The intrinsic cardiac plexus represents a major peripheral integration site for neuronal, hormonal, and locally produced neuromodulators controlling efferent neuronal output to the heart. This study examined the interdependence of norepinephrine, muscarinic agonists, and ANG II, to modulate intrinsic cardiac neuronal activity. Intracellular voltage recordings from whole-mount preparations of the guinea pig cardiac plexus were used to determine changes in active and passive electrical properties of individual intrinsic cardiac neurons. Application of either adrenergic or muscarinic agonists induced changes in neuronal resting membrane potentials, decreased afterhyperpolarization duration of single action potentials, and increased neuronal excitability. Adrenergic responses were inhibited by removal of extracellular calcium ions, while muscarinic responses were inhibited by application of TEA. The adrenergic responses were heterogeneous, responding to a variety of receptor-specific agonists (phenylephrine, clonidine, dobutamine, and terbutaline), although α-receptor agonists produced the most frequent responses. Application of ANG II alone produced a significant increase in excitability, while application of ANG II in combination with either adrenergic or muscarinic agonists produced a much larger potentiation of excitability. The ANG IIinduced modulation of firing was blocked by the angiotensin type 2 (AT 2) receptor inhibitor PD 123319 and was mimicked by the AT 2 receptor agonist CGP-42112A. AT 1 receptor blockade with telmasartin did not alter neuronal responses to ANG II. These data demonstrate that ANG II potentiates both muscarinically and adrenergically mediated activation of intrinsic cardiac neurons, doing so primarily via AT 2 receptor-dependent mechanisms. These neurohumoral interactions may be fundamental to regulation of neuronal excitability within the intrinsic cardiac nervous system.

Dysfunction of the intrinsic cardiac nervous system is implicated in the genesis of atrial and ventricular arrhythmias. While this system has been studied extensively in animal models, far less is known about the intrinsic cardiac nervous system of humans. This study was initiated to anatomically identify neurotransmitters associated with the right atrial ganglionated plexus (RAGP) of the human heart. Biopsies of epicardial fat containing a portion of the RAGP were collected from eight patients during cardiothoracic surgery and processed for immunofluorescent detection of specific neuronal markers. Colocalization of markers was evaluated by confocal microscopy. Most intrinsic cardiac neuronal somata displayed immunoreactivity for the cholinergic marker choline acetyltransferase and the nitrergic marker neuronal nitric oxide synthase. A subpopulation of intrinsic cardiac neurons also stained for noradrenergic markers. While most intrinsic cardiac neurons received cholinergic innervation evident as punctate immunostaining for the high affinity choline transporter, some lacked cholinergic inputs. Moreover, peptidergic, nitrergic, and noradrenergic nerves provided substantial innervation of intrinsic cardiac ganglia. These findings demonstrate that the human RAGP has a complex neurochemical anatomy, which includes the presence of a dual cholinergic/nitrergic phenotype for most of its neurons, the presence of noradrenergic markers in a subpopulation of neurons, and innervation by a host of neurochemically distinct nerves. The putative role of multiple neurotransmitters in controlling intrinsic cardiac neurons and mediating efferent signaling to the heart indicates the possibility of novel therapeutic targets for arrhythmia prevention.

Populations of intrathoracic extracardiac neurons transduce myocardial ischemia, thereby contributing to sympathetic control of regional cardiac indices during such pathology. Our objective was to determine whether electrical neuromodulation using spinal cord stimulation (SCS) modulates such local reflex control. In 10 anesthetized canines, middle cervical ganglion neurons were identified that transduce the ventricular milieu. Their capacity to transduce a global (rapid ventricular pacing) vs. regional (transient regional ischemia) ventricular stress was tested before and during SCS (50 Hz, 0.2 ms duration at 90% MT) applied to the dorsal aspect of the T1 to T4 spinal cord. Rapid ventricular pacing and transient myocardial ischemia both activated cardiac-related middle cervical ganglion neurons. SCS obtunded their capacity to reflexly respond to the regional ventricular ischemia, but not rapid ventricular pacing. In conclusion, spinal cord inputs to the intrathoracic extracardiac nervous system obtund the latter's capacity to transduce regional ventricular ischemia, but not global cardiac stress. Given the substantial body of literature indicating the adverse consequences of excessive adrenergic neuronal excitation on cardiac function, these data delineate the intrathoracic extracardiac nervous system as a potential target for neuromodulation therapy in minimizing such effects.

Ganglionated plexuses (GPs) are major constituents of the intrinsic cardiac nervous system, the final common integrator of regional cardiac control. We hypothesized that nicotinic stimulation of individual GPs exerts divergent regional influences, affecting atrial as well as ventricular functions. In 22 anesthetized canines, unipolar electrograms were recorded from 127 atrial and 127 ventricular epicardial loci during nicotine injection (100 mcg in 0.1 ml) into either the 1) right atrial (RA), 2) dorsal atrial, 3) left atrial, 4) inferior vena cava-inferior left atrial, 5) right ventricular, 6) ventral septal ventricular or 7) cranial medial ventricular (CMV) GP. In addition to sinus and AV nodal function, neural effects on atrial and ventricular repolarization were identified as changes in the area subtended by unipolar recordings under basal conditions and at maximum neurally-induced effects. Animals were studied with intact AV node or following ablation to achieve ventricular rate control. Atrial rate was affected in response to stimulation of all 7 GPs with an incidence of 50-95% of the animals among the different GPs. AV conduction was affected following stimulation of 6/7 GP with an incidence of 22-75% among GPs. Atrial and ventricular repolarization properties were affected by atrial as well as ventricular GP stimulation. Distinct regional patterns of repolarization changes were identified in response to stimulation of individual GPs. RAGP predominantly affected the RA and posterior right ventricular walls whereas CMVGP elicited biatrial and biventricular repolarization changes. Spatially divergent and overlapping cardiac regions are affected in response to nicotinic stimulation of neurons in individual GPs.

Chronic myocardial infarction (CMI) is associated with remodeling of the ventricle and evokes adaption in the cardiac neurohumoral control systems. To evaluate the remodeling of the intrinsic cardiac nervous system following myocardial infarction, the dorsal descending coronary artery was ligated in the guinea pig heart and the animals were allowed to recover for 7-9 wk. Thereafter, atrial neurons of the intrinsic cardiac plexus were isolated for electrophysiological and immunohistochemical analyses. Intracellular voltage recordings from intrinsic cardiac neurons demonstrated no significant changes in passive membrane properties or action potential configuration compared with age-matched controls and sham-operated animals. The intrinsic cardiac neurons from chronic infarcted hearts did demonstrate an increase in evoked action potential (AP) frequency (as determined by the number of APs produced with depolarizing stimuli) and an increase in responses to exogenously applied histamine compared with sham and age-matched controls. Conversely, pituitary adenylate cyclase-activating polypeptide (PACAP)-induced increases in intrinsic cardiac neuron-evoked AP frequency were similar between control and CMI animals. Immunohistochemical analysis demonstrated a threefold increase in percentage of neurons immunoreactive for neuronal nitric oxide synthase (NOS) in CMI animals compared with control and the additional expression of inducible NOS by some neurons, which was not evident in control animals. Finally, the density of mast cells within the intrinsic cardiac plexus was increased threefold in preparations from CMI animals. These results indicate that CMI induces a differential remodeling of intrinsic cardiac neurons and functional upregulation of neuronal responsiveness to specific neuromodulators.

The upper cervical spinal region functions as an intraspinal controller of thoracic spinal reflexes and contributes to neuronal regulation of the ischemic myocardium. Our objective was to determine whether stimulation of the C2 cervical spinal cord (SCS) of rats modified the input signal at the thoracic spinal cord when cardiac ischemia-sensitive (sympathetic) afferents were activated by transient occlusion of the left anterior descending coronary artery (CoAO). Changes in c-Fos expression were used as an index of neuronal activation within the spinal cord and brain stem. The pattern of substance P (SP) release, a putative nociceptive transmitter, was measured using antibody-coated microprobes. Two SCS protocols were used: reactive SCS, applied concurrently with intermittent CoAO and preemptive, sustained SCS starting 15 min before and continuing during the repeated intermittent CoAO. CoAO increased SP release from laminae I and II in the T4 spinal cord above resting levels. Intermittent SCS with CoAO resulted in greater levels of SP release from deeper laminae IV-VII in T4 than CoAO alone. In contrast, SP release from laminae I and II was inhibited when CoAO was applied during preemptive, sustained SCS. Preemptive SCS likewise reduced c-Fos expression in the T4 spinal cord (laminae I-V) and nucleus tractus solitarius but increased expression in the intermediolateral cell column of T4 compared with CoAO alone. These results suggest that preemptive SCS from the high cervical region modulates sensory afferent signaling from the ischemic myocardium.

To quantify the concurrent transduction capabilities of spatially distributed intrinsic cardiac neurons, the activities generated by atrial vs. ventricular intrinsic cardiac neurons were recorded simultaneously in 12 anesthetized dogs at baseline and during alterations in the cardiac milieu. Few (3%) identified atrial and ventricular neurons (2 of 72 characterized neurons) responded solely to regional mechanical deformation, doing so in a tightly coupled fashion (cross-correlation coefficient r = 0.63). The remaining (97%) atrial and ventricular neurons transduced multimodal stimuli to display stochastic behavior. Specifically, ventricular chemosensory inputs modified these populations such that they generated no short-term coherence among their activities (cross-correlation coefficient r = 0.21 ± 0.07). Regional ventricular ischemia activated most atrial and ventricular neurons in a noncoupled fashion. Nicotinic activation of atrial neurons enhanced ventricular neuronal activity. Acute decentralization of the intrinsic cardiac nervous system obtunded its neuron responsiveness to cardiac sensory stimuli. Most atrial and ventricular intrinsic cardiac neurons generate concurrent stochastic activity that is predicated primarily upon their cardiac chemotransduction. As a consequence, they display relative independent short-term (beat-to-beat) control over regional cardiac indexes. Over longer time scales, their functional interdependence is manifest as the result of interganglionic interconnections and descending inputs.

Antibody-coated microprobes were inserted into the thoracic (T3-4) spinal cord in urethane-anesthetized Sprague-Dawley rats to detect the differences in the release of immunoreactive substance P-like (irSP) substances in response to differential activation of cardiac nociceptive sensory neurons (CNAN). CNAN were stimulated either by intrapericardial infusion of an inflammatory ischemic exudate solution (IES) containing algogenic substances (i.e., 10 mM each of adenosine, bradykinin, prostaglandin E2, and 5-hydroxytryptamine), or by transient occlusion of the left anterior descending coronary artery (CoAO). There was widespread basal release of irSP from the thoracic spinal cord. Stimulation of the CNAN by IES did not alter the pattern of release of irSP. Conversely, CoAO augmented the release of irSP from T3-4 spinal segments from laminae I-VII. This CoAO-induced irSP release was eliminated after thoracic dorsal rhizotomy. These results indicate that heterogeneous activation of cardiac afferents, as with focal coronary artery occlusion, represents an optimum input for activation of the cardiac neuronal hierarchy and for the resultant perception of angina. Excessive stimulation of cardiac nociceptive afferent neurons elicited during regional coronary artery occlusion involves the release of SP in the thoracic spinal cord and suggests that local spinal cord release of SP may be involved in the neural signaling of angina.

Electrostimulatory forms of therapy can reduce angina that arises from activation of cardiac nociceptive afferent fibers during transient ischemia. This study sought to determine the effects of electrical stimulation of left thoracic vagal afferents (C8-T1 level) on the release of putative nociceptive [substance P (SP)] and analgesic [dynorphin (Dyn)] peptides in the dorsal horn at the T4 spinal level during coronary artery occlusion in urethane-anesthetized Sprague-Dawley rats. Release of Dyn and SP was measured by using antibody-coated microprobes. While Dyn and SP had a basal release, occlusion of the left anterior descending coronary artery only affected SP release, causing an increase from lamina I-VII. Left vagal stimulation increased Dyn release, inhibited basal SP release, and blunted the coronary artery occlusion-induced release of SP. Dyn release reflected activation of descending pathways in the thoracic spinal cord, because vagal afferent stimulation still increased the release of Dyn after bilateral dorsal rhizotomy of T2-T5. These results indicate that electrostimulatory therapy, using vagal afferent excitation, may induce analgesia, in part, via inhibition of the release of SP in the spinal cord, possibly through a Dyn-mediated neuronal interaction.

Background Autonomic regulation therapy involving either vagus nerve stimulation (VNS) or spinal cord stimulation (SCS) represents emerging bioelectronic therapies for heart disease. The objective of this study was to determine if VNS and/or SCS modulate primary cardiac afferent sensory transduction of the ischemic myocardium. Methods Using extracellular recordings in 19 anesthetized canines, of 88 neurons evaluated, 36 ventricular-related nodose ganglia sensory neurons were identified by their functional activity responses to epicardial touch, chemical activation of their sensory neurites (epicardial veratridine) and great vessel (descending aorta or inferior vena cava) occlusion. Neural responses to 1 min left anterior descending (LAD) coronary artery occlusion (CAO) were then evaluated. These interventions were then studied following either: i) SCS [T1-T3 spinal level; 50 Hz, 90% motor threshold] or ii) cervical VNS [15–20 Hz; 1.2 × threshold]. Results LAD occlusion activated 66% of identified nodose ventricular sensory neurons (0.33 ± 0.08–0.79 ± 0.20 Hz; baseline to CAO; p < 0.002). Basal activity of cardiac-related nodose neurons was differentially reduced by VNS (0.31 ± 0.11 to 0.05 ± 0.02 Hz; p < 0.05) as compared to SCS (0.36 ± 0.12 to 0.28 ± 0.14, p = 0.59), with their activity response to transient LAD CAO being suppressed by either SCS (0.85 ± 0.39–0.11 ± 0.04 Hz; p < 0.03) or VNS (0.75 ± 0.27–0.12 ± 0.05 Hz; p < 0.04). VNS did not alter evoked neural responses of cardiac-related nodose neurons to great vessel occlusion. Conclusions Both VNS and SCS obtund ventricular ischemia induced enhancement of nodose afferent neuronal inputs to the medulla.