Circadian clock protein Bmal1 regulates respiratory motor plasticity in male 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 respiratory function in people with neuromuscular disorders that compromise breathing. Although we recently reported that time-of-day regulates moderate AIH (PaO 2 ∼40-50 mmHg) induced respiratory motor plasticity, it is unknown if diurnal effects on AIH-induced phrenic (pLTF) or ventilatory (vLTF) long-term facilitation are mediated via the endogenous circadian clock vs other factors. Since many biological rhythms are driven by the endogenous clock, and clock genes (including the essential clock gene Bmal1) are rhythmically expressed in the phrenic motor system, we hypothesized that the molecular clock within respiratory motor neurons exerts 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 by ∼30%. AIH consisting of 15, 1-min hypoxic episodes (FIO 2 = 0.09) was delivered in the mid-rest ( i.e. light) or mid-active ( i.e. dark) phases, and pLTF (Δintegrated phrenic burst amplitude) and vLTF (ΔV̇E/V̇CO 2 ) were assessed in rats given siRNAs targeting Bmal1 vs non-targeting controls. In mid-rest phase, pLTF was reduced and vLTF abolished in rats given siBmal1 vs non-targeting siRNA. However, siBmal1 had no significant effect on either pLTF or vLTF in mid-active phase. Thus, the phrenic motor neuron circadian clock 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.