REVIEWHeart Development and Regeneration via Cellular Interaction
and Reprogramming
Masaki Ieda1–3

1Department of Clinical and Molecular Cardiovascular Research, School of Medicine, Keio University, Tokyo, Japan
2Department of Cardiology, School of Medicine, Keio University, Tokyo, Japan
3The Japan Science and Technology Agency, CREST, Tokyo, Japan(Received for publication on December 28, 2012)(Accepted for publication on June 28, 2013)(Published online in advance on September 10, 2013)

The heart consists of many types of cells, including cardiomyocytes, vascular cells, neural cells, and cardiac fibroblasts. Adult cardiomyocytes are terminally differentiated cells, and loss of cardiomyocytes
as a result of heart damage is irreversible. To regenerate damaged hearts and restore cardiac function,
understanding the cellular and molecular basis of heart development is of considerable importance.
Although it is well known that heart function is tightly regulated by cell–cell interactions, their roles
in heart development are not clear. Recent studies, including ours, identified important roles of cell–
cell interactions in heart development and function. The balance between neural chemoattractants andchemorepellents secreted from cardiomyocytes determines cardiac nervous development. Nerve growth
factor is a potent chemoattractant synthesized by cardiomyocytes, whereas Sema3a is a neural chemorepellent expressed specifically in the subendocardium. Disruption of this molecular balance induces
disorganized cardiac innervation and may lead to sudden cardiac death due to lethal arrhythmias. Cardiac fibroblasts, of which there are large populations in the heart, secrete high levels of specific extracellular matrix and growth factors. Embryonic cardiac fibroblast-specific secreted factors collaboratively
promote mitotic activity of embryonic cardiomyocytes and expansion of ventricular chambers during
cardiogenesis. More recently, utilizing knowledge of the regulatory mechanisms of heart development,
we found that cardiac fibroblasts can be directly reprogrammed into cardiomyocyte-like cells in vitro
and in vivo by gene transfer of cardiac-specific transcription factors. Understanding the mechanisms ofheart development and cardiac reprogramming technology may provide new therapeutic approaches
for heart disease in the future. (doi: 10.2302/kjm.2012-0020-RE; Keio J Med 62 (4) : 99–106, December
2013)

Keywords: sympathetic nervous system, growth factor, fibroblasts, reprogramming, regeneration

Introduction

anisms during heart development might provide insightto new therapeutic approaches for heart disease.
Compared with other organs, heart is extensively innervated via the autonomic nervous system, which comprises sympathetic and parasympathetic nerves. The
sympathetic nervous system produces norepinephrine,
which increases the heart rate, conduction velocity ofcardiac cell excitation, and myocardial contraction andrelaxation. It is well known that sympathetic innervation

Heart consists not only of cardiomyocytes, but also ofmany additional types of cells, including vascular cells,
neural cells, and cardiac fibroblasts. Heart function istightly regulated by the interactions between cardiomyocytes and other types of cells through the secretion ofmolecules and extracellular matrix (ECM). Understanding these cell–cell interactions and their molecular mech-

Reprint requests to: Masaki Ieda, MD, PhD, Department of Clinical and Molecular Cardiovascular Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan, E-mail: mieda@z8.keio.jp
Copyright © 2013 by The Keio Journal of Medicine
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density is strictly determined within the heart, and is highin the subepicardium and the central conduction system.1
The regional differences in sympathetic innervation influence specific cardiac functions, effectively controlling heart rate and myocardial contraction. Despite theclinical importance of cardiac innervation density, little
is known about the regulatory mechanisms underlying
sympathetic nerve development.
Cardiac fibroblasts are found throughout cardiac tissues, along with cardiomyocytes, and account for morethan half of the cells in the heart.1,2 Under physiological conditions, fibroblasts provide a mechanical scaffold
for cardiomyocytes and coordinate the pump function of
the heart.3 In diseased hearts, fibroblasts proliferate andsecrete ECM and growth factors that promote cardiomyocyte hypertrophy, leading to myocardial remodelingand heart failure.4,5 Although these findings demonstratethat cardiac fibroblasts are critical in adult heart function,
little is known about their developmental roles in the embryonic heart.
In contrast to cardiac fibroblasts, post-natal cardiomyocytes have little or no regenerative capacity. Loss of terminally differentiated cardiomyocytes as a result of heartdisease is irreversible; consequently, new therapeutic approaches are demanded. The large population of endogenous cardiac fibroblasts might be a potential source of
cardiomyocytes for regenerative purposes, if it proves
possible to directly convert resident fibroblasts into beating cardiomyocytes. The generation of induced pluripotent stem cells (iPSCs) by Dr. Yamanaka and colleagues
suggested that a specific combination of defined factors
could alter the global gene expression profile of a cell andallow greater plasticity of cell type than was previously
appreciated.
We and others demonstrated that cellular interactions
during development are critical for proper heart function, and that their disruption may lead to various kindsof heart disease. This article first reviews the molecularmechanisms of heart development, focusing on the crosstalk among cardiac sympathetic nerves, fibroblasts, andcardiomyocytes. Based on these studies, we recently
found that overexpression of cardiac-specific transcription factors can convert cardiac fibroblasts into cardiomyocyte-like cells in vitro and in vivo.
Nerve Growth Factor is Critical for
Development of the Cardiac Sympathetic andSensory Nervous Systems
The heart is highly innervated by autonomic nervous
systems, including sympathetic, parasympathetic, andsensory nerves, derived from neural crest cells. Cardiac
sympathetic nerves extend from sympathetic neurons inthe stellate ganglia, which are located bilateral to the thoracic vertebra. Sympathetic nerve fibers project from thebase of the heart into the myocardium, and are locatedpredominantly in the subepicardium of the ventricle.6,7
The central conduction system, which includes the sinoatrial node, atrioventricular node, and bundle of His,
is abundantly innervated compared with the working
myocardium.7–10 This regional difference in cardiac sympathetic innervation is highly conserved among mammals.7,8,11,12 Cardiac sensory neurons, which are locatedin the dorsal root ganglia, are derived from trunk neuralcrest cells, just as sympathetic neurons are. The cardiac
sensory nervous system is responsible for pain perception
and for initiating a protective cardiovascular response
during myocardial ischemia.
The growth-cone behavior of nerves is modulated by
the balance between neural chemoattractants and chemorepellents synthesized in the innervated tissue. Nerve
growth factor (NGF), a potent neural chemoattractant, isa prototypic member of the neurotrophin family, and thelevels of NGF expression within innervated tissues correspond approximately to the levels of sympathetic innervation density. Indeed, the sympathetic ganglion volume is reduced by 80% at postnatal day 3 in mice with
disruption of the NGF gene,13 while in mice lacking the
NGF receptor TrkA, no neurons remain at postnatal day
9.14,15 Deletion of a single copy of the NGF gene results ina 50% reduction in sympathetic neurons, whereas overexpression of NGF in the heart causes cardiac hyperinnervation.16,17 Despite the importance of NGF in sympathetic neural development, the upstream molecules that
regulate NGF expression in vivo remain unknown.18 Of
the several cardiac hypertrophic factors tested, only endothelin-1 (ET-1) specifically upregulated NGF expression in primary cultured cardiomyocytes.17 In addition,
NGF expression and cardiac sympathetic innervation
were reduced in ET-1-deficient mouse hearts, but not in
the hearts of angiotensinogen-deficient mice (Fig. 1).
In ET-1-deficient mice, the sympathetic stellate ganglia
also exhibited excessive apoptosis and neuronal loss.17,19
Moreover, we found that cardiac-specific overexpressionof NGF in ET-1-deficient mice reversed the sympathetic
nerve retardation. These findings indicated that ET-1 is a
key regulator of NGF expression in cardiomyocytes, andthat the ET-1/NGF pathway is critical for sympathetic innervation in the heart.17
In contrast to somatic tissues, visceral organs such as
the heart are believed to be rich in autonomic efferent
innervation, but poor in nociceptive afferent nerves.20
Zahner et al. reported that vanilloid receptor-1-immunopositive sensory nerves are enriched in the epicardium,but scarce in the myocardium.21 We reported that cardiac
sensory innervation is rich both at epicardial sites and in
the ventricular myocardium, and that sensory innervation
increases with heart development.22,23 In our screeningof several neurotrophic factors, we found that cardiac
sensory nerves develop in parallel with NGF synthesizedin the heart.23,24 Cardiac sensory nerves that are immunopositive for calcitonin gene-related peptide (the dorsal

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101

Fig. 1 Endothelin-1-deficient mouse hearts demonstrate reduced sympathetic innervation and downregulation of norepinephrine concentration.
(A) Immunostaining for nerve fibers with anti-GAP43, -PGP9.5, and -tyrosine hydroxylase (TH) antibodies in embryonic day 18.5
mouse hearts. Nerves were restricted to the epicardium in both endothelin-1 knockout (Edn1–/–) and angiotensinogen knockout (Atg–/–)mice , and levels of GAP43, PGP9.5, and TH were lower in Edn1–/– mice, but not in Atg–/– mice, compared with wild type littermates.
(B) Cardiac norepinephrine (NE) concentrations were reduced in Edn1–/– mice.17 **P<0.005; ns, not significant.

root ganglia and the dorsal horn) were markedly retardedin NGF-deficient mice, whereas cardiac-specific overexpression of NGF reversed these deficits. Thus, NGF synthesis in the heart is also critical for the development of
the sensory nervous system.23,25 We also found that the
reduced NGF expression in diabetic hearts might explain
the cardiac sensory denervation and neuropathy in diabetic mice, and overexpression of NGF in the hearts canreverse sensory denervation and diabetic neuropathy inthe mouse.23,26
Sema3a Determines Cardiac Sympathetic
Innervation Patterning and
Maintains Normal Heart Rhythm
As discussed above, NGF, a neural chemoattractant,plays critical roles in cardiac nerve development; however, no neural chemorepellent that induces growth-conecollapse and repels nerve axons has been identified in theheart. Sema3a is a class 3 secreted semaphorin that has
been cloned and identified as a potent neural chemorepellent and directional guidance molecule for nerve fibers
in the skin.27–29 However, it was not known until recently whether cardiomyocytes produce Sema3a, and if so,whether this protein affects sympathetic neural patterning and cardiac performance.
We found that Sema3a is strongly expressed in thedeveloping heart at embryonic day 12 in mice, and expression gradually fell with development.12 By analyzing

Sema3a knocked-in lacZ mice, we found Sema3a expression in the subendocardium, but not in the subepicardium of the atria and ventricles, the opposite pattern
to the epicardial-to-endocardial gradient of sympathetic
innervations.12,30 These results indicate that the distribution pattern of Sema3a expression is the opposite that ofsympathetic innervation in developing hearts, implicating Sema3a as a negative regulator of cardiac innervation. Sema3a knockout mice showed disrupted sympathetic innervation patterning and malformation of thestellate ganglia, which extend sympathetic nerves to theheart. Cardiac-specific Sema3a-overexpressing mice had
reduced sympathetic innervation and attenuation of theepicardial-to-endocardial innervation gradient. Importantly, both types of mutant mice were susceptible to sudden death, and Sema3a-overexpressing mice were highly
susceptible to ventricular tachyarrhythmias, whereas
Sema3a–/– mice developed sinus bradycardia and sinus
arrest. These results indicate that cardiomyocyte-derived
Sema3a plays critical roles in cardiac sympathetic innervation patterning and the maintenance of arrhythmia-freehearts (Fig. 2).
Cardiac Fibroblasts Regulate Heart Development by
Promoting Myocardial Proliferation throughβ1 Integrin Signaling
Cardiac ventricular formation involves growth of theheart muscle by proliferation of cardiomyocytes during

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Fig. 2 Sema3a expression in the developing heart and various kinds of arrhythmias in Sema3a-mutated mice.
(A) X-gal staining (blue) of Sema3alacZ/+ hearts at embryonic day 12.5 (E12.5) demonstrates strong Sema3a expression in the subendocardium. Bar, 100μm. (B) Electrocardiogram (ECG) recordings from wild-type (WT) and Sema3a –/– (KO) mice. Sema3a-deficient
mice display abrupt sinus slowing and bradycardia. Bars, 100ms. (C) ECG during programmed electrical stimulation (EPS) showing sustained ventricular tachyarrhythmias (VT) after administration of isoproterenol in cardiac-specific Sema3a-overexpressing(Sema3a TG) mice.12 Appropriate Sema3a-mediated sympathetic innervation patterning is critical for the maintenance of an arrhythmia-free heart. Bars, 500ms.

embryogenesis. Signals from the endocardium and epicardium may influence cardiomyocyte proliferation in aparacrine fashion,31,32 but how this proliferative activityof embryonic cardiomyocytes is regulated and subsequently terminated after birth remains unknown. Cardiac fibroblasts make up more than 50% of all the cells
in the heart, but their function during cardiogenesis has
not been determined. We demonstrated that embryoniccardiac fibroblasts develop coincident with expansion of
the ventricular compact layer and regulate the proliferation of cardiomyocytes.33 Embryonic cardiac fibroblasts
secrete high levels of fibronectin, collagen, and heparinbinding epidermal growth factor-like growth factor (HBEGF), which collaboratively interact and regulate mitotic
activity of cardiomyocytes through β1 integrin signaling.
β1 integrin is required for myocardial proliferation andventricular compaction during development, as indicatedby the smaller ventricles and interstitial fibrosis in thehearts of β1 integrin-deficient mice (Fig. 3). The bromo-

deoxyuridine labeling index was reduced in β1 integrindeficient hearts, and microarray analyses revealed thatthe cell-cycle promoters Ccnd1 and Ccne1 were strongly
downregulated in the mutant hearts. These results suggest that β1 integrin is required for cardiomyocyte proliferation and compact layer growth during embryogenesis,
coincident with the development of cardiac fibroblasts.
In contrast to embryonic fibroblasts, we found that adultcardiac fibroblasts had a unique function in promoting
hypertrophy rather than proliferation of cardiomyocytes,
suggesting that a fundamental switch in the cardiac fibroblast gene program may contribute to some of the physiological differences between embryonic and adult hearts.
Direct Reprogramming of Cardiac Fibroblasts into
Cardiomyocyte-like Cells by Defined Factors
In contrast to fibroblasts, adult cardiomyocytes are terminally differentiated cells and their regenerative capac-

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Fig. 3 β1 integrin signaling is critical for myocardial growth and muscle integrity.
(A) Wild-type and β1 integrin-deficient mouse hearts at postnatal day 1 (P1). Mutant ventricles were hypoplastic compared with wild
type hearts. Bars, 1mm. (B) Masson-trichrome staining in postnatal day 7 (P7) wild-type and β1 integrin-deficient hearts. Fibrosis
was detected in the mutant interventricular septum (IVS) and the right ventricular (RV) subendocardium.33 The arrow indicates thebundle of His. Bars, 100μm.

ity following injury is very limited; this fact is a major
contributor to mortality due to heart disease. Consequently, new cardiac regeneration therapy is demanded,
and cell replacement treatment using stem cell-derived
cardiomyocytes might be an attractive option to repair
injured myocardium. The ability to reprogram fibroblasts
into iPSCs using four defined factors might address someof the issues related to the cellular rejection and ethicalconcerns resulting from cell therapy, but generating sufficient numbers of iPSC-derived cardiomyocytes that are
pure and mature and that can be delivered safely remains
challenging. We hypothesized that if it were possible todirectly convert resident cardiac fibroblasts into cardiomyocytes in situ, the large population of endogenous
fibroblasts might be a potential source of new cardiomyocytes for regenerative purposes. To determine thecandidate factors of cardiac reprogramming, we used a
microarray to screen the genes that are specifically expressed in embryonic cardiomyocytes and selected 14
factors as candidates for cardiac reprogramming.
We generated α myosin heavy chain (αMHC) promoter-driven enhanced green fluorescent protein transgenic
mice (αMHC-GFP), in which only mature cardiomyocytes expressed GFP, and used this mouse for screening.12,34 Transduction of all 14 factors into fibroblasts induced 1.7% of GFP+ cells, and serial removal of individual
factors demonstrated that a combination of three factors[Gata4, Mef2c, and Tbx5, (GMT)] were sufficient for efficient GFP+ cell induction (around 15%) from cardiac fibroblasts. We designated these GFP+ cardiomyocyte-likecells induced cardiomyocytes (iCMs). These iCMs expressed several cardiac-specific marker proteins, such as
sarcomeric α-actinin, cardiac troponin T, and atrial natriuretic factor, and had well-defined sarcomeric structures
similar to neonatal cardiomyocytes (Fig. 4). The global
gene expression profile of the iCMs was not identical to,

but was similar to, neonatal cardiomyocytes, and was different from that of the original fibroblasts. The chromatinstate of iCMs was also similar to that of cardiomyocytes
but different from that of fibroblasts in terms of histone
modifications and DNA methylation patterns in several
cardiac gene promoters, at least.35 A subset of iCMs possessed functional properties of cardiomyocytes, including intracellular Ca2+ transients and action potentials after 2–4 weeks of culture. We also found that iCMs could
be derived from tail-tip fibroblasts, thereby excluding thepossibility that the iCMs arise from contamination ofcardiomyocytes or cardiac progenitors in the fibroblast
population.36,37 However, we also found that tail-tip fibroblasts are more resistant to cardiac reprogramming by
GMT, suggesting that some epigenetic blocks may exist
in some starting cell populations. We were also able togenetically map the “route” of cell fate alteration using a
Cre-loxP system. We tagged cardiac progenitors and theirderivatives with yellow fluorescent protein (YFP) using
Isl1-Cre-YFP and Mesp1-Cre-YFP mice, and found thatthe generation of iCMs from fibroblasts was a direct process that did not pass through cardiac progenitor states.38
More recently, we investigated whether direct genetransfer of GMT into mouse hearts could similarly induce
new cardiomyocyte generation from cardiac fibroblasts.39
Retrovirus was used as a vector for gene delivery after
myocardial infarction in the mouse because this type ofvirus infects mainly fibroblasts but not terminally differentiated cardiomyocytes. Injection of GMT retrovirus into αMHC-GFP transgenic mouse hearts induced
expression of GFP, a reporter of cardiomyocytes, in 3%
of virus-infected non-myocytes. GMT injection into thehearts of immunosuppressed nude mice induced cardiac protein expression in 1% of the transduced fibroblast
cells, although few cells showed sarcomeric structures.
We next developed a polycistronic vector expressing

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Fig. 4 Overexpression of Gata4, Mef2c, and Tbx5 induces cardiac gene expression and sarcomeric structures in fibroblasts.
Immunofluorescent staining for αMHC-GFP, α-actinin, and nuclei. The combination of the cardiac-specific factors Gata4, Mef2c,
and Tbx5 induced abundant αMHC-GFP and α-actinin expression in fibroblasts 2 weeks after transduction (middle row, bar 100μm).
High-magnification views in insets show sarcomeric organization (bottom row, bar 100μm). Fibroblasts without transduction wereused as controls.38

GMT separated by 2A “self-cleaving” peptides (3F2A)
to improve reprogramming efficiency. Injection of thispolycistronic retrovirus vector resulted in the generation in fibrotic tissues of iCMs that expressed sarcomeric
α-actinin, cardiac troponin T, and several cardiac-specific genes. Importantly, more iCMs had well-defined sarcomeric structures by using this system. These results
suggest that 3F2A-iCMs were more mature cardiomyocyte-like cells and that the polycistronic vector can beused for cellular reprogramming in vivo.
Following our first report of cardiac reprogramming,other groups also reported generation of cardiomyocytelike cells from mouse fibroblasts based on the same factors or microRNAs in vitro and in vivo.40–43 Althoughfurther work in larger animals is needed, as is the generation of iCMs from human fibroblasts and more efficient protocols of cardiac reprogramming, these reports
demonstrate that this new direct reprogramming strategy
might be a potential approach for heart regeneration in
the future (Fig. 5).44,45
Conclusions
During heart development, many cell types coalesce,interact, and develop into mature organs. In this process,cell function, proliferation, and differentiation are tightly regulated by cell–cell interactions to insure that theheart attains the necessary structure and function. Cardiac sympathetic innervation patterning is strictly controlled by the balance between neural chemoattractants(e.g., NGF) and chemorepellents (e.g., Sema3a) derived
from cardiomyocytes. Disruption of the sympathetic
innervation patterning may eventually lead to fatal arrhythmias in both diseased and developing hearts. Better
understanding of the mechanisms of cardiac sympathetic
innervation patterning may represent an important approach for future therapies to avoid sudden cardiac death.
Cardiac fibroblasts express specific ECM and growth factors that collaboratively promote proliferation of myocardial progenitors through β1 integrin signaling. Deficits ofthis interaction result in ventricular hypoplasia and heart
failure. Identification of the molecular mechanisms involved in the interactions between cardiomyocytes andother type of cells may enhance our understanding ofheart development, function, and disease. Direct reprogramming technology raises the possibility of converting
endogenous cardiac fibroblasts in injured hearts directly
into functional cardiomyocytes by gene transfer. Further

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Fig. 5 Possible future regenerative therapy using direct cardiac reprogramming technology.
In the future, direct cardiac reprogramming may be used to regenerate damaged myocardium. Gene transfer of Gata4, Mef2c and Tbx5
into infarcted hearts may be able to convert endogenous cardiac fibroblasts into induced cardiomyocytes in situ. To achieve this goal,
the following are needed: further work in larger animals, the generation of induced cardiomyocytes from human fibroblasts, and more
efficient and safe protocols for cardiac reprogramming.

refinements and understanding of the molecular basis ofcardiac reprogramming should be developed to enhance
reprogramming efficiency and advance this new technology to clinical application.
Acknowledgments
The author is supported in part by research grants fromJST CREST, JSPS, The Mitsubishi Foundation, AstraZeneca, Takeda Science Foundation, Banyu Life Science,
The Uehara Memorial Foundation, Kimura Memorial
Heart Foundation, Japan Research Foundation for Clinical Pharmacology, the Nateglinide Memorial Toyoshima
Research and Education Fund, and SENSHIN Medical
Research Foundation.
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