David Mendelowitz, Ph.D. - US grants

Heart rate is dominated by the tonic activity of the cardioinhibitory parasympathetic nervous system. Parasympathetic activity to the heart also influences coronary blood flow and the inotropic state of cardiac muscle. An impairment in parasympathetic innervation of the heart has been implicated in diseases such as hypertension, ischemic heart disease and sudden cardiac death. Surprisingly, however, little is known concerning the origin or control of parasympathetic cardiac activity within the central nervous system. In this study I will test a number of important hypotheses concerning the origin, and synaptic modulation of preganglionic parasympathetic cardiac activity. This project combines a novel technique that I have developed to identify and isolate preganglionic parasympathetic cardiac neurons with patch clamp methodology. The synthesis of these techniques will enable me to characterize the electrophysiological properties of identified cardiovascular neurons at a cellular level. There are 4 major and distinct aims. I will: 1) Test the hypothesis that preganglionic parasympathetic cardiac neurons, isolated from their surrounding tissue in the medulla, fire spontaneously and repetitively; 2) Characterize, using patch clamp techniques, the voltage-gated ionic currents in these cardiac neurons; 3) Test the hypothesis that acetylcholine and opioid neurotransmitters influence the activity of preganglionic parasympathetic cardiac neurons. I will also identify other neurotransmitters that are most likely to influence the activity of preganglionic cardiac neurons; and 4) Characterize the ionic currents that are evoked by these neurotransmitters, and the effects of these neurotransmitters on the voltage-gated currents. The results from this project will provide new and unique information, at the cellular level, concerning the origin, and synaptic regulation of parasympathetic activity that innervates the heart.[unreadable]GRANT=R15HL50123 Fitness enthusiasts, laborers, and athletes need activity guidelines during an upper respiratory illness (URI). Further, fitness levels may be demonstrated to impact on the severity and duration of an URI and the musculoskeletal and cardiorespiratory responses to an URI. Results of this study will be used to make recommendations regarding physical activity levels during an episode of URI. The objectives of the study are as follows: l) to determine the impact of an URI on exercise functional capacity; 2) to determine whether a person's fitness level is related to exercise functional capacity during an URI; 3) to determine the impact of exercise on both the severity and duration of an URI; 4) to determine whether a person's fitness level is related to the severity and duration of an URI. Subjects will be exercise tested and assigned to a fit or unfit group. Within each of these groups, subjects will be randomly assigned to either a control or experimental group. Subjects will be inoculated with rhinovirus 16 (RV16). In the first phase (year one) of the study, the subsequent impact of moderate exercise training on the severity and duration of an URI will be determined. In the second phase (year two) of the study, the impact of an URI during peak illness (days 2 and 3) on the exercise functional capacity of infected subjects will be determined. Differences in group data will be analyzed.

The neural control of the cardiovascular and respiratory systems are highly interrelated. For example, in each respiratory cycle the heart beats more rapidly in inspiration and slows during post-inspiration and expiration (often referred to as respiratory sinus arrhythmia). As another example, stimulation of sensory laryngeal receptors evokes a period of apnea and maintained a dramatic decrease in heart rate. This laryngeal reflex can be so exaggerated in newborns in can lead to death in neonatal animals, and has been suggested as a possible cause for sudden infant death syndrome (SIDS). This cardio-respiratory interactions occur within the central nervous system and are mediated largely, if not entirely, via the vagal innervation of the heart. Surprisingly, however, despite the physiological and clinical importance of cardiac vagal activity, little is known about its initiation and control within the central nervous system in neonates or adults, and no pathway from identified respiratory to cardiac vagal neurons within the medulla has yet been identified. This project will directly test the hypothesis that superior laryngeal motor-neurons excite vagal cardioinhibitory neurons via a direct monosynaptic pathway, and, in addition, act presynaptically to enhance other synapses impinging on cardiac vagal neurons. Superior laryngeal neurons are likely mediators of cardio-respiratory interaction because these neurons are active in post-inspiration, co-localized with cardiac vagal neurons, and have many axon collaterals within the nucleus ambiguus. To test this hypothesis, techniques that are quite new to this field, such as fluorescent retrograde identification of cardiac vagal and superior laryngeal motor-neurons, and dual patch clamp electrophysiological techniques will be used. This work will not only address issues and mechanisms fundamental to understanding the basis of cardio-respiratory rhythms in the neonatal medulla, but will also suggest which receptors and processes could be altered in diseases of the cardio-respiratory system such as SIDS.

The normal heart rate present in healthy animals and humans is determined primarily by the activity of parasympathetic cardiac neurons in the brainstem. Previous work has unequivocally demonstrated that the tonic parasympathetic activity that innervates the heart, and reflex changes in cardiac vagal activity, play an essential role in normal cardiovascular function. However in some hypotensive cardiovascular challenges, such as septic and hemorrhagic shock, there is a paradoxical increase in cardiac vagal activity and bradycardia which is detrimental to survival. This abnormal increase in cardiac vagal activity involves activation of central opioid receptors. The overall hypothesis guiding the current proposal is that activation of opioid receptors increase the excitability of cardiac vagal neurons by modulating critical synaptic inputs as well as directly altering voltage gated currents in cardiac vagal neurons. This hypothesis is supported both by our preliminary electrophysiological observations as well as our immunohistochemical and collaborative electron microscopic work. We will specifically 1) test whether activation of opioid receptors alters the critical synaptic innervation of cardiac vagal neurons. 2) examine whether activation of postsynaptic opioid receptors modifies the properties of voltage gated currents in cardiac vagal neurons, 3) identify the source of opioid pathways to cardiac vagal neurons and 4) characterize the electrophysiological properties of the opioid-containing neurons that monosynaptically project to cardiac vagal neurons. The anticipated results will provide information fundamental to understanding the basis and mechanisms of cardiac vagal activity that originate in the medulla, and will also suggest which receptors and processes could be involved in paradoxical and detrimental responses of the cardiovascular system.

DESCRIPTION (provided by applicant): The trigeminocardiac and diving reflexes are among the most powerful autonomic reflexes. Electrical or mechanical stimulation of the trigeminal nerve evokes a dramatic decrease in heart rate in animals including man, and has often been termed the `trigeminocardiac reflex' in the clinical literature, and `trigeminal depressor responses' in animal studies. A subset of the trigeminocardiac reflex is the diving reflex. Stimulation of the diving reflex by exposing the nasal mucosa to water or air-borne chemical irritants evokes a pronounced bradycardia with heart rate decreasing up to 51% upon a single facial submersion. However an exaggerated diving reflex has been implicated in sudden infant death syndrome (SIDS). SIDS is the leading cause of death among infants who are 1 month to 1 year old. One of the highest risk factors for SIDS is cigarette smoking and while tobacco smoke contains a number of deleterious agents in addition to nicotine, nicotine is of particular concern in relation to SIDS due to its ability to cross the placenta and concentrate in the fetus, and prenatal nicotine exposure impairs the ability of newborn animals to resuscitate from cardiorespiratory challenges. This project will directly test the hypotheses that activation of sensory neurons in the trigeminocardiac and diving reflexes excites cardiac vagal neurons, these reflex pathways are endogenously and differentially modulated by nicotinic and muscarinic cholinergic receptors, determine where in this reflex pathway this modulation occurs, and finally whether fetal exposure to nicotine exaggerates this excitation of cardiac vagal neurons. To test these hypotheses we will utilize techniques that are quite novel to this field. Two such new approaches include the identification of the synaptic terminals of sensory neurons originating in the nasal mucosa using lentivirus expression of enhanced yellow fluorescent protein (eYFP), as well as the expression of channelrhodopsin-2 (ChR2), a light activated cation channel, to selectively photoactivate the fibers and synaptic terminals of these nasal sensory neurons. PUBLIC HEALTH RELEVANCE: Project Narrative - Public Health Relevance Statement This work will address hypotheses fundamental to understanding the cellular basis and mechanisms by which nicotinic and muscarinic cholinergic receptors modulate the diving reflex within the brainstem, and will also suggest which receptors and processes are altered by fetal exposure to nicotine that increases the risk of cardiorespiratory diseases such as sudden infant death syndrome (SIDS).

DESCRIPTION (provided by applicant): One major, yet poorly understood cardiovascular health risk that occurs in as many as ~24% of males and 9% of females within the United States population is obstructive sleep apnea (OSA). OSA can participate in both the initiation and progression of several cardiovascular diseases including sudden death, hypertension, arrhythmias, myocardial ischemia and stroke. Treatment of OSA is primarily continuous positive airway pressure (CPAP), and while this treatment is marginally effective in reducing elevated arterial pressure (~2 mmHg) CPAP is intrusive, poorly tolerated and often discontinued despite the risks of OSA. Recent work has suggested activity in neurons within the paraventricular nucleus of the hypothalamus (PVN) that are critical for the cardiovascular responses to challenges such as stress and dehydration are not impaired, but rather possess augmented activity in models of OSA and are involved in the maintenance and/or generation of OSA induced hypertension. However the PVN is a heterogeneous nucleus. Whereas vasopressin (AVP) neurons in the PVN are sympathoexcitatory, and activation of vasopressin receptors inhibits cardioprotective parasympathetic cardiac vagal neurons (CVNs), recent work has provided exciting new evidence that the neuropeptide oxytocin, released from a different population of PVN neurons, is cardioprotective. Oxytocin reduces the adverse cardiovascular consequences of anxiety and stress and, as this study will test, perhaps the deleterious consequences of chronic nocturnal intermittent hypoxia/hypercapnia. This project challenges the paradigm that the PVN is solely sympathoexcitatory, and will test that there are two contrasting pathways from the PVN, one that co-releases oxytocin, activates CVNs and is cardioprotective, and another pathway in which vasopressin inhibits CVNs and increases adverse cardiovascular changes. Furthermore this work will test if these pathways are altered and if these two populations of PVN neurons can be differentially controlled to mitigate or enhance the adverse cardiovascular changes that occur in a model of OSA. This project will address major gaps in our knowledge and hopefully constitute a foundation for appraising new potential treatments and targets for patients with cardiovascular diseases including OSA.

ABSTRACT 22q11 Deletion Syndrome (22q11DS) patients have feeding and swallowing difficulties that compromise their nutritional status and increase nasal, ear, and respiratory infections due to aspiration and reflux. Unfortunately, there are few, if any, therapies to alleviate these significant health problems, and there is no focused basic research into defining the underlying pathology that will inform new therapeutic approaches. We will test the hypothesis that disrupted brainstem motor circuits are a primary site for dysphagia pathology and a primary target for therapeutic intervention. We will advance understanding of the pathology of feeding and swallowing dysfunction in dysphagia using the LgDel animal model for 22q11DS. These studies will provide the foundation for identifying feasible therapeutic approaches and novel drug targets that could improve feeding and swallowing in 22q11DS patients and other children with dysphagia. In Specific Aim 1 we will use a combination of quantitative 3D cellular imaging and cell-class specific analyses of transcriptional changes to assess how diminished 22q11 gene dosage disrupts motor neuron differentiation and innervation in brainstem cranial motor nuclei that regulate feeding and swallowing. In Specific Aim 2 we will use a combination of in vivo behavioral and pathology assays for feeding and swallowing difficulties and quantitative 3D cellular imaging of relevant brainstem CN motor neurons to test if a second mutation in Raldh2, a key synthetic enzyme for production of the developmental signal retinoic acid (RA), corrects feeding, swallowing, and cranial motor neuron differentiation anomalies and dysfunction in LgDel mice. In Specific Aim 3 we will test the hypothesis that altered excitability due to disrupted balance of excitation and inhibition of cranial motor neurons underlies dysphagia due to the 22q11 deletion. We will use a combination of patch-clamp and intracellular recordings to identify changes in the synaptic neurotransmission elicited upon fictive swallowing to brainstem cranial neurons essential for feeding and swallowing and, furthermore, test whether clinically useful drugs in children, such as GABA(A) receptor agonists (benzodiazepines), and/or NMDA receptor antagonists (ketamine) can be repurposed to restore normal activity of swallowing motor neurons. The results of PROJECT 1 will define pediatric dysphagia circuit pathology and provide new pharmacologic, genetic, and genomic insights that will provide a foundation for identifying new targets for novel therapeutic interventions. These results will define key outcomes whose developmental origins will be identified in PROJECT 2 and whose prevention will be the focus of PROJECT 3.

Heart failure (HF) is a widespread and debilitating cardiovascular disease that affects nearly 23 million people worldwide with approximately 2 million new patients diagnosed annually. A distinctive hallmark of heart failure is autonomic imbalance, consisting of increased sympathetic activity and decreased parasympathetic tone. Restoration of parasympathetic activity to the heart has recently emerged as a promising new therapeutic approach to inhibit the progression of heart failure and risk of sudden cardiac death. Our preliminary results provide critical new information for the field that identifies a novel target that could restore parasympathetic cardiac activity in an animal model of left ventricular hypertrophy that progresses to heart failure. The overarching hypothesis of the current proposal is that hypothalamic paraventricular nucleus of the hypothalamus (PVN) oxytocin neurons are essential for activating parasympathetic cardiac vagal neurons (CVNs) in the brainstem. In animals with trans-aortic compression (TAC), which leads to left ventricular hypertrophy that progresses to heart failure, the release of oxytocin, and activation of CVNs, is diminished. Perhaps more importantly, our preliminary results indicate selective restoration of oxytocin activity restores the synaptic release of oxytocin from PVN neurons, the excitatory neurotransmission from PVN to parasympathetic CVNs, improves cardiac function and favorably alters the indices of cardiac ischemia and damage that occurs in untreated animals. In this proposal we will build upon our preliminary results to address three Specific Aims: 1) Determine if there is reduced release of oxytocin from paraventricular neurons of the hypothalamus fibers in the brainstem and blunted excitation of cardiac vagal neurons in heart failure diseased animals. Furthermore test if selective chronic activation of oxytocin neurons in the PVN acts to restore both the release of oxytocin in the brainstem and activation of parasympathetic cardiac vagal neurons. 2) Test the hypothesis that chronic activation of PVN oxytocin neurons mitigates the progression of cardiac dysfunction that occurs in untreated HF disease animals. Left ventricular (LV) developed pressure, contractility, and electrical synchronization will be measured to assess mechanisms of improved cardiac function. 3) Examine if indices of cardiac ischemia, including in-vivo electrocardiograph (EKG) abnormalities, increased fluorescence of epicardial NADH (fNADH), and the formation of fibrotic (scar) tissue are absent or blunted in animals with chronic activation of hypothalamic PVN oxytocin neurons compared to untreated HF animals. The studies in this proposal will either support, or refute our hypothesis that PVN oxytocin neuron activation can restore diminished parasympathetic cardiac tone and blunt the deleterious progression of cardiac function alterations that occur in animals with LV hypertrophy, cardiac dysfunction and heart failure. This will provide an important foundation for future clinical studies, giving this work high translational potential and significance.

Sudden Cardiac Death (SCD) is responsible for between 15 and 20% of all deaths, and ~50% of all cardiovascular deaths in the United States. The most common cascade of events leading to SCD is acute coronary syndrome (ACS) progressing to acute myocardial ischemia and/or inflammation that triggers electrical instability and lethal arrhythmias. Preventing SCD is particularly difficult as approximately one-half of men and two-thirds of women who succumb to SCD had no known history of prior heart disease. Autonomic imbalance is a major risk factor for SCD. Augmented sympathetic activity induces changes in ECG repolarization and reduction of fibrillation threshold facilitating the initiation of ventricular fibrillation (VF). In contrast, the generation of fatal ventricular arrhythmias and risk of SCD is markedly reduced by increasing parasympathetic activity. However a rapid, safe and feasible approach to increase parasympathetic activity to the heart in patients at risk for fatal arrhythmias is severely lacking and is a major medical need. Our preliminary results provide critical new information for the field that identifies a novel target that could restore parasympathetic cardiac tone and reduce the incidence of arrhythmias and cardiac dysfunction following a MI. Our prior work in subjects with sleep apnea has shown intranasal (IN) application of oxytocin increases parasympathetic cardiac activity. In an animal model of ACS with ligation of the left anterior descending coronary artery (LAD) animals develop ischemia, arrhythmias and mortality similar to clinical studies. We show that LAD-ligated animals have reduced endogenous excitatory oxytocin-mediated neurotransmission to parasympathetic cardiac vagal neurons (CVNs) in the brainstem. We further show that selective and chronic activation of hypothalamic paraventricular nucleus (PVN) oxytocin neurons restores oxytocin release, increases parasympathetic activity to the heart and substantially reduces the incidence and initiation of arrhythmias, inflammation, fibrosis and other adverse cardiac outcomes. Based upon our novel results, our overall hypothesis is that chronic selective activation of PVN oxytocin neurons, as well as nasal oxytocin administration, markedly reduces arrhythmias and cardiac dysfunction in an animal model of ACS. In Aim 1 we will test the hypothesis that the critical excitatory pathway from PVN oxytocin neurons to CVNs that helps maintain protective parasympathetic activity to the heart is blunted in animals following LAD ligation, and that this key neurotransmission can be restored with nasal oxytocin treatment and chronic and selective activation of PVN oxytocin neurons. In Aim 2 we will test whether treatment by nasal oxytocin and chronic and selective activation of PVN oxytocin neurons increases parasympathetic activity to the heart in-vivo, reduce the incidence of arrhythmias, improves autonomic balance and effort capacity in exercise stress tests and cardiac function. In Aim 3 we will quantify the electrical and mechanical function of ex-vivo perfused hearts to identify the mechanisms responsible for the cardiac benefits of nasal oxytocin and selective activation of PVN oxytocin neurons in LAD-ligated animals.