K_V4 channels in isolectin B4 muscle dorsal root ganglion neurons of rats with experimental peripheral artery disease: effects of bradykinin B1 and B2 receptors

Abstract

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⁺ (K_V4) 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 K_V4 currents and engagement of bradykinin (BK) subtype receptors. Thus, we examined K_V4 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 K_V4 currents in IB4+ muscle DRG neurons and siRNA knockdown of K_V4 subunit K_V4.3 decreased the activity of K_V4 currents in IB4+ DRG neurons. In conclusion, our data suggest that limb ischemia and/or ischemia-induced BK inhibit activity of K_V4 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.

INTRODUCTION

Peripheral artery disease (PAD) is a common cardiovascular disease that affects a population of over 200 million globally and has a prevalence rate of 12%–20% in people over 60 yr in the United States (1, 2). The disease progressively narrows the lower extremity conduit vasculature and can lead to severe limb ischemia. One of the chief concerns impacting the daily life of patients with PAD is muscle pain caused by reduced blood flow to limb muscle, termed as “intermittent claudication,” which happens during exercise (e.g., walking) and stops with resting (1, 2). The limb ischemia is partly attributed to increases in responses of the arterial blood pressure (BP) and vasocontraction during exercise in patients with PAD (3, 4). In combination with the ischemia-induced leg pain and increased BP, the exercise capability of the patients with PAD is jeopardized. Of note, there is a high risk of cardiovascular events and reduced quality of life as well as all-cause mortality reported in the patients with PAD (5–7). Notably, augmented BP response during exercise may cause a higher incidence of cardiovascular events (8, 9), and the exaggerated sympathetic nervous activity (SNA)-regulated BP response during exercise contributes to poor clinical outcomes (10, 11). Epidemiology studies further suggest that an exaggerated BP response to exercise is associated with decreased survival in both asymptomatic normotensive subjects (12) and patients with PAD (13).

During exercise, two basic mechanisms contribute to the activation of the sympathetic nerve system and the subsequent increases in BP, heart rate (HR), myocardial contractility, and peripheral vasoconstriction (14, 15): 1) “Central Command” (16), which is initiated by a volitional signal emanating from central motor units and then induces the enhancement of SNA; and 2) “Exercise Pressor Reflex” (EPR) (17, 18), which is signaling inputs from the afferents of the contracting skeletal muscle and thereby increases autonomic activities. In specific, the EPR is activated due to metabolic stimulation (i.e., “metaboreceptor” stimulation in group IV afferents) and to mechanical deformation in the muscle afferents’ receptive field (i.e., “mechanoreceptor stimulation” in group III afferents) (19). The thin fiber muscle afferent nerves are engaged following stimulation of the receptors during exercise, and therefore induce activation of cardiovascular nuclei in the brainstem (18). Meanwhile, activation of the muscle afferent nerves modulates the arterial baroreflex functions in involvement of the SNA and cardiovascular responses to exercise (20). Although the baroreflex is important in contributing to an abnormal neural-hemodynamic response to exercise in PAD (21), it is generally accepted that an exaggerated EPR is a determinant of why BP rises with exercise in PAD (4). Thus, it is necessary to determine signaling pathways and molecular mediators involved in the exaggerated SNA and BP responses to stimulation of chemically and mechanically sensitive muscle afferents in PAD. Also, identifying the molecular mediators alleviating the exaggerated BP response to exercise in PAD is clinically significant.

A-type voltage-gated K⁺ channels (K_V channels) appear in various tissues of mammalian animals and are transmembrane channels for potassium and are sensitive to voltage changes in the cell membrane with fast inactivation and time-dependent properties. They play a crucial role in returning the depolarized cells to a resting state and are quintessential regulators of neuronal excitability (22). For those reasons, we previously examined the role played by K_V subtype K_V4 in regulating the excitability in muscle dorsal root ganglion (DRG) neurons of PAD rats (23). We also examined engagement of ischemia-induced metabolites [i.e., bradykinin (BK)] in the activities of K_V4 (23). Results of this published work suggest that BK inhibits the activities of K_V4 currents to a greater degree in muscle DRG neurons of PAD rats and a decrease in expression of K_V4.3 subunit in DRG neurons is a main contributor to the attenuated K_V4 activity in PAD rats. Nonetheless, it is still noteworthy to determine the characteristics of K_V4 current activity in muscle DRG neurons of PAD rats and the effects of BK receptors on K_V4 current.

There are two distinct DRG neurons, namely isolectin B₄-positive and negative- (IB4+ and IB4−) DRG neurons, because of their distinct neurochemical and neurotrophic characteristics (24–27); i.e., IB4+ neurons are a group of thin fiber neurons that express receptors for glial cell line-derived neurotrophic factor (GDNF), depending on GDNF for survival during postnatal development, and are relatively “peptide poor” but express a surface carbohydrate-binding IB₄. In contrast, IB4− DRG neurons express trkA receptors for nerve growth factor (NGF), depending on NGF for survival, and contain neuropeptides such as calcitonin gene-related peptide and substance P. In addition, IB4+ and IB4− DRG neurons of the thin fiber sensory nerves have distinct sensitivity to metabolic and mechanical stimulation (28–30). Both IB4+ and IB4− DRG neurons display hypersensitivity to acidic products in injured tissues and mechanical stimulation (28, 30). Results also showed that long-lasting visceral hypersensitivity induced by acetic acid is associated with A-type K_V currents and greater excitability in IB4+ DRG neurons but not in IB4− neurons (30). Moreover, A-type K_V currents alter the characteristics of neuronal firing in IB4+ nociceptive DRG neurons (31). Among nociceptive DRG neurons, IB4− neurons are mechanically sensitive with an increase in spontaneous activity and hyperexcitability (29).

According to those previous findings and our previous work (23), the current study was designed to examine K_V4 currents in IB4+ and IB4− muscle DRG neurons and the effects of BK B1 and B2 receptor activation on K_V4 currents in two different subpopulations of muscle DRG neurons. Using siRNA knockdown approach, in the current study we also examined the role of K_V4.1 and K_V4.3 subunits in regulating the activities of K_V4 currents in IB4+ and IB4− muscle DRG neurons. Overall, the purposes of our current study were to determine if 1) K_V4 currents are inhibited in a selective subpopulation of muscle DRG neurons (IB4+ and/or IB4−) of PAD rats; 2) activation of respective B1 and B2 receptors leads to a greater inhibition on K_V4 currents in IB4+ and/or IB4− muscle DRG neurons of PAD; and 3) K_V4.3 subunit plays a central role in regulating K_V4 current activity in involvement of the effects of ischemia and BK in PAD.

MATERIALS AND METHODS

Ethical Approval

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Penn State College of Medicine (Protocol No.: PRAMS201147671) and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats (4–6 wk old) were housed in accredited temperature and ventilation controlled facilities with a 12:12-h light-dark cycle and ad libitum access to standard rat chow and water.

Femoral Artery Occlusion

The rats were anesthetized by inhalation of an isoflurane-oxygen mixture (2%–5% isoflurane in 100% oxygen). The femoral artery on one limb was surgically exposed, dissected, and ligated ∼3 mm distal to the inguinal ligament as described previously (23, 32). As the control, the contralateral limb was dealt with the same procedure except for the suture below the femoral artery was not tied. After the surgery, all the rats were returned to the cage for regular housing for 3 days before experiments. Note that buprenorphine hydrochloride (0.05 mg/kg, sc) was administered before the surgery for postoperative pain relief. After the surgery, the animals were kept in the surgery room for 2–3 h for observation and then returned to the animal facility.

Electrophysiology

Labeling of hindlimb muscle afferent DRG neurons.

As described previously (23, 32), 2 days before the femoral artery ligation was performed, an incision in the calf area of one limb was made and the gastrocnemius muscle was exposed after rats were anesthetized. The lipophilic dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, 60 mg/mL) was injected into the white portion of the gastrocnemius muscle. A total volume of 1 µL DiI tracer was administered at different locations, with the needle left in the muscle for 1 min to prevent the tracer leakage. The same procedure was made in the contralateral limb. The skin incision overlying the muscle was then sutured and closed using surgical staples. The rats were then returned to their cages to wait for the fluorescent DiI retrograde transported to DRGs to label muscle DRG neurons. Note that no analgesics were given after making a skin incision for the injection of DiI since no pain behavior was observed in rats after the injection.

Culture of DRG neurons.

As described previously (23, 32), the rats were euthanatized by decapitation after inhalation of an overdose of isoflurane, and rat’s L4-L6 DRGs in both control limb and occluded limb were removed and dissected, immediately transferred into ice-cold Hank’s balanced salt solution. After being freed from the connective tissues, the ganglia were enzymatically digested and dissociated in Earle’s balanced salt solution (Sigma Aldrich) containing collagenase Type D (0.6 mg/mL; Roche), trypsin (0.30 mg/mL; Worthington), and DNase (0.1 mg/mL; Alfa Aesar), followed by shaking for 40 min at 34°C. The dissociated neurons were seeded on 10% poly-l-lysine–coated coverslips (Dia No. 8 mm) in 35-mm culture dish containing 2 mL DMEM medium (Thermo) supplemented with 10% FBS, 1% glutamine, and 1% penicillin-streptomycin. Then, the neurons were cultured at 37°C with 5% CO₂, 95% air in a cell culture incubator (VWR).

Recording of K⁺ currents.

The recording was performed on the neurons within 24 h after DRG dissociation. Immediately before the recording, neurons were incubated with IB4− Alexa Fluor 488 (3 µg/mL; Invitrogen) in the extracellular solution for 10 min and then rinsed for at least 3 min (33). Thus, we examined two distinct subpopulations of thin fiber afferent neurons, namely, IB4+ and IB4− muscle DRG neurons. DRG neurons were first visualized using differential interference contrast (DIC; ×20–40) optics and then an IB4+ neuron was visualized (as green color) using a combination of fluorescence illumination and DIC optics on a Nikon TE2000 inverted microscope. Meanwhile, those DRG neurons were also identified as DiI-positive (red color) under an inverted microscope with a fluorescent filter, and images were displayed on a video monitor. Figure 1 shows that IB4+ muscle DRG neurons were identified.

Figure 1.

A-type voltage-gated K⁺ (K_V4) currents in different phenotypes of muscle dorsal root ganglion (DRG) neurons. A: representative of isolectin B₄-positive (IB4+) and isolectin B₄-negative (IB4−) muscle DRG neurons. Scale bar: 50 µm. B: representative traces of K_V4 currents in muscle neurons of the control limbs. Protocol 1 was used to record the total tetraethylammonium (TEA)-resistant K⁺ currents (I_Ktotal), and protocol 2 to record the delayed rectifying K+ current (I_{K DR}), Kv4 currents = I_Ktotal − I_{K DR}, calculated by pClampfit 10.1. Representative traces for I_Ktotal, I_{K DR}, and K_V4 currents are indicated by red, black, and blue color, respectively. C: histogram of averaged K_V4 current density at 60 mV (Density_{60 mV}) in IB4+ and IB4− muscle DRG neurons. Averaged data showing that density of K_V4 currents is greater in IB4+ DRG neurons than that in IB4− DRG neurons. *P = 0.002 between two groups of neurons. Open circles, individual data. “n” in all the figures of this study indicates the number of the recorded DRG neurons.

K⁺ currents of IB4+ and IB4− muscle DRG neurons (DiI positive and cell diameters ≤ 35 µM) were recorded in the whole cell configuration using a MultiClamp 700B amplifier supplied with Digitizer 1440 A (Axon, Inc.). Signals were acquired with pClamp10.1 and analyzed with pClampfit10.7 software. All experiments were performed at room temperature of 20°C–22°C.

The extracellular solution contained (in mM) the following: 110 choline chloride, 5 KOH, 1 MgCl₂, 20 tetraethylammonium (TEA), 10 HEPES, 2 CdCl₂, and 10 d-glucose (pH 7.4, osmolality 310 mosmol). The electrode was filled with a solution containing (in mM) the following: 120 KCl, 2.5 MgCl₂, 10 EGTA, 10 HEPES, 0.3 Li-GTP, 2 MgATP, and 1 CaCl₂ (pH 7.3 adjusted with KOH, osmolality 290 mosmol).

Holding at −65 mV, after the seals (2–8 GΩ) obtained with 2–4 MΩ resistance of glass electrodes filled with internal solution, the whole cell configuration was applied. In voltage-clamp mode, two separate protocols were used to record tetraethylammonium (TEA) resistant A-type-K⁺ channel currents (TEA-R-IKA) in muscle DRG neurons as previously reported (30, 34). Total K⁺ currents (I_Ktotal) were recorded with the protocol 1: a 1-s conditioning pulse of −100 mV before 500-ms step depolarizations from −100 mV to 60 mV with an increment of 10 mV per step. The protocol 2 was similar to protocol 1 except for 1-s conditioning pulse of −30 mV for the delayed rectifier K⁺ currents (I_{K DR}). The recording current was filtered at 2 kHz and sampled at 10 kHz. Voltage errors were minimized by 80% series resistance compensation and linear leak subtraction was used for all recordings. All signals were acquired with pClamp10.1 and analyzed with Clampfit10.7 software. K_V4 currents = I_Ktotal − I_{K DR}, calculated by pClampfit 10.1. 20 μM TEA was applied to the external solution to block the TEA-sensitive K⁺ channels for ∼5 min before recording of current activities.

Chemicals, BK (Biotechne No. 3004), Lys-[Des-Arg⁹]-bradykinin (Biotechne No. 3225; Lys-BK, agonist to B1 receptor), and Phe⁸Ψ [(CH-NH)-Arg⁹]-bradykinin (Biotechne No. 3229; phe-BK, agonist to B2 receptor) were stored in the stock solutions and diluted in extracellular solution (1 µM) immediately before being used and individually held in a series of independent syringes of the pressurized VC3-8MP perfusion system (ALA). The distance from the outlet tip mouth to the neuron examined was within 100 µm. All the agonists were applied to the recorded neurons 5 min before recording of currents.

Immunohistochemistry

In situ DRG tissue.

The rats were anesthetized with an isoflurane-oxygen mixture and then perfused transcardially with 200 mL of ice-cold saline containing 1,000 U heparin followed by 500 mL of 4% freshly prepared, ice-cold paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4). Rats’ L4-L6 DRGs were immediately dissected out and immersed in the same fixative at 4°C for 2 h. The tissues were then stored in PBS containing 30% sucrose overnight, and a cryostat was used to obtain DRG sections (10 µm).

To examine localization of K_V4.1 and K_V4.3 channels within IB4+ DRG neurons, DRG sections on slides were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. After being washed with PBS, the tissue was permeabilized with 0.3% Triton X-100 in PBS for 10 min. The sections were then washed twice by PBS (5 min per time) and blocked with PBS supplemented with 0.2% Tween-20, and 5% BSA for 1 h. After the blocking step, the sections were incubated with the primary antibody of IB4 Alexa Fluor-488 Conjugate (1:200, Invitrogen Molecular Probes), rabbit polyclonal anti-K_V4.1, and anti-K_V4.3 (1:100, Alomone) overnight at 4°C. The specificity of the antibodies was verified by the manufacturer using the blocking peptides with K_V4.1 (Alomone No. BLP-PC119) and K_V4.3 (Alomone No. BLP-PC017). After being washed in PBS, the sections were incubated with the goat anti-rabbit fluorescein Alexa Fluor-594-labeled secondary antibody (1: 200, Invitrogen Molecular Probes) in PBS supplemented with 0.2% Tween-20 and 5% BSA for 2 h at room temperature. After that, the sections were washed in PBS, mounted by Flouromount-G Mounting Medium (Fisher Scientific), and coverslipped. Then, Alexa Fluor-594-labeled K_V4.1/K_V4.3 and Alexa Fluor-488-labeled IB4+ DRG neurons were examined using a Nikon Eclipse 80i microscope with rhodamine (red) and FITC (green) filters, respectively, and the images were captured using a Nikon DS-QiMc digital camera assisted with the NIS-Elements BR 3.1 software.

Dissociated DRG cells.

After rat’s L4-L5 DRG tissues were dissociated, DRG neurons were seeded on the coverslip coated with 0.1% poly-d-lysine solution and cultured for 3 h for attachment. After being fixed and permeabilized with 100% methanol at −20°C for 20 min, the neurons were blocked with 10% BSA in TPBS (containing 0.1% Tween-20 in 1× PBS solution) for 1 h at room temperature. Then, rabbit anti-K_V4.1 and anti-K_V4.3 primary antibodies (1:500, Alomone No. APC-119 and Alomone No. APC-017) were individually incubated overnight at 4°C, and then blotted with Alexa Fluor 594 anti-rabbit secondary antibody (1:3,000, Thermo Fisher Scientific No. A32740). After this, the stained neurons were treated with IB4− Alexa Fluor 488 (1 µg/mL, Thermo Fisher Scientific No. I21411) in PBS for 5 min and rinsed for another 3 min. After being mounted, the images of the targets were examined using a Nikon Eclipse 80i microscope with appropriate filters, and the images were captured using a Nikon DS-QiMc digital camera assisted with the NIS-Elements BR 3.1 software.

siRNA Knockdown in DRGs

The siRNA oligo duplex of rat K_V4.1 (NM-001105748.1) and rat K_V4.3 (NM-001270962.1) were designed to target three unique gene sequences for each gene: rat K_V4.1 siRNA (5′- AAGACCAACTTCACAAGCATC-3′; 5′- AAGACTACGTGTCATGAGTTC-3′; 5′- AAGAACACGCTGGATCGCTAC-3′) and rat K_V4.3 siRNA: (5′- AAGACCACGTCACTCATCGAG-3′; 5′- AAGAACCACGAGTTTATTGAT-3′; 5′- AAGGAGTTCTTCTTCAACGAG-3′). All siRNA oligo duplex and the negative control siRNA (No. SIC007) were obtained from Sigma.

As described previously (35, 36), L4-L5 DRGs were dissected, cut with three to four slides, and incubated in cold HBSS before transfection. The DRGs were first electroporated using Neon Electroporation System (Thermo Fisher Scientific) with a protocol of 1,000-V pulses/20 ms/3 times in a 100-µL electroporation transfection system, containing T solution, 3 µL of 100 µM siRNA oligos, and 3 µL of 100 mM 2,3-butanedione monoxime (BDM) (Abcam No. ab120616). Then, the DRGs were transferred onto a 22-mm dish containing 1 mL transfection mixture in Opti-MEM medium (20 µL of 100 µM siRNA oligos, 20 µL of BDM, 10 µL of Lipofectamine RNAiMAX, Thermo Fisher Scientific No. 13778030). After this, the DRGs were cultured in a CO₂ incubator for 4 hs. The transfection mixture was removed and changed into final DMEM growth medium to be cultured for 2 days. Another transfection was applied for 2 days before the patch-clamp experiments were performed.

Real-Time PCR

The total RNA of rat DRGs was extracted with RNeasy Micro Kit (Qiagen No. 74004). After being quantified, 200 ng of RNA was reverse-transcribed into 1st cDNA with SuperScript III First-Strand Synthesis SuperMix (Thermo No. 18080400). Then, the levels of K_V4.1 and K_V4.3 mRNA were respectively determined with FAM-MGB TaqMan primer assay: K_V4.1 (Thermo No. Rn01525167), K_V4.3 (Thermo No. Rn04339183), and rat GAPDH (Thermo No. Rn04339183) as an internal control. After the PCR reaction mixture was prepared with TaqMan Gene Expression Master Mix (Thermo No.4369016), PCR was run using QuantStudio 12 K Flex Real-Time PCR System (Thermo Fisher Scientific) with the protocol of preheating at 50°C/2 min and polymerase activation at 95°C/10 min, followed by 40 cycles of denaturing at 95°C/15 s and annealing at 60°C/1 min. Ct value of PCR reaction in each sample was detected, and the mRNA level was determined with 2^−ΔΔCt method.

Statistical Analysis

All data in this study were presented as means ± SD (standard deviation). An unpaired t test was used to analyze the difference in the K_V4 currents density of IB4+ and IB4− muscle DRG neurons in control limbs (Fig. 1). A two-way ANOVA was applied for all other experiments, and as appropriate, post hoc analysis with Tukey’s tests were applied to compare the difference between specific groups. In the event, the distribution of mean changes was not normally distributed, the non-parametric test was used to analyze the data. All statistical analyses were performed using SPSS v26, and the significant differences were considered at P < 0.05.

RESULTS

K_V4 Currents in Different Phenotypes of Muscle DRG Neurons

To explore the characteristics of K_V4 currents in two subgroups (IB4+ and IB4−) of rat muscle DRG neurons, we identified rat muscle DRG neurons stained with IB4 before K_V4 currents recording. As shown in Fig. 1A, the muscle DRG neurons containing DiI appeared to be IB4+ and IB4−. K_V4 currents were then recorded from IB4+ and IB4− muscle DRG neurons. To minimize the diversity of neurons, K_V4 currents in DRG neurons with a diameter of ≤35 µm were determined. Figure 1, B and C show typical traces and averaged data of K_V4 currents in two different phenotypes of muscle DRG neurons. A greater density of K_V4 currents was observed in IB4+ DRG neurons than that in IB4− DRG neurons; i.e., the current density at depolarization of 60 mV from −100 mV (Density_{60 mV}) was 232 ± 136 pA/pF (n = 66) in IB4+ group and 132 ± 94 pA/pF (n = 35; P = 0.002 between two groups).

K_V4 Currents in Muscle DRG Neurons of Control Limbs and Occluded Limbs

We further investigated the effect of femoral artery occlusion on K_V4 currents in IB4+ and IB4− groups of rat muscle DRG neurons. Figure 2, A–C, shows typical traces and averaged data of K_V4 currents in IB4+ and IB4− muscle DRG neurons of control rats and occluded rats. The results are consistent with our published data showing the smaller amplitude of K_V4 currents and decreased current density in DRG neurons of occluded rats (23) and this particular was observed in IB4+ DRG neurons. As shown in Fig. 2B, after 72 h of femoral artery occlusion, K_V4 current density in IB4+ muscle DRG neurons was decreased with depolarized voltage reaching to −20 mV, but this was not observed in the IB4− neurons. There was a significant difference in Density_{60 mV} of K_V4 currents in IB4+ muscle DRG neurons between the control and occluded group (P < 0.05), and between IB4+ and IB4− groups (P < 0.05). Density_{60 mV} of K_V4 currents in IB4+ muscle DRG neurons was decreased to 150 ± 94 pA/pF in the occluded limbs (n = 32, P = 0.004 compared with IB4+ group of the control limbs, Fig. 2C). K_V4 currents were also decreased in IB4− muscle DRG neurons of the occluded limbs but no statistical difference was observed between the control and occluded groups; i.e., Density_{60 mV} of K_V4 currents in IB4− DRG neurons was 103 ± 77 pA/pF in the occluded limbs (n = 22) and 132 ± 94 pA/pF (n = 35) in the control limbs (P = 0.176 between two groups; Fig. 2C). Percent reduction of K_V4 current density was 37% in IB4+ DRG neurons of occluded rats compared with control rats, whereas percent reduction of K_V4 current density was 22% in IB4− DRG neurons.

Figure 2.

A-type voltage-gated K⁺ (K_V4) currents in isolectin B₄-positive (IB4+) and isolectin B₄-negative (IB4−) dorsal root ganglion (DRG) neurons of control limbs and occluded limbs. A: representative traces of K_V4 currents in muscle DRG neurons of both control limbs and occluded limbs. Two individual step depolarization protocols with an increment of 10 mV were used to record Kv4 currents. Protocol 1 was for tetraethylammonium (TEA)-resistant I_Ktotal, and protocol 2 for I_Ktotal. B: current densities of muscle DRG neurons with different voltages applied. *P < 0.05 between control and occlusion groups. C: histogram of averaged data showing that Density_{60 mV} of K_V4 currents is smaller in IB4+ muscle DRG neurons of occluded limbs (n = 32) than that in those neurons of control limbs (n = 56). *P = 0.004 between control and occlusion groups. No significant difference in Density_{60 mV} of K_V4 currents was observed in IB4− DRG neurons between the control (n = 35) and occluded groups (n = 22; P = 0.176 between two groups). Open circles, individual data.

Effects of BK B1 and B2 Activation on K_V4 Currents

To identify the mechanisms of BK involved in the inhibition of K_V4 currents in rat muscle DRG neurons, we examined the effect of BK on K_V4 currents in IB4+ and IB4− muscle DRG neurons. The representative traces are illustrated in Fig. 3A. As shown in Fig. 3B, there was a significant difference in the inhibitory effect of BK on K_V4 currents between IB4+ and IB4− neurons, whereas no difference was seen between the control and occluded group. BK (1 µM) inhibited K_V4 currents in muscle DRG neurons of both the control and the occluded limbs, especially in IB4+ muscle DRG neurons. Percent inhibitory efficiency on K_V4 currents in IB4+ muscle DRG neurons was 33 ± 26% (n = 16) in the control limbs and 26 ± 17% (n = 18) in the occluded limbs (P = 0.605). The inhibitory efficiency in IB4− muscle DRG neurons was significantly lower than in IB4+ muscle DRG neurons; they were 6 ± 18% (n = 13, P = 0.005 compared with the IB4+ group) in the control limbs and 1 ± 25% (n = 17, P = 0.002 compared with the IB4+ group) in the occluded limbs. In addition, no significant difference in BK inhibition of K_V4 currents in IB4− DRG neurons was observed between the control and the occluded limbs (P = 0.895). Note that the current density before BK administration was comparable with that seen in Figs. 1 and 2. The current density in IB4+ muscle DRG neurons before BK was 275 ± 100 pA/pF for the control limbs (n = 16) and 157 ± 69 pA/pF for the occluded limbs (n = 18). The current density in IB4− muscle DRG neurons before BK was 134 ± 30 pA/pF for the control limbs (n = 13) and 91 ± 39 pA/pF for the occluded limbs (n = 17).

Figure 3.

Percentage inhibition of bradykinin (BK), and BK B1 and B2 receptor agonists on A-type voltage-gated K⁺ (K_V4) currents in isolectin B₄-positive (IB4+) and isolectin B₄-negative (IB4−) muscle dorsal root ganglion (DRG) neurons of control limbs and occluded limbs. A: representative traces of K_V4 currents following application control vehicle, BK, Lys-BK, and Phe-BK onto muscle DRG neurons of control limbs and occluded limbs. B: BK led to a greater inhibitory effect on IB4+ DRG neurons than that on IB4− muscle DRG neurons in both control limbs and occluded limbs. *P = 0.005 between two groups of neurons (IB4+ and IB4−) of the control limbs; and P = 0.002 between two groups of DRG neurons of the occluded limbs. There was no significant difference observed in the inhibition of K_V4 currents by BK in IB4+ and IB4− DRG neurons between control limbs and occluded limbs. Open circles, individual data. C: *P = 0.002 between two groups of neurons (IB4+ and IB4−). No significant difference was observed in inhibition of Lys-BK (B1 receptor agonist) on K_V4 currents in muscle IB4+ and IB4− DRG neurons between control limbs and occluded limbs. D: Phe-BK (B2 receptor agonist) inhibited K_V4 currents to a greater degree in muscle IB4+ and IB4− DRG neurons of occluded limbs than in control limbs. *P = 0.032 between IB4+ and IB4− in the control limbs; and *P = 0.049 between IB4+ and IB4− in the occluded limbs. *P = 0.045 between control limbs and occluded limbs for IB4+ neurons; and *P = 0.012 between control limbs and occluded limbs for IB4− neurons. “n”, number of recorded DRG neurons is indicated in B–D.

Moreover, we examined the effects of BK B1 and B2 receptor agonists on K_V4 currents to clarify the engagement of BK receptors. We used the specific agonists to B1 and B2 receptors, namely, Lys-BK and Phe-BK, to perform additional experiments. There was the similar current density before administration of Lys-BK and Phe-BK and in Figs. 1 and 2. The current density in IB4+ muscle DRG neurons before Lys-BK was 241 ± 133 pA/pF for the control limbs (n = 17) and 185 ± 117 pA/pF for the occluded limbs (n = 22); the current density in IB4− muscle DRG neurons before Lys-BK was 119 ± 86 pA/pF for the control limbs (n = 16) and 114 ± 83 pA/pF for the occluded limbs (n = 18). As shown in Fig. 3C, a distinct difference in inhibition of Lys-BK on K_V4 current was observed between IB4+ and IB4− neurons but not between the control and occluded groups. Lys-BK (1 µM) also had a prominent inhibition on K_V4 currents in IB4+ muscle DRG neurons of both the control and the occluded limbs; i.e., percent inhibitory efficiency was 20 ± 22% (n = 15 in control) and 14 ± 26% (n = 22 in occlusion), respectively. No significant difference was found between the two groups (P = 0.404). Also, percent inhibitory efficiency on K_V4 currents was greater in IB4+ DRG neurons than that in IB4− DRG neurons in the control limbs.

The current density in IB4+ muscle DRG neurons before Phe-BK administration was 221 ± 134 pA/pF for the control limbs (n = 20) and 172 ± 121 pA/pF for the occluded limbs (n = 25); the current density in IB4− muscle DRG neurons before Phen-BK was 121 ± 88 pA/pF for the control limbs (n = 15) and 119 ± 75 pA/pF for the occluded limbs (n = 21). An inhibition of K_V4 currents in muscle DRG neurons was observed in both the control limbs and the occluded limbs when they were treated with 1 µM Phe-BK (P = 0.032 between IB4+ and IB4− neurons in control limbs; P = 0.049 between IB4+ and IB4− neurons in occluded limbs). Interestingly, percent inhibitory efficiency of Phe-BK on K_V4 currents in IB4+ muscle DRG neurons was greater in the occluded limbs than that in the control limbs; i.e., it was 8 ± 33% (n = 20 in control) and 27 ± 26% (n = 25 in occlusion; P = 0.045 between the two groups) (Fig. 3D).

K_V4.1 and K_V4.3 Contribution to K_V4 Currents in DRG Neurons

To determine KV4.1 and KV4.3 distribution in different phenotypes of DRG neurons (IB4+ vs. IB4−), in additional groups we examined protein expression of K_V4.1 and K_V4.3 channels within DRG neurons with IB4+ staining in healthy control rats. The results of Fig. 4 illustrate that both K_V4.1 and K_V4.3 subunits were present in IB4+ DRG neurons of in situ tissue of rats. Some DRG neurons containing K_V4.1 and K_V4.3 subunits also appeared to be IB4− in situ tissue.

Figure 4.

Distribution of A-type voltage-gated K⁺ (K_V4.1) and K_V4.3 subunits in dorsal root ganglion (DRG) neurons in situ. Immunofluorescence was used to examine the presence of K_V4.1/K_V4.3 channels within isolectin B₄-positive (IB4+) dorsal root ganglion (DRG) neurons of healthy control rats. Representative photomicrographs show that K_V4.1 (top) and K_V4.3 (bottom) exist within IB4+ DRG neurons. After the images were merged, neuronal cells positive for both K_V4.1/K_V4.3 and IB4+ appear to be yellow color. Scale bar = 50 µm.

In addition, Fig. 5A shows the existence of K_V4.1 and K_V4.3 subunits within IB4+-dissociated DRG neuronal cells.

Figure 5.

Contribution of A-type voltage-gated K⁺ (K_V)4.1 and K_V4.3 to K_V4 currents in muscle dorsal root ganglion (DRG) neurons. A: representatives of K_V4.1 and K_V4.3 subunits’ distribution in rat dissociated DRG neurons. The isolectin B₄-positive (IB4+) DRG neurons were labeled green and the neurons containing K_V4.1 (top) and K_V4.3 (bottom) labeled red. The DRG neurons appear yellow as colocalization of K_V4 subunits and IB4+. Scale bar = 50 µm. B and C: approach of siRNA-K_V4.1/siRNA-K_V4.3 knockdown was used to decrease the expression levels of K_V4.1/K_V4.3 in DRG neurons and then the specific contribution of K_V4.1 and/or K_V4.3 subunit to K_V4 currents was determined. The effectiveness of siRNA-K_V4.1 and siRNA-K_V4.3 knockdown was verified (B). Histogram of averaged Density_{60 mV} of K_V4 currents in both IB4+ and isolectin B₄-negative (IB4−) muscle DRG neurons (C) showing that after knockdown of K_V4.3 the density of K_V4 currents was significantly decreased in IB4+ muscle DRG neurons compared with scramble control group, but this was not observed after the knockdown of K_V4.1 (P = 0.5002 between scramble and knockdown). *P = 0.0019 between K_V4.3 knockdown and scramble in IB4+ muscle DRG neurons. Open circles, individual data. “n,” number of recorded DRG neurons.

To minimize the confounding issues caused by nonspecificity of pharmacological agents, in this experiment we used approach of siRNA-K_V4.1/siRNA-K_V4.3 knockdown to decrease expression levels of K_V4.1/K_V4.3 in DRG neurons and then determined the specific contribution of K_V4.1 and/or K_V4.3 subunit to K_V4 currents. Data of Fig. 5B have verified that the effectiveness of siRNA-K_V4.1 and siRNA-K_V4.3 knockdown was >80% as the levels of K_V4.1 or K_V4.3 subunits’ mRNA were assessed. After the knockdown of siRNA-K_V4.3, density of K_V4 currents was significantly decreased in IB4+ muscle DRG neurons compared with scramble control group (Fig. 5C). Density_{60 mV} was 42 ± 23 pA/pF (n = 21) in IB4+ muscle DRG neurons with K_V4.3 knockdown and 82 ± 69 pA/pF with application of scramble (n = 48; P = 0.0019 between the two groups). In contrast, Density_{60 mV} was 62 ± 37 pA/pF in IB4+ muscle DRG neurons with siRNA-K_V4.1 knockdown (n = 31; P = 0.5002 compared with scramble; Fig. 5C). In contrast, K_V4 currents in IB4− DRG neurons muscle DRG neurons were not significantly altered after K_V4.3 or K_V4.1 subunit was knocked down.

DISCUSSION

The key findings of the current study include that 1) femoral artery occlusion decreases K_V4 current density in muscle DRG neurons, and this particularly appears to be greater in IB4+ DRG neurons as compared with IB4− DRG neurons; 2) activation of BK B2 receptor has a greater inhibitory effect on K_V4 currents in IB4+ muscle DRG neurons of PAD rats; and 3) siRNA knockdown of K_V4.3 channels decreases the activities of K_V4 currents in IB4+ muscle DRG neurons.

K_V4 Channels in Sensory Neurons

When the K_V4 channels are activated, a rapid transmembrane K⁺ efflux generates a transient outward A-type-K⁺ current, named as K_V4 current, which is resistant to TEA but sensitive to 4-aminopyridine (4-AP). K_V4 currents are also involved in the regulation of rest membrane potential (RMP) and action potential (AP) firing, thereby influencing neuronal excitability and signal integration (37). Because of its roles in the neuronal activity regulation in the DRG cells, expression and function of K_V4 channels have been largely investigated in the pain-related animal models. The dysfunction of K_V4 channels in DRG neurons is associated with persistent pain sensitization (38–42). In specific, the expression and distribution of K_V4 were decreased in DRG neurons of animal models such as chronic constriction sciatic nerve injury, vibration-induced muscle pain, and mechanical hypersensitivity (39, 40, 43–45). With approach of the whole cell patch clamp, a robust decrease in K_V4 currents was also observed in nociceptive neurons of rats with streptozotocin- or oxaliplatin-induced neuropathic pain (42, 46). In the previous work, we have shown that K_V4 currents in muscle DRG neurons of PAD rats were inhibited (23).

IB4+ and IB4− Muscle DRG Neurons

Our previous finding suggests that femoral artery occlusion and ischemia-induced-NGF selectively affect a subpopulation of IB4+ and IB4− muscle afferent neurons (33). For example, the femoral artery occlusion increased NGF levels in DRG neurons of rats, and this augmented capsaicin-induced currents of both IB4+ and IB4− muscle DRG neurons. Interestingly, infusion of NGF in the muscles as well as addition of NGF to the culture dish containing muscle DRG neurons increased the magnitude of capsaicin receptor response in IB4− DRG neurons but not in IB4+ DRG neurons (33). The data suggest both IB4+ and IB4− muscle DRG neurons are engaged in neuronal activities following femoral artery occlusion, likely dependent on the abnormalities in ischemia-induced products.

In the current study, our data demonstrated that femoral artery occlusion inhibited the current density of K_V4 to a greater degree in IB4+ muscle DRG neurons, providing evidence that a subpopulation of IB4+ muscle afferent neurons is mainly involved in inhibited activity of K_V4 currents in PAD rats. This further suggests that muscle DRG neurons responsive to muscle metabolites are likely involved in functions of K_V4 for activation of the thin fiber muscle afferent nerves in PAD.

Role of BK B1 and B2 Receptors in Regulating Activity of K_V4 Channels in Muscle DRG Neurons

BK is synthesized from its precursor kininogen via activation of the enzyme kallikrein and produced within the interstitium of the various tissues (47). Two subtypes of BK receptors in the peripheral neurons, namely kinin B1 and B2, are responsive to BK for its functional regulation. During exercise, BK production is increased in active muscles (48–50). In specific, B2 receptor plays a major role in the regulatory process of BK during the EPR response (51). Our previous study also indicates that the B2 receptor expression is increased in DRGs of PAD rats with femoral artery occlusion (52). The application of B2 receptor antagonist (HOE-140) induces a greater inhibition of sympathetic nerve and BP responses to muscle -endon stretch in PAD rats (52). HOE-140 also inhibits the response of group III afferents to contraction (53). It is therefore generally considered that the increased BK in the interstitial space of the ischemic muscle, the enhanced expression, and function of B2 receptor in muscle afferents contribute to the exaggerated EPR in PAD.

BK increases the excitability of sensory neurons by suppressing K_V channels activity via the kinin receptors (54). Similar to the previous finding, data of our current study showed that stimulation of BK B1 and B2 receptors using their respective agonists leads to a greater inhibitory effect on K_V4 currents in IB4+ muscle DRG neurons of control limbs and occluded limbs as compared with IB4− muscle DRG neurons. Notably, BK B2 agonist inhibits K_V4 currents in IB4+ muscle DRG neurons to a larger degree in occluded rats than that in control animals. This result supports the notion that B2 receptor plays a major role in regulating the exaggerated EPR observed in PAD using the whole animal preparations.

Nonetheless, it should be noted that there are general differences in the results of in vitro and in vivo experiments, i.e., endogenous activation of BK pathways during muscle contraction is likely lacking or less in in vivo whole animal experiments. In contrast, in our current patch-clamp experiments, application of BK onto muscle DRG neurons directly stimulates both BK B1 and B2 signaling pathways linked to the activities of K_V4 channels. Consistent with our previous result (23), BK was observed to inhibit K_V4 currents and this appear to be a greater degree in occluded limbs than in control limbs. Additional data of the current study suggest that BK has a greater inhibitory effect on K_V4 currents in IB4+ muscle DRG neurons. To better clarify the effects of B1 and B2 receptors on K_V4 currents, in the present study we applied respective B1 and B2 receptor agonists onto muscle DRG neurons. We found that B2 agonist inhibited K_V4 currents in IB4+ muscle DRG neurons to a greater degree in occluded limbs than that in control limbs. It needs to be mentioned that the inhibition of BK on K_V4 currents in DRG neurons was decreased by R-715, a B1 receptor antagonist, in the previous study (23). This difference is likely due to that previously IB4+ or IB4− was not identified in the recorded muscle DRG neurons.

Contribution of K_V4.1 and K_V4.3 Subunits to Activity of K_V4 Channels in Muscle DRG Neurons

Although K_V4.1 and K_V4.3 subunits appear in the DRG neurons, K_V4.3 has been reported to play a major role in regulating nociceptive sensation (34). Particularly, it is noted that K_V4.3 knockdown in rat spinal cord with antisense oligonucleotides leads to mechanical allodynia and hypersensitivities to vibration (39, 40). In our previous study, the effects of antagonist to K_V4.1 and K_V4.3 subunits on the activities of K_V4 currents were examined (23). Results showed that blocking K_V4.3 subunits using PaTx1 (a specific antagonist to Kv4.3 channels) had a less inhibitory effect on K_V4 currents in DRG neurons of occluded limbs than its effect in DRG neurons of control limbs. In contrast, there were no significant differences observed in inhibitory effects of blocking K_V4.1 subunit using JZX-XII (a specific antagonist to Kv4.1 channels) on K_V4 currents in DRG neurons of control limbs and occluded limbs. This previous result suggests a contribution of the downregulation of K_V4.3 subunit to the decreased K_V4 current activity in muscle DRG neurons of occluded limbs (23).

To minimize the confounding issues caused by nonspecificity of pharmacological agents, in the current study we used approach of siRNA-K_V4.1/siRNA-K_V4.3 knockdown to decrease the expression levels of K_V4.1/K_V4.3 in DRG neurons, and then determined the specific contribution of K_V4.1 and/or K_V4.3 subunit to K_V4 currents. Our data have verified the effectiveness of siRNA-K_V4.1/K_V4.3 knockdown and further showed that siRNA knockdown of K_V4.3 channels decreases activity of K_V4 currents in IB4+ muscle DRG neurons. We provided evidence for distribution of K_V4.1 and K_V4.3 subunits in DRG neurons and their contribution to K_V4 currents, suggesting that the potential cellular mechanisms of K_V4.3 channels are responsible for the role played by K_V4 in PAD. This is consistent with our previous result showing that K_V4.3 has a major contribution to the activities of K_V4 currents (23).

Study Limitation

In the current study, BK inhibited the activity of K_V4 channels in a selective subpopulation of neurons in PAD animals, but the physiological impact of these findings during exercise is still required to explore in the future study. A study using the whole animal preparations to examine the role played by K_V4 channels in regulating the exaggerated EPR in PAD is necessary.

In addition, 4- to 6-wk-old rats were included in the current study to obtain comparable data since animals at this age were used in the previously published work (23). However, it should be noted that data of the EPR using the whole animal study were obtained from 6- to 10-wk-old rats. An additional limitation of this study is that only male animals were included because sex discrepancies were beyond the scope of our current study. To address the issue of sex discrepancies requires further studies.

In conclusion, our data suggest that femoral artery occlusion induced-limb ischemia and/or ischemia induced-metabolites (i.e., BK) inhibit the activity of K_V4 channels in a selective subpopulation of the thin fiber muscle afferent neurons expressing receptors for GDNF, which is likely a part of sensory signaling pathways involved in the exaggerated EPR in PAD.

Perspectives and Significance

As a metabolic product in ischemic limb muscles of PAD, BK exaggerates autonomic responses to activation of muscle afferent nerves during exercise. Electrophysiological data of the current study showed that BK inhibits the activity of K_V4 current in a subpopulation of muscle afferent neurons depending on GDNF and this effect particularly appears to be a greater degree in PAD rats. As K_V4 channels have a major contribution to the excitability of muscle sensory nerves, it is speculated that GDNF signaling pathways play an important role in regulating the exaggerated blood pressure response during activation of muscle afferent nerves in PAD. Thus, interventions affecting BK-Kv4 channel activity in the process of GDNF signaling have significant implications for alleviating the exaggerated autonomic responsiveness in PAD.

GRANTS

This study was supported by the National Institutes of Health P01 HL134609, R01 HL141198, and R01 HL164571 (to J. Li), American Heart Association Career Development Award Grant 940567 (to L. Qin), and Penn State College of Medicine Departmental DOM Innovation and Inspiration Award INNOVQLI Fall2021 (to Q. Li).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.L. conceived and designed research; Q.L. and L.Q. performed experiments; Q.L., L.Q., and J.L. analyzed data; Q.L., L.Q., and J.L. interpreted results of experiments; Q.L. and L.Q. prepared figures; Q.L., L.Q., and J.L. drafted manuscript; Q.L., L.Q., and J.L. edited and revised manuscript; Q.L., L.Q., and J.L. approved final version of manuscript.