Cardiac vanilloid receptor-1 afferent depletion enhances stellate ganglion neuronal activity and efferent sympathetic response to cardiac stress

Abstract

Afferent fibers expressing the vanilloid receptor 1 (VR1) channel have been implicated in cardiac nociception; however, their role in modulating reflex responses to cardiac stress is not well understood. We evaluated this role in Yorkshire pigs by percutaneous epicardial application of resiniferatoxin (RTX), a toxic activator of the VR1 channel, resulting in the depletion of cardiac VR1-expressing afferents. Hemodynamics, epicardial activation recovery intervals, and in vivo activity of stellate ganglion neurons (SGNs) were recorded in control and RTX-treated animals. Stressors included inferior vena cava or aortic occlusion and rapid right ventricular pacing (RVP) to induce dyssynchrony and ischemia. In the epicardium, stellate ganglia, and dorsal root ganglia, immunostaining for the VR1 channel, calcitonin gene-related peptide, and substance P was significantly diminished by RTX. RTX-treated animals exhibited higher basal systolic blood pressures and contractility than control animals. Reflex responses to epicardial bradykinin and capsaicin were mitigated by RTX. Cardiovascular reflex function, as assessed by inferior vena cava or aortic occlusion, was similar in RTX-treated versus control animals. RTX-treated animals exhibited resistance to hemodynamic collapse induced by RVP. Activation recovery interval shortening during RVP, a marker of cardiac sympathetic outflow, was greater in RTX-treated animals and exhibited significant delay in returning to baseline values after cessation of RVP. The basal firing rate of SGNs and firing rates in response to RVP were also greater in RTX-treated animals, as was the SGN network activity in response to cardiac stressors. These data suggest that elimination of cardiac nociceptive afferents reorganizes the central-peripheral nervous system interaction to enhance cardiac sympathetic outflow. NEW & NOTEWORTHY Our work demonstrates a role for cardiac vanilloid receptor-1-expressing afferents in reflex processing of cardiovascular stress. Current understanding suggests that elimination of vanilloid receptor-1 afferents would decrease reflex cardiac sympathetic outflow. We found, paradoxically, that sympathetic outflow to the heart is instead enhanced at baseline and during cardiac stress.

Keywords: cardiac afferent nerves; sympathoexcitation; transient receptor potential vanilloid 1; vanilloid receptor 1.

Fig. 1.

Percutaneous application of resiniferatoxin (RTX) and baseline hemodynamic indexes in treated subjects. A, top: schematic depiction of the protocol for percutaneous epicardial application of RTX. Bottom, stepwise approach to percutaneously accessing the intrapericardial space and instillation of RTX. B: acute hemodynamic responses to RTX application. C–F: baseline hemodynamic measurements in control and RTX-treated animals 4 wk after percutaneous application. Shown are heart rate [HR; in beats/min (bpm)], systolic and diastolic blood pressures (SBP and DBP, respectively), and contractility (dP/dt_max and dP/dt_min, respectively). G: mean activation recovery interval (ARI) in control and RTX-treated animals. All values shown are means ± SE; n = 6–10 animals/group. *P < 0.05 and **P < 0.01 by Mann-Whitney test.

Fig. 2.

Structural and functional depletion of cardiac afferent fibers by intrapericardial resiniferatoxin (RTX). A: representative images demonstrating depletion of the transient receptor potential vanilloid 1 (TRPV1) channel and calcitonin gene-related peptide (CGRP), a marker of sensory afferent nerves, by RTX in T1 dorsal root ganglia (DRG). TRPV1 immunoreactivity in the cell membrane is indicated by arrows; fibers are shown by arrowheads. The lack of staining for the fibers for neurofilament heavy (NFH) can also be appreciated. TRPV1 and CGRP were significantly diminished in RTX-treated animals. Scale bar = 50 µm. B: left ventricular epicardium showing double immunostaining for protein gene product 9.5 (PGP9.5) and CGRP (top) and in chronic RTX-treated animals (bottom). C: representative images of CGRP and substance P immunoreactivity in DRG from control (top) and RTX-treated (bottom) animals. In both B and C, depletion of afferent nerve fibers can be appreciated. Scale bar = 50 µm. D and E: Hemodynamic responses to epicardial application of 20 μg/ml bradykinin (D) and 20 μg/ml capsaicin (E). Shown are percent change in heart rate [HR; in beats/min (bpm)], left ventricular end-systolic pressure (LVESP), diastolic blood pressure (DBP), and inotropy (dP/dt_max) in response to bradykinin (D) and capsaicin (E) (n = 6–8 animals/group). *P < 0.05 by Mann-Whitney test. F: representative images of CGRP and PGP9.5 immunoreactivity in T1 DRG from control and RTX-treated animals.

Fig. 3.

Vanilloid receptor 1 (VR1) afferent depletion does not modulate reflex responses to cardiovascular mechanoreceptor stimuli. A: representative hemodynamic tracings from control and resiniferatoxin (RTX)-treated animals during increased afterload induced by occlusion of the aorta distal to the great vessels. The duration of aortic occlusion is indicated by the solid horizontal bars. Shown are heart rate [HR; in beats/min (BPM)], arterial blood pressure (ABP), left ventricular contractility (dP/dt), and left ventricular pressure (LVP). During aortic occlusion, ABP recorded in the femoral artery plummeted. The increase in left ventricular end systolic pressure (LVESP; B), decrease in femoral systolic blood pressure (SBP) during occlusion of the aorta (C), percent decrease in HR (reflex bradycardia; D), and activation recovery interval (ARI; E) during aorta occlusion in control and RTX-treated animals are shown. F: representative hemodynamic tracings from control and RTX-treated animals during preload reduction induced by occlusion of the inferior vena cava (IVC) just below its junction with the right atrium. The duration of IVC occlusion is indicated by the solid horizontal bars. G and H: percent decrease in LVESP (G) and percent decrease in femoral SBP (H) during occlusion of the IVC. I and J: percent decrease in HR (I) and ARI (J) during IVC occlusion in control and RTX-treated animals. Data are for n = 5–8 animals/group and were evaluated by a Mann-Whitney test.

Fig. 4.

Transient receptor potential vanilloid 1 (TRPV1) depletion enhances resistance to multimodal stress induced by rapid right ventricular pacing (RVP). A: representative hemodynamic tracings from control and resiniferatoxin (RTX)-treated animals during rapid RVP. The solid horizontal bars indicate the duration of RVP. Shown are heart rate [HR; in beats/min (BPM)], arterial blood pressure (ABP), left ventricular contractility (dP/dt), and left ventricular pressure (LVP). B and C: systolic blood pressure (SBP; B) and diastolic blood pressure (DBP; C) [SBP: P < 0.0001 for treatment, P = 0.0006 for paced cycle length, and P = 0.969 for interaction; DBP: P < 0.0001 for treatment, P = 0.003 for paced cycle length, and P = 0.9 for interaction]. D: contractility [dP/dt_max: P < 0.0001 for treatment, P = 0.006 for paced cycle length, and P = 0.93 for interaction; dP/dt_min: P < 0.0001 for treatment, P = 0.03 for paced cycle length, and P = 0.92 for interaction]. E: activation recovery interval (ARI; E) shortening during rapid cardiac pacing at gradually decreasing cycle lengths from 600 ms (100 BPM) to 300 ms (200 BPM) (P < 0.0001 for treatment, P < 0.0001 for paced cycle length, and P = 0.2 for interaction). F: representative sinus rhythm ARI maps from the anterior left ventricle before and after rapid pacing at 300-ms cycle length in control and RTX-treated animals. Two-way ANOVA with Sidak’s multiple comparison test was used. G: summary data of percent ARI shortening of the sinus rhythm before and after RVP. H: recovery of ARI to baseline values after cessation of cardiac pacing. RTX-treated animals took substantially longer to return to baseline. n = 5–8 animals/group, *P < 0.05 by Mann-Whitney test.

Fig. 5.

Cardiac transient receptor potential vanilloid 1 (TRPV1) depletion increases stellate ganglion neuron (SGN) responsiveness to stress. A: 16-channel linear microelectrode array used for recording SGNs. B: a raw data channel and individual SGNs comprising the channel. Inset: the unique morphology of four SGNs is highlighted. C: mean basal SGN firing frequency in control and resiniferatoxin (RTX)-treated animals. D: mean firing rates of SGNs after termination of rapid right ventricular pacing (RVP) in control and RTX-treated animals. E: responsiveness of recorded SGNs to the stressors instituted. F: summary of changes in activity of SGNs recorded before and during each stressor. *P < 0.05; **P < 0.01; ***P < 0.001. C and D: unpaired t-test; E: χ²-test; F: Skellam-based maximum likelihood decoding.

Fig. 6.

Efferent sympathetic function of right stellate ganglia after sensory afferent depletion. Representative images of stellate ganglia stained with calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH), and peptide gene product 9.5 (PGP9.5) from control (A) and resiniferatoxin (RTX) (B)-treated animals are shown. Images show individual CGRP, TH, PGP9.5 staining and a merge of all three as indicated. An extensive network of CGRP-positive afferent fibers can be readily identified in A coursing through the stellate ganglion; however, after epicardial RTX application, these fibers were severely depleted, as shown in B. Scale bar = 100 µm. C: quantification of CGRP-positive afferent fiber depletion after RTX treatment. D: representative hemodynamic tracings during right stellate ganglion stimulation (RSGS) in control and RTX-treated animals. The duration of RSGS is indicated by the solid horizontal bars. Shown are heart rate [HR; in beats/min (BPM)], arterial blood pressure (ABP), left ventricular contractility (dP/dt), and left ventricular pressure (LVP). E: the HR response to RSGS was not significantly different between control and RTX-treated animals. F: the increase in mean arterial pressure (MAP) during RSGS was not significantly different between control and RTX-treated animals. G: the activation recovery interval (ARI) shortening in response to RSGS was similar between control and RTX-treated animals. ***P < 0.001 and †††P < 0.001 by two-way ANOVA with Sidak’s multiple comparison test.

Fig. 7.

Schematic representation of the impact of cardiac vanilloid receptor 1 (VR1) depletion on central-peripheral interactions for cardiac control. Illustrations of neural circuits involved in cardiac control are shown. Left: connectivity in control animals. Right: theorized changes suggested by data in the present article. DH, dorsal horn; NTS, nucleus of the solitary tract; RTX, resiniferatoxin; RVLM, rostral ventrolateral medulla.