Intrinsic Cardiac Nervous System Research Articles

Activation of PACAP+ vagal motoneurons following remote ischemic pre-conditioning

The Dorsal Motor Nucleus of the Vagus nerve (DMV) provides parasympathetic innervation of the intrinsic cardiac nervous system (ICN), and it has been shown that DMV activity is required for the cardioprotection induced by remote ischemic pre-conditioning (RIPC) against the pathological effects of myocardial infarction. However, the mechanism behind the DMV’s cardioprotective effects is not well understood. Previous literature has shown that the DMV and the other vagal motor nuclei, the nucleus ambiguus (NA), innervate distinct cells within the ICN. DMV neurons project to many other organs in addition to the heart. The cardioprotective neuropeptide Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) has been found to be expressed in the fibers innervating the ICN, where it is co-localized with the parasympathetic marker, ChAT. While we have found PACAP to be expressed in cardiac-projecting DMV neurons, it is unknown whether these neurons are preferentially activated in response to RIPC. To assess the activity of these neurons following RIPC, we performed immunofluorescence staining of brainstem tissue containing DMV neurons for both PACAP, and c-FOS, a neuronal activity-dependent marker. We hypothesized that PACAP+ DMV neurons would have increased activity following RIPC, and therefore increased c-FOS intensity and increased co-localization with PACAP compared to sham control. Our results suggest that PACAP+ neurons in the DMV show increased activity following RIPC compared to sham, as defined by increased c-FOS fluorescence intensity and co-localization with PACAP. These results suggest that PACAP+ neurons are activated during the cardioprotective response to remote ischemic pre-conditioning. Funding was provided by the R01 HL161696 grant through National Heart, Lung, and Blood institute and the National Institutes of Health Common Fund SPARC Program Grant OT2OD030534. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Intrinsic Cardiac Nervous System: From Pathophysiology to Therapeutic Implications.

The cardiac autonomic nervous system (CANS) plays a pivotal role in cardiac homeostasis as well as in cardiac pathology. The first level of cardiac autonomic control, the intrinsic cardiac nervous system (ICNS), is located within the epicardial fat pads and is physically organized in ganglionated plexi (GPs). The ICNS system does not only contain parasympathetic cardiac efferent neurons, as long believed, but also afferent neurons and local circuit neurons. Thanks to its high degree of connectivity, combined with neuronal plasticity and memory capacity, the ICNS allows for a beat-to-beat control of all cardiac functions and responses as well as integration with extracardiac and higher centers for longer-term cardiovascular reflexes. The present review provides a detailed overview of the current knowledge of the bidirectional connection between the ICNS and the most studied cardiac pathologies/conditions (myocardial infarction, heart failure, arrhythmias and heart transplant) and the potential therapeutic implications. Indeed, GP modulation with efferent activity inhibition, differently achieved, has been studied for atrial fibrillation and functional bradyarrhythmias, while GP modulation with efferent activity stimulation has been evaluated for myocardial infarction, heart failure and ventricular arrhythmias. Electrical therapy has the unique potential to allow for both kinds of ICNS modulation while preserving the anatomical integrity of the system.

Reduction in atrial and ventricular electrical heterogeneity following pulmonary vein isolation in patients with atrial fibrillation

Pulmonary vein isolation (PVI) modulates the intrinsic cardiac autonomic nervous system and reduces atrial fibrillation (AF) recurrence. In this retrospective analysis, we investigated the impact of PVI on ECG interlead P-wave, R-wave, and T-wave heterogeneity (PWH, RWH, TWH) in 45 patients in sinus rhythm undergoing clinically indicated PVI for AF. We measured PWH as a marker of atrial electrical dispersion and AF susceptibility and RWH and TWH as markers of ventricular arrhythmia risk along with standard ECG measures. PVI acutely (16 ± 8.9h) reduced PWH by 20.7% (from 31 ± 1.9 to 25 ± 1.6µV, p < 0.001) and TWH by 27% (from 111 ± 7.8 to 81 ± 6.5µV, p < 0.001). RWH was unchanged after PVI (p = 0.068). In a subgroup of 20 patients with longer follow-up (mean = 47 ± 3.7days after PVI), PWH remained low (25 ± 1.7µV, p = 0.01), but TWH partially returned to the pre-ablation level (to 93 ± 10.2, p = 0.16). In three individuals with early recurrence of atrial arrhythmia in the first 3months after ablation, PWH increased acutely by 8.5%, while in patients without early recurrence, PWH decreased acutely by 22.3% (p = 0.048). PWH was superior to other contemporary P-wave metrics including P-wave axis, dispersion, and duration in predicting early AF recurrence. The rapid time course of decreased PWH and TWH after PVI suggests a beneficial influence likely mediated via ablation of the intrinsic cardiac nervous system. Acute responses of PWH and TWH to PVI suggest a favorable dual effect on atrial and ventricular electrical stability and could be used to track individual patients' electrical heterogeneity profile.

Denervation or stimulation? Role of sympatho-vagal imbalance in HFpEF with hypertension.

Heart failure (HF) in the elderly is an increasingly large and complex problem in modern society. Notably, the cause of HF with preserved ejection fraction (HFpEF) is multifactorial and its pathophysiology is not fully understood. Among these, hypertension has emerged as a pivotal factor in the pathophysiology and therapeutic targets of HFpEF. Neuronal elements distributed throughout the cardiac autonomic nervous system, from the level of the central autonomic network including the insular cortex to the intrinsic cardiac nervous system, regulate the human cardiovascular system. Specifically, increased sympathetic nervous system activity due to sympatho-vagal imbalance is suggested to be associated the relationship between hypertension and HFpEF. While several new pharmacological therapies, such as sodium-glucose cotransporter 2 inhibitors, have been shown to be effective in HFpEF, neuromodulatory therapies of renal denervation and vagus nerve stimulation (VNS) have received recent attention. The current review explores the pathophysiology of the brain-heart axis that underlies the relationship between hypertension and HFpEF and the rationale for therapeutic neuromodulation of HFpEF by non-invasive transcutaneous VNS.

Recording plasticity in neuronal activity in the rodent intrinsic cardiac nervous system using calcium imaging techniques.

The intrinsic cardiac nervous system (ICNS) is composed of interconnected clusters of neurons called ganglionated plexi (GP) which play a major role in controlling heart rate and rhythm. The function of these neurons is particularly important due to their involvement in cardiac arrhythmias such as atrial fibrillation (AF), and previous work has shown that plasticity in GP neural networks could underpin aberrant activity patterns that drive AF. As research in this field increases, developing new techniques to visualize the complex interactions and plasticity in this GP network is essential. In this study we have developed a calcium imaging method enabling the simultaneous recording of plasticity in neuronal activity from multiple neurons in intact atrial GP networks. Calcium imaging was performed with Cal-520 AM labeling in aged spontaneously hypertensive rats (SHRs), which display both spontaneous and induced AF, and age-matched Wistar Kyoto (WKY) controls to determine the relationship between chronic hypertension, arrhythmia and GP calcium dynamics. Our data show that SHR GPs have significantly larger calcium responses to cholinergic stimulation compared to WKY controls, as determined by both higher amplitude and longer duration calcium responses. Responses were significantly but not fully blocked by hexamethonium, indicating multiple cholinergic receptor subtypes are involved in the calcium response. Given that SHRs are susceptible to cardiac arrhythmias, our data provide evidence for a potential link between arrhythmia and plasticity in calcium dynamics that occur not only in cardiomyocytes but also in the GP neurons of the heart.

Closed-loop modeling of central and intrinsic cardiac nervous system circuits underlying cardiovascular control.

The baroreflex is a multi-input, multi-output control physiological system that regulates blood pressure by modulating nerve activity between the brainstem and the heart. Existing computational models of the baroreflex do not explictly incorporate the intrinsic cardiac nervous system (ICN), which mediates central control of the heart function. We developed a computational model of closed-loop cardiovascular control by integrating a network representation of the ICN within central control reflex circuits. We examined central and local contributions to the control of heart rate, ventricular functions, and respiratory sinus arrhythmia (RSA). Our simulations match the experimentally observed relationship between RSA and lung tidal volume. Our simulations predicted the relative contributions of the sensory and the motor neuron pathways to the experimentally observed changes in the heart rate. Our closed-loop cardiovascular control model is primed for evaluating bioelectronic interventions to treat heart failure and renormalize cardiovascular physiology.

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.

Cryoballoon ablation for atrial fibrillation: Effects on neuromodulation.

Pulmonary vein isolation (PVI) represents the mainstay of atrial fibrillation (AF) ablation, and PVI with cryoballoon catheter (CB) ablation (CB-A) has proven to be as effective and safe as radiofrequency ablation (RF-A). Although AF is initiated by triggers arising from the pulmonary veins (PV) and non-PV foci, the intrinsic cardiac nervous system (ICNS) plays a significant role in the induction and maintenance of AF. The ICNS is an epicardial neural system composed of ganglionated plexi (GPs) and a complex network of interconnecting neurons. In the left atrium, the major GPs are located in proximity to the PV-left atrial junction. Vagal reactions have been described as markers of autonomic modulation during PVI with both RF-A and CB-A. The occurrence of neuromodulation during PVI with CB-A may be explained by both the anatomical relationship between the GPs and the PVs and the characteristics of the CB. Due to the CB/PV size mismatch, the CB creates a wide ablation area that extends from the PV ostium toward the antrum, possibly including the GPs. Although targeted GPs ablation, as a supplemental strategy to PVI, has been associated with a better AF outcome in patients undergoing RF-A, the additional clinical benefit of neuromodulation during PVI with CB-A remains a matter of debate. In this review, we provide an overview of the anatomy of the ICNS, the relationship between the ICNS and AF pathophysiology, and the current evidence on the clinical relevance of neuromodulation during PVI with CB-A.

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.

The triangle correlation among the impact on serum S100B protein release, incidence of the silent cerebral ischemic phenomena and pulsed field ablation for patients with atrial fibrillation

Abstract Funding Acknowledgements Type of funding sources: None. Backgrounds: Pulsed field ablation (PFA) represents a cardiac specific energy source for atrial fibrillation (AF) ablation. The correlation among this remarkable technique, the incidence of silent cerebral ischemic phenomena (SCIP), and the clinical impact on serum S100B release, a well-known neural injury relevant biomarker concomitant to denervation of the intrinsic cardiac autonomic nervous system during AF ablation, has been unclear. Aims This study thought to reveal the "triangle correlation" by investigating the serum S100B increase, incidence of SCIP on cerebral magnetic resonance imaging (MRI), and its interactive correlation in PFA using the FARAPULSE system. Methods and Results Pre- and postprocedural serum S100B was analyzed in a total of 85 patients who underwent AF ablation using the FARAPULSE PFA device. Cerebral MRI was performed subsequently 24-48 hours after procedure. All PVs (332 PVs) were successfully isolated solely with PFA. No additional left atrial mapping using other catheters was performed. The median overall serum S100B increase from the preprocedural value was 0.01μg/l (-0.02 – 0.04μg/l). Asymptomatic SCIP were detected on MRI in 10/53 patients (18.9 %). Of those, the patients with SCIP demonstrated a significantly higher S100B release (0.04, 0.02 – 0.16μg/l vs. without SCIP:0.01, -0.04 – 0.03μg/l, p = 0.0038). Receiver operating characteristic curve revealed a predictive cut-off point for SCIP of 0.04 μg/l (sensitivity:0.7, specificity: 0.49). Conclusions We revealed no clear detectable impact of PFA on S100B release, highlighting the cardioselectivity with limited concomitant denervation. SCIP was detected approximately in a one-fifth after procedures and was a potential confounder of the higher S100B increase.

Anatomical and Molecular Phenotypes of Fast and Slow Vagal Targets in the Intrinsic Cardiac Nervous System

The vagus nerve coordinates the communication between the brain and most of the visceral organs. Vagus nerve activity is critical to promote cardiac health and cardioprotective response in heart failure. The vagal motor outflow involves two nuclei, the nucleus ambiguous (NA) and the dorsal motor nucleus of the vagus (DMV). NA acts via nicotinic cholinergic processes to elicit fast kinetics of the intrinsic cardiac nervous system (ICN) response at the heart. By contrast, the DMV influences the ICN neurons via muscarinic cholinergic processes yielding slower kinetics of response. Interestingly, NA and DMV project to distinct neuronal populations in the ICN, providing an anatomical basis for the fast versus slow vagal action on the heart. We aim to delineate the ICN neuronal network mediating the two lanes of vagal inputs for regulating distinct cardiac functions with lane‐specific transcriptomic identities of ICN neuron targets. We performed immunohistochemistry (IHC) on the rat heart tissue sections embedded in the optimal cutting temperature (OCT) followed by confocal microscopy imaging. We found that while the ICN neurons express muscarinic and nicotinic acetylcholine receptors (AChRs) broadly across the ICN, with several neurons predominantly expressing either muscarinic or nicotinic AChRs. We used Laser Capture Microscopy (LCM) to acquire neurons that predominantly express muscarinic or nicotinic AChRs. We are profiling the transcriptomic identities of these LCM‐captured neuron classes. Our results suggest that mAChR‐only and nAChR‐only neurons differentially express neuromodulatory processes, including receptors, neurotransmitter synthesis enzymes, and neuropeptides. Further, we incorporate a systems approach combining spatial single‐cell transcriptomics, neuronal network modeling, and physiological control systems modeling to identify selective targets for novel neuromodulatory interventions that improve heart health.

Modeling and Analysis of Closed‐Loop Control of the Cardiovascular System

The baroreceptor reflex is a multi‐input, multi‐output physiological control system that regulates short‐term blood pressure by modulating parasympathetic and sympathetic nerve activity between the brainstem and the heart. The opposing effects of parasympathetic and sympathetic nerve activity work in conjunction to maintain cardiovascular homeostasis, with imbalances in activity associated with cardiovascular disease. Recently, attention has focused on the role of the intrinsic cardiac nervous system (ICN) in local control of nervous regulation of the heart and its role in balancing parasympathetic and sympathetic nerve activity. However, it is unknown how the ICN network structure contributes to integrative control of the heart. We formulated multiple alternative network options based on the anatomical, molecular and physiological evidence. We extended a quantitative closed‐loop computational model of the baroreceptor reflex by incorporating a high‐fidelity representation of the ICN to evaluate the impact of altered ICN network structures on overall cardiovascular control. The present computational model consists of (1) a system of ordinary differential equations to represent blood flow in the cardiovascular system, and (2) transfer function representations of sensory neurons, central nervous system neuronal groups, and ICN neuronal groups, connected in a closed‐loop control circuit. We use this model to investigate, via simulation, the role of the intrinsic cardiac nervous system in integrating and modulating parasympathetic and sympathetic nerve activity in healthy and diseased states. Our results show that the local circuit neurons may modulate the ICN network response to distinct vagal inputs towards the integrative control of local cardiac function.

Biophysical Models of Minipig Right Atrial Ganglionic Plexus Principal Neurons Identified from Transcriptomics Data

The Right Atrial Ganglionic Plexus (RAGP) is a component of the intrinsic cardiac nervous system. Its neurons are critical to the cardiac pacemaker response to vagal stimulation, regulating sino‐atrial node activity. We developed single‐compartment computational models of RAGP principal neurons by incorporating ion channels identified from transcriptomics data from HT‐qPCR (High Throughput quantitative Polymerase Chain Reaction) and RNA‐sequencing of 405 principal neurons in female Yucatan minipig RAGP. Out of the ~200 genes in the transcriptomics map, we identified 13 genes that encode ion channel proteins, namely: Naf (Scn1a, Nav 1.1), KDR (Kcnc1, Kv 3.1; Kcnab1, Kv 1.1), P‐, N‐, L‐, T‐type Ca2+ (Cacna1a, Cav 2.1; Cacna1b, Cav 2.2; Cacna1c, Cav 1.2; Cacna1g, Cav 3.1; Cacna1i, Cav 3.3), KA (Kcnd2) and HCN1, HCN2, HCN3, HCN4. The corresponding Hodgkin‐Huxley‐based kinetic models of these ion channel isoforms were mined from databases: Ion Channel Genealogy, Channelpedia, and ModelDB. Since each ion channel gene had several kinetic models associated with it, we ran multiple simulations on the NetPyNE software to identify a single model per gene that would best represent the channel's dynamic behaviour in the assembled electrophysiological model. A binary transcriptomic map of the 13 genes showed 101 distinct ion channel combinations. Our 505 electrophysiological models (5 sets of the 101 cells) elicited two primary firing profiles. 65% elicited a single‐spike phasic response, ~32% elicited a tonic firing in response to a current clamp stimulus, and ~3% produced tonic firing that continued after the stimulus. The firing frequencies increased by ~13 Hz for every 0.1 nA increase in the current clamp stimulus and decreased by ~11 Hz for every 0.01 S/cm2 increase in KA channel conductance. Our electrophysiological models are available to the SPARC community in NetPyNE and NIH’s O2S2PARC platform. The models exhibit phasic and tonic excitability profiles with phasic being more prevalent. We next aim to incorporate neuromodulatory inputs and simulate a network of these neuronal models.

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, 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.

Identification of Mineralocorticoid Receptors, Aldosterone, and Its Processing Enzyme CYP11B2 on Parasympathetic and Sympathetic Neurons in Rat Intracardiac Ganglia.

Recent interest has focused on the mineralocorticoid receptor (MR) and its impact on the myocardium and the performance of the heart. However, there is a lack of evidence about MR expression and its endogenous ligand aldosterone synthesis with specific regard to the intrinsic cardiac nervous system. Therefore, we looked for evidence of MR and aldosterone in sympathetic and parasympathetic neurons of intracardiac ganglia. Tissue samples from rat heart atria were subjected to conventional reverse-transcriptase polymerase chain reaction (PCR), Western blot, and double immunofluorescence confocal analysis of MR, corticosterone-inactivating enzyme 11β-hydroxysteroid-dehydrogenase-2 (11β-HSD2), aldosterone, and its processing enzyme CYP11B2 together with the neuronal markers vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH). Our results demonstrated MR, 11β-HSD2, and CYP11B2 specific mRNA and protein bands in rat heart atria. Double immunofluorescence labeling revealed coexpression of MR immunoreactivity with VAChT in large diameter parasympathetic principal neurons. In addition, MR immunoreactivity was identified in TH-immunoreactive small intensely fluorescent (SIF) cells and in nearby VAChT- and TH-immunoreactive nerve terminals. Interestingly, the aldosterone and its synthesizing enzyme CYP11B2 and 11β-HSD2 colocalized in MR– immunoreactive neurons of intracardiac ganglia. Overall, this study provides first evidence for the existence of not only local expression of MR, but also of 11β-HSD2 and aldosterone with its processing enzyme CYP11B2 in the neurons of the cardiac autonomic nervous system, suggesting a possible modulatory role of the mineralocorticoid system on the endogenous neuronal activity on heart performance.

Modeling and Analysis of the Intrinsic Cardiac Nervous System in Closed-Loop Cardiovascular Control

The baroreceptor reflex is a multi-input, multi-output physiological control system that regulates short-term blood pressure by modulating nerve activity between the brainstem and the heart. The computational model by Park et al. (2020) is the most recent iteration in our exploration of the system. However, the contributions of ”the little brain of the heart”, the intrinsic cardiac nervous system (ICN), to local control of the heart and to the integration of sensory information is unknown and has been overlooked in previous models. We have incorporated a high-fidelity representation of the ICN into a model of the baroreceptor reflex based on anatomical, molecular, and physiological evidence. The model consists of (1) differential equations to represent the cardiovascular system, and (2) transfer functions to represent neural control components, connected in a closed-loop control circuit. We use the model to evaluate the impact of alternative ICN network structures on overall cardiovascular control in response to mean arterial pressure and lung tidal volume perturbations. Our results show that the local circuit neurons that integrate sensory information into the ICN strengthen the response of ICN neuron activity, especially at low blood pressures, suggesting that the ICN amplifies the brainstem's response to perturbations.

Electrical properties and synaptic transmission in mouse intracardiac ganglion neurons in situ.

The intrinsic cardiac nervous system represents the final site of signal integration for neurotransmission to the myocardium to enable local control of cardiac performance. The electrophysiological characteristics and ganglionic transmission of adult mouse intrinsic cardiac ganglion (ICG) neurons were investigated using a whole‐mount ganglion preparation of the excised right atrial ganglion plexus and intracellular microelectrode recording techniques. The passive and active electrical properties of ICG neurons and synaptic transmission including synaptic response strength and efficacy as a function of stimulation frequency were examined. The resting membrane potential and input resistance of ICG neurons were −47.9 ± 4.0 mV and 197.2 ± 81.5 MΩ, respectively. All neurons had somatic action potentials with overshoots of >+15 mV and after‐hyperpolarizations having an average of 10 mV amplitude and ~45 ms half duration. Phasic discharge activities were recorded from the majority of neurons studied and several types of excitatory synaptic responses were recorded following inputs from the vagus or interganglionic nerve trunk(s). Most postganglionic neurons (>75%) received a strong, suprathreshold synaptic input and reliably followed high‐frequency repetitive nerve stimulation up to at least 50 Hz. Nerve‐evoked synaptic transmission was blocked by extracellular Cd2+, ω‐conotoxin CVIE, or α‐conotoxin RegIIA, a selective α3‐containing nicotinic acetylcholine receptor antagonist. Synaptic transmission and the electrical properties of murine ICG neurons contribute to the pattern of discharge which regulates chronotropic, dromotropic, and inotropic elements of cardiac function.

3D single cell scale anatomical map of sex-dependent variability of the rat intrinsic cardiac nervous system

SummaryWe developed and analyzed a single cell scale anatomical map of the rat intrinsic cardiac nervous system (ICNS) across four male and three female hearts. We find the ICNS has a reliable structural organizational plan across individuals that provide the foundation for further analyses of the ICNS in cardiac function and disease. The distribution of the ICNS was evaluated by 3D visualization and data-driven clustering. The pattern, distribution, and clustering of ICNS neurons across all male and female rat hearts is highly conserved, demonstrating a coherent organizational plan where distinct clusters of neurons are consistently localized. Female hearts had fewer neurons, lower packing density, and slightly reduced distribution, but with identical localization. We registered the anatomical data from each heart to a geometric scaffold, normalizing their 3D coordinates for standardization of common anatomical planes and providing a path where multiple experimental results and data types can be integrated and compared.

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.

Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice.

The tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b/PEX5R) is an interaction partner and auxiliary subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are key for rhythm generation in the brain and in the heart. Since TRIP8b is expressed in central neurons but not in cardiomyocytes, the TRIP8b-HCN interaction has been studied intensely in the brain, but is deemed irrelevant in the cardiac conduction system. Still, to date, TRIP8b has not been studied in the intrinsic cardiac nervous system (ICNS), a neuronal network located within epicardial fat pads. In vitro electrophysiological studies revealed that TRIP8b-deficient mouse hearts exhibit increased atrial refractory and atrioventricular nodal refractory periods, compared to hearts of wild-type littermates. Meanwhile, heart rate, sino-nodal recovery time, and ventricular refractory period did not differ between genotypes. Trip8b mRNA was detected in the ICNS by quantitative polymerase chain reaction. RNAscope in situ hybridization confirmed Trip8b localization in neuronal somata and nerve fibers. Additionally, we found a very low amount of mRNAs in the sinus node and atrioventricular node, most likely attributable to the delicate fibers innervating the conduction system. In contrast, TRIP8b protein was not detectable. Our data suggest that TRIP8b in the ICNS may play a role in the modulation of atrial electrophysiology beyond HCN-mediated sino-nodal control of the heart.