretically restores SERCA2a activity, has
been achieved by the knockout transgenic approach
and the virally mediated antisense or dominant
negative strategy for PLB. In the comparative study
between PLB-knockout and PLB-overexpressing
transgenic mice, the hearts from PLB-knockout mice
exhibited increased contractility of the left ventricle
compared with wild-type mice, whereas PLB-overexpressing mice showed the more severely depressed
contractility even though the level of SERCA2a
activity remained normal [14]. The study shows,
therefore, that the expression ratio of PLB to
SERCA2a is a critical regulator of cardiac function
and could be a potential target to treat heart failure.
This result also is supported by studies of in vivo
gene transfer of the antisense message for PLB.
He et al [15] first demonstrated the significant effect
of mutant and antisense RNA of PLB on SERCA2a
activity. The reduced expression of PLB resulted
in increased Ca2+ affinity of the SR Ca2+ pump and
decreased time to 50% recovery of Ca2+ transients in
neonatal cardiomyocytes. In this study, the PLB level
was not affected significantly in adult rat myocytes
by the antisense strategy, most likely because in adult
myocytes that have a high abundance of endogenous
PLB, a more effective antisense oligonucleotide of
PLB might be required to reduce PLB protein levels.
In another in vitro study [16], the antisense strategy
that used adenoviral vectors restored the frequency
response to a normal positive force frequency
relationship and enhanced SR Ca2+ release in failing
human myocytes from nine patents with end-stage
heart failure. Sato et al [17] and Hoshijima et al [18]
reported that in vivo gene transfer of the antisense
of PLB had a significant stimulatory effect on cardiac function in normal rat heart and cardiomyopathic hamster models, respectively. The in vivo
study demonstrated that the functional effect of PLB
antisense in myocytes isolated from the heart was
similar to Hajjar and colleagues [19] previous result
in vitro that showed a significant increase in the contractility and a reduction of relaxation time. Of note,
the study by Hoshijima et al [18] that used recom-

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binant adeno-associated virus expressing a pseudophosphorylated mutant of human PLB (S16EPLN)

showed a protective effect on cardiomyocytes and a
preventive effect on progressive cardiac dysfunction
for 28 to 30 weeks. These experimental studies have
indicated that in vivo overexpression of the antisense
of PLB could be an alternative to improving cardiac
function by the ablation of PLB. The antisense
strategy for PLB and in vivo SERCA2a overexpression, therefore, have the potential to treat human
heart failure.
Manipulating b-adrenergic receptor signaling
The principal role of b-ARs in the heart is to
regulate heart rate and contractility in response to
catecholamines such as epinephrine and norepinephrine. The b-AR family is divided into three subtypes:
b1-AR, b2-AR, and b3-AR. b1-AR is quantitatively
in the majority, accounting for 75% to 80% of the
total receptors in the heart, but b2-AR also is present.
When agonists activate b-ARs in the heart, b-ARs
binds to a heterotrimeric guanine nucleotide-binding
protein (G protein). The G protein (Gs) stimulates
adenylyl cyclase to produce cAMP within the cell,
which goes on to regulate several important downstream targets [20]. In heart failure, there are some
compensatory changes to preserve the function (eg, a
large sympathetic outflow of norepinephrine) [21].
The chronic stimulation of b-ARs leads to altered
b-AR systems, termed desensitization, and then to
aggravation of cardiac dysfunction. More precisely,
at the level of the b-AR signaling pathway, the
desensitization is characterized mainly by downregulation of myocardial b-ARs [22], b-AR uncoupling [23], and upregulation of the b-AR kinase
(b-ARK1) [24]. Hence, these three factors could
be targets for manipulating b-AR signaling, which
increases cAMP levels and improves the contractile function.
Restoration of b-adrenergic receptor density
To date, it has been reported that in the human
failing heart, the b1-AR contribution was reduced
following prolonged sympathetic activation, whereas
b2-AR numbers were preserved. In addition, inhibitory G protein (Gi) was increased and acted to
suppress the contractile effects of b-AR stimulation
[25]. Restoration of b1-AR, therefore, seemingly was
a main target. In contrary to the expectation, transgenic studies showed that overexpression of b1-ARs
was pathologic and involved in the development of
cardiomyopathy and apoptosis with catecholamines
[26], whereas overexpression of b2-AR worked pro-

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tectively. Milano et al [27] demonstrated that transgenic overexpression of b2-ARs enhanced cardiac
function and, importantly, these mice showed normal
lifespan and minimum negative effects. A later study
showed that although b2-AR produced some cardiomyopathy at higher levels, the effects largely were
beneficial at lower levels [28]. In a study on isolated
myocytes, the stimulation of b2-AR signaling was
antiapoptotic, ameliorating the proapoptotic effects of
b1/Gs activation in the adult rat heart [29]. In addition,
the improvement of the contractility by chronic
b2-AR/Gi stimulation also is supported by the recent
study with b-blockers showing a possible stimulation
of the b2-AR/Gi pathway [30,31].
Inhibition of b-adrenergic receptor kinase
Another alternative is inhibition of b-ARK1. Koch
et al [32] reported that overexpression of b-ARKct,
which competitively inhibits b-ARK1, could result in
enhanced inotropic responsiveness. The efficacy of
b-ARKct also has been investigated in several heart
failure animal models [33,34] and has shown protective effects on the contractile function. In particular, in MLP / (muscle LIM protein knockout)
mice, which show a dilated cardiomyopathy with
impaired contractility, overexpression of b-ARKct
preserved the cardiac function, with normal size of
the ventricular chamber [35]. Moreover, in transgenic
calsequestrin-overexpressing mice, which are characterized by severe dilated cardiomyopathy and premature death, overexpression of b-ARKct also
showed additional benefit to b-blocker therapy and
prolonged survival, with improvement of the cardiac
function [34]. It is particularly noteworthy that this
study showed the possibility of combined therapy of
traditional drug therapy and gene therapy. The alteration of b-AR signaling is of importance in the pathogenesis of heart failure and, obviously, targeting the
b-AR signaling pathway has massive potential to restore cardiac function in heart failure. Like the result
of b1-AR overexpressing transgenic mice, however,
inotropic therapy to stimulate the b-AR signaling
pathway in the clinical setting is associated with
decreases in the survival rate of patients with CHF.
Augmenting cardiomyocyte resistance to apoptosis
In recent years, it has been widely recognized that
cell death is not just the response to an injury but
can be a highly regulated and controlled process
(eg, apoptosis). This programmed cell death usually
brings about a characteristic morphologic change, in
which the cell and the nucleus shrink and condense
and frequently fragment into membrane-neighboring

cells. In the process of apoptosis, there is no leakage

of cytosolic components and no inflammatory response. In theory, apoptosis is associated with tissue, whereby cells progress through the cell cycle. It
once was thought that apoptosis could not occur in
terminally differentiated cells such as cardiomyocytes and brain cells; however, recent studies have
indicated that myocardial renewal occurs throughout life in the heart and is partly involved in cardiac
homeostasis [26,36] and apoptosis in the human heart
[37,38]. Condorelli et al [39] reported that apoptosis increased with the progression of the contractile
dysfunction in pressure-overload heart failure rat
models and that dysfunction was accompanied by
high levels of the proapoptotic protein Bax and a
reduced ratio of the antiapoptotic protein Bcl-2/Bax.
Other studies also indicated that the decrease in the
Bcl-2/Bax ratio was responsible for the increased
susceptibility of stretched myocytes to undergo apoptosis [40]. Overexpression of Bcl-2, therefore, may
be a target to increase resistance to apoptosis. This
concept is supported by several in vivo studies
from transgenic approaches [41] and gene transfer
using adenoviral vectors [42]. In rabbit ischemia
reperfusion models, despite no effect in the acute
stage, Bcl-2-overexpressing rabbits showed better
heart function and smaller ventricular size, with
greater preservation of ventricular free-wall thickness 6 weeks after reperfusion injury. Now it is
widely accepted that continued ventricular dilatation
occurs after myocardial infarction because thinning
of ventricular walls occurs not only in the infarcted
zone but also in the border zone region between
ischemic segments and nonischemic viable myocardium by increased tension load. This study, therefore,
may indicate the possibility that overexpression of
Bcl-2 prevents mechanical stretch induced apoptosis in the border zone. The following points, however,
should be mentioned. First, overexpression of Bcl-2
can prevent heart function from deteriorating further
due to apoptosis of viable myocytes but cannot restore the contractile function. Second, it is uncertain
whether blocking apoptosis in such a cell may prove
to be detrimental because the cell may go on to
necrosis instead of apoptosis. Recent studies have
indicated another possibility: cytokines may be a
crucial mediator of the transition from hypertrophy to
apoptosis in response to biomechanical stress. In
particular, gp130, which is a common receptor of the
interleukin-6 family of cytokinesis, is thought to play
an important role in the transition [43]. This study
demonstrated that in the ventricular muscle specific
gp130-knockout mice having normal cardiac structure and function, pressure overload by aortic

hf progression: targeting genes

banding rapidly led to dilated cardiomyopathy with

massive cardiac apoptosis. Hence, overexpression of
gp130-dependent signaling may be an additional
therapeutic option to prevent heart failure. Furthermore, although the precise mechanism by which
growth factors exhibit their antiapoptotic activity is
not completely understood, there now are numerous
studies showing that growth factors, including transforming growth factor b-1, insulin, insulin-like
growth factor, cardiotrophin-1, and fibroblast growth
factor, inhibit apoptosis in a variety of tissues and
organs [44].

Cell therapy in heart failure

Although the target of cell therapy (also known as
cellular cardiomyoplasty) now has spread to nonischemic cardiomyopathy, the basic concept of cell
therapy is to replace postinfarction scar tissue with
contractile cells and to restore contractile function to
the area. There seems to be three distinct approaches:
(1) reinduction of residual cardiomyocytes to a mitotic cycle and expansion of the number of contractile
elements, (2) transformation of in-scar fibroblasts into
contractile cells, and (3) injection of exogenous contractile cells into the scar [45].

Reinduction of residual cardiomyocytes to a mitotic

cycle and expansion of the number of contractile
elements
Recently, it has been shown that most adult
myocytes are differentiated terminally and withdraw
irreversibly from the cell cycle, but there are some
myocytes able to re-enter the cell cycle with a probability that increases significantly in certain pathologic
conditions [36,46]. It is now likely that even the adult
heart contains cardiomyocytes that have a proliferative capacity. Nevertheless, the expression of Ki-67
(a marker of cell cycle entry) was detected in only a
small population of myocytes in the region adjacent
to the infarct. The proliferation is extremely limited:
only 0.08% of mitotic index in the adjacent region
to the infarct [46]. Consequently, the proliferative
capacity obviously is not enough to compensate for
the massive cell loss by a large infarct. This evidence,
however, also raises the possibility that increasing the
number of remaining cardiomyocytes may be a therapeutic alternative to replacing damaged myocardium.
To date, some attempts to stimulate cardiomyocyte
DNA replication have been reported. It is known that

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progression of the mammalian cell cycle is regulated

by a family of cyclins and cyclin-dependent kinases
(CDKs). In particular, the cyclin D1 plays an important role for promoting the cell cycle G1-to-S phase
progression by inactivating the action of the retinoblastoma protein through phosphorylation. TamamoriAdach et al [47] showed that the nuclear import of
the cyclin D1/CDK4 complex was inhibited tightly
in postmitotic cardiomyocytes, and the nucleocytoplasmic transport machinery of cyclin D1 played a
critical role for determining the proliferative capacity
of cardiomyocytes. This study also demonstrated
dual gene transfer in vivo with cyclin D1 and
CDK4 induced cell cycle progression, leading to
cell division of neonatal cardiomyocytes. Although
the nuclear import of cyclin D1/CDK4 certainly
shows the possibility for induction of the cell cycle
re-entry of cardiomyocytes in the adult heart, it is
uncertain whether the re-entry into the cell cycle is
due to a stimulatory effect on the ability of cardiomyocytes that retain proliferative capacity or to a
direct effect to promote the cell cycle re-entry. Moreover, this strategy raises major safety issues because
of the stimulation of the cell cycle by overexpression
of viral oncoproteins.

Transformation of in-scar fibroblasts into contractile

cells by overexpression of MyoD
So far, no specific transcription factor for cardiac
myogenesis exists. Meanwhile, in skeletal muscle,
the MyoD family of basic helix-loop-helix proteins
are well known to function as master genes for the
induction of the skeletal muscle differentiation program [48]. Therefore, studies on the transformation of
scar tissue have focused on the conversion into
skeletal muscle. Tam et al [49] first indicated the
possibility that overexpression of the MyoD gene
in vitro may convert cardiac fibroblasts into skeletal
muscle cells showing myotubes, myosin heavy chain,
and myocyte-specific enhancer factor 2. Although the
in vivo conversion of fibroblasts to skeletal muscles
required high doses of adenoviral vectors, MyoD
gene transfer induced skeletal muscle differentiation
in the scar formation of infarcted heart [49]. In canine
myocardial infarction models, however, the transfection of MyoD was limited and the converted cells
showed the expression of skeletal myosin heavy
chain but no morphologic myotubes [51]. Accordingly, this concept still is controversial. In addition, it
should be mentioned that further investigation of
specific cardiac myogenic factors must be required
for a successful transformation strategy.

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Injection of exogenous contractile cells into the scar

From a clinical standpoint, transplantation of cells
has looked more realistic than other approaches.
Although most studies have focused on ischemic,
segmental cardiomyopathies, some reports have suggested that this technique can apply to CHF due to
idiopathic or doxorubicin-induced globally dilated
cardiomyopathies. The candidates for implanted
cells can be divided broadly into noncontractile cells
(eg, fibroblasts and smooth muscle cells) and contractile cells. Noncontracting cells are implanted for
the purpose of preventing the deterioration of postinfarct diastolic function, whereas contracting cells
are expected to improve systolic and diastolic function [52]. Furthermore, contractile cells are divided
into two cell types: naturally contractile cells and
potentially contractile cells. The former includes fetal
and neonatal cardiomyocytes and skeletal myoblasts.
The latter is represented by embryonic stem (ES) cells
and bone marrow cells (also known as adult stem
cells) [45].
Noncontracting cell transplantation
Fibroblast cells as a cell source for cell transplantation. Fibroblast cells are an attractive cell source
for cell transplantation and the target for transformation strategy because fibroblasts are autologous,
abundant, and easily expandable. It also is easy to
harvest fibroblast cells from several organs including
skin and pericardium. These properties are important
particularly for elderly patients because of their
limited availability of adult stem cells and myoblasts.
As previously mentioned in the discussion of transformation to skeletal muscles, several attempts with
in vivo gene transfer of MyoD using direct injection
(DI) required a high dose of viral vectors and yielded
a low efficiency of the conversion into skeletal muscle. Etzion et al [53] combined the concept of the
transformation strategy with cell transplantation to
avert the resistance of the necrotic myocardium with
immune reaction. This study indicated the possibility
of a two-staged procedure (MyoD-based therapy)
composed of ex vivo transformation of cardiac fibroblasts with MyoD and cell transplantation.
Smooth muscle cell transplantation. Mickle and
colleagues [54,55] demonstrated that smooth muscle
cell transplantation improved systolic and diastolic
function in rat myocardial infarction models and in
hamster dilated cardiomyopathy models because implanted cells increased wall tension and elasticity.
These studies indicated that injected smooth muscle

cells may prevent the deterioration of systolic dysfunction by attenuating systolic overstretch of native viable cardiomyocytes and diastolic dysfunction
(ventricular enlargement) by limiting ventricular expansion. In addition, smooth muscle cells have the
following technical and biologic advantages: the possibility of autotransplantation, the ability to proliferate more easily than skeletal myoblasts, the property
of elasticity, the long-term steadiness of the contractile property (termed tonus contraction), and the
hyperplasic response of smooth muscle cells to the
increased stretch.
Naturally contractile cells
Fetal and neonatal cardiomyocytes. In cellular
cardiomyoplasty, the primary candidates are fetal
and neonatal myocytes because these cells have the
ability to differentiate into an adult, matured cardiomyocyte phenotype but, at the same time, still retain
a certain ability to proliferate [56]. Soonpaa et al [57]
first demonstrated that grafted fetal cardiomyocytes
were detected over 2 months in normal mouse hearts.
Interestingly, in this fundamental study, grafted fetal
cardiomyocytes in normal mice hearts showed nascent intercalated disks connected with the host
myocardium. Similarly, Murry and colleagues [58]
indicated that grafted fetal and neonatal cardiomyocytes formed matured myocardium in rat hearts
2 months after the injection. In some cases in this
study, graft cells formed gap and adhesion junctions
with host cardiomyocytes, suggesting electromechanical coupling. Theoretically, successful cell transplantation, which contributes to the improvement of
cardiac function, depends crucially on integration into
the host and differentiation toward the adult phenotype. In this regard, fetal and neonatal cardiomyocytes seem to be promising candidates. It must be
mentioned, however, that as far as a clinical perspective is concerned, ethical issues could hinder the application severely, with other hurdles to overcome,
including availability and immunogenicity.
Skeletal myoblasts. Skeletal myoblasts, also known
as satellite cells, are myogenic precursors and
normally exist in a quiescent state under the basal
membrane of skeletal muscle fibers. These cells are
mobilized rapidly into the injured area and proliferate and fuse to regenerate the damaged fibers. Skeletal myoblasts have some advantages as a source for
cell transplantation: (1) autologous origin, (2) relatively easy to multiply to large numbers from a small
biopsy, (3) low tumoregenicity because of welldifferentiated myogenic lineage, and (4) high resistance to ischemia. In experimental studies, it has been

hf progression: targeting genes

shown that grafted myoblasts differentiated into typical multinucleated myotubes and substituted the
postinfarct fibrosis; however, no studies to date have
shown reliable evidence of transdifferentiation of injected myoblasts into cardiomyocytes, despite some
forms of phenotype (slow myosin pattern) that can
adapt to the myocardial environment. In addition,
most studies of skeletal myoblast transplantation have
failed to show the existence of gap junction of injected cells with host myocytes in vivo [50,59,60].
Furthermore, the in vitro study by Murry and colleagues [60] reported that although cultured skeletal myoblasts express N-cadherin and connexin 43
(Cx43), which is known as the major protein for gap
junctions, the expression decreased after intramyocardial implantation in vivo. Recently, Menasche and
colleagues [54,61] showed conclusive evidence that
skeletal myoblasts retained their typical electrical
membrane properties and functional and electrophysiologic independence after transplantation. In
contrast to these negative results of the graft host
coupling, the improvement of cardiac function after
skeletal myoblast transplantation has been demonstrated continuously in many animal models and even
in clinical cases in the short- and long-term [61,62].
Menasche and colleagues [45] repeatedly indicated
that functional improvement may be linked mechanistically to engrafted skeletal myoblasts and proposed three possible mechanisms. First, the elastic
properties of implanted cells may provide a scaffold,
strengthening the ventricular wall and subsequently
limiting postinfarct scar expansion in a similar way to
the smooth muscle cell implants [54]. In this regard,
it is consistent that the grafted skeletal myoblasts
have a protective effect against excessive remodeling.
Second, intrinsic contractile function of skeletal myoblasts may be directly involved in the improvement
of the systolic function. In a 1-year follow-up study,
functional parameters of sheep hearts remained significantly improved in systolic and diastolic function,
detected 4 months after the transplantation [61].
Similarly, Menasche et al [63] reported that postoperative echocardiography showed that 63% of the
myoblast-transplanted infarcted segments demonstrated a new-onset systolic thickening; therefore,
despite many negative results of skeletal myoblastto-host cardiomyocyte coupling, this possibility remains. Third, the engrafted myoblast may act as a
source of growth or angiogenic factors. It has been
reported that hepatocyte growth factor/scatter factor
was detected in skeletal myoblasts [64]. In addition,
because the expression of hepatocyte growth factor
in myocardial ischemia and reperfusion has antiapoptotic and antifibrotic effects [65], this factor has

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been targeted as a new therapeutic alternative for

ischemic heart disease [66,67]. The precise role of
hepatocyte growth factor is not completely clear, but
it is possible that grafted skeletal myoblasts secrete
hepatocyte growth factor and have a positive effect
on the host cardiomyocytes, including recruiting resident cardiac stem cells and stimulating regeneration
of cardiomyocytes. This hypothesis also is supported
by the result of long-term follow-up after skeletal
transplantation that shows that, despite the decrease
in the number of grafted cells in the heart, the functional improvement remains 1 year after transplantation. Since the first clinical report of cell-based
therapy in 2001 by Menasche et al [68], the results
of the first phase I trial were much awaited. This trial
served a major dual purpose: the feasibility and the
safety of autologous skeletal myoblast transplantation in patients with severe ischemic cardiomyopathy. The results and the protocol are mentioned only
briefly here; the reader is referred to the excellent
clinical report and editorial comment recently published [63,69]. The cell-based therapy was performed
in 10 patients. The criteria of patient selection were
(1) impairment of left ventricular function: ejection
fraction 0.35; (2) previous history of myocardial
infarction, with a residual discrete, akinetic, and nonviable scar in the left ventricle; and (3) indication for
concomitant coronary artery bypass graft in another
segment of the ventricle that is dependent and viable but ischemic and not in the cell-implanted area.
Skeletal muscle was harvested from the patients
thigh under local anesthesia. The sample was upscaled to at least 5  108 cells containing at least
50% myoblasts and 70% viable cells. All patients had cell transplantation of 8.7  108 cells on
average, which was concomitant with two or three
bypass grafts. The whole process, from the muscle
biopsy to the operation, was done within 2 to 3 weeks.
Among 10 patients, there was one early death and
one noncardiac death during the follow-up, but no
perioperative complication related to the cell preparation and the transplantation, except for ventricular
arrhythmia. It is fair to say, therefore, that the results are encouraging in terms of feasibility and
safety of the procedure. The investigators carefully
concluded that the scale-up of cell numbers was
more than enough to improve the cardiac function
and that the transplantation was relatively safe. It is
difficult to estimate the efficacy of the procedure
because this trial had a limited number of subjects
and no control subjects; however, determining efficacy was not main purpose of the trial. In addition, it
would be impossible to completely rule out the effect
of the concomitant revascularization. Nevertheless, it

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is noteworthy that at about 11 months of follow-up,

63% of cases showed a new-onset systolic wall
thickening in the implanted scar area. In addition,
patients also showed a significant improvement of
New York Heart Association functional class (from
2.7 to 1.6) and left ventricular ejection fraction
(from 24% to 32%). Concerning ventricular tachycardia after the transplantation, whether the arrhythmia directly connects with the transplantation is not
clear. It should be mentioned, however, that four of
nine cases showed sustained ventricular tachycardia
and had the implantation of an automatic internal
cardiodefibrillator. This phase I trial has shown the
real possibility of cell-based therapy to treat human
heart failure and, at the same time, it has opened our
eyes to the drawbacks such as the possibility of arrhythmia after transplantation. Now the clinical trial
has moved to phase II, which aims to assess the
safety and efficacy with a placebo-control group in
multiple centers.
Potentially contractile cells
Embryonic stem cells. Myogenic stem cells that
have the potential for differentiation into cardiomyocytes can be divided into two categories: ES cells
and adult stem cells, which are contained in bone
marrow. ES cells derived from the inner cell mass
of blastocyst-stage embryos are characterized by the
ability to proliferate unlimitedly and undifferentiatedly and to be pluripotent, which means the capability to differentiate into every somatic cell type
of the adult organism. These cells, therefore, are
expected to be an ideal source of cells for the reproduction of damaged myocardium. Mouse ES cells
in vitro form an aggregate termed embryonic bodies.
In general, within 10 days, 80% to 100% of them
have spontaneously contracting areas, but the fraction
of the contracting areas is small. Methods to generate essentially pure cardiomyocyte cultures have been
reported by several groups. Klug et al [70] reported
that more than 99% purified cultured and genetically
selected mouse cardiomyocytes formed stable intracardiac grafts with a normal myocardial structure,
showing the formation of intercalated discs and gap
junctions when transplanted into the healthy mouse
heart. Like mouse ES cells, human ES cells are
characterized by immortality, expression of specific
transcription factors and cell surface molecules, and
the ability to differentiate into all cell types. The
pluripotent stem cells express a variety of receptors
for growth factors. In particular, transforming growth
factor b-1 and activin-A are known to promote differentiation into mesodermal derivatives such as
muscle cells. In 2001, Kehat et al [71] reported the

differentiation of cardiomyocytes from human ES

cells and the electrical coupling and synchronous
contraction of the human stem cell derived cardiomyocytes with rat cardiomyocytes in coculture [72].
In addition, in rat heart infarction models, implanted
ES cells survived and differentiated into mature
cardiomyocytes 6 weeks after transplantation and,
as a result, cardiac function was improved [73].
Moreover, a recent study reported that the improvement remained in the long-term (32 weeks) [74].
Undoubtedly, these studies have indicated that ES
cells could provide enormous potential for cell therapy in heart failure because ES cells theoretically
have the unlimited availability as a source of cardiomyocytes even for humans. Grafted ES cells also are
expected to form electrical coupling with the host
cardiomyocytes and improve global cardiac function.
It is fair to say, however, that there remain a number
of major issues to overcome for the clinical application of human ES cells. First, the allogeneic origin
of these cells raises immunologic problems, and antirejection strategies including immunosuppressive
drugs may be required. Second, the efficacy and culture conditions for cardiomyocyte differentiation
should be more optimized. Third, the immortality of
undifferentiated stem cells, which can form cell types
other than cardiomyocytes, is a huge benefit but also
may cause tumorigenicity. Last but not least, like fetal
and neonatal cardiomyocytes, moral and ethical issues
could be the greatest obstacle to clinical application.
Bone marrow stem cells (adult stem cells). Recently, bone marrow cells, which means stem cells
derived from the bone marrow, have been focused
on as a source of cell transplantation into the heart.
Like ES cells, the so-called adult stem cell is
expected to have the capacity of unlimited, undifferentiated proliferation and to be able to develop
into different types of cells including cardiomyocytes.
To date, because there is no universally acceptable
characterization and definition of stem cells and progenitor cells, these cells have a variety of names
(eg, marrow progenitor cells, stromal stem cells,
marrow stromal cells, marrow mononuclear cells, and
mesenchymal stem cells). In this review, the authors
use bone marrow stem cells (BMSCs) as the term
for cells that seem pluripotent and have the ability
to differentiate into various phenotypes. In contrast
to ES cells, BMSCs appear to be an ideal cell source
for cardiac repair because these cells have great plasticity and can be collected from patients without
evoking ethical and moral questions or creating problems of immunologic reaction. Although the precise
angiogenic and myogenic effects after the trans-

hf progression: targeting genes

plantation of BMSCs still are unclear, most experimental studies have shown differentiation into cardiomyocytes with the improvement of global cardiac
function in acute or chronic infarcted heart models
[75 77] and even in the nonischemic heart [78].
There are several studies that show the induction of
differentiation of BMSCs into cardiomyocytes by
treating with 5-azacytidine [77,79], and the investigation of additional inducing factors for the differentiation has been explored. The recent study by
Fukuhara and Tomita [80] showed that only mouse
BMSCs cocultured with neonatal rat cardiomyocytes
showed synchronous contraction with cardiomyocytes and cardiac-specific proteins including connexion 43 and troponin 1. This result is consistent with
the previous study by Wang et al [81] that reported
that BMSCs differentiated into myofibroblasts in the
scar tissue, whereas they became cardiomyocytes in
viable myocardium. Direct cell-to-cell interaction,
therefore, may be one of the most important factors
for BMSCs to differentiate into cardiomyocytes. To
date, results of several clinical phase I trials of autologous BMSC transplantation have been published
[82 84]. Although the aim of these trials was to assess the feasibility and safety, and although the
number of patients was limited and there was no
control group (except for Perins study [85]), all of
these studies indicated that this procedure was safe
and appeared to improve symptoms and myocardial
perfusion. In particular, it is noteworthy that in contrast to clinical studies of skeletal myoblast transplantation, these studies reported no patients with
ventricular arrhythmia including sustained ventricular
tachycardia. Still, it also should be mentioned that
the condition and cell types of these clinical studies
varied and long-term follow-up must be done. In
addition, the following questions must be answered
for the clinical application to treating heart failure:
Which component of BMSCs, such as stromal cells
or hematopoietic progenitors, is suitable for a cell
source of cardiomyocytes? How should the population of BMSCs be scaled up without affecting cell
plasticity? According to the study by Jackson et al
[86], the percentage of the donor stem cell derived
cardiomyocytes is 0.02%obviously not enough to
improve the damaged heart function. These results
are consistent with recent reports that suggest that
the main effect of BMSC transplantation on the improvement of regional heart function is angiogenesis
by the paracrine effect of BMSCs, not direct effects
on contraction. It is certain that the paracrine effects
are likely to trigger the differentiation of adult stem
cells into cardiomyocytes, stimulate host myocytes to
re-entry of a cell cycle, and induce a cardiomyogenic

&

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295

lineage of BMSCs or circulating progenitor cells to

injured sites. Even so, the scale-up of BMSCs will
be vital to improve cardiac function directly.

Delivery systems for gene and cell therapy in heart

failure
Theoretically, the fundamental basis of delivery
systems to targeted cardiovascular tissues is not
different between gene therapy and cell therapy. So
far, in experimental and clinical studies of gene and
cell therapy for heart disease, a variety of delivery
methods has been reported, but the methods are
physiologically categorized into two major methods:
injection using coronary circulation, including intracoronary injection (ICI) and retrograde injection by
way of the coronary sinus, or direct injection (DI)
including epicardial and endocardial approaches. In
addition, each delivery method can be applicable to
interventional and less invasive or ordinary surgical
approaches. Whether the delivered material is genes
or cells, the functional efficacy to treat heart disease
depends on how efficiently they can settle in the
targeted tissue and how well the settled materials can
function as expected. In this regard, gene and cell
therapy in heart disease still have no sufficient delivery systems. Furthermore, all systems have the
same potential risk, namely, untoward effects on
nontarget organs. Therefore, it is of a paramount importance to obtain biodistribution data from clinical
studies, and defining biodistribution and excretion
of injected material is essential for the safe progression of clinical studies.

The transduction efficiency and functional effect

of the gene product in cardiac gene delivery
To date, the feasibility of in vivo gene transfer
mainly has been investigated experimentally and
clinically in terms of viral vectorsin particular, recombinant adenovirusesbecause the vector has
several advantages including relative safety and the
ability to transduce genes into non-replicating cells
like myocytes. The virus, however, also is known to
induce a robust immune reaction. Hence, it is obvious
that in successful clinical application, other vectors or
further-refined adenoviral vectors may be required. In
gene therapy, the retention and effect of vectors
carrying genes are revealed as the transduction efficiency and the functional effect of the gene product,
respectively. In addition, in contrast to cell therapy,
gene therapy needs not only to settle into the injected

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area but also to cross the membrane of targeted cells

and transduce genes. Because the factors involved in
the transduction efficiency are numerous and no
gold standard evaluation method exists, it would
be hard to generalize the optimal condition for all
mechanical gene delivery systems from experimental
studies. So far, however, DI shows significant transfection and gene expression, with limited extent of
gene expression surrounding injection sites, whereas
the transfection efficiency of ICI is low [16,87 89],
except for a few studies [90,91]. The difference in the
transfection efficiency between DI and ICI is thought
to be attributable to the dynamic feature of injected
vectors by way of ICI, which requires breaching the
endothelial barrier to transduce cardiomyocytes. This
idea is supported by the result that adeno-associated
virus (18 26 nm in diameter), which is smaller than
adenovirus (~70 100 nm in diameter) [92] and
endothelial vesicles (<50 nm) [93], showed better
transfection efficiency with ICI [94]. Regarding optimal conditions for gene delivery by ICI, the study
by Donahue et al [95] using explanted hearts indicated that the critical factors for the transfection efficiency were (1) washing blood out of the heart by
perfusing crystalloid solution; (2) high coronary flow
rate, with high pressure in coronary arteries; (3) long
exposure time; (4) high virus concentration; and
(5) body temperature (37C). Among these parameters, high-pressure infusion or long exposure time are
supported by other studies [89,96]. Furthermore, the
importance of the endothelial barrier as an obstacle
to successful transfection is consistent with the results
of retrograde injection by way of the coronary sinus,
which can reach cardiomyocytes without passing
through endothelial barriers, and showed significant
transgene expression without any assistance [97,98].
These factors may be applicable to those for DI. Sato
et al [17] already reported that a modified DI method,
named the immobilization technique, which blocks
the coronary flow in the targeted area during the injection, could improve the transfection efficiency of
gene transfer using adenoviral PLB-mediated vectors
[17]. This study indicates that even in DI, which can
reach cells directly, coronary circulation may be one
of the major inhibitors for successful gene delivery,
and injected vectors could be washed out to systemic
circulation rapidly if there is no concomitant suspension of coronary circulation. This situation may explain why gene expression is limited surrounding
needle sites when using DI. Some attempts to improve the efficiency are mainly aimed at enhancing
endothelial permeability to make vectors transduce
into cells easily and quickly. The approaches to date
can be divided into two major methods: ultrasonic

and pharmacologic pretreatment. The ultrasonic destruction method is based on the hypothesis that
albumin-coated microbubbles could be used to deliver an adenoviral transgene effectively through
microvessel rupture, with local extravasation of blood
cells. This method showed a 2.5- to ~10-fold higher
transgene expression compared with the control group
[98 100]. Another ultrasonic method, sonoporation,
recently has gained popularity. Sonoporation is a general phenomenon of creating holes or pores in the cell
membrane by way of intense acoustic forces created
by ultrasonic cavitations [101]. The authors reported
that the efficacy of gene transfer by way of the combination of sonoporation and adenoviral vectors was
sevenfold higher than that of DI [17]. On the other
hand, several drugs that may increase permeability
agents (eg, histamine, serotonin, bradykinin, vascular
endothelial growth factor [VEGF-A165], and low calcium solution) have been investigated as pretreatment agents. Although these drugs, to some extent,
have a supplementary effect to shorten the required
exposure time of vectors, the major factors still are
high-pressure infusion, long exposure time, or both
[96,102]. Effective therapy in heart failure is likely
to require a gene delivery method that is capable
of globally transducing the heart. Although globally transducing the heart theoretically is possible
by multiple DIs, gene delivery by way of the coronary circulation may be better. Therefore, optimizing conditions for gene transfer using ICI could be
vital for the clinical application of gene therapy in
heart failure.

The survival and functional effect in cell delivery

In cell therapy, there is no significant difference
between delivery methods in experimental studies.
Today, the delivery of skeletal myoblasts in clinical
trials has been done by multiple Dls by way of
epicardial approach under direct vision, whereas in
BMSC transplantation, various methods including
multiple DIs by way of epicardial approach [82],
ordinary coronary transluminal angioplasty catheter
[83,84], and endoventricular injection using the
NOGA myostar injection catheter (BioscienceWebster, Inc., Johnson & Johnson) [85] have been
used. Not surprisingly, clinical delivery procedures
have a tendency to become less invasive. It should be
mentioned, however, that whatever the method for
cell delivery, the obstacle still is the lack of sufficient
functional efficacy. The settlement of injected cells
seems to be easier than that of vectors in cardiac gene
delivery because injected cells, which are biologically

hf progression: targeting genes

bigger than virus, may have the ability to permeate

through endothelial barriers. Therefore, the survival
rate may be involved mainly with the low functional
efficacy. Theoretically, the survival of grafted cells
is influenced by physical and biologic factors. The
former may include cell loss during injection due to
technical errors and cell death due to high-pressure
injection. In addition, washing-out by coronary flow
and contractile force within the injected area may
affect the survival of grafted cells. Biologic factors
may be composed of inflammatory reaction, environmental hypoxia, and apoptosis. Among studies that
have investigated the survival of grafted cells, some
discrepancies have existed regarding the major
factors or cell survival. Several quantitative studies
after cell therapy (of which most used neonatal cardiomyocytes) have been reported. Zhang et al [103]
reported that the survival rate was reduced by 53%
in granulation tissue and by 86% in normal myocardium at day 1, and eventually, 90% of cells were lost
over the first week, with half the cell death occurring
over the first 24 hours. These results are similar to
another study by Murry et al [104] who reported that
the survival rate 1 day after transplantation (32%)
rapidly decreased to 1% to 10% at 7 days. In addition,
this study showed that dead cells in the graft had
several electron microscopic findings of irreversible
ischemic injury and apoptosis and that there was an
increase in the fraction of dying cells with increasing cell number. These results indicate that ischemia
may play a major role in cell death despite the fact
that acute host inflammation remains another possibility [103]. In contrast to these studies, some studies
demonstrated that viability of engrafted cells in infarcted myocardium has been demonstrated for up to
6 to 7 months after transplantation of fetal and neonatal cardiomyocytes, with a survival rate of 60% in
infarcted rat heart models [105,106]. These studies
appear to support the hypothesis that physical factors
mainly affect the fate of grafted cells and can explain
why grafted cells in the infarct model showed a better
survival rate (60% at 6 months after transplantation)
[106] than the noninfarcted model [57]. Moreover,
in skeletal myoblast implantation, the increase in the
number of injected cells correlated with the improvement of cardiac function [107].

Summary
The experimental studies highlighted in this review consistently have shown that gene therapy and
cell transplantation are promising therapeutic approaches for the treatment of heart failure. Their

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297

concepts basically are simple and logical. Some concepts, such as angiogenesis using adenoviral vectors
and BMSCs and skeletal myoblast transplantation,
already have been applied in clinical trials. So far,
results mainly are encouraging. We must realize, however, that our expectations should be tempered
by a realistic appreciation of the hurdles to overcome. For example, improvements in the delivery
system must be made, with investigation of optimal
conditions for injected vectors carrying genes or
cells. As yet, there are no conclusive answers about
ideal gene targets in gene therapy and optimal cell
types for cell transplantation. The recent study by
Chien and colleagues [108] showed that gene therapy
has the potential to treat heart failure. Even so, its
application to clinical trials should be set up more
carefully than that of cell transplantation because
when a persistently expressing gene is transfected
into a human heart, it must be the appropriate amount
and injected correctly into the target. In other words,
after the gene is inserted, the transgene product cannot be controlled, as is done easily with conventional
drugs. It is indispensable, therefore, to develop techniques for regulating the expression of the gene. In
this respect, improvements in vector technology, with
investigation of the factors that have effects on transfection efficiency, must be central to research for gene
therapy in heart failure. On the other hand, unsolved
questions in cell therapy mainly are connected with
the dynamics and kinetics of grafted cells, such as
the mechanism of the improvement of the contractile
function and the optimal condition for the survival of
implanted cells. As previously mentioned, ischemia
or apoptosis may be major impact factors for grafted
cell death. The strategy, which combines gene therapy with cell transplantation (eg. Akt-overexpressing
neonatal cardiomyocytes [103]) showed attenuated
apoptosis after the transplantation. Therefore, geneoverexpressing cell therapy may be an alternative
therapeutic approach for the treatment of heart failure. In addition, the precise mechanism of arrhythmia
after cell transplantation, which was realized in clinical trials of skeletal myoblast transplantation, must
be investigated intensively to justify this innovative
treatment. Despite no clinical cases with ventricular
arrhythmia in BMSC transplants, the possibility of
the arrhythmogenicity of stem cell derived cardiomyocytes already has been indicated [109,110]. Despite these formidable gaps between basic research
and clinical treatment, it is fair to say that every basic
and clinical study of gene and cell therapy highlighted in this review is important and a significant
step in the steady progression toward the new revolution of meaningful therapies for heart failure.

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sato et al

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