Igor Simões Assunção Felippe

Aims: The carotid body (CB) is a multimodal chemosensory organ, yet how it encodes differential external stimuli to drive distinct peripheral chemoreflex responses remains unclear. This study aimed to define differences in intrinsic cellular mechanisms underlying CB signalling and to investigate how these processes are altered in pre-hypertension. Methods and results: We used potassium cyanide (KCN) as a hypoxia mimetic to activate the CB. K+ channels implicated in hypoxia sensing were pharmacologically inhibited, with TWIK-related acid-sensitive K+ (TASK) channels targeted using Ba2+ or ML365 and BK/Kv channels targeted using TEA + 4AP or iberiotoxin. We combined in vitro Ca2+ imaging of dissociated glomus cells, ex vivo carotid sinus nerve (CSN) recordings, and an in situ double-perfused working heart-brainstem preparation to investigate chemosensory signalling in juvenile Wistar and age-matched pre-hypertensive spontaneously hypertensive rats (SHRs). KCN application to the CB ex vivo evoked a biphasic CSN response, consisting of two temporally distinct components. This biphasic response was exaggerated in SHRs, with disproportionate changes in the magnitude of each component. Ca2+ imaging revealed that these components reflect distinct glomus cell subpopulations, characterized by transient vs. prolonged KCN-evoked Ca2+ events, with their relative proportions shifted in hypertension. Pharmacological dissection suggested that differential inactivation of K+ channels contributes to these divergent Ca2+ dynamics. In situ, we confirmed that localized Ba2+ applied to the CB facilitated stronger tachypnoeic and bradycardic chemoreflex evoked responses than TEA + 4AP, consistent with functional heterogeneity among glomus cell populations. Compared with Wistar rats, in vitro TASK and BK/Kv channel expression and activity were differentially altered in SHRs, accompanied by corresponding changes in in situ chemoreflex characteristics. Conclusion: We identify, for the first time, distinct glomus cell subpopulations defined by their K+ channel expression that differentially contribute to CB signalling patterns and distinct chemoreflex pathways. These findings provide mechanistic support for the recently proposed ‘ribbon cable’ hypothesis, whereby discrete glomus cell populations couple to specific chemoreflex outputs. These findings offer new insight into CB signalling complexity and its role in autonomic dysfunction during the early stages of hypertension.

Cardiorespiratory homeostasis is maintained by carotid body (CB) peripheral chemoreceptors that monitor blood oxygen and stimulate breathing. While proportionality between peripheral chemoreflex sensitivity and respiratory response is relative to hypoxia severity, the fundamental regulatory mechanisms remain unknown. We hypothesized that intercellular amino acid transmission between glomus cells modulates CB sensitivity. We identified a full complement of glutamate and gamma‐aminobutyric acid (GABA) synaptic signalling machinery in the CB. During hypoxia stimulation, glutamate acting via NMDA receptors (NMDA‐R) stimulated CB afferents while releasing GABA, which acted on GABA _A receptors to rapidly attenuate afferent drive mediated by purinergic P2X receptors. We propose that this intrinsic ‘accelerator–brake’ mechanism regulates CB sensitivity to hypoxia. Further, glutamate and NMDA‐R were essential for long‐term facilitation (LTF) of respiratory chemoreflex responses induced by repeated hypoxia or glutamate exposures. The accelerator–brake mechanism establishes the set‐point of chemoreflex gain and provides plasticity for modulation, explaining ventilatory LTF to repeated bouts of hypoxia. image Key points: An intra‐carotid body interaction between glutamate and GABA transmission is crucial for the generation of the biphasic carotid body afferent nerve response when stimulated with low doses of sodium cyanide. Glutamate stimulates the carotid body chemoreflex to increase inspiratory drive without affecting sympathetic activity, heart rate or perfusion pressure, indicating selective activation of a carotid body respiratory reflex arc. Repetitive application of glutamate to the carotid body sensitizes chemoreflex phrenic nerve responses producing long‐term facilitation.

General anesthetics enable invasive experimentation but can affect cardiovascular and respiratory physiology, biasing preclinical outcomes. We compared five anesthetic regimens in adult male Wistar rats, tribromoethanol (TBE, 250 mg/kg i.p.), chloral hydrate (CH, 400 mg/kg i.p.), ketamine–xylazine (KX, 80/10 mg/kg i.p.), thiopental (TP, 80 mg/kg i.p.), and isoflurane (ISO, 4% induction, 2% maintenance), to investigate integrated cardiorespiratory and biochemical markers. Femoral arterial catheterization allowed continuous blood pressure (BP) and derived heart rate (HR) recordings, while ventilation was assessed through pletysmography at baseline (awake), during induction, and recovery phases of anesthesia. Variability was evaluated in the time and frequency domains, including HR, systolic blood pressure (SBP), and spontaneous baroreflex sensitivity. In an independent cohort of rats, butyrylcholinesterase (BChE), CK-MB, cTnI, and LDH were measured. Baseline BP was unchanged by TBE and TP, whereas all anesthetics affected HR. Minute ventilation and breathing frequency were reduced with all agents, while tidal volume decreased with KX and TBE only. LDH and cTnI were unaffected, BChE was reduced by KX, TBE, and ISO, and CK-MB increased with CH and KX. Variability analysis showed that all anesthetics depressed pulse-interval and SBP variability and shifted spectral power toward higher frequencies, while baroreflex sensitivity and effectiveness were consistently reduced. During recovery, KX and TP restored most variability indices, whereas CH, TBE, and ISO showed persistent suppression. These findings highlight distinct profiles of cardiovascular depression and biomarker responses across anesthetics and underscore the importance of accounting for autonomic variability when selecting different anesthetics in experimental protocols. Highlights: Five anesthetic regimens were tested in rats. All anesthetics reduced ventilation, and KX and TBE also reduced tidal volume. CH and KX increased CKMB, while KX, TBE and ISO reduced BChE. All anesthetics reduced blood pressure variability and baroreflex sensitivity. Variability recovered with TP and KX, whereas CH, TBE and ISO showed persistent suppression.

Aims: Elevated sympathetic nerve activity (eSNA) is a hallmark of cardiovascular disease and represents an important clinical target for disease management. A known driver of eSNA is aberrant signalling from, and sensitization of, the carotid body (CB) arterial chemoreceptors. Sensitization is coupled with CB hypertrophy, the cause of which remains unclear. Here, we set out to characterize the morphological basis of CB hypertrophy in hypertension to understand its aberrant activity. Methods and results: Using high-throughput fluorescence microscopy, we mapped the neurovascular interface and chemoafferent innervation of the CB of spontaneously hypertensive rats (SHR) across multiple age groups. We show that CB hypertrophy driven by chemosensory (type I) cell hyperplasia and an expanded vascular network is evident in 4–6-week-old SHR without established hypertension. Specifically, CB hypertrophy in the SHR is linked to increased chemoafferent innervation and an age-dependent remodelling of nerve fibre composition. Conclusions: CB hypertrophy in hypertension is associated with chemosensory hyperplasia and angiogenesis, likely mediated by impaired HIF-PHD signalling in the SHR. We propose that CB size may serve as a candidate marker of chemoafferent sensitivity and the efficacy of therapies targeting the CB. However, further validation in humans is needed to support this link. Neurotrophic pathways promoting increased chemoafferent innervation in hypertension are proposed as a potential target for modulating CB activity in sympathetically mediated diseases.

Aims: ATP acting on P2X3 receptors (P2X3R) within carotid bodies (CBs) underpins chemoreflex-mediated sympathetic overactivity in spontaneously hypertensive rats (SHR). Pyridoxal 5′ phosphate (PLP), the active form of vitamin B6, has been reported to act as a non-selective P2X receptor blocker. Hence, we hypothesised that PLP antagonism of P2X3R in the CB would treat hypertension. Methods and results: Herein, we employed a multipronged approach to investigate PLP’s capability to attenuate CB hyperexcitability in hypertension. First, PLP inhibited Ca2+ responses evoked by α, β-methylene ATP in cell lines expressing human (h) P2X3R with an IC50 of 8.7 µM. Next, in-silico data predicted that PLP binds to the same site of Gefapixant, supporting an allosteric antagonism. Using an isolated perfused carotid artery bifurcation-CB preparation, arterial infusion of PLP (50 µM; 15 min) attenuated CBs sensory firing in SHR (P = 0.012). Using the in situ working-heart brainstem preparation, carotid artery injections of PLP (1–5 mM) attenuated the chemoreflex-evoked sympathetic (P = 0.023) but not phrenic (P = 0.62) responses; the CB was stimulated with potassium cyanide (KCN,50 µL; 0.04%). In awake telemetered SHR (n = 6), intravenous infusion of PLP (48 mg/Kg/h; 30 min) attenuated KCN-evoked chemoreflex responses and reduced systolic, diastolic, and mean blood pressures (ΔMBP = −15.6 mmHg; P = 0.025). Translating our results, we performed a small double-blind, randomised clinical trial. In volunteers with hypertension (n = 14), oral supplementation with pyridoxine hydrochloride (600 mg) attenuated the hypoxic ventilatory response only in patients with high peripheral chemoreflex sensitivity (P = 0.021). Conclusion: Our findings suggest that PLP binds to and antagonises P2X3R and is a viable candidate for larger clinical trials to treat CB dysregulation in cardiovascular diseases.

BACKGROUND: The internal milieu of the body is controlled by a system of interoceptors coupled to motor outflows that drive compensatory adaptive responses. These include the arterial chemoreceptors, best known for sensing arterial oxygen. In cardiometabolic diseases, such as essential hypertension, the carotid bodies (CB) exhibit heightened reflex sensitivity and tonic activity without an apparent stimulus. The mechanisms behind CB sensitization in these conditions are not well understood. METHODS: Guided by functional genomics, a range of functional assays is used to interrogate downstream intracellular and interorgan signaling pathways involved in arterial chemosensory function. RESULTS: Here, we report the presence of the MC4R (melanocortin 4 receptor) in the mammalian CB and show its elevated expression in experimental hypertension. We demonstrate that melanocortin agonists activate arterial chemosensory cells, modulating CB chemosensory afferent drive to influence chemoreflex-evoked sympathetic and ventilatory activity. Transcriptional analysis of hypertensive CB implicates the activation of the Mash1 (mammalian achaete-scute homolog 1; Ascl1 ) regulatory network in driving elevated Mc4r expression. CONCLUSIONS: Collectively, our data indicate a primarily pathophysiological role of melanocortin signaling in arterial chemosensation, contributing to excess sympathetic activity in cardiometabolic disease.

Background: The body’s internal milieu is controlled by a system of interoceptors coupled to motor outflows that drive compensatory adaptive responses. These include the arterial chemoreceptors, best known for sensing arterial oxygen. In cardiometabolic diseases, such as essential hypertension, the carotid bodies (CB) exhibit heightened reflex sensitivity and tonic activity without an apparent stimulus. The mechanisms behind CB sensitization in these conditions are not well understood. Methods: Guided by functional genomics, a range of functional assays is used to interrogate downstream intracellular and interorgan signalling pathways involved in arterial chemosensory function. Results: Here, we report the presence of the Melanocortin 4 receptor (MC4R) in the mammalian CB and show its elevated expression in experimental hypertension. We demonstrate that melanocortin agonists activate arterial chemosensory cells, modulating CB chemosensory afferent drive to influence both resting and chemoreflex-evoked sympathetic and ventilatory activity. Transcriptional analysis of hypertensive CB implicates the activation of the Mash1 ( Ascl1 ) regulatory network in driving elevated Mc4r expression. Conclusions: Collectively, our data indicate a primarily pathophysiological role of melanocortin signalling in arterial chemosensation, contributing to excess sympathetic activity in cardiometabolic disease.

Carotid body pathophysiology is associated with many cardiovascular–respiratory–metabolic diseases. This pathophysiology reflects both hyper‐sensitivity and hyper‐tonicity. From both animal models and human patients, evidence indicates that amelioration of this pathophysiological signalling improves disease states such as a lowering of blood pressure in hypertension, a reduction of breathing disturbances with improved cardiac function in heart failure (HF) and a re‐balancing of autonomic activity with lowered sympathetic discharge. Given this, we have reviewed the mechanisms of carotid body hyper‐sensitivity and hyper‐tonicity across disease models asking whether there is uniqueness related to specific disease states. Our analysis indicates some commonalities and some potential differences, although not all mechanisms have been fully explored across all disease models. One potential commonality is that of hypoperfusion of the carotid body across hypertension and HF, where the excessive sympathetic drive may reduce blood flow in both models and, in addition, lowered cardiac output in HF may potentiate the hypoperfusion state of the carotid body. Other mechanisms are explored that focus on neurotransmitter and signalling pathways intrinsic to the carotid body (e.g. ATP, carbon monoxide) as well as extrinsic molecules carried in the blood (e.g. leptin); there are also transcription factors found in the carotid body endothelium that modulate its activity (Krüppel‐like factor 2). The evidence to date fully supports that a better understanding of the mechanisms of carotid body pathophysiology is a fruitful strategy for informing potential new treatment strategies for many cardiovascular, respiratory and metabolic diseases, and this is highly relevant clinically. image