Medullary astrocytes mediate irregular breathing patterns generation in chronic heart failure through purinergic P2X7 receptor signalling

Abstract

Background: Breathing disorders (BD) (apnoeas/hypopneas, periodic breathing) are highly prevalent in chronic heart failure (CHF) and are associated with altered central respiratory control. Ample evidence identifies the retrotrapezoid nucleus (RTN) as an important chemosensitivity region for ventilatory control and generation of BD in CHF, however little is known about the cellular mechanisms underlying the RTN/BD relationship. Within the RTN, astrocyte-mediated purinergic signalling modulates respiration, but the potential contribution of RTN astrocytes to BD in CHF has not been explored.

Methods: Selective neuron and/or astrocyte-targeted interventions using either optogenetic and chemogenetic manipulations in the RTN of CHF rats were used to unveil the contribution of the RTN on the development/maintenance of BD, the role played by astrocytes in BD and the molecular mechanism underpinning these alterations.

Findings: We showed that episodic photo-stimulation of RTN neurons triggered BD in healthy rats, and that RTN neurons ablation in CHF animals eliminates BD. Also, we found a reduction in astrocytes activity and ATP bioavailability within the RTN of CHF rats, and that chemogenetic restoration of normal RTN astrocyte activity and ATP levels improved breathing regularity in CHF. Importantly, P"X/ P2X7 receptor (P2X7r) expression was reduced in RTN astrocytes from CHF rats and viral vector-mediated delivery of human P2X7 P2X7r into astrocytes increases ATP bioavailability and abolished BD.

Interpretation: Our results support that RTN astrocytes play a pivotal role on BD generation and maintenance in the setting CHF by a mechanism encompassing P2X7r signalling.

Funding: This study was funded by the National Research and Development Agency of Chile (ANID).

Keywords: Astrocyte; Chronic heart failure; Disordered breathing; P2X7 receptor; Retrotrapezoid nucleus.

Figure 1

RTN neurones are required for the enhanced central chemoreflex drive and active expiration in chronic heart failure rats. (a) representative traces of one rat per group showing tidal volume (V_T) and respiratory frequency (Rf) at rest and during central chemoreflex stimulation with hypercapnia (F_iCO₂ 7%) measured by unrestrained whole-body plethysmography. (b,c) quantification of central chemoreflex sensitivity in Sham control rats and CHF rats treated with vehicle or SSP immunotoxin, (b) measurement of minute ventilation (V_E) in hypercapnia vs. normocapnia and (c) hypercapnic ventilatory response (HCVR). (d) representative traces of ventilation showing the early (E1) and late expiratory (E2) phases. (e) measurement of active expiration as the late-to-early (E2/E1) expiratory ratio. (f) representative images of histological sections (30 μm) of the ventral surface of the brainstem of rats receiving SSP toxin or vehicle (0.9% NaCl) in the RTN (scale bar 200 µm). (g) quantification of the total number of RTN chemosensory neurons (Phox2b⁺ TH^- phenotype) in Sham control rats and CHF rats treated with Vehicle or with the SSP toxin. Data show mean ± s.e.m.; Data was analysed using one-way ANOVA; *: p<0.05 vs. Sham+Veh, +: p<0.05 vs. CHF+Veh; #: p<0.05 vs. Sham+SSP). n=8 animals per group.

Figure 2

Partial RTN neurones ablation in chronic heart failure rats improve resting breathing pattern. (a), representative traces of resting ventilation in one rat per group. Arrowheads highlight respiratory disorders such as apnoeas and hypopneas. (b,c) representative plots of ventilatory variability in one rat per group. (B) Poincaré plots showing breath-to-breath T_TOT variability. (c) histograms of V_T cycle amplitudes. (d-f) measurements of respiratory instability during resting ventilation. (d) irregularity score (IS), (e) coefficient of variation of V_T respiratory cycles, (f) apnoea-hypopnea index (AHI). Data show mean ± s.e.m.; Data was analysed using one-way ANOVA; *: p<0.05 vs. Sham+Veh, +: p<0.05 vs. CHF+Veh; #: p<0.05 vs. Sham+SSP). n=8 animals per group.

Figure 3

Repetitive optogenetic photo-stimulation of RTN neurons elicits breathing disorders. (a) representative traces of ventilatory flow, respiratory frequency (Rf) and tidal volume (V_T) at rest, before, during and after unilateral high frequency photo-stimulation of RTN neurons measured by unrestrained whole-body plethysmography (460 nm, 30 s trains of 10 ms pulses delivered at 20 Hz spaced by 6 min during 1 h). (b) representative picture of one rat during optogenetic stimulation. (c) Neuromedin B positive neurons within the RTN co-localized with channelrhodopsin2 (ChR2). RTN neurons were transduced in-vivo with a lentiviral vector to express ChR2 (LVV-PRSX8-ChR2-YFP). RNAscope against neuromedin B (Nmb) revealed that YFP⁺ cells within the RTN co-express Nmb, a biomarker of RTN chemoreceptor neurons (scale bar 50 µm). (d,e) representative plots of ventilatory variability before and after optogenetic RTN photo-stimulation. (d) Poincare plots showing breath-to-breath variability. (e) histograms of V_T cycle amplitudes. (f) minute ventilation (V_E) during photo-stimulation. (g-i) quantification of ventilatory variability parameters at different laser gains during the experimental protocol. (g) short-term variability (SD1) and long-term variability (SD2, h) of breath-to-breath variability. (i) coefficient of variation of V_T, (j) representative traces of ventilation during the first and last train of light pulse stimulation (pre and post light ON). (k) quantification of V_E and (l) irregularity score (IS) before (∼200 breaths), during (∼50 breaths) and after (∼200 breaths) photo-stimulation. Data show mean ± s.e.m.; Data was analyzed using one-way analysis of variance (ANOVA) (f-i: *: p<0.05 vs. Pre-stimulation, #: p<0.05 vs. post-stimulation) and paired t-test (k, *: p<0.05 vs. baseline, +: p<0.05 vs. Pre-stimulation; l, *: p<0.05 vs. Pre-stimulation). n=4 animals.

Figure 4

Contribution of RTN astrocytes to breathing disorders in heart failure. (a) RTN relative protein expression levels of FosB, Glial fibrillary acidic protein (GFAP, b) and S100β (c) in Sham rats (n=8) and CHF rats (n=8). (d) measurements of RTN ATP levels in Sham and CHF rats. (e) representative traces of V_T and Rf at rest and during central chemoreflex stimulation by hypercapnia (F_iCO₂ 7%) in CHF rats transfected with an excitatory Designer Receptors Exclusively Activated by Designer Drugs (CHF+DREADD_Gq, n=6) and control empty vector (CHF+Ctrl, n=6) following chronic (for 2 weeks) DREADD activation with clozapine-N-oxide (CNO). (f,g) quantification of central chemoreflex sensitivity in CHF+Ctrl and CHF+DREADD_Gq rats, (f) the difference in minute ventilation (V_E) in hypercapnia vs. baseline ventilation and (g) the hypercapnic ventilatory response (HCVR). (h) representative traces of ventilation of one CHF+Ctrl and one CHF+DREADD_Gq rat. Arrowheads showing apnoeas and hypopneas. (i,j), representative plots of ventilatory variability at rest. (I) Poincare plots showing breath-to-breath T_TOT variability. (j) histograms of V_T. (k-m) measurements of respiratory instability during resting ventilation. (k), irregularity score (IS), (l), coefficient of variation of V_T. (m) histological confirmation of RTN astrocytic viral transfection of the excitatory DREADD (mCherry fluorescence) (scale bar 100 µm). (n) western blots experiments showing relative GFAP protein levels in the RTN of CHF+Ctrl (n=6) and CHF+DREADD_Gq rats (n=5). (o) measurement of RTN ATP levels. (p) Correlation analysis between GFAP expression and ATP levels. (q) apnoea-hypopnea index (AHI) Data show mean ± s.e.m.; Data was analysed using unpaired t-tests (a-d *: p<0.05 vs. Sham; e-q *: p<0.05 vs. CHF+Ctrl). (a-d).

Figure 5

Lack of P2X7r in the RTN is linked to disordered breathing. (a) relative P2X7r mRNA and (b) protein expression in Sham control rats (n=6) and CHF rats (n=6) by qPCR and western blot, respectively. (c) representative image of astrocytic P2X7r mRNA immunolabelling in brainstem sections containing the RTN in one Sham and CHF rat (scale bar 20 µm). (d) quantification of P2X7r RNA spots detected (e) representative traces of V_T and Rf in one wild-type mouse (WT; n=9), P2X7r KO mouse (P2X7r^−/−; n=9) and one KO mouse transfected with an adenoviral vector expressing the P2X7 receptor (P2X7r^−/−+AAV_P2X7r n=4) into the RTN at baseline normoxic conditions and during hypercapnic stimulation. (f) measurements of V_E at baseline and during hypercapnia (F_iCO₂ 7%) in WT, P2X7r^−/− and P2X7r^−/−+AAV_P2X7r mice. (g) representative traces of resting ventilation in one WT, P2X7r^−/− and P2X7r^−/−+ AAV_P2X7r mouse. (h-i), representative plots of ventilatory variability at rest. (h) Poincare plots showing breath-to-breath variability. (i) histograms of V_T. (j-m) measurements of respiratory instability during resting ventilation in all experimental groups. (j-k) SD1 and SD2 of the breath-to-breath variability, respectively, (l) coefficient of variation of V_T cycle amplitudes, (m) apnoea-hypopnea index (AHI). Data show mean ± s.e.m.; Data was analyzed using unpaired t-tests (a,b, *: p<0.05 vs. Sham), two-way ANOVA (f, #: p<0.05 vs. its own V_E in baseline) and one-way ANOVA (g-m, *: p<0-05 vs. WT, +: p<0.05 vs. P2X7r^−/−+ AAV_P2X7r).

Figure 6

P2X7r upregulation in RTN astrocytes restores normal breathing patterns in heart failure. (a) representative traces of one CHF+Ctrl and one CHF rat after transfection with AAV5- GFAP-P2X7r-P2A-GFP (CHF+P2X7r) showing tidal volume (V_T) and respiratory frequency (Rf) at rest and during central chemoreflex stimulation with hypercapnia (F_iCO₂ 7%). (b) quantification of V_E at baseline and during central chemoreceptor stimulation with hypercapnia. (c) representative traces of resting ventilation obtained in one CHF+Ctrl rat and one CHF+P2X7r rat. Arrowheads shows breathing disorders. (d,e) representative plots of ventilatory variability at rest in one CHF+Ctrl rat (n=7) and one CHF+P2X7r rat (n=7) . (d) Poincare plots showing breath-to-breath T_TOT variability. (e) histograms of V_T. (f,h) measurements of respiratory instability during resting ventilation in the 2 groups. (f) irregularity score (IS), (g) coefficient of variation of V_T, (h) apnoea-hypopnea index (AHI). (i) visual confirmation of astrocytic viral transfection in the RTN by GFP fluorescence and GFAP immunolabeling (scale bar 100 µm). (j-k), relative protein expression levels of the P2X7r (j) and GFAP (k) in RTN micro-punches. (l) quantification of RTN ATP levels. Data show mean ± s.e.m.; Data was analysed using unpaired t-tests (c-l *: p<0.05 vs. CHF+Ctrl).