Cardiac Ganglia Research Articles

Does Differential Receptor Distribution Underlie Variable Responses to a Neuropeptide in the Lobster Cardiac System?

Central pattern generators produce rhythmic behaviors independently of sensory input; however, their outputs can be modulated by neuropeptides, thereby allowing for functional flexibility. We investigated the effects of C-type allatostatins (AST-C) on the cardiac ganglion (CG), which is the central pattern generator that controls the heart of the American lobster, Homarus americanus, to identify the biological mechanism underlying the significant variability in individual responses to AST-C. We proposed that the presence of multiple receptors, and thus differential receptor distribution, was at least partly responsible for this observed variability. Using transcriptome mining and PCR-based cloning, we identified four AST-C receptors (ASTCRs) in the CG; we then characterized their cellular localization, binding potential, and functional activation. Only two of the four receptors, ASTCR1 and ASTCR2, were fully functional GPCRs that targeted to the cell surface and were activated by AST-C peptides in our insect cell expression system. All four, however, were amplified from CG cDNAs. Following the confirmation of ASTCR expression, we used physiological and bioinformatic techniques to correlate receptor expression with cardiac responses to AST-C across individuals. Expression of ASTCR1 in the CG showed a negative correlation with increasing contraction amplitude in response to AST-C perfusion through the lobster heart, suggesting that the differential expression of ASTCRs within the CG is partly responsible for the specific physiological response to AST-C exhibited by a given individual lobster.

Clinical analysis of cardiac autonomic ganglion plexus ablation for bradyarrhythmia: Research protocol for an intervention study.

This study aimed to explore the safety and effectiveness of selective cardiac autonomic ganglion plexus (GP) ablation on patients with bradyarrhythmia. The heart is controlled by its own intrinsic and central autonomic nerves. Increased cardiac vagal tone leads to sinus node dysfunction and atrioventricular conduction disorders, resulting in bradyarrhythmia. Pacemaker implantation can relieve the symptoms of arrhythmia caused by bradycardia, but it is not easy for patients to accept a pacemaker implantation as a form of treatment. Therefore, more and more attention has been paid to cardiac vagus nerve ablation. In this study, 20 patients who met the inclusion criteria of GP ablation in the First Affiliated Hospital of Xinjiang Medical University from November 2019 to June 2020 were enrolled. Biochemical and other related examinations along with electrophysiological examinations were conducted before ablation, and then cardiac GP ablation was performed. The patients were followed up 3 times at 3, 6, and 12 months after the operation. The minimum HR and mean HR were significantly increased after treatment with cardiac autonomic GP ablation (p<0.01). Moreover, the SDNN (Standard deviation of Normal-to-Normal Intervals) and RMSSD (Root mean square successive differences between successive R-R intervals) was significantly decreased after treatment with cardiac autonomic ganglion plexus ablation for 6 months and 12 months (p<0.01). Cardiac GP ablation is relatively simple and easy to implement in units that have performed radiofrequency ablation for bradyarrhythmias. This procedure can be performed without any new equipment. Some patients with bradycardia may not have a permanent pacemaker implantation and may go in for additional treatment options.

A single cell transcriptomics map of paracrine networks in the intrinsic cardiac nervous system

SummaryWe developed a spatially-tracked single neuron transcriptomics map of an intrinsic cardiac ganglion, the right atrial ganglionic plexus (RAGP) that is a critical mediator of sinoatrial node (SAN) activity. This 3D representation of RAGP used neuronal tracing to extensively map the spatial distribution of the subset of neurons that project to the SAN. RNA-seq of laser capture microdissected neurons revealed a distinct composition of RAGP neurons compared to the central nervous system and a surprising finding that cholinergic and catecholaminergic markers are coexpressed, suggesting multipotential phenotypes that can drive neuroplasticity within RAGP. High-throughput qPCR of hundreds of laser capture microdissected single neurons confirmed these findings and revealed a high dimensionality of neuromodulatory factors that contribute to dynamic control of the heart. Neuropeptide-receptor coexpression analysis revealed a combinatorial paracrine neuromodulatory network within RAGP informing follow-on studies on the vagal control of RAGP to regulate cardiac function in health and disease.

What Is the Clinical Correlation of Cardiac Noradrenergic Denervation in Parkinson Disease?

The autonomic nervous system controls cardiac performance on a beat-to-beat basis in order to match regional blood flow demand. To that end, there is continuous interaction among central autonomic areas (anterior insula, anterior cingulate cortex, amygdala, hypothalamus, periaqueductal gray matter, parabrachial nucleus, and medulla), preganglionic sympathetic and parasympathetic efferent neurons, and intrinsic cardiac ganglia.1,2 Cardiac sympathetic and parasympathetic outputs have been classically thought to act in a reciprocal antagonistic fashion, whereby sympathetic stimulation increases heart rate (chronotropy), atrioventricular conduction (dromotropy), and myocardial contractility (inotropy), whereas parasympathetic stimulation reduces heart rate and atrioventricular conduction. There is evidence, however, for a complex and redundant organization, as cardiac control is mediated by feedback loops organized at 3 main levels: the intrinsic cardiac nervous system, the intrathoracic extracardiac ganglia, and the central autonomic nervous system. These systems are in constant communication, and at each level, afferent signals provide feedback information to neurons that in turn affect efferent control of the heart.1,3

Suppression of Cardiac Sympathetic Ganglion Attenuates Cardiac Remodeling in a Circadian Disruption Model

Background: Circadian rhythms have a considerable impact on the daily physiology of the heart, and their disruption causes pathology. Sympathetic nervous system is essential for transmitting circadian information from central clock to peripheral organs. The role of cardiac sympathetic ganglion, a key target of cardiac sympathetic outflow, in circadian disruption-related cardiovascular diseases is not completely understood. Thereby, we aim todiscuss whether cardiac sympathetic ganglion plays an important role in the development of cardiac remodeling after myocardial infarction (MI)in a circadian disruption model. Methods: Rats were randomly divided into three groups: Sham group (Sham), MI group (MI), and MI+ circadian disruption group (MI+Dis); rats were treated with PRV injections for trans-synaptic retrograde tracing; rats were randomly divided into two groups: MI+ circadian disruption+ Empty Vector+ CNO (Empty Vector) and MI+ circadian disruption+ hM4D(Gi)+ CNO (hM4D(Gi)). Findings: Circadian disruption significantly facilitated cardiac remodeling after MI with lower systolic function, larger left ventricular volume, increased infarct size and aggravated cardiac fibrosis. Serum norepinephrine levels and cardiac sympathetic remodeling makers were also significantly increased by disruption. PRV virus-labeled neurons were identified in the SCG, PVN, and SCN regions. Ganglionic blockade via DREADD technique suppressed the cardiac sympathetic outflow and significantly alleviated the disruption-related cardiac dysfunction. Interpretation: Circadian disruption adversely affected cardiac remodeling after MI, possibly by increasing the cardiac sympathetic outflow, and cardiac sympathetic ganglionic blockade canattenuatethis disruption-related cardiac dysfunction. Funding Statement: National Key R&D Program of China, and National Natural Science Foundation of China. Declaration of Interests: None. Ethics Approval Statement: Experimental protocols were conducted under the guidelines of the National Institutes of Health and were approved by the Animal Care and Use Committees of Renmin Hospital of Wuhan University.

Cloning of the first cDNA encoding a putative CCRFamide precursor: identification of the brain, eyestalk ganglia, and cardiac ganglion as sites of CCRFamide expression in the American lobster, Homarus americanus.

Over the past decade, many new peptide families have been identified via in silico analyses of genomic and transcriptomic datasets. While various molecular and biochemical methods have confirmed the existence of some of these new groups, others remain in silico discoveries of computationally assembled sequences only. An example of the latter are the CCRFamides, named for the predicted presence of two pairs of disulfide bonded cysteine residues and an amidated arginine-phenylalanine carboxyl-terminus in family members, which have been identified from annelid, molluscan, and arthropod genomes/transcriptomes, but for which no precursor protein-encoding cDNAs have been cloned. Using routine transcriptome mining methods, we identified four Homarus americanus (American lobster) CCRFamide transcripts that share high sequence identity across the predicted open reading frames but more limited conservation in their 5' terminal ends, suggesting the Homarus gene undergoes alternative splicing. RT-PCR profiling using primers designed to amplify an internal fragment common to all of the transcripts revealed expression in the supraoesophageal ganglion (brain), eyestalk ganglia, and cardiac ganglion. Variant specific profiling revealed a similar profile for variant 1, eyestalk ganglia specific expression of variant 2, and an absence of variant 3 expression in the cDNAs examined. The broad distribution of CCRFamide transcript expression in the H. americanus nervous system suggests a potential role as a locally released and/or circulating neuropeptide. This is the first report of the cloning of a CCRFamide-encoding cDNA from any species, and as such, provides the first non-in silico support for the existence of this invertebrate peptide family.

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.

Genome-Wide Analysis Identifies an Essential Human TBX3 Pacemaker Enhancer.

The development and function of the pacemaker cardiomyocytes of the sinoatrial node (SAN), the leading pacemaker of the heart, are tightly controlled by a conserved network of transcription factors, including TBX3 (T-box transcription factor 3), ISL1 (ISL LIM homeobox 1), and SHOX2 (short stature homeobox 2). Yet, the regulatory DNA elements (REs) controlling target gene expression in the SAN pacemaker cells have remained undefined. Identification of the regulatory landscape of human SAN-like pacemaker cells and functional assessment of SAN-specific REs potentially involved in pacemaker cell gene regulation. We performed Assay for Transposase-Accessible Chromatin using sequencing on human pluripotent stem cell-derived SAN-like pacemaker cells and ventricle-like cells and identified thousands of putative REs specific for either human cell type. We validated pacemaker cell-specific elements in the SHOX2 and TBX3 loci. CRISPR-mediated homozygous deletion of the mouse ortholog of a noncoding region with candidate pacemaker-specific REs in the SHOX2 locus resulted in selective loss of Shox2 expression from the developing SAN and embryonic lethality. Putative pacemaker-specific REs were identified up to 1 Mbp upstream of TBX3 in a region close to MED13L harboring variants associated with heart rate recovery after exercise. The orthologous region was deleted in mice, which resulted in selective loss of expression of Tbx3 from the SAN and (cardiac) ganglia and in neonatal lethality. Expression of Tbx3 was maintained in other tissues including the atrioventricular conduction system, lungs, and liver. Heterozygous adult mice showed increased SAN recovery times after pacing. The human REs harboring the associated variants robustly drove expression in the SAN of transgenic mouse embryos. We provided a genome-wide collection of candidate human pacemaker-specific REs, including the loci of SHOX2, TBX3, and ISL1, and identified a link between human genetic variants influencing heart rate recovery after exercise and a variant RE with highly conserved function, driving SAN expression of TBX3.

Multiple transcriptome mining coupled with tissue specific molecular cloning and mass spectrometry provide insights into agatoxin-like peptide conservation in decapod crustaceans

Over the past decade, in silico genome and transcriptome mining has led to the identification of many new crustacean peptide families, including the agatoxin-like peptides (ALPs), a group named for their structural similarity to agatoxin, a spider venom component. Here, analysis of publicly accessible transcriptomes was used to expand our understanding of crustacean ALPs. Specifically, transcriptome mining was used to investigate the phylogenetic/structural conservation, tissue localization, and putative functions of ALPs in decapod species. Transcripts encoding putative ALP precursors were identified from one or more members of the Penaeoidea (penaeid shrimp), Sergestoidea (sergestid shrimps), Caridea (caridean shrimp), Astacidea (clawed lobsters and freshwater crayfish), Achelata (spiny/slipper lobsters), and Brachyura (true crabs), suggesting a broad, and perhaps ubiquitous, conservation of ALPs in decapods. Comparison of the predicted mature structures of decapod ALPs revealed high levels of amino acid conservation, including eight identically conserved cysteine residues that presumably allow for the formation of four identically positioned disulfide bridges. All decapod ALPs are predicted to have amidated carboxyl-terminals. Two isoforms of ALP appear to be present in most decapod species, one 44 amino acids long and the other 42 amino acids in length, both likely generated by alternative splicing of a single gene. In carideans, a gene or terminal exon duplication appears to have occurred, with alternative splicing producing four ALPs, two 44 and two 42 amino acid isoforms. The identification of ALP precursor-encoding transcripts in nervous system-specific transcriptomes (e.g., Homarus americanus brain, eyestalk ganglia, and cardiac ganglion assemblies, finding confirmed using RT-PCR) suggests that members of this peptide family may serve as locally-released and/or hormonally-delivered neuromodulators in decapods. Their detection in testis- and hepatopancreas-specific transcriptomes suggests that members of the ALP family may also play roles in male reproduction and innate immunity/detoxification.

Estresse térmico agudo altera epicárdio, vasos epicárdicos, aspectos morfológicos, morfométricos e quantitativos do plexo cardíaco de frangos de granja

O aprimoramento genético de aves tem sido usado com o objetivo de maximizar a produção de carne de frango. Assim, técnicas de manejo têm se destacado, pois melhoram a rentabilidade e reduzem os custos de produção, uma vez que otimizam o ganho de peso das aves e aceleram o abate. Todavia, o acelerado ganho de peso pode tornar os animais suscetíveis a transtornos metabólicos em decorrência da falta de adaptação do aparelho cardiorrespiratório cujo funcionamento se relaciona à temperatura ambiente e à inervação cardíaca. Desta forma, o objetivo deste estudo foi avaliar a influência do estresse térmico agudo sobre o coração de frangos de granja, avaliando o epicárdio, os vasos epicárdicos e os aspectos quantitativos, morfológicos, morfométricos e histopatológicos do plexo cardíaco. Para tanto, foram utilizados 14 animais machos divididos em dois grupos (n=7): animais mantidos em ambiente de conforto térmico (18oC) e animais em ambiente de estresse térmico (mantidos a 32o C nas 12 horas prévias ao abate). Os resultados mostraram que o estresse térmico diminuiu a área dos adipócitos, a área e o comprimento dos gânglios cardíacos, aumentou o colágeno total dos adipócitos e diminuiu o colágeno total dos vasos e gânglios do coração. Pode-se concluir que de fato o estresse térmico agudo interferiu nos parâmetros avaliados, pois várias alterações histopatológicas foram identificadas tais como a presença de infiltrados linfocitários nos adipócitos do tecido epicárdico, edema, congestão vascular, infiltração de fibrina nos gânglios do plexo cardíaco e ganglionite.

Differential neuropeptide modulation of premotor and motor neurons in the lobster cardiac ganglion.

The American lobster, Homarus americanus, cardiac neuromuscular system is controlled by the cardiac ganglion (CG), a central pattern generator consisting of four premotor and five motor neurons. Here, we show that the premotor and motor neurons can establish independent bursting patterns when decoupled by a physical ligature. We also show that mRNA encoding myosuppressin, a cardioactive neuropeptide, is produced within the CG. We thus asked whether myosuppressin modulates the decoupled premotor and motor neurons, and if so, how this modulation might underlie the role(s) that these neurons play in myosuppressin’s effects on ganglionic output. Although myosuppressin exerted dose-dependent effects on burst frequency and duration in both premotor and motor neurons in the intact CG, its effects on the ligatured ganglion were more complex, with different effects and thresholds on the two types of neurons. These data suggest that the motor neurons are more important in determining the changes in frequency of the CG elicited by low concentrations of myosuppressin, whereas the premotor neurons have a greater impact on changes elicited in burst duration. A single putative myosuppressin receptor (MSR-I) was previously described from the Homarus nervous system. We identified four additional putative MSRs (MSR-II–V) and investigated their individual distributions in the CG premotor and motor neurons using RT-PCR. Transcripts for only three receptors (MSR-II–IV) were amplified from the CG. Potential differential distributions of the receptors were observed between the premotor and motor neurons; these differences may contribute to the distinct physiological responses of the two neuron types to myosuppressin.NEW & NOTEWORTHY Premotor and motor neurons of the Homarus americanus cardiac ganglion (CG) are normally electrically and chemically coupled, and generate rhythmic bursting that drives cardiac contractions; we show that they can establish independent bursting patterns when physically decoupled by a ligature. The neuropeptide myosuppressin modulates different aspects of the bursting pattern in these neuron types to determine the overall modulation of the intact CG. Differential distribution of myosuppressin receptors may underlie the observed responses to myosuppressin.

Neuropeptidomic Profiling and Localization in the Crustacean Cardiac Ganglion Using Mass Spectrometry Imaging with Multiple Platforms.

The crustacean cardiac neuromuscular system is a useful model for studying how neural circuits generate behavior, as it is comprised of a simple ganglion containing nine neurons, yet acts as a robust central pattern generator. The crustacean heart is neurogenic, receiving input from neuropeptides. However, the specific effects of neuropeptides on cardiac output is not fully understood, and the large degree of comodulation between multiple neuropeptides makes studying these effects more challenging. To address this challenge, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) imaging was used to localize neuropeptides within the cardiac ganglion (CG), providing information about the identity and localization of neuropeptides being present. CG extracts were also profiled using liquid chromatography coupled to tandem mass spectrometry (MS/MS) with a data independent acquisition method, resulting in the confirmation of 316 neuropeptides. Two MS imaging (MSI) platforms were compared to provide comprehensive results, including a MALDI-Orbitrap instrument for high mass spectral resolution for accurate identifications and a MALDI TOF/TOF instrument for improved spatial resolution and sensitivity, providing more descriptive MS images. MS images for 235 putative neuropeptides were obtained, with the identification of 145 of these being confirmed by either complementary MS/MS data or accurate mass matching. The MSI studies demonstrate the sensitivity and power of this MALDI-based in situ analytical strategy for unraveling the chemical complexity present in a small nine-cell neuronal system. The results of this study will enable more informative assays of the functions of neuropeptides within this important neural circuit.

Molecular Regulation of α3β4 Nicotinic Acetylcholine Receptors by Lupeol in Cardiovascular System.

Cardiovascular disease (CVD) occurs globally and has a high mortality rate. The highest risk factor for developing CVD is high blood pressure. Currently, natural products are emerging for the treatment of hypertension to avoid the side effects of drugs. Among existing natural products, lupeol is known to be effective against hypertension in animal experiments. However, there exists no study regarding the molecular physiological evidence against the effects of lupeol. Consequently, we investigated the interaction of lupeol with α3β4 nicotinic acetylcholine receptors (nAChRs). In this study, we performed a two-electrode voltage-clamp technique to investigate the effect of lupeol on the α3β4 nicotine acetylcholine receptor using the oocytes of Xenopus laevis. Coapplication of acetylcholine and lupeol inhibited the activity of α3β4 nAChRs in a concentration-dependent, voltage-independent, and reversible manner. We also conducted a mutational experiment to investigate the influence of residues of the α3 and β4 subunits on lupeol binding with nAChRs. Double mutants of α3β4 (I37A/N132A), nAChRs significantly attenuated the inhibitory effects of lupeol compared to wild-type α3β4 nAChRs. A characteristic of α3β4 nAChRs is their effect on transmission in the cardiac sympathetic ganglion. Overall, it is hypothesized that lupeol lowers hypertension by mediating its effects on α3β4 nAChRs. The interaction between lupeol and α3β4 nAChRs provides evidence against its effect on hypertension at the molecular-cell level. In conclusion, the inhibitory effect of lupeol is proposed as a novel therapeutic approach involving the antihypertensive targeting of α3β4 nAChRs. Furthermore, it is proposed that the molecular basis of the interaction between lupeol and α3β4 nAChRs would be helpful in cardiac-pharmacology research and therapeutics.

235Anatomic guided ablation of the right ganglionated plexus is enough for cardiac autonomic denervation in patients with significant bradyarrhythmias

Abstract Background In patients with significant bradyarrhytmias, cardiac denervation is an alternative therapeutic approach. Previous reports proposed different methods (as high frequency ednocardial stimulation of ganglionated plexus and specific atrial electrogram identification) and targets (right and left atrial ganglionated plexus) for adequate denervation. There is no consensus on the best way to perform these procedures, in spite the right atrial ganglia plexus (GP) ablation seeming to be the most contributive to its success. Purpose To assess the results of a purely anatomic approach for ablation of just the right atrial plexus in patients with severe vagal bradyarrhytmias. Methods We enrolled patients referred for ablation of cardiac parasympathetic ganglia, with or without atrial fibrillation ablation. We performed eletroanatomic mapping of the right and left atria and used an irrigated tip catheter for ablation, aiming at the anterior right GP at the right pulmonary veins antrum along with ablation at the superior vena cava junction and the inferior right GP at the posterior aspect of the right inferior pulmonary vein along with ablation of the right aspect of the interatrial septum, between the posterior wall and coronary sinus ostium (Figure 1). We assessed the PW and Wenckenback cycle lengths (CL) pre and post procedure in patients with sinus arrest or AV block, respectively, and the patients had new 24h holter readings at least 30 days from the index procedure. Results We enrolled 12 patients: 9 males (75%), median age of 49,5 years (IQR 36-61,75). All patients had structurally normal hearts. 7 patients had only ablation of the parasympathetic ganglia and 5 patients had simultaneous pulmonary vein isolation for previously documented atrial fibrillation. 7 patients (58,3%) had sinus bradycardia (2 patients had sinus arrest with pauses of 8 and 13 seconds), 2 patients with cardioinhibitory syncope (with pauses of 23 and 28 seconds) and 3 patients had transient high degree AV block. The ablation procedure led to a median sinus rate acceleration of 15 bpm (IQR 3-29), a median decrease of 320 ms in PW (IQR 23,75-609,5) in patients with sinus arrest and a decrease of 80 ms in wenckenback CL (IQR 60-200) in patients with AV block. With a median follow up of 133,50 days (IQR 36-61,75), no patient had recurrence of symptoms or conduction disturbances. Conclusions In selected patients with severe functional paroxysmal bradyarrhytmias, cardiac denervation using an ablation strategy purely based on anatomic aspects and targeting only the right GP, seems to be an effective therapeutic approach. Abstract Figure 1: Abl of right ganglionated plex

Cardiac Neuroanatomy for the Cardiac Electrophysiologist.

The cardiac neuraxis is integral to cardiac physiology, and its dysregulation is implicated in cardiovascular disease. Neuromodulatory therapies are being developed that target the cardiac autonomic nervous system (ANS) to treat cardiac pathophysiology. An appreciation of the cardiac neuroanatomy is a prerequisite for development of such targeted therapies. Here, we provide a review of the current understanding of the cardiac ANS. The parasympathetic and sympathetic nervous system are composed of higher order cortical centers, brainstem, spinal cord, intrathoracic extracardiac ganglia and intrinsic cardiac ganglia. A series of interacting feedback loops mediates reflex pathways to exert control over the cardiac conduction system and contractile tissue. Further exploration of this complex regulatory system promises to yield neuroscience-based therapeutics for cardiac disease.

Strategies for Autonomic Nervous System Mapping of Fiber Composition and Neural Circuitry Using Transmission Electron Microscopy in Support of SPARC Projects

The optimization of neuromodulation strategies is important for a development and refinement of therapies. The design of customized tools, such as electrical stimulating devices, depends on the detailed knowledge of nerve fiber composition and inter‐relationships between target organs, nerve populations, and synaptic circuitry. The autonomic nervous system gives support to organs and are composed by myelinated and unmyelinated fibers as well as pre‐ and post‐ganglionic neurons. Mapping an autonomic nerve such as the pelvic or vagus nerve that innervates peripheral tissues and understanding the synaptology of neurons in the cardiac and stellate ganglia may provide advanced knowledge for improved neuromodulation strategies. Transmission electron microscopy (TEM) is a high‐level technology that helps to identify and characterize axonal myelination and ultrastructure of unmyelinated fibers. TEM also allows for fine structural analysis of neural circuitry and innervation of autonomic targets. Immunogold detection techniques using TEM permits the identification and characterization of neuronal tracers in the nervous system. With the support from the Stimulating Peripheral Activity to Relieve Conditions (SPARC) program at NIH, we developed customized ultrastructural studies of the autonomic nervous system to facilitate refinement of neuromodulation strategies related to the autonomic control of cardiac, gastrointestinal, and lower urinary tract systems. Morphometric analysis is performed by automatic segmentation, and the development of a new strategy to calculate measurements of axon diameter, fiber diameter, myelin thickness, and G‐ratio provides a better understanding of fiber size for modelling studies. Both males and females from different species, including humans, are analyzed and compared. Implementation of common sampling techniques for nerves and ganglia across species provides increased rigor and reproducibility. TEM studies promote an improved understanding of functional studies and provide critical ultrastructural information for the development of new neuromodulation strategies.Support or Funding InformationSPARC/NIH 1OT2OD026585‐01 and Dr Miriam and Sheldon G. Adelson Medical Research Foundation (AMRF)

Evidence that Sympathetic Activity is Increased Selectively to the Heart in a Mouse Model of Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM), the most common genetic heart disease, is characterized by cardiac hypertrophy, fibrosis, and increased risk of arrhythmias and sudden death, even in the absence of heart failure. We have studied young (&lt;20 wks) transgenic mice with cardiac‐specific expression of mutated alpha‐tropomyosin (Glu180Gly), an established model of HCM, to investigate autonomic mechanisms. In early experiments, abnormal heart rate and blood pressure responses to β‐adrenergic receptor blockade and ganglionic blockade indicated high cardiac accompanied by low vasomotor sympathetic tone in HCM vs. wild‐type (WT) mice. The aim of this study was to further characterize regional sympathetic activity in HCM vs. WT littermates by measuring expression/activity of tyrosine hydroxylase (TH, rate‐limiting enzyme for norepinephrine (NE) synthesis) and the norepinephrine transporter (NET) in sympathetic ganglia (stellate, superior cervical, celiac/mesenteric), and expression of β1 receptors in left ventricle (LV). Results (below) are expressed relative to WT (n=4 male mice each group). TH protein expression (Western blot) (standardized to neuronal content with PGP9.5) was significantly higher in stellate (cardiac) ganglia in HCM mice (HCM 162±12 vs. WT 100±7; p&lt;0.05), but not significantly different in superior cervical (HCM 82±4 vs. WT 100±14; p&gt;0.05) or celiac‐mesenteric (HCM 95±7 vs. WT 100±4; p&gt;0.05) ganglia. TH/PGP9.5 protein expression was also higher in the LV of HCM (135±15) vs. WT (100±1) mice (p&lt;0.05). LV β1 receptor protein expression was significantly lower in HCM (60±1) vs. WT (100±4) mice (p&lt;0.05), consistent with increased local NE levels. NET protein (immunofluorescence) was lower in stellate ganglia from HCM mice vs. WT (n=3 mice per group, 3 sections/mouse, p&lt;0.05). NET activity was assessed by measuring the rate of uptake of a fluorescent NET substrate (Molecular Devices) into individual sympathetic neurons isolated from stellate and superior cervical ganglia. NET activity measured in stellate neurons was significantly lower in HCM (4.5±0.3 a.u.*min−1, n=115 neurons, from 4 mice) vs. WT (5.3±0.4 a.u.*min−1, n=98 neurons, from 4 mice). NET activity in superior cervical ganglia did not differ between genotypes. In conclusion, increased TH expression (enhanced ‐NE synthesis) and decreased NET expression/activity (reduced NE reuptake) in stellate ganglia are consistent with increased cardiac sympathetic activity in HCM, whereas the lack of differences between HCM and WT in other sympathetic ganglia are consistent with normal sympathetic activity to non‐cardiac target organs in HCM.Support or Funding InformationNIH F32HL140880

In silico analyses suggest the cardiac ganglion of the lobster, Homarus americanus, contains a diverse array of putative innexin/innexin-like proteins, including both known and novel members of this protein family.

Gap junctions are physical channels that connect adjacent cells, permitting the flow of small molecules/ions between the cytoplasms of the coupled units. Innexin/innexin-like proteins are responsible for the formation of invertebrate gap junctions. Within the nervous system, gap junctions often function as electrical synapses, providing a means for coordinating activity among electrically coupled neurons. While some gap junctions allow the bidirectional flow of small molecules/ions between coupled cells, others permit flow in one direction only or preferentially. The complement of innexins present in a gap junction determines its specific properties. Thus, understanding innexin diversity is key for understanding the full potential of electrical coupling in a species/system. The decapod crustacean cardiac ganglion (CG), which controls cardiac muscle contractions, is a simple pattern-generating neural network with extensive electrical coupling among its circuit elements. In the lobster, Homarus americanus, prior work suggested that the adult neuronal innexin complement consists of six innexins (Homam-Inx1-4 and Homam-Inx6-7). Here, using a H. americanus CG-specific transcriptome, we explored innexin complement in this portion of the lobster nervous system. With the exception of Homam-Inx4, all of the previously described innexins appear to be expressed in the H. americanus CG. In addition, transcripts encoding seven novel putative innexins (Homam-Inx8-14) were identified, four (Homam-Inx8-11) having multiple splice variants, e.g., six for Homam-Inx8. Collectively, these data indicate that the innexin complement of the lobster nervous system in general, and the CG specifically, is likely significantly greater than previously reported, suggesting the possibility of expanded gap junction diversity and function in H. americanus.

Predestinating Role of Cardiac Ganglia on Heart Life Expectancy in Rabbits After Brain Death Following Subarachnoid Hemorrhage: An Experimental Study

BackgroundCardiac ganglia are rechargeable batteries of the heart. The essential role of cardiac ganglia on cardiac life expectancy has not been examined following brain death. The aim of this study was to determine cardiac ganglia numbers and neuron density following subarachnoid hemorrhage (SAH). MethodsTwenty-five hybrid rabbits were grouped as control (n = 5), sham (n = 5), and SAH (n = 15). The SAH groups’ animals were subjected to injections of lethal dose of 2.00 cc autologous blood into their cisterna magna until linear EEG was obtained. The hearts of all animals were extracted following intracardiac formalin injection and examined. Cardiac ganglia and normal/degenerated neuron densities of cardiac neurons were recorded. ResultsThe mean volume of normal neuron density of ganglia was 6.980 ± 830/mm3, and the degenerated neuron density of ganglia was 3 ± 1/mm3 in the control group, 6134 ± 712/mm3; 23 ± 9/mm3 in the sham group, 3456 ± 589; 1161 ± 72/mm3 in the surviving group; and 1734 ± 341/mm3, 4259 ± 865/mm3 in the dead animals in the SAH group. The algebraic results of heart work capacity (Wh) were estimated as 1375 ± 210 Wh in the control group, 1036 ± 225 in the sham group, 800 ± 110 Wh in the surviving group, and < 100 ± 20 in the dead animals in the SAH group. Degenerated cardiac neuron density/Wh correlation is statistically meaningful between the dead in the SAH group versus the SAH-surviving, sham, and control groups (P < .0005). ConclusionsNormal cardiac ganglia numbers and/or cardiac ganglia neuron density may be related to cardiac survival following brain death after subarachnoid hemorrhage.

IL-1-dependent electrophysiological changes and cardiac neural remodeling in a mouse model of Kawasaki disease vasculitis.

Kawasaki disease (KD) is the leading cause of acquired heart disease in children. In addition to coronary artery abnormalities, aneurysms and myocarditis, acute KD is also associated with echocardiogram (ECG) abnormalities in 40-80% of patients. Here, we show that these ECG changes are recapitulated in the Lactobacillus casei cell wall extract (LCWE)-induced KD vasculitis mouse model. LCWE-injected mice developed elevated heart rate and decreased R wave amplitude, with significant differences in prolonged ventricular repolarization. LCWE-injected mice developed cardiac ganglion inflammation, that may affect the impulse-conducting system in the myocardium. Furthermore, serum nerve growth factor (NGF) was significantly elevated in LCWE-injected mice, similar to children with KD vasculitis, associated with increased neural remodeling of the myocardium. ECG abnormalities were prevented by blocking interleukin (IL)-1 signaling with anakinra, and the increase in serum NGF and cardiac neural remodeling were similarly blocked in Il1r1-/- mice and in wild-type mice treated with anakinra. Thus, similar to clinical KD, the LCWE-induced KD vasculitis mouse model also exhibits electrophysiological abnormalities and cardiac neuronal remodeling, and these changes can be prevented by blocking IL-1 signaling. These data support the acceleration of anti-IL-1 therapy trials to benefit KD patients.