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Aims: The behavior of pacemaker cardiomyocytes (PCs) in the sinoatrial node (SAN) is modulated by neurohormonal and paracrine factors, many of which signal through G-protein coupled receptors (GPCRs). The aims of the present study are to catalog GPCRs that are differentially expressed in the mammalian SAN and to define the acute physiological consequences of activating the cholecystokinin-A signaling system in isolated PCs. Methods and results: Using bulk and single cell RNA sequencing datasets, we identify a set of GPCRs that are differentially expressed between SAN and right atrial tissue, including several whose roles in PCs and in the SAN have not been thoroughly characterized. Focusing on one such GPCR, Cholecystokinin-A receptor (CCKAR), we demonstrate expression of Cckar mRNA specifically in mouse PCs, and further demonstrate that subsets of SAN fibroblasts and neurons within the cardiac intrinsic nervous system express cholecystokinin, the ligand for CCKAR. Using mouse models, we find that while baseline SAN function is not dramatically affected by loss of CCKAR, the firing rate of individual PCs is slowed by exposure to sulfated cholecystokinin-8 (sCCK-8), the high affinity ligand for CCKAR. The effect of sCCK-8 on firing rate is mediated by reduction in the rate of spontaneous phase 4 depolarization of PCs and is mitigated by activation of beta-adrenergic signaling. Conclusion: (1) PCs express many GPCRs whose specific roles in SAN function have not been characterized, (2) Activation of the cholecystokinin-A signaling pathway regulates PC automaticity. [ABSTRACT FROM AUTHOR]

Aims: The behavior of pacemaker cardiomyocytes (PCs) in the sinoatrial node (SAN) is modulated by neurohormonal and paracrine factors, many of which signal through G-protein coupled receptors (GPCRs). The aims of the present study are to catalog GPCRs that are differentially expressed in the mammalian SAN and to define the acute physiological consequences of activating the cholecystokinin-A signaling system in isolated PCs.Methods and results: Using bulk and single cell RNA sequencing datasets, we identify a set of GPCRs that are differentially expressed between SAN and right atrial tissue, including several whose roles in PCs and in the SAN have not been thoroughly characterized. Focusing on one such GPCR, Cholecystokinin-A receptor (CCKAR), we demonstrate expression of Cckar mRNA specifically in mouse PCs, and further demonstrate that subsets of SAN fibroblasts and neurons within the cardiac intrinsic nervous system express cholecystokinin, the ligand for CCKAR. Using mouse models, we find that while baseline SAN function is not dramatically affected by loss of CCKAR, the firing rate of individual PCs is slowed by exposure to sulfated cholecystokinin-8 (sCCK-8), the high affinity ligand for CCKAR. The effect of sCCK-8 on firing rate is mediated by reduction in the rate of spontaneous phase 4 depolarization of PCs and is mitigated by activation of beta-adrenergic signaling.Conclusion: (1) PCs express many GPCRs whose specific roles in SAN function have not been characterized, (2) Activation of the cholecystokinin-A signaling pathway regulates PC automaticity.

Aims The response to high frequency stimulation (HFS) is used to locate putative sites of ganglionated plexuses (GPs), which are implicated in triggering atrial fibrillation (AF). To identify topological and immunohistochemical characteristics of presumed GP sites functionally identified by HFS. Methods and results Sixty-three atrial sites were tested with HFS in four Langendorff-perfused porcine hearts. A 3.5 mm tip quadripolar ablation catheter was used to stimulate and deliver HFS to the left and right atrial epicardium, within the local atrial refractory period. Tissue samples from sites triggering atrial ectopy/AF (ET) sites and non-ET sites were stained with choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH), for quantification of parasympathetic and sympathetic nerves, respectively. The average cross-sectional area (CSA) of nerves was also calculated. Histomorphometry of six ET sites (9.5%) identified by HFS evoking at least a single atrial ectopic was compared with non-ET sites. All ET sites contained ChAT-immunoreactive (ChAT-IR) and/or TH-immunoreactive nerves (TH-IR). Nerve density was greater in ET sites compared to non-ET sites (nerves/cm2: 162.3 ± 110.9 vs. 69.65 ± 72.48; P = 0.047). Overall, TH-IR nerves had a larger CSA than ChAT-IR nerves (µm2: 11 196 ± 35 141 vs. 2070 ± 5841; P < 0.0001), but in ET sites, TH-IR nerves were smaller than in non-ET sites (µm2: 6021 ± 14 586 vs. 25 254 ± 61 499; P < 0.001). Conclusions ET sites identified by HFS contained a higher density of smaller nerves than non-ET sites. The majority of these nerves were within the atrial myocardium. This has important clinical implications for devising an effective therapeutic strategy for targeting autonomic triggers of AF. [ABSTRACT FROM AUTHOR]

Stellate ganglia within the intrathoracic cardiac control system receive and integrate central, peripheral, and cardiopulmonary information to produce postganglionic cardiac sympathetic inputs. Pathological anatomical and structural remodeling occurs within the neurons of the stellate ganglion (SG) in the setting of heart failure (HF). A large proportion of SG neurons function as interneurons whose networking capabilities are largely unknown. Current therapies are limited to targeting sympathetic activity at the cardiac level or surgical interventions such as stellectomy, to treat HF. Future therapies that target the SG will require understanding of their networking capabilities to modify any pathological remodeling. We observe SG networking by examining cofluctuation and specificity of SG networked activity to cardiac cycle phases. We investigate network processing of cardiopulmonary transduction by SG neuronal populations in porcine with chronic pacing-induced HF and control subjects during extended in-vivo extracellular microelectrode recordings. We find that information processing and cardiac control in chronic HF by the SG, relative to controls, exhibits: (i) more frequent, short-lived, high magnitude cofluctuations, (ii) greater variation in neural specificity to cardiac cycles, and (iii) neural network activity and cardiac control linkage that depends on disease state and cofluctuation magnitude.

Heart failure (HF) is associated with significant morbidity and mortality. The disease is characterised by autonomic imbalance with increased sympathetic activity and withdrawal of parasympathetic activity. Despite the use of medical therapies that target, in part, the neurohormonal axis, rates of HF progression, morbidity and mortality remain high. Emerging therapies centred on neuromodulation of autonomic control of the heart provide an alternative device-based approach to restoring sympathovagal balance. Preclinical studies have proven favourable, while clinical trials have had mixed results. This article highlights the importance of understanding structural/functional organisation of the cardiac nervous system as mechanistic-based neuromodulation therapies evolve.

Neural control of the heart involves continuous modulation of cardiac mechanical and electrical activity to meet the organism's demand for blood flow. The closed-loop control scheme consists of interconnected neural networks with central and peripheral components working cooperatively with each other. These components have evolved to cooperate control of various aspects of cardiac function, which produce measurable "functional" outputs such as heart rate and blood pressure. In this review, we will outline fundamental studies probing the cardiac neural control hierarchy. We will discuss how computational methods can guide improved experimental design and be used to probe how information is processed while closed-loop control is operational. These experimental designs generate large cardio-neural datasets that require sophisticated strategies for signal processing and time series analysis, while presenting the usual large-scale computational challenges surrounding data sharing and reproducibility. These challenges provide unique opportunities for the development and validation of novel techniques to enhance understanding of mechanisms of cardiac pathologies required for clinical implementation. [ABSTRACT FROM AUTHOR]

Neural control of the heart involves continuous modulation of cardiac mechanical and electrical activity to meet the organism’s demand for blood flow. The closed-loop control scheme consists of interconnected neural networks with central and peripheral components working cooperatively with each other. These components have evolved to cooperate control of various aspects of cardiac function, which produce measurable “functional” outputs such as heart rate and blood pressure. In this review, we will outline fundamental studies probing the cardiac neural control hierarchy. We will discuss how computational methods can guide improved experimental design and be used to probe how information is processed while closed-loop control is operational. These experimental designs generate large cardio-neural datasets that require sophisticated strategies for signal processing and time series analysis, while presenting the usual large-scale computational challenges surrounding data sharing and reproducibility. These challenges provide unique opportunities for the development and validation of novel techniques to enhance understanding of mechanisms of cardiac pathologies required for clinical implementation.

Our objective was to determine whether atrial fibrillation (AF) results from excessive activation of intrinsic cardiac neurons (ICNs) and, if so, whether select subpopulations of neurons therein represent therapeutic targets for suppression of this arrhythmogenic potential. Trains of five electrical stimuli (0.3-1.2 mA, 1 ms) were delivered during the atrial refractory period to mediastinal nerves (MSN) on the superior vena cava to evoke AF. Neuroanatomical studies were performed by injecting the neuronal tracer DiI into MSN sites that induced AF. Functional studies involved recording of neuronal activity in situ from the right atrial ganglionated plexus (RAGP) in response to MSN stimulation (MSNS) prior to and following neuromodulation involving either preemptive spinal cord stimulation (SCS; T1-T3, 50 Hz, 200-ms duration) or ganglionic blockade (hexamethonium, 5 mg/kg). The tetramethylindocarbocyanine perchlorate (DiI) neuronal tracer labeled a subset (13.2%) of RAGP neurons, which also colocalized with cholinergic or adrenergic markers. A subset of DiI-labeled RAGP neurons were noncholinergic/nonadrenergic. MSNS evoked an ~4-fold increase in RAGP neuronal activity from baseline, which SCS reduced by 43%. Hexamethonium blocked MSNS-evoked increases in neuronal activity. MSNS evoked AF in 78% of right-sided MSN sites, which SCS reduced to 33% and hexamethonium reduced to 7%. MSNS-induced bradycardia was maintained with SCS but was mitigated by hexamethonium. We conclude that MSNS activates subpopulations of intrinsic cardiac neurons, thereby resulting in the formation of atrial arrhythmias leading to atrial fibrillation. Stabilization of ICN local circuit neurons by SCS or the local circuit and autonomic efferent neurons with hexamethonium reduces the arrhythmogenic potential. [ABSTRACT FROM AUTHOR]

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 TI 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. [ABSTRACT FROM AUTHOR]

Richer LP, Vinet A, Kus T, Cardinal R, Ardell JL, Armour JA. α-Adrenoceptor blockade modifies neurally induced atrial arrhythmias. Am J Physiol Regul Integr Comp Physiol 295: RI 175-Ri 180, 2008. First published August 20, 2008; doi: 10.11 52/ajpregu.00840.2007.-Our objective was to determine whether neuronally induced atrial arrhythmias can be modified by α-adrenergic receptor blockade. In 30 anesthetized dogs, trains of five electrical stimuli (1 mA; I ms) were delivered immediately after the P wave of the ECG to mediastinal nerves associated with the superior vena cava. Regional atrial electrical events were monitored with 191 atrial unipolar electrodes. Mediastinal nerve sites were identified that reproducibly initiated atrial arrhythmias. These sites were then restimulated following 1 h (time control, n = 6), or the intravenous administration of naftopidil (α[sub1]-adrenergic blocker: 0.2 mg/kg, n = 6), yohimbine (α[sub2]-adrenergic blocker: 1 mg/kg, n 6) or both (n = 8). A ganglionic blocker (hexamethonium: 1 mg/kg) was tested in four dogs. Stimulation of mediastinal nerves sites consistently elicited atrial tachyarrhythmias. Repeat stimulation after 1 h in the time-control group exerted a 19% decrease of the sites still able to induce atrial tachyarrhythmias. Hexamethonium inactivated 78% of the previously active sites. Combined α-adrenoceptor blockade inactivated 72% of the previously active sites. Bradycardia responses induced by mediastinal nerve stimulation were blunted by hexamethonium, but not by α[sub1,2]-adrenergic blockade. Naftopidil or yohimbine alone eliminated atnal arrhythmia induction from 31% and 34% of the sites (similar to time control). We conclude that heterogeneous activation of the intrinsic cardiac nervous system results in atrial arrhythmias that involve intrinsic cardiac neuronal α-adrenoceptors. In contrast to the global suppression exerted by hexamethonium, we conclude that α-adrenoceptor blockade targets intrinsic cardiac local circuit neurons involved in arrhythmia formation and not the flow-through efferent projections of the cardiac nervous system. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Antibody-coated microprobes were inserted into the thoracic (T3–4) spinal cord in urethane-anesthetized Sprague­Dawley rats to detect the differences in the release of immunoreactive substance P-like (irSP) substances in response to differential activation of cardiac nocicep0ve 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–T4 spinal segments from laminae I–VII This CoAO-induce 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. [ABSTRACT FROM AUTHOR]