Cardiac Ganglia Research Articles

Molecular profiling of single neurons of known identity in two ganglia from the crab Cancer borealis

Understanding circuit organization depends on identification of cell types. Recent advances in transcriptional profiling methods have enabled classification of cell types by their gene expression. While exceptionally powerful and high throughput, the ground-truth validation of these methods is difficult: If cell type is unknown, how does one assess whether a given analysis accurately captures neuronal identity? To shed light on the capabilities and limitations of solely using transcriptional profiling for cell-type classification, we performed 2 forms of transcriptional profiling-RNA-seq and quantitative RT-PCR, in single, unambiguously identified neurons from 2 small crustacean neuronal networks: The stomatogastric and cardiac ganglia. We then combined our knowledge of cell type with unbiased clustering analyses and supervised machine learning to determine how accurately functionally defined neuron types can be classified by expression profile alone. The results demonstrate that expression profile is able to capture neuronal identity most accurately when combined with multimodal information that allows for post hoc grouping, so analysis can proceed from a supervised perspective. Solely unsupervised clustering can lead to misidentification and an inability to distinguish between 2 or more cell types. Therefore, this study supports the general utility of cell identification by transcriptional profiling, but adds a caution: It is difficult or impossible to know under what conditions transcriptional profiling alone is capable of assigning cell identity. Only by combining multiple modalities of information such as physiology, morphology, or innervation target can neuronal identity be unambiguously determined.

Respiratory syncytial virus-associated mortality in a healthy 3-year-old child: a case report

BackgroundRespiratory syncytial virus (RSV) is the most frequently identified pathogen in children with acute lower respiratory tract infection. Fatal cases have mainly been reported during the first 6 months of life or in the presence of comorbidity.Case presentationA 47-month-old girl was admitted to the pediatric intensive care unit following sudden cardiopulmonary arrest occurring at home. The electrocardiogram showed cardiac asystole, which was refractory to prolonged resuscitation efforts. Postmortem analyses detected RSV by polymerase chain reaction in an abundant, exudative pericardial effusion. Histopathological examination was consistent with viral myoepicarditis, including an inflammatory process affecting cardiac nerves and ganglia. Molecular analysis of sudden unexplained death genes identified a heterozygous mutation in myosin light chain 2, which was also found in two other healthy members of the family. Additional expert interpretation of the cardiac histology confirmed the absence of arrhythmogenic right ventricular dysplasia or hypertrophic cardiomyopathy.ConclusionsRSV-related sudden death in a normally developing child of this age is exceptional. This case highlights the risk of extrapulmonary manifestations associated with this infection, particularly arrhythmia induced by inflammatory phenomena affecting the cardiac autonomic nervous system. The role of the mutation in this context is uncertain, and it is therefore necessary to continue to assess how this pathogenic variant contributes to unexpected sudden death in childhood.

The Role of Cardiac Ganglia in the Prevention of Coronary Atherosclerosis: An Analytical Examination of Cholesterol-fed Rabbits

Background:The heart is innervated by the autonomic nervous system, which contributes to the control of the heart’s rhythm and coronary circulation. It has been suggested that the cardiac fibers of the vagus nerve play important roles in controlling circulatory functions and in protecting against atherosclerotic pathologies in coronary arteries.Aims:To investigate the presence of atherosclerotic differences in the coronary arteries of cholesterol-fed rabbits by measuring the density of cardiac ganglia neurons.Study Design:Animal experiment.Methods:This study was conducted using 45 male rabbits. Over a period of 16 weeks, they were kept on an atherogenic diet of water ad libitum and high fat (8.6%) containing saturated fatty acids with 205 mg/kg of cholesterol (1%) per day. Then, their hearts were removed and examined by histopathological methods. Atherosclerotic plaques of the main coronary arteries were examined using the Cavalieri method. Atherosclerosis index values (AIVs) were estimated as the wall surface area/plaque surface area, and the results were analyzed with the Kruskal-Wallis and Mann-Whitney U tests.Results:While the average atherosclerosis index value was estimated to be ≤8% in 21 animals, the atherosclerosis index value was 9-20% in animals with minor plaque detection (n=11) and ≥20% in animals with major plaque detection (n=10). Increased atherosclerosis index values were more common in animals with low neuron densities than in animals with high neuron densities (p<0.017).Conclusion:The low neuron density of the cardiac ganglia in cholesterol-fed rabbits is associated with an increased atherosclerotic plaque incidence and volume.

Fate Tracing of Isl1+Cells in Adult Mouse Hearts under Physiological and Exercise Conditions.

Myocardial damage due to dysfunctional myocardium has been increasing, and the prognosis of pharmacological and device-based therapies remain poor. Isl1-expressing cells were thought to be progenitor cells for cardiomyocyte proliferation after specific stimuli. However, the true origin of the proliferating myocardiac cells and the role of Isl1 in adult mammals remain unresolved. In this study, Isl1-CreERT2 knock-in mouse model was constructed using CRISPR/Cas9 technology. Using tamoxifen-inducible Isl1-CreERT/Rosa26R-LacZ system, Isl1+cells and their progeny were permanently marked by lacZ-expression. X-gal staining, immunostaining, and quantitative PCR were then used to reveal the fate of Isl1+cells under physiological and exercise conditions in mouse hearts from embryonic stage to adulthood. Isl1+cells were found to localize to the sinoatrial node, atrioventricular node, cardiac ganglia, aortic arch, and pulmonary roots in adult mice heart. However, they did not act as cardiac progenitor cells under physiological and exercise conditions. Although Isl1+cells showed progenitor cell properties in early mouse embryos (E7.5), this ability was lost by E9.5. Furthermore, although the proliferation and regeneration of heart cell was observed in response to exercise, the cells associated were not Isl1 positive.

Cardiac neural crest contributes to cardiomyocytes in amniotes and heart regeneration in zebrafish.

Cardiac neural crest cells contribute to important portions of the cardiovascular system including the aorticopulmonary septum and cardiac ganglion. Using replication incompetent avian retroviruses for precise high-resolution lineage analysis, we uncover a previously undescribed neural crest contribution to cardiomyocytes of the ventricles in Gallus gallus, supported by Wnt1-Cre lineage analysis in Mus musculus. To test the intriguing possibility that neural crest cells contribute to heart repair, we examined Danio rerio adult heart regeneration in the neural crest transgenic line, Tg(-4.9sox10:eGFP). Whereas the adult heart has few sox10+ cells in the apex, sox10 and other neural crest regulatory network genes are upregulated in the regenerating myocardium after resection. The results suggest that neural crest cells contribute to many cardiovascular structures including cardiomyocytes across vertebrates and to the regenerating heart of teleost fish. Thus, understanding molecular mechanisms that control the normal development of the neural crest into cardiomyocytes and reactivation of the neural crest program upon regeneration may open potential therapeutic approaches to repair heart damage in amniotes.

Identification of peripheral neural circuits that regulate heart rate using optogenetic and viral vector strategies

Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we develop a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs.

Intrinsic Cardiac Ganglionic Neurons Projecting to the SA node in the Rat and Pig Hearts: Retrograde Labeling and Neurolucida 3‐D Reconstruction

Intrinsic cardiac ganglionic neurons (ICNs) within the heart are essential for regulation of cardiac function. ICNs include afferent, efferent neurons and interneurons and constitute a local integration center. In rat and pig hearts, there are multiple cardiac ganglia. Among these ganglia, the largest ganglionated plexus in the rat heart and the right atrial ganglionic plexus (RAGP) in the pig heart are near the sinoatrial node (SAN). However, it is not clear whether both plexi project to the SAN. In this study, retrograde tracer Fast DiI was injected into the SAN in rats (n=5) and a pig. SAN cells were identified by immunostaining for pacemaker channel HCN4. Fluorogold (FG) was injected (i.p.) to label all of the ICNs in rats. FG‐ and DiI‐labeled ICNs were identified and analyzed using a confocal microscopic system and a Neurolucida Tracing and Digitization System. Sixteen days after Fast DiI injection in rats, the animals were perfused with paraformaldehyde, and the whole‐mount atria were prepared. There were multiple ganglionated plexi and the DiI‐labeled neurons were mainly found in the largest ganglionated plexus near the SAN in all these rats. Twenty‐one days after Fast DiI injection in the pig, the animal was sacrificed, and the RAGP was removed and sectioned at 40 μm. Numerous DiI‐labeled neurons were found in the RAGP of this pig. Interestingly, these DiI‐labeled neurons were organized in five spatially‐separated columns perpendicular to the epicardial surface. Our data suggest that the largest ganglionated plexus in rat indeed projects mainly to the SAN, and the pig RAGP projects very heavily to the SAN. The functional significance of this large ganglionated plexus in rat and the five spatially‐separated columns of RAGP ICN projecting neurons in the pig will be elucidated in the future.Support or Funding InformationSupported by NIH SPARC grant OT2OD023848, NIH R15HL137143‐01A1.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Two central pattern generators from the crab, Cancer borealis, respond robustly and differentially to extreme extracellular pH.

The activity of neuronal circuits depends on the properties of the constituent neurons and their underlying synaptic and intrinsic currents. We describe the effects of extreme changes in extracellular pH - from pH 5.5 to 10.4 - on two central pattern generating networks, the stomatogastric and cardiac ganglia of the crab, Cancer borealis. Given that the physiological properties of ion channels are known to be sensitive to pH within the range tested, it is surprising that these rhythms generally remained robust from pH 6.1 to pH 8.8. The pH sensitivity of these rhythms was highly variable between animals and, unexpectedly, between ganglia. Animal-to-animal variability was likely a consequence of similar network performance arising from variable sets of underlying conductances. Together, these results illustrate the potential difficulty in generalizing the effects of environmental perturbation across circuits, even within the same animal.

Foxc2 is required for proper cardiac neural crest cell migration, outflow tract septation, and ventricle expansion.

Proper development of the great vessels of the heart and septation of the cardiac outflow tract requires cardiac neural crest cells. These cells give rise to the parasympathetic cardiac ganglia, the smooth muscle layer of the great vessels, some cardiomyocytes, and the conotruncal cushions and aorticopulmonary septum of the outflow tract. Ablation of cardiac neural crest cells results in defective patterning of each of these structures. Previous studies have shown that targeted deletion of the forkhead transcription factor C2 (Foxc2), results in cardiac phenotypes similar to that derived from cardiac neural crest cell ablation. We report that Foxc2-/- embryos on the 129s6/SvEv inbred genetic background display persistent truncus arteriosus and hypoplastic ventricles before embryonic lethality. Foxc2 loss-of-function resulted in perturbed cardiac neural crest cell migration and their reduced contribution to the outflow tract as evidenced by lineage tracing analyses together with perturbed expression of the neural crest cell markers Sox10 and Crabp1. Foxc2 loss-of-function also resulted in alterations in PlexinD1, Twist1, PECAM1, and Hand1/2 expression in association with vascular and ventricular defects. Our data indicate Foxc2 is required for proper migration of cardiac neural crest cells, septation of the outflow tract, and development of the ventricles. Developmental Dynamics 247:1286-1296, 2018. © 2018 Wiley Periodicals, Inc.

Dopamine maintains network synchrony via direct modulation of gap junctions in the crustacean cardiac ganglion.

The Large Cell (LC) motor neurons of the crab cardiac ganglion have variable membrane conductance magnitudes even within the same individual, yet produce identical synchronized activity in the intact network. In a previous study we blocked a subset of K+ conductances across LCs, resulting in loss of synchronous activity (Lane et al., 2016). In this study, we hypothesized that this same variability of conductances makes LCs vulnerable to desynchronization during neuromodulation. We exposed the LCs to serotonin (5HT) and dopamine (DA) while recording simultaneously from multiple LCs. Both amines had distinct excitatory effects on LC output, but only 5HT caused desynchronized output. We further determined that DA rapidly increased gap junctional conductance. Co-application of both amines induced 5HT-like output, but waveforms remained synchronized. Furthermore, DA prevented desynchronization induced by the K+ channel blocker tetraethylammonium (TEA), suggesting that dopaminergic modulation of electrical coupling plays a protective role in maintaining network synchrony.

HNK-1 in Morphological Study of Development of the Cardiac Conduction System in Selected Groups of Sauropsida.

Human natural killer (HNK)-1 antibody is an established marker of developing cardiac conduction system (CCS) in birds and mammals. In our search for the evolutionary origin of the CCS, we tested this antibody in a variety of sauropsid species (Crocodylus niloticus, Varanus indicus, Pogona vitticeps, Pantherophis guttatus, Eublepharis macularius, Gallus gallus, and Coturnix japonica). Hearts of different species were collected at various stages of embryonic development and studied to map immunoreactivity in cardiac tissues. We performed detection on alternating serial paraffin sections using immunohistochemistry for smooth muscle actin or sarcomeric actin as myocardial markers, and HNK-1 to visualize overall staining pattern and then positivity in specific myocyte populations. We observed HNK-1 expression of various intensity distributed in the extracellular matrix and mesenchymal cell surface of cardiac cushions in most of the examined hearts. Strong staining was found in the cardiac nerve fibers and ganglia in all species. The myocardium of the sinus venosus and the atrioventricular canal exhibited transitory patterns of expression. In the Pogona and Crocodylus hearts, as well as in the Gallus and Coturnix ones, additional expression was detected in a subset of myocytes of the (inter)ventricular septum. These results support the use of HNK-1 as a conserved marker of the CCS and suggest that there is a rudimentary CCS present in developing reptilian hearts. Anat Rec, 302:69-82, 2019. © 2018 Wiley Periodicals, Inc.

Molecular evidence for an intrinsic circadian pacemaker in the cardiac ganglion of the American lobster, Homarus americanus - Is diel cycling of heartbeat frequency controlled by a peripheral clock system?

Whether cardiac output in decapod crustaceans is under circadian control has long been debated, with mixed evidence for and against the hypothesis. Moreover, the locus of the clock system controlling cardiac activity, if it is under circadian control, is unknown. However, a report that the crayfish heart in organ culture maintains a circadian oscillation in heartbeat frequency suggests the presence of a peripheral pacemaker within the cardiac neuromuscular system itself. Because the decapod heart is neurogenic, with contractions controlled by the five motor and four premotor neurons that make up the cardiac ganglion (CG), a likely locus for a circadian clock is the CG itself. Here, a CG-specific transcriptome was generated for the lobster, Homarus americanus, and was used to assess the presence/absence of transcripts encoding putative clock-related proteins in the ganglion. Using known Homarus brain/eyestalk ganglia clock-related proteins as queries, BLAST searches of the CG transcriptome were conducted for the five proteins that form the core clock, i.e., clock, cryptochrome 2, cycle, period and timeless, as well as for a variety of clock-associated, clock input pathway and clock output pathway proteins. With the exception of pigment dispersing hormone receptor [PDHR], a putative clock output pathway protein, one or more transcripts encoding each of the proteins searched for were identified from the CG assembly; no PDHR-encoding transcripts were found. RT-PCR confirmed the expression of all core clock transcripts in multiple independent CG cDNAs; RNA-Seq data suggest that both the motor and premotor neurons could contribute to the cellular locus of a pacemaker. These data provide support for the possible existence of an intrinsic circadian clock in the H. americanus CG, and form a foundation for guiding future anatomical, molecular and physiological investigations of circadian signaling in the lobster cardiac neuromuscular system.

Functional Identification and Characterization of the Diuretic Hormone 31 (DH31) Signaling System in the Green Shore Crab, Carcinus maenas.

The functional characterization of crustacean neuropeptides and their cognate receptors has not kept pace with the recent advances in sequence determination and, therefore, our understanding of the physiological roles of neuropeptides in this important arthropod sub-phylum is rather limited. We identified a candidate receptor-ligand pairing for diuretic hormone 31 (DH31) in a neural transcriptome of the crab, Carcinus maenas. In insects, DH31 plays species -specific but central roles in many facets of physiology, including fluid secretion, myoactivity, and gut peristalsis but little is known concerning its functions in crustaceans. The C. maenas DH31 transcript codes for a 147 amino acid prepropeptide, and a single receptor transcript translates to a secretin-like (Class B1) G protein-coupled receptor (GPCR). We used an in vitro aequorin luminescence Ca2+ mobilization assay to demonstrate that this candidate DH31R is activated byCarcinus and insect DH31s in a dose-dependent manner (EC50 15–30 nM). Whole mount immunohistochemical and in situ hybridization localization revealed extensive DH31 expressing neurons throughout the central nervous system, most notably in the abdominal ganglion where large, unpaired cells give rise to medial nerves, which terminate in extensive DH31 immunopositive dendritic fields intimately associated with oesophageal musculature. This system constitutes a large and hitherto undescribed neurohemal area adjacent to key muscle groups associated with the gastric system. DH31 expressing neurons were also seen in the cardiac, commissural, oesophageal, and stomatogastric ganglia and intense labeling was seen in dendrites innervating fore- and hindgut musculature but not with limb muscles. These labeling patterns, together with measurement of DH31R mRNA in the heart and hindgut, prompted us test the effects of DH31 on semi-isolated heart preparations. Cardiac superfusion with peptide evoked increased heart rates (10–100 nM). The neuroanatomical distribution of DH31 and its receptor transcripts, particularly that associated with gastric and cardiac musculature, coupled with the cardio- acceleratory effects of the peptide implicate this peptide in key myoactive roles, likely related to rhythmic coordination.

Treatment of Neurocardiogenic Syncope: From Conservative to Cutting-edge.

Neurocardiogenic syncope is the most frequent cause of syncope in the general population. Many years have been spent on determining an effective treatment for this condition. Conventional treatment usually follows a tiered approach for neurocardiogenic syncope, as follows: first, lifestyle modification, including increased fluid intake and the introduction of physical counterpressure maneuvers, is tried; then the use of targeted pharmacologic therapy, particularly agents that support blood pressure or that drive blood pressure is attempted; and, finally, pacemaker implantation in patients with a predominant cardioinhibitory component to their syncopal episodes is performed. More recently, autonomic modulation with cardiac ganglion ablation has emerged as a promising treatment modality for patients refractory to traditional approaches. In this review, we sought to summarize the existing therapies for neurocardiogenic syncope and explore the latest research on new modalities of treatment.

No effect of thymosin beta-4 on the expression of the transcription factor Islet-1 in the adult murine heart.

The transcription factor Islet‐1 marks a progenitor cell population of the second heart field during cardiogenesis. In the adult heart Islet‐1 expression is limited to the sinoatrial node, the ventricular outflow tract, and parasympathetic ganglia. The regenerative effect in the injured mouse ventricle of thymosin beta‐4 (TB4), a 43‐aminoacid peptide, was associated with increased Islet‐1 immunostaining, suggesting the induction of an Islet‐1‐positive progenitor state by TB4. Here we aimed to reassess this effect in a genetic model. Mice from the reporter mouse line Isl1‐nLacZ were primed with TB4 and subsequently underwent myocardial infarction. Islet‐1 expression was assessed 2, 7, and 14 days after infarction. We detected only a single Islet‐1+ cell in 8 TB4 treated and infarcted hearts which located outside of the sinoatrial node, the outflow tract or cardiac ganglia (in ~2500 sections). Two cells were identified in 5 control infarcted hearts. TB4 did not induce LacZ positivity in ventricular explants cultures of Isl1‐nLacZ mice nor did it affect the density of LacZ+ cells in explant cultures of nLacZ+ regions of the heart. In summary, we found no evidence that TB4 reactivates Islet‐1 expression in adult mouse ventricle.

Electrophysiological effects of nicotinic and electrical stimulation of intrinsic cardiac ganglia in the absence of extrinsic autonomic nerves in the rabbit heart

BackgroundThe intrinsic cardiac nervous system is a rich network of cardiac nerves that converge to form distinct ganglia and extend across the heart and is capable of influencing cardiac function.ObjectiveThe goals of this study were to provide a complete picture of the neurotransmitter/neuromodulator profile of the rabbit intrinsic cardiac nervous system and to determine the influence of spatially divergent ganglia on cardiac electrophysiology.MethodsNicotinic or electrical stimulation was applied at discrete sites of the intrinsic cardiac nerve plexus in the Langendorff-perfused rabbit heart. Functional effects on sinus rate and atrioventricular conduction were measured. Immunohistochemistry for choline acetyltransferase (ChAT), tyrosine hydroxylase, and/or neuronal nitric oxide synthase (nNOS) was performed using whole mount preparations.ResultsStimulation within all ganglia produced either bradycardia, tachycardia, or a biphasic brady-tachycardia. Electrical stimulation of the right atrial and right neuronal cluster regions produced the largest chronotropic responses. Significant prolongation of atrioventricular conduction was predominant at the pulmonary vein-caudal vein region. Neurons immunoreactive (IR) only for ChAT, tyrosine hydroxylase, or nNOS were consistently located within the limits of the hilum and at the roots of the right cranial and right pulmonary veins. ChAT-IR neurons were most abundant (1946 ± 668 neurons). Neurons IR only for nNOS were distributed within ganglia.ConclusionStimulation of intrinsic ganglia, shown to be of phenotypic complexity but predominantly of cholinergic nature, indicates that clusters of neurons are capable of independent selective effects on cardiac electrophysiology, therefore providing a potential therapeutic target for the prevention and treatment of cardiac disease.

Single Cell Gene Expression Analysis and 3‐D Mapping of Cardiac Ganglia

The intrinsic nervous system within the heart influences cardiac function at both physiological and molecular levels. The human cardiac ganglionic innervation organized into functionally distinct ganglia is composed of 14,000 neurons. Recent studies suggest that ganglia work together in an interaction network to control cardiac function. It has been shown that cardiac neurons are functionally heterogeneous and that the specific neuronal network that influences each neuron can also be broadly heterogeneous. Functional understanding of the complexity within cardiac intrinsic nervous system and its communication to and from the autonomic centers of the central nervous system is incomplete. Additionally, there is sparse knowledge of the spatial organization of these neuronal phenotypes within and among cardiac ganglia.In this study we aim to identify distinct spatially organized neuronal transcriptional phenotypes with high resolution using a novel combination of immunohistochemistry ‐ labeling and 3‐D reconstruction of the whole rat heart along with transcriptomic data of single neuron samples collected from rat hearts. Block face imaging of several rat hearts along with cresyl‐violet stained sections is being used to construct a 3‐D atlas of the rat heart with ontological information and spatial neuronal locational information (in collaboration with MBF Biosciences). We are collecting single neurons as well as neuron pools (comprising of 5 or 10 cells) using Laser Capture Microdissection from rat hearts. These samples are being processed for transcriptome analysis (multiplexed RNA‐seq). We are using BioMark multiplex RT‐qPCR system to validate our transcriptional data obtained from RNA Seq. Transcriptional data along with spatial location of hundreds neurons per heart thus obtained will enable us to map molecular profiles of cardiac ganglia heterogeneity.We have demonstrated the ability to identify molecular phenotypes of neurons through preliminary examination of ~250 laser captured neurons using multiplex RT‐qPCR. We have also generated a more targeted list of cardiac ganglia specific genes through microarray experiments comparing neuron‐enriched sections of the heart with unenriched myocardium. Multiplexed RT‐qPCR data shows distinct molecular phenotypes emerge even with consideration of only ~40 genes. We were able to identify dopaminergic, catecholaminergic, and two types of cholinergic neurons from these data. Within each larger phenotype there exists heterogeneity of transcriptional states that support the need for further investigation.Along with circuit relevance of ganglia projections we will be able to generate a cardiac neuro‐reference framework atlas with neuronal distribution, density of innervation, receptive (sensory) or motor fields and terminal structures of axons from different peripheral ganglia and vagal nuclei. Our study will provide a new holistic view of vagal and spinal afferent, autonomic efferent (sympathetic and parasympathetic), and cardiac ganglionic innervation of the heart in 3‐D whole heart qualitatively and quantitatively.Support or Funding InformationSPARC‐OT2‐OD023848This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of Novel mRNA Transcripts in the Sympathetic Stellate Ganglia using RNA Sequencing

IntroductionSympathetic hyperactivity is a key feature of human hypertension as well as animal models of disease. In the spontaneously hypertensive rat (SHR), a genetic‐based model of hypertension, dysautonomia occurs prior to increases in blood pressure.AimIn this study, we aimed to establish the genetic changes in post‐ganglionic sympathetic neurons (PGSNs) obtained from SHR rats and to confirm the presence of these transcripts in stellate ganglia from human donor patients.MethodPGSNs were obtained from 16‐week normotensive Wistar rats and age‐matched hypertensive SHR with known sympathetic hyper‐responsiveness. RNA sequencing was used to investigate the transcriptomic changes within the cardiac sympathetic stellate ganglia in disease.ResultsRNAseq data were analysed using a Salmon‐DESeq2 pipeline1,2. We observed 768 differentially expressed mRNA transcripts in SHR compared with Wistar PGSNs, using the Benjamini‐Hochberg adjusted p&lt;0.01 level. Gene ontology (GO) analysis of the rat dataset revealed extracellular ligand‐gated ion channels (GO:0005302) to be the most over‐represented ‘molecular function’ GO group. There were 15 differentially expressed genes in SHR stellate neurons within this group; including nicotinic, purinergic, glutamatergic, serotonergic, GABAergic and glycinergic receptor subunits. The presence of these transcripts was confirmed using qRT‐PCR in stellate ganglia obtained from human and rat.ConclusionRNAseq transcriptomics has revealed differential gene expression profiles in PGSNs obtained from SHR and normotensive Wistar rats. These key transcripts were also present in human stellate ganglia. Taken together, these genes require further investigation for their role in sympathetic hyperactivity and dysautonomias.Support or Funding InformationThis project was funded by the Wellcome Trust OXION initiative (105409/Z/14/Z), the British Heart Foundation, NIH SPARC (OT2OD023848) and new innovator (DP2HL142045) initiatives.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Three members of a peptide family are differentially distributed and elicit differential state-dependent responses in a pattern generator-effector system.

C-type allatostatins (AST-Cs) are pleiotropic neuropeptides that are broadly conserved within arthropods; the presence of three AST-C isoforms, encoded by paralog genes, is common. However, these peptides are hypothesized to act through a single receptor, thereby exerting similar bioactivities within each species. We investigated this hypothesis in the American lobster, Homarus americanus, mapping the distributions of AST-C isoforms within relevant regions of the nervous system and digestive tract, and comparing their modulatory influences on the cardiac neuromuscular system. Immunohistochemistry showed that in the pericardial organ, a neuroendocrine release site, AST-C I and/or III and AST-C II are contained within distinct populations of release terminals. Moreover, AST-C I/III-like immunoreactivity was seen in midgut epithelial endocrine cells and the cardiac ganglion (CG), whereas AST-C II-like immunoreactivity was not seen in these tissues. These data suggest that AST-C I and/or III can modulate the CG both locally and hormonally; AST-C II likely acts on the CG solely as a hormonal modulator. Physiological studies demonstrated that all three AST-C isoforms can exert differential effects, including both increases and decreases, on contraction amplitude and frequency when perfused through the heart. However, in contrast to many state-dependent modulatory changes, the changes in contraction amplitude and frequency elicited by the AST-Cs were not functions of the baseline parameters. The responses to AST-C I and III, neither of which is COOH-terminally amidated, are more similar to one another than they are to the responses elicited by AST-C II, which is COOH-terminally amidated. These results suggest that the three AST-C isoforms are differentially distributed in the lobster nervous system/midgut and can elicit distinct behaviors from the cardiac neuromuscular system, with particular structural features, e.g., COOH-terminal amidation, likely important in determining the effects of the peptides.NEW & NOTEWORTHY Multiple isoforms of many peptides exert similar effects on neural circuits. In this study we show that each of the three isoforms of C-type allatostatin (AST-C) can exert differential effects, including both increases and decreases in contraction amplitude and frequency, on the lobster cardiac neuromuscular system. The distribution of effects elicited by the nonamidated isoforms AST-C I and III are more similar to one another than to the effects of the amidated AST-C II.

Simulated Microgravity Impairs Cardiac Autonomic Neurogenesis from Neural Crest Cells.

Microgravity-induced alterations in the autonomic nervous system (ANS) contribute to derangements in both the mechanical and electrophysiological function of the cardiovascular system, leading to severe symptoms in humans following space travel. Because the ANS forms embryonically from neural crest (NC) progenitors, we hypothesized that microgravity can impair NC-derived cardiac structures. Accordingly, we conducted in vitro simulated microgravity experiments employing NC genetic lineage tracing in mice with cKitCreERT2/+, Isl1nLacZ, and Wnt1-Cre reporter alleles. Inducible fate mapping in adult mouse hearts and pluripotent stem cells (iPSCs) demonstrated reduced cKitCreERT2/+-mediated labeling of both NC-derived cardiomyocytes and autonomic neurons (P < 0.0005 vs. controls). Whole transcriptome analysis, suggested that this effect was associated with repressed cardiac NC- and upregulated mesoderm-related gene expression profiles, coupled with abnormal bone morphogenetic protein (BMP)/transforming growth factor beta (TGF-β) and Wnt/β-catenin signaling. To separate the manifestations of simulated microgravity on NC versus mesodermal-cardiac derivatives, we conducted Isl1nLacZ lineage analyses, which indicated an approximately 3-fold expansion (P < 0.05) in mesoderm-derived Isl-1+ pacemaker sinoatrial nodal cells; and an approximately 3-fold reduction (P < 0.05) in cardiac NC-derived ANS cells, including sympathetic nerves and Isl-1+ cardiac ganglia. Finally, NC-specific fate mapping with a Wnt1-Cre reporter iPSC model of murine NC development confirmed that simulated microgravity directly impacted the in vitro development of cardiac NC progenitors and their contribution to the sympathetic and parasympathetic innervation of the iPSC-derived myocardium. Altogether, these findings reveal an important role for gravity in the development of NCs and their postnatal derivatives, and have important therapeutic implications for human space exploration, providing insights into cellular and molecular mechanisms of microgravity-induced cardiomyopathies/channelopathies.