Table of Contents
The study complied with all relevant ethical regulations. The Institutional Ethics Committee in Animal Experimentation-CEUA of the Ribeirão Preto Medical School, University of São Paulo approved the experimental protocols (Protocol number 033/2017). The experiments were carried out in adult (7-8 weeks old) and juvenile (4 weeks old) male Wistar rats supplied by the Animal Facility of the Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil. The animals were housed under standard conditions with 24 h free access to food and water, on a 12 h light 12 h dark cycle.
Experimental heart failure
HF was induced by myocardial infarction as described previously by us56. Briefly, animals were anesthetized with ketamine (50 mg/kg, im; União Química Farmacêutica Nacional S/A, Embu-Guaçu, SP, Brazil) and Xylazine (10 mg/kg im; Hertape Calier Saúde animal S/A, Juatuba, MG, Brazil), submitted to orotracheal intubation and ventilated mechanically (Advanced Safety Ventilator, Harvard Apparatus, MA1 55-7059, Holliston, MA, USA). The depth of anesthesia was assessed frequently by a noxious pinch to the tail or a paw to check for a withdrawal response. Supplemental doses of anesthesia were given as required. The heart was exposed by an incision in the third intercostal space, and the anterior descending branch of the left coronary artery was identified and ligated with a silk suture (4-0). Sham rats underwent a similar surgical procedure but without coronary ligation.
In situ working heart–brainstem preparation
These experiments could not be performed blind because the heart is exposed and visualized in the preparation. Juvenile male Wistar rats, 4 weeks, weighing 40–60 g were anesthetized deeply with isoflurane (Baxter Hospitalar, São Paulo, SP, Brazil, 5% induction, maintenance 1.5–3%) and submitted to myocardial infarction as described above. The depth of anesthesia was assessed frequently by a noxious pinch to the tail or a paw to check for a withdrawal response. Supplemental doses of anesthesia were given as required. Ten days later, rats were anesthetized deeply using isoflurane (5%), such that breathing was depressed and there was no withdrawal response to a noxious pinch to the tail or a paw, and were prepared as originally described57. In brief, rats were bisected below the diaphragm and made insentient via decerebration at the pre-collicular level. The carotid body and petrosal ganglion were isolated on the right side of the preparation. Preparations were transferred to a recording chamber, and a double lumen catheter was placed into the descending aorta for retrograde perfusion with a Ringer solution containing in mM: NaCl (125), NaHCO3 (24), KCl (3), CaCl2 (2.5), MgSO4 (1.25), KH2PO4 (1.25), D-glucose (10), and an oncotic agent (1.25% polyethylene glycol, Sigma), saturated with 95% O2−5% CO2 (pH, 7.35-7.4) and warmed to 31 °C. Activation of the chemoreflex was evaluated by administration of potassium cyanide (KCN; 0.05 mL, i.v., 0.05%)57. A neuromuscular blocking agent (vecuronium bromide, 3-4 μg/mL, Cristália Produtos Químicos Farmacêuticos) was added to prevent respiratory-related movement. Recordings from the PN, tSN, and AbN were made simultaneously using custom bipolar glass suction electrodes. The activity of the tSN was recorded from levels T8-T12 and AbN at the thoraco–lumbar level. HR was derived from the inter R wave of the ECG. All signals were amplified (10X), band-pass filtered (1700 amplifier; A-M Systems, Sequim, WA, USA; 0.1 Hz–5 kHz), and acquired (5 kHz) with an A/D converter (CED 1401, Cambridge Electronic Design, CED) controlled by a computer running Spike 2 software (Cambridge Electronic Design, CED). The noise level from the sympathetic chain was measured after the application of lidocaine (2%) at the end of each experiment and subtracted. All nerves were recorded in absolute units (μV), and analyses were performed off-line. Signals were rectified and integrated (50 ms time constant). Whole-cell current clamp recordings (Axopatch-200B integrating amplifier; Molecular Devices) of chemoreceptive petrosal neurons were performed18 using electrodes filled with a solution containing in mM: K-gluconate (130), MgCl2 (4.5), trisphosphocreatine (14), HEPES (10), EGTA (5), Na-ATP (5), Na-GTP (0.3). This solution had an osmolarity of ~ 300 mOsmol/Kg.H2O, pH 7.39, and resistance of 6–8 MΩ. Electrodes were positioned into the petrosal ganglion along its lateral aspect using a micromanipulator (PatchStar; Scientifica, Uckfield, UK) under visual control (microscope; Seiler, St Louis, MO, USA). The chemoreceptive petrosal neurons were functionally identified by their excitatory response to KCN18. The signals were amplified (10X), filtered (low pass filter 2 kHz), and acquired (10 kHz) with an A/D converter (Axon Digidata 1550B; Molecular Devices) controlled by a computer running pClamp software (Molecular Devices).
Baseline PN activity was assessed by burst frequency (Hz). To perform comparisons of the tSN recordings between groups, changes in activity were expressed as percentage changes in accordance with a scale (0–100%) determined for each preparation, as previously described58,59. Briefly, the maximal level of tSN produced by carotid body stimulation was used as 100%. Respiratory sinus arrhythmia was evaluated by the peak-to-trough difference in HR between inspiration and expiration. The tSN (averaged across all respiratory phases and during expiration only) and AbN expiratory responses to KCN was assessed by the measurement of the area under the curve and expressed as percentage values relative to baseline (Δ tSN and Δ AbN in percentage). PN response to KCN was assessed by the difference between baseline PN frequency and the peak of response observed after the KCN (Δ PN in Hz). Rat groups included: Sham coronary ligation, Sham + AF-353 injected into the carotid bodies, HF and HF + AF-353 injected into the carotid bodies. The electrophysiological properties of petrosal neurons measured were: (a) baseline membrane potential; (b) baseline firing frequency, and; (c) firing response to chemoreflex activation. The baseline membrane potential was assessed using a cumulative histogram (bin width 0.5 s) from the membrane potential recordings. Their firing response to chemoreflex activation was assessed by the difference between baseline firing frequency and the peak of response observed after KCN. Note: carotid body excitability is defined as either the level of carotid sinus nerve activity recorded at baseline (after background has been subtracted) or the change in carotid sinus nerve activity to stimulation with KCN. Carotid body/chemoreflex hyperreflexia refers to the magnitude of the reflex evoked response in tSN or PN activities.
Chronic AF-130 treatment
Graphic timeline of the experimental protocol is displayed in the supplemental material (Fig S9). Either AF-130 administration (Afferent Pharmaceuticals, San Mateo, California, USA), 30 mg/kg s.c. per day or vehicle (dimethylsulfoxide 99.9%, DMSO, Sigma-Aldrich, St. Louis, MO, USA) started three days after myocardium infarction surgery and lasted for 7-8 weeks. Rat groups included: sham coronary ligation treated with vehicle (Sham), CHF treated with vehicle (CHF + vehicle) and CHF treated with AF-130 (HF + AF-130).
Respiratory and blood gases measurements in conscious rats
The femoral artery was catheterized 24 h before the arterial blood gases measurements. Rats were anaesthetized with ketamine and xylazine and a catheter was inserted into the femoral artery, directed to the abdominal aorta (PE-10 connected to PE-50 tubing; Clay Adams, Parsippany, NJ, USA). The depth of anesthesia was assessed frequently by a noxious pinch to the tail or a paw to check for a withdrawal response. Supplemental doses of anesthesia were given as required. Samples of arterial blood (100 μl) were collected using the femoral catheter before and during the animals’ respiratory irregularities to analyze the PaCO2 and PaO2 (gas analyzer; Cobas b121; Roche Diagnostics GmbH, Germany).
Tidal volume (Vt), respiratory rate (RR), and minute ventilation (VE) were studied by whole-body plethysmography in conscious rats. Pressure oscillations caused by respiratory movements were detected by a differential pressure transducer (ML141, ADInstruments, Sydney, Australia) and were digitally recorded in an IBM/PC connected to a PowerLab System (ML866, ADInstruments, Sydney, Australia). Vt was calculated using the formula described by Bartlett and Tenney60. RR was calculated from the excursion of the Vt signal using the cyclic rate built into the computer software LabChart v7.2 (ADInstruments, Sydney, Australia). VE was calculated as the product of Vt and RR. Breathing interval variability was assessed from resting breathing recordings by Poincaré plots and analysis of SD1 and SD261. Apnea and hypopnea incidence, considered as cessation (for a period greater than a control respiratory cycle length at rest) or 50% reduction in Vt over 3 consecutive breaths, were calculated and reported as apnea and hypopnea index (events/h). Post-sigh apneas numbers were also measured.
Chemoreflex function and respiratory measurements in anesthetized animals
Animals were anesthetized with urethane (1 g/kg, i.p., Sigma Chemical, St. Louis, MO) and the depth of anesthesia was assessed frequently by a noxious pinch to the tail or a paw to check for a withdrawal response. Supplemental doses of anesthesia were given as required. Rats were placed on a heating pad (ALB 200 RA; Bonther, Ribeirão Preto, Brazil), and core body temperature maintained at 37 °C via a heating blanket with feedback from a rectal thermocouple (MLT1403; Harvard Apparatus, Holliston, MA, USA). A polyethylene catheter (Intramedic, Clay Adams, Parsippany, NJ) was inserted into the femoral vein. The carotid bifurcation was exposed and the carotid sinus nerve isolated, as we previously described57. Briefly, the carotid sinus nerve was traced from its point of convergence with the glossopharyngeal nerve and traced caudally towards the common carotid artery bifurcation. All measurements were performed in spontaneous breathing animals with or without the trachea cannulated breathing room air and vagus nerves intact. Teflon-coated bipolar stainless steel electrodes were implanted in the Dia and the Abd muscles for EMG recordings62. Activation of the chemoreflex was evaluated by administration of KCN (0.05 mL, i.v., 0.05%)58. All recorded signals were amplified (10X; 1700 amplifier; A-M Systems, Sequim, WA, USA), band-pass filtered (0.3 Hz – 5 kHz), and acquired by a data acquisition system (5 kHz; ML866; ADInstruments) controlled by a computer running LabChart software (v.5.0; ADInstruments). The recorded signal from the carotid sinus nerve was fed to a spike amplitude discriminator and counter, which digitally counted in 1 s intervals to assess its discharge frequency (spikes per second). Changes in carotid sinus nerve in response to KCN were assessed by the difference between baseline and the peak of response observed after the stimulus (Δ CSN). EMGs were recorded in absolute units (μV) and analyses were performed off-line from rectified and integrated (∫) signals (time constant: 50 ms). DiaEMG burst frequency was assessed as RR. Changes in the AbdEMG activity during baseline condition were expressed in µV. Based upon absolute values, we determined percentage changes in order to compare their activities in each animal. At the end of the experimental procedures, blood samples were collected for further analysis of plasma N-Terminal Pro-B-Type natriuretic peptide (NT-proBNP; see below) concentration. Rats were euthanized with a high dose of pentobarbital (100 mg/kg, i.v.) and once breathing had ceased the lungs and hearts were removed, rinsed in ice-cold 0.9% NaCl solution, dried, and weighed. The heart was fixed in 3.7% formaldehyde, embedded in paraffin, and the sections were stained with Masson’s trichrome to reveal the infarct size and measured using the NIH ImageJ software (developed by National Institutes of Health and available on the internet site rsb.info.nih.gov/nih-image/). Infarct size was calculated by dividing the length of the infarcted area by the total circumference of the LV and expressed as a percentage56.
The echocardiographic evaluation was performed one day before the myocardial surgery (control), and repeated three days and seven weeks after the myocardial infarction in chronic HF rats. In juvenile rats, the echocardiographic analysis was performed ten days after the myocardial infarction. Rats were anesthetized with ketamine (50 mg/kg) and Xylazine (10 mg/kg, i.m.), and the depth of anesthesia was assessed frequently by a noxious pinch to the tail or a paw to check for a withdrawal response. Supplemental doses of anesthesia were given as required. Body temperature was monitored and maintained, and cardiac parameters were obtained through a VEVO2100® (Fuji) machine using a 30 MHz transducer. Diastolic left ventricle diameter, and ventricular posterior wall thickness were evaluated in M-mode; end systolic volume, stroke volume, and ejection fraction were calculated using a bidimensional mode.
Analysis of R-R wave interval variability
The rats were anesthetized transiently with isoflurane (Baxter Hospitalar, São Paulo, SP, Brazil, 5% induction, maintenance 1.5–3%) and subcutaneous electrocardiogram (ECG) electrodes were implanted. After 48 h, the ECG signal was recorded for 1 h in the conscious state. R-R wave interval variability analysis was performed in the frequency domain using CardioSeries software (v2.7, www.danielpenteado.com). The R-R interval time series were resampled at 10 Hz (1 value every 100 ms) by cubic spline interpolation, to regularize the time interval between beats. The R-R interval time series with 15 min duration were divided into 34 half-overlapping (Welch protocol) segments, each one with 512 values. Next, Hanning windowing was employed and each stable segment was subjected to spectral analysis using Fast Fourier Transform. Pulse interval spectra were integrated into low (LF: 0.20–0.75 Hz) and high frequency (HF: 0.75–3.00 Hz) frequency bands. LF and HF powers are expressed in normalized units (nu) and the LF/HF ratio is also shown.
Plasma NT-proBNP concentration was measured using AssayMax™ immunoenzymatic assay kit following the manufacturers instructions (St. Charles, MO, USA, catalogue number: ERB1202-1).
In the single-cell RT-qPCR experiments, the pipette solution containing the cytoplasmatic material of the recorded petrosal neuron was collected from the patch pipette. The High Capacity cDNA Reverse Transcription Kit reagents (Life Technologies) and nuclease-free water were used for subsequent transcription in a thermocycler (ProFlex PCR System; Applied Biosystems, Foster City, CA, USA). cDNA pre-amplification was performed in the single-cell RT-qPCR experiments using the TaqMan PreAmp Master Mix Kit (Life Technologies) using the P2X2 (Rn04219592_g1), P2X3 (Rn00579301_m1) and β-actin (NM_031144.2) probes. The reactions for the RT-qPCR were performed in singleplex and triplicate (StepOnePlus System, Applied Biosystems) using the same probes described above and the TaqMan Universal PCR Master Mix kit (Life Technologies) according to the manufacture’s recommendations. β-actin was used as a house keeping control gene to normalize reactions. The relative quantitation was determined by the ΔΔCt method. For each sample, the threshold cycle (Ct) was determined and normalized relative to β-actin (ΔCt = Ct Unknown – Ct referencegene). The fold change of mRNA content from the petrosal ganglia chemoreceptive neurons from HF relative to the sham animals was determined by 2 − ΔΔCt, where ΔΔCt = ΔCt Unknown – ΔCt Control. Data are presented as mRNA expression relative to the sham animals.
Carotid bifurcations from HF rats were surgically removed immediately after the in situ experiments and transferred into ice cold Ringer. Carotid bodies were dissected, fixed overnight with 4% formaldehyde, and submerged in sucrose solution (30%) for 24 h. Coronal sections (40 μm thick) were washed three times in phosphate-buffered saline (PBS 0.1 M) for 5 min and then blocked and permeabilized in PBS, 10% normal horse serum, and 0.1% Triton X-100 for one hour (room temperature). The sections were incubated in mouse anti-tyrosine hydroxylase (TH; 1:1000; Millipore, Burlington, MA, USA) and in rabbit anti-P2X3 receptor (1:500; Abcam, Waltham, MA, USA) primary antibodies overnight. In sequence, they were washed three times with PBS for 5 min, followed by incubation in goat anti-mouse Alexa Fluor 488 (1:500; Thermo Fisher Scientific, Waltham, MA, USA) and goat anti-rabbit Alexa 647 (1:500; Thermo Fisher Scientific) for 4 h. We performed negative controls to show an absence of non-specific staining from secondary antibodies (Fig S12). Subsequently, sections and cells were mounted onto glass slides with Fluoromount (Sigma-Aldrich). The images were acquired using a Leica TCS SP5 (Wetzlar, Germany) confocal microscope equipped with 488 and 633 nm lasers and detection of tunable emission wavelengths.
Infarct size analysis
The hearts were fixed in phosphate-buffered 4% formalin and mounted in paraffin blocks. Each block was serially cut at 6 μm from the midventricular surface. The sections were stained with Masson’s trichrome, and the infarct size was measured using the NIH ImageJ software (developed by the National Institutes of Health; rsb.info.nih.gov/nih-image/). Infarct size was calculated by dividing the length of the infarcted area by the total circumference of the LV and expressed as a percentage56.
In this protocol, rats were submitted to myocardial infarction and the administration of vehicle (DMSO) or AF-130 (2 mg/kg/h) started four weeks after the surgical procedure. For vehicle or AF-130 infusion, a polyethylene catheter was inserted in the jugular vein and connected with a programmable iPRECIO SMP-300 pump (Primetech Corporation, Tokyo, JP) placed under the skin of the back. The animals were treated for 3 weeks. At the end of the treatment, blood samples were collected from the tail vein. The cells from the experimental groups were placed in 96-well round-bottom plates for cytofluorometric analysis. Following Fc receptor blocking, cells were incubated with colour combinations of the monoclonal antibodies (BD Biosciences, San Jose, CA, USA). Stained cells were stored for analysis in PBS containing 1% paraformaldehyde, in sealed tubes held in the dark. All steps were performed at 4 °C. Analysis of these cells was performed using a Becton Dickinson FACScan flow cytometer with DIVA-BD software (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). Representative plots of gating strategy are showed in figures S13 and S14.
Plasma cytokine (TNF-α, IL-1β, and IL-10) levels were analyzed by the immune-enzymatic ELISA method, using Duo set kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturers’ instructions.
Two antagonists with very similar P2X3 and P2X2/3 selectivity were used. AF-353 (Afferent Pharmaceuticals) has a low polar surface area and as a result, crosses the blood-brain barrier63, however AF-130 (Afferent Pharmaceuticals), with a methyl sulfone substitution (Supplementary Fig. S10, making the overall selectivity/affinity profile similar, has a much higher polar surface area, and does not cross the blood-brain barrier64,65. The latter was used in the in vivo studies. AF-130 data were generated by Afferent Pharmaceuticals, are unpublished and include that this antagonist is a highly selective and potent inhibitor of P2X3 and P2X2/3 channels showing greater potency at P2X3 homotrimers than P2X2/3 heterotrimers by around eight-fold. The potency of AF-130 is reflected by the IC50 ranges of 126–407 nM for P2X3 receptors and 240–5670 nM for P2X2/3 receptors. AF-130 has >25-fold selectivity over other P2X channels tested (including P2X1, P2X2, P2X4, P2X5 and P2X7). It has been tested on 73 non-purinergic targets (e.g, ion channels, GPCR, transporters, and enzymes). Only when doses were 25–100 fold above the IC50 range for P2X3 and P2X2/3 receptors was a partial (20%) antagonism of some tested processes observed (e.g. adenosine 3 receptors, 5-HT6 receptors, and dopamine transporter). See Supplementary Figure 10 for additional information on AF-130.
Results are expressed as the mean ± standard deviation (SD). Data were tested for normality using Kolmogorov–Smirnov test and compared using unpaired t-test with Welch’s correction, One-way ANOVA or repeated measures two-way ANOVA, with Student-Newman-Keuls, Bonferroni or Tukey post hoc comparisons. Correlations were assessed using Pearson’s correlation coefficients. The type of statistical test performed is indicated in the figure legends. Differences were considered to be statistically significant with p < 0.05.
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