The Molecular Circadian Clock of Phox2b-expressing Cells Drives Daily Variation of the Hypoxic but Not Hypercapnic Ventilatory Response in Mice

Abstract

While the suprachiasmatic nucleus (SCN) controls 24-hour rhythms in breathing including minute ventilation (VE), the mechanisms by which the SCN drives these daily changes are not well understood. Moreover, the extent to which the circadian clock regulates hypercapnic and hypoxic ventilatory chemoreflexes is unknown. We hypothesized that the SCN regulates daily breathing and chemoreflex rhythms by synchronizing the molecular circadian clock of cells. We used whole-body plethysmography to assess ventilatory function in transgenic BMAL1 knockout (KO) mice to determine the role of the molecular clock in regulating daily rhythms in ventilation and chemoreflex. Unlike their wild-type littermates, BMAL1 KO mice exhibited a blunted daily rhythm in VE and failed to demonstrate daily variation in hypoxic (HVR) and hypercapnic (HCVR) chemoreflexes. To determine if the observed phenotype was mediated by the molecular clock of key respiratory cells, we then assessed ventilatory rhythms in BMAL1fl/fl; Phox2bCre/+ mice which lack BMAL1 in all Phox2b-expressing chemoreceptor cells (hereafter called BKOP). BKOP mice lacked daily variation in HVR, similar to BMAL1 KO mice. However, unlike BMAL1 KO mice, BKOP mice exhibited circadian variations in VE and HCVR. These data indicate that the SCN regulates daily rhythms in VE, HVR, and HCVR, in part, through the synchronization of the molecular clock. Moreover, the molecular clock of Phox2b-expressing cells is specifically necessary for daily variation in the hypoxic chemoreflex. These findings suggest that disruption of circadian biology may undermine respiratory homeostasis which, in turn, may have clinical implications for respiratory disease.

In light of breathing: environmental light is an important modulator of breathing with clinical implications

“…This indicates that light has the ability to immediately drive behavior independently of the SCN (also referred to as “light masking”). In line with this notion, we recently identified that mice lacking a functional molecular clock continue to exhibit a detectable daily rhythm in minute ventilation when housed on a standard light:dark cycle ( Jones et al, 2023 ). This suggests that, like some other behaviors, light can synchronize daily breathing rhythms independent of the circadian clock.…”

“…The SCN appears to entrain airway tissues via the vagal nerve ( Bando et al, 2007 ), however, it is currently unknown how the SCN confers timing to other respiratory structures. While it is unclear whether these other mechanisms contribute to daily breathing behavior, we recently demonstrated that eliminating molecular time-keeping specifically within respiratory cells of the autonomic nervous system abolishes circadian variation in the hypoxic ventilatory response ( Jones et al, 2023 ). This finding suggests that the circadian clock may organize daily breathing rhythms, in part, via the organization of clock genes in the neural respiratory network.…”

“…The DRN has the potential to modulate breathing Brn3bDTA (Opn4 cre/+ ; Brn3b DTA/+ ) and wild-type littermates (Opn4 cre/+ ; Brn3b +/+ ) were bred in-house from founders provided by Dr. Tiffany Schmidt (Northwestern University) according to the breeding strategy of prior studies (Chen et al, 2011;Fernandez et al, 2018). Minute ventilation was measured continuously for 37 h at 11-24 weeks of age by means of whole-body plethysmography (SCIREQ/Emka Technologies, Paris, France) using previously described parameters (Jones et al, 2023). All methods were reviewed, approved by, and performed according to the guidelines of the Institutional Animal Care and Use Committee of Marquette University (Milwaukee, WI).…”