The vagus nerve in cardiovascular physiology and pathophysiology: From evolutionary insights to clinical medicine.

The parasympathetic nervous system via the vagus nerve exerts profound influence over the heart. Together with the sympathetic nervous system, the parasympathetic nervous system is responsible for fine-tuned regulation of all aspects of cardiovascular function, including heart rate, rhythm, contractility, and blood pressure. In this review, we highlight vagal efferent and afferent innervation of the heart, with a focus on insights from comparative biology and advances in understanding the molecular and genetic diversity of vagal neurons, as well as interoception, parasympathetic dysfunction in heart disease, and the therapeutic potential of targeting the parasympathetic nervous system in cardiovascular disease.

Combination transarterial chemoembolization and microwave ablation vs. microwave ablation monotherapy for hepatocellular carcinomas greater than 3 cm: a comparative study.

PURPOSE: To evaluate the efficacy of combination therapy using transarterial chemoembolization with microwave ablation (MWA) therapy vs. MWA monotherapy for hepatocellular carcinomas (HCCs) >3 cm in size. METHODS: This two-arm retrospective observational study included patients with HCCs >3 cm who underwent either combination therapy (29 patients) or MWA monotherapy (35 patients) between 2014 and 2020. The treatment outcomes related to primary treatment efficacy, local tumor progression (LTP), tumor control rate, and overall survival were compared between each cohort. RESULTS: The technical success and primary efficacy were 96.56% and 100.00% in the combination therapy cohort, and 91.42% and 100.00% in the MWA cohort, respectively, over a mean follow-up period of 27.6 months. The 1- and 3-year rates of LTP-free survival were 78.57% and 69.56% in the combination therapy cohort, vs. 72.45% and 35.44% in the MWA cohort, respectively (P = 0.001). The overall progression-free survival was longer in the combination therapy cohort compared with the MWA cohort (median: 56.0 vs. 13.0 months; P = 0.017). With the incorporation of additional locoregional therapy, the overall survival rates were not significantly different, with 1- and 3-year overall survival rates of 100.00% and 88.71% in the combination therapy cohort and rates of 90.15% and 82.76% in the MWA cohort, respectively (P = 0.235). CONCLUSION: The combination therapy provided significantly longer upfront LTP-free survival in HCCs >3 cm when compared with the MWA treatment alone, albeit with similar local tumor control and overall survival rates when accounting for additional locoregional therapies.

CLSTN3ß enforces adipocyte multilocularity to facilitate lipid utilization.

Multilocular adipocytes are a hallmark of thermogenic adipose tissue1,2, but the factors that enforce this cellular phenotype are largely unknown. Here, we show that an adipocyte-selective product of the Clstn3 locus (CLSTN3ß) present in only placental mammals facilitates the efficient use of stored triglyceride by limiting lipid droplet (LD) expansion. CLSTN3ß is an integral endoplasmic reticulum (ER) membrane protein that localizes to ER-LD contact sites through a conserved hairpin-like domain. Mice lacking CLSTN3ß have abnormal LD morphology and altered substrate use in brown adipose tissue, and are more susceptible to cold-induced hypothermia despite having no defect in adrenergic signalling. Conversely, forced expression of CLSTN3ß is sufficient to enforce a multilocular LD phenotype in cultured cells and adipose tissue. CLSTN3ß associates with cell death-inducing DFFA-like effector proteins and impairs their ability to transfer lipid between LDs, thereby restricting LD fusion and expansion. Functionally, increased LD surface area in CLSTN3ß-expressing adipocytes promotes engagement of the lipolytic machinery and facilitates fatty acid oxidation. In human fat, CLSTN3B is a selective marker of multilocular adipocytes. These findings define a molecular mechanism that regulates LD form and function to facilitate lipid utilization in thermogenic adipocytes.

Proarrhythmic Effects of Sympathetic Activation Are Mitigated by Vagal Nerve Stimulation in Infarcted Hearts.

OBJECTIVES: The goal of this study was to evaluate whether intermittent VNS reduces electrical heterogeneities and arrhythmia inducibility during sympathoexcitation. BACKGROUND: Sympathoexcitation increases the risk of ventricular tachyarrhythmias (VT). Vagal nerve stimulation (VNS) has been antiarrhythmic in the setting of ischemia-driven arrhythmias, but it is unclear if it can overcome the electrophysiological effects of sympathoexcitation in the setting of chronic myocardial infarction (MI). METHODS: In Yorkshire pigs after chronic MI, a sternotomy was performed, a 56-electrode sock was placed over the ventricles (n = 17), and a basket catheter was positioned in the left ventricle (n = 6). Continuous unipolar electrograms from sock and basket arrays were obtained to analyze activation recovery interval (ARI), a surrogate of action potential duration. Bipolar voltage mapping was performed to define scar, border zone, or viable myocardium. Hemodynamic and electrical parameters and VT inducibility were evaluated during sympathoexcitation with bilateral stellate ganglia stimulation (BSS) and during combined BSS with intermittent VNS. RESULTS: During BSS, global epicardial ARIs shortened from 384 ± 59 milliseconds to 297 ± 63 milliseconds and endocardial ARIs from 359 ± 36 milliseconds to 318 ± 40 milliseconds. Dispersion in ARIs increased in all regions, with the greatest increase observed in scar and border zone regions. VNS mitigated the effects of BSS on border zone ARIs (from -18.3% ± 6.3% to -2.1% ± 14.7%) and ARI dispersion (from 104 ms2 [1 to 1,108 ms2] to -108 ms2 [IQR: -588 to 30 ms2]). VNS reduced VT inducibility during sympathoexcitation (from 75%-40%; P < 0.05). CONCLUSIONS: After chronic MI, VNS overcomes the detrimental effects of sympathoexcitation by reducing electrophysiological heterogeneities exacerbated by sympathetic stimulation, decreasing VT inducibility.

High-resolution structure-function mapping of intact hearts reveals altered sympathetic control of infarct border zones.

Remodeling of injured sympathetic nerves on the heart after myocardial infarction (MI) contributes to adverse outcomes such as sudden arrhythmic death, yet the underlying structural mechanisms are poorly understood. We sought to examine microstructural changes on the heart after MI and to directly link these changes with electrical dysfunction. We developed a high-resolution pipeline for anatomically precise alignment of electrical maps with structural myofiber and nerve-fiber maps created by customized computer vision algorithms. Using this integrative approach in a mouse model, we identified distinct structure-function correlates to objectively delineate the infarct border zone, a known source of arrhythmias after MI. During tyramine-induced sympathetic nerve activation, we demonstrated regional patterns of altered electrical conduction aligned directly with altered neuroeffector junction distribution, pointing to potential neural substrates for cardiac arrhythmia. This study establishes a synergistic framework for examining structure-function relationships after MI with microscopic precision that has potential to advance understanding of arrhythmogenic mechanisms.

Lamina-specific properties of spinal astrocytes.

Astrocytes are indispensable for proper neuronal functioning. Given the diverse needs of neuronal circuits and the variety of tasks astrocytes perform, the perceived homogeneous nature of astrocytes has been questioned. In the spinal dorsal horn, complex neuronal circuitries regulate the integration of sensory information of different modalities. The dorsal horn is organized in a distinct laminar manner based on termination patterns of high- and low-threshold afferent fibers and neuronal properties. Neurons in laminae I (L1) and II (L2) integrate potentially painful, nociceptive information, whereas neurons in lamina III (L3) and deeper laminae integrate innocuous, tactile information from the periphery. Sensory information is also integrated by an uncharacterized network of astrocytes. How these lamina-specific characteristics of neuronal circuits of the dorsal horn are of functional importance for properties of astrocytes is currently unknown. We addressed if astrocytes in L1, L2, and L3 of the upper dorsal horn of mice are differentially equipped for the needs of neuronal circuits that process sensory information of different modalities. We found that astrocytes in L1 and L2 were characterized by a higher density, higher expression of GFAP, Cx43, and GLAST and a faster coupling speed than astrocytes located in L3. L1 astrocytes were more responsive to Kir4.1 blockade and had higher levels of AQP4 compared to L3 astrocytes. In contrast, basic membrane properties, network formation, and somatic intracellular calcium signaling were similar in L1-L3 astrocytes. Our data indicate that the properties of spinal astrocytes are fine-tuned for the integration of nociceptive versus tactile information.

Innervation and Neuronal Control of the Mammalian Sinoatrial Node a Comprehensive Atlas.

[Figure: see text].

Optical vagus nerve modulation of heart and respiration via heart-injected retrograde AAV.

Vagus nerve stimulation has shown many benefits for disease therapies but current approaches involve imprecise electrical stimulation that gives rise to off-target effects, while the functionally relevant pathways remain poorly understood. One method to overcome these limitations is the use of optogenetic techniques, which facilitate targeted neural communication with light-sensitive actuators (opsins) and can be targeted to organs of interest based on the location of viral delivery. Here, we tested whether retrograde adeno-associated virus (rAAV2-retro) injected in the heart can be used to selectively express opsins in vagus nerve fibers controlling cardiac function. Furthermore, we investigated whether perturbations in cardiac function could be achieved with photostimulation at the cervical vagus nerve. Viral injection in the heart resulted in robust, primarily afferent, opsin reporter expression in the vagus nerve, nodose ganglion, and brainstem. Photostimulation using both one-photon stimulation and two-photon holography with a GRIN-lens incorporated nerve cuff, was tested on the pilot-cohort of injected mice. Changes in heart rate, surface electrocardiogram, and respiratory responses were observed in response to both one- and two-photon photostimulation. The results demonstrate feasibility of retrograde labeling for organ targeted optical neuromodulation.

Cardiac TRPV1 afferent signaling promotes arrhythmogenic ventricular remodeling after myocardial infarction.

Chronic sympathoexcitation is implicated in ventricular arrhythmogenesis (VAs) following myocardial infarction (MI), but the critical neural pathways involved are not well understood. Cardiac adrenergic function is partly regulated by sympathetic afferent reflexes, transduced by spinal afferent fibers expressing the transient receptor potential cation subfamily V member 1 (TRPV1) channel. The role of chronic TRPV1 afferent signaling in VAs is not known. We hypothesized that persistent TRPV1 afferent neurotransmission promotes VAs after MI. Using epicardial resiniferatoxin (RTX) to deplete cardiac TRPV1-expressing fibers, we dissected the role of this neural circuit in VAs after chronic MI in a porcine model. We examined the underlying mechanisms using molecular approaches, IHC, in vitro and in vivo cardiac electrophysiology, and simultaneous cardioneural mapping. Epicardial RTX depleted cardiac TRPV1 afferent fibers and abolished functional responses to TRPV1 agonists. Ventricular tachycardia/fibrillation (VT/VF) was readily inducible in MI subjects by programmed electrical stimulation or cesium chloride administration; however, TRPV1 afferent depletion prevented VT/VF induced by either method. Mechanistically, TRPV1 afferent depletion did not alter cardiomyocyte action potentials and calcium transients, the expression of ion channels, or calcium handling proteins. However, it attenuated fibrosis and mitigated electrical instability in the scar border zone. In vivo recordings of cardiovascular-related stellate ganglion neurons (SGNs) revealed that MI enhances SGN function and disrupts integrated neural processing. Depleting TRPV1 afferents normalized these processes. Taken together, these data indicate that, after MI, TRPV1 afferent-induced adrenergic dysfunction promotes fibrosis and adverse cardiac remodeling, and it worsens border zone electrical heterogeneity, resulting in electrically unstable ventricular myocardium. We propose targeting TRPV1-expressing afferent to reduce VT/VF following MI.

Increased arrhythmia susceptibility in type 2 diabetic mice related to dysregulation of ventricular sympathetic innervation.

Patients with type 2 diabetes mellitus (T2DM) have a greater risk of developing life-threatening cardiac arrhythmias. Because the underlying mechanisms and potential influence of diabetic autonomic neuropathy are not well understood, we aimed to assess the relevance of a dysregulation in cardiac autonomic tone. Ventricular arrhythmia susceptibility was increased in Langendorff-perfused hearts isolated from mice with T2DM (db/db). Membrane properties and synaptic transmission were similar at cardiac postganglionic parasympathetic neurons from diabetic and control mice; however, a greater asynchronous neurotransmitter release was present at sympathetic postganglionic neurons from the stellate ganglia of db/db mice. Western blot analysis showed a reduction of tyrosine hydroxylase (TH) from the ventricles of db/db mice, which was confirmed with confocal imaging as a heterogeneous loss of TH-immunoreactivity from the left ventricular wall but not the apex. In vivo stimulation of cardiac parasympathetic (vagus) or cardiac sympathetic (stellate ganglion) nerves induced similar changes in heart rate in control and db/db mice, and the kinetics of pacing-induced Ca2+ transients (recorded from isolated cardiomyocytes) were similar in control and db/db cells. Antagonism of cardiac muscarinic receptors did not affect the frequency or severity of arrhythmias in db/db mice, but sympathetic blockade with propranolol completely inhibited arrhythmogenicity. Collectively, these findings suggest that the increased ventricular arrhythmia susceptibility of type 2 diabetic mouse hearts is due to dysregulation of the sympathetic ventricular control.NEW & NOTEWORTHY Patients with type 2 diabetes mellitus have greater risk of suffering from sudden cardiac death. We found that the increased ventricular arrhythmia susceptibility in type 2 diabetic mouse hearts is due to cardiac sympathetic dysfunction. Sympathetic dysregulation is indicated by an increased asynchronous release at stellate ganglia, a heterogeneous loss of tyrosine hydroxylase from the ventricular wall but not apex, and inhibition of ventricular arrhythmias in db/db mice after ß-sympathetic blockade.

Neuromodulation for Ventricular Tachycardia and Atrial Fibrillation: A Clinical Scenario-Based Review.

Autonomic dysregulation in cardiovascular disease plays a major role in the pathogenesis of arrhythmias. Cardiac neural control relies on complex feedback loops consisting of efferent and afferent limbs, which carry sympathetic and parasympathetic signals from the brain to the heart and sensory signals from the heart to the brain. Cardiac disease leads to neural remodeling and sympathovagal imbalances with arrhythmogenic effects. Preclinical studies of modulation at central and peripheral levels of the cardiac autonomic nervous system have yielded promising results, leading to early stage clinical studies of these techniques in atrial fibrillation and refractory ventricular arrhythmias, particularly in patients with inherited primary arrhythmia syndromes and structural heart disease. However, significant knowledge gaps in basic cardiac neurophysiology limit the success of these neuromodulatory therapies. This review discusses the recent advances in neuromodulation for cardiac arrhythmia management, with a clinical scenario-based approach aimed at bringing neurocardiology closer to the realm of the clinical electrophysiologist.

Publisher Correction: Systemic AAV vectors for widespread and targeted gene delivery in rodents.

During the production process, the authors of this paper supplied revised versions of Figs. 2-5, Supplementary Tables 1-4, and Supplementary Videos 1-3, but because of publisher error, these revised items were not included in the final published version of the protocol. The figures have been updated in the PDF and HTML versions of the paper, and the revised Supplementary Information files are now available online. We note that the figures have been revised to improve their resolution only; the content of the figures and the data reflected remain unchanged. Also, print requirements impose some limits on figure resolution, but the authors have made very high-resolution versions of Figs. 2-5 available at as Source data.

Anesthetizing the Fibrillating Heart.

See Article Lee et al.

Premature ventricular contraction diurnal profiles predict distinct clinical characteristics and beta-blocker responses.

INTRODUCTION: Frequent premature ventricular complexes (PVCs) can lead to symptoms, such as cardiomyopathy and increased mortality. Beta-blockers are recommended as first-line therapy to reduce PVC burden; however, the response is unpredictable. The objective of this study is to determine whether PVC diurnal-variability patterns are associated with different clinical profiles and predict drug response. METHODS: Consecutive patients with frequent PVCs (burden ≥ 1%), referred for Holter monitoring between 2014 and 2016, were included. Follow-up Holters, when available, were assessed after beta-blocker initiation to assess response (≥50% reduction). Patients were divided into three groups on the basis of relationship between hourly PVC count and mean HR during each of the 24 Holter hours: (1) fast-HR-dependent-PVC (F-HR-PVC) for positive correlation (Pearson, P < 0.05), (2) slow-HR-dependent-PVC (S-HR-PVC) for a negative, and (3) independent-HR-PVC (I-HR-PVC) when no correlation was found. RESULTS: Of the 416 patients included, 50.2% had F-HR-PVC, 35.6% I-HR-PVC, and 14.2% S-HR-PVC with distinct clinical profiles. Beta-blocker therapy was successful in 34.0% patients overall: patients with F-HR-PVC had a decrease in PVC burden (18.8 ± 10.4% to 9.3 ± 6.6%, P < 0.0001; 62% success), I-HR-PVC had no change (18.4 ± 17.9% to 20.6 ± 17.9%, P = 0.175; 0% success), whereas S-HR-PVC had an increase in burden (14.6 ± 15.3% to 20.8 ± 13.8%, P = 0.016; 0% success). The correlation coefficient was the only predictor of beta-blocker success (AUC = 0.84, sensitivity = 100%, specificity = 67.7%; r ≥ 0.4). CONCLUSIONS: A simple analysis of Holter PVC diurnal variability may provide incremental value to guide clinical PVC management. Only patients displaying a F-HR-PVC profile benefited from beta-blockers. An alternative strategy should be considered for others, as beta-blockers may have no effect or can even be harmful.

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

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

Systemic AAV vectors for widespread and targeted gene delivery in rodents.

We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.