Episodic stimulation of central chemoreceptor neurons elicits disordered breathing and autonomic dysfunction in volume overload heart failure

Abstract

Enhanced central chemoreflex (CC) gain is observed in volume overload heart failure (HF) and is correlated with autonomic dysfunction and breathing disorders. The aim of this study was to determine the role of the CC in the development of respiratory and autonomic dysfunction in HF. Volume overload was surgically created to induce HF in male Sprague-Dawley rats. Radiotelemetry transmitters were implanted for continuous monitoring of blood pressure and heart rate. After recovering from surgery, conscious unrestrained rats were exposed to episodic hypercapnic stimulation [EHS; 10 cycles/5 min, inspiratory fraction of carbon dioxide (${\text{F}I}_{{\text{CO}}_{\text{2}}}$) 7%] in a whole body plethysmograph for recording of cardiorespiratory function. To determine the contribution of CC to cardiorespiratory variables, selective ablation of chemoreceptor neurons within the retrotrapezoid nucleus (RTN) was performed via injection of saporin toxin conjugated to substance P (SSP-SAP). Vehicle-treated rats (HF+Veh and Sham+Veh) were used as controls for SSP-SAP experiments. Sixty minutes post-EHS, minute ventilation was depressed in sham animals relative to HF animals (ΔV̇e: -5.55 ± 2.10 vs. 1.24 ± 1.35 mL/min 100 g, P < 0.05; Sham+Veh vs. HF+Veh). Furthermore, EHS resulted in autonomic imbalance, cardiorespiratory entrainment, and ventilatory disturbances in HF+Veh but not Sham+Veh rats, and these effects were significantly attenuated by SSP-SAP treatment. Also, the apnea-hypopnea index (AHI) was significantly lower in HF+SSP-SAP rats compared with HF+Veh rats (AHI: 5.5 ± 0.8 vs. 14.4 ± 1.3 events/h, HF+SSP-SAP vs. HF+Veh, respectively, P < 0.05). Finally, EHS-induced respiratory-cardiovascular coupling in HF rats depends on RTN chemoreceptor neurons because it was reduced by SSP-SAP treatment. Overall, EHS triggers ventilatory plasticity and elicits cardiorespiratory abnormalities in HF that are largely dependent on RTN chemoreceptor neurons.

Keywords: breathing disorders; chemoreflex; heart failure; retrotrapezoid nucleus; ventilatory plasticity.

Fig. 1.

Substance P-conjugated saporin (SSP-SAP) toxin destroys Phox2b-positive neurons but not tyrosine-hydroxylase (TH)-positive neurons within the retrotrapezoid nucleus (RTN). A: representative images of histological sections (40 μm) of the ventral surface of the brainstem (bregma level: −11.6 mm) of rats receiving SSP-SAP or vehicle (0.9% NaCl) in the RTN. B: quantification of the total number of Phox2b+ TH⁻ neurons in Sham and heart failure (HF) rats treated with vehicle or with the SSP-SAP toxin. One-way ANOVA followed by the Holm-Sidak post hoc test. Box and whiskers represent median ± range. *P < 0.05 vs. Sham+Veh +P < 0.05 vs. HF+Veh; n = 6 rats per group.

Fig. 2.

Ventilatory plasticity induced by episodic hypercapnic stimulation in heart failure (HF) is retrotrapezoid nucleus (RTN) chemoreceptor neuron-dependent. A: schematic of episodic hypercapnic stimulation (EHS) paradigm (top panel). Effect of EHS on ventilation (ΔR_f) during pre- and post-EHS phases. Note that during the post-EHS phase, Sham+Veh rats showed ventilatory depression, and this effect was absent in HF+Veh rats. Selective ablation of RTN neurons by substance P-conjugated saporin (SSP-SAP) toxin normalizes the post-EHS ventilatory response in HF rats. B: representative traces of ventilation pre- and post-EHS in all experimental groups. C–E: summary data of tidal volume (ΔV_T), respiratory frequency (ΔR_f), and minute ventilation (ΔV̇e) during post-EHS phase, respectively. Data represent mean ± SE (A) while box and whiskers represent median ± range (C–E). One-way ANOVA followed by Holm-Sidak post hoc test; n = 6 rats per group. *P < 0.05 vs. Sham+Veh; +P < 0.05 vs. HF+Veh.

Fig. 3.

Selective ablation of retrotrapezoid nucleus (RTN) chemoreceptor neurons blunts the hypercapnic ventilatory response (HCVR). A: representative traces of ventilation during normoxia ($F{I}_{O_2}$ 21%), hypoxia ($F{I}_{O_2}$ 10%), and hypercapnia (${\text{F}\mathrm{I}}_{{\text{CO}}_{\text{2}}}$ 7%). B and C: respiratory frequency (ΔR_f) during hypoxia (B) and the hypoxic ventilatory response (HVR) (C) were not different between groups. D and E: the respiratory frequency (ΔR_f) during hypercapnia (D) and the hypercapnic ventilatory response (HCVR) (E) were increased in heart failure (HF) rats and substance P-conjugated saporin (SSP-SAP) toxin reduced both. Data represent mean ± SE (B, C), and box and whiskers represent median ± range (D, E). One-way ANOVA followed by Holm-Sidak post hoc test; n = 6 rats per group. *P < 0.05 vs. Sham+Veh; +P < 0.05 vs. HF+Veh. n.s., not significant.

Fig. 4.

Selective ablation of retrotrapezoid nucleus (RTN) chemosensory neurons prevents ventilatory disturbances elicited by episodic hypercapnic stimulation. A: representative traces of ventilation during pre- and post-episodic hypercapnic stimulation (EHS) phases. Arrows point to disturbances in breathing patterns such as apneas/hypopneas. B and C: representative Poincaré plots and histograms during pre- and post-EHS phases. D and E: summary data of short-term (SD1) (D) and long-term variability (SD2) (E), and coefficient of variation (CV) of tidal volume (V_T) (F) during pre- and post-EHS phases. Substance P-conjugated saporin (SSP-SAP) toxin injection in the RTN diminished breath-to-breath and V_T amplitude variability in the post-EHS phase in heart failure (HF) rats. Box and whiskers represent median ± range. Two-way ANOVA followed by Holm-Sidak post hoc tests; n = 6 rats per group. *P < 0.05 vs. Sham+Veh Pre; #P < 0.05 vs. HF+Veh Pre; +P < 0.05 vs. Sham+Veh Post; †P < 0.05 vs. HF+Veh Post.

Fig. 5.

Ablation of retrotrapezoid nucleus (RTN) chemosensory neurons attenuates autonomic imbalance in heart failure (HF) rats. A: representative time-varying heart rate variability (HRV) analysis during pre- and post-episodic hyperapnic stimulation (EHS) period in 1 rat per group. Note that HF rats displayed a marked increase in the low-frequency HRV component (0.04–0.6 Hz), and substance P-conjugated saporin (SSP-SAP) toxin injection in the RTN blunted this enhanced sympathetic response during the post-EHS period. B: representative power spectral density (PSD) analysis of HRV during pre- and post-EHS phases. C–E: summary data of low frequency (LF) (C), high-frequency (HF, 0.6–2.4 Hz) (D), and LF/HF ratio (E) during pre- and post-EHS phases. Note that the autonomic imbalance during post-EHS phase was blunted by SSP-SAP toxin delivery into the RTN. Box and whiskers represent median ± range. Two-way ANOVA followed by Holm-Sidak post hoc analysis; n = 6 rats per group. *P < 0.05 vs. Sham+Veh Pre; #P < 0.05 vs. HF+Veh Pre; ┴P < 0.05 vs. Sham+SSP-SAP Pre; ‡P < 0.05 vs. HF+SSP-SAP Pre; +P < 0.05 vs. Sham+Veh Post; †P < 0.05 vs. HF+Veh Post. n.u., normalized units.

Fig. 6.

Episodic hypercapnic stimulation further increases active expiration in heart failure (HF) rats. A: representative traces of one breathing cycle pre- (continuous traces) and post- (segmented traces)-episodic hypercapnic stimulation (EHS) in 1 Sham+Veh rat, 1 Sham+SSP-SAP-treated rat, 1 HF+Veh rat, and 1 HF+SSP-SAP-treated rat. In HF rats, the early expiratory flow (E1) was reduced, whereas the late expiratory flow (E2) was increased compared with Sham rats. B: summary data showing E2/E1 ratio. C: summary of expiratory time obtained in 20 consecutive respiratory cycles in all groups, pre- and post-EHS. Box and whiskers represent median ± range. Two-way ANOVA followed by post hoc analysis of Holm-Sidak; n = 6 rats per group. *P < 0.05 vs. Sham+Veh; +P < 0.05 vs. Sham+SSP-SAP; †P < 0.05 vs. HF+SSP-SAP; @P < 0.05 vs. HF+Veh post-EHS.

Fig. 7.

Episodic hypercapnic stimulation-induced respiratory-cardiovascular coupling in heart failure depends on intact retrotrapezoid nucleus (RTN) chemoreceptor neurons. A: representative traces of respiratory flow, respiratory frequency (R_f), blood pressure (BP), and systolic blood pressure (SBP) in 1 rat per group during pre- and post-episodic hypercapnic stimulation (EHS) phase. Segments where coupling between ventilatory and cardiovascular signals was observed are highlighted (gray). B: summary data of coherence analysis. Note that following EHS, heart failure (HF) rats displayed an increased coherence between tidal volume (V_T) oscillation and SBP, and this was blunted in HF rats treated with substance P-conjugated saporin (SSP-SAP) toxin. Box and whiskers represent median ± range. Two-way ANOVA followed by Holm-Sidak post hoc analysis; n = 6 rats per group. #P < 0.05 vs. HF+Veh Pre-EHS. LF, low frequency.