Review article

franklin h. epstein lecture

Franklin H. Epstein, M.D., served the New England Journal of Medicine for more than 20 years.
A keen clinician, accomplished researcher, and outstanding teacher, Dr. Epstein was Chair and Professor of
Medicine at Beth Israel Deaconess Medical Center, Boston, where the Franklin H. Epstein, M.D., Memorial
Lectureship in Mechanisms of Disease has been established in his memory.

Cardiac Development and Implications

for Heart Disease
Jonathan A. Epstein, M.D.
From the Department of Cell and Developmental Biology and the Cardiovascular
Institute, University of Pennsylvania School
of Medicine, Philadelphia. Address reprint
requests to Dr. Epstein at the Department
of Cell and Developmental Biology, University of Pennsylvania School of Medicine,
421 Curie Blvd., 1154 BRB II, Philadelphia,
PA 19104, or at epsteinj@mail.med.upenn
.edu.
N Engl J Med 2010;363:1638-47.
Copyright 2010 Massachusetts Medical Society.

uring the past decade, our understanding of the development

of the embryonic heart has been improved by a number of discoveries. These
new findings will require changes in standard teachings of how the fourchambered heart forms, and they have implications for the management of congenital and acquired heart disease. In the coming years, additional advances in our
knowledge of cardiac development are likely to further influence the classification
and treatment of congenital heart disease, inform clinicians on the best uses of regenerative treatment (e.g., stem-cell therapy), and revise our understanding of some
cardiovascular disorders in adults. This review gives examples of recent findings in
the field of cardiac development, with an emphasis on those likely to have the
greatest effect on clinical practice.

Ov erv ie w
Classic teaching holds that cardiovascular development proceeds from the early specification of bilateral clusters of progenitor cells that coalesce to form a cardiac
crescent and a midline linear heart tube. This tube, which consists of an inner cell
layer of endothelium surrounded by myocardial precursor cells,1 undergoes a series
of looping or bending events followed by ballooning or expansion of regions destined to become cardiac chambers. Subsequently, a series of septation events results
in a four-chambered heart with parallel systemic and pulmonary circulations.
Additional cell types that lie outside the heart tube are important in the development of the heart and influence its morphogenesis (Fig. 1). For example, neuralcrest cells, which form components of the peripheral nervous system and the craniofacial regions, migrate to the heart, where they are essential for septation of the
cardiac outflow tract.2 The link between neural-crest cells3 and septation helps to
explain the association of craniofacial defects with some forms of congenital heart
disease.

L ine age R e s t r ic t ion

The mature heart consists of many different cell types, including myocardial cells,
endothelial cells, smooth-muscle cells, fibroblasts, and specialized conducting cells.
Until recently, the origins and lineages of these various cell types were unclear.
Recently, however, the technique of gene targeting has allowed rigorous fate mapping (a method used to determine the cellular derivatives of a cell or population of
cells) and lineage analysis in mammalian embryos and adults.4-7 The results of
these studies, and of clonal analyses of embryonic stem-cell differentiation in vi1638

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Fr anklin H. Epstein Lecture

Figure 1. Overview of Cardiac Development.

Traditional descriptions of cardiac development include progression from the cardiac crescent to the linear heart
tube, which loops and becomes septated as it develops into the mature heart. Multiple cell types extrinsic to the initial cardiac crescent, including neural-crest cells, cells arising from the second heart field, and epicardial progenitors, contribute to cardiac morphogenesis.

tro,7-10 convincingly document a progressive lineage restriction of cells engaged in cardiac development (Fig. 2). It is now clear that precursor cells
in the embryo have the potential to differentiate
into various types of cardiac cells. As a particular
lineage develops, however, the potential of its
member cells to deviate into alternative lineages
becomes progressively restricted.
Embryonic stem cells are pluripotent that is,
they have the ability to become nearly any kind of
cell. Such stem cells can differentiate into spontaneously beating myocardial cells when grown in
tissue culture in the presence of specific growth
factors and under particular conditions.12-14 Cardiac precursor cells that arise in vitro from em-

bryonic stem cells express kinase-domainrelated

(KDR) receptor (a receptor for vascular endothelial growth factor) and NKX2-5 (a transcription
factor with a role in cardiac development).8,10
These early cardiac precursor cells have the potential to become endothelium, smooth muscle, or
myocardium. In vivo genetic studies suggest that
similar precursor cells contribute to multiple lineages in the developing heart.
The phenomenon of progressive lineage restriction during cardiac development has important implications for the use of stem-cell therapy
in cardiac disease. For treatments intended to
enhance endogenous myocardial repair or to
generate new heart muscle through the delivery

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Figure 2. Progressive Lineage Restriction.

In the hematopoietic system, stem cells become progressively restricted during differentiation. Similarly, cardiac progenitors, which may
share a common precursor with hematopoietic stem cells, become progressively restricted in terms of the types of potential mature derivatives that can ultimately be produced. Adapted from Wu et al.11

of appropriate cardiac progenitor cells, or to grow

bioprostheses of contractile myocardial patches,8
researchers and practitioners must consider which
cells can best meet these goals.8,15,16 For example,
the regenerated tissue that results from treatment
with a progenitor cell that has the potential to
produce only cardiac myocytes will be unlike normal cardiac tissue, which has multiple cellular
components. Progenitors with a restricted differentiation capacity may have to be replaced with
multipotent progenitor cells if the goal is to regenerate multilineage tissue composed of endothelium, smooth muscle (regenerating vasculature), and contractile myocardium. At this time,
the most appropriate type of progenitor cell to be
used in stem-cell therapy for ischemic cardiomy1640

opathy and other forms of heart failure is unknown, and the markers and gene-expression
signatures that characterize various progenitors
are only now being elucidated.
Progressive lineage restriction is also a feature
of the differentiation of a multipotent hematopoietic stem cell into various blood-cell lineages.17
The delineation of each stage of lineage restriction
in blood-cell precursors has allowed for identification of clinically useful growth factors, such as
granulocyte colony-stimulating factor, granulocytemacrophage colony-stimulating factor, and
erythropoietin, each of which affects a different
progenitor. As is the case with hematopoietic stem
cells, the identification and characterization of
specific cardiac progenitor cells and cardiac

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Fr anklin H. Epstein Lecture

growth factors may lead to useful treatments for tal abnormality, or similar developmental abnormyocardial infarction or heart failure.
malities, can underlie anatomically distinct congenital heart disorders. An example is the group
of clinically dissimilar congenital heart defects
The Sec ond He a r t Fiel d
(e.g., a double-outlet right ventricle and right venNot all precursors of cardiac muscle reside in the tricular hypoplasia) that are related through their
cardiac crescent and the early linear heart tube. association with abnormalities of second-heart
Many right ventricular myocytes and, to a variable field progenitor cells.25 Aberrations of these predegree, myocytes in the atria, left ventricle, and cursor cells may also cause anatomical abnorcardiac inflow and outflow tracts enter the devel- malities of the left or right side of the heart (e.g.,
oping heart after its initial looping stages are com- defects of atrial septation, ventricular septation,
plete.18-20 These additional cells arise from a sec- conus positioning, and great-vessel alignment)
ond heart field that is medial and ventral to the because the cells contribute to both the inflow
primary cardiac crescent. (In the embryo, a field and the outflow tracts of the heart.26 Moreover,
consists of a group of related cells within a de- recent studies of what have come to be known as
fined boundary.) Cells in the second heart field second-heartfield cardiac defects suggest that inmigrate first to the pharyngeal regions, where flow and outflow abnormalities frequently coexthey can be identified in mouse embryos in early ist.26 Anatomical classification is undoubtedly
gestation or midgestation according to the prod- clinically useful, but it is likely that classification
ucts of specific marker genes, including the tran- systems based on developmental relationships and
scription factor islet 1.20 These second-heartfield genetic causes will provide additional diagnostic
cardiac precursors in the pharyngeal regions invade and prognostic information. Consensus opinions
the developing heart and migrate along its inflow that explicitly define developmentally based clasand outflow tracts. Second-heartfield progeni- sifications of congenital heart disorders will also
tors that express islet 1 are multipotent cells that promote enhanced communication among basic
can give rise to smooth-muscle cells at the base researchers and their clinical colleagues.
of the aorta and pulmonary arteries, to endothelial cells, or to myocardium.7
The Epic a r dium in C a r di ac
The existence of a second heart field has imDe v el opmen t a nd R epa ir
portant implications for understanding congenital heart diseases. For example, abnormalities in The epicardium, a layer of connective tissue lothe distinct genetic pathways that mediate for- cated between the myocardium and the pericarmation of myocytes in either the right or the left dium,27 arises from a transient embryonic strucventricle could explain congenital defects whose ture called the proepicardial organ (Fig. 3). Cells
predominant effect is on the right or left ventri- in this organ derive from the septum transvercle. The results of induced genetic perturbations in sum, which separates the embryos thorax from
only second-heartfield cells of embryonic mice its abdomen, and to the diaphragm and liver. Some
suggest that abnormalities in this population can proepicardial cells migrate to the developing heart
cause double-outlet right ventricle, right ventricu- and contribute to the formation of the epicardial
lar hypoplasia, pulmonic stenosis, and tetralogy of layer. Descendants of proepicardial cells invade
Fallot.21,22 Moreover, genetic studies in humans the myocardium, where they develop into fibrohave shown that haplotypes within the ISL1 lo- blasts in the heart and smooth-muscle cells of
cus are strongly associated with these forms of the coronary arteries.29-32 Signals from the epicongenital heart disease.23 Perhaps other right- cardium are required for proper maturation of the
sided disorders, such as hypoplastic right ventri- myocardium and normal development of the corcle, Ebsteins anomaly, and some forms of arrhyth- onary arteries.33-35
mogenic right ventricular dysplasia,24 are also the
Recent studies suggest that epicardial proresult of abnormalities in second-heartfield cells. genitor cells are multipotent, with the ability to
The usual classification of congenital heart differentiate into smooth muscle, fibroblasts, and
diseases depends on the anatomical characteris- perhaps also cardiac muscle and endothelium36,37
tics of the abnormality. These traditional classi- (although the idea that these cells form coronary
fication systems may have to be changed in light endothelium has been challenged38). These studof emerging evidence that the same developmen- ies used genetic markers expressed by the proepin engl j med 363;17 nejm.org october 21, 2010

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Figure 3. Development and Derivatives of the Epicardium.

The epicardium derives from the proepicardial organ (Panel A), a multipotent cluster of cells that arises dorsal to
the looped heart. Epicardial progenitors migrate to and encompass the developing heart and form the mature epicardium (Panel B). Some epicardial progenitors undergo an epithelial-to-mesenchymal transition, invade the myocardium, and differentiate into various mature cardiac cell types, which may include vascular smooth-muscle cells,
fibroblasts, endothelial cells, and cardiac myocytes. Adapted from Schlueter.28

cardial organ the Wilms tumor 1 gene (Wt1)37

and the T-box 18 gene (Tbx18)36 to map the
fate of epicardial precursor cells throughout the
course of their differentiation. The results suggest that some epicardial precursors contribute
to myocardium, an indication of an additional
developmental avenue for the generation of cardiac muscle. In adult zebrafish, which can regenerate myocardium after injury,39 the epicardium becomes activated by surgical resection of
heart muscle40; the activated epicardium expresses fetal genes, including wt1 and tbx18. It remains
unclear whether these activated epicardial cells
contribute directly to the regeneration of myocardium or produce signals that cause cardiac myocytes to enter the cell cycle.41 Since epicardium
normally gives rise to cardiac fibroblasts, the main
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components of scars, it is possible that the ability of mammals to grow new heart muscle was
lost during evolution in a trade-off for the ability to rapidly form a scar after injury.
Epicardium-derived progenitor cells (EPDCs)
have been isolated from human, rat, and mouse
hearts and have been grown in tissue culture.42
In rodents (as in zebrafish), these cells reactivate
fetal genes after myocardial infarction and proliferate.43,44 Ex vivo, EPDCs can differentiate into
multiple cell types and express contractile proteins
of myocytes. Various growth factors, including
thymosin 4, have been found to promote the proliferation of EPDCs and to improve recovery and
myocardial function after injury in animals45-47;
it remains unclear whether these changes are due
to myocardial regeneration or to factors secreted

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Fr anklin H. Epstein Lecture

by EPDCs that have paracrine effects influencing

myocyte survival or function. These studies suggest that interventions involving the manipulation
of epicardial activation and EPDCs after injury
whether through the use of systemic therapy, treatments administered within the pericardial space,
or the application of a drug-eluting patch to the
epicardial surface of the damaged heart may
be worthwhile avenues for further investigation.

The C a r di ac C onduc t ion S ys tem

The specialized cells of the cardiac conduction system arise from myocardial precursor cells. The
mature cells have relatively poor contractility and
express specialized ion channels and gap-junction
proteins, including connexins, that mediate electrical coupling with neighboring cells.48,49 Early
in development, at the time of chamber specification, the myocardium between the developing
atria and ventricles has slow conduction characteristics and other properties reminiscent of the
atrioventricular node. Similarly, the myocardium
of the inflow tract acquires autonomous activity
and develops pacemaker function. The sinus node
develops from this tissue. The cells that give rise
to the sinus node express the fetal TBX18 gene,
whereas the cells that give rise to the atrioventricular node and the Purkinje system express the
NKX2-5 transcription factor.48 The possibility that
precursors of pacemaker cells of the sinus node
are related to the myocardium surrounding the
pulmonary veins could be important (and is currently under investigation) because atrial fibrillation commonly arises from an arrhythmia within
the pulmonary veins. The myocardium of the posterior wall of the left atrium extends to and ensheathes the proximal pulmonary vein, thereby
providing electrical continuity, and atrial fibrillation can be successfully treated through electrical
isolation of the pulmonary veins.50 The development of pulmonary-vein myocardium requires
the PITX2 transcription factor,51 and recent genomewide association studies have identified
haplotypes at 4q25, near PITX2, that are associated
with atrial fibrillation.52,53 Thus, the response of
pulmonary-vein myocardial cells to altered PITX2
function may underlie the susceptibility to atrial
fibrillation. It is also possible that melanocytelike cells in the heart,54 which are present in the
atrioventricular ring, the atria, and the pulmonary veins in the developing embryo, play a role
in atrial fibrillation. In animal models, genetic

abnormalities induced in these cells increase the

susceptibility to atrial arrhythmia.
The region of slow-conducting myocardium
that separates atria from ventricles in the embryo
and provides atrioventricular delay initially occupies the entire atrioventricular ring.48,49,55 As
cardiac development proceeds, fibroblasts derived
from the epicardium invade the atrioventricular
sulcus and form the annulus fibrosis,56,57 which
isolates the atria from the ventricles electrically.
The atrioventricular canal myocardium regresses,
and the property of slow conduction becomes
restricted to the specialized cells of the atrioventricular node. In animal models, deficient development of the annulus fibrosis causes abnormal
electrical connectivity between the atria and ventricles, pre-excitation, and characteristics of the
WolffParkinsonWhite syndrome.56,58 Ectopic
myocardium that bridges the atrioventricular region is, however, more common than breakdown
of the annulus fibrosis in humans with this syndrome. Consequently, some forms of the syndrome may result when the normal regression of
the atrioventricular canal myocardium fails to occur during development.
Proper formation of the atrioventricular node
depends on transcription factors that play reiterated roles during cardiac development. These factors include NKX2-5, TBX5, and GATA4.48 In mice,
Tbx5 and Gata4 regulate the expression of connexin 30.2, which is required for slow conduction
in the atrioventricular node.59 Haploinsufficiency
of Gata4 in mice causes a short PR interval.59 Mutation of NKX2-5, TBX5, or GATA4 has been associated with an atrial septal defect in humans as well
as in animal models.60-63 Hence, structural interference of conducting fibers by the septal defect
may not entirely explain the association between
atrial septal defects and conduction abnormalities.
Rather, a single underlying genetic defect may
affect septal closure and specialized conduction
cells independently.64 Indeed, patients with
NKX2-5 mutations can have isolated conduction
defects. These factors, and the cellular processes
that these factors regulate, may continue to play
important roles in conduction tissues throughout adult life. For example, progressive degeneration of the atrioventricular node and heart block
develop when the Nkx2-5 gene has been inactivated in adult mice.65 It seems likely that an association between certain risk alleles of this gene
or related genes will be found in and may
predict heart block in elderly patients.

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C a r diova scul a r M at ur at ion

At the time of birth, the cardiovascular system undergoes a series of abrupt and critical changes.
Blood must be diverted to the lungs for oxygenation, and the portal circulation must perfuse the
liver when enteric feeding begins. Increased oxygen tension associated with a babys first breaths,
coupled with withdrawal from exposure to maternal prostaglandins, stimulates closure of the ductus arteriosus, sending blood from the right ventricle to the lungs and establishing the parallel
pulmonary and systemic circulations. The foramen
ovale of the atrial septum closes. The ductus venosus constricts, sending portal blood to the liver.
Less obvious but equally important changes
occur in myocardial cells during the neonatal period. There is a shift in gene-expression profiles
within the heart; many fetal isoforms of genes
become down-regulated or replaced by their adult
counterparts. Examples include genes encoding
contractile components of the sarcomere, calciumhandling machinery, energy-utilization enzymes,
and natriuretic factors.66-68 Re-expression of fetal genes occurs in nearly every form of heart
failure in adults, and this phenomenon is thought
to contribute to the progression of heart failure.
Elucidation of the mechanisms that regulate the
re-expression of fetal genes during disease states
may reveal new targets for the treatment of heart
failure. In addition, an improved understanding
of the genetic programs governing myocyte maturation should inform the development of regenerative treatments; the use of such treatments
for cardiac disease has been hampered by a limited ability to engineer fully mature adult cardiac
myocytes from progenitor cells.
Our knowledge of the mechanisms that regulate gene expression has increased considerably
in recent years. Gene expression requires specific transcription factors proteins that activate or inhibit transcription from genomic DNA
to messenger RNA (mRNA) by binding to promoter or enhancer regions of genes. Changes in
DNA packaging that are effected by chromatin
and the enzymatic modification of histones, the
principal protein component of chromatin, also
influence gene expression through a mechanism
termed epigenetic modification. This mechanism
regulates gene expression by affecting the enzymatic acetylation of histones (and causing other
chemical modifications) and triggering the unwinding of chromatin (which exposes actively
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transcribed loci); it also represses gene transcription by enzymatically deacetylating histones and
condensing chromatin. Epigenetic control of chromatin structure, a mechanism that mediates global changes in gene-expression programs, is critical
to cellular reprogramming (the directed alteration in which one cell type is changed to another
e.g., a fibroblast becomes a pluripotent stem
cell) and to the determination of cell fate.
There are indications that the activities of specific histone deacetylase enzymes are necessary
for regulation of the expression of the fetal gene
program in the heart during development and
that these enzymes are involved in heart failure
in adults. Genetic inactivation of histone deacety
lase 2 in mice, for example, decreases the expression of fetal cardiac genes and inhibits reactivation of the fetal gene program in situations
involving cardiac stress in adults.69 Chemical inhibitors of this enzyme, now being studied in
phase 3 clinical trials for the treatment of certain cancers, can prevent reactivation of the fetal
gene program, cardiac hypertrophy, and heart
failure in animal models (e.g., ClinicalTrials.gov
numbers NCT00773747 and NCT01023308).70-75
These results suggest that epigenetic mechanisms
regulate the transition of fetal cardiac gene programs to adult programs and that these mechanisms could be targets for new treatments of
heart failure.
MicroRNA (miRNA) molecules are short, single
strands of RNA that modulate gene expression
by binding to complementary regions in mRNA
transcripts. The miRNA genes in chromosomal
DNA can be expressed as independent genes under the control of their own promoters or can be
coexpressed with other genes in which they are
embedded. In mice, the fetal heart expresses betamyosin heavy chain (-MHC), whereas the adult
heart expresses alpha-myosin heavy chain (-MHC).
In adult mice, cardiac stressors such as pressure
overload or chronic adrenergic stimulation induce re-expression of -MHC and suppression of
-MHC.66 The basis of this reciprocal regulation
is the embedding of a regulatory miRNA gene in
the intron of the genes for each of the two MHC
isoforms.76 The expression of -MHC results in
coexpression of miR-208 (a cardiac-specific miRNA
gene), which, in the presence of stress-induced
signals, indirectly activates transcription of -MHC
(Fig. 4). In the absence of miR-208, stress signals
fail to activate -MHC and other fetal genes, and
both the hypertrophic response and associated

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Fr anklin H. Epstein Lecture

Figure 4. MicroRNA-Mediated Regulation of Myosin Heavy-Chain Gene Isoform Expression.

MicroRNA-208 (miRNA-208), which is embedded within an intron of the gene encoding alpha-myosin heavy chain
(-MHC), indirectly regulates the expression of beta-myosin heavy chain (-MHC) in response to stress-induced
signals. AAA represents the polyadenylated tail of messenger RNA; LA denotes left atrium, LV left ventricle, RA right
atrium, and RV right ventricle. Adapted from a drawing provided courtesy of Dr. E. Olson.

fibrosis are blunted. The possibility of targeting only partially known, and specific stages of the
miRNA genes with therapeutic agents is an progressive lineage restriction of these cardiac
progenitors require further characterization. The
emerging area of investigation.77-79
ability to expand cardiac progenitor populations,
either in situ or ex vivo, and to direct cell fate
Sum m a r y
will have important implications for regeneraRecent studies have revealed a surprising num- tive cardiovascular therapies. The cell biology of
ber of previously unappreciated aspects of car- myocyte maturation and the effects of the trandiac morphogenesis that are relevant to both sition from the fetal heart to the adult heart recongenital and adult cardiovascular disease. It main areas of investigation that are likely to
is now clear that cell populations extrinsic to inform our understanding of heart failure. Studthe primary heart field and the linear heart tube ies of the regulation of gene-expression programs
of the embryo contribute to the development of in the heart are likely to suggest new therapeuthe mature heart and modulate cardiac mor- tic targets for cardiovascular disease.
phogenesis. These cell populations include neuSupported by a grant from the National Institutes of Health
ral-crest cells, the cells arising from a second (U01 HL100405), a DeHaan Cardiac Myogenesis Award from the
heart field, and epicardial cells. The full devel- American Heart Association, and the W.W. Smith endowed chair
for cardiovascular research.
opmental potential and unique defining characDisclosure forms provided by the author are available with the
teristics of various cardiac progenitor cells are full text of this article at NEJM.org.
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