Circadian clock protein Bmal1 within respiratory motor neurons regulates rest phase acute intermittent hypoxia-induced phrenic and ventilatory long-term facilitation in rats

Acute intermittent hypoxia (AIH) elicits respiratory motor plasticity in the phrenic, intercostal, and hypoglossal motor pools, and has emerged as a promising therapeutic strategy to improve breathing function in people with a range of neuromuscular disorders that compromise breathing. Although we recently reported that time-of-day is a powerful regulator of moderate AIH (PaO 2 ~40-50 mmHg) induced respiratory motor plasticity, it is not known if these diurnal effects on AIH-induced phrenic (pLTF) or ventilatory (vLTF) long-term facilitation are mediated by endogenous circadian clock mechanisms vs other factors. Since many biological rhythms are driven by the endogenous circadian clock, and clock genes (including the core clock gene Bmal1) are rhythmically expressed in the phrenic motor system, we hypothesized that the molecular clock within respiratory motor neurons underlies time-of-day effects on pLTF and vLTF in Sprague-Dawley rats (3-6 month old males). Intrapleural injections of small-interfering RNAs (siRNAs) were used to selectively knock down Bmal1 within respiratory motor neurons. AIH consisting of 15, 1-min hypoxic episodes (FIO 2 = 0.09) was delivered in the diurnal mid-rest (i.e. light) or mid-active (i.e. dark) phase, and pLTF (Δintegrated phrenic burst amplitude) and vLTF (ΔFormula: see textE/Formula: see textCO 2 via whole body plethysmography) were assessed in rats given siRNAs targeting Bmal1 mRNA vs non-targeting controls (n = 7/group). Bmal1 protein knockdown within phrenic motor neurons was verified via immunofluorescence. In the mid-rest phase, pLTF was reduced (p < 0.0001) and vLTF abolished (p = 0.0114) in rats given siBmal1 vs controls. However, in the mid-active phase, siBmal1 had no effect on pLTF (p = 0.537) or vLTF (p = 0.975). Thus, the circadian clock within phrenic motor neurons regulates AIH-induced respiratory motor plasticity in a time-of-day-dependent manner. It is important to consider circadian biology in future studies of AIH-induced respiratory motor plasticity. Funding: NIH F32HL176044 (AAJ); R01HL148030 T32HL134621-5 & K99HL175029 (ABM) This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.