Cardiac Sympathetic Neurons Research Articles

Ganglion-Specific Impairment of the Norepinephrine Transporter in the Hypertensive Rat

Hypertension is associated with enhanced cardiac sympathetic transmission, although the exact mechanisms underlying this are still unknown. We hypothesized that defective function of the norepinephrine uptake transporter (NET) may contribute to the sympathetic phenotype of the spontaneously hypertensive rat, and that this may occur before the development of hypertension itself. The dynamic kinetics of NET were monitored temporally using a novel fluorescent assay of the transporter in cultured postganglionic sympathetic neurons from the cardiac stellate ganglion, the superior cervical ganglion, the celiac ganglia/superior mesenteric ganglia, and the renal sympathetic chain. All NET activity was blocked by desipramine. NET rate was significantly impaired in cardiac stellate sympathetic neurons from the prehypertensive spontaneously hypertensive rat compared with age-matched normotensive Wistar-Kyoto rats. A similar response was seen in hypertensive spontaneously hypertensive rats stellate sympathetic neurons. However, no reduction in transporter rate was observed at either age in the other major noncardiac sympathetic ganglia. Depolarization of cardiac stellate neurons by electrical field stimulation further potentiated the difference in transporter rate observed between the hypertensive and normotensive rats at both developmental ages. In conclusion, dysregulation of the norepinephrine transporter in the hypertensive rat is ganglion-specific, where NET impairment in the stellate neurons may contribute to the increased cardiac norepinephrine spillover seen in hypertension.

Cardiac sympathetic neurons provide trophic signal to the heart via β2-adrenoceptor-dependent regulation of proteolysis

Increased cardiac sympathetic neuron (SN) activity has been associated with pathologies such as heart failure and hypertrophy, suggesting that cardiac innervation regulates cardiomyocyte trophism. Whether continuous input from the SNs is required for the maintenance of the cardiomyocyte size has not been determined thus far. To address the role of cardiac innervation in cardiomyocyte size regulation, we monitored the effect of pharmacological sympathetic denervation in mice on cardiac structure, function, and signalling from 24 h to 30 days in the absence of other pathological stimuli. SN ablation caused an immediate reduction in the cardiomyocyte size with minimal consequences on the resting contractile function. Atrophic remodelling was mediated by the ubiquitin-proteasome system through FOXO-dependent early induction of the muscle-specific E3 ubiquitin ligases Atrogin-1/MAFbx and MuRF1, which was followed by activation of the autophagy-lysosome system. MuRF1 was found to be determinant in denervation atrophy as remodelling did not develop in denervated MuRF1 knock-out (KO) hearts. These effects were caused by decreased basal stimulation of cardiomyocyte β2-adrenoceptor (AR), as atrophy was prevented by treatment of denervated mice with the β2-AR agonist clenbuterol. Consistent with these data, we also observed that β2-AR KO mice showed cardiac atrophy at rest. Cardiac SNs are strong regulators of the cardiomyocyte size via β2-AR-dependent repression of proteolysis, demonstrating that the neuro-cardiac axis operates constitutively for the determination of the physiological cardiomyocyte size. These results are of great clinical relevance given the role of β-AR in cardiovascular diseases and their modulation in therapy.

P2X receptors in the cardiovascular system

AbstractP2X receptors are ligand‐gated ion channels activated by ATP. They are trimeric receptors formed from P2X subunit proteins (P2X1–7), which can associate as homomers or heteromers. Among the P2X receptors, the P2X1 receptor is most widely distributed in the cardiovascular system, being expressed on the smooth muscle of most blood vessels investigated. It mediates contraction to ATP coreleased with noradrenaline as a sympathetic neurotransmitter. P2X receptors are also expressed in the heart and mediate an increase in myocyte contractility, an effect in which the P2X4 receptor is important. Prejunctional P2X3 and P2X2/3 receptors on cardiac sympathetic neurones may, through positive feedback, amplify sympathetic neurotransmission. More recently, P2X receptors have been discovered on endothelial cells with immunoreactivity for P2X1–7 receptor proteins variously having been shown, and a role identified for the P2X4 receptor as a mediator of endothelium‐dependent vasodilatation during shear stress. Their widespread expression in the heart and vasculature identifies P2X receptors as potential targets for the development of drugs to treat cardiovascular disease. WIREs Membr Transp Signal 2012, 1:663–674. doi: 10.1002/wmts.58For further resources related to this article, please visit the WIREs website.

Aldehyde Dehydrogenase Type 2 Activation by Adenosine and Histamine Inhibits Ischemic Norepinephrine Release in Cardiac Sympathetic Neurons: Mediation by Protein Kinase Cε

During myocardial ischemia/reperfusion, lipid peroxidation leads to the formation of toxic aldehydes that contribute to ischemic dysfunction. Mitochondrial aldehyde dehydrogenase type 2 (ALDH2) alleviates ischemic heart damage and reperfusion arrhythmias via aldehyde detoxification. Because excessive norepinephrine release in the heart is a pivotal arrhythmogenic mechanism, we hypothesized that neuronal ALDH2 activation might diminish ischemic norepinephrine release. Incubation of cardiac sympathetic nerve endings with acetaldehyde, at concentrations achieved in myocardial ischemia, caused a concentration-dependent increase in norepinephrine release. A major increase in norepinephrine release also occurred when sympathetic nerve endings were incubated in hypoxic conditions. ALDH2 activation substantially reduced acetaldehyde- and hypoxia-induced norepinephrine release, an action prevented by inhibition of ALDH2 or protein kinase Cε (PKCε). Selective activation of G(i/o)-coupled adenosine A(1), A(3), or histamine H(3) receptors markedly inhibited both acetaldehyde- and hypoxia-induced norepinephrine release. These effects were also abolished by PKCε and/or ALDH2 inhibition. Moreover, A(1)-, A(3)-, or H(3)-receptor activation increased ALDH2 activity in a sympathetic neuron model (differentiated PC12 cells stably transfected with H(3) receptors). This action was prevented by the inhibition of PKCε and ALDH2. Our findings suggest the existence in sympathetic neurons of a protective pathway initiated by A(1)-, A(3)-, and H(3)-receptor activation by adenosine and histamine released in close proximity of these terminals. This pathway comprises the sequential activation of PKCε and ALDH2, culminating in aldehyde detoxification and inhibition of hypoxic norepinephrine release. Thus, pharmacological activation of PKCε and ALDH2 in cardiac sympathetic nerves may have significant protective effects by alleviating norepinephrine-induced life-threatening arrhythmias that characterize myocardial ischemia/reperfusion.

Histamine 3 Receptor Activation Reduces the Expression of Neuronal Angiotensin II Type 1 Receptors in the Heart

In severe myocardial ischemia, histamine 3 (H₃) receptor activation affords cardioprotection by preventing excessive norepinephrine release and arrhythmias; pivotal to this action is the inhibition of neuronal Na⁺/H⁺ exchanger (NHE). Conversely, angiotensin II, formed locally by mast cell-derived renin, stimulates NHE via angiotensin II type 1 (AT₁) receptors, facilitating norepinephrine release and arrhythmias. Thus, ischemic dysfunction may depend on a balance between the NHE-modulating effects of H₃ receptors and AT₁ receptors. The purpose of this investigation was therefore to elucidate the H₃/AT₁ receptor interaction in myocardial ischemia/reperfusion. We found that H₃ receptor blockade with clobenpropit increased norepinephrine overflow and arrhythmias in Langendorff-perfused guinea pig hearts subjected to ischemia/reperfusion. This coincided with increased neuronal AT₁ receptor expression. NHE inhibition with cariporide prevented both increases in norepinephrine release and AT₁ receptor expression. Moreover, norepinephrine release and AT₁ receptor expression were increased by the nitric oxide (NO) synthase inhibitor N(G)-methyl-L-arginine and the protein kinase C activator phorbol myristate acetate. H₃ receptor activation in differentiated sympathetic neuron-like PC12 cells permanently transfected with H₃ receptor cDNA caused a decrease in protein kinase C activity and AT₁ receptor protein abundance. Collectively, our findings suggest that neuronal H₃ receptor activation inhibits NHE by diminishing protein kinase C activity. Reduced NHE activity sequentially causes intracellular acidification, increased NO synthesis, and diminished AT₁ receptor expression. Thus, H₃ receptor-mediated NHE inhibition in ischemia/reperfusion not only opposes the angiotensin II-induced stimulation of NHE in cardiac sympathetic neurons, but also down-regulates AT₁ receptor expression. Cardioprotection ultimately results from the combined attenuation of angiotensin II and norepinephrine effects and alleviation of arrhythmias.

Altered norepinephrine content and ventricular function in p75NTR−/− mice after myocardial infarction

Cardiac sympathetic neurons stimulate heart rate and the force of contraction through release of norepinephrine. Nerve growth factor modulates sympathetic transmission through activation of TrkA and p75NTR. Nerve growth factor plays an important role in post-infarct sympathetic remodeling. We used mice lacking p75NTR to examine the effect of altered nerve growth factor signaling on sympathetic neuropeptide expression, cardiac norepinephrine, and ventricular function after myocardial infarction. Infarct size was similar in wildtype and p75NTR−/− mice after ischemia–reperfusion surgery. Likewise, mRNAs encoding vasoactive intestinal peptide, galanin, and pituitary adenylate cyclase activating peptides were identical in wildtype and p75NTR−/− cardiac sympathetic neurons, as was expression of the TrkA neurotrophin receptor. Norepinephrine content was elevated in the base of the p75NTR−/− ventricle compared to wildtype, but levels were identical below the site of occlusion. Left ventricular pressure, dP/dtMAX, and dP/dtMIN were measured under isoflurane anesthesia 3 and 7days after surgery. Ventricular pressure decreased significantly 3days after infarction, and deficits in dP/dtMAX were revealed by stimulating beta receptors with dobutamine and release of endogenous norepinephrine with tyramine. dP/dtMIN was not altered by genotype or surgical group. Few differences were observed between genotypes 3days after surgery, in contrast to low pressure and dP/dtMAX previously reported in control p75NTR−/− animals. Seven days after surgery ventricular pressure and dP/dtMAX were significantly lower in p75NTR−/− hearts compared to WT hearts. Thus, the lack of p75NTR did not enhance cardiac function after myocardial infarction.

Cardiac ischemia-reperfusion regulates sympathetic neuropeptide expression through gp130-dependent and independent mechanisms

Cardiac function is regulated by a balance of sympathetic and parasympathetic transmission. Neuropeptide Y (NPY) and galanin (GAL) released from cardiac sympathetic neurons inhibits parasympathetic transmission in the heart. Sympathetic peptides may contribute to autonomic imbalance, which is characterized by increased sympathetic and decreased parasympathetic transmission and contributes to life threatening cardiovascular pathologies. Several gp130 cytokines are increased in the heart after myocardial infarction (MI), and these cytokines stimulate neuropeptide expression in sympathetic neurons. We used mice whose sympathetic neurons lack the gp130 receptor (gp130(DBH-Cre/lox) mice) to ask if cytokine activation of gp130 regulated neuropeptide expression in cardiac sympathetic nerves after MI. Myocardial infarction decreased NPY mRNA through a gp130 independent mechanism and increased VIP and PACAP mRNA via gp130, while GAL mRNA was unchanged. Immunohistochemistry revealed a gp130-dependent increase in PACAP38 in cells of the stellate ganglion after MI, and PACAP was detected in pre-ganglionic fibers of all genotypes and surgical groups. VIP was identified in a few sympathetic nerve fibers in all genotypes and surgical groups. GAL and PACAP38 were not detected in sham hearts, but peptide immunoreactivity was high in the infarct three days after MI. Surprisingly, peptides were abundant in cells that co-labeled with macrophage markers F4/80 and MAC2, but were not detected in sympathetic axons. PACAP protects cardiac myocytes from apoptosis, and GAL stimulates axon regeneration in addition to inhibiting parasympathetic transmission. Thus, these peptides may play an important role in cardiac and neuronal remodeling after ischemia-reperfusion.

Structural neuroplasticity following T5 spinal cord transection: increased cardiac sympathetic innervation density and SPN arborization

When the spinal cord is injured at or below thoracic level 5 (T5), cardiovascular control is markedly unbalanced as the heart and blood vessels innervated by upper thoracic segments remain under brain stem control, whereas the vasculature of the lower body is affected by unregulated spinal reflexes. Importantly, the regulation of heart rate and cardiac function is abnormal after spinal cord injury (SCI) at T5 because sympathetic outflow to the heart is increased. An increase in tonic sympathetic outflow may be attributable to multiple mechanisms, such as increases in cardiac sympathetic innervation density, altered morphology of stellate ganglia neurons, and/or structural neuroplasticity of cardiac sympathetic preganglionic neurons (SPNs). Furthermore, these neuroplastic changes associated with SCI may be mediated by nerve growth factor (NGF). NGF is a neurotrophin that supports the survival and differentiation of sympathetic neurons and enhances target innervation. Therefore, we tested the hypothesis that T5 spinal cord transection (T5X) is associated with an increased left ventricular (LV) NGF content, LV sympathetic innervation density, and cardiac SPN arborization. In intact and paraplegic (9 wk posttransection) rats, LV NGF content (ELISA), LV sympathetic innervation density (tyrosine hydroxylase immunohistochemistry), and cardiac SPN arborization (cholera toxin B immunohistochemistry and Sholl Analysis) were determined. Paraplegia, compared with intact, significantly increased LV NGF content, LV sympathetic innervation density, and cardiac SPN arborization. Thus, altered autonomic behavior following SCI is associated with structural neuroplastic modifications.

Identification of μ‐ and κ‐opioid receptors as potential targets to regulate parasympathetic, sympathetic, and sensory neurons within rat intracardiac ganglia

Recent interest has been focused on the opioid regulation of heart performance; however, specific allocation of opioid receptors to the parasympathetic, sympathetic, and sensory innervations of the heart is scarce. Therefore, the present study aimed to characterize such specific target sites for opioids in intracardiac ganglia, which act as a complex network for the integration of the heart's neuronal in- and output. Tissue samples from rat heart atria were subjected to RT-PCR, Western blot, radioligand-binding, and double immunofluorescence confocal analysis of mu (M)- and kappa (K)-opioid receptors (ORs) with the neuronal markers vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), and substance P (SP). Our results demonstrated MOR- and KOR-specific mRNA, receptor protein, and selective membrane ligand binding. By using immunofluorescence confocal microscopy, MOR and KOR immunoreactivity were colocalized with VAChT in large-diameter parasympathetic principal neurons, with TH-immunoreactive small intensely fluorescent (SIF) cells, and on nearby TH-IR varicose terminals. In addition, MOR and KOR immunoreactivity were identified on CGRP- and SP-IR sensory neurons throughout intracardiac ganglia and atrial myocardium. Our findings show that MOR and KOR are expressed as mRNA and translated into specific receptor proteins on cardiac parasympathetic, sympathetic, and sensory neurons as potential binding sites for opioids. Thus, they may well play a role within the complex network for the integration of the heart's neuronal in- and output.

Spinal cord stimulation (SCS) modifies cardiac‐related dorsal root ganglia (DRG) afferent neurons in response to myocardial ischemia

ObjectiveTo determine if SCS modulates the capacity of cardiac sympathetic afferent neurons to transduce regional myocardial ischemia.MethodsUsing extracellular recording techniques in anesthetized canines, cardiac‐related DRG neurons (T1‐T3 spinal levels) were identified by multimodal stimulation of the left anterior descending (LAD) region. Then neuronal responses to transient (1 min) LAD coronary artery occlusion (CAO) were evaluated prior to and following SCS (T1‐T3 spinal level; 50Hz, 90% motor threshold; 20 min).ResultsLAD CAO caused a ~4 fold increase in cardiac afferent neuronal activity that persisted for at least 30 minutes following reperfusion. SCS doubled basal cardiac afferent neuronal activity, doing so in a stochastic manner. Following SCS, LAD CAO evoked no further increase in such activity.ConclusionTransient myocardial ischemia activates primary afferent inputs to the spinal cord well beyond stressor application. Pre‐emptive SCS obtunds sensory transduction of the ischemic myocardium to spinal cord neurons. (Supported by HL71830.)

Heterogeneous ventricular sympathetic innervation, altered β-adrenergic receptor expression, and rhythm instability in mice lacking the p75 neurotrophin receptor

Sympathetic nerves stimulate cardiac function through the release of norepinephrine and the activation of cardiac beta(1)-adrenergic receptors. The sympathetic innervation of the heart is sculpted during development by chemoattractive factors including nerve growth factor (NGF) and the chemorepulsive factor semaphorin 3a. NGF acts through the TrkA receptor and the p75 neurotrophin receptor (p75(NTR)) in sympathetic neurons. NGF stimulates sympathetic axon extension into the heart through TrkA, but p75(NTR) modulates multiple coreceptors that can either stimulate or inhibit axon outgrowth. In mice lacking p75(NTR), the sympathetic innervation density in target tissues ranges from denervation to hyperinnervation. Recent studies have revealed significant changes in the sympathetic innervation density of p75NTR-deficient (p75(NTR-/-)) atria between early postnatal development and adulthood. We examined the innervation of adult p75(NTR-/-) ventricles and discovered that the subendocardium of the p75(NTR-/-) left ventricle was essentially devoid of sympathetic nerve fibers, whereas the innervation density of the subepicardium was normal. This phenotype is similar to that seen in mice overexpressing semaphorin 3a, and we found that sympathetic axons lacking p75(NTR) are more sensitive to semaphorin 3a in vitro than control neurons. The lack of subendocardial innervation was associated with decreased dP/dt, altered cardiac beta(1)-adrenergic receptor expression and sensitivity, and a significant increase in spontaneous ventricular arrhythmias. The lack of p75(NTR) also resulted in increased tyrosine hydroxylase content in cardiac sympathetic neurons and elevated norepinephrine in the right ventricle, where innervation density was normal.

Targeted ablation of cardiac sympathetic neurons reduces the susceptibility to ischemia-induced sustained ventricular tachycardia in conscious rats

The Cardiac Arrhythmia Suppression Trial demonstrated that antiarrhythmic drugs not only fail to prevent sudden cardiac death, but actually increase overall mortality. These findings have been confirmed in additional trials. The "proarrhythmic" effects of most currently available antiarrhythmic drugs makes it essential that we investigate novel strategies for the prevention of sudden cardiac death. Targeted ablation of cardiac sympathetic neurons may become a therapeutic option by reducing sympathetic activity. Thus cholera toxin B subunit (CTB) conjugated to saporin (a ribosomal inactivating protein that binds to and inactivates ribosomes; CTB-SAP) was injected into both stellate ganglia to test the hypothesis that targeted ablation of cardiac sympathetic neurons reduces the susceptibility to ischemia-induced, sustained ventricular tachycardia in conscious rats. Rats were randomly divided into three groups: 1) control (no injection); 2) bilateral stellate ganglia injection of CTB; and 3) bilateral stellate ganglia injection of CTB-SAP. CTB-SAP rats had a reduced susceptibility to ischemia-induced, sustained ventricular tachycardia. Associated with the reduced susceptibility to ventricular arrhythmias were a reduced number of stained neurons in the stellate ganglia and spinal cord (segments T(1)-T(4)), as well as a reduced left ventricular norepinephrine content and sympathetic innervation density. Thus CTB-SAP retrogradely transported from the stellate ganglia is effective at ablating cardiac sympathetic neurons and reducing the susceptibility to ventricular arrhythmias.

Electrical stimulation of sympathetic neurons induces autocrine/paracrine effects of NGF mediated by TrkA

Neuronal remodeling with increased sympathetic innervation density has been implicated in the pathogenesis of atrial fibrillation (AF). Recently, increased transcardiac nerve growth factor (NGF) levels were observed in a canine model of AF. Whether atrial myocytes or cardiac sympathetic neurons are the source of neurotrophins, and whether NGF is the main neurotrophic factor contributing to sympathetic nerve sprouting (SNS) in AF still remains unclear. Therefore, neonatal rat atrial myocytes were cultured under conditions of high frequency electrical field stimulation (HFES) to mimic rapid atrial depolarization. Likewise, sympathetic neurons from the superior cervical ganglia of neonatal rats were exposed to HFES to simulate the physiological effect of sympathetic stimulation. Real-time PCR, ELISA and Western blots were performed to analyze the expression pattern of NGF and neurotrophin-3 (NT-3). Baseline NGF and NT-3 content was 3-fold higher in sympathetic neurons than in atrial myocytes (relative NGF protein expression: 1±0.0 vs. 0.37±0.11, all n=5, p<0.05). HFES of sympathetic neurons induced a frequency dependent NGF and NT-3 gene and protein up-regulation (relative NGF protein expression: 0Hz=1±0.0 vs. 5Hz=1.13±0.19 vs. 50Hz=1.77±0.08, all n=5, 0Hz/5Hz vs. 50Hz p<0.05), with a subsequent increase of growth associated protein 43 (GAP-43) expression and morphological SNS. Moreover, HFES of sympathetic neurons increased the tyrosine kinase A (TrkA) receptor expression. HFES induced neurotrophic effects could be abolished by lidocaine, TrkA blockade or NGF neutralizing antibodies, while NT-3 neutralizing antibodies had no significant effect on SNS. In neonatal rat atrial myocytes, HFES resulted in myocyte hypertrophy accompanied by an increase in NT-3 and a decrease in NGF expression. In summary, this study provides evidence that high-rate electrical stimulation of sympathetic neurons mediates nerve sprouting by an increase in NGF expression that targets the TrkA receptor in an autocrine/paracrine manner.

Atrial Fibrillation: Focal or Reentrant or Both?

Ablation for atrial fibrillation (AF) is a genuinely viable and increasingly used option for improving quality of life in patients with symptomatic drug-refractory AF. What is remarkable about the limited success achieved with these procedures is that progress has occurred despite a near complete lack of knowledge about what causes or maintains AF. Focal origins for AF were first demonstrated decades ago1–4 with topical application of acetylcholine to junctional tissues and atrial sites triggering AF. More recently, there has been convincing documentation of rapid tachycardia arising from the pulmonary veins inciting AF, thus supporting the concept of “trigger elimination” contributing to the success of pulmonary vein isolation. However, studies also support the presence of multiple reentrant circuits maintaining and possibly initiating AF.5, 6 The ablation era has taught us7–9 that in persistent and chronic AF, trigger elimination alone is not enough and that substrate modification is probably necessary to decrease the likelihood of reentrant wavelet continuance and thereby increase success rates. Even the most ardent supporter of either hypothesis recognizes the limitations of each. How can a focal source exist in this milieu indefinitely, producing and maintaining chronic AF, and how do multiple reentrant wavelets arise spontaneously? Article see p 384 In this issue of Circulation: Arrhythmia and Electrophysiology , Niu et al10 provide important, interesting, and novel insights into solving this age-old riddle using a previously unexplored angle-autonomic manipulation. Acetylcholine was used to induce AF in 31 canines. In 12 of the animal studies, propafenone was administered. They then ablated the ganglionated plexi (GP). Before ablation, AF could still be induced despite administration of propafenone. After ablation, there was a consistent pattern of electrogram organization and atrial synchronization. In addition, subsequent administration of propafenone resulted in the inability to induce atrial tachyarrhythmia. There are several …

Comparison of the effects of agonists and antagonists at peroxissome proliferator‐activated receptor gamma and angiotensin II receptors on murine preadipocytes and cardiac sympathetic neurons

The angiotensin II (AngII) receptor antagonist telmisartan, activates the peroxisome proliferator‐activated receptor gamma (PPARγ) which is a binding site for the antidiabetic thiazolidinedione. The aim of this study was to characterize the action of different AT1 receptor antagonists on PPARγ‐mediated effects and, reversely, to characterize the action of the PPARγ agonist, rosiglitazone (Rz), on AngII receptor‐mediated effects. Adipogenesis (Oil red O staining) in a preadipocyte cell line (3T3‐L1) was used to test the actions of several AT1 antagonists on PPARγ. Facilitation of tritiated noradrenaline (3H‐NA) release induced by rat sympathetic nerve stimulation was studied to test the actions of Rz on AngII receptors. Rz (300 nM) but also ZD7155 (1 μM) and candesartan (10 μM) promoted adipogenesis which was blocked by the PPARγ antagonist GW9662 (3 μM). Neither losartan nor eprosartan had any effect on adipogenesis. Rz (30‐300 nM) caused a concentration‐dependent facilitation on the evoked overflow of 3H‐NA. These results suggest that ZD7155 and candesartan promote PPARγ‐mediated adipogenesis, while losartan and eprosartan are devoided of this action. Conversely, the facilitatory action of Rz on 3H‐NA release from sympathetic nerve terminals suggests that Rz and AngII may share binding sites or signal transduction pathways.Supported by "Investigação na Pré‐graduação, Universidade do Porto‐CGD"

Targeted ablation of cardiac sympathetic neurons reduces resting, reflex and exercise-induced sympathetic activation in conscious rats

Cholera toxin B subunit conjugated to saporin (SAP, a ribosomal inactivating protein that binds to and inactivates ribosomes) was injected in both stellate ganglia to evaluate the physiological response to targeted ablation of cardiac sympathetic neurons. Resting cardiac sympathetic activity (cardiac sympathetic tonus), exercise-induced sympathetic activity (heart rate responses to graded exercise), and reflex sympathetic activity (heart rate responses to graded doses of sodium nitroprusside, SNP) were determined in 18 adult conscious Sprague-Dawley male rats. Rats were randomly divided into the following three groups (n = 6/group): 1) control (no injection), 2) bilateral stellate ganglia injection of unconjugated cholera toxin B (CTB), and 3) bilateral stellate ganglia injection of cholera toxin B conjugated to SAP (CTB-SAP). CTB-SAP rats, compared with control and CTB rats, had reduced cardiac sympathetic tonus and reduced heart rate responses to graded exercise and graded doses of SNP. Furthermore, the number of stained neurons in the stellate ganglia and spinal cord (segments T(1)-T(4)) was reduced in CTB-SAP rats. Thus CTB-SAP retrogradely transported from the stellate ganglia is effective at ablating cardiac sympathetic neurons and reducing resting, exercise, and reflex sympathetic activity. Additional studies are required to further characterize the physiological responses to this procedure as well as determine if this new approach is safe and efficacious for the treatment of conditions associated with excess sympathetic activity (e.g., autonomic dysreflexia, hypertension, heart failure, and ventricular arrhythmias).

Cardiac sympatho-excitatory action of PVN-spinal oxytocin neurones

A significant proportion of the spinally projecting neurones in the paraventricular nucleus are immunoreactive for oxytocin. Some of these oxytocin neurones terminate on sympathetic preganglionic neurones in the upper thoracic spinal cord, a region from which cardiac sympathetic neurones originate. No studies have so far identified a cardiac action of the supraspinal oxytocin neurones. The present study was designed to test the hypothesis that these oxytocin neurones excite spinal cardiac sympathetic neurones. This was done by measuring heart rate changes in response to intrathecal oxytocin and a selective agonist, and to stimulation of paraventricular neurones before and during blockade of spinal sites with selective antagonists. Rats were anaesthetised with chloralose and urethane (50 mg and 650 mg/kg) and recordings were made of heart rate and blood pressure. Drugs in a volume of 10 µl were applied to the upper thoracic spinal cord via a catheter placed intrathecally with its tip at T2. The paraventricular nucleus was explored with a glass micropipette, placed stereotaxically, and filled with d, l-homocysteic acid (DLH, 200 mM) for exciting neurones and pontamine sky blue for marking the position. Oxytocin (0.002 mM) applied to the spinal cord elicited increases in heart rate (26 ± 5 beats per minute). This was mimicked by a highly selective oxytocin agonist. These heart rate increases were blocked selectively by two different oxytocin antagonists but unaffected by a V 1a vasopressin antagonist. Excitation of sites in dorsal and medial parvocellular sub-nuclei of the paraventricular nucleus elicited increases in heart rate (36 ± 3 bpm) which were significantly reduced by oxytocin antagonists but not affected by V 1a antagonist. Also these induced increases in heart rate were unaffected by vagotomy or i.v. atropine but were abolished by i.v. esmolol. It is concluded that there is a population of paraventricular-spinal oxytocin neurones that excite cardiac sympathetic preganglionic neurones controlling heart rate.

Subclinical REM Sleep Behavior Disorder and Its Clinical and Research Implications

The recent report by Miyamoto et al.1 on comparably reduced cardiac 123I-MIBG accumulation in idiopathic REM sleep behavior disorder (iRBD), Parkinson's disease (PD), and Dementia with Lewy bodies, which implicates parkinsonian pathology (Lewy bodies; α-synuclein) in cardiac postganglionic sympathetic and intrinsic neurons, contains the following comment on the 31 iRBD patients (mean age, 66 yrs; 23 males): “In most cases, a marked reduction in 123I-MIBG accumulation occurred soon after the onset of the disease.” Therefore, most iRBD patients presumably had cardiac neuronal parkinsonian pathology present around the time that their RBD emerged. This finding can be considered together with findings from a SPECT brain imaging study of iRBD and subclinical iRBD by Eisensehr et al.2 in which decreased striatal presynaptic dopamine transporters correlated negatively with increased electromyographic (EMG) muscle activity during REM sleep. In that study, significant differences in striatal presynaptic dopamine transporters were found across a gradient in 4 groups: PD patients, iRBD patients, subclinical iRBD patients (i.e., those with increased EMG activity in REM sleep without clinical RBD behaviors), and normal controls.2 The 8 subclinical iRBD patients (mean age, 62 years; 6 males) had a significant decrease of striatal presynaptic dopamine transporters compared to normal controls. These findings in a small sample, though not yet replicated, represent an objective marker of presumed parkinsonian involvement in subclinical iRBD, which carries clinical and research implications, particularly since most middle aged and older patients with iRBD will eventually develop a parkinsonian disorder. 3,4 PSG scoring criteria for identifying subclinical RBD exist, which utilize a threshold value of 15%–20% of REM sleep with increased EMG muscle activity.2,5 Subclinical RBD includes either PSG abnormalities alone or with non-clinical behaviors in REM sleep, such as limb twitching and jerking, and simple behaviors. Since subclinical RBD is usually an incidental finding during a clinical PSG study, sleep clinicians who review and interpret PSG studies should be aware that subclinical RBD may carry a risk not only for future clinical RBD but also for parkinsonism in middle-aged and older patients. Therefore, we recommend that patients with subclinical RBD undergo a neurologic history and exam, and have their primary care physicians be informed about the possible implications associated with age-related subclinical RBD, with the caveat that medication-induced cases may not carry a parkinsonian risk.3 The longitudinal course of subclinical RBD is unknown. Subclinical RBD (also called preclinical RBD, and REM sleep without atonia [RWA]) may remain asymptomatic or resolve partially or completely, or it could progress to i) clinical iRBD; ii) clinical iRBD with progression to PD/other parkinsonism; or iii) PD/other parkinsonism directly without any clinical RBD. The RBD and parkinsonian implications are currently not known for subclinical RBD associated with younger age groups, and also for all age groups with subclinical RBD associated with medications, alcohol and drug abuse, narcolepsy, and other neurologic disorders. A growing number of non-motor abnormalities found in PD are also found in iRBD, besides the two mentioned above, including olfactory dysfunction, visuospatial constructional dysfunction and visuospatial memory impairment, etc.4 Middle-aged and older adults with subclinical iRBD may be suitable candidates for research studies probing for the non-motor abnormalities already found in iRBD and PD. Results of such studies may indicate that a future clinical research goal already recognized for iRBD also pertains to subclinical iRBD (in middle-aged and older adults), viz. to enroll patients in studies utilizing neuro-protective agents that would retard or prevent progression to future parkinsonism.3 As yet, no effective neuroprotective agent has been found, however.

Methanandamide Allosterically Inhibits in Vivo the Function of Peripheral Nicotinic Acetylcholine Receptors Containing the α7-Subunit

Methanandamide (MAEA), the stable analog of the endocannabinoid anandamide, has been proven in Xenopus oocytes to allosterically inhibit the function of the alpha7-nicotinic acetylcholine receptors (nAChRs) in a cannabinoid (CB) receptor-independent manner. The present study aimed at demonstrating that this mechanism can be activated in vivo. In anesthetized and vagotomized pithed rats treated with atropine, we determined the tachycardic response to electrical stimulation of preganglionic sympathetic nerves via the pithing rod or to i.v. nicotine (0.7 micromol/kg) activating nAChRs on the cardiac postganglionic sympathetic neurons. MAEA (3 and 10 micromol/kg) inhibited the electrically induced tachycardia (maximally by 15-20%; abolished by the CB(1) receptor antagonist AM 251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide]; 3 micromol/kg) in pentobarbitone-anesthetized pithed rats, but not in urethane-anesthetized pithed rats, which, thus, are suitable to study the CB(1) receptor-independent inhibition of nicotine-evoked tachycardia. The subunit-nonselective nAChR antagonist hexamethonium (100 micromol/kg) and the selective alpha7-subunit antagonist methyllycaconitine (MLA; 3 and 10 micromol/kg) decreased the nicotine-induced tachycardia by 100 and 40%, respectively (maximal effects), suggesting that nAChRs containing the alpha7-subunit account for 40% of the nicotine-induced tachycardia. MAEA (3 micromol/kg) produced an AM 251-insensitive inhibition (maximum again by 40%) of the nicotine-induced tachycardia. Simultaneous or sequential coadministration of MLA and MAEA inhibited the nicotine-induced tachycardia to the same extent (maximally by 40%) as each of the drugs alone. In conclusion, according to nonadditivity of the effects, MAEA mediates in vivo inhibition by the same receptors as MLA, namely alpha7-subunit-containing nAChRs, although at an allosteric instead of the orthosteric site.

Post-infarct cardiac sympathetic hyperactivity regulates galanin expression

The neuropeptide galanin is elevated in the cardiac sympathetic innervation after myocardial infarction (MI). Galanin inhibits vagal transmission and may support the regeneration of sympathetic nerves, thereby contributing to the development of arrhythmia and sudden cardiac death after MI. The reason for increased galanin production in sympathetic neurons after myocardial infarction is not known. Cardiac sympathetic neurons are activated chronically after cardiac ischemia-reperfusion, and activation of sympathetic neurons in culture stimulates galanin expression. Therefore, we tested the hypothesis that increased sympathetic nerve activity stimulates galanin expression in cardiac sympathetic neurons after myocardial infarction. To test this hypothesis we used TGR(ASrAOGEN) transgenic rats, which lack brain angiotensinogen and do not exhibit post-infarct sympathetic hyperactivity. Hearts and stellate ganglia were collected 1 week after ischemia-reperfusion. Galanin mRNA was quantified by real-time PCR and peptide content was assayed by enzyme-linked immunosorbent assay. Galanin mRNA increased approximately 3-fold after MI in cardiac sympathetic neurons of both genotypes compared to unoperated and sham controls. Left ventricular galanin content, however, increased after MI only in Sprague-Dawley rats and not in AOGEN rats. These data suggest that post-infarct cardiac sympathetic hyperactivity stimulates galanin peptide production but is not required for increased galanin mRNA expression.