Astrocytes within the retrotrapezoid nucleus maintain irregular breathing patterns in heart failure: role of P2X7 receptors
Authors: Del Rio Rodrigo , Toledo Camilo , Andrade David C. , Pereyra Katherin , Schwarz Karla G. , Díaz-Jara Esteban 
Published: April 2020

Irregular breathing (IB) patterns are a hallmark of heart failure (HF). The mechanisms underlying these alterations are not known. Interestingly, it has been described that purinergic signaling (PurS) in the retrotrapezoid nucleus (RTN), a main central chemoreceptor area, modulate ventilatory chemoreflex control in healthy conditions. However, nothing is known about the role of the RTN on IB in HF nor the precise molecular mechanisms underpinning the development/maintenance of IB in HF. Accordingly, we aimed to determine in HF rats: i) the contribution of the RTN on IB; ii) the alterations in PurS in the RTN; iii) the activation level of RTN chemoreceptor neurons/astrocytes; iv) the effects of chronic RTN chemogenetic manipulation using DREADDs on the maintenance of IB; and v) if astrocyte‐targeted expression of the P2X7 receptor (P2X7r) normalizes breathing. Sprague‐Dawley rats underwent volume overload to induce HF. IB was assessed by whole‐body plethysmography. RTN micropunches were used to determine neuron/astrocyte activation, P2X7r gene/protein expression and ATP levels. Adeno‐associated vector (AAV) expressing an excitatory Designer Receptor Exclusively Activated by Designer Drugs (DREADD) or an AAV carrying the P2X7r under the control of GFAP promoter were stereotaxically injected into the RTN of HF rats. Compared to control rats, HF rats (Sham vs. HF, p<.05) displayed increases in the apnea/hypoapnea index (AHI) (3.5±1 vs. 8.7±2 events/hr), breath‐to‐breath interval variability (SD1: 38.5±8 vs. 83.6±8 ms; SD2: 48.5±5 vs. 107.4±10 ms) and in the coefficient of variation (CV) of VTamplitudes (8.7±2 vs. 14.3±3 %). In addition, both ATP levels (78±2 vs. 21±10 pmoles/100μg of protein) and P2X7r gene/protein expression were reduced in the RTN of HF rats. Importantly, we found that P2X7r expression was restricted only to astrocytes within the RTN. No chronic activation of RTN neurons were found in HF as evidenced by no changes in fosB expression compared to control rats. Contrarily, GFAP expression was significantly lower in the RTN from HF rats compared to controls. Chronic chemogenetic activation of RTN astrocytes in HF increases ATP levels (28±3 vs. 71±4 pmoles/100μg of protein; HF vs. HFDREADD; p<.05, respectively) and improve breathing regularity (Irregularity score: 25±3 vs. 12±1%; HF vs. HFDREADD; p<.05, respectively). Finally, increasing the expression of P2X7r in RTN astrocytes markedly reduced AHI (8.7±2 vs. 3,7±1 events/hr; HF vs. HFP2X7r; p<.05, respectively) and decreases breath‐to‐breath (SD1 83.6±8 vs. 53.6±4 ms; SD2 107.4±10.1 vs 68.5±5.0 ms; HF vs. HFP2X7r; p<.05, respectively) and VTvariability (CV: 25±1 vs. 19±2; HF vs. HFP2X7r; p<.05, respectively). Our results show that the RTN is necessary for the maintenance of IB in HF and that RTN astrocytes play a pivotal role on breathing rhythm regulation by a mechanism encompassing ATP release and the P2X7r.Support or Funding InformationSupported by FONDECYT 1180172 and the basal Center of Excellence in Aging and Regeneration (AFB 170005).