chronic heart failure
dorsal root ganglion
exercise pressor reflex
left ventricular end-diastolic pressure
left ventricular systolic pressure
mean arterial pressure
An exaggerated exercise pressor reflex (EPR) contributes to exercise intolerance and excessive sympatho-excitation in the chronic heart failure (CHF) state. However, the components of this reflex that are responsible for the exaggerated EPR in CHF remain unknown. To determine whether muscle afferent function is altered in CHF, we recorded the discharge of group III and IV afferents in response to static contraction, passive stretch and hindlimb intra-arterial injection of capsaicin in sham and CHF rats. We also investigated the roles of purinergic 2X receptor (P2X) and the transient receptor potential vanilloid 1 (VR1) in mediating the altered sensitivity of muscle afferents. Compared with sham rats, CHF rats exhibited greater responses of group III afferents to contraction and stretch whereas the responses of group IV afferents to contraction and capsaicin were blunted. Hindlimb intra-arterial infusion of pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), a P2X antagonist, attenuated the responses of group III afferents to contraction and stretch in CHF rats to a greater extent than in sham rats. Western blot data showed that P2X3 receptors were significantly upregulated in doral root ganglion (DRG) of CHF rats whereas VR1 receptors were significantly downregulated. Immunohistochemical evidence showed that immunostaining of the P2X3 receptors was more intense in both IB4-positive (C-fibre) and NF200-positive (A-fibre) neurons in DRG of CHF rats whereas the immunostaining of the VR1 receptors was decreased in IB4-positive neurons. These data suggest that group III afferents are sensitized whereas group IV afferents are desensitized in CHF, which is related to the dysfunction of P2X and VR1 receptors.
Static contraction of skeletal muscle reflexively activates the sympathetic nervous system and increases arterial pressure and heart rate. Two distinct neural control mechanisms have been postulated to explain the increases in cardiovascular function during exercise: central command and the exercise pressor reflex (EPR) (Sinoway & Li, 2005; Smith et al. 2006). Central command is a mechanism whereby signals from a central site responsible for recruiting motor units activate cardiovascular control areas in the brain stem (Goodwin et al. 1972; Eldridge et al. 1985). The EPR is a peripheral neural reflex originating in skeletal muscle which contributes to the regulation of the cardiovascular system during exercise. Substantial evidence has been accrued to show that stimulation of group III (predominately mechanically sensitive) and group IV (predominately metabolically sensitive) afferents, but not group I and II afferents, is responsible for the activation of the EPR (McCloskey & Mitchell, 1972; Perez-Gonzalez & Coote, 1972; Kaufman et al. 1983).
In some diseases such as chronic heart failure (CHF), the EPR is activated to cause exercise intolerance and excessive sympatho-excitation during exercise (Middlekauff et al. 2000; Smith et al. 2003; Koba et al. 2008; Piepoli et al. 2008). However, the mechanisms underlying the exaggerated EPR in the CHF state remain to be determined. For instance, it is not yet clear which components of the muscle reflex arch mediate the exaggerated EPR in the CHF state. Considering that peripheral skeletal myopathy develops in CHF including muscle atrophy, decreased peripheral blood flow, fibre-type transformation and reduced oxidative capacity (Sullivan et al. 1990; Sullivan et al. 1991; Drexler et al. 1992; Mancini et al. 1992; Toth et al. 1997; Vescovo et al. 1998; Duscha et al. 1999), muscle afferent function may be affected by the abnormal skeletal muscle milieu likely to be present in CHF. However, this hypothesis has not been verified by direct evidence from muscle afferent recording experiments. In addition, the role of mechanical or metabolic components in the evolution of the exaggerated EPR in the CHF state remains controversial. On one hand, evidence from experimental and human studies (Middlekauff et al. 2000; Smith et al. 2003; Li et al. 2004; Smith et al. 2005a) suggest that the mechanoreceptor reflex is enhanced, which predominantly contributes to the exaggerated EPR in the CHF state whereas the metaboreceptor reflex is blunted (Sterns et al. 1991; Li et al. 2004; Smith et al. 2005b). In contrast, evidence from other studies suggests that the metaboreceptor reflex but not the mechanoreceptor reflex is enhanced, which is responsible for the exaggerated EPR in the CHF state (Piepoli & Coats, 2007; Piepoli et al. 2008; Jankowska et al. 2009; Li et al. 2009). The reason for this discrepancy is not clear. However, direct electrophysiolgical recording from group III and IV afferents in CHF animals is necessary in order to address this issue. Based on these considerations, in the present study, we took advantage of the technique of single fibre recording to test the hypotheses that (1) the sensitivity of muscle afferents is altered in the CHF state and (2) group III afferents are sensitized whereas group IV afferents are blunted in the CHF state.
Furthermore, in order to determine the underlying mechanisms by which the sensitivity of muscle afferents is altered, we evaluated the roles of purinergic 2X receptors (P2X) and capsaicin-sensitive receptors (transient receptor potential vanilloid 1, TRPV or VR1) in mediating the altered sensitivity of group III and IV afferents in rats with CHF. P2X has been reported to mediate the sensitization of group III afferents in the normal state (Kindig et al. 2006, 2007). However, there is no direct evidence showing that P2X receptors also mediate the abnormal sensitization of group III afferents in the CHF state. On the other hand, VR1 receptors are primarily localized to metabolically sensitive afferent fibres (group IV) in skeletal muscle (Smith et al. 2005b, 2010). In the present study, we further determined whether these receptors are responsible for the altered sensitivity of group III and IV afferents in CHF.
Experiments were performed on male Sprague–Dawley rats weighing 360–450 g. These experiments were approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center and carried out under the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Model of CHF
Heart failure was produced by coronary artery ligation as previously described (Wang et al. 2008, 2010). Briefly, rats were ventilated at a rate of 60 breaths min−1 with 2–3% isoflurane during the surgical procedure. A left thoracotomy was performed through the fifth intercostal space, the pericardium was opened, the heart was exteriorized, and the left anterior descending coronary artery was ligated. Sham-operated rats were prepared in the same manner but did not undergo coronary artery ligation. All the rats survived from the sham surgery and ∼70% of rats from the coronary artery ligation surgery.
In this study, the cardiac function in all experimental animals was measured by echocardiography as previously described (Wang et al. 2008, 2010). In addition, at the beginning of acute experiments, a Millar catheter (SPR 524; size, 3.5-Fr; Millar Instruments, Houston, TX, USA) was advanced through the right carotid artery into the left ventricle (LV) to determine LV end-diastolic pressure (LVEDP) and LV systolic pressure (LVSP). The transducer was then pulled back into the aorta and left in place to record blood pressure. At the end of the acute experiment, the rats were killed with an overdose of pentobarbital sodium. The hearts and lungs were removed, and the ratio of the infarct area to LV was measured.
Rats were initially anaesthestized with 5% isoflourane and maintained on 2–3% isoflourane in oxygen. A jugular vein and the trachea were cannulated. As indicated above, the right carotid artery was catheterized for measurement of mean arterial pressure (MAP) and heart rate (HR). Body temperature was maintained between 37°C and 38°C by a heating pad. A catheter was placed in the right iliac artery with its tip advanced to the abdominal aortic bifurcation, ensuring that the drugs were delivered to the left hindlimb through the left iliac artery without interrupting flow. Muscle afferent function was compared before and after administration of drugs.
The decerebration procedure was performed as described by us and others (Smith et al. 2001; Wang et al. 2009, 2010). Briefly, rats were placed in a stereotaxic apparatus (Stoelting Co., Chicago, IL, USA) and customized spinal frame. The head and pelvis were stabilized. Before decerebration, the lungs were ventilated with an isoflourane–oxygen mixture. Dexamethasone (0.2 mg i.v.) was given to reduce brain oedema and inflammatory responses from the decerebration. The remaining intact carotid artery was isolated and ligated to reduce bleeding during decerebration. Subsequently, a portion of bone superior to the central sagittal sinus was removed. The dura mater was breached and reflected. The cerebral cortex was gently aspirated to visualize the superior and inferior colliculi. Using a blunt instrument, the brain was perpendicularly sectioned pre-collicularly and the transected forebrain aspirated. The cranial vault was filled with warm agar (37°C). After the decerebration had been completed, the lungs were ventilated with a mixture of room air and oxygen instead of the anaesthetic gas. A minimum recovery period of 1.25 h was employed post-decerebration before data collection began.
Recording of GROUP III and group IV afferent impulse activity
In order to directly address the issue of afferent sensitivity in the CHF state, we recorded the impulse activity of thinly myelinated group III (Aδ) and group IV (C) fibres by using the technology of single fibre recording. Group III afferents were activated by either passive stretch of the triceps surae muscles using a calibrated rack and pinion system (Harvard Apparatus, Inc., Holliston, MA, USA) or static contraction induced by electrical stimulation of the peripheral end of L4 or L5 ventral roots whereas group IV were activated by either static contraction or hindlimb intra-arterial (i.a.) injection of capsaicin. For electrical stimulation of ventral roots, a laminectomy exposing the lower lumbar portions of the spinal cord (L2–L6) was performed. The dura of the cord was cut and reflected, allowing visual identification of the L4–L6 spinal roots. The dorsal and ventral roots of L4 and L5 were carefully separated. The ventral roots were sectioned and the cut peripheral ends were positioned on insulated bipolar platinum electrodes. The exposed neural tissue was covered in a pool of warm mineral oil (37°C). The animals were secured within the spinal adaptor (Stoelting Co., Wood Dale, IL, USA) by clamps placed on rostral lumbar vertebrae. Further, the pelvis was stabilized with steel posts within the frame and the hindlimbs containing the triceps surae muscles under study were fixed in one position with clamps. The angle of the hip and knee was 120 and 80 deg respectively. The calcaneal bone was sectioned and the Achilles’ tendon connected to a force transducer (Model FT-03, Grass Instruments, West Warwick, RI, USA) for the measurement of muscle tension. Electrical stimulation was performed using a Grass Instruments S88 stimulator. Electrically induced static muscle contraction of the triceps surae was performed by stimulating the L4 or L5 ventral roots for about 30–35 s. Constant-current stimulation was used at 2.5 times motor threshold (defined as the minimum current required to produce a muscle twitch) with a pulse duration of 0.1 ms at 30–40 Hz (Kaufman et al. 1983; Hayes et al. 2005). In order to minimize or eliminate the stimulus artifact, we recorded the group III afferent impulse from L4 dorsal root in response to static contraction induced by electrical stimulation of the L5 ventral root, and vice versa.
The procedure used for single fibre recording was similar to that described by Kaufman et al. (1983), Mense & Meyer (1985) and Hoheisel et al. (2004). Group III and IV fibres with endings in the triceps surae muscle were recorded from the cut peripheral ends of L4 or L5 dorsal roots. First, we determined the fibres whose receptive fields were in the triceps surae muscle. Only those group III and IV afferents whose receptive fields had been located in the triceps surae muscle were studied. The receptive fields of group III and IV muscle afferents were located by several methods as described previously (Mense & Meyer, 1985). In brief, (1) by touch: slightly touching or stroking the surface of the tissue with a blunt forceps; this stimulus was repeated about once every second; this stimulus is most likely to activate mechanoreceptors in the receptive field; (2) by moderate pressure: innocuous steady deformation of the tissue for 15 s using a forceps with broadened tips; and (3) by noxious pressure: squeezing the tissue with the same forceps for 15 s; this stimulus is perceived as painful, and is likely to activate a metaboreceptor.
Group III and IV fibres were then defined by conduction velocity. Group III fibres conduct at 1.5–15 m s−1 and group IV fibres conduct at less than 1.5 m s−1 (Lynn & Carpenter, 1982; Kaufman et al. 1983; Hoheisel et al. 2004). To measure the conduction velocities of fibres with endings in the triceps surae muscle, we stimulated the tibial nerves and recorded the conduction time from the stimulating electrode to the recording electrode in the dorsal root by using Scope software (ADInstruments, Colorado Springs, CO, USA). The conduction distance between the two points was measured and conduction velocity was calculated by dividing the conduction distance by the conduction time. Finally, we examined 10 sweeps from one spike in order to identify whether it came from a single muscle afferent or not (Fig. 1). In some cases, if the spikes came from two separate group III afferents, we used a window discriminator (Model 121, World Precision Instruments, Sarasota, FL, USA) to isolate and record the impulse of the individual fibre.
Drugs and injected solutions
In order to determine whether P2X receptors mediate the abnormal sensitization of group III in the CHF state, pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), an antagonist of the P2X receptor, was administrated via hindlimb intra-arterial (i.a.) infusion (10 mg kg−1, 0.2 ml, 10 min) by using a syringe pump delivery system (model 310; Stoelting Co.). The responses of group III afferents to either static contraction or passive stretch were compared before and after i.a. treatment with PPADS in sham and CHF rats.
Hindlimb i.a. bolus injections of capsaicin (1.0 or 20 μg kg−1, 0.15 ml) were used to activate the group IV afferents in sham and CHF rats. Briefly, a catheter was placed in the right iliac artery with its tip advanced to the abdominal aortic bifurcation, ensuring that capsaicin was delivered to the left hindlimb through the left iliac artery.
PPADS was purchased from Fisher Scientific Corporation, and was dissolved in saline before use. Capsaicin was purchased from Sigma, and was dissolved in alcohol and then diluted with saline.
Western blot analysis
For Western blot analysis, six sham and six CHF rats were anaesthetized with pentobarbital sodium (40 mg kg−1, i.p.). Cardiac functions such as LVEDP and LVSP were first determined as described above. Then the rats were killed with an overdose of pentobarbital sodium (150 mg kg−1, i.v.). The L4/L5 dorsal root ganglia (DRGs) were rapidly removed and lysed with 20 mm Tris-HCl buffer, pH 8.0, containing 1% NP-40, 150 mm NaCl, 1 mm EDTA, 10% glycerol, 0.1%β-mercaptoethanol, 0.5 mm dithiothreitol, and a mixture of proteinase and phosphatase inhibitors (Sigma). The hearts and lungs were also removed, and infarct size measured as indicated above. Protein concentration was measured by the BCA protein assay method using bovine serum albumin as standard. The proteins were loaded onto a 10% SDS-PAGE gel along with protein standards (Bio-Rad Laboratories, Hercules, CA, USA) in a separate lane for electrophoresis and then transferred to polyvinylidene fluoride membrane. The membrane was probed with rabbit polyclonal antibody against P2X3 (1:1000 dilutions; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or VR1 receptors (1:1000 dilutions, Santa Cruz Biotechnology) and secondary antibody of goat anti-rabbit IgG (1:5000 dilutions, Pierce/Thermo Fisher Scientific, Rockford, IL, USA). The protein signals were detected by enhanced chemiluminescence reagent (Pierce/Thermo Fisher Scientific) and analysed using a bioimaging system (UVP, Upland, CA, USA). GAPDH (1:1000, Santa Cruz Biotechnology) was used to verify equal protein loading, and the densitometric results of P2X3 and VR1 receptors were reported as the ratio to GAPDH.
Rats (n= 5 per group) were anaesthetized with pentobarbital sodium (40 mg kg−1, i.p.). Cardiac function such as LVEDP and LVSP were first determined as described above. At the end of the experiment the rats were perfused through the aorta, first with 100 ml heparinized saline followed by 500 ml 4% paraformaldehyde in 0.1 mol l−1 phosphate buffered saline (PBS, pH 7.4). The L4/L5 DRGs were immediately dissected and immersed in 4% paraformaldehyde in 0.1 mol l−1 PBS (pH 7.2) overnight at 4°C. The tissues were then transferred to 30% sucrose in PBS and kept in the solution until they sank to the bottom. Thereafter, the blocks were rapidly frozen and 10 μm sections were cut on a Leica cryostat and thawed onto gelatin-coated slides. Since the slices were 10 μm thick, and the DRG neuron soma ranged from less than 10 μm to over 100 μm, the same neuron could potentially be counted twice. Therefore, we counted every 10th slice.
For triple-immunostaining of P2X3 or VR1 receptors, sections were stained with the isolectin IB4 (a C-fibre neuron marker; Wang et al. 1994) and NF200 (an A-fibre neuron marker; Finkel, 1998). Ganglionic sections, after being pre-incubated in 10% goat serum for 60 min, were incubated with rabbit anti-P2X3 or anti-VR1 antibody (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-NF200 antibody (1:200 dilution, Abcam, Cambridge, MA, USA) overnight at 4°C. Sections were then washed with PBS and incubated with fluorescence-conjugated secondary antibody (Alexa 488-conjugated goat anti-rabbit IgG and Pacific blue-conjugated goat anti-mouse IgG, 1:200, Invitrogen, Carlsbad, CA, USA) and Alexa FluorR 568 conjugated isolectin-B4 (1:200, Invitrogen) for 60 min at room temperature. After three washes with PBS, the sections were mounted on pre-cleaned microscope slides. Slides were observed under a Leica fluorescence microscope with corresponding filters. Pictures were captured by a digital camera system. No staining was seen when a negative control was performed with PBS instead of the primary antibody (data not shown).
Counts were obtained from the total number of IB4- (C-fibre) or NF200-positive (A-fibre) neurons. The percent of neurons positive for P2X3 or VR1 for A or C neurons was calculated. A neuron was considered to be ‘positive” when the measured intensity of immunostaining was more than five times greater than the background. Ten sections from each DRG were analysed for a total of five sham and five CHF DRGs.
Data acquisition and statistical analysis
Muscle tension and impulse activity of group III and IV afferents were acquired using PowerLab software (ADInstruments). Baseline values were determined by analysing at least 30 s of the data before muscle contraction. The peak response was determined during the period of the greatest change from baseline. The tension–time index (TTI) was calculated by integrating the area between the tension trace and the baseline level and is expressed in kilogram seconds. Peak developed tension was calculated by subtracting the resting tension from the peak tension and is expressed in grams. All values are expressed as means ± standard error of the mean (s.e.m.). Differences between groups were determined by a two-way ANOVA followed by Tukey's post hoc test. Changes in TTI, peak developed tension and discharges of group III and IV afferents before and after arterial administration of chemicals were determined by Student's paired t test. P < 0.05 was considered statistically significant.
Evaluation of body weight, organ weight, and baseline haemodynamics
Echocardiographic and hemodynamic measurements of sham-operated and CHF rats are summarized in Table 1. The heart weight and lung weight to-body weight ratios were significantly higher in CHF rats than in sham-operated rats, suggesting cardiac hypertrophy and substantial pulmonary congestion in the CHF state. Moreover, in rats with CHF, a gross examination revealed a dense scar in the anterior ventricular wall. The mean infarct area was 43.4 ± 2.5% of the LV area. No infarcts were identified in sham-operated rats. Pleural fluid and ascites were also found in the CHF rats but none in the sham-operated rats. Compared to sham rats, there was a slight but significant decrease in baseline MAP in CHF rats. However, there was no significant difference in baseline HR between sham and CHF rats. Furthermore, CHF rats exhibited elevated LVEDP and reduced ejection fraction and fractional shortening compared with sham rats, indicating decreased cardiac function.
|Sham (n= 62)||CHF (n= 63)|
|Body weight (g)||459 ± 10||482 ± 7|
|Heart weight (mg)||1523 ± 20||2532 ± 30*|
|HW/BW (mg g−1)||3.3 ± 0.1||5.3 ± 0.1*|
|WLW/BW (mg g−1)||5.1 ± 0.3||9.2 ± 0.5*|
|MAP (mmHg)||101.1 ± 4.0||88.3 ± 4.1*|
|HR (bpm)||365.5 ± 6.4||380.1 ± 8.5|
|LVEDP (mmHg)||−0.8 ± 0.4||15.7 ± 1.5*|
|LVSP (mmHg)||129.1 ± 5.0||96.2 ± 4.5*|
|LVESD (mm)||3.7 ± 0.1||7.9 ± 0.2*|
|LVEDD (mm)||7.1 ± 0.2||10.5 ± 0.2*|
|LVESV (μl)||70.2 ± 2.7||370.5 ± 17.5*|
|LVEDV (μl)||330.3 ± 16.2||614.6 ± 19.1*|
|EF (%)||78.4 ± 1.3||39.7 ± 1.0*|
|FS (%)||47.3 ± 1.5||24.7 ± 1.0*|
|Infarct size (%)||0||43.4 ± 2.5%*|
Conduction velocity of group III and group IV afferents in sham and CHF rats
We recorded the impulse activity of 87 group III afferents (42 fibres from 26 sham rats and 45 fibres from 29 CHF rats) and 71 group IV afferents (37 fibres from 25 sham rats and 34 fibres from 23 CHF rats), each of which had its receptive field in the triceps surae muscle including the calcaneal tendon, the junction and the belly of this muscle. There were no significant differences in conduction velocities of group III and IV afferents between sham and CHF rats (group III: 6.9 ± 1.1 vs. 7.2 ± 0.8 m s−1, sham vs. CHF; group IV: 0.7 ± 0.1 vs. 0.8 ± 0.1 m s−1, sham vs. CHF, P > 0.05). For the most part, group III afferents responded to light mechanical probing of their receptive fields, whereas group IV afferents responded to noxious pinching of their receptive fields. Group IV afferents did not respond to gentle stroking or non-noxious pinching of the muscles.
The discharges of group III afferents in response to either static contraction or passive stretch in sham and CHF rats
In sham rats, 30 of 42 group III fibres were activated by static contraction. Furthermore, 22 of 30 group III fibres were also responsive to passive stretch, a purely mechanical stimulus. Of the remaining 12 group III fibres that were non-responsive to static contraction, five were still activated by stretch, which is consistent with the finding of Hayes et al. (2005) suggesting that there is not complete overlap between stretch-sensitive and contraction-sensitive group III afferent populations. Finally, the other seven group III fibres did not respond to either static contraction or passive stretch and were consequently excluded from the data analysis. In CHF rats, 34 of 45 group III afferents were activated by static contraction. Twenty-six of 34 group III fibres were also sensitive to passive stretch. In the remaining 11 group III fibres that were non-responsive to static contraction, six were activated by stretch whereas five did not respond to either stimulus and were excluded from the data analysis.
A common characteristic of a group III afferent response to static contraction was a sudden explosive burst of discharge during the onset of contraction, followed by an adaptive decrease during the steady state period of contraction (Fig. 2A–D). A similar phenomenon was seen in stretch-induced discharge of group III afferents. Compared with sham rats, CHF rats exhibited an exaggerated activity of group III afferents in response to either static contraction (Figs 2 and 5) or passive stretch (Figs 3 and 5), indicating that these group III afferents were more sensitive to mechanical stimuli in the CHF state.
Discharge of group IV afferents in response to either static contraction or capsaicin in sham and CHF rats
Thirty-five of 37 group IV fibres in sham rats and 30 of 34 group IV fibres in CHF rats were activated by static contraction. In all contraction-sensitive group IV afferents, 30 fibres in sham rats and 26 fibres in CHF rats were also responsive to hindlimb i.a. injection of capsaicin. In the remaining six group IV fibres that were non-responsive to static contraction (2 from sham rats and 4 from CHF rats), three were still activated by capsaicin whereas the other three did not respond to either static contraction or capsaicin and were consequently excluded from the data analysis.
Although both respond to static contraction, group IV afferents often do not discharge at the onset of contraction as do group III afferents. Instead, the onset latencies of group IV afferents responding to static contraction were longer than those of group III afferents (Fig. 2E–H). Compared with sham rats, CHF rats exhibited decreased activity of group IV afferents in response to either static contraction (Figs 2 and 5) or capsaicin (Figs 4 and 5), indicating that the sensitivity of group IV afferents are blunted rather than enhanced in the CHF state.
Effect of PPADS on the discharge of group III afferents in response to either static contraction or passive stretch in sham and CHF rats
In order to determine whether P2X receptors mediate the abnormal sensitization of group III afferents in CHF, PPADS, an antagonist of P2X receptors, was administrated via hindlimb i.a. infusion. As mentioned above, static contraction of the triceps surae muscles increased the discharge rate of most group III afferents tested in sham and CHF rats. After treatment with PPADS, the discharge of group III afferents in response to contraction was significantly attenuated in both sham and CHF rats (n= 18 per group, Fig. 6B). Furthermore, compared to sham rats, PPADS had a greater inhibitory effect on the discharge of group III afferents in response to static contraction in CHF rats (Fig. 6B). Similarly, PPADS also had a greater inhibitory effect on the discharge of group III afferents in response to passive stretch in CHF rats than in sham rats (Fig. 6C). PPADS did not change the baseline activity of group III afferents in sham or CHF rats (Fig. 6A). There was no significant difference in TTI generated by static contraction or passive stretch before and after PPADS administration in sham and CHF rats (Fig. 6D).
Protein expression of P2X3 receptors in the dorsal root ganglion of sham and CHF rats
Western blot analysis was used to detect P2X3-receptor protein levels in the intact DRGs from sham rats and CHF rats. As shown in Fig. 7A (left panel), the P2X3 protein level was significantly increased by ∼2.8-fold in the L4/L5 DRG of CHF rats (n= 6) compared with the sham control (n= 6).
Immunohistochemical labelling was performed on DRG sections to assess which subpopulation of DRG cells had increased expression of P2X3 receptors as indicated by the Western blot analysis. In both sham and CHF DRGs, immunostaining with the antibody to the P2X3 receptor was seen in IB4-positive (C-fibre) and NF200-positive (A-fibre) neurons (Fig. 7B). Compared with sham rats, the immunostaining was generally more intense in both A- and C-fibre neurons in DRG of CHF rats (Fig. 7B, third panels, green). Compared with sham rats, the percentage of DRG neurons positive for P2X3 against for A and C neurons was significantly greater in CHF rats (Table 2).
|Total IB4-positive||2536 ± 154||2218 ± 155|
|P2X3 with IB4||1120 ± 141||44.3 ± 2.2||1470 ± 146||66.3 ± 2.1*|
|Total NF200-positive||1690 ± 112||1545 ± 102|
|P2X3 with NF200||779 ± 100||46.1 ± 1.8||1134 ± 98||73.4 ± 1.9*|
|Total IB4-positive||2447 ± 145||2100 ± 155|
|VR1 with IB4||1250 ± 141||50.8 ± 1.9||552 ± 35||26.0 ± 1.3*|
|Total NF200-positive||1643 ± 112||1434 ± 92|
|VR1 with NF200||160 ± 20||9.7 ± 1.1||123 ± 19||8.5 ± 0.8|
Protein expression of VR1 receptors in dorsal root ganglion of sham and CHF rats
In contrast to the P2X3 protein expression in DRG, the VR1 protein was significantly decreased by 51% in the L4/L5 DRG of CHF rats compared with the sham control (Fig. 8A). Furthermore, immunohistochemical data showed that immunostaining with the antibody to the VR1 receptor was predominantly seen in IB4-positive (C-fibre) neurons and a much smaller proportion of NF200-positive (A-fibre) neurons (Fig. 8B). Compared with sham rats, immunostaining was decreased in IB4-positive neurons in DRG of CHF rats (Fig. 8B, third panels, green). Compared with sham rats, the percentage of DRG neurons positive for VR1 for C neurons was significantly decreased in CHF rats (Table 2).
Muscle tension produced by static contraction or passive stretch
In the present study, muscle peak developed tension induced by static contraction ranged from 400 g to 500 g in sham and CHF rats. There was no significant difference in TTI during static contraction between sham and CHF rats (Table 3). In the experiments in which passive stretch was performed, we used two levels of stretch to activate the stretch-sensitive group III afferents in sham and CHF rats. The low level of peak tension was about 200 g and the high level was about 500 g, which matched the peak tension developed by static contraction. Again, there were no significant differences in TTI during both levels of stretch between sham and CHF rats (Table 3).
|Group||TTI (kg s)|
|Contraction||Low stretch||High stretch|
|Sham||15.6 ± 1.2||6.7 ± 0.8||16.8 ± 1.6|
|CHF||15.3 ± 1.1||6.6 ± 0.7||16.6 ± 1.5|
In the current study, we provide direct evidence that (1) skeletal muscle afferent function is altered in rats with CHF and (2) that group III afferents are sensitized whereas group IV afferents are blunted in CHF. Another important finding of this study is that P2X and VR1 receptors are involved in the mechanisms underlying the sensitization of the group III afferents and the desensitization of group IV afferents, respectively, in CHF.
Altered function of muscle afferents in the CHF state
Exercise intolerance is a hallmark feature of CHF patients. However, the degree of exercise intolerance is not directly related to the degree of cardiac dysfunction (Ventura-Clapier et al. 2002; Sinoway & Li, 2005; Witte & Clark, 2007; Duscha et al. 2008), indicating that abnormalities of the skeletal musculature may play an important role in mediating exercise intolerance. Early in the 1990s, Coats and colleagues developed the ‘Muscle Hypothesis’, in which they postulated that overactivity of muscle afferents caused the exaggerated ventilation and sympatho-excitation during exercise in CHF patients (Clark et al. 1996). In the following 20 years, evidence from experimental and clinical studies (Middlekauff et al. 2000; Smith et al. 2003; Piepoli et al. 2008; Koba et al. 2008) supports the concept that the exercise pressor reflex is exaggerated in the CHF state. However, due to the absence of electrophysiological recording from muscle afferents, these studies were unable to determine if increased sensitivity of muscle afferent endings or other components of the reflex arc contributed to the exaggerated EPR in CHF. In the present study, we first demonstrated that there are significant differences in the sensitivity of group III and IV afferents between sham and CHF rats. These data provide the most direct evidence in support of the ‘Muscle Hypothesis’ expressed by Coats and colleagues (Clark et al. 1996). Nonetheless, we cannot exclude the possibility that other components of the muscle reflex arc also mediate the exaggerated EPR in the CHF state. For example, Toney & Mifflin (1994) and Potts and Paton (Potts et al. 2003) have published a series of studies demonstrating inhibitory interactions in the NTS between contraction sensitive muscle afferents and arterial baroreceptor inputs. In accordance with the current study, exaggerated muscle afferent inputs in CHF would be expected to produce greater inhibition of the baroreflex, possibly allowing for a greater EPR. This question needs to be clarified by additional experiments.
Mechanoreceptor reflex and metaboreceptor reflex in CHF
Although the exaggerated EPR in CHF has been well accepted, the role of mechanical or metabolic components (mechanoreceptor reflex or metaboreceptor reflex) in the evolution of the exaggerated EPR in CHF remains a controversial issue. Based on measurements of ventilation, the studies of Piepoli and colleagues (Piepoli & Coats, 2007; Piepoli et al. 2008) showed that CHF patients have an overactive metaboreceptor reflex compared with control subjects. However, based on measurements of blood pressure or sympatho-excitatory responses to post-handgrip circulatory arrest (PHG-CA, an isolated activator of the muscle metaboreceptor reflex), the studies of Sterns et al. (1991), and Middlekauff et al. (2000) showed that the metaboreceptor reflex control of blood pressure and sympathetic nerve activity was reduced in patients with CHF (New York Heart Association (NYHA) class II to IV), indicating that metaboreceptor reflex function is blunted rather than exaggerated in CHF patients. In animal experiments, the studies of Li et al. (2004) and Smith et al. (2005a,b) reported that the pressor response to hindlimb injection of capsaicin (an exogenous metabolite to activate the metaboreceptor reflex) was attenuated in CHF rats, supporting the idea that the metaboreceptor reflex control of cardiovascular function was blunted in CHF. However, in parallel studies, activating the muscle metaboreceptor reflex via a reduction of blood flow to the exercising muscles (hindlimbs) during dynamic exercise, O’Leary and colleagues (Jankowska et al. 2009; Li et al. 2009) demonstrated that muscle metaboreceptor reflex activation in CHF induces an augmented HR response, exaggerated peripheral vasoconstriction and secretion of renin, vasopressin and noradrenaline indicating that the muscle metaboreceptor reflex-induced sympathoexcitation is augmented but not decreased in CHF. With regard to the mechanoreceptor reflex, the studies by Mostoufi-Moab et al. (2000), Middlekauff et al. (2001, 2004) and Negrao et al. (2001) suggested that the enhanced mechanoreceptor reflex contributes to the exaggerated EPR in CHF patients whereas Carrington et al. (2001) reported that the mechanoreceptor reflex contributes little to the EPR activity in CHF patients. In animal experiments, the studies of Smith et al. (2005a) and Li et al. (2004) demonstrated that the mechanoreceptor reflex, which was activated by either static contraction or passive stretch, is enhanced in rats with large myocardial infarcts. Various approaches used to activate the metaboreceptor or mechanoreceptor reflex in these studies may contribute to the dispute concerning the blunted or enhanced mechanoreceptor or metaboreceptor reflex in the CHF state. Most importantly, these studies have not provided direct evidence from group III and group IV afferent recordings to address this issue. In the present study, we provide evidence that group III afferents are sensitized whereas group IV afferents are blunted during muscle contraction in CHF rats. These data support the view that the mechanoreceptor reflex mainly contributes to the exaggerated EPR whereas the metaborecptor reflex is blunted in CHF.
The roles of P2X and VR1 receptors in the abnormal sensitization of muscle afferents in CHF rats
The mechanisms underlying the abnormal sensitization of muscle afferents in CHF remain unclear. Previous studies reported that group III afferents can be sensitized by muscle metabolites during muscle contraction, such as prostaglandin and ATP (Hayes et al. 2006; Kindig et al. 2006). Of these metabolites, ATP acts on the group III afferent ending via the P2X receptor (Hayes et al. 2008). Kindig et al. (2006) reported that PPADS, a P2X antagonist, attenuated the responses of groups III muscle afferents to static contraction as well as to tendon stretch in decerebrate cats, suggesting that P2X activation sensitizes group III afferents in the normal state. Recent studies of Li and colleagues (Li et al. 2004; Gao et al. 2007) reported that (1) the P2X activation-induced pressor response to hindlimb i.a. injection of α,β-methylene-ATP was exaggerated in rats with myocardial infarction (MI), and the responsiveness was related to the degree of left ventricular dysfunction; (2) P2X receptor stimulation by α,β-methylene-ATP enhanced the pressor response to muscle stretch greater in rats with large MI than in control sham rats; and (3) the percentage of P2X immunostaining-positive neurons in the DRG was markedly greater in large MI rats compared with sham rats. These findings suggest that ATP and P2X receptors are involved in the mechanism underlying the exaggerated EPR and enhanced mechanoreceptor reflex in the CHF state. In the present study, we provided several new findings: (1) PPADS (P2X antagonist) attenuated the response of muscle group III afferents to either static contraction or passive stretch in CHF rats to a greater extent than in sham rats; (2) protein expression of P2X3 receptors in DRG was significantly increased in CHF rats compared with sham rats; and (3) increased protein expression of P2X3 receptors in DRG was located on both IB4-positive (C-fibre marker) and NF200-positive (A-fibre marker) neurons. Our data suggest that the P2X receptor is responsible for the exaggerated EPR and enhanced mechanoreceptor reflex in CHF rats via sensitizing muscle group III afferents.
In order to explore the underlying mechanism by which group IV afferents were blunted in CHF, we investigated the role of VR1 receptors in mediating the desensitization of group IV afferents. VR1 receptors are primarily localized to metabolically sensitive afferent fibres (group IV) in skeletal muscle (Smith et al. 2005b), which is believed to be closely related to the group IV afferent activation. VR1 receptors are stimulated by capsaicin and hydrogen ions, the latter being a by-product of muscular contraction (Kaufman et al. 1983; Rotto & Kaufman, 1988). An earlier study from Kindig et al. (2005) demonstrated that VR1 blockade failed to prevent the pressor response to static contraction in decerebrated cats, indicating that VR1 plays little role in evoking the exercise pressor reflex in this decerebrate cat model. However, a recent study of Smith et al. (2010) reported that VR1 blockade did attenuate the pressor response to static contraction in decerebrate rats, indicating that VR1 plays an important role in evoking the exercise pressor reflex in the decerebrate rat model. Furthermore, in CHF rats, several studies by this group (Smith et al. 2005b, 2006, 2010) reported that (1) VR1 activation by capsaicin caused a blunted cardiovascular response in CHF rats compared to sham rats, which was confirmed by our recent study (Wang et al. 2010); (2) chronic deletion of VR1 receptors in normal rats reproduces the exaggerated EPR observed in CHF rats; and (3) the mRNA level of VR1 in DRG and skeletal muscle was decreased in CHF rats compared to sham rats. These findings suggest that the VR1 receptor plays an important role in the genesis of the EPR in the normal and CHF states. In the present study, we further provided new findings that (1) the response of group IV afferents to exogenous VR1 activation by capsaicin was blunted in CHF rats compared with sham rats; (2) the protein expression of VR1 receptor in DRG was significantly decreased in CHF state; and (3) the percentage of VR1-positive neurons in the group IV neurons (IB4-positive) was significantly reduced in DRG of CHF rats. We believe that the current finding provide a reasonable explanation for the blunted sensitivity of group IV afferents in the CHF state.
The exaggerated EPR is an important contributor to exercise intolerance and excessive sympatho-excitation during exercise in the CHF state. The present study provides direct evidence that mechanically sensitive afferents (group III) are sensitized, whereas metabolically sensitive afferents (group IV) are desensitized in CHF rats, both of which contribute to the genesis of the exaggerated EPR in CHF. In addition, we found that the abnormal sensitization of muscle afferents in CHF rats is related to the altered levels of P2X and VR1 receptors. These findings have broad implications for understanding the mechanisms of exercise intolerance and the exaggerated sympatho-excitation during exercise in CHF. They provide a potential therapeutic target for exercise intolerance in this disease.
H.-J.W. designed and performed the majority of the experiments including recording the group III and IV afferent impulse, measuring protein expression by Western blot and immunostaining the P2X3 and VR1 receptors in dorsal root ganglia. Also, he drafted this manuscript. Y.L. and L.G. were involved in some data collection, analysis and interpretation of data. W.W. and I.H.Z. are responsible for conception and design of the experiments, analysis and interpretation of data, and revision of the draft manuscript. All authors approved the final version of the manuscript.
We thank Johnnie Hackley, Pamela Curry and Richard Robinson for their expert technical assistance. This work was supported, in part, by a grant from the National Heart, Lung, and Blood Institute (PO1 HL62222). H.-J.W. was supported by a postdoctoral fellowship from the American Heart Association, Heartland Affiliate.