Intracardiac Neurons Research Articles

Altered firing activity of intracardiac neurons isolated from mice carrying the Scn5a-1798insD mutation

Abstract Funding Acknowledgements None. Background Dysfunction of the cardiac sodium channel Nav1.5 (encoded by SCN5A) is associated with arrhythmias and sudden cardiac death, the mechanisms of which have been extensively studied in cardiomyocytes. However, patients carrying an SCN5A mutation often display clinical symptoms which are not readily explained solely by alterations in cardiomyocyte electrophysiology. For instance, carriers of the SCN5A-1795insD mutation, which is associated with features of Brugada syndrome and Long QT syndrome type 3 (LQT3), display severe bradycardia leading to excessive QT-prolongation and sudden death especially occurring at rest or during the night, suggesting autonomic triggers. Interestingly, SCN5A is not only present in cardiomyocytes, but also in intracardiac neurons (ICN). ICN modulate cardiac electrophysiological properties and their dysregulation may contribute to arrhythmogenesis. Purpose To explore whether ICN from mice carrying the Scn5a-1798insD mutation (the murine equivalent of SCN5A-1795insD) display electrophysiological alterations that may (in part) contribute to the arrhythmogenic phenotype. Methods and Results ICN were isolated by enzymatic dissociation from fat pads located in the pulmonary veins and sinoatrial node regions of the hearts of wild-type (WT) and Scn5a-1798insD mice. Immunodetection of the cholinergic marker ChAT confirmed the prominent cholinergic phenotype of ICN. In agreement with previous reports in other species, we also detected the presence of Nav1.5 in murine ICN. Nav1.5 expression was mainly observed in ChAT positive cells. To evoke repetitive action potential (AP) firing in single ICN, 500 ms-sustained depolarizing current pulses were applied of increasing amplitude (from 0 pA to 150 pA). Two types of neurons were identified on the basis of firing pattern characteristics: accommodating (neurons that discharged multiple APs decrementing in frequency during the pulse) and tonic (neurons that discharged multiple APs at a high frequency). APs firing frequency did not differ between WT and Scn5a-1798insD accommodating ICN, while it was significantly increased in Scn5a-1798insD tonic ICN as compared to WT (at 100 pA, AP count/500ms in WT CMs: 29.5±1.7, n=24; at 100 pA, AP count/500ms in Scn5a-1798insD: 36.2±2.0, n=18, p=0.02). Conclusions The impact of SCN5A mutations on ICN function are unexplored, but may play an essential role in arrhythmogenesis. The increased AP firing frequency observed in Scn5a-1798insD ICN may lead to increased release of neurotransmitters, such as acetylcholine, that in turn may contribute to the bradycardia observed in patients. Overall, further elucidation of the electrophysiological consequences of inherited Nav1.5 dysfunction in ICN might provide novel therapeutic targets to prevent lethal arrhythmias.

Ventricular hyperinnervation in a mouse model of catecholaminergic polymorphic ventricular tachycardia

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): European Society of Cardiology Basic Research Fellowship. Dutch Heart Foundation Dekker Grant Background Inherited arrhythmia syndromes are a leading cause of sudden cardiac death in young and otherwise healthy patients. They are often caused by mutations in genes that encode ion channels or transporters, leading to conditions such as Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). CPVT is caused by mutations in RYR2 (encoding ryanodine receptor 2, RyR2). In arrhythmia research, investigation into the effects of these mutations have exclusively been performed in cardiac cells. However, RyR2 is also present in neuronal tissue, including intracardiac neurons [1] (which modulate cardiac function). Moreover, patients often present with clinical signs of autonomic nervous system (ANS) dysfunction, and treatment strategies frequently target the ANS. Aim Our novel research line aims to investigate the neuronal phenotype induced by RyR2 mutations, using a CPVT mouse model to assess functional alterations as well as broader autonomic remodelling. Results Using immunocytochemistry, we reveal for the first time that RyR2 is also expressed in mouse stellate ganglia (crucial autonomic modulators of cardiac function). Through analysis of published RNA sequencing datasets, we find that RYR2 is more abundantly expressed in both stellate ganglion and intracardiac neurons than characteristic neuronal markers such as CHAT, SYN1 and SCN8A [2,3]. Functional patch clamp investigation of isolated stellate ganglion neurons have revealed two distinct populations based on their patterns of action potential (AP) firing, identifying both phasic-firing and tonic-firing type neurons. Investigation of an established CPVT mouse model (RyR2-R2474S) has thus far not revealed any differences in the AP firing frequency of isolated stellate ganglion neurons during sustained depolarising stimuli of various amplitudes. Assessment of calcium homeostasis and neurotransmitter release is currently ongoing. We have furthermore investigated broader autonomic remodelling using immunohistochemistry in mouse heart cryosections. Neuronal innervation density was assessed by staining 10 µm sections of mid-ventricular myocardium for the sympathetic neuron marker tyrosine hydroxylase (TH). Quantification of the TH-positive area revealed a significant increase in the percentage of sympathetic neuronal innervation in ventricular tissue of CPVT mice (5.7% wild-type vs 7.2% RyR2-R2474S, p=0.0253, N=6). Conclusions The neurons that modulate heart function (stellate ganglion and intracardiac neurons) abundantly express RyR2. The RyR2-R2474S mutation leads to significant sympathetic hyper-innervation of ventricular myocardium, which may have important consequences for neurotransmitter release and hence cardiac (electrical) function and arrhythmogenesis.

Electrophysiology and 3D-imaging reveal properties of human intracardiac neurons and increased excitability with atrial fibrillation.

Altered autonomic input to the heart plays a major role in atrial fibrillation (AF). Autonomic neurons termed ganglionated plexi (GP) are clustered on the heart surface to provide the last point of neural control of cardiac function. To date the properties of GP neurons in humans are unknown. Here we have addressed this knowledge gap in human GP neuron structure and physiology in patients with and without AF. Human right atrial GP neurons embedded in epicardial adipose tissue were excised during open heart surgery performed on both non-AF and AF patients and then characterised physiologically by whole cell patch clamp techniques. Structural analysis was also performed after fixation at both the single cell and at the entire GP levels via three-dimensional confocal imaging. Human GP neurons were found to exhibit unique properties and structural complexity with branched neurite outgrowth. Significant differences in excitability were revealed between AF and non-AF GP neurons as measured by lower current to induce action potential firing, a reduced occurrence of low action potential firing rates, decreased accommodation and increased synaptic density. Visualisation of entire GPs showed almost all neurons are cholinergic with a small proportion of noradrenergic and dual phenotype neurons. Phenotypic distribution differences occurred with AF including decreased cholinergic and dual phenotype neurons, and increased noradrenergic neurons. These data show both functional and structural differences occur between GP neurons from patients with and without AF, highlighting that cellular plasticity occurs in neural input to the heart that could alter autonomic influence on atrial function. KEY POINTS: The autonomic nervous system plays a critical role in regulating heart rhythm and the initiation of AF; however, the structural and functional properties of human autonomic neurons in the autonomic ganglionated plexi (GP) remain unknown. Here we perform the first whole cell patch clamp electrophysiological and large tissue confocal imaging analysis of these neurons from patients with and without AF. Our data show human GP neurons are functionally and structurally complex. Measurements of action potential kinetics show higher excitability in GP neurons from AF patients as measured by lower current to induce action potential firing, reduced low firing action potential rates, and decreased action potential accommodation. Confocal imaging shows increased synaptic density and noradrenergic phenotypes in patients with AF. Both functional and structural differences occur in GP neurons from patients with AF that could alter autonomic influence on atrial rhythm.

Molecular and functional characterization of the mouse intrinsic cardiac nervous system.

The intrinsic cardiac nervous system (ICNS) refers to clusters of neurons, located within the heart, that participate in the neuronal regulation of cardiac functions and that are involved in the initiation of cardiac arrhythmias. Therefore, deciphering its role in cardiac physiology and physiopathology is mandatory. The aim of this study was to provide a phenotypic, electrophysiological, and pharmacological characterization of the mouse ICNS, which is still poorly characterized. Global cardiac innervation and phenotypic diversity were investigated using immunohistochemistry on cleared murine hearts and on tissue sections. The patch clamp technique was used for the electrophysiological and pharmacological characterization of isolated mouse intracardiac neurons. We have identified the expression of 7 distinct neuronal markers within the mouse ICNS, thus proving the neurochemical diversity of this network. Of note, it was the first time that the existenceof neurons expressing the calcium-binding protein calbindin, neuropeptide Y, and cocaine and amphetamine regulated transcript peptide was described in the mouse. Electrophysiology studies also revealed the existence of 4 different neuronal populations on the basis of their electrical behavior. Finally, we showed that these neurons can be modulated by several neuromodulators. This study showed that the mouse ICNS presents a molecular and functional complexity similar to other species and is therefore a suitable model to decipher the role of individual neuronal subtypes regarding the modulation of cardiac function and the initiation of cardiac arrhythmias.

Electrophysiological characterization of mouse intracardiac calbindin neurons

Neural control of the heart involves central and peripheral neurons that act interdependently to modulate cardiac parameters such as heart rate, conduction velocity or contractility. Within this cardiac neuronal regulation, the intrinsic cardiac nervous system (ICNS), which correspond to clusters of neurons found on the dorsal atrial surface of the heart, is receiving growing attention. Indeed, whereas they were initially considered as simple parasympathetic postganglionic neurons, studies conducted over the past 30 years suggested a more complex organization, involving the existence of sensory, local regulatory and motor neurons within intracardiac ganglia. Moreover, growing evidence suggest the implication of this neural network in the initiation and maintenance of cardiac arrhythmias. However, the functional organization of this intracardiac neural network, as well as its involvement in cardiac diseases have not been fully elucidated. This study aims to decipher the complexity of this intracardiac nervous system by examining the phenotypic and electrophysiological properties of mouse intracardiac neurons. Global cardiac innervation and phenotypic diversity of mouse intracardiac neurons were investigated by performing immunohistochemistry on cleared heart and on tissue sections. The patch clamp technique (current and voltage clamp) was used for the electrophysiological characterization of these neurons. Calbindin expressing neurons were specifically studied by using cre transgenic mice and targeted viral transduction strategy. We identified 6 distinct neuronal markers within mouse intracardiac neurons revealing the neurochemical diversity of this intracardiac neural network. The characterization of passive and active electrical membrane properties of these neurons gave rise to the identification of two distinct firing profiles within intracardiac neurons. The first group was classified as phasic due to its limited firing activity while the second was defined as adapting. Phasic neurons were also characterized by a higher rheobase compared to the adapting one. In addition, intracardiac neurons could also be distinguished by the presence or absence of an afterhyperpolarization. We also identified calbindin expressing neurons as a population with a distinct electrophysiological signature. This could be explained by the differential expression of several ionic channels including the N-type voltage-gated calcium channel. This study demonstrates the molecular and functional complexity of the mouse ICNS.

Intrinsic cardiac neurons of the adult pigs: chemical types, abundance, parameters and distribution within ganglionated plexus

Intracardiac neurons (ICNs) have a pivotal role in the regulation of heart rhythm and myocardial contractility. However, comparative data about the precise distribution of the chemically phenotypically distinct ICNs in the heart as well as their interrelations within intracardiac ganglia (ICG) are limited. In this study, we employed the whole-mount approach to characterise immunohistochemically the porcine atrial ICG. We found that almost all, 99%, of ICNs express cholinergic neuronal marker in the adult pig. Some cholinergic ICNs were simultaneously positive for adrenergic (3.6%) or nitrergic (3.3%) neuronal markers as well. These biphenotypic adrenergic ICNs were more abundant within the right atrial ganglionated subplexi (sGPi), while the biphenotypic nitrergic ICNs were more numerous in the dorsal sGPi compared with the ventral sGPi. Non-cholinergic as well as entirely adrenergic and nitrergic ICNs were seldom and compiled respectively 0.1% and 0.4% of all intracardiac neuronal population. In contrast to neuronal somata, nerve fibres observed in the adult pig atrial sGPi were also positive for vesicular glutamate transporter 2 and calcitonin gene-related peptide (CGRP). Receptor protein for CGRP, CRLR, was found on cholinergic neuronal somata within ICG as well as the putative neuro-somatic contact sites with peptidergic and adrenergic nerve fibres. Morphological analysis of neuronal somata disclosed that the largest somata were nitrergic. This study demonstrates the scanty chemical variety of ICNs in the adult porcine atrial ICG that possess mainly cholinergic neuronal somata with the few adrenergic and nitrergic ones which are patchily distributed within sGPi. Inside the ICG, cholinergic ICNs are wrapped by a complex network of chemically diverse nerve terminals, some of which contain varicosities with probable passage type synapses.

From Mice to Mainframes: Experimental Models for Investigation of the Intracardiac Nervous System.

The intracardiac nervous system (IcNS), sometimes referred to as the “little brain” of the heart, is involved in modulating many aspects of cardiac physiology. In recent years our fundamental understanding of autonomic control of the heart has drastically improved, and the IcNS is increasingly being viewed as a therapeutic target in cardiovascular disease. However, investigations of the physiology and specific roles of intracardiac neurons within the neural circuitry mediating cardiac control has been hampered by an incomplete knowledge of the anatomical organisation of the IcNS. A more thorough understanding of the IcNS is hoped to promote the development of new, highly targeted therapies to modulate IcNS activity in cardiovascular disease. In this paper, we first provide an overview of IcNS anatomy and function derived from experiments in mammals. We then provide descriptions of alternate experimental models for investigation of the IcNS, focusing on a non-mammalian model (zebrafish), neuron-cardiomyocyte co-cultures, and computational models to demonstrate how the similarity of the relevant processes in each model can help to further our understanding of the IcNS in health and disease.

Structural Identification of the Pacemaker Cells and Expression of Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channels in the Heart of the Wild Atlantic Cod, Gadus morhua (Linnaeus, 1758).

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are proteins that contain highly conserved functional domains and sequence motifs that are correlated with their unique biophysical activities, to regulate cardiac pacemaker activity and synaptic transmission. These pacemaker proteins have been studied in mammalian species, but little is known now about their heart distribution in lower vertebrates and c-AMP modulation. Here, we characterized the pacemaker system in the heart of the wild Atlantic cod (Gadus morhua), with respect to primary pacemaker molecular markers. Special focus is given to the structural, ultrastructural and molecular characterization of the pacemaker domain, through the expression of HCN channel genes and the immunohistochemistry of HCN isoforms, including the location of intracardiac neurons that are adjacent to the sinoatrial region of the heart. Similarly to zebrafish and mammals, these neurons are immunoreactive to ChAT, VAChT and nNOS. It has been shown that cardiac pacemaking can be modulated by sympathetic and parasympathetic pathways, and the existence of intracardiac neurons projecting back to the central nervous system provide a plausible link between them.

The Intrinsic Cardiac Nervous System and Its Role in Cardiac Pacemaking and Conduction.

The cardiac autonomic nervous system (CANS) plays a key role for the regulation of cardiac activity with its dysregulation being involved in various heart diseases, such as cardiac arrhythmias. The CANS comprises the extrinsic and intrinsic innervation of the heart. The intrinsic cardiac nervous system (ICNS) includes the network of the intracardiac ganglia and interconnecting neurons. The cardiac ganglia contribute to the tight modulation of cardiac electrophysiology, working as a local hub integrating the inputs of the extrinsic innervation and the ICNS. A better understanding of the role of the ICNS for the modulation of the cardiac conduction system will be crucial for targeted therapies of various arrhythmias. We describe the embryonic development, anatomy, and physiology of the ICNS. By correlating the topography of the intracardiac neurons with what is known regarding their biophysical and neurochemical properties, we outline their physiological role in the control of pacemaker activity of the sinoatrial and atrioventricular nodes. We conclude by highlighting cardiac disorders with a putative involvement of the ICNS and outline open questions that need to be addressed in order to better understand the physiology and pathophysiology of the ICNS.

The Zebrafish Heart: an Archetype for Neurocardiology

The intracardiac nervous system (ICNS) is the final common pathway for reflex control of cardiac output by the autonomic nervous system (ANS). The normal operation of this system is key to supporting cardiovascular homeostasis, and ANS dysfunction or remodelling can contribute to the development of multiple cardiopathies. Thus the role of the ICNS in cardiac control is essential, however our current understanding of this role is incomplete. The majority of work on the ICNS to date has been in mammalian models, but because the neural elements comprising this network are embedded in cardiac tissues, visualization and experimental access to the entire network for integrative studies of neuronal function has been limiting. To address these issues, we have developed the zebrafish, a cyprinid teleost, as an alternate model for cardiac anatomical and neurophysiological studies. In this ancestral vertebrate the two‐chambered heart, although anatomically distinct from the mammalian four chamber arrangement, is dually innervated by sympathetic and parasympathetic limbs of the ANS and has a well‐developed ICNS which is functional in spontaneously beating isolated preparations. Activation of extrinsic cardiac nerves evokes tachycardia (via B2‐adrenoceptors) and bradycardia (M2 muscarinic receptors). Thus the essential features of ANS control of the heart are conserved across the vertebrates. We have mapped the general innervation patterns of the sinoatrial valve region (SAR), atrium and ventricle. A ganglionated plexus in the SAR surrounds the valve and contains most of the intracardiac neuronal somata; the majority is cholinergic with the remainder adrenergic, nitrergic and serotonergic. Furthermore, peptidergic axon terminals contact plexal somata. Optical mapping of the isolated heart confirms the site of origin of normal primary pacemaker activity within the SAR, where cells in the base of the valve leaflets express Islet‐1 (Isl‐1) and hyperpolarization‐activated, cyclic nucleotide‐gated channel 4 (HCN4), known markers for mammalian pacemaker cells. In addition, intracellular recordings from these cells display spontaneous diastolic membrane depolarization patterns typical of pacemaker cells. Extrinsic cardiac nerve stimulation, in addition to evoking bradycardia, can displace the pacemaker site to a secondary pacemaker in the atrioventricular region (AVR), where a population of cells also expresses Isl‐1 and HCN4. A small ganglion containing cholinergic somata is present in the AVR, suggesting local neural control of atrioventricular pacemaker activity. We have thus established the zebrafish heart as a viable model for studies of integrative neural control of pacemaker function. In particular, this model provides a platform to use optogenetic techniques to target intracardiac neurons and pacemaker cells, in combination with traditional electrophysiological methods, to perform detailed analyses of the intracardiac neuronal circuitry involved in cardiac control. This work will provide a solid foundation for further studies of ANS‐related cardiac pathophysiologies in zebrafish analogs of human cardiac disease states.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (FMS, TAQ, RPC)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Substance P Receptor in the Rat Heart and Regulation of Its Expression in Long-Term Diabetes.

Substance P (SP) is a neuropeptide engaged in the signal transmission of neural C fibers afferents in the myocardium. The actions of SP in the heart are extensive and they are mediated by the neurokinin 1 receptor (NK1R), a member of the tachykinin subfamily of G-protein coupled receptors. The receptors have been found in the heart, but to our knowledge, their exact localization in the heart has not been described yet. Here, we investigated the presence of NK1R protein in separate rat heart compartments by means of western blot and its tissue distribution by means of immunofluorescence. Specificity of NK1R immunolabeling was controlled by preabsorption of the antiserum with its corresponding peptide. Additionally, we investigated abundance of gene for NK1R in separated heart chambers by means of quantitative real-time PCR (RT-PCR). Relative abundance of NK1R mRNA was expressed as a ratio of target gene Cq value to Cq value of control gene – beta-actin. Finally, we studied abundance of NK1R mRNA in different cell types of heart isolated by laser capture microdissection. Immunofluorescence showed NK1R immunoreactivity on the surface of some intracardiac neurons and smooth muscle cells of coronary vessels. The results of quantitative RT-PCR indicate abundance of mRNA for NK1R in all heart chambers with highest level in the left atrium. The presence of NK1R mRNA was detected in some samples of dissected intracardiac neurons, but not in cardiomyocytes or smooth muscle cells of coronary vessels. In the course of long-term diabetes, a significant downregulation of the NK1R mRNA was seen in the right atrium and upregulation in the right ventricle 53 weeks after the induction of diabetes. Our results indicate localization of NK1R in some intracardiac neurons and smooth muscle cells. Impaired transcription of the NK1R gene in the diabetic heart may be induced by unidentified genes or factors involved in the development of diabetic cardiomyopathy.

Ganglion cardiacum or juxtaductal body of human fetuses.

The ganglion cardiacum or juxtaductal body is situated along the left recurrent laryngeal nerve in the aortic window and is an extremely large component of the cardiac nerve plexus. This study was performed to describe the morphologies of the ganglion cardiacum or juxtaductal body in human fetuses and to compare characteristics with intracardiac ganglion. Ganglia were immunostained in specimens from five fetuses of gestational age 12–16 weeks and seven fetuses of gestational age 28–34 weeks. Many ganglion cells in the ganglia were positive for tyrosine hydroxylase (TH; sympathetic nerve marker) and chromogranin A, while a few neurons were positive for neuronal nitric oxide synthase (NOS; parasympathetic nerve marker) or calretinin. Another ganglion at the base of the ascending aorta carried almost the same neuronal populations, whereas a ganglion along the left common cardinal vein contained neurons positive for chromogranin A and NOS but no or few TH-positive neurons, suggesting a site-dependent difference in composite neurons. Mixtures of sympathetic and parasympathetic neurons within a single ganglion are consistent with the morphology of the cranial base and pelvic ganglia. Most of the intracardiac neurons are likely to have a non-adrenergic non-cholinergic phenotype, whereas fewer neurons have a dual cholinergic/noradrenergic phenotype. However, there was no evidence showing that chromogranin A- and/or calretinin-positive cardiac neurons corresponded to these specific phenotypes. The present study suggested that the ganglion cardiacum was composed of a mixture of sympathetic and parasympathetic neurons, which were characterized the site-dependent differences in and near the heart.

Quantitative study of synaptic boutons on frog intracardiac neurons

The frog is a useful model to study the structure and function of intracardiac neurons. The goal of this study was to evaluate the size and distribution of synaptic boutons on the intracardiac neurons in the frog Rana temporaria. Interatrial septa from four animals were double-labelled immunohistochemically for the cholinergic marker choline acetyltransferase (ChAT) and the marker of synaptic vesicles synaptophysin (SYP). One hundred intracardiac neurons were analysed by confocal microscopy. Terminals of preganglionic axons were strongly positive for ChAT, while synaptic boutons were strongly positive for both ChAT and SYP. The number of synaptic boutons per neuron ranged from 2 to 121 and was 10±2 (mean±SE). The total area of synaptic boutons ranged from 6 μm2 to 270 μm2 and was 98±6 μm2. The largest total area of synaptic boutons was found on the axonal half of neuronal soma (59±4 μm2). The total areas of synaptic boutons on both the non-axonal half of soma and the proximal axon were smaller (36±3 μm2; 6±2 μm2; P<0.001). Synaptic boutons occupied 13±1% of the area of the neuronal soma profile. Conclusions: 1) The axonal half of the soma of the frog intracardiac neuron is more densely innervated than the non-axonal half of the soma. 2) The axosomatic and axoaxonic synapses are present on frog intracardiac neurons. The study provides a framework for further experimental studies on the formation and rearrangement of synapses on frog intracardiac neurons.

Ganglionic and non-ganglionic neurons in frog heart: do they differ?

This study was designed to compare the morphology of neurons in relation to their distance from the major nerve trunks in the heart of the frog Rana temporaria. Seventy-nine intracardiac neurons were labelled intracellularly with fluorescent markers Lucifer Yellow CH and Alexa Fluor 568. The neurons located on the extensions of the vagus nerve were considered as ganglionic, while neurons spread loosely at further distance from these extensions were considered as non-ganglionic. The mean area of the soma in ganglionic neurons was about 25% larger than in non-ganglionic neurons. Ganglionic neurons had a higher soma area/nucleus area ratio than non-ganglionic neurons. Although both the total number and the total length of dendrite-like processes was similar between the two groups, ganglionic neurons had significantly fewer dendrite-like processes from the soma (1.5±0.3 vs. 3.9±1.0; P<0.05) and shorter total length of these processes from the soma (63±18 μm vs. 178±51 μm; P<0.05). In conclusion, ganglionic and non-ganglionic frog intracardiac neurons exhibit substantial morphological differences. We hypothesize that these differences may indicate different projections or variations in the number of their preganglionic inputs.

Distribution and chronotropic effects of serotonin in the zebrafish heart.

Several lines of evidence suggest that serotonin (5-HT) has a regulatory role in cardiovascular function from embryogenesis through adulthood. However, the reported actions of 5-HT are often contradictory and include bradycardia or tachycardia, hypotension or hypertension, and vasodilation or vasoconstriction. Clarifying such cardiac effects requires further research and may benefit from utilizing a model simpler than the mammalian hearts traditionally used in these studies. In the present study, we describe the cardiac distribution and chronotropic responses of 5-HT in the zebrafish heart. A combined anatomical, electrophysiological, and pharmacological approach was used to investigate the involvement of 5-HT pathways, and to compare neural and direct myocardial pathways of biological action. Immunohistochemical methods revealed 5-HT in endocardial cells, glial-like cells, and intracardiac neurons in the atrium. Electrocardiogram (ECG) recordings combined with the administration of pharmacological agents demonstrated that 5-HT acted predominantly through direct myocardial pathways resulting in a reduction of heart rate. Overall, the results of this study contribute significant advances in the establishment of the zebrafish as a new model for studies of the role of 5-HT in autonomic cardiac control.

Purinergic Signaling in the Cardiovascular System.

There is nervous control of the heart by ATP as a cotransmitter in sympathetic, parasympathetic, and sensory-motor nerves, as well as in intracardiac neurons. Centers in the brain control heart activities and vagal cardiovascular reflexes involve purines. Adenine nucleotides and nucleosides act on purinoceptors on cardiomyocytes, AV and SA nodes, cardiac fibroblasts, and coronary blood vessels. Vascular tone is controlled by a dual mechanism. ATP, released from perivascular sympathetic nerves, causes vasoconstriction largely via P2X1 receptors. Endothelial cells release ATP in response to changes in blood flow (via shear stress) or hypoxia, to act on P2 receptors on endothelial cells to produce nitric oxide, endothelium-derived hyperpolarizing factor, or prostaglandins to cause vasodilation. ATP is also released from sensory-motor nerves during antidromic reflex activity, to produce relaxation of some blood vessels. Purinergic signaling is involved in the physiology of erythrocytes, platelets, and leukocytes. ATP is released from erythrocytes and platelets, and purinoceptors and ectonucleotidases are expressed by these cells. P1, P2Y1, P2Y12, and P2X1 receptors are expressed on platelets, which mediate platelet aggregation and shape change. Long-term (trophic) actions of purine and pyrimidine nucleosides and nucleotides promote migration and proliferation of vascular smooth muscle and endothelial cells via P1 and P2Y receptors during angiogenesis, vessel remodeling during restenosis after angioplasty and atherosclerosis. The involvement of purinergic signaling in cardiovascular pathophysiology and its therapeutic potential are discussed, including heart failure, infarction, arrhythmias, syncope, cardiomyopathy, angina, heart transplantation and coronary bypass grafts, coronary artery disease, diabetic cardiomyopathy, hypertension, ischemia, thrombosis, diabetes mellitus, and migraine.

Zebrafish heart as a model to study the integrative autonomic control of pacemaker function

The cardiac pacemaker sets the heart's primary rate, with pacemaker discharge controlled by the autonomic nervous system through intracardiac ganglia. A fundamental issue in understanding the relationship between neural activity and cardiac chronotropy is the identification of neuronal populations that control pacemaker cells. To date, most studies of neurocardiac control have been done in mammalian species, where neurons are embedded in and distributed throughout the heart, so they are largely inaccessible for whole-organ, integrative studies. Here, we establish the isolated, innervated zebrafish heart as a novel alternative model for studies of autonomic control of heart rate. Stimulation of individual cardiac vagosympathetic nerve trunks evoked bradycardia (parasympathetic activation) and tachycardia (sympathetic activation). Simultaneous stimulation of both vagosympathetic nerve trunks evoked a summative effect. Effects of nerve stimulation were mimicked by direct application of cholinergic and adrenergic agents. Optical mapping of electrical activity confirmed the sinoatrial region as the site of origin of normal pacemaker activity and identified a secondary pacemaker in the atrioventricular region. Strong vagosympathetic nerve stimulation resulted in a shift in the origin of initial excitation from the sinoatrial pacemaker to the atrioventricular pacemaker. Putative pacemaker cells in the sinoatrial and atrioventricular regions expressed adrenergic β2 and cholinergic muscarinic type 2 receptors. Collectively, we have demonstrated that the zebrafish heart contains the accepted hallmarks of vertebrate cardiac control, establishing this preparation as a viable model for studies of integrative physiological control of cardiac function by intracardiac neurons.

Cardiac purinergic signalling in health and disease.

This review is a historical account about purinergic signalling in the heart, for readers to see how ideas and understanding have changed as new experimental results were published. Initially, the focus is on the nervous control of the heart by ATP as a cotransmitter in sympathetic, parasympathetic, and sensory nerves, as well as in intracardiac neurons. Control of the heart by centers in the brain and vagal cardiovascular reflexes involving purines are also discussed. The actions of adenine nucleotides and nucleosides on cardiomyocytes, atrioventricular and sinoatrial nodes, cardiac fibroblasts, and coronary blood vessels are described. Cardiac release and degradation of ATP are also described. Finally, the involvement of purinergic signalling and its therapeutic potential in cardiac pathophysiology is reviewed, including acute and chronic heart failure, ischemia, infarction, arrhythmias, cardiomyopathy, syncope, hypertrophy, coronary artery disease, angina, diabetic cardiomyopathy, as well as heart transplantation and coronary bypass grafts.

Nav-igating through a complex landscape: SCN10A and cardiac conduction

Genome-wide association studies (GWAS) have implicated SCN10A, which encodes a nociceptor-associated voltage-gated sodium channel subunit, as a modulator of cardiac conduction; however, this role has traditionally been ascribed to SCN5A, which is highly expressed in cardiac muscle. SCN10A is believed to affect cardiac conduction either directly through cardiomyocytes or indirectly via intracardiac neurons. In this issue of the JCI, van den Boogaard and colleagues introduce a third possibility: that the SCN10A locus acts as an enhancer of SCN5A gene expression. The authors demonstrate that SCN10A expression is negligible within human and murine hearts, and that a T-box enhancer within the SCN10A locus drives SCN5A expression within cardiomyocytes. This work reasserts SCN5A as the key determinant of cardiac conduction and highlights the importance of deciphering the functionality of coding versus noncoding regions when interpreting GWAS data.

Regional innervation of the heart in the goldfish, Carassius auratus: A confocal microscopy study

The intracardiac nervous system represents the final common pathway for autonomic control of the vertebrate heart in maintaining cardiovascular homeostasis. In teleost fishes, details of the organization of this system are not well understood. Here we investigated innervation patterns in the heart of the goldfish, a species representative of a large group of cyprinids. We used antibodies against the neuronal markers zn-12, acetylated tubulin, and human neuronal protein C/D, as well as choline acetyltransferase, tyrosine hydroxylase, nitric oxide synthetase, and vasoactive intestinal polypeptide (VIP) to detect neural elements and their transmitter contents in wholemounts and sections of cardiac tissue. All chambers of the heart were innervated by choline acetyltransferase-positive axons, implying cholinergic regulation; and by tyrosine hydroxylase-containing axons, implying adrenergic regulation. The mean total number of intracardiac neurons was 713 ± 78 (SE), nearly half of which were cholinergic. Neuronal somata were mainly located in a ganglionated plexus around the sinoatrial valves. Somata were contacted by cholinergic, adrenergic, nitrergic, and VIP-positive terminals. Putative pacemaker cells, identified by immunoreactivity for hyperpolarization activated, cyclic nucleotide-gated channel 4, were located in the base of the sinoatrial valves, and this region was densely innervated by cholinergic and adrenergic terminals. We have shown that the goldfish heart possesses the necessary neuroanatomical substrate for fine, region-by-region autonomic control of the myocardial effectors that are involved in determining cardiac output.