P2X3 receptor antagonism attenuates the progression of heart failure

Abstract

Despite advances in the treatment of heart failure, prognosis is poor, mortality high and there remains no cure. Heart failure is associated with reduced cardiac pump function, autonomic dysregulation, systemic inflammation and sleep-disordered breathing; these morbidities are exacerbated by peripheral chemoreceptor dysfunction. We reveal that in heart failure the carotid body generates spontaneous, episodic burst discharges coincident with the onset of disordered breathing in male rats. Purinergic (P2X3) receptors were upregulated two-fold in peripheral chemosensory afferents in heart failure, and when antagonized abolished these episodic discharges, normalized both peripheral chemoreceptor sensitivity and the breathing pattern, reinstated autonomic balance, improved cardiac function, and reduced both inflammation and biomarkers of cardiac failure. Aberrant ATP transmission in the carotid body triggers episodic discharges that via P2X3 receptors play a crucial role in the progression of heart failure and as such offer a distinct therapeutic angle to reverse multiple components of its pathogenesis.

Fig. 1. Episodic carotid sinus nerve discharge in chronic heart failure (CHF) rats is mediated by P2X3 receptors and causes respiratory disturbance via activation of active expiration.

a RT–qPCR indicated upregulation of P2X3 but not P2X2 receptors in the petrosal ganglion chemoreceptive neurons from HF rats. β-actin was used as a house keeping control gene to normalize reactions. The relative quantitation was determined by the ΔΔCt method. Data shown as mean ± SD; n = 10 and 6 for sham and HF group, respectively. Two-way ANOVA Bonferroni post-test. **P < 0.01, ***P < 0.001. b Immunofluorescence of P2X3 receptors (green) and glomus cells expressing tyrosine hydroxylase (TH; red) within the carotid body of a heart failure rat. The top panel is a conventional image and two orthogonal views taken at positions X and Y are shown beneath. The absence of P2X3 receptor immunofluorescence superimposition with TH staining supports the viewpoint that P2X3 receptors are on sensory afferent fibres juxtaposed to the glomus cells. This was repeated in three rats. Scale bar 5 μm. For additional P2X3/TH immunofluorescence images see Fig S11. c Raw and integrated (∫) simultaneous recordings of carotid sinus nerve (CSN) activity, electromyographic (EMG) recordings from expiratory (Abd_EMG) and inspiratory (Dia_EMG) muscles in anaesthetized rats. Note the presence of episodic CSN discharge (blue arrows) coincident with both breathing irregularity and onset of active expiration in CHF rats. These changes were all prevented by AF-130 treatment (CHF + AF-130) as was the tonic CSN activity (arrowed). d and e show a correlation between the level of CSN activity with respiratory rate (Resp. Rate, n = 7) and changes in activity of Abd_EMG (n = 7) over 10 min from chronic HF rats, respectively. f CSN tonic discharge is enhanced in chronic CHF rats and normalized by AF-130. Data shown are mean ± SD; n = 7 for sham vehicle; n = 11 for CHF vehicle and n = 7 for CHF AF-130 group. Data were tested for normality (Kolmogorov–Smirnov test) and compared using One-way ANOVA Bonferroni post-test. ***P < 0.001. Correlations were assessed using Pearson’s correlation coefficients, two-sided. Source data are provided as a Source Data file.

Fig. 2. Chronic P2X3-receptor antagonism restored normal breathing pattern in chronic heart failure (CHF) rats.

a Representative tracings displaying tidal volume (V_t) and respiratory frequency (RR) recorded in conscious rats using plethysmography and b Poincaré plots for breath-to-breath interval (BB_N) versus the subsequent interval (BB_N+1). Breathing instability in CHF rats is demonstrated in V_t and RR tracings, and higher breathing variability in CHF rats. P2X3-receptor antagonism restored normal breathing rhythm in HF rats (a, b) and reduced minute ventilation (V_E), respiratory frequency (RR), short- and long-term breathing interval variability (SD1 and SD2), and the incidence of apnoea and hypopnoea (AHI) in CHF rats (c–f). Data are shown as mean ± SD. One-way ANOVA Tukey post-test; n = 8 for sham vehicle, n = 8 for CHF vehicle, and n = 9 for CHF AF-130 group. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig. 3. P2X3-receptor antagonism in heart failure (HF) rats is associated with restoring autonomic balance and respiratory activity in the in situ preparation.

a Raw (∫) and integrated records of thoracic sympathetic (tSN), phrenic nerve (PN), and heart rate (HR, bpm) in sham and HF rats before and after P2X3 receptor inhibition using AF-353. Note the drug was infused via a micropipette into the carotid bodies directly. In HF rats, there was increased activity of tSN and PN, associated with a reduced magnitude of respiratory sinus arrhythmia (RSA) compared to shams. Average values of b tSN, c respiratory sinus arrhythmia (RSA), and d PN frequency from sham and HF rats before and after AF-353 delivery. Data are shown as mean ± SD; n = 10 and 16 for sham and HF group, respectively. One-way ANOVA Bonferroni post-test. **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig. 4. Carotid body P2X3-receptors mediate afferent hypersensitivity and chemoreflex hyperreflexia in heart failure (HF) rats.

a Integrated (∫) recordings of carotid sinus nerve (CSN) activity during activation of peripheral chemoreceptors (potassium cyanide - KCN; 0.05 ml, i.v., 0.05%) in anaesthetized rats. Note presence of elevated tonicity (relative to sham and dotted line) and augmented evoked response in CSN b in the chronic HF (CHF) rats and that these were both normalized after P2X3 receptor blockade (a, b; drug was given systemically). Data are shown as mean ± SD and were tested for normality (Kolmogorov–Smirnov test) and compared using One-way ANOVA Bonferroni post-test. n = 7 per group; ***P < 0.001. The chemoreflex hyperreflexia of both the thoracic sympathetic (tSN) (c, d), and abdominal nerve (AbN) activity (c. e), was normalized by P2X3 receptor blockade in the carotid bodies performed by microperfusion of antagonist in the in situ preparation. ∫PN integrated phrenic nerve activity. Data are shown as mean ± SD; n = 10 and 16 for sham and HF group, respectively. One-way ANOVA Bonferroni post-test. **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig. 5. Chronic treatment with AF-130 improved heart rate variability (HRV) and reduced heart failure (HF) severity in chronic HF (CHF) rats.

HRV was examined in the frequency domain (Hz) by spectral analysis. AF-130 treatment improved the cardiac sympathovagal modulation in CHF rats. a Lf: Low-frequency power; b Hf: high-frequency power and c Lf/Hf; nu: normalized units. P2X3-receptor antagonism attenuated the increase in d heart weights/body weight ratio, and prevented the rise in e lung /body weight ratio and f plasma N-Terminal Pro-B-Type natriuretic peptide (NT-proBNP), indicating that the treatment with AF-130 attenuated the HF progression in these animals. Data are shown as mean ± SD. One-way ANOVA Tukey post-test; (a–e) n = 6 for sham vehicle, n = 6 for CHF vehicle and n = 5 for CHF AF-130 group; (f) n = 5 for sham vehicle, n = 6 for CHF vehicle and n = 6 for CHF AF-130 group. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig. 6. P2X3-receptor antagonism improves cardiac function in chronic heart failure (CHF) rats.

a Representative images of echocardiography in rats submitted to myocardial infarction (MI), before and after 7 weeks of treatment with vehicle or AF-130. Red arrows indicate diastolic ventricular diameter and yellow arrows indicate diastolic ventricular wall thickness. P2X3-receptor antagonism prevented the reduction of ejection fraction (b) and stroke volume (c) during HF development, and reduced left ventricular (LV) end-systolic volume (d). The parameters were analyzed before MI surgery, three days after MI and after 7 weeks of vehicle (n = 13) or AF-130 (n = 8) administration. IVS: interventricular septum. Data are mean ± SD. Repeated measures two-way ANOVA, with Student-Newman-Keuls post hoc comparison. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig. 7. P2X3 receptor blockade reduces the immune cell response and plasma cytokine levels in chronic heart failure (CHF) rats.

Quantitative data for natural killer cells (CD161, a), B cells (CD45+, b) and T cells (CD3 + CD4 + CD25+, c). These specific immune cell types were suppressed by antagonizing P2X3 receptors systemically in CHF rats. The immune cells were analyzed after three weeks of vehicle (n = 5) or AF-130 (n = 4) administration. Two-way repeated measures ANOVA, with Tukey post hoc comparison. Plasma levels of IL-1β (d), TNF-α (e), and IL-10 (f) in sham vehicle (n = 9) and CHF rats treated with either vehicle (n = 4, 9, and 8, respectively) or AF-130 (n = 6) are shown; administrations lasted for seven weeks. One-way ANOVA, with Tukey post hoc comparison. Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001. Source data are provided as a Source Data file.