Clinical Anatomy 22:36–46 (2009)

REVIEW

Autonomic Innervation of the Developing Heart:

Origins and Function
VICTORIA HILDRETH,* ROBERT H. ANDERSON, AND DEBORAH J. HENDERSON
Institute of Human Genetics, Newcastle University, Centre for Life, Central Parkway,
Newcastle upon Tyne, United Kingdom

Maintenance of homeostatic circulation in mammals and birds is reliant upon

autonomic innervation of the heart. Neural branches of mixed cellular origin
and function innervate the heart at the arterial and venous poles as it
matures, eventually coupling autonomic output to the cardiac components,
including the conduction system. The development of neural identity is con-
trolled by specific networks of genes and growth factors, whereas functional
properties are governed by the use of different neurotransmitters. In this
review, we summarize briefly the anatomic arrangement of the vertebrate au-
tonomic nervous system and describe, in detail, the innervation of the heart.
We discuss the timing of cardiac innervation in the chick and mouse, empha-
sizing the relationship of the cardiac neural networks to the anatomical struc-
tures within the heart. We also discuss the variable contribution of the neural
crest to vagal cardiac nerves, and summarize the main neurotransmitters
secreted by the developing sympathetic and parasympathetic autonomic
divisions. We provide an overview of the main growth factor and gene fami-
lies involved in neural development, discussing how these factors may
impact upon the development of cardiac abnormalities in congenital syn-
dromes associated with autonomic dysfunction. Clin. Anat. 22:36–46,
2009. V 2008 Wiley-Liss, Inc.
C

Key words: cardiac development; neurons; neural crest; cardiac plexus;

cardiac ganglia; neurotransmitters

ORGANIZATION OF THE VERTEBRATE Sympathetic efferent nerves containing pregan-

NERVOUS SYSTEM
The vertebrate nervous system is divided into two
major components: the central nervous system, con-
sisting of the spinal cord and brain, and the periph-
eral nervous system, which can be subdivided into
somatic and autonomic components. The autonomic
division of the peripheral system exerts functional
control over internal organs by way of efferent nerve
fibers originating in the central system. Sympathetic
and parasympathetic efferent nerves generate com-
plementary motor outputs in response to sensory
inputs directed to the brain from internal organs via
mechanoreceptive and chemoreceptive afferent
neurons. Both divisions of the autonomic system use
preganglionic to postganglionic relays, although
they differ in their organization and biochemical
properties.
glionic neurons arise segmentally from the central
portion of the spine, and terminate in sympathetic
ganglia, which are organized into two bilateral chains
that run parallel to the spinal cord. Here, they syn-
apse with postganglionic sympathetic neurons that
travel to the effector organ (Fig. 1). These neurons,
in the case of the heart, serve to increase heart rate
and contraction. The main neurotransmitter of the
sympathetic nervous system is norepinephrine,
*Correspondence to: Victoria Hildreth, Institute of Human Genet-
ics, Newcastle University, Centre for Life, Central Parkway, New-
castle upon Tyne, United Kingdom.
E-mail: Victoria.Hildreth@ncl.ac.uk
Received 27 March 2008; Accepted 2 July 2008
Published online 8 October 2008 in Wiley InterScience (www.
interscience.wiley.com). DOI 10.1002/ca.20695

C 2008
V Wiley-Liss, Inc.

Fig. 1. Innervation of the heart. The heart is inner-
vated by sympathetic (dark grey) and parasympathetic
(black) efferent nerves that originate from the spinal cord
and brain stem respectively. Afferent nerves (dashed
lines) also utilize these pathways to relay sensory inputs
from the heart to the brain. Pre- to post-ganglionic sym-
pathetic transfer occurs in the sympathetic ganglion
chain adjacent to the spinal cord. Post-ganglionic sympa-
thetic neurons travel to the cardiac plexus, where pre-
ganglionic parasympathetic neurons synapse to post-
ganglionic elements. From the cardiac plexus, autonomic
nerves can synapse with intrinsic cardiac neurons (light
grey), located within the network of cardiac ganglia.
although acetylcholine is used by sympathetic post-
ganglionic neurons.
Postganglionic efferent nerves of the parasympa-
thetic division differ in that they arise from the brain
stem and spinal cord and synapse with post-
ganglionic neurons located near to, or within, the
effector organ (Fig. 1). Acetylcholine is the main
neurotransmitter used in the preganglionic and post-
ganglionic neurons of the parasympathetic system,
with parasympathetic activity slowing the heart rate
and reducing the force of contraction.
Sensory afferent pathways involve both sympa-
thetic and parasympathetic nerves to relay informa-
tion from the organs to the brain (Fig. 1). Cardiac
afferent pathways made up of parasympathetic
nerves are responsible for controlling sympathetic
and parasympathetic output, while afferents associ-
ated with sympathetic nerves do not influence auto-
Autonomic Innervation of the Developing Heart 37
nomic output in the heart, and are thought to have
alternative functions. The majority of sensory
impulses are relayed to motor neurons within the
central system. Parasympathetic afferent pathways
transverse the nodose ganglion, connecting to effer-
ent pathways via interneurons located within the
brainstem. Sympathetic afferent nerves travel
through the stellate ganglia of the sympathetic
chain, and connect with motor neurons in the spinal
cord. Sympathetic afferent reflex responses can also
occur through direct interactions with motor nerves
in the stellate ganglia, while it is thought that some
sensory afferent pathways are intrinsic to the heart
and can direct reflex responses through sympathetic
and parasympathetic neurons and interneurons
located in the network of cardiac ganglia.
INNERVATION OF THE HEART
Parasympathetic activity in the heart is controlled
by the vagus nerve, which originates in the medulla
oblongata. In the human, it is the superior, inferior,
and thoracic branches of the vagus nerve that inner-
vate the heart (Kawashima, 2005). In the chick, car-
diac branches of the developing right and left para-
sympathetic vagal nerves pass along the arteries
within the 6th pharyngeal arches before entering
into the arterial pole of the heart between the aorta
and pulmonary trunk (Kuratani and Tanaka, 1990;
Verberne et al., 1998). Staining with an antibody
against HNK1, a carbohydrate moiety originally iden-
tified on human natural killer cells (Abo et al., 1982),
has shown that innervation of the outflow tract takes
place at stage 29 of Hamburger and Hamilton (HH).
More caudally, positive HNK1 staining is first
observed around the 24th stage in the dorsal meso-
cardium, reaching the systemic venous sinus by the
26th stage, and forming the cardiac sinal branch of
the vagus nerve (Kuratani and Tanaka, 1990; Ver-
berne et al., 1998). Between the 28th and 29th
stages, further branches from the vagus extend to-
ward the arterial and venous pole, entering along
the arteries coursing through the 3rd and 4th pha-
ryngeal arches, and the right and left cardinal veins,
respectively (Verberne et al., 1998). In the mouse,
nerve cells are first seen in the dorsal mesocardium
at E10.5 (10.5 days after fertilization). This is
approximately equivalent to HH20 in the chick. Well-
developed nerve tracts can be identified using the
neurofilament marker NF160D that extend through
this region and reach the heart by E12.5 (HH24),
(Hildreth et al., 2008) (Fig. 2). As in the chick heart,
innervation of the outflow tract also occurs slightly
later in the mouse, with the first neural elements
first seen between the separating aorta and pulmo-
nary trunk at E11.5 (Hildreth et al., 2008) (Fig. 3).
Sympathetic innervation has been shown to occur
at later stages compared with parasympathetic
innervation in both avian and mammalian species
(Kirby et al., 1980; Shoba and Tay, 2000). Postgan-
glionic sympathetic neurons join the vagus nerve at
the nodose ganglion, and then use the established
vagal tracts to gain entry into the heart. Studies in
the chick using silver staining, histofluorescence, and
38 Hildreth et al.

nerve fibers in comparison to neuropeptide-Y immu-

noactivity in the rat (Shoba and Tay, 2000).

THE CARDIAC PLEXUS AND THE

INTRINSIC CARDIAC NEURAL SUPPLY
The nerves supplying the heart join in the cardiac
plexus, an accumulation of mixed neurons located

Fig. 2. Innervation of the venous pole of the heart in

the mouse. a,b: NF160D antibody staining reveals the
presence of neurofilaments in the dorsal mesocardium at
E10.5. c,d: By E12.5, neurofilaments are seen extending
into the heart through the dorsal mesocardium and pri-
mary atrial septum toward the atrioventricular cushions.
AVC, atrioventricular cushion; LA, left atrium; LV, left
ventricle; DM, dorsal mesocardium; PAS, primary atrial
septum; RA, right atrium; RV, right ventricle; SG, sym-
pathetic ganglion; V, vagus nerve branches.

immunohistochemistry with the sympathetic nerve

marker tyrosine hydroxylase have determined that
bilateral sympathetic nerves from the first thoracic
ganglia, along with branches from the superior, mid-
dle, and lower cervical ganglia, all contribute to car-
diac innervation (Kirby et al., 1980; Verberne et al.,
1999). In the human, superior, middle, and inferior
cardiac nerves provide the supply to the heart, col-
lectively arising from regions of the sympathetic
chain spanning the superior cervical to the inferior
cervical ganglia (Kawashima, 2005). Additional sym-
pathetic innervation is provided by nerves originating
from the region of the thoracic sympathetic trunk
below the inferior cervical ganglia (Kawashima,
2005). In the mouse, staining against tyrosine
hydroxylase and the parasympathetic neuron marker
vasoactive cholinesterase transporter protein
(VAChT) reveals the presence of sympathetic and
parasympathetic neurons within the main nerves
innervating the arterial and venous poles of the
heart at E11.5 and E12.5 (Hildreth et al., 2008)
(Fig. 3). Autonomic efferent innervation precedes
sensory innervation, as shown by the later appear-
ance of calcitonin gene-related peptide-positive
Fig. 3. At E11.5, NF160D staining reveals the pres-
ence of the first neurofilaments innervating the outflow
tract of the heart between the developing aorta and pul-
monary trunk (a). These nerves display parasympa-
thetic and sympathetic activity, as shown using antibod-
ies raised against VAChT (c) and TH (e). Nerves stained
by NF160D innervating the venous pole of the heart at
E12.5 (b), also show parasympathetic and sympathetic
activity (d, f), although the cardiac plexus displays a
notable reduction in TH staining (f) when compared
with VAChT staining (d), indicating a smaller proportion
of sympathetic nerves in the cardiac plexus at this
stage. Ao, aorta; CP, cardiac plexus; NF160D, neurofila-
ment160D; PT, pulmonary trunk; TH, tyrosine hydroxy-
lase; VAChT, vasoactive cholinesterase transporter pro-
tein; V, vagus nerve branches.
 Autonomic Innervation of the Developing Heart 39

believed to develop in response to cardiac innerva-

Fig. 4. a: At E14.5, parasympathetic nerves are
abundant in the cardiac plexus, as shown by VAChT
antibody staining. b: TH staining shows that sympa-
thetic nerves are present but are much reduced in the
cardiac plexus at the same stage, reflecting similar find-
ings at earlier embryonic stages. CP, cardiac plexus;
TH, tyrosine hydroxylase; VAChT, vasoactive cholines-
terase transporter protein; V, vagus nerve branches.
tion from migratory neural precursor cells that travel
along the established nerve tracts.
Recent studies have shown the importance of the
intrinsic cardiac ganglia in modulating relay between
extrinsic autonomic nerves and the nodal tissues of
the cardiac conduction system. Specifically, auto-
nomic inputs from the left and right vagal branches
are regulated to varying degrees by different cardiac
ganglia, revealing complex interlinking pathways in
the control of heart rate. These pathways have been
revealed in studies of atrial fibrillation, which can be
induced through electrical stimulation of autonomic
nerves or cardiac ganglia. (Patterson et al., 2005;
Scherlag et al., 2005a,b). A study in the dog (Hou
et al, 2007) revealed that the right and left vagal
trunks exert sympathetic control over heart rate, but
that both inputs are modulated through specific,
interlinking pathways, with these pathways differing
between each node (Fig. 6). There is, however, a

cranial and dorsal to the heart (Fig. 1). In the human

heart, left and right-sided cardiac plexuses surround
the brachiocephalic trunk and the aortic arch,
respectively, and form part of a larger cardiac plexus
that lies between the aorta and pulmonary trunk
(Kawashima, 2005). Immunohistochemical staining
against VAChT has shown that parasympathetic neu-
rons are abundant the murine cardiac plexus at
E12.5 and later stages, with a smaller number of
sympathetic neurons present, visualized with anti-
body staining to tyrosine hydroxylase (Hildreth
et al., 2008) (Figs. 3 and 4). The cardiac plexus also
contains sensory afferent elements associated with
these autonomic neural components (Crick et al.,
2000). From the cardiac plexus, the majority of au-
tonomic neurons travel between the aorta and pul-
monary trunk, extending along the coronary arteries
before branching to innervate the atria and ven-
tricles (Kawashima, 2005). Neurons can innervate
the heart directly, or synapse with intrinsic inter-
neurons located in cardiac ganglia that are distri-
buted within the neuronal network of the heart
(Fig. 5). Cardiac ganglia differ in number and distri-
bution between species, but are mostly found within
the subepicardial layer. In mammals, the cardiac
ganglia are predominantly located around the atria
at the roots of caval and pulmonary veins, found
particularly within the infolded atrial walls often
described inappropriately as the ‘‘septum secun-
dum,’’ this area being better described as the supe- Fig. 5. Schematic representation of the locations of
rior interatrial fold. From these ganglia, nerves cardiac ganglia in the human heart. a: Cross sectional
extend to reach the sinus and atrioventricular nodes view of the heart showing cardiac ganglia (white circles)
of the conduction system (Armour et al., 1997; Bap- distributed throughout the atria. Ganglia can be found
tista and Kirby, 1997). In the human, it is estimated near the nodal tissues of the conduction system and
that there are, on average, around 700–900 cardiac around the entry points of the pulmonary veins and
ganglia, contained within seven separate subplex- caval veins. b: Rostral view of the heart showing cardiac
uses located in different regions of the heart. The left ganglia clustered around the aorta, pulmonary trunk,
atrium contains three of the subplexuses. Around and pulmonary veins, as well as the superior caval vein.
half of all the cardiac ganglia are located on the dor- Ao, Aorta; AVN, atrioventricular node; ICV, inferior
sal and dorsolateral surface of this chamber (Pauza caval vein; LA, left atrium; LV, left ventricle; PT, pulmo-
et al., 2000). Morphologically distinct at stage 35 in nary trunk; PV, pulmonary veins; RA, right atrium; RV,
the chick (Verberne et al., 1998), the cardiac ganglia right ventricle; SCV, superior caval vein; SN, sinus
and their associated postganglionic fibers are node.
40 Hildreth et al.
Fig. 6. Schematic drawing showing variable modu-
lation of sympathetic (vagal) control of sinus node and
atrioventricular node rate by three cardiac ganglionated
plexuses in the heart. Control of the sinus node (grey
arrows) predominantly occurs through the anterior right
ganglionated plexus, which receives similar inputs from
the right vagal branch and the left vagal branch directly,
and via the superior left ganglionated plexus. Control of
the atrioventricular node (dashed arrows) predomi-
nantly occurs through the inferior right ganglionated
plexus. The majority of input from the left and right
vagal branches is directed through the superior left gan-
glionated plexus to the anterior right ganglionated
plexus before modulation by the inferior right ganglion-
ated plexus. ARGP, anterior right ganglionated plexus;
AVN, atrioventricular node; IRGP, inferior right gangli-
onated plexus; LV, left vagus nerve; RV, right vagus
nerve; SLGP, superior left ganglionated plexus; SN,
sinus node. Figure adapted from Hu et al. (2007).
degree of variation in these pathways between dif-
ferent individuals. Failure completely to attenuate
the autonomic responses following ablation of these
ganglia suggested that the modulation involves other
ganglia within the cardiac network (Hou et al.,
2007).
CARDIAC INNERVATION
AND THE NEURAL CREST
The majority of the autonomic nervous system is
thought to arise from the neural crest (Fig. 7), with
additional contributions from other sources to the
glia and a proportion of the neurons of the sensory
ganglia (D’Amico-Martel and Noden, 1983). Origins
of parasympathetic nerves from the neural crest
were first suggested in early ablation studies, when
disrupted cardiac innervation was observed in addi-
tion to defects within the developing outflow tracts
(Kirby and Stewart, 1983; Kirby et al., 1985). Using
chimeral and retroviral methods in the chick, it was
shown that the precursors gained entry to the heart
by migration along the arterial and sinal branches of
the vagus nerve (Kirby and Stewart, 1983; Poel-
mann et al., 1998; Verberne et al., 1998, 2000). The
advent of reliable systems for marking the neural
crest has since confirmed that the cardiac ganglia
also take their origin from the neural crest in mam-
mals (Waldo et al., 1999; Pietri et al., 2003).
Compensation from other parts of the neural crest
has been suggested for the limited loss of parasym-
pathetic activity in response to ablation of the car-
diac component of the crest in the chick (Kirby et al.,
1987). In embryos where the cardiac neural crest
has been ablated; however, chimeral marking has
shown that parasympathetic neurons persisting in
the heart originate from the nodose placode, empha-
sizing the ability of nonneural crest-derived precur-
sors to form functional cholinergic neurons within the
heart (Kirby, 1988). Wnt1-cre lineage tracing of neu-
ral crest cells, in combination with NF160D staining,
in the mouse has shown that the vagal nerves con-
tain a large proportion of nonneural crest-derived
neurons in addition to neurons of neural crest cell or-
igin, supporting a role for neurons derived from
sources other than the neural crest in parasympa-
thetic relay (Hildreth et al., 2008) (Fig. 8). In the
cranial region, sensory ganglia within the vagus
nerve are also of mixed origin, containing neurons
derived from the neural crest and the nodose pla-
code (D’Amico-Martel and Noden, 1983).
Sympathetic cardiac innervation is thought to
originate from more caudal levels of the neural crest
than do the parasympathetic nerves (Kirby and
Bockman, 1984; Kirby et al., 1987). The loss of sym-
pathetic innervation is accompanied by an expansion
of the parasympathetic population, suggesting a
possible compensatory effect (Kirby et al., 1987). In
the Splotch2H mouse, development of the vagal
nerves, cardiac plexus, and innervation at the ve-
nous pole of the heart appears normal (Fig. 9), de-
spite dramatic reductions in the number of cardiac
neural crest cells reaching the outflow tract, support-
ing a possible alternative origin for sympathetic and
parasympathetic elements at the venous pole (Hil-
dreth et al., 2008).
NEUROTRANSMITTERS IN CARDIAC
INNERVATION
Regulation of cardiac function by the autonomic
nervous system plays crucial roles in the response of
the organism to external stimulation. The sympa-
thetic nervous system increases heart rate and the
force of contraction via effects on the function of the
sarcoplasmic reticulum within the cardiomyocytes
and on ion channel activity. The parasympathetic
system, in contrast, counteracts the action of the

Fig. 7. Neural crest contributions to the heart. The
neural crest (dark blue) is divided into cranial, cardiac,
vagal, trunk, and sacral segments. Cells from the cardiac
division of the neural crest, located between the otic vesi-
cle and the 3rd somite, migrate to the heart via the 3rd,
4th, and 6th pharyngeal arches and are involved in out-
flow tract septation and arch artery remodeling. Neural
crest cells also contribute to the formation of major auto-
nomic nerves as well as the cardiac plexus and the car-
diac ganglia. Vagus nerve formation and parasympa-
thetic activity is disturbed when the cardiac neural crest
is ablated, and cardiac ganglia are reduced in size and
number, suggesting overlap between the vagal and car-
diac divisions. Cells from the trunk neural crest contrib-
ute to the formation of sympathetic nerves.
Autonomic Innervation of the Developing Heart 41
Fig. 9. Neural crest cell derivatives at the venous
pole of the Splotch2H (Sp2H) mouse. a: Wnt1-cre neural
crest cell lineage tracing in the Sp2H heterozygote
mouse, which undergoes normal development, reveals
a large contribution of neural crest cells in the cardiac
plexus and in the nerves innervating the venous pole of
the heart at E13.5 (blue). b: In the Sp2H homozygote
mutant mouse, which is null for the Pax3 gene and
shows reduced numbers of neural crest cells at the arte-
rial pole of the heart in addition to cardiovascular
defects, neural crest cell contributions to the cardiac
plexus and cardiac innervation appear similar to that of
the control at the venous pole, indicating that the neural
crest origin of nerves in this region may differ from
those innervating the arterial pole. CP, cardiac plexus;
V, vagus nerve branches.
sympathetic nerves, slowing the heart rate. Under
normal conditions, the balance between these two
opposing systems is well maintained.
The local environment plays an important role in
determining the specific neurotransmitter used by
presumptive autonomic postganglionic neurons.
Most sympathetic neurons express norepinephrine as
a primary neurotransmitter, along with a range of
peptides that modulate signaling. Norepinephrine
binds to a range of adrenergic receptors, broadly
classified as a and b. Under normal circumstances, a
high affinity b-adrenoceptor binds to multiple G pro-
teins, activating adenylyl cyclase to produce cAMP.
This then improves excitation-contraction coupling
within the cardiac muscle, increasing heart rate and
force. Norepinephrine is produced from its precursor,
Fig. 8. Contributions to cardiac innervation from
the neural crest and the nodose placode. a: Cells from
the hindbrain region of the cranial neural crest, the car-
diac neural crest, and the vagal neural crest contribute
to the formation of parasympathetic nerves and the car-
diac ganglia, as well some sensory nerves. Precursors
from the trunk neural crest form sympathetic nerves
that innervate the heart. The ectodermal nodose pla-
code also gives rise to sensory neural elements, and
may also contribute to the formation of parasympa-
thetic cardiac nerves. b: Wnt1-cre neural crest cell line-
age tracing in the embryonic mouse reveals that the
vagus nerve contains a mixed population of cells. A pro-
portion of the vagus nerve is derived from the neural
crest (blue), but many neurofilaments (brown, NF160D
antibody) do not stain blue, indicating an alternative or-
igin. These elements may be placodally derived. NP,
nodose placode.
42 Hildreth et al.
L-tyrosine, by the action of three enzymes, tyrosine
hydroxylase, dopamine b-hydroxylase, and phenyle-
thanolamine N-methyl transferase, all found within
the cell body of sympathetic neurons. Once pro-
duced, the norepinephrine is transported along the
axon to nerve terminals where it is stored. In all spe-
cies studied to date, maturation of the sympathetic
innervation and onset of function occurs late in the
fetal, or early in the neonatal, heart. Despite this,
response to catecholamines, in the form of tachycar-
dia, occurs well before birth, suggesting that b-adre-
nergic receptors must be present during the fetal
period, perhaps as early as E9.5 in the mouse (Liu
et al., 1999). Indeed, although few tyrosine hydrox-
ylase-positive neurons are found in the heart until
late in gestation, myocardial cells expressing tyro-
sine hydroxylase, dopamine b-hydroxylase, and phe-
nylethanolamine N-methyl transferase are found
interspersed throughout the myocardium in the
mouse early in its gestation. By mid-gestation, they
have localized to the regions of formation of the
sinus and atrioventricular nodes (Ebert and Thomp-
son, 2001). These data are supported by the finding
that, following sympathectomy, tyrosine hydroxylase
activity can still be detected in the chick heart
(Stewart and Kirby, 1985). Although the roles of
these myocardially produced catecholamines remain
unclear, the fact that the heart can respond to these
factors from early in gestation suggests that they
may be playing an important role.
A wide range of cotransmitters are found within
the sympathetic nervous system, functioning along-
side norepinephrine. Although the mechanisms
underlying the specification of these cotransmitters
remain unclear, cell lineage plays an important role.
Of these cotransmitters, the adenosinergic system,
using adenosine, is the earliest functionally respon-
sive system in the rodent heart, with activation of
the adenosine receptor slowing the heart rate from
E8 in the rat embryo (Porter and Rivkees, 2001).
Adenosine acts to inhibit norepinephrine release
from sympathetic nerve endings and is important
both as a vasodilator and for its anti-arrhythmic
properties. Similar to catecholamines, cardiac adeno-
sine receptors are functional well before sympathetic
innervation occurs. It has been suggested that fetal
plasma may be the source early in gestation (Sawa
et al., 1991). Neuropeptide Y is the most abundant
peptide in the heart, being found in postganglionic
sympathetic neurons synapsing on postganglionic
parasympathetic neurons in the cardiac ganglia, en-
docardium, and myocardium (Palmiter et al., 1998).
It has been shown to inhibit release of catechol-
amines and regulate the vasoconstrictor action of
norepinephrine. Neuropeptide Y has also been shown
to produce vasoconstriction of coronary vessels in
many species, including human (Michel et al., 1989).
Neuropeptide Y is first seen late in development in
the region of the sinus and atrioventricular nodes
and in the intrinsic cardiac ganglia. Levels increase in
the immediate postnatal period, and then remain
steady thereafter. Some studies have suggested that
the peptide can produce long-term inhibition of the
responses to vagal stimulation in the sinus and atrio-
ventricular nodes (Potter, 1987). Neuropeptide Y has
been suggested to play a crucial role as a neuromo-
dulator in the complex interactions that take place
between different branches of the sensory and auto-
nomic innervation of the heart (reviewed in Crick
et al., 2000).
Acetylcholine is the main neurotransmitter
released by preganglionic and postgangionic vagal
nerve terminals. Two types of cholinergic receptor
have been described, namely muscarinic and nico-
tinic. Muscarinic receptors are the main type found
on cardiac effector cells, whereas nicotinic receptors
are found on postganglionic neurons. Acetylcholine is
mainly synthesized at the nerve endings where it is
stored until release (Loffelholz and Pappano, 1985).
Acetycholine can generally be detected in the heart
at an earlier stage than can norepinephrine, reflect-
ing the earlier onset of parasympathetic innervation
than sympathetic ingrowth (Pappano, 1977).
Vasoactive intestinal peptide is a peptidergic
cotransmitter in cholinergic parasympathetic neu-
rons. Neurons expressing this peptide are localized
primarily in relation to blood vessels in microvascular
beds (Della et al., 1983), and to the sinus and atrio-
ventricular nodes and coronary vessels (Weihe et al.,
1984). The peptide has direct effects on heart rate.
In the dog, it has been reported to be twice as
potent as norepinephrine in increasing heart rate
(Rigel, 1988). It has also been suggested to modu-
late the activity of acetylcholine and norepinephrine
(Ferron et al., 1985).
Somatostatin immunoreactivity is associated with
postganglionic parasympathetic neurons, localizing
to nerve fibers in the myocardium, endocardium,
and the conduction system. The actions of somato-
statin, slowing the heart rate and decreasing cardiac
output, closely resemble those caused by vagal stim-
ulation. It exerts its suppressing cardiac effects by
acting on calcium channels excited by b-adrenocep-
tor activity (Diez et al., 1985) and/or by inducing the
release of acetylcholine from intracardiac parasym-
pathetic neurons (Wiley et al., 1989), but may also
modulate sympathetic neurotransmission by acting
at a postjunctional site.
The neurotransmitters substance P and calcitonin
gene-related peptide act on the heart via the sen-
sory-motor pathway. Both transmitters are stored in
granules within the nerve terminal, are coreleased
on stimulation, and interact with parasympathetic
nerves (Armour et al., 1993). Substance P-contain-
ing nerve fibers are found surrounding blood vessels
within the myocardium, and within the sinus and
atrioventricular nodes (Crick et al., 1994). Substance
P, a potent vasodilator, although it has no effect on
heart rate, may control parasympathetic activity
(Corr, 1992). Calcitonin gene-related peptide is
scarce in the human heart (Crick et al., 1994). It is a
potent vasodilator of peripheral blood vessels,
including the coronary arteries. In the mouse, it is
expressed in sensory nerves within the heart,
appearing around the time of birth. These, with other
data, suggest that the sensory innervation of the
heart occurs later than autonomic innervation
(Gordon et al., 1993).

The myocardium also secretes endocrine factors
that regulate the cardiac nervous system. The two
most abundantly expressed proteins are the atrial
and brain natriuretic peptides, which exert an inhibi-
tory effect on sympathetic input to the heart. These
factors are mainly expressed in the atria, although
they are also expressed in other regions of the heart.
They have additional roles, such as diuretic and na-
triuretic homeostatic function in the kidney, and in-
hibitory effects on the renin-angiotensin-aldosterone
system (McGrath et al., 2005).
A number of other nonadrenergic noncholinergic
transmitters have been localized within the heart of
various species, including opioids, neurotensin, and
peptide histidine isoleucine, albeit that their actions
remain poorly defined. They will not be discussed
further here. For a more detailed review of these
transmitters, see Crick et al. (2000).
GENES AND GROWTH FACTORS
REGULATING NEURAL DEVELOPMENT
Development of the peripheral nervous system is
reliant upon the interactions of several genes and
growth factor families. The neurotrophin family of
growth factors are particularly important, influencing
autonomic and sensory neurogenesis and function
(Huang and Reichardt, 2001). Nerve growth factor
(NGF), brain-derived neurotrophic factor (BDNF),
and neutrophins 3 and 4 (NT3 and NT4) make up the
family, acting through the high-affinity Trk receptor
tyrosine kinases A, B, and C, and lower-affinity neu-
rotrophin receptor p75 (p75NTR).
NGF primarily acts through TrkA, and promotes
growth, survival, and maintenance of sympathetic
nerves (Crowley et al., 1994; Ieda et al., 2006). It is
expressed by cardiomyocytes, and its overexpres-
sion in the hearts of mice results in hyperinnervation
(Hassankhani et al., 1995). Its function is independ-
ent of its role in neuronal survival (Glebova and
Ginty, 2004). BDNF also supports the survival of a
subset of sensory neurons and is released by sympa-
thetic nerves (Causing et al., 1997; Kohn et al.,
1999; Jahed and Kawaja, 2005).
Like NGF, NT3 is implicated in the development
and maintenance of sensory and sympathetic neural
elements before birth, and for sympathetic innerva-
tion after birth (Farinas et al., 1994; ElShamy and
Ernfors, 1996a,b; ElShamy et al., 1996). NT3 is also
thought to act as a target-derived growth factor in
mature neurons (Zhou et al., 1997, Zhang and Rush,
2001). The increase in sensory ganglion loss in
TrkC / mice in comparison to NT3 / mice sug-
gests that this growth factor signals through other
receptors during neural development (Tessarollo
et al., 1997).
The glial cell line-derived (GDNF) family of neuro-
receptors are also implicated in neural development,
signaling through a complex of RET tyrosine kinase
and GDNF family (GFR)a receptors (Hiltunen et al.,
2000; Mabe et al., 2006). Both are required for nor-
mal parasympathetic innervation of the organ (Hil-
tunen et al., 2000). In Ret / mice, migration of
Autonomic Innervation of the Developing Heart 43
sympathetic neuron precursors is disturbed (Eno-
moto et al., 2001) Normal control of these precur-
sors may be controlled by the activity of the GDNF
family member artemin, expressed outside of the
nervous system (Enomoto et al., 2001).
Another growth factor known to affect neural de-
velopment is neuregulin-1 (NRG1), which signals
through heterodimers of ErbB tyrosine kinase recep-
tors, principally ErbB2/ErbB3 or ErbB2/ErbB4 (Car-
raway and Cantley, 1994). Studies on the develop-
ment of the sympathetic ganglia in NGR1 / ,
ErbB2 / , and ErbB3 / mice also reveal a critical
role for NGR1 in the migration of neural crest cells
destined to develop a sympathetic neuronal pheno-
type (Britsch et al., 1998).
Basic helix-loop-helix (HLH) genes are important
in the early determination of neural cell fate and
neural identity. In the central and peripheral compo-
nents of the nervous system in mammals, the HLH
gene Mash1, and another HLH gene neurogenin-1
(Ngr-1), are expressed in complementary fashion,
and promote the development of autonomic and sen-
sory neurons, respectively (Ma et al., 1996, 1998;
Lo et al., 1998, 1999). Mash1 is necessary for the
development of all sympathetic and parasympathetic
neurons and, in vitro, is expressed in neural crest
cells, which display neuronal commitment but not a
neuronal phenotype, suggesting that it promotes dif-
ferentiation of neuronal precursors rather than multi-
potent uncommitted cells (Sommer et al., 1995).
Proneural genes such as Mash1 also appear to have
their activities balanced by negative regulators dur-
ing normal neurogenesis. An example of a negative
regulator is Hes1. Hes1 null mice exhibit acceleration
of neural development, which is associated with the
upregulation of Mash1 and other proneuronal factors
(Ishibashi et al., 1995; Hatakeyama et al., 2004). It
inhibits the function of Mash1 at the protein level
(Sasai et al., 1992; Chen et al., 1997).
Early development of sensory lineages is con-
trolled by the neurogenin (Ngn) family (Ma et al.,
1999). Early lineage specification of multipotent neu-
ral crest cells is influenced by genes outside of the
HLH family such as Sry-related HMG box (Sox)10. In
vitro, Sox10 can prevent Tgf-b-induced smooth mus-
cle differentiation of neural crest progenitors in favor
of neuronal differentiation (Cheng et al., 2000;
Britsch et al., 2001; Kim et al., 2003).
CONGENITAL DISORDERS RELATED
TO ABNORMALITIES IN
CARDIAC INNERVATION
Dysautonomias are a group of diseases that result
from impaired autonomic interactions. The most well
known of these is familial dysautonomia (Riley-Day
syndrome; OMIM 223900). A decrease in the synthe-
sis of norepinephrine has been reported in patients
with familial dysautonomia, possibly due to reduced
levels of dopamine-beta-hydroxylase, the enzyme
that converts dopamine to norepinephrine. Some
dysautonomic children had no plasma dopamine-
beta-hydroxylase activity and their mothers had
44 Hildreth et al.
decreased activity (Goodall et al., 1971). In 2001, the
syndrome was shown to result from mutations in the
IKBKAP gene (Anderson et al., 2001; Slaugenhaupt et
al., 2001). Depletion has been associated with
reduced transcription of a number of genes and defec-
tive cell motility (Hawkes et al., 2002; Close et al.,
2006). Patients with familial dysautonomia have an
increased risk of sudden death and have abnormal or-
thostatic blood pressure, along with responses of
heart rate and cardiac tone. Sudden Infant Death
Syndrome (SIDS) has also been linked to abnormal
development of the autonomic nervous system. SIDS
is a multifactorial condition thought to be caused by
abnormalities in the control of the heart, breathing,
autonomic nervous system, and patterns of arousal
during sleep. Physiological studies suggest that there
may be a delay in the maturation of the autonomic
nervous system, which results in a decreased propen-
sity to arouse from sleep and a decreased sympatho-
vagal cardiac balance (Kahn et al., 2003).
CLINICAL SIGNIFICANCE OF
CARDIAC INNERVATION
From our review, it is clear that much more is
known about the development of the cardiac nerves,
and their related growth factors and gene products,
than is known about the specific distribution of the
autonomic nerves in the human heart. It is also
clear, nonetheless, that these autonomonic nerves
play a crucial role in multiple diseases, such as the
congenital malformations discussed earlier, and
other anomalies such as atrial fibrillation, also dis-
cussed previously. The role of disordered cardiac
innervation in syndromes such as cardiac failure,
however, remains to be elucidated. There is much to
be done, therefore, in matching the knowledge of
the distribution of the various elements of the auto-
nomic nervous system in clinical disease states to
that now existing for development of these nerves in
experimental animals.
SUMMARY
We have provided an account of autonomic and
sensory innervation in the vertebrate heart, empha-
sizing the importance of the intrinsic cardiac nerves
in the control of cardiac conduction. We have
emphasized the contribution of the neural crest to
the development of the autonomic nerves, along
with the roles of specifying genes, growth factors,
and neurotransmitters in the establishment of car-
diac innervation. Future research into these factors,
and the interactions between them, is imperative if
we are to elucidate the etiology of clinical disorders
associated with autonomic aberrations, and the
potential significance of abnormal aoutonomic inner-
vation in clinical cardiac disease.
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