Muscle Afferent Neurons Research Articles

NEURAL INFLAMMATION CONTRIBUTES TO VOLTAGE-GATED POTASSIUM CHANNELS DYSFUNCTION IN LUMBAR DORSAL ROOT GANGLIA IN CHRONIC HEART FAILURE RATS

Objective: Skeletal muscle afferent sensitization causes excessive sympatho-excitation and exercise intolerance during physical activity in the chronic heart failure (CHF) state. Our recent study reported that downregulated voltage-gated potassium (Kv) channels in lumbar dorsal root ganglia (DRGs) contribute to muscle afferent sensitization in CHF rats. However, the cellular mechanisms underlying downregulated Kv channels in CHF remain unknown. We hypothesized that chronic macrophage activation contributes to Kv channels dysfunction in the lumbar DRGs in CHF. Design and method: Myocardial infarction (MI) was produced by left coronary artery ligation in rats. Molecular experiments were carried out to assess macrophage activation, cytokines and Kv channel expression in L4-L6 DRGs post MI. In vitro co-culture of lipopolysaccharide (LPS)-pretreated RAW264.7 (activated M1 macrophage) with 50B11 DRG neurons were performed. Patch clamp experiments were performed to assess Kv channel activity in acutely isolated muscle afferent DRG neurons. Chronic oral administration of minocycline (Mino, 20 mg/kg/day) was used to suppress macrophage activation in L4-L6 DRGs. Results: Compared to sham rats, male MI rats exhibited a robust increase in the number of Iba1-positive macrophages and the protein expression of Interferon regulatory factor 8 (IRF8, a pro-inflammatory macrophage marker) in L4-L6 DRGs at 8 weeks post MI, which was largely prevented by Mino (Figure 1). SEM showed macrophage infiltration into lumbar DRGs 8 weeks post MI. PCR data suggested that the mRNA expressions of pro-inflammatory cytokines including IL1beta and IL6 in lumbar DRGs were significantly elevated in male rats at 8-weeks post MI compared to sham rats. Protein expression of Kv channels including Kv1.4, 3.4, 4.2 and 4.3, was significantly downregulated in male rat L4-L6 DRGs at 8 weeks post MI. This was largely attenuated by Mino. Patch clamp data demonstrated decreased Ito current in isolated male rat muscle afferent DRG neurons 8 weeks post MI, also attenuated by Mino. Moreover, LPS-pretreated BV2 cells or RAW264.7 (LPS was washed out 4 hours after exposure, co-cultured with 50B11 DRG neurons significantly downregulated several Kv isoforms in DRG neurons. Conclusions: Macrophage activation contributes to Kv channels dysfunction in lumbar DRGs in CHF male rats.

Amplified P2X3 pathway activity in muscle afferent dorsal root ganglion neurons and exercise pressor reflex regulation in hindlimb ischaemia-reperfusion.

Hindlimb ischaemia-reperfusion (IR) is among the most prominent pathophysiological conditions observed in peripheral artery disease (PAD). An exaggerated arterial blood pressure (BP) response during exercise is associated with an elevated risk of cardiovascular events in individuals with PAD. However, the precise mechanisms leading to this exaggerated BP response are poorly elucidated. The P2X3 signalling pathway, which plays a key role in modifying the exercise pressor reflex (EPR), is the focus of the present study. We determined the regulatory role of P2X3 on the EPR in a rat model of hindlimb IR. In vivo and in vitro approaches were used to determine the expression and functions of P2X3 in muscle afferent nerves and EPR in IR rats. We found that in IR rats there was (1) upregulation of P2X3 protein expression in the L4-6 dorsal root ganglia (DRG); (2) amplified P2X currents in isolated isolectin B4 (IB4)-positive muscle DRG neurons; and (3) amplification of the P2X-mediated BP response. We further verified that both A-317491 and siRNA knockdown of P2X3 significantly decreased the activity of P2X currents in isolated muscle DRG neurons. Moreover, inhibition of muscle afferents' P2X3 receptor using A-317491 was observed to alleviate the exaggerated BP response induced by static muscle contraction and P2X-induced BP response by α,β-methylene ATP injection. P2X3 signalling pathway activity is amplified in muscle afferent DRG neurons in regulating the EPR following hindlimb IR.

Predictive analytics identifies key factors driving hyperalgesic priming of muscle sensory neurons.

Hyperalgesic priming, a form of neuroplasticity induced by inflammatory mediators, in peripheral nociceptors enhances the magnitude and duration of action potential (AP) firing to future inflammatory events and can potentially lead to pain chronification. The mechanisms underlying the development of hyperalgesic priming are not well understood, limiting the identification of novel therapeutic strategies to combat chronic pain. In this study, we used a computational model to identify key proteins whose modifications caused priming of muscle nociceptors and made them hyperexcitable to a subsequent inflammatory event. First, we extended a previously validated model of mouse muscle nociceptor sensitization to incorporate Epac-mediated interaction between two G protein-coupled receptor signaling pathways commonly activated by inflammatory mediators. Next, we calibrated and validated the model simulations of the nociceptor's AP response to both innocuous and noxious levels of mechanical force after two subsequent inflammatory events using literature data. Then, by performing global sensitivity analyses that simulated thousands of nociceptor-priming scenarios, we identified five ion channels and two molecular processes (from the 18 modeled transmembrane proteins and 29 intracellular signaling components) as potential regulators of the increase in AP firing in response to mechanical forces. Finally, when we simulated specific neuroplastic modifications in Kv1.1 and Nav1.7 alone as well as with simultaneous modifications in Nav1.7, Nav1.8, TRPA1, and Kv7.2, we observed a considerable increase in the fold change in the number of triggered APs in primed nociceptors. These results suggest that altering the expression of Kv1.1 and Nav1.7 might regulate the neuronal hyperexcitability in primed mechanosensitive muscle nociceptors.

Identification of key factors driving inflammation-induced sensitization of muscle sensory neurons.

Sensory neurons embedded in muscle tissue that initiate pain sensations, i.e., nociceptors, are temporarily sensitized by inflammatory mediators during musculoskeletal trauma. These neurons transduce peripheral noxious stimuli into an electrical signal [i.e., an action potential (AP)] and, when sensitized, demonstrate lower activation thresholds and a heightened AP response. We still do not understand the relative contributions of the various transmembrane proteins and intracellular signaling processes that drive the inflammation-induced hyperexcitability of nociceptors. In this study, we used computational analysis to identify key proteins that could regulate the inflammation-induced increase in the magnitude of AP firing in mechanosensitive muscle nociceptors. First, we extended a previously validated model of a mechanosensitive mouse muscle nociceptor to incorporate two inflammation-activated G protein-coupled receptor (GPCR) signaling pathways and validated the model simulations of inflammation-induced nociceptor sensitization using literature data. Then, by performing global sensitivity analyses that simulated thousands of inflammation-induced nociceptor sensitization scenarios, we identified three ion channels and four molecular processes (from the 17 modeled transmembrane proteins and 28 intracellular signaling components) as potential regulators of the inflammation-induced increase in AP firing in response to mechanical forces. Moreover, we found that simulating single knockouts of transient receptor potential ankyrin 1 (TRPA1) and reducing the rates of Gαq-coupled receptor phosphorylation and Gαq subunit activation considerably altered the excitability of nociceptors (i.e., each modification increased or decreased the inflammation-induced fold change in the number of triggered APs compared to when all channels were present). These results suggest that altering the expression of TRPA1 or the concentration of intracellular Gαq might regulate the inflammation-induced increase in AP response of mechanosensitive muscle nociceptors.

Composition of TTX-S Nav current in rat muscle afferent neurons.

Group III and IV muscle afferents carry pain and exercise pressor reflex (EPR) signals. However, aberrant signaling of the EPR in patients with cardiovascular disease can increase their risk of a myocardial infarction. Thus, blocking action potentials that signal this reflex could benefit these patients. Immunohistochemistry studies have shown that group III/IV neurons isolated from Sprague Dawley rats express tetrodotoxin-sensitive (TTX-S) voltage-dependent sodium (NaV) channels rNaV1.6 and rNaV1.7, but the contribution of these channels to the TTX-S current is unknown. We initially tested several blockers for selectivity against rNaV1.6 and rNaV1.7 channels expressed in HEK cells and found that µ-conotoxin PIIIA (PIIIA, 500 nM) selectively blocked rNaV1.6 current, while Protoxin II (PTxII, 10 nM) was more selective for rNaV1.7 channels. 4,9-Anhydrotetrodotoxin was found to block non-selectively. To evaluate the TTX-S component currents in muscle afferent neurons, we sequentially added the NaV1.6-selective PIIIA, followed by PTxII to block NaV1.7 current, and then 300 nM TTX to block the remaining TTX-S current. On average PIIIA blocked 14 ± 13% (mean ± SD) and PTxII blocked 70 ± 17% of the TTX-S NaV current in rat muscle afferent neurons. However, the NaV1.6 current fraction significantly varied with cell diameter with mean inhibitions of 0% for neurons < 27 µm, 17% between 27-32 µm (p < 0.05), 5% between 32-27 µm and 21% for neurons > 37 µm (p < 0.05), suggesting differential expression among group III/IV neurons. The TTX-S NaV current in group III/IV neurons is primarily comprised of NaV1.7 >> NaV1.6 = unidentified TTX-S channel activity. Blocking group III/IV action potentials with a selective NaV1.7 channel blocker should inhibit the EPR to reduce the myocardial risk in patients with cardiovascular disease and provide analgesia in patient with severe muscle pain.

KV4 channels in isolectin B4 muscle dorsal root ganglion neurons of rats with experimental peripheral artery disease: effects of bradykinin B1 and B2 receptors.

Muscle afferent nerve-activated reflex sympathetic nervous and blood pressure responses are exaggerated during exercise in peripheral artery diseases (PAD). However, the precise signaling pathways and molecular mediators responsible for these abnormal autonomic responses in PAD are poorly understood. Our previous study suggests that A-type voltage-gated K+ (KV4) channels regulate the excitability in muscle dorsal root ganglion (DRG) neurons of PAD rats; however, it is still lacking regarding the effects of PAD on characteristics of KV4 currents and engagement of bradykinin (BK) subtype receptors. Thus, we examined KV4 currents in two distinct muscle DRG neurons, namely isolectin B4-positive and B4-negative (IB4+ and IB4-) DRG neurons. IB4+ neurons express receptors for glial cell line-derived neurotrophic factor (GDNF), whereas IB4- DRG neurons are depending on nerve growth factors for survival. Our data showed that current density in muscle DRG neurons of PAD rats was decreased and this particularly appeared in IB4+ DRG neurons as compared with IB4- DRG neurons. We also showed that stimulation of BK B1 and B2 receptors led to a greater inhibitory effect on KV4 currents in IB4+ muscle DRG neurons and siRNA knockdown of KV4 subunit KV4.3 decreased the activity of KV4 currents in IB4+ DRG neurons. In conclusion, our data suggest that limb ischemia and/or ischemia-induced BK inhibit activity of KV4 channels in a subpopulation of the thin fiber muscle afferent neurons depending on GDNF, which is likely a part of signaling pathways involved in the exaggerated blood pressure response during activation of muscle afferent nerves in PAD.

NaV1.9 current in muscle afferent neurons is enhanced by substances released during muscle activity.

Skeletal muscle contraction triggers the exercise pressor reflex (EPR) to regulate the cardiovascular system response to exercise. During muscle contraction, substances are released that generate action potential activity in group III and IV afferents that mediate the EPR. Some of these substances increase afferent activity via G-protein-coupled receptor (GPCR) activation, but the mechanisms are incompletely understood. We were interested in determining if tetrodotoxin-resistant (TTX-R) voltage-dependent sodium channels (NaV) were involved and investigated the effect of a mixture of such compounds (bradykinin, prostaglandin, norepinephrine, and ATP, called muscle metabolites). Using whole cell patch-clamp electrophysiology, we show that the muscle metabolites significantly increased TTX-R NaV currents. The rise time of this enhancement averaged ∼2 min, which suggests the involvement of a diffusible second messenger pathway. The effect of muscle metabolites on the current-voltage relationship, channel activation and inactivation kinetics support NaV1.9 channels as the target for this enhancement. When applied individually at the concentration used in the mixture, only prostaglandin and bradykinin significantly enhanced NaV current, but the sum of these enhancements was <1/3 that observed when the muscle metabolites were applied together. This suggests synergism between the activated GPCRs to enhance NaV1.9 current. When applied at a higher concentration, all four substances could enhance the current, which demonstrates that the GPCRs activated by each metabolite can enhance channel activity. The enhancement of NaV1.9 channel activity is a likely mechanism by which GPCR activation increases action potential activity in afferents generating the EPR.NEW & NOTEWORTHY G-protein-coupled receptor (GPCR) activation increases action potential activity in muscle afferents to produce the exercise pressor reflex (EPR), but the mechanisms are incompletely understood. We provide evidence that NaV1.9 current is synergistically enhanced by application of a mixture of metabolites potentially released during muscle contraction. The enhancement of NaV1.9 current is likely one mechanism by which GPCR activation generates the EPR and the inappropriate activation of the EPR in patients with cardiovascular disease.

High resolution retrograde tracing, morphological analysis, RNA sequencing and differential expression analysis of identified vagal afferent neurons innervating the muscle or mucosa in 3 different stomach regions in the rat

The functions of the stomach are powerfully influenced by the vagus nerve and vago‐vagal reflexes. The vagal afferent neurons that innervate the stomach and monitor its functional state have clearly demonstrated roles in appetite regulation, gastric accommodation and gastric emptying. In mammals with single compartment stomachs, including humans, the stomach can be divided into functional regions and the muscle and mucosal layers show region‐specific specializations. Electrophysiological and morphological studies suggest that the vagal sensory axons innervating the different regions and layers of the stomach wall have distinct properties and participate in different types of reflex controls. We therefore hypothesized that these functional and structural differences would be reflected in distinct gene expression profiles of afferent neurons, depending on the stomach region and tissue innervated. We developed methods to selectively retrogradely label afferent neurons innervating the muscle or mucosa in the corpus and antrum and the muscle in the fundus in the rat, using local microinjections of fluorescent nanospheres that diffuse minimally. Up to 30 microinjections of 30‐100nL of tracer beads were made into either the muscle or the mucosa in one stomach region in each rat under anesthesia. The animal was allowed to recover and kept for 1‐2 weeks before being humanely killed and tissues harvested. Labelled neurons were studied in situ in the nodose ganglion using confocal microscopy, immunohistochemistry and fluorescence in situ hybridization, and individual neurons collected after dissociation for RNAseq analysis. RNA sequencing was performed using pooled samples of 5‐12 neurons labelled from each region and deep sequencing (30‐50 million reads per sample), and then analyzed using minimal filtering to allow detection of genes expressed at low copy number.Nodose neurons labelled from the mucosa were significantly larger than those labelled from the muscle in both the corpus and antrum, whether quantified in situ or after dissociation, confirming that microinjections into the different stomach wall layers labelled different populations of neurons. Pairwise differential expression analyses of RNAseq data showed distinct mRNA expression profiles depending on the region innervated, and between muscle and mucosal innervating neurons in each region, based on Benjamini/Hochberg adjusted P values (Limma analysis tool). Some genes displayed region specific expression patterns while others showed tissue specific patterns (eg muscle vs mucosa) and some showed both. Among differentially expressed genes were some previously identified to specifically label gastric mucosal and muscle afferent neurons, such as somatostatin, CCKB receptor and GRP65. In addition, many other genes were differentially expressed and mRNA for genes known to be functional in vagal afferent neurons, such as ghrelin, leptin and galanin receptors, were detected. Differential expression patterns for various genes were confirmed in labelled neurons in situ using immunohistochemistry and/or in situhybridization. The data provide a rich resource for further studies of the visceral sensation in the different stomach regions and how region‐specific signaling might regulate appetite and gastric function in both normal and disease states.

ASIC3 knockout alters expression and activity of P2X3 in muscle afferent nerves of rat model of peripheral artery disease.

In peripheral artery disease (PAD), the metaboreceptor and mechanoreceptor in muscle afferent nerves contribute to accentuated sympathetic outflow via a neural reflex termed exercise pressor reflex (EPR). Particularly, lactic acid and adenosine triphosphate (ATP) produced in exercising muscles respectively stimulate acid sensing ion channel subtype 3 (ASIC3) and P2X3 receptors (P2X3) in muscle afferent nerves, inducing the reflex sympathetic and BP responses. Previous studies indicated that those two receptors are spatially close to each other and AISC3 may have a regulatory effect on the function of P2X3. This inspired our investigation on the P2X3‐mediated EPR response following AISC3 abolished, which was anticipated to shed light on the future pharmacological and genetic treatment strategy for PAD. Thus, we tested the experimental hypothesis that the pressor response to P2X3 stimulation is greater in PAD rats with 3 days of femoral artery occlusion and the sensitizing effects of P2X3 are attenuated following ASIC3 knockout (KO) in PAD. Our data demonstrated that in wild type (WT) rats femoral occlusion exaggerated BP response to activation of P2X3 using α,β‐methylene ATP injected into the arterial blood supply of the hindlimb, meanwhile the western blot analysis suggested upregulation of P2X3 expression in dorsal root ganglion supplying the afferent nerves. Using the whole cell patch‐clamp method, we also showed that P2X3 stimulation enhanced the amplitude of induced currents in muscle afferent neurons of PAD rats. Of note, amplification of the P2X3 evoked‐pressor response and expression and current response of P2X3 was attenuated in ASIC3 KO rats. We concluded that the exaggerated P2X3‐mediated pressor response in PAD rats is blunted by ASIC3 KO due to the decreased expression and activities of P2X3 in muscle afferent neurons.

Abstract 9053: ASIC3 Knockout Alleviates Exaggerated Blood Pressure Response Induced by P2X3 Activation in Hindlimb Muscle Afferent Nerves of Rats With Experimental Peripheral Artery Disease

Background: In peripheral artery disease (PAD), the metaboreceptor and mechanoreceptor in muscle afferent nerves contribute to accentuated sympathetic outflow via a neural reflex. Particularly, lactic acid and ATP produced in exercising muscles respectively stimulate acid sensing ion channel subtype 3 (ASIC3) and purinergic subunit P2X3 receptors in muscle afferent nerves, inducing the reflex sympathetic and arterial blood pressure responses during exercise. Methods: We examined that the P2X3 protein expression in the dorsal root ganglion (DRG) and P2X currents in DRG neurons using western blotting and patch-clamp methods, and the pressor response to P2X3 stimulation using the whole animal preparations. Four groups were included: 1. Wild type (WT) rats; 2. ASIC3 knockout (KO) rats; 3. WT-PAD rats with three days of femoral artery occlusion; 4. KO-PAD rats with three days of femoral artery occlusion. Results: First, P2X3 expression was significantly increased in KO rats compared with that in WT rats (optical density: 6.51±2.20/n=5; P &lt;0.05 vs. WT). However, the levels of P2X3 expression were not significantly increased in KO-PAD rats compared with the KO rats (optical density: 7.99±1.99/n=6; P &gt;0.05 vs. KO) . Second, in WT rats, as 10, 30 and 50 μM of αβ-me ATP (P2X3 agonist) were applied, peak amplitude of both transient and sustained P2X currents was increased by femoral artery occlusion (all P &lt;0.05 WT-PAD vs. WT). Insignificant differences in both transient and sustained P2X currents were found between KO rats and KO-PAD rats (all P &gt;0.05 vs. KO) when αβ-me ATP was applied except of the transient P2X current amplitude at 50 μM of αβ-me ATP. Third, there were insignificant differences in the mean arterial pressure response to 0.0625, 0.125 and 0.25 mM of αβ-me ATP injected into the hindlimb circulation between KO rats and KO-PAD rats (all P &gt;0.05 vs. KO). Conclusions: Compared with WT rats, augmented P2X3-mediated pressor response is blunted by ASIC3 KO in PAD rats and this is accompanied with downregulated P2X3 expression and suppressed activities in P2X3 signaling pathways in muscle afferent neurons.

In silico Identification of Key Factors Driving the Response of Muscle Sensory Neurons to Noxious Stimuli.

Nociceptive nerve endings embedded in muscle tissue transduce peripheral noxious stimuli into an electrical signal [i.e., an action potential (AP)] to initiate pain sensations. A major contributor to nociception from the muscles is mechanosensation. However, due to the heterogeneity in the expression of proteins, such as ion channels, pumps, and exchangers, on muscle nociceptors, we currently do not know the relative contributions of different proteins and signaling molecules to the neuronal response due to mechanical stimuli. In this study, we employed an integrated approach combining a customized experimental study in mice with a computational model to identify key proteins that regulate mechanical nociception in muscles. First, using newly collected data from somatosensory recordings in mouse hindpaw muscles, we developed and then validated a computational model of a mechanosensitive mouse muscle nociceptor. Next, by performing global sensitivity analyses that simulated thousands of nociceptors, we identified three ion channels (among the 17 modeled transmembrane proteins and four endoplasmic reticulum proteins) as potential regulators of the nociceptor response to mechanical forces in both the innocuous and noxious range. Moreover, we found that simulating single knockouts of any of the three ion channels, delayed rectifier voltage-gated K+ channel (Kv1.1) or mechanosensitive channels Piezo2 or TRPA1, considerably altered the excitability of the nociceptor (i.e., each knockout increased or decreased the number of triggered APs compared to when all channels were present). These results suggest that altering expression of the gene encoding Kv1.1, Piezo2, or TRPA1 might regulate the response of mechanosensitive muscle nociceptors.

Voltage-gated potassium channel dysfunction in dorsal root ganglia contributes to the exaggerated exercise pressor reflex in rats with chronic heart failure.

An exaggerated exercise pressor reflex (EPR) causes excessive sympathoexcitation and exercise intolerance during physical activity in the chronic heart failure (CHF) state. Muscle afferent sensitization contributes to the genesis of the exaggerated EPR in CHF. However, the cellular mechanisms underlying muscle afferent sensitization in CHF remain unclear. Considering that voltage-gated potassium (Kv) channels critically regulate afferent neuronal excitability, we examined the potential role of Kv channels in mediating the sensitized EPR in male rats with CHF. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting experiments demonstrate that both mRNA and protein expressions of multiple Kv channel isoforms (Kv1.4, Kv3.4, Kv4.2, and Kv4.3) were downregulated in lumbar dorsal root ganglions (DRGs) of CHF rats compared with sham rats. Immunofluorescence data demonstrate significant decreased Kv channel staining in both NF200-positive and IB4-positive lumbar DRG neurons in CHF rats compared with sham rats. Data from patch-clamp experiments demonstrate that the total Kv current, especially IA, was dramatically decreased in medium-sized IB4-negative muscle afferent neurons (a subpopulation containing mostly Aδ neurons) from CHF rats compared with sham rats, indicating a potential functional loss of Kv channels in muscle afferent Aδ neurons. In in vivo experiments, adenoviral overexpression of Kv4.3 in lumbar DRGs for 1 wk attenuated the exaggerated EPR induced by muscle static contraction and the mechanoreflex by passive stretch without affecting the blunted cardiovascular response to hindlimb arterial injection of capsaicin in CHF rats. These data suggest that Kv channel dysfunction in DRGs plays a critical role in mediating the exaggerated EPR and muscle afferent sensitization in CHF.NEW & NOTEWORTHY The primary finding of this manuscript is that voltage-gated potassium (Kv) channel dysfunction in DRGs plays a critical role in mediating the exaggerated EPR and muscle afferent sensitization in chronic heart failure (CHF). We propose that manipulation of Kv channels in DRG neurons could be considered as a potential new approach to reduce the exaggerated sympathoexcitation and to improve exercise intolerance in CHF, which can ultimately facilitate an improved quality of life and reduce mortality.

Effects of bradykinin on voltage-gated KV 4 channels in muscle dorsal root ganglion neurons of rats with experimental peripheral artery disease.

During exercise, bradykinin (BK), a muscle metabolite in ischaemic muscles, exaggerates autonomic responses to activation of muscle afferent nerves in peripheral artery disease (PAD). We examined whether BK inhibits activity of KV 4 channels in muscle afferent neurons of PAD rats induced by femoral artery occlusion. We demonstrated that: 1) femoral occlusion attenuates KV 4 currents in dorsal root ganglion (DRG) neurons innervating the hindlimb muscles and decreases the threshold of action potential firing; 2) BK has a greater inhibitory effect on KV 4 currents in muscle DRG neurons of PAD rats; and 3) expression of KV 4.3 is downregulated in DRGs of PAD rats and inhibition of KV 4.3 significantly decreases activity of KV 4 currents in muscle DRG neurons. Femoral artery occlusion-induced limb ischaemia and/or ischaemia-induced metabolites (i.e. BK) inhibit activity of KV 4 channels in muscle afferent neurons and this is likely involved in the exaggerated exercise pressor reflex in PAD. Muscle afferent nerve-activated reflex sympathetic nervous and blood pressure responses are exaggerated during exercise in patients with peripheral artery diseases (PAD) and in PAD rats induced by femoral artery occlusion. However, the precise signalling pathways and molecular mediators responsible for these abnormal autonomic responses in PAD are poorly understood. A-type voltage-gated K+ (KV ) channels are quintessential regulators of cellular excitability in the various tissues. Among KV channels, KV 4 (i.e. KV 4.1 and KV 4.3) in primary sensory neurons mainly participate in physiological functions in regulation of mechanical and chemical sensation. However, little is known about the role of KV 4 in regulating neuronal activity in muscle afferent neurons of PAD. In addition, bradykinin (BK) is considered as a muscle metabolite contributing to the exaggerated exercise pressor reflex in PAD rats with femoral artery occlusion. Our data demonstrated that: 1) KV 4 currents are attenuated in dorsal root ganglion (DRG) neurons innervating the hindlimb muscles of PAD rats, along with a decreasing threshold of action potential firing; 2) KV 4 currents are inhibited by application of BK onto muscle DRG neurons of PAD rats to a greater degree; and 3) expression of KV 4.3 is downregulated in the DRGs of PAD rats and KV 4.3 channel is a major contributor to the activity of KV 4 currents in muscle DRG neurons. In conclusion, data suggest that femoral artery occlusion-induced limb ischaemia and/or ischaemia-induced metabolites (i.e. BK) inhibit the activity of KV 4 channels in muscle afferent neurons likely leading to the exaggerated exercise pressor reflex observed in PAD.

Nerve growth factor in muscle afferent neurons of peripheral artery disease and autonomic function.

In peripheral artery disease patients, the blood supply directed to the lower limbs is reduced. This results in severe limb ischemia and thereby enhances pain sensitivity in lower limbs. The painful perception is induced and exaggerate during walking, and is relieved by rest. This symptom is termed by intermittent claudication. The limb ischemia also amplifies autonomic responses during exercise. In the process of pain and autonomic responses originating exercising muscle, a number of receptors in afferent nerves sense ischemic changes and send signals to the central nervous system leading to autonomic responses. This review integrates recent study results in terms of perspectives including how nerve growth factor affects muscle sensory nerve receptors in peripheral artery disease and thereby alters responses of sympathetic nerve activity and blood pressure to active muscle. For the sensory nerve receptors, we emphasize the role played by transient receptor potential vanilloid type 1, purinergic P2X purinoceptor 3 and acid sensing ion channel subtype 3 in amplified sympathetic nerve activity responses in peripheral artery disease.

Effects of Lactate on One Class of Group III (CT3) Muscle Afferents

A class of Group III muscle afferent neurons has branching sensory terminals in the connective tissue between layers of mouse abdominal muscles (“CT3 muscle afferents”). These sensory endings are both mechanosensitive and metabosensitive. In the present study, responses of CT3 afferents to lactate ions and changes in temperature were recorded. Raising muscle temperature from 32.7°C to 37°C had no consistent effects on CT3 afferent basal firing rate or responses to either von Frey hair stimulation or to an applied load. Superfusion with lactate ions (15 mM, pH 7.4) was associated with an increase in firing from 6 ± 0.7 Hz to 11.7 ± 6.7 Hz (14 units, n = 13, P < 0.05, P = 0.0484) but with considerable variability in the nature and latency of response. Reducing the concentration of extracellular divalent cations, which mimicked the chelating effects of lactate, did not increase firing. Raised concentrations of divalent cations (to compensate for chelation) did not block excitatory effects of lactate on CT3 afferents, suggesting that effects via ASIC3 were not involved. Messenger RNA for the G-protein coupled receptor, hydroxyl carboxylic acid receptor 1 (HCAR1) was detected in dorsal root ganglia and HCAR1-like immunoreactivity was present in spinal afferent nerve cell bodies retrogradely labeled from mouse abdominal muscles. HCAR1-like immunoreactivity was also present in axons in mouse abdominal muscles. This raises the possibility that some effects of lactate on group III muscle afferents may be mediated by HCAR1.

Sensitization of group lV skeletal muscle afferents in type 1 diabetic rats

The blood pressure (BP) response to exercise is abnormally exaggerated in type 1 diabetes mellitus (T1DM). However, the mechanisms underlying the potentiated BP response remain largely unclear. Sensory information from working muscle, generated by activation of mechanically and metabolically sensitive muscle afferents, is known to contribute importantly to this exercise‐induced exaggeration in BP. Moreover, receptors for advanced glycation end products (RAGE) and protein kinase C (PKC) interact to modulate neuronal function, including muscle afferents. It is possible that the interactive relationship between RAGE and PKC is altered in T1DM. Therefore, it was hypothesized that the potentiated exercise‐induced BP response in T1DM is mediated by sensitization of skeletal muscle afferent fibers via an overactive RAGE/PKC signaling pathway.PURPOSETo investigate 1) action potential responses to mechanical and chemical stimulation in muscle afferents of healthy and T1DM rats in vitro, 2) the impact of T1DM on plasma levels of high‐mobility group box‐protein 1 (HMGB‐1), a RAGE ligand, and 3) the impact of T1DM on PKC activation in dorsal root ganglion (DRG) subserving skeletal muscle afferent neurons.METHODST1DM was induced in Sprague‐Dawley rats by intraperitoneally injecting a single dose of 50 mg/kg streptozotocin. In healthy control animals, only saline was injected. At 1–3 weeks after the injection, the function of mechanically (application of a 0–196 mN ramped stimulus) or chemically (administration of 1 μM capsaicin) activated group IV fibers were assessed by obtaining single‐fiber recordings using the extensor digitorum longus muscle‐nerve preparation. Plasma HMGB1 was determined using ELISA. Phosphorylated PKC protein levels were quantified in DRG (L3‐L5) by western blotting.RESULTST1DM rats exhibited hyperglycemia after overnight fasting (P&lt;0.05). Plasma insulin in T1DM tended to be lower as compared to control animals (P=0.09). Spontaneous activity in group IV fibers was significantly larger in diabetic rats than in control animals (P&lt;0.05). Compared to control, response magnitudes to mechanical stimulation (P&lt;0.01) and capsaicin exposure (P=0.01) in group IV afferents were significantly greater in T1DM. Plasma HMGB1 levels were significantly higher in T1DM as compared with control (P&lt;0.01). T1DM showed markedly increased phosphorylated PKC levels in DRGs compared with control (P&lt;0.05).CONCLUSIONSIn T1DM, the sensitivity of group IV skeletal muscle afferent fibers to mechanical and chemical stimuli is abnormally increased. The data likewise suggest this enhanced afferent sensitivity may be due to augmented activation of the RAGE/PKC pathway. Importantly, these changes likely contribute significantly to the exaggerated pressor response to exercise characteristic of T1DM.Support or Funding InformationSupported by Lawson &amp; Rogers Lacy Research Fund and SHP Interdisciplinary Research Grant Program.

Heat treatment improves the exaggerated exercise pressor reflex in rats with femoral artery occlusion via a reduction in the activity of the P2X receptor pathway.

During exercise, the blood pressure (BP) response is exaggerated in peripheral artery disease (PAD). We examined whether heat treatment (HT) has beneficial effects on the exaggerated exercise pressor reflex in PAD rats. With HT (increase in basal muscle temperature of ∼1.5°C for 30min, twice daily for three continuous days), the amplified BP response to muscle contraction is alleviated in PAD. We demonstrated that HT attenuates the enhancement of the BP response induced by stimulation of P2X in muscle afferent nerves of PAD rats. HT also attenuates the upregulation of the P2X3 and the increase in P2X currents in the muscle afferent neurons of PAD rats. Previous heat exposure plays a beneficial role in modifying the exaggeration of the exercise pressor reflex in PAD and a reduction in the activity of the P2X receptor pathway is probably a part of the mechanism mediating this improvement. The current study was performed to examine if heat treatment (HT) has beneficial effects on the exaggerated exercise pressor reflex in rats with peripheral artery disease (PAD). We further determined if the temperature-sensitive P2X receptor is involved in the effects of HT. The pressor response to static muscle contraction and α,β-methylene ATP (αβ-me ATP, a P2X agonist) was examined. Western blot analysis was used to determine the protein levels of P2X3 in the dorsal root ganglion (DRG), and the whole cell patch clamp was used to examine the amplitude of P2X currents in the DRG neurons. The basal muscle temperature (Tm ) was lower in PAD rats than in control rats. Tm was increased by ∼1.5°C and this increase was maintained for 30min. This HT protocol was performed tweice daily for three continuous days. A greater blood pressure (BP) response to contraction was observed in PAD rats. HT attenuated the amplification of the BP response in PAD rats. HT also attenuated the enhancement of the BP response induced by the arterial injection of αβ-me ATP in PAD rats. In addition, HT attenuated the upregulation of the P2X3 and increased P2X currents in the DRG neurons of PAD rats. In conclusion, previous heat exposure plays an inhibitory role in modifying the exaggeration of the exercise pressor reflex in PAD and a reduction of the activity of the P2X receptor pathway is probably a part of mechanisms leading to the beneficial effects of HT.

Enhancement by TNF-α of TTX-resistant NaV current in muscle sensory neurons after femoral artery occlusion.

Femoral artery occlusion in rats has been used to study human peripheral artery disease (PAD). Using this animal model, a recent study suggests that increases in levels of tumor necrosis factor-α (TNF-α) and its receptor lead to exaggerated responses of sympathetic nervous activity and arterial blood pressure as metabolically sensitive muscle afferents are activated. Note that voltage-dependent Na+ subtype NaV1.8 channels (NaV1.8) are predominately present in chemically sensitive thin fiber sensory nerves. The purpose of this study was to examine the role played by TNF-α in regulating activity of NaV1.8 currents in muscle dorsal root ganglion (DRG) neurons of rats with PAD induced by femoral artery occlusion. DRG neurons from control and occluded limbs of rats were labeled by injecting the fluorescent tracer DiI into the hindlimb muscles 5 days before the experiments. A voltage patch-clamp mode was used to examine TTX-resistant (TTX-R) NaV currents. Results were as follows: 72 h of femoral artery occlusion increased peak amplitude of TTX-R [1,922 ± 139 pA in occlusion (n = 11 DRG neurons) vs. 1,178 ± 39 pA in control (n = 10), means ± SE; P < 0.001 between the 2 groups] and NaV1.8 currents [1,461 ± 116 pA in occlusion (n = 11) and 766 ± 48 pA in control (n = 10); P < 0.001 between groups] in muscle DRG neurons. TNF-α exposure amplified TTX-R and NaV1.8 currents in DRG neurons of occluded muscles in a dose-dependent manner. Notably, the amplification of TTX-R and NaV1.8 currents induced by TNF-α was attenuated in DRG neurons with preincubation with respective inhibitors of the intracellular signaling pathways p38-MAPK, JNK, and ERK. In conclusion, our data suggest that NaV1.8 is engaged in the role of TNF-α in amplifying muscle afferent inputs as the hindlimb muscles are ischemic; p38-MAPK, JNK, and ERK pathways are likely necessary to mediate the effects of TNF-α.

Peripheral EphrinB1/EphB1 signalling attenuates muscle hyperalgesia in MPS patients and a rat model of taut band-associated persistent muscle pain.

BackgroundMyofascial pain syndrome (MPS) is an important clinical condition that is characterized by chronic muscle pain and a myofascial trigger point (MTrP) located in a taut band (TB). Previous studies showed that EphrinB1 was involved in the regulation of pathological pain via EphB1 signalling, but whether EphrinB1-EphB1 plays a role in MTrP is not clear.MethodsThe present study analysed the levels of p-EphB1/p-EphB2/p-EphB3 in biopsies of MTrPs in the trapezius muscle of 11 MPS patients and seven healthy controls using a protein microarray kit. EphrinB1-Fc was injected intramuscularly to detect EphrinB1s/EphB1s signalling in peripheral sensitization. We applied a blunt strike to the left gastrocnemius muscles (GM) and eccentric exercise for 8 weeks with 4 weeks of recovery to analyse the function of EphrinB1/EphB1 in the muscle pain model.ResultsP-EphB1, p-EphB2, and p-EphB3 expression was highly increased in human muscles with MTrPs compared to healthy muscle. EphB1 (r = 0.723, n = 11, P < 0.05), EphB2 (r = 0.610, n = 11, P < 0.05), and EphB3 levels (r = 0.670, n = 11, P < 0.05) in the MPS group were significantly correlated with the numerical rating scale (NRS) in the MTrPs. Intramuscular injection of EphrinB1-Fc produces hyperalgesia, which can be partially prevented by pre-treatment with EphB1-Fc. The p-EphB1 contents in MTrPs of MPS animals were significantly higher than that among control animals (P < 0.01). Intramuscular administration of the EphB1 inhibitor EphB1-Fr significantly suppressed mechanical hyperalgesia.ConclusionsThe present study showed that the increased expression of p-EphB1/p-EphB2/p-EphB3 was related to MTrPs in patients with MPS. This report is the first study to examine the function of EphrinB1-EphB1 signalling in primary muscle afferent neurons in MPS patients and a rat animal model. This pathway may be one of the most important and promising targets for MPS.

An Exaggerated Muscle Mechanoreflex in Type 2 Diabetic Rats Is Mediated by Potentiated Skeletal Muscle Afferent Discharge to Mechanical Stimulation

The blood pressure response to exercise is heightened in patients with type 2 diabetes (T2D). Since such elevations in blood pressure increase the risk for developing an adverse cardiovascular event, elucidating the underlying mechanisms is clinically relevant. The exercise pressor reflex (EPR) plays an important role in regulating the cardiovascular system during exercise. Our group has previously demonstrated that the EPR is exaggerated in T2D rats contributing importantly to the augmented pressor response to physical activity. However, the mechanisms responsible for this abnormal EPR function in T2D remain to be elucidated. Sensory information from working muscle is generated, in part, by stimulation of mechanically sensitive afferent neurons (i.e. the skeletal muscle mechanoreflex, a component of the EPR). In this study, it was hypothesized that the EPR dysfunction characteristic of T2D is mediated by an exaggerated muscle mechanoreflex resulting from an increase in muscle afferent responsiveness to mechanical stimulation.PURPOSEIn T2D rats, to 1) investigatethe pressor and sympathetic responses to stimulation of the muscle mechanoreflex in vivo and 2) examine neuronal discharge to mechanical stimulation in skeletal muscle thin fiber afferents in vitro.METHODSFor 14–16 weeks, rats were given either a normal diet (control group) or a high fat diet in combination with a low dose (35 mg/kg) of streptozotocin (T2D group). In vivo, we measured changes in renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) during passive stretch of hindlimb muscle in decerebrate rats. In vitro, muscle afferent neurons were mechanically activated via ramp‐shaped mechanical stimulation (0–196 mN) with the resulting action potentials assessed by obtaining single‐fiber recordings using an extensor digitorum longusmuscle‐nerve preparation.RESULTSCompared to control, induction of T2D evoked hyperglycemia (104±5 vs. 161±10 mg/dL, P&lt;0.05). Similarly, T2D elicited significant increases in plasma insulin (111±10 vs. 150±13 pmol/L, P&lt;0.05) and HOMA‐IR (3.9±0.3 vs. 9.3±1.2, P&lt;0.01). Mechanoreflex activationby passive stretch evoked significantly greater increases in RSNA and MAP in T2D (13±3 vs. 81±16 %, P&lt;0.01 and 7±3 vs. 29±7 mmHg, P&lt;0.05, respectively). Further, the response magnitude to mechanical stimulation tended to be greater in muscle afferents from T2D compared to control (20±7 vs. 40±8 spikes, P=0.10).CONCLUSIONSThese findings suggest that the muscle mechanoreflex is exaggerated in T2D resulting, at least in part, from augmentations in the responsiveness of thin muscle afferent fibers to mechanical stimuli. Importantly, these changes likely contribute significantly to the EPR overactivity manifest in T2D.Support or Funding InformationSupported by Lawson &amp; Rogers Lacy Research Fund in Cardiovascular Disease and the Southwestern School of Health Professions Interdisciplinary Research Grant Program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.