Chronic ablation of TRPV1-sensitive skeletal muscle afferents attenuates the muscle metaboreflex

Abstract

Exercise intolerance is a hallmark symptom of cardiovascular disease and likely occurs via enhanced activation of muscle metaboreflex-induced vasoconstriction of the heart and active skeletal muscle which, thereby limits cardiac output and peripheral blood flow. Muscle metaboreflex vasoconstrictor responses occur via activation of metabolite-sensitive afferent fibers located in ischemic active skeletal muscle, some of which express transient receptor potential vanilloid 1 (TRPV1) cation channels. Local cardiac and intrathecal administration of an ultrapotent noncompetitive, dominant negative agonist resiniferatoxin (RTX) can ablate these TRPV1-sensitive afferents. This technique has been used to attenuate cardiac sympathetic afferents and nociceptive pain. We investigated whether intrathecal administration (L4–L6) of RTX (2 µg/kg) could chronically attenuate subsequent muscle metaboreflex responses elicited by reductions in hindlimb blood flow during mild exercise (3.2 km/h) in chronically instrumented conscious canines. RTX significantly attenuated metaboreflex-induced increases in mean arterial pressure (27 ± 5.0 mmHg vs. 6 ± 8.2 mmHg), cardiac output (1.40 ± 0.2 L/min vs. 0.28 ± 0.1 L/min), and stroke work (2.27 ± 0.2 L·mmHg vs. 1.01 ± 0.2 L·mmHg). Effects were maintained until 78 ± 14 days post-RTX at which point the efficacy of RTX injection was tested by intra-arterial administration of capsaicin (20 µg/kg). A significant reduction in the mean arterial pressure response (+45.7 ± 6.5 mmHg pre-RTX vs. +19.7 ± 3.1 mmHg post-RTX) was observed. We conclude that intrathecal administration of RTX can chronically attenuate the muscle metaboreflex and could potentially alleviate enhanced sympatho-activation observed in cardiovascular disease states.

INTRODUCTION

Whole body dynamic exercise presents one of the greatest challenges to cardiovascular control as heart rate and cardiac output rise from resting values to maximal levels during peak workloads (1–13). Blood flow to inactive vascular beds is reduced in many species and vasodilation in the active skeletal muscle and even the coronary circulation is restrained by the substantial increases in sympathetic activity (14–17). These marked changes in autonomic activity occur through the action and likely interaction between activation of central command, resetting of the arterial baroreflex, and activation of skeletal muscle afferents. In cardiovascular dysfunction, these challenges often become exacerbated as systemic perfusion and cardiac function may be compromised resulting in massive sympathetic activation. One potential mechanism mediating the enhanced sympathetic activity during exercise in cardiovascular disease is overactivation of skeletal muscle afferents, in particular those activated via accumulation of metabolites due to suboptimal oxygen delivery—termed as the muscle metaboreflex (18–33). Even in normal subjects this reflex is thought to become tonically active at relatively modest workloads and activation of this reflex can cause substantial increases in sympathetic activity, which elicits increases in cardiac output via tachycardia, increased ventricular contractility, and central blood volume mobilization (5–7, 9–13, 34–41). Arterial elastance rises in parallel with ventricular maximal elastance, thereby optimizing ventricular-vascular coupling and energy transfer from the left ventricle to the systemic circulation (11, 42, 43). Peripheral vasoconstriction can also occur which is countered by adrenal epinephrine release and β₂-mediated vasodilation (16, 34, 38, 40). Normally, via the large increases in total blood flow (e.g., cardiac output) coupled with increased O₂-carrying capacity of the blood (via splanchnic vasoconstriction causing red blood cell mobilization), the muscle metaboreflex acts to correct deficits in blood flow and O₂ delivery to the active muscle (7, 29, 44). In pathophysiological states, even during moderate workloads this reflex likely becomes markedly overactivated and often profound vasoconstriction occurs in inactive vascular beds and even the ischemic active muscle and heart are vasoconstricted (15, 17, 41, 45–49). This exacerbates an already precarious situation leading to a positive feedback amplification of sympathetic activity (16, 17). The potentially extreme levels of sympathetic activity may pose increased risks for adverse cardiovascular events such as stroke, myocardial infarction, ventricular arrhythmia, and sudden cardiac death. Thus, mechanisms to ameliorate overactivation of skeletal muscle afferents during exercise in patients with cardiovascular disease may be beneficial.

Skeletal muscle metabo-sensitive afferents respond to several substances via stimulation of a variety of receptors. These receptors include TRPV1 channels which are nonselective cation channels known to be stimulated by a variety of stimuli such as temperature, and H⁺ ion concentration (18, 50–53). Recent studies have investigated the impact of stimulating or antagonizing TRPV1-expressing afferents using capsaicin, capsazepine, their analogs, and other various pharmaceutical compounds (33, 54–61). These studies found that activation of TRPV1 receptors has been linked to not only initiation of blood pressure responses but also the sensation of nociceptive pain (56, 57, 62–68). Short-term attenuation of groups III and IV afferents including those expressing TRPV1, during exercise, decreases the sensation of muscle fatigue and the pressor responses during exercise via activation of μ opioid channels. (69–71). To what extent TRPV1 receptors are stimulated during exercise and mediate the muscle metaboreflex responses is controversial (23, 33, 57, 58, 72–75). However, group IV afferents contain TRPV1 receptors (76), and thus this can be used as a tool to alter their activity. Our goal was to assess if metaboreflex responses can be chronically attenuated in a conscious model using the availability of TRPV1 receptors on metabo-sensitive afferents as a tool for ablation. We hypothesized that chronic ablation of TRPV1-expressing afferent neurons in the hindlimbs could be used to chronically reduce the strength or gain of the muscle metaboreflex. In a longitudinally designed study, we first established the strength of the muscle metaboreflex in chronically instrumented canines during mild exercise. We then used an ultrapotent-dominant negative agonist of capsaicin, resiniferatoxin (RTX) injected into the intrathecal space to partially destroy afferents expressing TRPV1 receptors. We repeated the experiments from 7 to 78 ± 14 days after injection and found that the strength of the muscle metaboreflex was chronically attenuated by over 50%. We conclude that this approach could be useful to attenuate exaggerated sympathetic activation during exercise in patients with cardiovascular disease.

METHODS

Experimental Subjects

Six adult mongrel canines (1 male, 5 females) of ∼19–25 kg were selected based on their willingness to walk on a motor-driven treadmill. We have previously shown that sex does not affect the strength or mechanisms of muscle metaboreflex activation (77). Canines were acclimatized to exercise at a workload of 3.2 km/h with a 0% grade. All experimental and surgical procedures used for this study comply with the National Institutes of Health Guide to the Care and Use of Laboratory Animals and were approved by the Wayne State University Institutional Animal Care and Use Committee (IACUC). Animals used in this study underwent a 14-day acclimation period with the laboratory surroundings and personnel and exercised on their own volition during all experiments.

Surgical Instrumentation

All animals underwent a series of surgical procedures over a period of 4 wk using standard aseptic surgical techniques. Before each surgery, animals were sedated with an intramuscular injection of acepromazine (0.4–0.5 mg/kg) and administered a subcutaneous injection of slow-release analgesic buprenorphine SR (0.03 mg/kg) 30 min before induction of anesthesia. Induction was achieved by intravenous administration of ketamine (5 mg/kg) and diazepam (0.2–0.3 mg/kg). Anesthesia was maintained pre and intra-operatively with (1%–3%) isoflurane gas. Additional preoperative analgesic included intravenous administration of carprofen (4.4 mg/kg). Postoperatively acepromazine (0.2–0.3 mg/kg iv) and buprenorphine (0.01–0.03 mg/kg im) were administered as needed. Acute proactive antibiotic (cephalexin 30 mg/kg iv) was administered pre- and postoperatively. Prophylactic antibiotic (cephalexin 30 mg/kg orally twice a day) were administered to prevent microbial infection.

The first surgical procedure was a left thoracotomy through the left 3rd/4th intercostal space to expose the heart. The pericardium was incised and parted for placement of a telemetric pressure transducer tip (TA11 PA-D70, DSI) into the apex of the left ventricle that was tunneled from the telemeter body tethered subcutaneously caudal to the thoracotomy incision site. The upper section of pericardium was then retracted dorsally to expose the aorta and pulmonary vein. A section of the aortic trunk was used for placement of a blood flow transducer (20 PAU, Transonic Systems) for measure of cardiac output. Unrelated to the investigations of this study, four stainless steel pacing leads (0-FLexon, Ethicon) were attached to the right ventricle free wall. All leads and cables were tunneled subcutaneously and exteriorized between the scapulae. The pericardium and ribs were reapproximated and the chest was closed in layers.

The final surgical procedure occurred at a minimum of 14 days postthoracotomy. A left retroperitoneal approach was used to access the terminal aorta. All arterial blood vessels caudal to the renal artery and cranial to the edge of the incision above the iliac crest were ligated. The most accessible cranial arterial branch caudal to the renal artery was catheterized with a 19-gauge polyvinyl catheter (Tygon, S54-HL, Norton) for measure of arterial pressure. In order, cranial to caudal, placed caudal to the arterial catheter, a blood flow transducer (10 PAU, Transonic Systems) and two 8–10 mm hydraulic occluders (DocXS Biomedical Products) were placed to measure and manipulate hindlimb blood flow. All cables and occluder lines were tunneled subcutaneously and exteriorized between the scapulae.

Intrathecal RTX Administration and Terminal Capsaicin Response

Administration of RTX was performed after completion of all control experiments. Before this procedure animals were sedated, induced, anesthetically maintained, and treated with the same analgesics and aseptic techniques as described in Surgical Instrumentation. Flow probes and pressure transducers were connected. Heart rate and mean arterial pressure responses were taken during a 1-min steady state before administration of each pharmaceutical agent, and peak responses postadministration for analysis. Before placement of the spinal needle, intra-arterial capsaicin (20 µg/kg) was administered through the catheter placed in the terminal aorta. After recovery from capsaicin administration a 20- to 22-gauge spinal needle was placed into the intrathecal space between the L4–L5 and L5–L6 vertebrae. Placement of the spinal needle was done based on animal positioning, alignment, and successful observation of cerebrospinal fluid through the needle. Spinal fluid equal to the volume amount of intrathecal RTX administered to reach the desired dose volume (2 µg/kg) plus 0.5 mL to use as a flush was removed. RTX was then administered through the spinal needle at a concentration of 2 µg/kg followed by flush of 0.5 mL spinal fluid and removal of the spinal needle. After ∼30–60 min and recovery from responses to RTX, a second intra-arterial injection of capsaicin (20 µg/kg) was performed. Animals were monitored postoperatively and administered acepromazine (0.2–0.3 mg/kg iv) and buprenorphine (0.01–0.03 mg/kg im) as needed. Metaboreflex experiments were repeated after a 7- to 10-day recovery period. After completion of all experiments a final terminal anesthetic procedure was performed to assess the long-term efficacy of the RTX injection. Animals were sedated, induced, treated with analgesics, and anesthetically maintained as described above in the surgical procedures. All equipment was connected and steady-state values of in vivo hemodynamics were taken during a 1-min steady-state before intra-arterial administration of capsaicin (20 µg/kg).

Data Acquisition

Animals were acclimated 10–20 min in the laboratory setting and then led to the treadmill and equipment. Arterial pressure was measured by connection of the 19-gauge polyvinyl catheter (Tygon, S54-HL, Norton) to a pressure transducer (Transpac iv, ICU Medical). Left ventricular pressure telemeter (DSI) and blood flow transducers were connected to their appropriate instrumentation. All flows and pressures were monitored and recorded in real time through an A/D converter (IWorx). A/D output was run through Labscribe4 acquisition and analysis software (IWorx).

Experimental Procedures

All hemodynamic parameters were measured under steady-state conditions during rest, mild free flow exercise (3.2 km/h 0% grade), and sustained mild free flow exercise with successive reductions in hindlimb blood flow used to activate the muscle metaboreflex. Hydraulic vascular occluders (DocXS) were used to achieve reductions in hindlimb blood flow to ∼30% of free flow conditions during exercise for both control and RTX-treated animals. Experiments were repeated after RTX, thus each animal served as its own control in this longitudinally designed study.

Analysis

All in vivo hemodynamic parameters were continuously recorded during each experiment. One-minute averaged steady-state values were taken after a 3-min acclimation period at rest, free flow exercise, and during steady-state at each reduction of hindlimb blood flow (HLBF). Measures of mean arterial pressure, cardiac output, and hindlimb blood flow were measured in vivo. Heart rate was derived from the cardiac output waveform. All other hemodynamic parameters were mathematically derived from in vivo measurements.

Statistical Analysis

Systat Software (Systat 13.0) was used for statistical analysis. Data are reported as means ± SE and statistical significance was determined by an α level of P < 0.05. Data were analyzed using a two-way ANOVA for repeated measures, and when a significant interaction was observed, individual means were compared using C-matrix test for simple effects. Direct observations on changes in muscle metaboreflex gain, and comparison in changes observed during surgical procedures were assessed by a Student’s paired t test.

RESULTS

Figure 1A shows the pressor responses to intra-arterial capsaicin (20 µg/kg) before and ∼30 min post-RTX injection. RTX markedly attenuated the pressor and tachycardic responses to capsaicin. Figure 1B shows the responses to intra-arterial capsaicin performed in a terminal procedure 78 ± 14 days after intrathecal administration of RTX. Compared to the responses observed before RTX, the pressor and tachycardic responses to capsaicin were significantly attenuated by ∼50%.

Figure 1.

A: intraoperative responses to intra-arterial administration of capsaicin (20 µg/kg) while under isoflurane anesthesia. Pre-RTX (gray) and post-RTX (red) are the Δ responses comparing a 1-min steady-state before capsaicin administration to the maximal response for each variable after administration. B: intraoperative responses to intra-arterial capsaicin (20 µg/kg) before administration of RTX and during a terminal experiment performed 78 ± 14 days postinjection of RTX. Bar graphs show standard error of the mean with individual data points overlain. Standard error shown on bar graphs with actual data points overlain. †Statistical significance is shown compared with previous condition (P < 0.05, Student’s paired t test), n = 6 animals, RTX, resiniferatoxin.

Figure 2 shows that in control experiments, heart rate (HR), stroke volume (SV), cardiac output (CO), and stroke work (SW) increased significantly from rest to exercise, whereas there were no significant changes in mean arterial pressure (MAP) or effective arterial elastance [E_aZ, an index of vascular load (42, 78)] likely as a result of the significant increases in nonischemic vascular conductance (NIVC, vascular conductance to all vascular beds except the hindlimbs calculated as (CO-HLBF)/MAP). During muscle metaboreflex activation HR, SV, CO, SW, E_aZ, and MAP, all increased significantly with no significant increases in NIVC when compared to the free flow exercise condition. Intrathecal RTX injection had no effect on MAP, HR, SV, CO, NIVC, or E_aZ at rest and during steady-state exercise. The significant increase in stroke work from rest to steady state exercise was lost. However, after RTX muscle metaboreflex responses were virtually abolished: only SW showed a small but statistically significant increases in response to similar reductions in HLBF.

Figure 2.

One-minute averaged hemodynamic responses taken during steady states at rest, exercise (3.2 km/h, 0% grade), and exercise with muscle metaboreflex activation before (gray) and after administration of intrathecal RTX (2 µg/kg) (L4–L6) (red). Bar graphs show means ± SE with individual data points overlain. Standard error shown on bar graphs with actual data points overlain. *Statistical significance (P < 0.05) compared with previous workload. †Significance to previous condition (P < 0.05, Two way ANOVA), n = 6 animals, Ex, exercise; MMA, muscle metaboreflex activation; RTX, resiniferatoxin.

Figure 3 shows a comparison of changes observed in the hemodynamic values between steady-state mild exercise (3.2 km/h 0% grade) and steady-state maximal muscle metaboreflex activation. Increases in HR and SV were significantly attenuated which thereby lowered CO after intrathecal administration of RTX. The significant reduction in the increase of MAP after intrathecal RTX is primarily a result of the lack of CO response and a reduction in stroke work. The rise in E_aZ was not significantly different before or after administration of RTX.

Figure 3.

Average change between 1-min steady-state values taken during exercise (3.2 km/h, 0% grade) and during exercise with muscle metaboreflex activation before (gray) and after administration of intrathecal RTX (2 µg/kg) (L4–L6) (red). Standard error shown on bar graphs with actual data points overlain. †Statistical significance (P < 0.05, Student’s paired t test) compared with previous condition (n = 6 animals). RTX, resiniferatoxin.

Figure 4 is a comparison of the reflex gain defined as the slope of the regression line from the threshold of reflex to peak muscle metaboreflex activation of between MAP, CO, and HR versus hindlimb blood flow before and after RTX. The slopes of MAP, CO, and HR were significantly attenuated after treatment with RTX. The slope of CO relationship showed the largest reduction by ∼70% compared with reductions of MAP and HR of ∼50%.

Figure 4.

Assessment of changes in the gain of the muscle metaboreflex determined by the slope of the linear regression taken from 1-min averaged steady states at each successive reduction in hindlimb blood flow from threshold to peak reflex activation before (black circles, gray bars) and after administration of RTX (red circles, red bars). Line graphs should be observed from the right point (exercise) to the middle point (reflex threshold) to the final point left (peak muscle metaboreflex activation) as hindlimb blood flow is reduced. Standard error shown on bar graphs with actual data points overlain. †Statistical significance between control (gray) and after administration of intrathecal RTX (2 µg/kg) (L4–L6) (red) (P < 0.05, Student’s paired t test), n = 6 animals. RTX, resiniferatoxin.

DISCUSSION

This is the first study to demonstrate that the cardiovascular responses to muscle metaboreflex activation evoked during volitional treadmill exercise in conscious animals, can be chronically attenuated by ablation of TRPV1-sensitive afferents via intrathecal administration of RTX. We determined that intrathecal RTX attenuates the muscle metaboreflex primarily through significant reductions in heart rate and stroke volume and attenuation of ventricular performance as indexed by stroke work. These reduced responses lower the reflex increase in cardiac output. Inasmuch as in normal subjects during submaximal dynamic exercise, the increase in cardiac output is the major mechanism mediating the rise in arterial pressure, with the reduced rise in cardiac output the metaboreflex pressor response was significantly attenuated.

Traditionally, the muscle metaboreflex increases cardiac output via the combined effects of tachycardia, increased ventricular contractility, and enhanced central blood volume mobilization (10, 11, 34, 39). This reflex arises due to the activation of metabo-sensitive skeletal muscle afferents (19, 23, 24, 26, 27, 32, 79). These afferents are primarily group IV, but some group III afferents, which primarily respond to mechanical stimulation, are also metabo-sensitive (23, 24, 33, 58, 76, 80). These metabo-sensitive afferents respond to TRPV1 agonists (33, 50, 56, 76, 81). Although, these afferents respond to TRPV1 receptor activation, there is controversy regarding whether these receptors mediate the muscle metaboreflex under physiological conditions. Studies in unanesthetized, acutely decerebrated rats (58, 72, 74, 75) and cats (73) concluded that TRPV1 receptors are not responsible for activation of the muscle metaboreflex. Most of the evidence in these studies is centered around the nonphysiological conditions required to activate TRPV1, limited activators, and the inability of TRPV1 inhibition to prevent reflex responses to static muscle contraction and skeletal muscle circulatory occlusion (58, 72, 73, 75). Alternatively, others have reported that muscle metaboreflex responses can be attenuated by antagonism of TRPV1 receptors (33, 57). TRPV1 receptor sensitivity can vary across species (52) and substances related to tissue injury (82–84) can interact with TRPV1 receptors and possibly reduce activation thresholds (85). Therefore, there is potential for TRPV1 activation to elicit or enhance pressor responses via an altered inflammatory state such as heart failure and peripheral arterial disease. In chronic heart failure, TRPV1 expression is downregulated in the skeletal muscle (86), and this may be a result of overactivation due to tissue injury as a result of hypoperfusion, although no study to date has addressed this aspect of TRPV1 receptors. Our study neither supports nor refutes the possible implications of TRPV1 activation eliciting the muscle metaboreflex. We used the ability to selectively ablate capsaicin-sensitive skeletal muscle afferents that express ASIC, P2X, and EP receptors that are known modulators of the muscle metaboreflex (21, 81, 87–91). These receptors are coexpressed with the TRPV1 receptor and thus are also eliminated with the use of resiniferatoxin. Thus, the existence of TRPV1 receptors on group IV afferents served as a convenient tool to ablate these neurons which contain a variety of other receptors likely involved in triggering metaboreflex responses.

In the present study, we used a noncompetitive agonist to ablate TRPV1-sensitive afferent fibers in the hindlimb skeletal muscle. Intrathecal RTX itself usually caused a pressor response likely due to irreversible activation of the TRPV1 receptors. Thirty minutes to one hour later, the reflex responses to intra-arterial capsaicin were significantly attenuated. Metaboreflex experiments were performed 7 to 78 ± 14 days after RTX and the responses were consistently reduced. In the terminal experiment, the responses to intra-arterial capsaicin was reduced by ∼50%, indicating sustained significant ablation of afferents containing TRPV1 receptors after 78 ± 14 days. The lack of complete ablation is likely a result of multiple factors such as toxin volume delivered, intrathecal flow, and the levels of TRPV1 receptors available per neuron to accept RTX and achieve the level of cation flow required for cytotoxicity. Furthermore, expression patterns of TRPV1 neurons likely vary per neuron and their upregulation and downregulation from various stimuli may play a role in injection efficacy. Wang et al. (86) showed that TRPV1 protein levels can vary based on pathological state possibly acting as a compensatory mechanism in response to enhanced levels of metabolites in muscle with reduced blood supply.

Peripheral vascular responses were altered post-RTX. We observed a significant reduction in nonischemic vascular resistance during muscle metaboreflex activation likely as a result of reduced β₂-mediated vasodilation observed in previous studies (16,17, 38). We have shown that muscle metaboreflex activation enhances ventricular maximal elastance and effective arterial elastance such that ventricular-vascular coupling is maintained, and stroke work is optimal (11, 42). In this study, intrathecal RTX significantly attenuated increases in stroke work with no significant changes in effective arterial elastance. Reductions in stroke work is probable evidence of some degree of ventricular-vascular uncoupling, likely a result of reductions in ventricular maximal elastance during muscle metaboreflex activation post-RTX. In addition to overall reductions in hemodynamic responses to graded reductions in hindlimb blood flow, we also observed significant reductions in the gain of the muscle metaboreflex. Calculation of muscle metaboreflex gain (strength) is quantified via the slope of the regression line between a given hemodynamic parameter versus the reduction in hindlimb blood flow once beyond metaboreflex threshold. We observed that not only are maximum hemodynamic values attenuated but also the slope of mean arterial pressure, heart rate, and cardiac output relationships versus hindlimb blood flow are significantly reduced with no change in threshold, indicating that reflex gain is reduced for all observed hemodynamic parameters. These experiments were performed only during mild exercise, and to what extent metaboreflex responses after RTX are different at higher workloads remains to be investigated.

Limitations

This study used resiniferatoxin and the availability of TRPV1 receptors on skeletal muscle afferents to achieve a chronic ablation. We did not address the role of TRPV1 receptors in mediating the muscle metaboreflex. Furthermore, no specific compounds traditionally used to assess metaboreflex activation in cats and rats such as lactic acid, adenosine, or potassium were used to assess any residual metaboreflex capabilities with or without intact TRPV1-expressing afferents.

We did not quantify exercise capacity or tolerance before or after administration of RTX. The animals exercised voluntarily and received no negative stimuli to reinforce exercise. Anecdotally, we did observe what appeared to be improved exercise performance during occlusions after administration of RTX. Amann et al. (69, 92–94) concluded that anesthetization of skeletal muscle afferents via intrathecal fentanyl infusion in humans lessened the sensation of fatigue during leg exercise, which could improve exercise tolerance.

In this study, we were unable to directly address if ablation of group IV afferents causes enhanced group III mechanosensitive afferent activation as was concluded by Smith et al. (58). However, RTX did not affect the normal cardiovascular responses to steady-state mild exercise before metaboreflex activation indicating that there was no enhanced activation of skeletal muscle mechano-receptors at this workload. We observed that the muscle metaboreflex was significantly reduced after application of RTX; however, there was not a complete loss of the response. This could be a result of group III afferent compensation, or more likely a result of still intact group IV afferents due to inefficient toxin delivery, lack of TRPV1 quantity and availability to interact with RTX and induce neuronal toxicity, or lastly compensation from alternative cardiovascular reflexes such as the baroreflex.

No morphological analyses of dorsal root ganglia were conducted during this study to show any potential off-target sensory neuron ablation. Sensory afferent function was conducted after ablation by veterinary staff to address any loss of proprioception in the hindlimb and none was noted. Morphological analyses have been conducted using this technique and found that RTX ablates dorsal root ganglia in a canine model of bone cancer (95, 96) and no off-target effects were noted in those studies. We are confident that our functional assessments (responses to capsaicin infusion and metaboreflex activation) strongly support the conclusions that RTX administration partially ablated group IV afferents which mediate the muscle metaboreflex.

Perspectives and Significance

Several cardiovascular diseases (e.g., heart failure, hypertension, and peripheral vascular disease) alter the strength and mechanisms of the muscle metaboreflex (15, 34, 43, 46–49). Impaired ventricular function can occur not only from the direct effect of the disease, but also from enhanced reflex coronary vasoconstriction (15, 48), which thereby contributes to the attenuated ability to raise cardiac output and thus correct metabolic mismatches in ischemic active skeletal muscle. Not only the heart, but also the ischemic muscle becomes a target for metaboreflex-induced vasoconstriction (17). Inability to improve perfusion of ischemic active skeletal muscle in heart failure causes enhanced sympathetic activation due to overactivation of the muscle metaboreflex (15, 17). In heart failure, ventricular-vascular interaction is already uncoupled and enhanced sympathetic outflow further decouples this relationship, which thereby lessens the ability to sustain or improve skeletal muscle blood flow (42). Impaired ventricular function and ventricular-vascular interactions leads to an enhanced vasoconstriction even within the ischemic muscle (17). Limitations in raising skeletal muscle blood flow are thus both the cause and effect of an enhanced metabolic mismatch that causes the enhanced sympathetic activation in heart failure and likely other cardiovascular pathologies such as hypertension (48, 49, 70, 97, 98). To date, no study has assessed the effect of chronic chemical ablation of skeletal muscle afferents in cardiovascular disease during exercise. A common symptom of these pathologies is exercise intolerance and fatigue, such that quality of life and the will to exercise are reduced. Studies have shown that exercise training regimens are capable of improving, and in some cases, correcting abnormal cardiovascular responses during exercise (62, 99–106). Furthermore, multiple studies have shown that attenuation of skeletal muscle afferents by way of activation of μ opioid receptors located on lower body afferents improves cardiovascular responses during exercise and reduces fatigue (69, 70, 79, 92, 107). In the present study, we observed that a reduction of skeletal muscle afferents via intrathecal RTX was capable of significantly attenuating muscle metaboreflex responses during exercise. In addition, anecdotally we observed animals treated with RTX could maintain workload performance with greater reduction in hindlimb blood flow. Direct experimental assessments of fatigue and performance were not measured and need to be addressed in future studies involving RTX to address if treatment may also lessen the sensation of fatigue during exercise as were observed in studies utilizing intrathecal opioids (57, 68–70, 79, 90–94). Thus, chronic ablation of skeletal muscle afferents may allow greater exercise-induced increases in cardiovascular fitness, less exercise intolerance, and improved overall quality of life for patients with various cardiovascular pathologies.

GRANTS

This work was supported by the National Heart, Lung, and Blood Institute Grants HL-55473, HL-126706, and HL-120822, and in part supported by NIGMS/NIH R25-GM058905.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

J.M. and D.S.O'L. conceived and designed research; J.M., M.-H.A., B.L., A.A., D.S., and D.S.O'L. performed experiments; J.M., M.-H.A., B.L., A.A., D.S., and D.S.O'L. analyzed data; J.M. and D.S.O'L. interpreted results of experiments; J.M. and D.S.O'L. prepared figures; J.M. and D.S.O'L. drafted manuscript; J.M. and D.S.O'L. edited and revised manuscript; J.M. and D.S.O'L. approved final version of manuscript.