A Mechanistic Understanding of Vagus Nerve Stimulation Therapy: Investigating the Effects of Parasympathetic Modulation on Cardiac Dynamics

University of Minnesota Ph.D. dissertation.March 2018. Major: Biomedical Engineering. Advisor: Elena Tolkacheva. 1 computer file (PDF); xii, 129 pages.Vagus nerve stimulation (VNS) is a neuromodulatory approach that involves delivering electrical impulses to the vagus nerve to modulate the autonomic nervous system. In addition to being FDA-approved for use in epilepsy and depression, a plethora of both experimental and clinical studies also supports the potential of VNS to treat cardiovascular diseases. While promising, the precise mechanism by which this therapy exerts its cardioprotective effects are not well-established. The central dogma states that vagus nerves primarily innervate the atria with very minimal to no innervation into the ventricles. Based on this concept, parasympathetic system activation by VNS should not significantly affect ventricular properties. This is not the case, however, as VNS has been extensively shown to exert marked anti-arrhythmic effects in the ventricles. Furthermore, this supposition that there is minimal vagal innervation into the ventricular myocardium has been challenged by a series of immunohistochemical, histological, and western blot experiments. The first aim of my dissertation was to investigate the potential for a direct effect of long-term VNS on ventricular electrophysiology and test the hypothesis that VNS induces electrical remodeling of the ventricles to render the therapy’s reported anti-arrhythmic effects. Secondly, I then examined the direct contributions of the M2 muscarinic receptor activated potassium channel (M2R-IKACh) in mediating the chronotropic effects of VNS. For this second aim, I applied VNS on several transgenic mice that lack the IKACh channel constitutively, and selectively in the atria or the ventricles. Thirdly, there still does not exist a universally accepted published prospective VNS paradigm, which further highlights the complexity of parameter optimization. This work introduced an innovative concept of incorporating stochasticity when stimulating the vagus nerve (stochastic VNS, S-VNS) and I evaluated the effects of S-VNS on acute heart rate (HR) dynamics in comparison to traditional, periodic VNS. Collectively, the work performed in this thesis contribute to the mechanistic understanding of VNS therapy, in particular to the fundamental role of the parasympathetic nervous system in ventricular electrophysiology, and facilitate the optimization and improvement of VNS efficacy

Novel therapies for hypertension and associated cardiovascular risk

University of Minnesota Ph.D. dissertation. August 2018. Major: Biomedical Engineering. Advisor: Alena Talkachova. 1 computer file (PDF); xvii, 134 pages.This thesis is comprised of two parts. The first part investigates a novel therapy, vagus nerve stimulation, for hypertension and hypertension-induced heart disease. Hypertension impacts over 1 billion people worldwide, and clinical management is challenging. Left uncontrolled, high blood pressure can significantly increase the risk of cardiovascular events. The majority of hypertensive patients are treated with anti-hypertensive drugs to control blood pressure, but many limitations exist including resistant hypertension, inability to tolerate therapy, and non-compliance with the medication regime. For these patients, an alternative approach is needed to control blood pressure. Recently, the imbalance in the autonomic nervous system, evident in hypertension, has been the target of novel device-based therapies such as vagus nerve stimulation. The main goal of this research is to evaluate the efficacy of vagus nerve stimulation to treat hypertension and hypertension-induced heart disease. This thesis investigates the impact of vagus nerve stimulation on disease progression, survival, and cardiovascular remodeling in Dahl salt-sensitive hypertensive rats. Overall, the results of this work provide evidence for the beneficial therapeutic effects of vagus nerve stimulation in hypertension and motivate future studies to optimize therapy parameters and further understand therapeutic mechanisms. The second part of this thesis focuses on atrial fibrillation and the evaluation of new mapping techniques for improving rotor localization for ablation procedures. Currently, success rates for ablation procedures for non-paroxysmal atrial fibrillation are low and require repeat procedures or a lifetime of pharmacological agents to reduce the risk of stroke. Improved signal processing techniques for mapping electrical activity in the atrium can help further our understanding of the generation and maintenance of atrial fibrillation and ultimately improve ablation procedure success rates and terminate the arrhythmia. The main goal of this work was to validate new signal processing techniques – multiscale frequency, kurtosis, Shannon entropy, and multiscale entropy – to identify regions of abnormal electrical activity. The results of this work demonstrate improved accuracy of these novel techniques in mapping rotors in cardiac arrhythmias and motivates further studies evaluating more complex arrhythmias and human intracardiac electrograms

Cardiac Arrhythmias

The most intimate mechanisms of cardiac arrhythmias are still quite unknown to scientists. Genetic studies on ionic alterations, the electrocardiographic features of cardiac rhythm and an arsenal of diagnostic tests have done more in the last five years than in all the history of cardiology. Similarly, therapy to prevent or cure such diseases is growing rapidly day by day. In this book the reader will be able to see with brighter light some of these intimate mechanisms of production, as well as cutting-edge therapies to date. Genetic studies, electrophysiological and electrocardiographyc features, ion channel alterations, heart diseases still unknown , and even the relationship between the psychic sphere and the heart have been exposed in this book. It deserves to be read

Chronic Low-Level Vagus Nerve Stimulation Improves Long-Term Survival in Salt-Sensitive Hypertensive Rats

Chronic hypertension (HTN) affects more than 1 billion people worldwide, and is associated with an increased risk of cardiovascular disease. Despite decades of promising research, effective treatment of HTN remains challenging. This work investigates vagus nerve stimulation (VNS) as a novel, device-based therapy for HTN treatment, and specifically evaluates its effects on long-term survival and HTN-associated adverse effects. HTN was induced in Dahl salt-sensitive rats using a high-salt diet, and the rats were randomly divided into two groups: VNS (n = 9) and Sham (n = 8), which were implanted with functional or non-functional VNS stimulators, respectively. Acute and chronic effects of VNS therapy were evaluated through continuous monitoring of blood pressure (BP) and ECG via telemetry devices. Autonomic tone was quantified using heart rate (HR), HR variability (HRV) and baroreflex sensitivity (BRS) analysis. Structural cardiac changes were quantified through gross morphology and histology studies. VNS significantly improved the long-term survival of hypertensive rats, increasing median event-free survival by 78% in comparison to Sham rats. Acutely, VNS improved autonomic balance by significantly increasing HRV during stimulation, which may lead to beneficial chronic effects of VNS therapy. Chronic VNS therapy slowed the progression of HTN through an attenuation of SBP and by preserving HRV. Finally, VNS significantly altered cardiac structure, increasing heart weight, but did not alter the amount of fibrosis in the hypertensive hearts. These results suggest that VNS has the potential to improve outcomes in subjects with severe HTN

Topographical Distribution and Morphology of Sympathetic Postganglionic Innervation and Chronic Intermittent Hypoxia (CIH) Induced Remodeling of the Whole Heart at Single Cell/Axon/Varicosity Scale

The sympathetic nervous system is crucial for controlling multiple cardiac functions and its overactivity is associated with many cardiovascular diseases (CVD). Chronic intermittent hypoxia (CIH) is a current model for sleep apnea, which constitutes a major risk factor for CVD through sympathetic overactivity. However, a comprehensive neuroanatomical map of the sympathetic innervation of the heart is unavailable which impedes our understanding of the remodeling of this map in pathological conditions. First, we used a combination of state-of-the-art techniques, including flat-mount tissue processing, immunohistochemistry for tyrosine hydroxylase (TH, a sympathetic marker), confocal microscopy and Neurolucida 360 software to trace, digitize, and quantitatively map the topographical sympathetic innervation in the whole heart of mice. Then we integrated our tracing data onto a 3D heart scaffold. Second, we determined the remodeling of sympathetic innervation in CIH, by exposing mice to either room air or CIH for 8-10 weeks. We found that (1) 4–5 extrinsic TH-IR nerve bundles entered the right atrium from the superior vena cava and the left atrium from the left precaval vein. Although these bundles projected to different areas of the atria, their projection fields partially overlapped. (2) TH-IR axon and terminal density varied considerably between different sites of the heart with the greatest density of innervation near the sinoatrial node region (P \u3c 0.05, n = 6). (3) TH-IR axons also innervated blood vessels and adipocytes. (4) In ventricles: TH-IR axons formed dense terminal networks in the epicardium, myocardium, and vasculature. (5) TH-IR axons were traced and integrated into 3D heart scaffolds. (6) CIH significantly increased TH-IR innervation and complexity in the heart. Collectively, this work provided detailed mapping of catecholaminergic axons and terminal structures in the whole heart at single-cell/axon/varicosity scale in normal and CIH conditions. This work may provide a foundation for the functional study of sympathetic control of the heart and valuable neuromodulation strategies to treat CVD

Advances in Electrocardiograms

Electrocardiograms have become one of the most important, and widely used medical tools for diagnosing diseases such as cardiac arrhythmias, conduction disorders, electrolyte imbalances, hypertension, coronary artery disease and myocardial infarction. This book reviews recent advancements in electrocardiography. The four sections of this volume, Cardiac Arrhythmias, Myocardial Infarction, Autonomic Dysregulation and Cardiotoxicology, provide comprehensive reviews of advancements in the clinical applications of electrocardiograms. This book is replete with diagrams, recordings, flow diagrams and algorithms which demonstrate the possible future direction for applying electrocardiography to evaluating the development and progression of cardiac diseases. The chapters in this book describe a number of unique features of electrocardiograms in adult and pediatric patient populations with predilections for cardiac arrhythmias and other electrical abnormalities associated with hypertension, coronary artery disease, myocardial infarction, sleep apnea syndromes, pericarditides, cardiomyopathies and cardiotoxicities, as well as innovative interpretations of electrocardiograms during exercise testing and electrical pacing

Mechanisms of Atrial Arrhythmia: Investigations of the Neuro-Myogenic Interface in the Mouse

Arrhythmia mechanisms rely on multiple factors including structural (myogenic), nervous (neurogenic), and interrelated (the neuro-myogenic interface) factors. I hypothesized that due to this neuro-myogenic interface, the intrinsic cardiac autonomic nervous system (ICANS) is involved in most atrial arrhythmias. This thesis also provides a Threshold Model as a tool to assess the role of different physiological factors influencing arrhythmia. This model allows relative comparison and interpretation of the role of various factors influencing arrhythmogenesis. The mouse allows relatively simple manipulation of genes to determine their role in arrhythmia. This thesis determined what atrial arrhythmias are inducible in the mouse (in vivo) and how to systematically study those arrhythmias. I found that atrial tachycardia/fibrillation (AT/F) and junctional tachycardia (JT) are inducible in the mouse. AF and JT pose significant clinical challenges as many patients do not respond well to current interventions. Neurogenic AF relies on acetylcholine, while myogenic AF relies, in part on gap junctions formed by connexins (Cxs). The atria has muscarinic M2 and M3 receptors. The duration of M2R/M3R G protein signalling is regulated by GTP hydrolysis, a process accelerated by the regulators of G protein signalling (RGS). RGS2 deficient (RGS2-/-) mice had reduced refractory periods that were normalized with a selective M3R blocker (Darifenacin) and increased susceptibility to AT/F induction compared to littermates. For the first time, this showed a role of M3 and RGS in atrial arrhythmia. Cx40 deficient (Cx40-/-) mice were protected from carbachol induced AT/F, while Cx43 G60S mutant (Cx43G60S/+) mice, with an 80% reduction in phospho-Cx43 in the atria were highly susceptible to AT/F that was terminated by darifenacin. This shows a novel neurogenic component to what was previously described as myogenic arrhythmia. Another novel finding was that JT has a neurogenic component, resulting from inappropriate AV nodal pacemaker activation initiated by autonomics. Ivabradine hydrochloride, a selective pacemaker channel blocker, prevented JT and may be useful in patients with JT. In conclusion, this thesis has provided novel findings of the vital role of the neuro-myogenic interface in atrial arrhythmias and has provided the basis for future investigations of potential therapeutic options for patients

Analysis of intrinsic cardiac neuron activity in relation to neurogenic atrial fibrillation and vagal stimulation

La fibrillation auriculaire est le trouble du rythme le plus fréquent chez l'homme. Elle conduit souvent à de graves complications telles que l'insuffisance cardiaque et les accidents vasculaires cérébraux. Un mécanisme neurogène de la fibrillation auriculaire mis en évidence. L'induction de tachyarythmie par stimulation du nerf médiastinal a été proposée comme modèle pour étudier la fibrillation auriculaire neurogène. Dans cette thèse, nous avons étudié l'activité des neurones cardiaques intrinsèques et leurs interactions à l'intérieur des plexus ganglionnaires de l'oreillette droite dans un modèle canin de la fibrillation auriculaire neurogène. Ces activités ont été enregistrées par un réseau multicanal de microélectrodes empalé dans le plexus ganglionnaire de l'oreillette droite. L'enregistrement de l'activité neuronale a été effectué continument sur une période de près de 4 heures comprenant différentes interventions vasculaires (occlusion de l'aorte, de la veine cave inférieure, puis de l'artère coronaire descendante antérieure gauche), des stimuli mécaniques (toucher de l'oreillette ou du ventricule) et électriques (stimulation du nerf vague ou des ganglions stellaires) ainsi que des épisodes induits de fibrillation auriculaire. L'identification et la classification neuronale ont été effectuées en utilisant l'analyse en composantes principales et le partitionnement de données (cluster analysis) dans le logiciel Spike2. Une nouvelle méthode basée sur l'analyse en composante principale est proposée pour annuler l'activité auriculaire superposée sur le signal neuronal et ainsi augmenter la précision de l'identification de la réponse neuronale et de la classification. En se basant sur la réponse neuronale, nous avons défini des sous-types de neurones (afférent, efférent et les neurones des circuits locaux). Leur activité liée à différents facteurs de stress nous ont permis de fournir une description plus détaillée du système nerveux cardiaque intrinsèque. La majorité des neurones enregistrés ont réagi à des épisodes de fibrillation auriculaire en devenant plus actifs. Cette hyperactivité des neurones cardiaques intrinsèques suggère que le contrôle de cette activité pourrait aider à prévenir la fibrillation auriculaire neurogène. Puisque la stimulation à basse intensité du nerf vague affaiblit l'activité neuronale cardiaque intrinsèque (en particulier pour les neurones afférents et convergents des circuits locaux), nous avons examiné si cette intervention pouvait être appliquée comme thérapie pour la fibrillation auriculaire. Nos résultats montrent que la stimulation du nerf vague droit a été en mesure d'atténuer la fibrillation auriculaire dans 12 des 16 cas malgré un effet pro-arythmique défavorable dans 1 des 16 cas. L'action protective a diminué au fil du temps et est devenue inefficace après ~ 40 minutes après 3 minutes de stimulation du nerf vague.Atrial fibrillation is the most frequent sustained rhythm disorder in humans and often leads to severe complications such as heart failure and stroke. A neurogenic mechanism of atrial fibrillation has been hypothesized. Tachyarrhythmia induction by mediastinal nerve stimulation has been proposed as a model to study neurogenic atrial fibrillation. In this thesis, we studied the activity of intrinsic cardiac neurons and their interactions inside the right atrium ganglionated plexus in a canine model of neurogenic atrial fibrillation. These activities were recorded by a multichannel microelectrode array that was paled into the right atrium ganglionated plexus. The recording was done for up to 4 hours and it covered the neuronal activity during different interventions such as vascular (aorta occlusion, inferior vena cava occlusion, left anterior descending coronary artery occlusion), mechanical (touching atrium and ventricle) and electrical (stimulating of vagus nerve or stellate ganglion) stimuli as well as atrial fibrillation induction. Neuronal identification and classification were done using the principal component analysis and cluster on measurements analysis in Spike2 software. New method based on principal component analysis was proposed to cancel superimposed atrial activity on neuronal signal to increase the accuracy of the neuronal response identification and classification. Based on the neuronal response, we defined subtypes of neurons (afferent, efferent and local circuit neurons) and their related activity to different stressors which provided a more detailed description of the intrinsic cardiac nervous system. The majority of recorded neurons reacted to episodes of atrial fibrillation by becoming more active. This hyperactivity of intrinsic cardiac neurons during atrial fibrillation suggested that controlling that activity might help preventing neurogenic atrial fibrillation. Since low-level vagus nerve stimulation obtunds the intrinsic cardiac neuronal activity (especially for afferent and convergent local circuit neurons), we investigated whether this intervention could be applied as a therapy for atrial fibrillation. Our results showed that right vagus nerve stimulation was able to mitigate atrial fibrillation in 12 of 16 cases and showed an adverse pro-arrhythmic effect in 1 of 16 cases. The protective action however decreased over time and became ineffective after ~40 minutes for 3 minutes vagus nerve stimulation