Decreased purinergic signaling in the RTN is associated with respiratory disorders in HFpEF rats
Authors: Toledo Camilo , Andrade David Cristóbal , Lucero Claudia , Diaz Hugo , Arce Alexis , Del Rio Rodrigo 
Published: November 2023

Heart failure with preserved ejection fraction (HFpEF) is characterized by the presence of respiratory disorders. Interestingly, it has been described that ATP‐mediated purinergic signaling (PSATP), in chemosensitive areas from the brainstem, contribute to central respiratory chemoreception. We recently described that HFpEF rats displays both resting breathing pattern irregularities and enhanced central chemoreflex drive. Therefore, we aimed to determine if HFpEF rats display alterations in the PSATP in the Retrotrapezoid nucleus (RTN), a major central chemosensitive area, and its possible role in the development of irregular breathing patterns in HFpEF. Adult male Sprague‐Dawley rats were subjected to volume overload to induce HFpEF. Whole body plethysmography was used to assess the ventilatory reflex response to hypercapnia to determine central chemoreceptors (CC) sensitivity. Resting breathing pattern was analyzed to score apneas/hypopneas incidence (AHI), as well as breath‐to‐breath (BB) interval variability. RTN micropunches were obtained for the study of PSATP protein expression and molecular markers of neuronal/astrocyte activation using immunoblot. ATP bioavailability in the RTN was determined using a high sensitive bioluminescence assay. Compared to control rats, HFpEF rats (Sham vs. CHF) display: increased AHI (9,7±1,1 vs. 20,2±1,6 events/hour, p<.05), increased both the short‐term (SD1) and long‐term (SD2) BB interval variability (SD1: 42.8±8.0 vs.115,6±28,2; SD2: 54.5±9.0 vs. 138.4±33.1, p<.05) and enhanced CC sensitivity (127.3±10.3 vs. 165.3±9.1 ml/min/100g, p<.05). No changes in normoxic minute ventilation was found between sham and CHF condition. Also, we found that HFpEF rats compared to control rats (Sham vs. CHF) display similar FosB protein expression (p>.05), decreased expression of GFAP (100±2.0% vs. 65±7.0%, p<.05) without changes in S100β expression (85±11.8% vs. 100±9.5%, p>.05) and decreased ATP bioavailable (78±2 vs. 21±10 pmoles/100μg of protein, p<.05) in the RTN. In addition, we found that the P2X7 receptor was constitutively expressed both at the mRNA and protein levels in the RTN from sham rats and that CHF rats showed a significant decrease in the P2X7 receptor expression (45±8% vs. 100±15%, Sham vs. CHF, respectively, p<.05). Remarkably, stereotaxic injection (2.5mm caudal to lambda, 1.8mm lateral to the midline and 7.5mm below the dura mater) of BzATP (200 μM/100 nl) into the RTN, a selective agonist of P2X7 receptor, markedly decrease the BB interval variability in sham rats (SD1: 100±8% vs. 45.7±5%; SD2: 100±8% vs. 40.7±5% pre vs. post BzATP, respectively, p<.05) without changing resting tidal volume and/or respiratory frequency (p>.05). In summary, our results show that HFpEF rats displayed astrocyte inhibition, reduced ATP bioavailability and decreased P2X7 receptor expression in the RTN along with a higher incidence of breathing irregularities compared to healthy animals. Together, our results suggest that altered breathing patterns in HFpEF may be associated with a decrease purinergic signalling in the RTN acting preferentially on P2X7 receptor within the RTN.

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FONDECYT 1140275