Special Issue "Sympathetic Nerves and Cardiovascular Diseases".

Cardiovascular diseases (CVDs) constitute a spectrum of disorders affecting the heart and blood vessels, which include coronary heart disease, cerebrovascular disease, and peripheral artery disease [...].

The relevance of the superior cervical ganglion for cardiac autonomic innervation in health and disease: a systematic review.

PURPOSE: The heart receives cervical and thoracic sympathetic contributions. Although the stellate ganglion is considered the main contributor to cardiac sympathetic innervation, the superior cervical ganglia (SCG) is used in many experimental studies. The clinical relevance of the SCG to cardiac innervation is controversial. We investigated current morphological and functional evidence as well as controversies on the contribution of the SCG to cardiac innervation. METHODS: A systematic literature review was conducted in PubMed, Embase, Web of Science, and COCHRANE Library. Included studies received a full/text review and quality appraisal. RESULTS: Seventy-six eligible studies performed between 1976 and 2023 were identified. In all species studied, morphological evidence of direct or indirect SCG contribution to cardiac innervation was found, but its contribution was limited. Morphologically, SCG sidedness may be relevant. There is indirect functional evidence that the SCG contributes to cardiac innervation as shown by its involvement in sympathetic overdrive reactions in cardiac disease states. A direct functional contribution was not found. Functional data on SCG sidedness was largely unavailable. Information about sex differences and pre- and postnatal differences was lacking. CONCLUSION: Current literature mainly supports an indirect involvement of the SCG in cardiac innervation, via other structures and plexuses or via sympathetic overdrive in response to cardiac diseases. Morphological evidence of a direct involvement was found, but its contribution seems limited. The relevance of SCG sidedness, sex, and developmental stage in health and disease remains unclear and warrants further exploration.

Functional and 123I-MIBG scintigraphy assessment of cardiac adrenergic dysfunction in diabetes.

OBJECTIVES: To assess the agreement between clinical cardiovascular adrenergic function and cardiac adrenergic innervation in type 2 diabetes patients (T2D). METHODS: Thirty-three patients with T2D were investigated bimodally through (1) a standardized clinical cardiovascular adrenergic assessment, evaluating adequacy of blood pressure responses to the Valsalva maneuver and (2) 123I-meta-iodobenzylguanidine (MIBG) scintigraphy assessing myocardial adrenergic innervation measured as early and delayed heart heart/mediastinum (H/M) ratio, and washout rate (WR). RESULTS: T2D patients had significantly lower early and delayed H/M-ratios, and lower WR, compared to laboratory specific reference values. Thirteen patients had an abnormal adrenergic composite autonomic severity score (CASS > 0). Patients with abnormal CASS scores had significantly higher early H/M ratios (1.76 [1.66-1.88] vs. 1.57 [1.49-1.63], p < 0.001), higher delayed H/M ratios (1.64 [1.51:1.73] vs. 1.51 [1.40:1.61] (p = 0.02)), and lower WR (-0.13(0.10) vs -0.05(0.07), p = 0.01). Lower Total Recovery and shorter Pressure Recovery Time responses from the Valsalva maneuver was significantly correlated to lower H/M early (r = 0.55, p = 0.001 and r = 0.5, p = 0.003, respectively) and lower WR for Total Recovery (r = -0.44, p = 0.01). CONCLUSION: The present study found impairment of sympathetic innervation in T2D patients based on parameters derived from MIBG cardiac scintigraphy (low early H/M, delayed H/M, and WR). These results confirm prior studies. We found a mechanistically inverted relationship with favourable adrenergic cardiovascular responses being significantly associated unfavourable MIBG indices for H/M early and delayed. This paradoxical relationship needs to be further explored but could indicate adrenergic hypersensitivity in cardiac sympathetic denervated T2D patients.

Sympathetic transduction of cardiac sympathetic nerve activity in healthy, conscious sheep.

Sympathetic transduction is the study of how impulses of sympathetic nerve activity (SNA) affect end-organ function. Recently, the transduction of resting bursts of muscle SNA (MSNA) has been investigated and shown to have a role in the maintenance of blood pressure through changes in vascular tone in humans. In the present study, we investigate whether directly recorded resting cardiac SNA (CSNA) regulates heart rate (HR), coronary blood flow (CoBF), coronary vascular conductance (CVC), cardiac output (CO) and mean arterial pressure. Instrumentation was undertaken to record CSNA and relevant vascular variables in conscious sheep. Recordings were performed at baseline, as well as after the infusion of a ß-adrenoceptor blocker (propranolol) to determine the role of ß-adrenergic signalling in sympathetic transduction in the heart. The results show that after every burst of CSNA, there was a significant effect of time on HR (n = 10, ∆: +2.1 ± 1.4 beats min-1 , P = 0.002) and CO (n = 8, ∆: +100 ± 150 mL min-1 , P = 0.002) was elevated, followed by an increase in CoBF (n = 9, ∆: +0.76 mL min-1 , P = 0.001) and CVC (n = 8, ∆: +0.0038 mL min-1  mmHg-1 , P = 0.0028). The changes in HR were graded depending on the size and pattern of CSNA bursts. The HR response was significantly attenuated after the infusion of propranolol. Our study is the first to explore resting sympathetic transduction in the heart, suggesting that CSNA can dynamically change HR mediated by an action on ß-adrenoceptors. KEY POINTS: Sympathetic transduction is the study of how impulses of sympathetic nerve activity (SNA) affect end-organ function. Previous studies have examined sympathetic transduction primarily in the skeletal muscle and shown that bursts of muscle SNA alter blood flow to skeletal muscle and mean arterial pressure, although this has not been examined in the heart. We investigated sympathetic transduction in the heart and show that, in the conscious condition, the size of bursts of SNA to the heart can result in incremental increases in heart rate and coronary blood flow mediated by ß-adrenoceptors. The pattern of bursts of SNA to the heart also resulted in incremental increases in heart rate mediated by ß-adrenoceptors. This is the first study to explore the transduction of bursts of SNA to the heart.

Ischemia-reperfusion myocardial infarction induces remodeling of left cardiac-projecting stellate ganglia neurons.

Neurons in the stellate ganglion (SG) provide sympathetic innervation to the heart, brown adipose tissue (BAT), and other organs. Sympathetic innervation to the heart becomes hyperactive following myocardial infarction (MI). The impact of MI on the morphology of cardiac sympathetic neurons is not known, but we hypothesized that MI would stimulate increased cell and dendritic tree size in cardiac neurons. In this study, we examined the effects of ischemia-reperfusion MI on sympathetic neurons using dual retrograde tracing methods to allow detailed characterization of cardiac- and BAT-projecting neurons. Different fluorescently conjugated cholera toxin subunit B (CTb) tracers were injected into the pericardium and the interscapular BAT pads, respectively. Experimental animals received a 45-min occlusion of the left anterior descending coronary artery and controls received sham surgery. One week later, hearts were collected for assessment of MI infarct and SGs were collected for morphological or electrophysiological analysis. Cardiac-projecting SG neurons from MI mice had smaller cell bodies and shorter dendritic trees compared with sham animals, specifically on the left side ipsilateral to the MI. BAT-projecting neurons were not altered by MI, demonstrating the subpopulation specificity of the response. The normal size and distribution differences between BAT- and cardiac-projecting stellate ganglion neurons were not altered by MI. Patch-clamp recordings from cardiac-projecting left SG neurons revealed increased spontaneous excitatory postsynaptic currents despite the decrease in cell and dendritic tree size. Thus, increased dendritic tree size does not contribute to the enhanced sympathetic neural activity seen after MI.NEW & NOTEWORTHY Myocardial infarction (MI) causes structural and functional changes specifically in stellate ganglion neurons that project to the heart, but not in cells that project to brown adipose fat tissue.

Respiratory sinus arrhythmia (RSA), vagal tone and biobehavioral integration: Beyond parasympathetic function.

Linchpin to the entire area of psychophysiological research and discussion of the vagus is the respiratory and cardiovascular phenomenon known as respiratory sinus arrhythmia (RSA; often synonymous with high-frequency heart-rate variability when it is specifically linked to respiratory frequency), i.e. rhythmic fluctuations in heart rate synchronized to inspiration and expiration. This article aims 1) to clarify concepts, terms and measures commonly employed during the last half century in the scientific literature, which relate vagal function to psychological processes and general aspects of health; and 2) to expand upon an earlier theoretical model, emphasizing the importance of RSA well beyond the current focus upon parasympathetic mechanisms. A close examination of RSA and its relations to the vagus may 1) dispel certain commonly held beliefs about associations between psychological functioning, RSA and the parasympathetic nervous system (for which the vagus nerve plays a major role), and 2) offer fresh perspectives about the likely functions and adaptive significance of RSA, as well as RSA's relationship to vagal control. RSA is neither an invariably reliable index of cardiac vagal tone nor of central vagal outflow to the heart. The model here presented posits that RSA represents an evolutionarily entrenched, cardiovascular and respiratory phenomenon that significantly contributes to meeting continuously changing metabolic, energy and behavioral demands.

Targeted ablation of the left middle cervical ganglion prevents ventricular arrhythmias and cardiac injury induced by AMI.

Cardiac sympathetic overactivation is a critical driver in the progression of acute myocardial infarction (AMI). The left middle cervical ganglion (LMCG) is an important extracardiac sympathetic ganglion. However, the regulatory effects of LMCG on AMI have not yet been fully documented. In the present study, we detected that the LMCG was innervated by abundant sympathetic components and exerted an excitatory effect on the cardiac sympathetic nervous system in response to stimulation. In canine models of AMI, targeted ablation of LMCG reduced the sympathetic indexes of heart rate variability and serum norepinephrine, resulting in suppressed cardiac sympathetic activity. Moreover, LMCG ablation could improve ventricular electrophysiological stability, evidenced by the prolonged ventricular effective refractory period, elevated action potential duration, increased ventricular fibrillation threshold, and enhanced connexin43 expression, consequently showing antiarrhythmic effects. Additionally, compared with the control group, myocardial infarction size, circulating cardiac troponin I, and myocardial apoptosis were significantly reduced, accompanied by preserved cardiac function in canines subjected to LMCG ablation. Finally, we performed the left stellate ganglion (LSG) ablation and compared its effects with LMCG destruction. The results indicated that LMCG ablation prevented ventricular electrophysiological instability, cardiac sympathetic activation, and AMI-induced ventricular arrhythmias with similar efficiency as LSG denervation. In conclusion, this study demonstrated that LMCG ablation suppressed cardiac sympathetic activity, stabilized ventricular electrophysiological properties and mitigated cardiomyocyte death, resultantly preventing ischemia-induced ventricular arrhythmias, myocardial injury, and cardiac dysfunction. Neuromodulation therapy targeting LMCG represented a promising strategy for the treatment of AMI.

Cardiac autonomic plexus neuromodulation for decompensated heart failure: An updated review on the positive inotropic technique based on the DRI2P2S classification.

Current pharmacological regimen is unable to improve adverse outcomes such as mortality post hospitalization for Acutely Decompensated Heart Failure (ADHF) patients. Ongoing research is directed towards managing ADHF patients with Cardiac Autonomic Nervous System (CANS) excitatory interventions having long-term prognosis benefits. Recently, a novel treatment coined as Cardiac Pulmonary Nerve Stimulation (CPNS) has reproducibly shown increased inotropy with no change in heart rate. However, there are some potential limitations associated with the neurostimulation of the parasympathetic component of the CANS plexus. The INOVATE-HF trial involved the vagus nerve only. The early termination of the INOVATE-HF trial gave valuable insights into the cardio-protective effect of simultaneously stimulating the sympathetic and parasympathetic components of the CANS plexus done in CPNS. It is essential to individualize the treatment protocol keeping in mind patient selection. Ongoing trials assessing the efficacy and safety of the CPNS technique in ADHF patients shall set the tone for such innovative techniques in times to come.

Dynamic changes in renal sodium handling during sympathetic stimulation in healthy human males.

The temporal response of changes in renal sodium reabsorption during increased renal sympathetic nerve activity has not been investigated. Central hypovolemia by application of lower-body negative-pressure (LBNP) elicits baroreceptor mediated sympathetic reflexes to maintain arterial blood pressure. We hypothesized, that during 90 min LBNP, the renal sodium retention would increase rapidly, remain increased during intervention, and return to baseline immediately after end of intervention. METHODS: 30 young, healthy, sodium loaded, non-obese males were exposed to -15 mmHg LBNP, -30 mmHg LBNP, -15 mmHg LBNP + renin blockade or time-control (0 mmHg LBNP) for 90 min. Urine was collected every 15 min during 90 min of intervention and 60 min of recovery to identify a possible relation between time of intervention and renal response. RESULTS: All intervention groups exhibited a comparable reduction in distal sodium excretion at the end of the intervention (P = 0.46 between groups; -15 mmHg: -3.1 ± 0.9 %, -30 mmHg: -2.9 ± 0.6 %, -15 mmHg + aslikiren: -1.8 ± 0.6 %). -15 mmHg+Aliskiren resulted in a slower onset, but all groups exhibited a continued reduction in sodium excretion after 1 h of recovery despite return to baseline of renin, aldosterone, diuresis and cardiovascular parameters. CONCLUSION: Sympathetic stimulation for 90 min via LBNP at -30 mmHg LBNP compared to -15 mmHg did not result in a greater response in fractional Na+ excretion, suggesting that additional baroreceptor unloading did not cause further increases in renal sodium reabsorption. Changes in distal Na+ excretion were linear with respect to time (dose) of intervention, but seem to exhibit a saturation-like effect at a level around 4 %. The lack of recovery after 1 h is also a new finding that warrants further investigation.

A multivariate physiological model of vagus nerve signalling during metabolic challenges in anaesthetised rats for diabetes treatment.

Objective.This study aims to develop a comprehensive decoding framework to create a multivariate physiological model of vagus nerve transmission that reveals the complex interactions between the nervous and metabolic systems.Approach.Vagus nerve activity was recorded in female Sprague-Dawley rats using gold hook microwires implanted around the left cervical vagus nerve. The rats were divided into three experimental cohorts (intact nerve, ligation nerve for recording afferent activation, and ligation for recording efferent activation) and metabolic challenges were administered to change glucose levels while recording the nerve activity. The decoding methodology involved various techniques, including continuous wavelet transformation, extraction of breathing rate (BR), and correlation of neural metrics with physiological signals.Main results.Decrease in glucose level was consistently negatively correlated with an increase in the firing activity of the intact vagus nerve that was found to be conveyed by both afferent and efferent pathways, with the afferent response being more similar to the one on the intact nerve. A larger variability was observed in the sensory and motor responses to hyperglycaemia. A novel strategy to extract the BR over time based on inter-burst-interval is also presented. The vagus afferent was found to encode breathing information through amplitude and firing rate modulation. Modulations of the signal amplitude were also observed due to changes in heart rate in the intact and efferent recordings, highlighting the parasympathetic control of the heart.Significance.The analytical framework presented in this study provides an integrative understanding that considers the relationship between metabolic, cardiac, and breathing signals and contributes to the development of a multivariable physiological model for the transmission of vagus nerve signals. This work progresses toward the development of closed-loop neuro-metabolic therapeutic systems for diabetes.

A bioelectrical phase transition patterns the first vertebrate heartbeats.

A regular heartbeat is essential to vertebrate life. In the mature heart, this function is driven by an anatomically localized pacemaker. By contrast, pacemaking capability is broadly distributed in the early embryonic heart1-3, raising the question of how tissue-scale activity is first established and then maintained during embryonic development. The initial transition of the heart from silent to beating has never been characterized at the timescale of individual electrical events, and the structure in space and time of the early heartbeats remains poorly understood. Using all-optical electrophysiology, we captured the very first heartbeat of a zebrafish and analysed the development of cardiac excitability and conduction around this singular event. The first few beats appeared suddenly, had irregular interbeat intervals, propagated coherently across the primordial heart and emanated from loci that varied between animals and over time. The bioelectrical dynamics were well described by a noisy saddle-node on invariant circle bifurcation with action potential upstroke driven by CaV1.2. Our work shows how gradual and largely asynchronous development of single-cell bioelectrical properties produces a stereotyped and robust tissue-scale transition from quiescence to coordinated beating.

Aging impairs the neurovascular interface in the heart.

Aging is a major risk factor for impaired cardiovascular health. Because the aging myocardium is characterized by microcirculatory dysfunction, and because nerves align with vessels, we assessed the impact of aging on the cardiac neurovascular interface. We report that aging reduces nerve density in the ventricle and dysregulates vascular-derived neuroregulatory genes. Aging down-regulates microRNA 145 (miR-145) and derepresses the neurorepulsive factor semaphorin-3A. miR-145 deletion, which increased Sema3a expression or endothelial Sema3a overexpression, reduced axon density, mimicking the aged-heart phenotype. Removal of senescent cells, which accumulated with chronological age in parallel to the decline in nerve density, rescued age-induced denervation, reversed Sema3a expression, preserved heart rate patterns, and reduced electrical instability. These data suggest that senescence-mediated regulation of nerve density contributes to age-associated cardiac dysfunction.

Spatially resolved multiomics of human cardiac niches.

The function of a cell is defined by its intrinsic characteristics and its niche: the tissue microenvironment in which it dwells. Here we combine single-cell and spatial transcriptomics data to discover cellular niches within eight regions of the human heart. We map cells to microanatomical locations and integrate knowledge-based and unsupervised structural annotations. We also profile the cells of the human cardiac conduction system1. The results revealed their distinctive repertoire of ion channels, G-protein-coupled receptors (GPCRs) and regulatory networks, and implicated FOXP2 in the pacemaker phenotype. We show that the sinoatrial node is compartmentalized, with a core of pacemaker cells, fibroblasts and glial cells supporting glutamatergic signalling. Using a custom CellPhoneDB.org module, we identify trans-synaptic pacemaker cell interactions with glia. We introduce a druggable target prediction tool, drug2cell, which leverages single-cell profiles and drug-target interactions to provide mechanistic insights into the chronotropic effects of drugs, including GLP-1 analogues. In the epicardium, we show enrichment of both IgG+ and IgA+ plasma cells forming immune niches that may contribute to infection defence. Overall, we provide new clarity to cardiac electro-anatomy and immunology, and our suite of computational approaches can be applied to other tissues and organs.

Cardiac Transplantation: Physiology and Natural History of the Transplanted Heart.

Heart transplantation (HT) is one of the prodigious achievements in modern medicine and remains the cornerstone in the treatment of patients with advanced heart failure. Advances in surgical techniques, immunosuppression, organ preservation, infection control, and allograft surveillance have improved short- and long-term outcomes thereby contributing to greater clinical success of HT. However, prolonged allograft and patient survival following HT are still largely restricted by the development of late complications, including allograft rejection, infection, cardiac allograft vasculopathy (CAV), and malignancy. The introduction of mTOR inhibitors early after HT has demonstrated multiple protective effects against CAV progression, renal dysfunction, and tumorigenesis. Therefore, several HT programs increasingly use mTOR inhibitors with partial or complete withdrawal of calcineurin inhibitor (CNI) in stable HT patients to reduce complications risk and improve long-term outcomes. Furthermore, despite a substantial improvement in exercise capacity and health-related quality of life after HT as compared to advanced heart failure patients, most HT recipients remain with a 30% to 50% lower peak oxygen consumption (Vo 2 ) than that of age-matched healthy subjects. Several factors, including alterations in central hemodynamics, HT-related complications and alterations in the musculoskeletal system, and peripheral physiological abnormalities, presumably contribute to the reduced exercise capacity following HT. Cardiac denervation and subsequent loss of sympathetic and parasympathetic regulation are responsible for various physiological alterations in the cardiovascular system, which contributes to restricted exercise tolerance. Restoration of cardiac innervation may improve exercise capacity and quality of life, but the reinnervation process is only partial even several years after HT. Multiple studies have shown that aerobic and strengthening exercise interventions improve exercise capacity by increasing maximal heart rate, chronotropic response, and peak Vo 2 after HT. Novel exercise modalities, such as high-intensity interval training (HIT), have been proven as safe and effective for further improvement in exercise capacity, including among de novo HT recipients. Further developments have recently emerged, including donor heart preservation techniques, noninvasive CAV and rejection surveillance methods, and improvements in immunosuppressive therapies, all aiming at increasing donor availability and improving late survival after HT. © 2023 American Physiological Society. Compr Physiol 13:4719-4765, 2023.

Transient Receptor Potential Canonical 5 (TRPC5) Channels Activator, BTD [N-{3-(adamantan-2-yloxy)-propyl}-3-(6-methyl-1,1-dioxo-2H-1λ6,2,4- benzothiadiazin-3-yl)-propanamide)] Ameliorates Diabetic Cardiac Autonomic Neuropathy in Rats.

BACKGROUND: Diabetic cardiac autonomic neuropathy (DCAN) is a serious diabetic complication with no approved pharmacological agents for its treatment. Parasympathetic system dysfunction characterized by vagal nerve damage is one of the major drivers of DCAN. The TRPC5 or transient receptor potential canonical 5 channel is a promising target in autonomic dysfunction; however, its role in vagal nerve damage and subsequent DCAN has not yet been elucidated. The present study investigated the role of the TRPC5 channel in DCAN using [N-{3-(adamantan-2-yloxy)-propyl}-3-(6-methyl-1,1-dioxo-2H-1λ6,2,4-benzothiadiazin-3-yl) propanamide)] or BTD, which is a potent TRPC5 activator. OBJECTIVES: The role of the TRPC5 channel and its activator, BTD, was investigated in the treatment of parasympathetic dysfunction associated with DCAN. METHODS: Type 1 diabetes was induced in male Sprague-Dawley rats using streptozotocin. The alterations in cardiac autonomic parameters in diabetic animals were assessed by heart rate variability, hemodynamic parameters, and baroreflex sensitivity. TRPC5's role in DCAN was investigated by treating diseased rats with BTD (1 and 3 mg/kg, i.p. for 14 days). BTD's beneficial effects in parasympathetic dysfunction were assessed by western blotting, estimating oxidative stress and inflammatory markers in the vagus nerve. RESULTS: BTD treatment (3 mg/kg, i.p.) once daily for 14 days ameliorated heart rate variability, hemodynamic dysfunction, and baroreflex sensitivity in diseased rats. BTD treatment down regulated TRPC5 expression by increasing the activity of protein kinase C in the vagus nerve. It also down-regulated the apoptotic marker CASPASE-3 and also exerted potent anti-inflammatory action on pro-inflammatory cytokines levels in the vagus. CONCLUSION: BTD ameliorated parasympathetic dysfunction associated with DCAN by virtue of its TRPC5 modulatory, anti-inflammatory, and anti-apoptotic properties.

Dysregulation of hypoxia-inducible factor 1α in the sympathetic nervous system accelerates diabetic cardiomyopathy.

BACKGROUND: An altered sympathetic nervous system is implicated in many cardiac pathologies, ranging from sudden infant death syndrome to common diseases of adulthood such as hypertension, myocardial ischemia, cardiac arrhythmias, myocardial infarction, and heart failure. Although the mechanisms responsible for disruption of this well-organized system are the subject of intensive investigations, the exact processes controlling the cardiac sympathetic nervous system are still not fully understood. A conditional knockout of the Hif1a gene was reported to affect the development of sympathetic ganglia and sympathetic innervation of the heart. This study characterized how the combination of HIF-1α deficiency and streptozotocin (STZ)-induced diabetes affects the cardiac sympathetic nervous system and heart function of adult animals. METHODS: Molecular characteristics of Hif1a deficient sympathetic neurons were identified by RNA sequencing. Diabetes was induced in Hif1a knockout and control mice by low doses of STZ treatment. Heart function was assessed by echocardiography. Mechanisms involved in adverse structural remodeling of the myocardium, i.e. advanced glycation end products, fibrosis, cell death, and inflammation, was assessed by immunohistological analyses. RESULTS: We demonstrated that the deletion of Hif1a alters the transcriptome of sympathetic neurons, and that diabetic mice with the Hif1a-deficient sympathetic system have significant systolic dysfunction, worsened cardiac sympathetic innervation, and structural remodeling of the myocardium. CONCLUSIONS: We provide evidence that the combination of diabetes and the Hif1a deficient sympathetic nervous system results in compromised cardiac performance and accelerated adverse myocardial remodeling, associated with the progression of diabetic cardiomyopathy.

Catecholaminergic axon innervation and morphology in flat-mounts of atria and ventricles of mice.

Sympathetic efferent axons regulate cardiac functions. However, the topographical distribution and morphology of cardiac sympathetic efferent axons remain insufficiently characterized due to the technical challenges involved in immunohistochemical labeling of the thick walls of the whole heart. In this study, flat-mounts of the left and right atria and ventricles of FVB mice were immunolabeled for tyrosine hydroxylase (TH), a marker of sympathetic nerves. Atrial and ventricular flat-mounts were scanned using a confocal microscope to construct montages. We found (1) In the atria: A few large TH-immunoreactive (IR) axon bundles entered both atria, branched into small bundles and then single axons that eventually formed very dense terminal networks in the epicardium, myocardium and inlet regions of great vessels to the atria. Varicose TH-IR axons formed close contact with cardiomyocytes, vessels, and adipocytes. Multiple intrinsic cardiac ganglia (ICG) were identified in the epicardium of both atria, and a subpopulation of the neurons in the ICG were TH-IR. Most TH-IR axons in bundles traveled through ICG before forming dense varicose terminal networks in cardiomyocytes. We did not observe varicose TH-IR terminals encircling ICG neurons. (2) In the left and right ventricles and interventricular septum: TH-IR axons formed dense terminal networks in the epicardium, myocardium, and vasculature. Collectively, TH labeling is achievable in flat-mounts of thick cardiac walls, enabling detailed mapping of catecholaminergic axons and terminal structures in the whole heart at single-cell/axon/varicosity scale. This approach provides a foundation for future quantification of the topographical organization of the cardiac sympathetic innervation in different pathological conditions.