The Journal of Physiology - 2016 - Habecker - Molecular and Cellular Neurocardiology Development and Cellular and

J Physiol 594.
14 (2016) pp 3853–3875 3853
WHITE PAPER
Neuroscience
Molecular and cellular neurocardiology: development,

and cellular and molecular adaptations to heart disease
Beth A. Habecker1 , Mark E. Anderson2 , Susan J. Birren3 , Keiichi Fukuda4 , Neil Herring5 ,
Donald B. Hoover6 , Hideaki Kanazawa4 , David J. Paterson5 and Crystal M. Ripplinger7
1
Department of Physiology and Pharmacology, Department of Medicine Division of Cardiovascular Medicine and Knight Cardiovascular Institute,
Oregon Health and Science University, Portland, OR 97239, USA
2
Johns Hopkins Department of Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21287, USA
3
Department of Biology, Volen Center for Complex Systems, Brandeis University, Waltham, MA 02453, USA
4
Department of Cardiology, Keio University School of Medicine, 35-Shinanomachi, Shinjuku-ku, Tokyo 160–8582, Japan
5
Burdon Sanderson Cardiac Science Centre, Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Road, Oxford OX1 3PT, UK
6
Department of Biomedical Sciences, Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine,
East Tennessee State University, Johnson City, TN 37614, USA
The Journal of Physiology
7
Department of Pharmacology, University of California, Davis, CA 95616, USA
Abstract The nervous system and cardiovascular system develop in concert and are functionally
interconnected in both health and disease. This white paper focuses on the cellular and molecular
mechanisms that underlie neural–cardiac interactions during development, during normal
physiological function in the mature system, and during pathological remodelling in cardio-
vascular disease. The content on each subject was contributed by experts, and we hope that this
will provide a useful resource for newcomers to neurocardiology as well as aficionados.
(Received 30 November 2015; accepted after revision 15 March 2016; first published online 6 April 2016)
Corresponding author B. A. Habecker: 3181 SW Sam Jackson Park Rd, Portland, OR 97239, USA.
Email: habecker@ohsu.edu
Abbreviations APD, action potential duration; β-AR, β-adrenergic receptor; AT II, angiotensin II; CaM, calmodulin;
CaMKII, calmodulin-dependent protein kinase II; CGRP, calcitonin gene related peptide; ChR2, channelrhodopsin-2;
ICG, intrinsic cardiac ganglia; NA, noradrenaline; nNOS, nitric oxide synthase; NGF, nerve growth factor; NPY,
neuropeptide Y; PKA, protein kinase A; RyR, ryanodine receptor; SHR, spontaneously hypertensive rat; SR, sarcoplasmic
reticulum; TH, tyrosine hydroxylase; VIP, vasoactive intestinal peptide.
Neural–cardiac interactions: co-maturation fibres innervate the developing heart (Hildreth et al. 2009).
in development This can be considered as a co-maturation system in
which signals from cardiac tissue regulate the growth,
Autonomic control of the heart occurs through trans- patterning and transmission properties of the innervating
mission by sympathetic and parasympathetic neurons, sympathetic neurons, while reciprocal signalling from the
which have a common neural crest origin. The anlagen nerve fibres influence the maturation of cardiac myocytes
of cardiac parasympathetic ganglia first appear around and adult cardiac properties.
the middle of embryonic development in rodents, and The role of cardiac target-derived factors as regulators
vagal input is already present as the immature neurons of neuronal maturation has been well established.
migrate to positions at the dorsal atrium (Hildreth Cardiac-derived neurturin is essential for the survival of
et al. 2009). Parasympathetic neurons and surrounding cardiac parasympathetic neurons (Hiltunen et al. 2000;
satellite glial cells mature postnatally in rodents, and the Mabe & Hoover, 2009), and cardiac signals control
neurons double in size as they develop connections with multiple events in sympathetic neuron maturation.
the myocardium (Fregoso & Hoover, 2012). The rodent Trophic signals that include nerve growth factor,
sympathetic–cardiac circuit is established in the late pre- neurotrophin-3, and cytokines regulate outgrowth of
natal and early postnatal period as growing sympathetic sympathetic axons within the heart, continued survival

C 2016 The Authors. The Journal of Physiology
C 2016 The Physiological Society DOI: 10.1113/JP271840
14697793, 2016, 14, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP271840 by Univ de Buenos Aires, Wiley Online Library on [14/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
3854 B. A. Habecker and others J Physiol 594.14
upon reaching the target (Chun & Patterson, 1977; cycle shortly after birth and, for the most part, remain
Landis & Keefe, 1983; Lockhart et al. 2000; Glebova & quiescent throughout an organism’s life. Following cell
Ginty, 2005), regulation of neurotransmitter phenotypes cycle withdrawal, heart size increases predominantly via
(Yamamori et al. 1989; Slonimsky et al. 2003), and cellular hypertrophy, or the enlargement of individual
synaptic transmission (Lockhart et al. 1997; Luther & cardiomyocytes (Li et al. 1996; Ahuja et al. 2007). Thus,
Birren, 2006, 2009; Hasan et al. 2012). Additional signals the number of postnatal cardiomyocyte divisions, and
from cardiac tissue regulate developmental transitions the timing of the developmental transition between
between different growth states in sympathetic neurons. proliferative and hypertrophic growth in the young animal
For example, contact with cardiac tissue results in a are significant factors for determining the total number of
change in the neuronal response to bone morphogenetic cardiomyocytes in the adult heart.
proteins (BMPs), providing a stop-growth signal for The rapid transition of cardiomyocytes from a
sympathetic axons that have reached their target (Moon & proliferative to a hypertrophic growth state (Li et al.
Birren, 2008). Interacting actions of neurotrophic factors 1996) corresponds to the postnatal period of sympathetic
(Habecker et al. 2008; Lorentz et al. 2010) and Sema3a ingrowth into the heart (Hildreth et al. 2009). Recent
signalling (Ieda et al. 2007) are also likely to act in the evidence has shown that neonatal sympathectomy
patterning of sympathetic fibres within the heart. Overall, results in smaller hearts in developing and adult rats,
a rich array of heart-derived signals drives the maturation demonstrating that early sympathetic innervation has
and function of the postganglionic sympathetic system. long-term effects on heart development (Kreipke &
While less is known of the effects of innervating Birren, 2015). In vitro experiments using neonatal
sympathetic fibres on cardiac development, the cardiomyocytes grown in the presence or absence
development of a number of cardiac properties are of innervating sympathetic fibres demonstrated that
regulated by sympathetic signals. Co-culture with sympathetic β-adrenergic signalling prolonged the period
sympathetic neurons regulates the expression of of postnatal cardiomyocyte proliferation, delaying the
angiotensin II and atrial natriuretic peptide (ANP) in period of hypertrophic growth. While, ultimately, the
developing cardiomyocytes (Hunt et al. 1995; Hansson size of individual cardiomyocytes was not altered in
et al. 2001). Additionally, sympathetic innervation has adult animals that were sympathectomized at birth,
been linked to functional alterations in a number the total number of these cells was reduced in the
of important cardiac ionic currents, including Na+ , sympathectomized animals (Kreipke & Birren, 2015). This
L-type Ca2+ , pacemaker, inward rectifier and transient work shows that early sympathetic signalling leads to the
outward K+ currents (Qu & Robinson, 2004). Recent development of larger hearts with more cardiomyocytes
studies also suggest that sympathetic interactions play in vivo and suggests that sympathetic signalling is part
an important developmental role in regulating cardio- of a homeostatic system that promotes cardiomyocyte
myocyte maturation during the postnatal period. proliferation and acts to set the final number of cardio-
myocytes in the adult heart.
Questions and controversies Sympathetic regulation of postnatal cardiomyocyte
proliferation may also have implications for heart
Can we exploit the fact that myocyte proliferation in regeneration following cardiac damage. The neonatal
pathological situations is influenced by the autonomic mouse heart undergoes regeneration following tissue
nervous system with new therapeutics or interventional damage, a transient capability that is greatly diminished
treatments in man? in older animals (Porrello et al. 2011; Bryant et al.
Is there a component of inherited structural and 2015). Recent studies have shown that disruption of
electrophysiological heart conditions that arises from peripheral nerves blocks this regeneration. In one study,
dysfunctional autonomic control during cardiac pharmacological inhibition of cholinergic signalling pre-
development? vented cardiomyocyte proliferation following neonatal
Does abnormal autonomic cardiovascular develop- resection of the mouse ventricle (Mahmoud et al. 2015).
ment contribute to human essential hypertension? This suggests a role for parasympathetic innervation in
heart regeneration, a model supported by the finding
One way that early sympathetic innervation regulates that vagal nerve ablation also decreased cardiomyocyte
cardiomyocyte development, and the properties of the proliferation following cardiac injury via resection or myo-
adult heart, is through the regulation of cell cycle cardial infarction (Mahmoud et al. 2015). Interestingly,
withdrawal and determination of cardiomyocyte cell another study showed that chemical sympathectomy
number (Kreipke & Birren, 2015). While some organs in young animals also blocked early regeneration of
are able to replenish cell number throughout life, the damaged ventricular tissue, while increasing scarring in
regenerative capacity of cardiomyocytes is, at best, limited the tissue (White et al. 2015). While additional work
(Zak, 1973). Cardiomyocytes withdraw from the cell defining the relative roles of cholinergic and noradrenergic

C 2016 The Physiological Society
J Physiol 594.14 Molecular and cellular neurocardiology 3855
signalling in the regeneration process is needed, together Parasympathetic nerves. Cardiac parasympathetic
this work defines a new role for cardiac innervation in neurons reside in intrinsic cardiac ganglia (ICG), which
regenerative processes. Genetic programmes associated have a diffuse distribution on the surface of the heart,
with proliferative cardiomyocytes are reactivated during are connected by an extensive plexus of nerve fibre
pathological hypertrophy (Komuro et al. 1988; Ahuja et al. bundles (O’Shea & Evans, 1985; Pauza et al. 2000; Ardell,
2007), suggesting that these reciprocal interactions have 2001; Hoover et al. 2004, 2009; Mabe et al. 2006), and
implications for later processes in the cardiac system. project to distinct regions of myocardium (Quan et al.
1999, 2002; Zarzoso et al. 2013). Cholinergic nerve fibres
are most abundant in sinoatrial and atrioventricular
Questions and controversies
nodes, atrial myocardium and the ventricular conducting
What are the best molecular targets to ensure optimal system (Hoover et al. 2004). Stimulation of cardiac
neural remodelling to promote myocyte survival and parasympathetic nerves projecting to these regions
minimize the risk of arrhythmia post myocardial leads to acetylcholine (ACh) release, which can evoke
infarction and during chronic heart failure? prominent bradycardia, negative dromotropic responses
and decreases in atrial contractility via activation of
postjunctional M2 muscarinic receptors. Cholinergic
innervation in less abundant in the ventricles, but studies
Normal neurotransmission and injury-induced
in humans and other large mammals have shown that
plasticity in cardiac nerves
these nerve fibres can trigger significant negative inotropic
The mature heart is densely innervated, with responses mediated by muscarinic receptors (DeGeest
parasympathetic, sympathetic and sensory neurons et al. 1965; Xenopoulos & Applegate, 1994; Ardell, 2001;
innervating regions of the myocardium and cardiac Lewis et al. 2001; Coote, 2013). Only two studies have
conduction system. Each of these populations of neurons evaluated vagal parasympathetic effects on ventricular
has a distinct distribution and phenotype that is critical to contractility in small mammals (Takahashi et al. 2003;
its function. Cardiac nerves undergo significant changes in Nalivaiko et al. 2010). While the first study failed to detect
morphology and phenotype in disease. Changes in growth a negative inotropic response to vagal stimulation, the
factor expression, oxidative stress, and inflammatory cyto- second study provided definitive evidence for a small
kines within the heart and vasculature contribute to neuro- decrease in contractility in a majority of experimental
nal remodelling. These issues have been reviewed in detail preparations. Based on the slow onset of these responses,
recently (Kimura et al. 2012; Fukuda et al. 2015; Gardner it was suggested that cholinergic inhibition might
et al. 2016), and will be summarized here briefly. occur indirectly though attenuation of noradrenaline
(norepinephrine) release and/or postjunctional signalling
Sensory nerves. Sensory innervation of the heart includes (Nalivaiko et al. 2010). Cardiac parasympathetic ganglia
both mechanosensitive and chemosensitive nerve endings integrate information from a variety of sources, including
(reviewed by Hainsworth, 1991; Schultz, 2001). Mechano- cholinergic preganglionic neurons (McAllen & Spyer,
sensitive afferents transduce pressure changes in the atria 1976; Hoover et al. 2004, 2009; Mabe et al. 2006),
and ventricles during the cardiac cycle, while chemo- presumptive noradrenergic nerve fibres (Leger et al. 1999;
sensitive nerve endings respond to endogenous substances Hoard et al. 2008; Hoover et al. 2009) and peptidergic
including adenosine and H+ and chemicals like capsaicin fibres (Calupca et al. 2000, 2001).
(Longhurst et al. 2001). Most of the sensory neurons Studies of human ICG have demonstrated that
innervating the heart reside in the nodose–petrosal ganglia detrimental changes can occur in cardiac disease.
and dorsal root ganglia, although some neurons within the Morphological studies of ganglia from ischaemic hearts
intrinsic cardiac ganglion are sensory afferents (Armour, revealed pathological changes in about 35% of the neurons
2008). Many cardiac sensory fibres that arise in dorsal evaluated (Hopkins et al. 2000). Abnormalities included
root ganglia produce nitric oxide synthase (nNOS), sub- various inclusions and vacuoles in somata, neuronal
stance P and/or calcitonin gene related peptide (CGRP), enlargement, loss of dendrites, and neuronal degeneration
distinguishing them from nodose–petrosal neurons which with the infiltration of phagocytes. Recently, another study
lack all three (Hoover et al. 2008). CGRP and substance P found significant hypertrophy of intrinsic cardiac neurons
levels are decreased in coronary artery disease and diabetes from patients with autopsy-confirmed heart failure (Singh
mellitus, and end stage heart failure (Taquet et al. 1992; et al. 2013). In contrast, mouse models of diabetes, which
Wang et al. 2012). The loss of neuropeptide content in have significantly impaired vagal control of the heart, do
diabetes corresponds with CGRP-immunopositive cardiac not exhibit any changes within cardiac parasympathetic
sensory denervation and atrophic changes in the dorsal neurons of the ICG (Mabe & Hoover, 2011). Decreased
root ganglia (Ieda et al. 2006). parasympathetic transmission in the heart, and its

role in cardiovascular pathology, is discussed further cholinergic transdifferentiation of cardiac adrenergic

in the section on ‘Neuromodulation and autonomic neurons into cholinergic neurons, which is observed in
imbalance’. animal models of heart failure and in autopsy specimens
from patients with heart failure (Kanazawa et al. 2010).
Sympathetic nerves. The sympathetic neurons Emerging evidence suggests that a similar cholinergic
innervating the heart reside predominantly in the transdifferentiation occurs transiently after acute myo-
stellate ganglia (Norris et al. 1974; Kostreva et al. cardial infarction (Olivas et al. 2016). Leukemia inhibitory
1977; Pardini et al. 1990) and produce the transmitter factor (LIF) and other members of the interleukin-6 (IL-6)
noradrenaline (NA) and the co-transmitter neuro- family, which can induce fetal gene expression (so-called
peptide Y (NPY). Sympathetic axons innervate the atria, rejuvenation) in adult cardiomyocytes, are upregulated
cardiac conduction system and ventricles, where they during heart failure (Kimura et al. 2007) and after myo-
stimulate increased heart rate (chronotropy), conduction cardial infarction (Aoyama et al. 2000; Gritman et al.
velocity (dromotropy), and contractility (inotropy) via 2006). IL-6 family cytokines secreted from the damaged
NA activation of β1 -adrenergic receptors. Sympathetic myocardium act as negative modulators of sympathetic
nerves are most dense in the atria and near the base of function by suppressing noradrenergic function (Kimura
the ventricles with fewer nerves near the apex of the et al. 2007; Parrish et al. 2009) and inducing cholinergic
heart. Stimulation of the stellate increases contractility differentiation (Kanazawa et al. 2010; Olivas et al. 2016)
throughout the heart (Szentivanyi et al. 1967), but a via a gp130 signalling pathway.
transmural gradient of axons accompanies an epicardial
to endocardial gradient in cardiac action potential Nerve growth factor. Many of the injury-induced
duration that is important for normal activation and changes in cardiac sympathetic and sensory nerves
repolarization of the left ventricle (Antzelevitch et al. are driven by alterations in cardiac nerve growth
1991; Nabauer et al. 1996; Brunet et al. 2004). Activation factor (NGF). NGF supports the development and
of cardiac β-adrenergic receptors (β-ARs) modulates maintenance of cardiac sympathetic axons as well as the
myocyte repolarization by altering transmembrane CGRP-immunoreactive cardiac sensory nerves that richly
currents and Ca2+ homeostasis (Thomas et al. 2004; Bers, innervate the epicardium and the ventricular myocardium
2008; Cutler et al. 2011), while it increases contractility (Ieda et al. 2006). Increased cardiac NGF is observed
by modulating calcium handling proteins so that Ca2+ after myocardial infarction (Zhou et al. 2004; Hasan et al.
release from the sarcoplasmic reticulum (SR) is increased 2006; Meloni et al. 2010; Gardner & Habecker, 2013) and
(Valdivia et al. 1995; Marx et al. 2000; Shannon et al. in compensated cardiac hypertrophy (Ieda et al. 2004;
2000). The effects of sympathetic stimulation allow myo- Kimura et al. 2007), where it is associated with regional
cytes to meet increased cardiac demands during stress sympathetic hyperinnervation (Cao et al. 2000b; Li et al.
or exercise, but changes to sympathetic transmission 2004b; Zhou et al. 2004; Hasan et al. 2006; Kimura et al.
in pathological conditions can contribute to cardiac 2012). Conversely, decreased cardiac NGF in experimental
dysfunction. and clinical heart failure (Kaye et al. 2000; Qin et al. 2002;
Cardiac sympathetic neurons can change their function Kimura et al. 2010) and diabetes mellitus (Hellweg &
in cardiovascular disease. Sympathetic hyperinnervation Hartung, 1990; Schmid et al. 1999; Ieda et al. 2006) is
(Zhou et al. 2004; Hasan et al. 2006; Meloni et al. 2010) and associated with the loss of sympathetic and sensory fibres
denervation (Barber et al. 1983; Stanton et al. 1989; Dae and reduced cardiac NA and CGRP levels (Taquet et al.
et al. 1995; Li et al. 2004b; Gardner & Habecker, 2013) have 1992; Qin et al. 2002; Kristen et al. 2006; Kimura et al.
both been observed after myocardial infarction. Hyper- 2007, 2010; Ieda & Fukuda, 2009; Kuehl & Stevens, 2012).
innervation has also been observed in compensatory Restoring NGF to the heart prevents sensory dysfunction
cardiac hypertrophy during the transition to overt heart in diabetes mellitus (Ieda et al. 2006). The regulation
failure (Kimura et al. 2007; Lu et al. 2012), but NA of NGF in the heart is not completely understood,
synthesis is paradoxically reduced due to downregulation but the hypertrophic factor endothelin-1 increases NGF
of tyrosine hydroxylase (TH), the rate-limiting enzyme expression in cardiomyocytes (Ieda et al. 2004; Kimura
in NA synthesis (Kimura et al. 2007, 2010). In heart et al. 2007), while mechanical stretch and α1 -adrenergic
failure highly polysialylated neural cell adhesion molecule stimulation attenuate myocyte NGF expression (Rana et al.
(PSA-NCAM), which is a marker of immature neurons, is 2009).
highly expressed in cardiac sympathetic neurons (Kimura
et al. 2007). Further, many neurons in the stellate ganglia
Neuromodulation and autonomic imbalance
and left ventricle have increased expression of cholinergic
markers such as choline transporter and choline acetyl- Emerging evidence suggests that factors produced within
transferase during heart failure. This is accompanied the microenvironment of the heart, its vasculature
by decreased TH expression and thought to represent and between neuronal populations can influence

sympatho-vagal balance. These neuromodulators can Proximal stimulation of the cut cervical vagus in vitro
be intrinsic to sympathetic ganglia or vagal neurons will therefore cause retrograde stimulation of sensory
(such as neuronal nitric oxide and its adaptor protein, afferents. Stimulation of the intact vagus nerve in vivo
CAPON), or may act as either autoinhibitory or will cause both anterograde and retrograde stimulation of
crosstalk communicating messengers between neuronal both afferent and efferent fibres. Recent work has shown
populations (for example the co-transmitters neuro- that this has the potential to elicit a complex profile of
peptide Y (NPY), galanin and vasoactive intestinal mediators and modulators from both efferent and afferent
peptide (VIP)). Substances released from cardiac myo- fibres engaging multiple levels of the cardiac neuraxis to
cytes and the coronary vasculature, including natriuretic alter peripheral neural processing and central–peripheral
peptides and angiotensin II (AT II), may also influence neural interactions, including central drive (Ardell et al.
neuronal function acting in a paracrine manner (see 2015). With the introduction of implantable cardiac
Fig. 1). These signalling pathways converge on neuronal vagal nerve stimulators in patients with heart failure,
calcium handling, transmitter release and/or re-uptake it is important to understand the differences in cellular
mechanisms. The plasma levels and protein expression mechanisms by which this intervention may influence
of many of these neuromodulators are increased in both the heart, compared to application of exogenous neuro-
animal models and patients with cardiovascular disease. transmitters, or stimulation of the cervical vagus removed
However, their functional significance and the potential to from the central nervous system, as these approaches may
exploit these pathways pharmacologically in disease states yield different results.
is yet to be established.
Studying the actions of cholinergic neuromodulators Intrinsic neuromodulators.
is particularly challenging. It should be noted that whilst
stimulation of the cervical vagus activates efferent pre-
ganglionic parasympathetic neurons, up to 70% of the Neuronal nitric oxide and its activator protein, CAPON.
fibres are sensory afferents (Berthoud & Neuhuber, 2000). Neuronal nitric oxide synthase (nNOS) is localized
in both intrinsic cardiac vagal neurons and post-
ganglionic sympathetic neurons of the stellate ganglia.
Nitric oxide acts via stimulation of soluble guanylate
Sympathetic Parasympathetic
cyclase, to generate cGMP, leading in parasympathetic
NPY/Gal neurons to inhibition of phosphodiesterase 3 (PDE3)
nNOS/CAPON nNOS
and an increase in cAMP–protein kinase A (PKA)
dependent phosphorylation of N-type calcium channels
and release of acetylcholine (Herring et al. 2000, 2001b;
ACh Herring & Paterson, 2001), whilst in sympathetic neurons
NA VIP stimulation of PDE2 and/or protein kinase G (PKG) leads
AT II
to inhibition of calcium influx and NA release (Schwarz
BNP/
CNP et al. 1995; Wang et al. 2007). Increasing nNOS expression
NPY using viral vectors can reverse impaired vagal (Heaton et al.
CNP NA
Microvasculature ACh 2007) and exaggerated sympathetic drive (Li et al. 2007) in
BNP/
CNP the spontaneously hypertensive rat (SHR), and improve
β M
acetylcholine release and mortality acutely following
myocardial infarction in the guinea pig (Dawson et al.
Cardiac myocyte
2008).
Genome wide association studies implicate a variant
Figure 1. Schematic representation of the neuromodulatory in the neuronal nitric oxide synthase adaptor protein
pathways influencing the release of noradrenaline (NA) and (CAPON) in electrocardiographic QT variation and
acetylcholine (ACh) from postganglionic sympathetic and
parasympathetic neurons
sudden cardiac death, both of which are strongly
Neuromodulators can arise from the coronary microvasculature (e.g. influenced by cardiac autonomic balance (Arking et al.
angiotensin II (AT II) and C-type natriuretic peptide (CNP)), myocytes 2006; Kao et al. 2009). Recently it was shown that
(B-type natriuretic peptide (BNP)) as well as between neurons CAPON resides in cardiac sympathetic neurons (Lu
(neuropeptide Y (NPY), galanin (Gal), acetylcholine (ACh) and et al. 2015) and its expression, along with nNOS
vasoactive intestinal peptide (VIP)) and within neurons (such as
neuronal nitric oxide synthase (nNOS) and its activator protein,
(Li et al. 2013a), is decreased in the pre-hypertensive
CAPON). Stimulatory pathways are represented by green arrows, SHR. Moreover, increasing its expression using a novel
and inhibitory pathways by red arrows. β, beta adrenergic receptor; noradrenergic specific adenoviral vector increases nNOS
M, muscarinic receptor. activity and normalizes sympathetic neurotransmission.

Many questions relating to the role of nNOS/CAPON in 2013) and during left ventricular failure in humans where
cardiac autonomic control remain. they correlate positively with one-year mortality (Hulting
et al. 1990; Ullman et al. 1994). In the presence of
Questions and controversies β-blockers, which improve mortality in these conditions,
both sympathetic release of NPY and galanin can produce
How is nNOS localized within postganglionic neurons crosstalk inhibition of vagal neurotransmission via the
and is its activity influenced by calcium influx triggering Y2 and GALR1 receptors in animal models resulting
neurotransmitter release at individual varicosities? in a shift in autonomic balance (Herring et al. 2008,
Is the main role of CAPON intracellular targeting of 2012). Moreover, NPY may also have a direct action
nNOS and/or to increase nNOS enzyme activity? on ventricular myocytes to increase susceptibility to
Are CAPON single nucleotide polymorphisms in ventricular arrhythmias (Herring, 2015) and also limit
neurons the trigger for sudden cardiac death in patients myocardial perfusion through vasoconstriction of the
with and without QT disturbances? coronary vasculature (Shanks & Herring, 2013).
Convergence of neuromodulators on calcium induced trans- Parasympathetic co-transmitters. Many parasympathetic

mitter release and re-uptake. The main trigger for the neurons within the heart also co-stain for different
exocytotic release of neurotransmitter is the binding co-transmitters including NPY, somatostatin, VIP and
of calcium to synaptotagmin to activate the SNARE endogenous opioids such as dynorphins (Steele & Choate,
complex and cause fusion of the vesicular and cellular 1994). Whether these co-transmitters are released during
membranes (Sudhof & Rothman, 2009). The local calcium physiological activation of the vagus nerve or change
signal is a function of the opening of neuronal calcium their expression profile in cardiovascular disease is not
channels and may also be influenced by calcium induced known. For example, inhibitors of VIP enhance vagally
calcium release from the endoplasmic reticulum (ER) mediated bradycardia in the rat although high frequency
and/or mitochondrial buffering of intracellular calcium. stimulation is required to observe these effects (Hogan
The contribution of these calcium sources has been & Markos, 2006). Several cardiac opioid analogues
studied in postganglionic sympathetic neurons from the have also been identified, including the heptapeptide
stellate ganglia at the whole cell level through patch methionine-enkephalin-arginine-phenylalanine (MEAP)
clamping and fluorescence imaging. The pre-hypertensive (Younes et al. 2000). MEAP has been shown to be vagolytic
SHR for example has a larger intracellular calcium (Farias et al. 2001), probably via δ2 receptors (Jackson
transient, associated with a higher ER calcium content, et al. 2001), whilst low doses of MEAP can augment vagal
lower mitochondrial membrane potential and reduced bradycardia, via δ1 receptors (Farias et al. 2003). Inter-
mitochondrial buffering of intracellular calcium (Li et al. estingly, repeated transient ischaemia to the sinoatrial
2012). The whole cell calcium current is also larger in node artery is associated with an increase in locally released
the pre-hypertensive SHR (Lu et al. 2015). Moreover MEAP and increased vagal bradycardia (Deo et al. 2009)
increased NA release (Shanks et al. 2013b) and impaired whilst δ1 receptor agonists significantly reduce infarct
NA re-uptake (Shanks et al. 2013a) is also observed in the size in the rat (Schultz et al. 1998). Nevertheless, many
SHR at this developmental stage which may contribute questions still remain regarding the pathophysiological
to a larger tachycardia during stimulation of the stellate role of different neuropeptides.
ganglia and increased heart rate observed in vivo (Shanks Nitric oxide generated by nNOS has also been suggested
et al. 2013b). to act as a co-transmitter responsible for the cholinergic
modulation of ventricular fibrillation threshold via
Co-transmitters as neuromodulators. NO acting independently of the muscarinic receptor
(Brack et al. 2011). Early studies demonstrated that the
Sympathetic co-transmitters. Sympathetic nerves throu- anti-fibrillatory effect of the vagus on the ventricle was
ghout the autonomic nervous system contain co- abolished by the muscarinic receptor antagonist atropine
transmitters such as ATP, neuropeptide-Y (NPY) and (Corr & Gillis, 1974; Yoon et al. 1977; Vanoli et al.
galanin. Both NA and ATP have relatively short half-lives as 1991). Muscarinic agonists such as oxotremorine and
NA is taken back into the nerve terminal via the NA trans- methacholine can also reduce ventricular arrhythmias
porter and ATP is rapidly metabolized. Conversely whilst following myocardial infarction (De Ferrari et al. 1992,
the half-lives of NPY and galanin are significantly longer, 1993). However, in an isolated Langendorff perfused
they are released in much smaller quantities (Burnstock, rabbit heart, recent work suggests the anti-fibrillatory
2009). The role of co-transmitters may therefore be more action of direct vagal stimulation appears to be preserved
important during conditions associated with high levels in the presence of atropine, abolished in the presence
of adrenergic drive. For example, plasma NPY levels are of hexamethonium (Brack et al. 2011), and dependent
elevated following myocardial infarction (Cuculi et al. on the generation of nitric oxide by nNOS acting in a

paracrine fashion (Ng et al. 2007; Brack et al. 2009). also found within vascular endothelium and the human
This seemed surprising given previous observations with ventricle (Wei et al. 1993). BNP is able to act directly
atropine and muscarinic agonists in vivo, and the fact on cardiac sympathetic neurons to reduce the intra-
that NO generated by nNOS can facilitate acetylcholine cellular calcium transient and release of NA during neuro-
release (Herring & Paterson, 2001). More recently, vagus nal depolarization via a cGMP–PKG dependent pathway
nerve stimulation has been shown to be anti-arrhythmic (Li et al. 2015). Conversely, exogenous BNP and CNP
in the setting of ischaemia–reperfusion via effects on are also capable of directly augmenting vagal neuro-
mitochondrial function with atropine abolishing this transmission in a manner similar to NO via generation
effect (Shinlapawittayatorn et al. 2014). Silencing vagal of cGMP (Herring et al. 2001a). BNP may therefore be
preganglionic neurons from the dorsal motor nucleus expected to result in a beneficial shift in cardiac autonomic
of the vagus using an optogenetic approach also limits balance towards sympathetic withdrawal and greater
infarct size during ischaemia–reperfusion via an atropine vagal tone during chronic heart failure, although cGMP
dependent pathway (Mastitskaya et al. 2012). Some dependent signalling may be limited by phosphodiesterase
beneficial changes in ventricular electrophysiology were 2A (PDE2A) that is upregulated in this condition (Li et al.
still observed, suggesting there might be a paracrine action 2015). This may explain the lack of success of recombinant
via NO, since this action was removed following NOS BNP (nesiritide), which has only been trialled in acute
inhibition (Machhada et al. 2015). However, in this issue heart failure where results were disappointing (O’Connor
of The Journal of Physiology, Kalla et al. (2016) challenge et al. 2011).
the functional significance of the paracrine effect of NO.
Whilst they demonstrate that the acetylcholine analogue Questions and controversies
carbamylcholine raises ventricular fibrillation threshold
(VFT), they observed its effect was prevented by atropine, Although a natriuretic peptide receptor agonist has
the nicotinic receptor antagonist mecamylamine, and been trialled unsuccessfully in acute decompensated
inhibitors of neuronal nitric oxide synthase (nNOS) heart failure, would their use as sympatholytic neuro-
and soluble guanylyl cyclase (sGC). Moreover, the modulating agents be beneficial in chronic stable heart
NO donor sodium nitroprusside could mimic the failure if neuronal PDE2A levels can be controlled?
anti-fibrillatory action of carbamylcholine, but its action
was again ultimately dependent on muscarinic receptor Ventricular myocytes as a source of acetylcholine. Rat
stimulation, since all effects were blocked by atropine ventricular myocytes express choline acetyltransferase
(Kalla et al. 2016). (ChAT) and vesicular acetylcholine transporter, and the
expression of these genes and the intracellular acetyl-
Questions and controversies choline content can be modulated by muscarinic receptor
inhibition (Kakinuma et al. 2009). This suggests that
What are the physiological roles of cardiac sympathetic cardiomyocytes possess an acetylcholine synthesis system
and parasympathetic co-transmitters? that is positively modulated by cholinergic stimuli and may
Are they just neuromodulators or do they play a major therefore amplify the beneficial effects of vagal stimulation
role as co-transmitters during cardiovascular disease? on the ventricles. In keeping with this, cardiac specific
Can these pathways be exploited therapeutically in overexpression of ChAT in mice improves survival post
patients receiving conventional pharmacological and myocardial infarction (Kakinuma et al. 2013). Whether
interventional treatments? such a system plays an important role in large mammals
and humans is yet to be determined.
Paracrine neuromodulators.
Angiotensin II. The renin–angiotensin system plays a
Natriuretic peptides. The natriuretic peptide family is key role in the regulation of blood pressure, circulating
made up of at least three distinct peptides: atrial natriuretic volume and neuronal function. In addition to the classical
peptide (ANP), brain derived natriuretic peptide (BNP), pathway via angiotensin converting enzyme (ACE) in the
and C-type natriuretic peptide (CNP) which share a pulmonary vasculature, the presence of local cardiac ACE
disulphide linked 17 amino acid ring structure giving (Saito et al. 1987) raises the possibility of local AT II
them their functionality. ANP is secreted from the atria synthesis and influence on neurotransmission. Inhibitors
in response to myocardial stretch producing natriuresis of ACE and AT1 receptors facilitate the heart rate response
and vasorelaxation, whilst BNP is synthesized and secreted to peripheral vagal nerve stimulation (Takata et al. 2004)
in the atria and ventricle, particularly during congestive whilst AT II inhibits myocardial acetylcholine release
heart failure where plasma levels are a prognostic indicator (Kawada et al. 2007) via the AT1 receptor, although
(Doust et al. 2005). CNP is the predominant natriuretic the intracellular pathway potentially linking the AT1
peptide of the nervous system (Sudoh et al. 1990) and is receptor to transmitter release has not been elucidated.

Moreover, cardiac AT II levels are higher in the SHR where (Li et al. 2013b). Experimentally, CGRP is cardioprotective
it potentiates NA release reinforcing the sympathetic by activating the PI3K/Akt and ERK1/2 mitogen-activated
hyperactivity (Herring et al. 2011). The use of ACE protein kinase (MAPK) kinase pathways and restoring
inhibitors post myocardial infarction (e.g. AIRE trial) and Bcl-2/Bax to inhibit apoptosis in cultured rat cardio-
in chronic heart failure (e.g. SOLVD and CONSENSUS myocytes (Schaeffer et al. 2003; Ma et al. 2013; Umoh
trials) is well established where they confer a mortality et al. 2014). CGRP may also have a protective function in
benefit. heart failure through decreased inflammation, cell death
and fibrosis.
Neurohumoral impact on cardiac adaptations
Role of sympathetic efferent nerves in the diseased heart.
in disease
The contributions of sympathetic dysfunction to cardio-
Role of sensory afferent nerves in the diseased heart. vascular pathology have been the subject of many reviews
The cardiac autonomic nervous system is composed of (Esler et al. 1997; Lefkowitz et al. 2000; Gourine & Gourine,
efferent and afferent nerves. In contrast to sympathetic 2014; Shen & Zipes, 2014; Fukuda et al. 2015; Gardner
innervation, which has well-defined roles in the et al. 2016). Under normal physiological conditions,
progression of cardiac disease, sensory innervation and NA released from sympathetic nerves impacts cardiac
its alteration in diseased hearts have not been elucidated ion channels and Ca2+ handling to enact prototypical
sufficiently. Sensory nerves within the heart express the chronotropic and inotropic responses. Briefly, NA binds
peptides substance P and CGRP, and those peptides to β-ARs on cardiac myocytes leading to phosphorylation
have differing effects in cardiovascular disease. Substance of several intracellular targets, including L-type Ca2+
P induces adverse myocardial remodelling (Melendez channels, K+ channels, phospholamban and ryanodine
et al. 2011), while CGRP has cardioprotective effects in receptors (RyRs) (Bers, 2002). Phosphorylation of the
pathophysiological conditions including cardiovascular L-type Ca2+ channel (Cav1.2) increases current, which
disease and diabetes mellitus (Brain & Grant, 2004; Chai by itself would be expected to increase the cardiac
et al. 2006) (Fig. 2). CGRP is also an important angiogenic action potential duration (APD). However, this increase is
factor for microvascular formation in the diseased heart counterbalanced by the effect of β-AR on delayed rectifier
K+ currents, most notably an increase in rapidly activating
IKs , but slowly activating IKr may also be involved (Kagan
Sudden cardiac death et al. 2002; Marx et al. 2002). The net effect is typically a
shortening of the APD, which is required to accommodate
Heart failure Diabetes fast heart rates during sympathetic activity.
mellitus
Phosphorylation of phospholamban leads to increased
sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA)
NGF
activity and an increase in the Ca2+ content of the SR.
Arrhythmia Silent Although increased SR Ca2+ content facilitates positive
CGRP ischaemia
inotropy, SR Ca2+ overload can also occur, resulting in
spontaneous opening of RyRs and Ca2+ release into the
p38/ERK1/2 Bcl-2/Bax cytosol that is not in response to an action potential.
This leads to Ca2+ extrusion from the cytosol via the
Fibrosis
PI3K/Akt
Vasoconstriction
Na+ /Ca2+ exchanger (NCX), which produces a net inward
current (Pogwizd et al. 2001). If the inward current
Inflammation Apoptosis is large enough, the cell membrane depolarizes and a
triggered action potential occurs, also known as a delayed
after-depolarization (DAD).
Long term remodelling of these prototypical cardio-
Figure 2. Diagram of the pathophysiological interactions myocyte responses occurs as a result of chronic
between the cardiac sensory nerves and diseased state
CGRP can cause activation of multiple signalling pathways and has
sympathetic hyperinnervation or denervation, and both
cardioprotective effects. Cardiovascular disease and autonomic of these conditions have been linked to increased risk
neuropathy can lead to downregulation of CGRP, which causes of ventricular arrhythmias and sudden cardiac death
inflammation, vasoconstriction, impairing angiogenesis, fibrosis and (Cao et al. 2000a,b; Liu et al. 2003; Li et al. 2004b;
apoptosis, and may ultimately lead to heart failure or sudden cardiac Boogers et al. 2010; Nishisato et al. 2010; Vaseghi et al.
death. CGRP, calcitonin gene-related peptide; ROS, reactive oxygen
species; PI3K, phosphatidylinositol-3 kinase; Akt,
2012; Fallavollita et al. 2014). A loss of cardiomyocyte
serine/threonine-specific protein kinase; ERK, extracellular β-AR responsiveness over time is a hallmark of sustained
signal-regulated kinases; Bcl-2/Bax, B-cell lymphoma adrenergic stimulation and hyperinnervation. Changes in
2/Bcl-2-associated X protein. β-AR sensitivity are probably due to altered expression

of either β-ARs or G-protein receptor kinase 2 (GRK2, 2010). This suggests that sympathetic cholinergic trans-
also known as βARK1) (Iaccarino et al. 1998; Yatani differentiation might be an adaptive response that protects
et al. 2006). In addition to decreased responsiveness the heart. Consistent with this idea, cholinergic signalling
to NA, APDs may be prolonged in hyperinnervated via cardiac muscarinic acetylcholine receptors decreases
regions due to NGF-induced downregulation of K+ the susceptibility of ventricular myocytes to chronic
currents (Heath et al. 1998). This increase in APD adrenergic stress, improving cardiac function (LaCroix
may also facilitate early after-depolarizations (EADs), et al. 2008). Furthermore, cardiomyocytes can also
in which a secondary depolarization occurs before synthesize and release ACh, which is positively modulated
final repolarization. Conversely, chronic sympathetic by cholinergic stimuli. This suggests a positive feed-
denervation can lead to β-AR supersensitivity, which back mechanism for ACh release, which may further
results from upregulation of β-ARs and increased any cardioprotective effects (Kakinuma et al. 2009).
responsiveness upon catecholamine binding (Vatner et al. Similarly, augmenting parasympathetic nerve activity
1985) due to downregulation of GRK2 (Yatani et al. decreases mortality rates in heart failure, suggesting a
2006). Denervation also leads to a loss of Ito , which is beneficial effect of cholinergic signalling (Li et al. 2004a).
responsible for the initial repolarization in phase 1 of However, it remains controversial if release of ACh from
the action potential (Bru-Mercier et al. 2003; Bai et al. sympathetic nerves, which are distributed differently from
2008). Gardner et al. recently showed in a murine model parasympathetic nerves within the myocardium, is a
that reperfused infarcts remain denervated and display favourable or unfavourable event for cardiac performance
β-adrenergic supersensitivity, calcium mishandling, and and prognosis (DeMazumder et al. 2015).
decreased Ito that are reversed by restoring the innervation Thus, the spatial and temporal innervation pattern
(Gardner et al. 2015). and activity of sympathetic nerves directly impacts
In addition to these cellular level responses, abnormally pathogenesis in the diseased heart (Fig. 3).
heterogeneous release of NA due to hyperinnervation
or denervation leads to tissue level responses that also Questions and controversies
facilitate arrhythmia. Heterogeneous sympathetic nerve
distribution may produce non-uniform shortening of Does sympathetic release of ACh protect the heart?
cardiac APD and increased dispersion of repolarization. Does sympathetic ACh have the same effect in heart
Although non-uniform APD shortening occurs even failure and acute myocardial infarction?
under normal sympathetic control (due to the normally
heterogeneous sympathetic nerve distribution), Ajijola Neurohumoral regulation of calmodulin-dependent protein
and colleagues found that following myocardial infarction, kinase II in health and disease. The multifunctional
sympathetic stimulation further increased the dispersion Ca2+ - and calmodulin-dependent protein kinase II
of repolarization and altered activation and propagation (CaMKII) is a serine/threonine kinase that is abundant
patterns (Ajijola et al. 2013b). These results were in myocardium. CaMKII appears to augment myo-
confirmed in human patients with myocardial infarction cardial performance in response to physiological
in which reflex sympathetic stimulation caused a stress, at least in part, by neurohumoral agonist
230% increase in dispersion compared to patients stimulation. CaMKII catalyses phosphorylation events
with structurally normal hearts (Vaseghi et al. 2012). that activate many of the voltage-gated sarcolemmal ion
Collectively, these increases in dispersion of repolarization channels and Ca2+ homeostatic proteins orchestrating
increase the likelihood of conduction block and reentrant excitation–contraction coupling (Rokita & Anderson,
arrhythmias. Furthermore, localized release of adrenergic 2012). The role of CaMKII-catalysed phosphorylation of
agonists (e.g. hyperinnervation) has been shown to induce the type 2 intracellular Ca2+ release ryanodine receptor
propagating premature ventricular complexes (PVCs) and (RyR2) seems to be particularly important in augmenting
sustained focal ventricular tachycardia in intact hearts release of myofilament activating Ca2+ from the SR
(Nash et al. 2001; Myles et al. 2012, 2015). during neurohumoral agonist stimulation (Grimm et al.
However, NA is not the only substance released by 2015; Polakova et al. 2015) and during oxidant stress
sympathetic nerves in the heart during pathological (Shirokova et al. 2014). In cardiac pacemaker cells,
conditions. The impact of sympathetic NPY on coronary CaMKII activation contributes to fight or flight heart
arteries was described previously, but the effect of ACh rate increases in response to physical activity and iso-
released from cardiac sympathetic nerves is not yet under- proterenol (isoprenaline) (Wu et al. 2009). Thus, CaMKII
stood. Mice with heart failure and cholinergic trans- activation downstream to neurohumoral agonists acts
mission from cardiac sympathetic nerves had significantly to augment myocardial performance under physiological
improved survival and ventricular function compared stress. However, a large body of evidence supports a
with sympathetic nerve-specific gp130-deficient mice, view that CaMKII is also a nodal signal for promoting
which lacked sympathetic ACh (Kanazawa et al. heart failure and arrhythmias (Swaminathan et al.

2012). Myocardial diseases are marked by sustained in the N-terminus. The regulatory domain is inter-
elevation of neurohumoral signalling and excessive posed between the kinase and association domains
CaMKII activity. Furthermore, CaMKII is activated and consists of an autoinhibitory region (N-terminus)
by neurohumoral agonist pathways that are amongst and a calmodulin (CaM) binding region (C-terminus).
the best clinically validated targets for heart failure Under resting conditions, CaMKII is largely inactive
therapeutics. Thus, it seems likely that high impact because the kinase domain engages the autoinhibitory
cardiovascular drugs, including β-adrenergic receptor region, which acts as a pseudosubstrate. However, when
antagonists (β-blockers), angiotensin II receptor blockers the concentration of intracellular Ca2+ ([Ca2+ ]i ) rises
(ARBs), angiotensin converting enzyme (ACE) inhibitors and binds to CaM the calcified CaM (Ca2+ /CaM)
and mineralocorticoid receptor (MR) antagonists are embraces the CaM binding region and induces a
beneficial, at least in part, because they mitigate against conformational change that distorts the relationship
CaMKII hyperactivation under conditions of sustained, between the catalytic domain and the autoinhibitory
pathological neurohumoral stress. region, leading to disinhibition and activation of
The CaMKII holoenzyme consists of two stacked CaMKII. Brief, low frequency [Ca2+ ]i elevations permit
hexamers and includes a total of 12 homologous or Ca2+ /CaM unbinding and a return to autoinhibition.
identical subunits (Chao et al. 2011). Each mono- However, when [Ca2+ ]i elevations are rapid or sustained
meric CaMKII subunit joins the holoenzyme by a CaMKII transitions between Ca2+ /CaM-dependent and
C-terminal association domain (Fig. 4). The kinase Ca2+ /CaM-autonomous conformations by an auto-
domain, containing the ATP binding pocket resides phosphorylation process (Kuret & Schulman, 1985;
Catecholaminergic Cholinergic differentiation Cholinergic
Rejuvenation
TH
PSA-NCAM ChAT/CHT
TH
Functional
denervation
Anatomical
denervation
Hyperinnervation
TrkA TrkA TrkA

LIFR
LIFR gp130 LIFR gp130 gp130
NGF LIF NGF LIF NGF
LIF LIF LIF

NGF NGF NGF
Figure 3. Crosstalk between cardiomyocyte and sympathetic nerve via humoral factors in diseased
heart
Failing cardiomyocytes induce NGF via an endothelin-1 mediated pathway and LIF. NGF leads to hyperinnervation
(anatomical modulation) and LIF leads to rejuvenation/cholinergic differentiation (functional modulation). This
phenomenon shows the expression of catecholaminergic marker such as reduced TH and increased cholinergic
(CHT, ChAT) and juvenile markers (PSA-NCAM). PSA-NCAM, polysialylated neural cell adhesion molecule; TrkA,
tropomyosin-related kinase A; NGF, nerve growth factor; LIF, leukemia inhibitory factor; LIFR, LIF receptor; TH,
tyrosine hydroxylase.

Schworer et al. 1986). Autophosphorylation of a conserved activity are O-GlcNAcylation (at serine 280) (Erickson
threonine at position 286 or 287 (Thr 287, numbering et al. 2013) and nitrosylation (at cysteine 290) (Gutierrez
varies slightly by isoform) in the regulatory domain et al. 2013; Curran et al. 2014). Cysteine 290 may show
enhances the avidity of Ca2+ /CaM binding (so called CaM more nitrosylation during catecholamine stimulation but
trapping) (Meyer et al. 1992), favouring sustained CaMKII other nitrosylation site(s) may lead to reduced CaMKII
activation, but also prevents effective autoinhibition even activity (Erickson et al. 2015). O-GlcNAcylation is not pre-
after Ca2+ /CaM unbinding, allowing for Ca2+ /CaM auto- sently known to be downstream to neurohumoral agonist
nomous CaMKII activity. More recently, we discovered stimulation. Thus, a rich variety of regulatory domain
a mechanism for Ca2+ /CaM independent CaMKII post-translational modifications account for important
activity by oxidation of a methionine pair (281/282) aspects of CaMKII activation in response to neurohumoral
(Erickson et al. 2008). Oxidized CaMKII (ox-CaMKII) agonist stimulation.
does not support CaM trapping and is reversible by The role of Ca2+ /CaM independent CaMKII activation
a methionine reductase (MsrA, methionine sulfoxide appears to be important for the role of CaMKII in
reductase A). Interestingly, CaMKII activation can be neurohumoral agonist induced myocardial disease. At this
sustained at very low [Ca2+ ]i in oxidative environments, time, neurohumoral agonist initiated pathways are best
suggesting that reactive oxygen species (ROS) sensitize understood to activate Ca2+ /CaM autonomous CaMKII
the Ca2+ /CaM dependence of CaMKII activation. Other activity by increasing Thr 287 autophosphorylation or Met
post-translational modifications to the regulatory domain 281/282 oxidation. Infusion of the mixed β1/2 receptor
shown to increase Ca2+ /CaM autonomous CaMKII agonist isoproterenol is a model for catecholamine
Isoproterenol Angiotensin II
Aldosterone
ARBs
β-Blockers ACE
MR antagonists
SMMHRQETVDC
281/282 287 290
+Ca2+/CaM PTM
CaM
Closed Extended Extended
(inactive) (Ca2+/CaM dependent) (Ca2+/CaM autonomous)
Figure 4. Neurohumoral activation of CaMKII

Catecholamines, angiotensin II and aldosterone are upstream activators of CaMKII by inducing post-translational
modifications (PTM) to the N-terminus of the regulatory domain (blue horizontal bar, see text). Catecholamine
agonists promote phosphorylation of threonine 287 and nitrosylation of cysteine 290. Angiotensin II and
aldosterone increase oxidant stress leading to oxidation of methionines 281 and 282. Hyperglycaemia can also
activate CaMKII directly (by O-Glycnacylation of serine 280) and by increasing reactive oxygen species and oxidizing
methionines 281 and 282. The inactive CaMKII holoenzyme exists in a compact configuration. Intracellular Ca2+
increases lead to calcification of calmodulin (Ca2+ /CaM) that binds to the C-terminus of the regulatory domain.
Ca2+ /CaM binding induces CaMKII into an extended, active conformation that is inactivated after Ca2+ /CaM
unbinding. Post-translational modifications to the regulatory domain keep CaMKII in a persistantly extended,
active state even after Ca2+ /CaM unbinding. ARB, angiotensin II receptor blockers; ACE, angiotensin converting
enzyme; MR, mineralocorticoid receptor.

induced cardiomyopathy. In our hands, isoproterenol to rapid pacing induced atrial fibrillation and knock-in
activates CaMKII and induces Ca2+ /CaM independent mice lacking Met 281/282 on CaMKIIδ or knock-in
CaMKII activity primarily by favouring Thr 287 mice lacking a validated CaMKII phosphorylation site
autophosphorylation (Erickson et al. 2008). Features of on RyR2 (serine 2814) are resistant to angiotensin II
isoproterenol induced myocardial toxicity include hyper- biasing of atrial fibrillation induction (Purohit et al.
trophy, cell death, fibrosis and depressed left ventricular 2013). Aldosterone infusion, to achieve blood levels
contraction (Zhang et al. 2005; Yang et al. 2006). Mice present in patients with heart failure after myocardial
with transgenic expression of a CaMKII inhibitory peptide infarction, increases ox-CaMKII and promotes myocardial
(AC3-I) (Zhang et al. 2005) or a protein kinase A (PKA) rupture by enhancing ox-CaMKII mediated expression
inhibitory peptide (PKI) (Zhang et al. 2013) are resistant of matrix metalloproteinase 9 in mice after myocardial
to CaMKII activation and isoproterenol infusion-induced infarction surgery (He et al. 2011). Taken together, these
myocardial injury. The precise nature of pathways for and other findings support a view that CaMKII is an
activating CaMKII in response to β-adrenergic agonist important component of physiological and pathological
stimulation remain controversial because some evidence pathways central to neurohumoral agonist signalling in
supports exclusive, or near exclusive, CaMKII activation myocardium.
as a consequence of PKA-induced intracellular Ca2+
mobilization (Zhang et al. 2013), while other studies Adapting novel molecular and cellular tools
support a role for the PKA-independent, exchange for neurocardiology research
proteins directly activated by cAMP (EPAC) pathway
(Grimm et al. 2015). Detailed studies of how whole-heart function is altered
by the activity of specific populations of neurons that
Questions and controversies innervate the heart provide insights into neurocardiac
physiology as well as disease mechanisms and potential
Do β-adrenergic receptors activate CaMKII solely therapies. However, controlled measurements of the inter-
through PKA, or does EPAC play a role? play between nerve activity and the myocardium can
be challenging, particularly for in vivo animal studies
Although detailed dose ranging studies are lacking, and in excised perfused heart experiments. For example,
isoproterenol appears to be a more toxic myocardial in a hybrid perfused heart approach, parasympathetic
agent compared to angiotensin II or aldosterone because neurons were activated by careful dissection and
these latter two agents do not induce substantial electrical stimulation of the left and right vagus nerve
loss of left ventricular contraction in the absence while sympathetic neurons were activated by electrical
of a ‘second hit’, such as myocardial infarction. We stimulation of the spinal canal at the second thoracic
found that mice with myocardial targeted expression vertebra (Ng et al. 2001; Brack et al. 2004). This approach
of a CaMKII inhibitory peptide, AC3-I, were protected is powerful because it provides for controlled activation
against isoproterenol infusion induced cardiomyocyte of the cardiac nerves in the absence of circulating
death (Yang et al. 2006), cardiomyopathy and myo- compounds, although spinal cord stimulation activates
cardial infarction (Zhang et al. 2005), a condition where both left and right sympathetic pathways, reducing
elevated neurohumoral signalling contributes to increased left–right specificity (Brack et al. 2004). The approach
mortality. Mice with myocardial and mitochondrial has been primarily used in rabbits and could be difficult
CaMKII inhibition by transgenic expression of another to implement in smaller species due to the additional
CaMKII inhibitory peptide, CaMKIIN (Chang et al. steps required to prepare the heart for perfusion, isolate
1998), are also protected against myocardial death after the nerve tissue, and position stimulation electrodes. For
isoproterenol injection or myocardial infarction (Joiner in vivo animal studies, electrodes are usually implanted
et al. 2012), suggesting CaMKII actions in mitochondria around the vagus nerve for chronic parasympathetic
contribute to catecholamine cardiotoxicity. CaMKIIδ stimulation (Li et al. 2004a), but this has the limitation of
knock-out mice are also protected from pathological potentially stimulating both efferent and afferent nerves.
stress responses after aortic banding (Backs et al. 2009; Exposing and electrically stimulating the stellate ganglia
Ling et al. 2009), a model of elevated afterload, where provides sympathetic stimulation in animals (Ajijola et al.
neurohumoral signalling probably contributes to adverse 2013a) while sympathetic cardiac neurons can be silenced
outcomes. Angiotensin II infusion, to approximate blood by removing or ablating the stellate ganglia (Schwartz,
concentrations present in patients with heart failure, 2014; Vaseghi et al. 2014).
oxidizes CaMKII at Met 281/282 (ox-CaMKII) leading to In other studies, exogenous neurotransmitters, or their
sinus node cell death (Swaminathan et al. 2011), inducing analogues, are administered to the coronary circulation to
a heart rate disease phenotype resembling ‘sick sinus activate postsynaptic receptors to study the downstream
syndrome.’ Angiotensin II infusion increases susceptibility effect of receptor activation on whole-heart function (Liu

et al. 1999; Lang et al. 2015). The neurotransmitters inhibitory postsynaptic currents in cardiac vagal neurons,
circulate through the myocardium to ubiquitously providing a circuit substrate for the withdrawal of
activate their receptors. Although this approach reveals, parasympathetic activity and increase in heart rate that
for example, important aspects of adrenergic receptor occurs during stressful events. The selective activation
sensitivity and expression within the myocytes, it does of noradrenergic cells using optogenetics was a critical
not recapitulate neurotransmitter release and the physio- component in the success of these studies.
logical kinetics and spatially dependent concentration
of transmitters released from cardiac neurons. This Questions and controversies
can be an important experimental limitation when
the spatial arrangement of innervation (Freeman et al. Could therapies that target the brainstem blunt auto-
2014), disparate activity levels of nerve bundles (Ng nomic imbalance that develops during heart failure?
et al. 2001), or altered autonomic tone (Thayer &
Lane, 2007; Cauley et al. 2015) influences myocardial In other studies, transgenic mice similar to those
function. of Wang et al. (2014) were used for expression
Optogenetics is a powerful way to selectively target of ChR2 in noradrenergic (TH) neurons to elicit
and activate cells in living tissue with unmatched a cardiac β-adrenergic response that originated
spatiotemporal specificity using light-actuated ion from sympathetic nerve terminals within the heart
channels, such as channelrhodopsin-2 (ChR2). Originally (Wengrowski et al. 2015). Hearts were excised and
developed for neuroscience (Boyden et al. 2005), with perfused using a standard Langendorff approach. The
ongoing developments in cardiac tissue (Ambrosi et al. epicardial surface was illuminated with blue light to
2014; Nussinovitch & Gepstein, 2015), optogenetics photo-stimulate noradrenergic neurons to generate a
provides unparalleled specificity for cellular activation. myocardial adrenergic response. Changes in contra-
This has created new opportunities for investigating the ctile force, heart rate, and electrical activity were
interaction between the cardiac nerves and the myo- measured before and after photo-stimulation. Results
cardium, overcoming previous approaches that required demonstrated pronounced increases in heart rate and
dissection and non-specific electrical stimulation of contractile force during photo-stimulation as well as
bundles of heterogeneous nerve fibres. Precise cell-type recovery of prestimulation function after cessation of
activation can be accomplished by regulating ChR2 photo-stimulation. Photo-stimulation also shortened
expression using Cre-Lox recombination and cell-specific myocardial action potential duration, consistent with
promoters. In a transgenic implementation of this system, β-adrenergic activation, and made hearts more susceptible
a Cre responder animal would have a loxP-flanked to arrhythmia. Photo-stimulation of sympathetic nerve
STOP cassette upstream of a ChR2-enhanced yellow terminals provided approximately half the increase in
fluorescent protein (EYFP) fusion gene. This animal contractile force and heart rate that resulted from
would then be mated with a Cre driver animal ubiquitous β-adrenergic receptor activation using iso-
that expresses Cre recombinase under the control proterenol (5 μM) (Wengrowski et al. 2015).
of a cell-specific promoter such as α-myosin heavy
chain (cardiomyocytes) (Zaglia et al. 2015), tyrosine Questions and controversies
hydroxylase (TH, sympathetic neurons) (Wang et al.
2014; Wengrowski et al. 2015), or choline acetyl- Could a premature ventricular contraction be initiated
transferase (ChAT, parasympathetic neurons) (Kalmbach by the simultaneous activation of a minimum number
et al. 2012). In the progeny, ChR2-EYFP would then of sympathetic nerve terminals?
have promoter-specific expression only in targeted How would increased fibrosis and scar tissue during
tissues. heart failure, or within an infarct, modulate that
This approach was recently used to study the neural minimum number?
control of parasympathetic tone in the brainstem of trans-
genic mice (Wang et al. 2014). ChR2 was expressed in Optogenetics has also been instrumental in studies of
noradrenergic cells using Cre-Lox recombination and cardiac ischaemia–reperfusion injury by providing for
a TH promoter. Parasympathetic cardiac vagal neurons selective stimulation of specific populations of cardiac
were labelled with the retrograde fluorescent tracer neurons. Using optogenetics to activate neurons in the
X-rhodamine isothiocyanate (XRITC). Brains were then brainstem, Mastitskaya et al. showed that cardioprotection
excised, sliced to reveal the locus coeruleus and cardiac provided by remote ischaemic preconditioning in rats
vagal neurons, and noradrenergic (TH) neurons were is dependent upon the activity of brainstem vagal pre-
photo-stimulated to study their influence on the cardiac ganglionic neurons (Mastitskaya et al. 2012). Lenti- and
vagal neurons. Photo-stimulated action potentials in adenoviral vectors were administered to specific locations
the noradrenergic neurons increased the frequency of within the dorsal motor nucleus of the vagus nerve

(DVMN) for selective expression of a channelrhodopsin Ajijola OA, Vaseghi M, Zhou W, Yamakawa K, Benharash P,
variant (ChIEF) (Lin et al. 2009) to activate neurons using Hadaya J, Lux RL, Mahajan A & Shivkumar K (2013a).
light and a Drosophila allatostatin receptor (AlstR) to Functional differences between junctional and
silence neurons using allatostatin (Tan et al. 2006). ChIEF extrajunctional adrenergic receptor activation in mammalian
and AlstR were selectively expressed in cholinergic vagal ventricle. Am J Physiol Heart Circ Physiol 304, H579–H588.
Ajijola OA, Yagishita D, Patel KJ, Vaseghi M, Zhou W,
preganglionic neurons of the DVMN by driving expression
Yamakawa K, So E, Lux RL, Mahajan A & Shivkumar K
with a PRSx8 promoter (Hwang et al. 2001), which is (2013b). Focal myocardial infarction induces global
activated by Phox2, a transcription factor expressed in remodeling of cardiac sympathetic innervation: neural
cholinergic vagal preganglionic neurons of the DVMN. remodeling in a spatial context. Am J Physiol Heart Circ
Cardioprotection provided by remote preconditioning Physiol 305, H1031–H1040.
was abolished when the brainstem DVMN neurons were Ambrosi CM, Klimas A, Yu J & Entcheva E (2014). Cardiac
silenced with allatostatin. Interestingly, photo-stimulation applications of optogenetics. Prog Biophys Mol Biol 115,
of the brainstem DVMN neurons before ischaemia 294–304.
and during reperfusion (in the absence of remote pre- Antzelevitch C, Sicouri S, Litovsky SH, Lukas A, Krishnan SC,
conditioning) effectively mimicked the effect of remote Di Diego JM, Gintant GA & Liu DW (1991). Heterogeneity
preconditioning. This work has provided new insights into within the ventricular wall. Electrophysiology and
pharmacology of epicardial, endocardial, and M cells. Circ
the mechanisms of preconditioning, which appear to be
Res 69, 1427–1449.
strongly mediated by vagal activity and cardiac muscarinic Aoyama T, Takimoto Y, Pennica D, Inoue R, Shinoda E,
receptors. Hattori R, Yui Y & Sasayama S (2000). Augmented
expression of cardiotrophin-1 and its receptor component,
Questions and controversies gp130, in both left and right ventricles after myocardial
infarction in the rat. J Mol Cell Cardiol 32, 1821–1830.
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Zaglia T, Pianca N, Borile G, Da Broi F, Richter C, Campione None declared.
M, Lehnart SE, Luther S, Corrado D, Miquerol L & Mongillo
M (2015). Optogenetic determination of the myocardial
requirements for extrasystoles by cell type-specific targeting Funding
of ChannelRhodopsin-2. Proc Natl Acad Sci USA 112, This work was supported, in part, by National Institutes
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and HL071140 (M.E.A.), HL068231 and HL093056 (B.A.H.),
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function. Cardiovasc Res 99, 566–575.
Zhang R, Khoo MS, Wu Y, Yang Y, Grueter CE, Ni G, Price EE, The authors thank Dr Rebecca Kreipke (Brandeis University)
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Lederer WJ, Colbran RJ & Anderson ME (2005). University) for contributions to the section on novel molecular
Calmodulin kinase II inhibition protects against structural tools and critical reading of the manuscript. Mr Timothy Phelps
heart disease. Nat Med 11, 409–417. contributed art work.


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J Physiol 594.

14 (2016) pp 3853–3875 3853

Molecular and cellular neurocardiology: development,

role in cardiovascular pathology, is discussed further cholinergic transdifferentiation of cardiac adrenergic

Convergence of neuromodulators on calcium induced trans- Parasympathetic co-transmitters. Many parasympathetic

Catecholaminergic Cholinergic differentiation Cholinergic

TrkA TrkA TrkA

LIF LIF LIF

281/282 287 290

Figure 4. Neurohumoral activation of CaMKII

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