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[Rationale:] Obesity leads to resistant hypertension and mechanisms are poorly understood, but high plasma levels of leptin have been implicated. Leptin increases blood pressure acting both centrally in the dorsomedial hypothalamus and peripherally. Sites of the peripheral hypertensive effect of leptin have not been identified. We previously reported that leptin enhanced activity of the carotid sinus nerve, which transmits chemosensory input from the carotid bodies (CBs) to the medullary centers, and this effect was abolished by nonselective blockers of Trp (transient receptor potential) channels. We searched our mouse CB transcriptome database and found that the Trpm7 (transient receptor potential melastatin 7) channel was the most abundant Trp channel., [Objective] To examine if leptin induces hypertension acting on the CB Trpm7., [Methods and Results] C57BL/6J (n=79), leptin receptor (LepRb) deficient db/db mice (n=22), and LepRb-EGFP (n=4) mice were used. CB Trpm7 and LepRb gene expression was determined and immunohistochemistry was performed; CB glomus cells were isolated and Trpm7-like current was recorded. Blood pressure was recorded continuously in (1) leptin-treated C57BL/6J mice with intact and denervated CB; (2) leptin-treated C57BL/6J mice, which also received a nonselective Trpm7 blocker FTY720 administered systemically or topically to the CB area; (3) leptin-treated C57BL/6J mice transfected with Trpm7 small hairpin RNA to the CB, and (4) Leprb deficient obese db/db mice before and after Leprb expression in CB. Leptin receptor and Trpm7 colocalized in the CB glomus cells. Leptin induced a nonselective cation current in these cells, which was inhibited by Trpm7 blockers. Leptin induced hypertension in C57BL/6J mice, which was abolished by CB denervation, Trpm 7 blockers, and Trpm7 small hairpin RNA applied to CBs. Leprb overexpression in CB of Leprb-deficient db/db mice demethylated the Trpm7 promoter, increased Trpm7 gene expression, and induced hypertension., [Conclusions] We conclude that leptin induces hypertension acting on Trmp7 in CB, which opens horizons for new therapy.

Leptin is a potent respiratory stimulant. The carotid bodies (CB) express the long functional isoform of leptin receptor, LepRb, but the role of leptin in CB has not been fully elucidated. The objectives of the current study were (1) to examine the effect of subcutaneous leptin infusion on minute ventilation (VE) and the hypoxic ventilatory response to 10% O2 (HVR) in C57BL/6J mice before and after CB denervation; (2) to express LepRb in CB of LepRb‐deficient obese db/db mice and examine its effects on breathing during sleep and wakefulness and on HVR. We found that leptin enhanced carotid sinus nerve activity at baseline and in response to 10% O2 in vivo. In C57BL/6J mice, leptin increased VE from 1.1 to 1.5 mL/min/g during normoxia (P < 0.01) and from 3.6 to 4.7 mL/min/g during hypoxia (P < 0.001), augmenting HVR from 0.23 to 0.31 mL/min/g/Δurn:x-wiley:00223751:media:tjp13266:tjp13266-math-0001 (P < 0.001). The effects of leptin on VE and HVR were abolished by CB denervation. In db/db mice, LepRb expression in CB increased VE from 1.1 to 1.3 mL/min/g during normoxia (P < 0.05) and from 2.8 to 3.2 mL/min/g during hypoxia (P < 0.02), increasing HVR. Compared to control db/db mice, LepRb transfected mice showed significantly higher VE throughout non‐rapid eye movement (20.1 vs. −27.7 mL/min respectively, P < 0.05) and rapid eye movement sleep (16.5 vs 23.4 mL/min, P < 0.05). We conclude that leptin acts in CB to augment VE and HVR, which may protect against sleep disordered breathing in obesity., This work was funded by the NIH R01s HL133100 to V.Y.P. and J.S.K.S., R01 HL128970 to V.Y.P. and A.R.S., R01 HL138932 to V.Y.P. V.Y.P. is also supported by NIEHS grant P50 ES018176 and EPA Agreements 83615201 & 83451001. C.C.E. was supported in part by ‘Beca de estancias formativas de la Consejería de Salud de Andalucía 2017. C. Caballero Eraso. Número de expediente: EF‐0128‐2016’.

Mice are the most suitable species for understanding genetic aspects of postnatal developments of the carotid body due to the availability of many inbred strains and knockout mice. Our study has shown that the carotid body grows differentially in different mouse strains, indicating the involvement of genes. However, the small size hampers investigating functional development of the carotid body. Hypoxic and/or hyperoxic ventilatory responses have been investigated in newborn mice, but these responses are indirect assessment of the carotid body function. Therefore, we need to develop techniques of measuring carotid chemoreceptor neural activity from young mice. Many studies have taken advantage of the knockout mice to understand chemoreceptor function of the carotid body, but they are not always suitable for addressing postnatal development of the carotid body due to lethality during perinatal periods. Various inbred strains with well-designed experiments will provide useful information regarding genetic mechanisms of the postnatal carotid chemoreceptor development. Also, targeted gene deletion is a critical approach.

We have previously shown that the adult DBA/2J and A/J strains of mice differ in carotid body volume and morphology. The question has arisen whether these differences develop during the prenatal or postnatal period. Investigating morphological development of the carotid body and contributing genes in these mice can provide further understanding of the appropriate formation of the carotid body. We examined the carotid body of these mice from 1 day to 4 wk old for differences in volume, morphology, and gene expression of Gdnf family, Dlx2, Msx2, and Phox2b. The two strains showed divergent morphology starting at 1 wk old. The volume of the carotid body increased from 1 wk up to 2 wk old to the level of 4 wk old in the DBA/2J mice but not in the A/J mice. This corresponds with immunoreactivity of LC3, an autophagy marker, in A/J tissues at 10 days and 2 wk. The differences in gene expression were examined at 1 wk, 10 days, and 2 wk old, because divergent growth occurred during this period. The DBA/2J's carotid body at 1 wk old showed a greater expression of Msx2 than the A/J's carotid body. No other candidate genes showed consistent differences between the ages and strains. The difference was not seen in sympathetic cervical ganglia of 1 wk old, suggesting that the difference is carotid body specific. The current study indicates the critical postnatal period for developing distinctive morphology of the carotid body in these mice. Further studies are required to further elucidate a role of Msx2 and other uninvestigated genes.

Defects in pharyngeal mechanical and neuromuscular control are required for the development of obstructive sleep apnea. Obesity and age are known sleep apnea risk factors, leading us to hypothesize that specific defects in upper airway neuromechanical control are associated with weight and age in a mouse model. In anesthetized, spontaneously breathing young and old wild-type C57BL/6J mice, genioglossus electromyographic activity (EMGGG) was monitored and upper airway pressure-flow dynamics were characterized during ramp decreases in nasal pressure (Pn, cmH2O). Specific body weights were targeted by controlling caloric intake. The passive critical pressure (Pcrit) was derived from pressure-flow relationships during expiration. The Pn threshold at which inspiratory flow limitation (IFL) developed and tonic and phasic EMGGG activity during IFL were quantified to assess the phasic modulation of pharyngeal patency. The passive Pcrit increased progressively with increasing body weight and increased more in the old than young mice. Tonic EMGGG decreased and phasic EMGGG increased significantly with obesity. During ramp decreases in Pn, IFL developed at a higher (less negative) Pn threshold in the obese than lean mice, although the frequency of IFL decreased with age and weight. The findings suggest that weight imposes mechanical loads on the upper airway that are greater in the old than young mice. The susceptibility to upper airway obstruction increases with age and weight as tonic neuromuscular activity falls. IFL can elicit phasic responses in normal mice that mitigate or eliminate the obstruction altogether.

We characterized the passive structural and active neuromuscular control of pharyngeal collapsibility in mice and hypothesized that pharyngeal collapsibility, which is elevated by anatomic loads, is reduced by active neuromuscular responses to airflow obstruction. To address this hypothesis, we examined the dynamic control of upper airway function in the isolated upper airway of anesthetized C57BL/6J mice. Pressures were lowered downstream and upstream to the upper airway to induce inspiratory airflow limitation and critical closure of the upper airway, respectively. After hyperventilating the mice to central apnea, we demonstrated a critical closing pressure (Pcrit) of −6.2 ± 1.1 cmH2O under passive conditions that was unaltered by the state of lung inflation. After a period of central apnea, lower airway occlusion led to progressive increases in phasic genioglossal electromyographic activity (EMGGG), and in maximal inspiratory airflow (V̇imax) through the isolated upper airway, particularly as the nasal pressure was lowered toward the passive Pcrit level. Moreover, the active Pcrit fell during inspiration by 8.2 ± 1.4 cmH2O relative to the passive condition ( P < 0.0005). We conclude that upper airway collapsibility (passive Pcrit) in the C57BL/6J mouse is similar to that in the anesthetized canine, feline, and sleeping human upper airway, and that collapsibility falls markedly under active conditions. Active EMGGG and V̇imax responses dissociated at higher upstream pressure levels, suggesting a decrease in the mechanical efficiency of upper airway dilators. Our findings in mice imply that anatomic and neuromuscular factors interact dynamically to modulate upper airway function, and provide a novel approach to modeling the impact of genetic and environmental factors in inbred murine strains.

Obesity is associated with alterations in upper airway collapsibility during sleep. Obese, leptin-deficient mice demonstrate blunted ventilatory control, leading us to hypothesize that ( 1) obesity and leptin deficiency would predispose to worsening neuromechanical upper airway function and that ( 2) leptin replacement would acutely reverse neuromuscular defects in the absence of weight loss. In age-matched, anesthetized, spontaneously breathing C57BL/6J (BL6) and ob− /ob− mice, we characterized upper airway pressure-flow dynamics during ramp decreases in nasal pressure (PN) to determine the passive expiratory critical pressure (PCRIT) and active responses to reductions in PN, including the percentage of ramps showing inspiratory flow limitation (IFL; frequency), the PN threshold at which IFL developed, maximum inspiratory airflow (Vimax), and genioglossus electromyographic (EMGGG) activity. Elevations in body weight were associated with progressive elevations in PCRIT (0.1 ± 0.02 cmH2O/g), independent of mouse strain. PCRIT was also elevated in ob− /ob− compared with BL6 mice (1.6 ± 0.1 cmH2O), independent of weight. Both obesity and leptin deficiency were associated with significantly higher IFL frequency and PN threshold and lower Vimax. Very obese ob− /ob− mice treated with leptin compared with nontreated mice showed a decrease in IFL frequency (from 63.5 ± 2.9 to 30.0 ± 8.6%) and PN threshold (from −0.8 ± 1.1 to −5.6 ± 0.8 cmH2O) and increase in Vimax (from 354.1 ± 25.3 to 659.0 ± 71.8 μl/s). Nevertheless, passive PCRIT in leptin-treated mice did not differ significantly from that seen in nontreated ob− /ob− mice. The findings suggest that weight and leptin deficiency produced defects in upper airway neuromechanical control and that leptin reversed defects in active neuromuscular responses acutely without reducing mechanical loads.

The role of the carotid body (CB) as a primary chemosensor is well established in large mammals. Due to our ability to better modify the genome the mouse has been used for hypoxic chemosensing rese...

Upper airway obstruction (UAO) can elicit neuromuscular responses that mitigate and/or compensate for the obstruction. It was hypothesised that flow-limited breathing elicits specific timing responses that can preserve ventilation due to increases in inspiratory duty cycle rather than respiratory rate. By altering nasal pressure during non-rapid eye movement (non-REM) sleep, similar degrees of UAO were induced in healthy males and females (n = 10 each). Inspiratory duty cycle, respiratory rate and minute ventilation were determined for each degree of UAO during non-REM sleep and compared with the baseline nonflow-limited condition. A dose-dependent increase in the inspiratory duty cycle and respiratory rate was observed in response to increasing severity of UAO. Increases in the inspiratory duty cycle, but not respiratory rate, helped to acutely maintain ventilation. Heterogeneity in these responses was associated with variable degrees of ventilatory compensation, allowing for the segregation of individuals at risk for hypoventilation during periods of inspiratory airflow limitation. Upper airway obstruction constitutes a unique load on the respiratory system. The inspiratory duty cycle, but not the respiratory rate, determine the individual9s ability to compensate for inspiratory airflow limitation during sleep, and may represent a quantitative phenotype for obstructive sleep apnoea susceptibility.

To examine the dynamic modulation of upper airway (UA) function during sleep, we devised a novel approach to measuring the critical pressure (Pcrit) within a single breath in tracheostomized sleep apnea patients. We hypothesized that the UA continuously modulates airflow dynamics during transtracheal insufflation. In this study, we examine tidal pressure-flow relationships throughout the respiratory cycle to compare phasic differences in UA collapsibility between closure and reopening. Five apneic subjects (with tracheostomy) were recruited (2 men, 3 women; 18–50 yr; 20–35 kg/m2; apnea-hypopnea index >20) for this polysomnographic study. Outgoing airflow through the UA (face mask pneumotachograph) and tracheal pressure were recorded during brief transtracheal administration of insufflated airflow via a catheter. Pressure-flow relationships were generated from deflation (approaching Pcrit) and inflation (after Pcrit) of the UA during non-rapid eye movement sleep. During each breath, UA function was described by a pressure-flow relationship that defined the collapsibility (Pcrit) and upstream resistance (Rus). UA characteristics were examined in the presence and absence of complete UA occlusion. We demonstrated that Pcrit and Rus changed dynamically throughout the respiratory cycle. The UA closing pressure (4.4 ± 2.0 cmH2O) was significantly lower than the opening pressure (10.8 ± 2.4 cmH2O). Rus was higher for deflation (18.1 ± 2.4 cmH2O·l−1·s) than during inflation (7.5 ± 1.9 cmH2O·l−1·s) of the UA. Preventing occlusion decreases UA pressure-flow loop hysteresis by ∼4 cmH2O. These findings indicate that UA collapsibility varies dynamically throughout the respiratory cycle and that both local mechanical and neuromuscular factors may be responsible for this dynamic modulation of UA function during sleep.