Cardiac Afferent Neurons Research Articles

Lysophosphatidic acid contributes to myocardial ischemia/reperfusion injury by activating TRPV1 in spinal cord.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that plays a crucial role in cardiovascular diseases. Here, we question whether LPA contributes to myocardial ischemia/reperfusion (I/R) injury by acting on transient receptor potential vanilloid 1 (TRPV1) in spinal cord. By ligating the left coronary artery to establish an in vivo I/R mouse model, we observed a 1.57-fold increase in LPA level in the cerebrospinal fluid (CSF). The I/R-elevated CSF LPA levels were reduced by HA130, an LPA synthesis inhibitor, compared to vehicle treatment (4.74 ± 0.34 vs. 6.46 ± 0.94μg/mL, p = 0.0014). Myocardial infarct size was reduced by HA130 treatment compared to the vehicle group (26 ± 8% vs. 46 ± 8%, p = 0.0001). To block the interaction of LPA with TRPV1 at the K710 site, we generated a K710N knock-in mouse model. The TRPV1K710N mice were resistant to LPA-induced myocardial injury, showing a smaller infarct size relative to TRPV1WT mice (28 ± 4% vs. 60 ± 7%, p < 0.0001). Additionally, a sequence-specific TRPV1 peptide targeting the K710 region produced similar protective effects against LPA-induced myocardial injury. Blocking the K710 region through K710N mutation or TRPV1 peptide resulted in reduced neuropeptides release and decreased activity of cardiac sensory neurons, leading to a decrease in cardiac norepinephrine concentration and the restoration of intramyocardial pro-survival signaling, namely protein kinase B/extracellular regulated kinase/glycogen synthase kinase-3β pathway. These findings suggest that the elevation of CSF LPA is strongly associated with myocardial I/R injury. Moreover, inhibiting the interaction of LPA with TRPV1 by blocking the K710 region uncovers a novel strategy for preventing myocardial ischemic injury.

Real-time in vivo thoracic spinal glutamate sensing during myocardial ischemia.

In the spinal cord, glutamate serves as the primary excitatory neurotransmitter. Monitoring spinal glutamate concentrations offers valuable insights into spinal neural processing. Consequently, spinal glutamate concentration has the potential to emerge as a useful biomarker for conditions characterized by increased spinal neural network activity, especially when uptake systems become dysfunctional. In this study, we developed a multichannel custom-made flexible glutamate-sensing probe for the large-animal model that is capable of measuring extracellular glutamate concentrations in real time and in vivo. We assessed the probe's sensitivity and specificity through in vitro and ex vivo experiments. Remarkably, this developed probe demonstrates nearly instantaneous glutamate detection and allows continuous monitoring of glutamate concentrations. Furthermore, we evaluated the mechanical and sensing performance of the probe in vivo, within the pig spinal cord. Moreover, we applied the glutamate-sensing method using the flexible probe in the context of myocardial ischemia-reperfusion (I/R) injury. During I/R injury, cardiac sensory neurons in the dorsal root ganglion transmit excitatory signals to the spinal cord, resulting in sympathetic activation that potentially leads to fatal arrhythmias. We have successfully shown that our developed glutamate-sensing method can detect this spinal network excitation during myocardial ischemia. This study illustrates a novel technique for measuring spinal glutamate at different spinal cord levels as a surrogate for the spinal neural network activity during cardiac interventions that engage the cardio-spinal neural pathway.NEW & NOTEWORTHY In this study, we have developed a new flexible sensing probe to perform an in vivo measurement of spinal glutamate signaling in a large animal model. Our initial investigations involved precise testing of this probe in both in vitro and ex vivo environments. We accurately assessed the sensitivity and specificity of our glutamate-sensing probe and demonstrated its performance. We also evaluated the performance of our developed flexible probe during the insertion and compared it with the stiff probe during animal movement. Subsequently, we used this innovative technique to monitor the spinal glutamate signaling during myocardial ischemia and reperfusion that can cause fatal ventricular arrhythmias. We showed that glutamate concentration increases during the myocardial ischemia, persists during the reperfusion, and is associated with sympathoexcitation and increases in myocardial substrate excitability.

Abstract 14090: Myocardial Infarction Selectively Decreases Cardiac Vagal Afferent Neurotransmission: Implications for Mechanisms Behind Cardiac Parasympathetic Dysfunction

Introduction: Parasympathetic dysfunction after myocardial infarction (MI) predisposes to arrhythmias and heart failure. Mechanisms behind this dysfunction are unclear. It is known that cardiac sensory afferent neurons in the vagal ganglia sense beat-to-beat changes at the level of the heart. This vagal afferent signaling/activation then increases central cardiac vagal efferent drive. Hypothesis: We hypothesized that MI causes a functional reduction in vagal afferent signaling that subsequently decreases efferent vagal tone, resulting in parasympathetic dysfunction. Methods: A transgenic mouse line was created by crossing mice expressing excitatory vesicular glutamate transporter 2, VGlut-ires-cre, with those expressing channel rhodopsin 2 EYFP (ChR2), resulting in mice with VGlut and ChR2 expression in afferent-specific vagal neurons and fibers. MI was created by left anterior descending artery ligation. Sham animals underwent thoracotomy only. Control animals did not undergo a thoracotomy. Two weeks post-MI or sham, the left cervical vagus nerve was isolated and optically stimulated in vivo using blue light laser (473 nm, 20 Hz, 10 &amp; 20 msec). Heart rate (HR), respiratory rate (RR), and time to activation of efferent vagal effects (time to nadir HR/RR) were assessed. Results: In response to optical stimulation, MI animals (n=5) demonstrated significantly reduced HR responses vs. sham (n=4) and control (n=7) animals at both 10 ms and 20 ms, figure. Time to nadir HR was also increased in MI animals (P&lt;0.05 vs. sham/control). RR decreased in all animals, without significant differences between groups. Conclusions: MI is associated with decreased efferent vagal responses to similar levels of parasympathetic afferent activation. These results suggests that mechanisms behind decreased vagal tone post-MI may be due altered vagal afferent neurotransmission and signaling capabilities. MI selectively affects cardiac, not respiratory, neural responses.

Spinal Anesthesia Reduces Myocardial Ischemia-triggered Ventricular Arrhythmias by Suppressing Spinal Cord Neuronal Network Interactions in Pigs.

Cardiac sympathoexcitation leads to ventricular arrhythmias. Spinal anesthesia modulates sympathetic output and can be cardioprotective. However, its effect on the cardio-spinal reflexes and network interactions in the dorsal horn cardiac afferent neurons and the intermediolateral nucleus sympathetic neurons that regulate sympathetic output is not known. The authors hypothesize that spinal bupivacaine reduces cardiac neuronal firing and network interactions in the dorsal horn-dorsal horn and dorsal horn-intermediolateral nucleus that produce sympathoexcitation during myocardial ischemia, attenuating ventricular arrhythmogenesis. Extracellular neuronal signals from the dorsal horn and intermediolateral nucleus neurons were simultaneously recorded in Yorkshire pigs (n = 9) using a 64-channel high-density penetrating microarray electrode inserted at the T2 spinal cord. Dorsal horn and intermediolateral nucleus neural interactions and known markers of cardiac arrhythmogenesis were evaluated during myocardial ischemia and cardiac load-dependent perturbations with intrathecal bupivacaine. Cardiac spinal neurons were identified based on their response to myocardial ischemia and cardiac load-dependent perturbations. Spinal bupivacaine did not change the basal activity of cardiac neurons in the dorsal horn or intermediolateral nucleus. After bupivacaine administration, the percentage of cardiac neurons that increased their activity in response to myocardial ischemia was decreased. Myocardial ischemia and cardiac load-dependent stress increased the short-term interactions between the dorsal horn and dorsal horn (324 to 931 correlated pairs out of 1,189 pairs, P < 0.0001), and dorsal horn and intermediolateral nucleus neurons (11 to 69 correlated pairs out of 1,135 pairs, P < 0.0001). Bupivacaine reduced this network response and augmentation in the interactions between dorsal horn-dorsal horn (931 to 38 correlated pairs out of 1,189 pairs, P < 0.0001) and intermediolateral nucleus-dorsal horn neurons (69 to 1 correlated pairs out of 1,135 pairs, P < 0.0001). Spinal bupivacaine reduced shortening of ventricular activation recovery interval and dispersion of repolarization, with decreased ventricular arrhythmogenesis during acute ischemia. Spinal anesthesia reduces network interactions between dorsal horn-dorsal horn and dorsal horn-intermediolateral nucleus cardiac neurons in the spinal cord during myocardial ischemia. Blocking short-term coordination between local afferent-efferent cardiac neurons in the spinal cord contributes to a decrease in cardiac sympathoexcitation and reduction of ventricular arrhythmogenesis.

Evaluation of the bilateral cardiac afferent distribution at the spinal and vagal ganglia by retrograde labeling

The identity of sensory neurons innervating the heart tissue and the extent of information reported to the brain via these neurons are poorly understood. In order to evaluate the multidimensional distribution and abundance of the cardiac spinal and vagal afferents, we assessed the retrograde labeling efficiency of various tracers, and mapped the cardiac afferents qualitatively and quantitatively at the bilateral nodose ganglia (NGs) and dorsal root ganglia (DRGs). From the five different retrograde tracers evaluated, Di-8-ANEPPQ yielded reproducibly the highest labeling efficiency of cardiac afferents. We demonstrated specific cardiac afferents at NGs and C4 to T11 DRG segments. Next, the 2D reconstruction of the tissue sections and 3D imaging of the whole NGs and DRGs revealed homogeneous and bilateral distribution of cardiac afferents. The quantitative analyses of the labeled cardiac afferents demonstrated approximately 5–6% of the soma in NGs that were equally distributed bilaterally. The neuronal character of Di-8-ANEPPQ labeled cells were validated by coimmunostaning with pan-neuronal marker Tuj-1. In addition, the cell diameters of labeled cardiac sensory neurons were found smaller than 20 μm, implying the nociceptor phenotype confirmed by co-labeling with TRPV1 and Di-8-ANEPPQ. Importantly, co-labeling with two distinct tracers Di-8-ANEPPQ and WGA-647 demonstrated exclusively the same cardiac afferents in DRGs and NGs, validating our findings. Collectively, our findings revealed the cardiac afferents in NGs bilaterally and DRGs with the highest labeling efficiency reported, spatial distribution and quantitation at both 2D and 3D levels, furthering our understanding of this novel neuron population.

Inhibition of DRG-TRPV1 upregulation in myocardial ischemia contributes to exogenous cardioprotection

Myocardium ischemia-reperfusion injury (IRI) is the major cause of postoperative cardiac dysfunction. While intrathecal morphine preconditioning (ITMP) can reduce IRI in animals, the molecular processes underlying IRI and ITMP remain elusive. Transient receptor potential vanilloid type 1 (TRPV1) is highly expressed in cardiac sensory neurons and has a crucial role in detecting myocardial ischemia. This study aimed to determine the role of up-regulated dorsal root ganglion (DRG)-TRPV1 in IRI and whether its inhibition contributes to ITMP-induced cardioprotection. Animal model of IRI was established by left coronary artery occlusion (30 min) and reperfusion (2 h) in rats. Intrathecal intubation was prepared for morphine preconditioning, TRPV1-shRNA or selective TRPV1 antagonist administration. After IRI, both protein and phosphorylation levels of TRPV1 were significantly increased, and the immunofluorescence intensity of TRPV1 was increased and colocalized with μ-opioid receptors in DRG. Intrathecal pre-administration of either TRPV1-shRNA or TRPV1 antagonist significantly reduced myocardial injury and the upregulation of TRPV1 in DRG induced by IRI. Simultaneously, ITMP significantly suppressed TRPV1 protein expression and phosphorylation in DRG, as well as the heart infarct size and arrhythmia score caused by IRI. The suppression of TRPV1 elevation and activation by ITMP were reversed by intrathecal injection of the selective μ receptor antagonist. Furthermore, IRI elevated DRG cAMP, while intrathecal administration of the selective cAMP-PKA inhibitor reduced myocardial injury. Finally, we showed that activation of opioid receptor by morphine inhibited PKA activator-induced TRPV1 channel activity at the cellular level. These findings suggest that the elevation and activation of TRPV1 in DRG during myocardial ischemia-reperfusion might be responsible for cardiac injury. ITMP exerts cardioprotection by inhibiting DRG-TRPV1 activity via modulation cAMP. Therefore, inhibition of TRPV1 upregulation in DRG might be used as a novel therapeutic mechanism for myocardium ischemia-reperfusion injury.

Cardiac Afferent Denervation Abolishes Ganglionated Plexi and Sympathetic Responses to Apnea: Implications for Atrial Fibrillation.

Background The autonomic nervous system response to apnea and its mechanistic connection to atrial fibrillation (AF) are unclear. We hypothesize that sensory neurons within the ganglionated plexi (GP) play a role. We aimed to delineate the autonomic response to apnea and to test the effects of ablation of cardiac sensory neurons with resiniferatoxin (RTX), a neurotoxic TRPV1 (transient receptor potential vanilloid 1) agonist. Methods Sixteen dogs were anesthetized and ventilated. Apnea was induced by stopping ventilation until oxygen saturations decreased to 80%. Nerve recordings from bilateral vagal nerves, left stellate ganglion, and anterior right GP were obtained before and during apnea, before and after RTX injection in the anterior right GP (protocol 1, n=7). Atrial effective refractory period and AF inducibility on single extrastimulation were assessed before and during apnea, and before and after intrapericardial RTX administration (protocol 2, n=9). GPs underwent immunohistochemical staining for TRPV1. Results Apnea increased anterior right GP activity, followed by clustered crescendo vagal bursts synchronized with heart rate and blood pressure oscillations. On further oxygen desaturation, a tonic increase in stellate ganglion activity and blood pressure ensued. Apnea-induced effective refractory period shortening from 110.20±31.3 ms to 90.6±29.1 ms ( P<0.001), and AF induction in 9/9 dogs versus 0/9 at baseline. After RTX administration, increases in GP and stellate ganglion activity and blood pressure during apnea were abolished, effective refractory period increased to 126.7±26.9 ms ( P=0.0001), and AF was not induced. Vagal bursts remained unchanged. GP cells showed cytoplasmic microvacuolization and apoptosis. Conclusions Apnea increases GP activity, followed by vagal bursts and tonic stellate ganglion firing. RTX decreases sympathetic and GP nerve activity, abolishes apnea's electrophysiological response, and AF inducibility. Sensory neurons play a role in apnea-induced AF.

Baicalin Depresses the Sympathoexcitatory Reflex Induced by Myocardial Ischemia via the Dorsal Root Ganglia

Myocardial ischemia (MI) is one of the major causes of death in cardiac diseases. Purinergic signaling is involved in bidirectional neuronal-glial communication in the primary sensory ganglia. The sensory neuritis of cardiac afferent neurons in cervical dorsal root ganglion (cDRG) interacts with cardiac sympathetic efferent postganglionic neurons, forming feedback loops. The P2Y12 receptor is expressed in satellite glial cells (SGCs) of DRG. Baicalin is a major active ingredient extracted from natural herbal medicines, which has anti-inflammatory and strong anti-oxidation properties. In this study we investigated the effect of baicalin on P2Y12 receptor in the cervical DRG SGC-mediated sympathoexcitatory reflex, which is increased during MI. The results showed that the expression of P2Y12 receptor mRNA and protein in DRG, and the co-localization values of P2Y12 receptor and glial fibrillary acidic protein (GFAP) in cDRG SGCs were increased after MI. The activated SGCs increased IL-1β protein expression and elevated Akt phosphorylation in cDRG. Baicalin treatment inhibited the upregulation of the P2Y12 receptor, GFAP protein and Akt phosphorylation in cDRG neurons/SGCs. The stellate ganglia (SG) affect cardiac sympathetic activity. Baicalin treatment also decreased the upregulation of the P2Y12 receptor, GFAP protein in the SG. The P2Y12 agonist, 2Me-SADP, increased [Ca2+]i in HEK293 cells transfected with the P2Y12 receptor plasmid and SGCs in cDRG. These results indicate that application of baicalin alleviates pathologic sympathetic activity induced by MI via inhibition of afferents in the cDRG.

Abstract 19432: Neuronal Isoform of Nitric Oxide Synthase is Upregulated in Cardiac Intrinsic, and Extrinsic Sympathetic Afferent and Efferent Ganglia Following Myocardial Infarction

Objective: The impact of myocardial infarction (MI) on neuronal nitric oxide synthase (nNOS), an important mediator of cardiac neurotransmission is not well understood. Methods: Yorkshire pigs (n=10) underwent anterior MI by LAD occlusion. After 6-8 weeks, bilateral stellate ganglia (SG), T1 dorsal root ganglia (DRG), right atrial (RAGP) and ventral inter-ventricular ganglionated plexi (VIVGP)were extracted, formalin-fixed, paraffin-embedded, and sectioned. Immno-staining for nNOS was performed on representative slides through the middle of the ganglia, and for calcitonin-gene related peptide (CGRP), a marker of sensory afferent neurons. The percentage of immunoreactive (IR) neurons was quantified. Results: An average of 63.1±40.7, 169.7±55.5, and 448.1±204.5 neurons were counted in the GPs, DRG, and SG respectively. There was no significant difference in the number of neurons counted in controls vs MIs. In the GPs, nNOS was differentially altered by MI, with significant upregulation in VIVGP but not RAGP of MI (13.8±2.8% vs 46.6±5.3%, p&lt;0.001; and 12.1±3.6% vs 12.5±2.9%, p=NS, respectively) compared to control animals. In the DRG, IR of both CGRP and nNOS was increased (36.3±6.2% vs 65.3±4.8%, p&lt;0,001 for nNOS; and 64.1±6.2% vs 85.2±5.6%, p&lt;0.001 for CGRP), with increased co-expression of both CGRP and nNOS in approximately 20% of DRG neurons. In the SG, the number of neurons demonstrating strong IR for nNOS was approximately 0.4% in control animals. After MI, this increased 8-fold to 3.12% (P&lt;0.01). Conclusions: nNOS expression is upregulated differentially following MI in the GPs, with increased expression in the ventricular GP innervating the region of MI, but not in the RAGP. Both cardiac sympathetic afferent (DRG) neurons, and efferent (SG) neurons increase expression of nNOS following MI. The impact of nNOS in these different sites warrants further investigation.

Abnormal cardiac autonomic regulation in mice lacking ASIC3.

Integration of sympathetic and parasympathetic outflow is essential in maintaining normal cardiac autonomic function. Recent studies demonstrate that acid-sensing ion channel 3 (ASIC3) is a sensitive acid sensor for cardiac ischemia and prolonged mild acidification can open ASIC3 and evoke a sustained inward current that fires action potentials in cardiac sensory neurons. However, the physiological role of ASIC3 in cardiac autonomic regulation is not known. In this study, we elucidate the role of ASIC3 in cardiac autonomic function using Asic3 −/− mice. Asic3 −/− mice showed normal baseline heart rate and lower blood pressure as compared with their wild-type littermates. Heart rate variability analyses revealed imbalanced autonomic regulation, with decreased sympathetic function. Furthermore, Asic3 −/− mice demonstrated a blunted response to isoproterenol-induced cardiac tachycardia and prolonged duration to recover to baseline heart rate. Moreover, quantitative RT-PCR analysis of gene expression in sensory ganglia and heart revealed that no gene compensation for muscarinic acetylcholines receptors and beta-adrenalin receptors were found in Asic3 −/− mice. In summary, we unraveled an important role of ASIC3 in regulating cardiac autonomic function, whereby loss of ASIC3 alters the normal physiological response to ischemic stimuli, which reveals new implications for therapy in autonomic nervous system-related cardiovascular diseases.

Cardiac Sensory Consequences of Low‐Amplitude Autonomic Regulation Therapy

ObjectiveTo determine if vagus nerve stimulation (VNS) or spinal cord stimulation (SCS) affects the capacity of cardiac afferent neurons to transduce myocardial ischemia (MI).MethodsUsing extracellular recordings in anesthetized canines, cardiac‐related dorsal root (DRG; T1‐T3 spinal levels) or nodose ganglia neurons were identified by touch/chemical activation in their epicardial sensory fields located adjacent to the left anterior descending (LAD) coronary artery. Neuronal responses to 1 min LAD coronary artery occlusion (CAO) were then evaluated prior to and following SCS [T1‐T3 spinal level; 50Hz, 90% motor threshold; 20 min] or left cervical VNS [20 Hz; current amplitudes (CA): CA1 (1–1.9 mA) or CA2 (2–3.5 mA); 3 min].ResultsLAD CAO activated cardiac afferent neurons in nodose and DRG ganglia, enhancement that persisted for up to 30 min into reperfusion. Following SCS or CA2 VNS, LAD CAO failed to alter their neural activity. VNS at CA1 did not mitigate MI‐induced activation of nodose neurons.ConclusionsTransient myocardial ischemia activates primary afferent inputs to the central nervous system (CNS), responses that persist into reperfusion. Pre‐emptive VNS or SCS obtunds sensory transduction of the ischemic myocardium to the CNS, in part accounting for the anti‐anginal and broader cardioprotective effects of such therapy being evaluated clinically. (HL71830)

TRPV1 receptor expression in cardiac vagal afferent neurons of infant rats

We examined in infant rats whether cardiac afferent neurons in the nodose ganglia (NG) express transient receptor potential vanilloid 1 (TRPV1) receptors. Anesthetized rats (9–11 days old) were injected with 2 μl of a fluorescent retrograde tracer (Fluoro-Gold, FG) into the ventricular wall through the anterior epicardial surface. After 2 days, the NG were excised under anesthesia for identification of FG-labeled neurons and immunohistochemical analysis. Of 3858 NG neurons, 5% (202 neurons) were labeled with FG. Among the FG-labeled neurons, 180 (89%) were TRPV1 positive, of which 123 co-expressed 200-kDa neurofilaments (NF200), which is specific for myelinated nerve fibers. Among the FG-labeled neurons that expressed both TRPV1 and NF200, 37 had relatively large cell diameters (>26 μm) (range: 12–38 μm). In conclusion, in infant rats, most cardiac afferent neurons (both myelinated and unmyelinated) in the NG may express TRPV1 receptors. Although functional properties such as those related to the arterial baroreflex may vary among the neurons, our results suggest that, in immature animals, TRPV1 receptors help convey cardiac sensations and control autonomic reflexes.

The cardiac neuronal hierarchy and susceptibility to arrhythmias

The cardiac neuronal hierarchy and susceptibility to arrhythmias

Endogenous endothelin stimulates cardiac sympathetic afferents during ischaemia

Myocardial ischaemia activates cardiac sympathetic afferents leading to chest pain and reflex cardiovascular responses. Previous studies have shown that a brief period of myocardial ischaemia increases endothelin in cardiac venous plasma draining ischaemic myocardium and that exogenous endothelin excites cutaneous group III and IV sensory nerve fibres. The present study tested the hypothesis that endogenous endothelin stimulates cardiac afferents during ischaemia through direct activation of endothelin A receptors (ET(A)Rs). Nerve activity of single unit cardiac sympathetic afferents was recorded from the left sympathetic chain or rami communicates (T(2)-T(5)) in anaesthetized cats. Single fields of 38 afferents (CV = 0.25-3.86 m s(-1)) were identified in the left or right ventricle with a stimulating electrode. Five minutes of myocardial ischaemia stimulated all 38 cardiac afferents (8 Adelta, 30 C-fibres) and the responses of these 38 afferents to chemical stimuli were further studied in the following protocols. In the first protocol, injection of endothelin 1 (ET-1, 1, 2 and 4 microg) into the left atrium (LA) stimulated seven ischaemically sensitive cardiac afferents in a dose-dependent manner. Second, BQ-123, a selective ET(A)R antagonist, abolished the responses of nine afferents to 2 microg of ET-1 injected into the left atrium and attenuated the ischaemia-related increase in activity of eight other afferents by 51%. In contrast, blockade of ET(B) receptors caused inconsistent responses to exogenous ET-1 as well as to ischaemia. Furthermore, in the absence of ET(A)R blockade, cardiac afferents responded consistently to repeated administration of ET-1 (n = 7) and to recurrent myocardial ischaemia (n = 7). Finally, using an immunocytochemical staining approach, we observed that ET(A) receptors were expressed in cardiac sensory neurons in thoracic dorsal root ganglia. Taken together, these data indicate that endogenous endothelin contributes to activation of cardiac afferents during myocardial ischaemia through direct stimulation of ET(A) receptors likely to be located in the cardiac sensory nervous system.

Abnormal Reactivity of Afferent Neuronal Pathway from the Heart to the Brain in Heart Failure Patients

BackgroundThe involvement of afferent neuronal pathway from the heart to the brain in heart failure (HF) remains unknown.Methods and ResultsWe recorded cardiac cerebral evoked potentials (CEPs) induced by ventricular (VVI) pacing in 21 patients with or without HF [64.5±2.6 yrs (SEM), 16 males and 5 females] within a week after pacemaker implantation or cardiac resynchronized therapy. Electroencephalogram (EEG) was simultaneously recorded from the vertex (Cz, international 10–20 system) during the VVI pacing of control stimulation (1.5 V) and an intense stimulation [6–8 (7.21±0.15) V] at 70–100 (80.5±1.9) bpm for 2 min. We averaged 100 EEG waves triggered by the VVI pacing to obtain CEPs. Cardiac CEPs were obtained by subtracting CEPs with control stimulation from those with intense stimulation. In Stage B/C HF patients (n=7 each), cardiac CEPs were reproducibly identified with first negative (N1) peak at 67–93 msec, first positive (P1) peak at 128–147 msec and second negative (N2) peak at 180–194 msec after the stimuli, whereas no such clear CEPs were detected in the remaining 7 patients without organic heart disease and HF.ConclusionsThese results suggest that the abnormal reactivity of cardiac afferent neurons from the heart to the brain is associated with development of HF.

ASIC2a and ASIC3 Heteromultimerize to Form pH-Sensitive Channels in Mouse Cardiac Dorsal Root Ganglia Neurons

Acid-sensing ion channels (ASICs) are Na+ channels that are activated by acidic pH. Their expression in cardiac afferents and remarkable sensitivity to small pH changes has made them leading candidates to sense cardiac ischemia. Four genes encode six different ASIC subunits, however it is not yet clear which of the ASIC subunits contribute to the composition of ASICs in cardiac afferents. Here, we labeled cardiac afferents using a retrograde tracer dye in mice, which allowed for patch-clamp studies of murine cardiac afferents. We found that a higher percentage of cardiac sensory neurons from the dorsal root ganglia respond to acidic pH and generated larger currents compared to those from the nodose ganglia. The ASIC-like current properties of the cardiac dorsal root ganglia neurons from wild-type mice most closely matched the properties of ASIC2a/3 heteromeric channels. This was supported by studies in ASIC-null mice: acid-evoked currents from ASIC3(-/-) cardiac afferents matched the properties of ASIC2a channels, and currents from ASIC2(-/-) cardiac afferents matched the properties of ASIC3 channels. We conclude that ASIC2a and -3 are the major ASIC subunits in cardiac dorsal root ganglia neurons and provide potential molecular targets to attenuate chest pain and deleterious reflexes associated with cardiac disease.

Undiscovered role of endogenous thromboxane A2 in activation of cardiac sympathetic afferents during ischaemia

Myocardial ischaemia activates blood platelets, which in turn stimulate cardiac sympathetic afferents, leading to chest pain and sympathoexcitatory reflex cardiovascular responses. Previous studies have shown that activated platelets stimulate ischaemically sensitive cardiac sympathetic afferents, and that thromboxane A(2) (TxA(2)) is one of the mediators released from activated platelets during myocardial ischaemia. The present study tested the hypothesis that endogenous TxA(2) stimulates cardiac afferents during ischaemia through direct activation of TxA(2) (TP) receptors coupled with the phospholipase C-protein kinase C (PLC-PKC) cellular pathway. Nerve activity of single unit cardiac sympathetic afferents was recorded from the left sympathetic chain or rami communicantes (T(2)-T(5)) in anaesthetized cats. Single fields of 39 afferents (conduction velocity = 0.27-3.65 m s(-1)) were identified in the left or right ventricle initially with mechanical stimulation and confirmed with a stimulating electrode. Five minutes of myocardial ischaemia stimulated all 39 cardiac afferents (8 Adelta-, 31 C-fibres) and the responses of these 39 afferents to chemical stimuli were further studied in the following four protocols. In the first protocol, 2.5, 5 and 10 microg of the TxA(2) mimetic, U46619, injected into the left atrium (LA), stimulated seven ischaemically sensitive cardiac afferents in a dose-dependent manner. Second, BM13,177, a selective TxA(2) receptor antagonist, abolished the responses of six afferents to 5 microg of U46619 injected into the left atrium and attenuated the ischaemia-related increase in activity of seven other afferents by 44%. In contrast, cardiac afferents, in the absence of TP receptor blockade responded consistently to repeated administration of U46619 (n = 6) and to recurrent myocardial ischaemia (n = 7). In the fourth protocol, administration of PKC-(19-36), a selective PKC inhibitor, attenuated the responses of six other cardiac afferents to U46619 by 38%. Finally, using an immunohistochemical staining approach, we observed that TP receptors were expressed in cardiac sensory neurons in thoracic dorsal root ganglia. Taken together, these data indicate that endogenous TxA(2) contributes to the activation of cardiac afferents during myocardial ischaemia through direct stimulation of TP receptors probably located in the cardiac sensory nervous system and that the stimulating effect of TxA(2) on cardiac afferents is dependent, at least in part, upon the PLC-PKC cellular pathway.

Role of TRPV1 and intracellular Ca2+ in excitation of cardiac sensory neurons by bradykinin

Bradykinin is an important mediator produced during myocardial ischemia and infarction that can activate and/or sensitize cardiac spinal (sympathetic) sensory neurons to trigger chest pain. Because a long-onset latency is associated with the bradykinin effect on cardiac spinal afferents, a cascade of intracellular signaling events is likely involved in the action of bradykinin on cardiac nociceptors. In this study, we determined the signal transduction mechanisms involved in bradykinin stimulation of cardiac nociceptors. Cardiac dorsal root ganglion (DRG) neurons in rats were labeled by intracardiac injection of a fluorescent tracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine percholate (DiI). Whole cell current-clamp recordings were performed in acutely isolated DRG neurons. In DiI-labeled DRG neurons, 1 microM bradykinin significantly increased the firing frequency and lowered the membrane potential. Iodoresiniferatoxin, a highly specific transient receptor potential vanilloid type 1 (TRPV1) antagonist, significantly reduced the excitatory effect of bradykinin. Furthermore, the stimulating effect of bradykinin on DiI-labeled DRG neurons was significantly attenuated by baicalein (a selective inhibitor of 12-lipoxygenase) or 2-aminoethyl diphenylborinate [an inositol 1,4,5-trisphosphate (IP(3)) antagonist]. In addition, the effect of bradykinin on cardiac DRG neurons was abolished after the neurons were treated with BAPTA-AM or thapsigargin (to deplete intracellular Ca(2+) stores) but not in the Ca(2+)-free extracellular solution. Collectively, these findings provide new evidence that 12-lipoxygenase products, IP(3), and TRPV1 channels contribute importantly to excitation of cardiac nociceptors by bradykinin. Activation of TRPV1 and the increase in the intracellular Ca(2+) are critically involved in activation/sensitization of cardiac nociceptors by bradykinin.

Sustained Currents Through ASIC3 Ion Channels at the Modest pH Changes That Occur During Myocardial Ischemia

Acid-sensing ion channel 3 (ASIC3) is highly expressed on sensory neurons that innervate heart and skeletal muscle and, therefore, is proposed to detect lactic acidosis and to transduce angina and muscle ischemic pain. A difficulty with this idea is that ASIC3 rapidly desensitizes. How can a desensitizing ion channel mediate a persisting sensation such as angina? Here, we show that rat ASIC3 produces a sustained current within the limited range of extracellular pH (7.3 to 6.7) that occurs during cardiac and skeletal muscle ischemia; experiments use patch clamp on transfected cell lines and on fluorescently tagged sensory neurons that innervate rat heart. No such sustained current occurs with ASIC1a (either as homomers or 1a/3 heteromers), whereas ASIC2a/3 heteromers give much larger currents than ASIC3 homomers. The sustained current persists even over tens of minutes because it is caused by a region of pH where there is overlap between inactivation and activation of the channel. Lactate, an anaerobic metabolite, allows the current to activate at slightly more basic pH. Surprisingly, amiloride, which blocks ASICs when they are activated at lower pH, increases ASIC3 current evoked at pH 7.0. Cardiac sensory neurons exhibit a small, perfectly sustained current when pH changes from 7.4 to 7.0. At least some of this current is carried by ASICs because the current is increased by both Zn(2+), an ASIC modulator, and amiloride. We suggest that this sustained mode is the most relevant form of ASIC3 gating for triggering angina and other ischemic pain.

Identification of an endogenous cardioprotective pathway involving 12‐lipoxygenase (12‐LOX), C‐fibers and the non‐selective cation channel TRPV1

Eicosanoids derived via 12-LOX metabolism of arachidonic acid (AA) are protective against myocardial ischaemia-reperfusion (I/R) injury, although the precise mechanism involved remains uncertain. To investigate the pathways involved we used male Wistar rat hearts perfused in the Langendorff mode. Infusion of 12-LOX (2 μg ml -1, 30 min) and AA (30 μM, 30 min) reduced infarct size (~50% control, P<0.01) and improved left ventricular developed pressure (P<0.001) after a 25 min/2 h I/R insult. This protection was abolished by the 12-LOX inhibitor baicalein (10 μM, 45 min, P<0.05) and by treatment of animals with capsaicin (50 mg kg-1, P<0.05), supporting a role for sensory C-fibers in 12-LOX-mediated cardioprotection. Analysis of TRPV1 in left ventricles of hearts subjected to I/R in vitro demonstrated significant reduction of protein but no change in mRNA levels. In contrast, using a similar model in vivo, cardiac TRPV1 mRNA levels were significantly reduced and protein expression elevated. Additionally in dorsal root ganglia (DRG, the cell bodies of cardiac sensory neurons), TRPV1 mRNA was unchanged whilst protein levels were elevated. Together these data suggest that I/R injury results in a decrease in TRPV1 expression locally within the heart, but an increase of TRPV1 synthesis in DRG and transport to cardiac nerve terminals occurs in vivo to maintain expression levels. Moreover, during I/R injury upregulation/activation of the 12-LOX/AA/TRPV1 pathway represents an endogenous damage-limiting mechanism. This research is funded by the British Heart Foundation.