NADPH oxidase 2 knockout prevents chronic intermittent hypoxia‐induced diaphragm muscle weakness but not increased propensity for apnoea in a mouse model of obstructive sleep apnoea syndrome
Authors: Drummond Sarah E. , Burns David P. , Ziegler Oscar , El Maghrani Sarah , Healy Vincent , O’Halloran Ken D. 
Published: April 2020

Obstructive sleep apnoea syndrome (OSAS) is characterized by exposure to chronic intermittent hypoxia (CIH), as a consequence of repetitive occlusions of the upper airway in patients during sleep. CIH evokes redox changes culminating in impaired diaphragm muscle function and aberrant respiratory plasticity which manifests as destabilised breathing during sleep. We sought to investigate the putative role of the superoxide‐generating NADPH oxidase 2 (NOX2) enzyme in CIH‐induced diaphragm muscle dysfunction and respiratory maladaptation.

A mouse model of CIH was generated by the cycling of gas from normoxia (21% O2) for 210 seconds to hypoxia (5% O2 at the nadir) over 90 seconds for 8hr/day during light hours for 2 weeks. Adult male (C57BL/6J) mice were assigned to one of 5 groups: normoxic controls, CIH‐exposed and CIH+apocynin (NADPH oxidase 2 inhibitor, 2mM) given in the drinking water throughout the CIH exposure, NOX2 null (B6.129S‐Cybbtm1Din/J) assigned to a sham or CIH exposure. On day 15, whole body plethysmography was used to measure breathing parameters on a breath‐by‐breath basis in room air. An apnoea was defined as ≥ 2 missed breaths. Diaphragm muscle contractile function was examined ex vivo. Gene expression was examined by qRT‐PCR. Markers of oxidative stress were determined using spectrophotometric assays. Values are expressed as mean ± SD and data were statistically compared by unpaired Student t‐test.

Exposure to CIH significantly decreased diaphragm muscle peak specific force by ~45% compared with sham exposure. Apocynin and NOX2 gene knockout completely prevented CIH‐induced diaphragm muscle weakness. CIH resulted in no overt oxidative stress in diaphragm evidenced by unaltered thiobarbituric acid reactive substances (TBARS) and citrate synthase activity. CIH increased mRNA expression of NRF2, MuRF1, BNIP3, LC3B, GABARAPL1, PARK2, Myostatin, Myogenin, MEF2C, MyoD. Apocynin and NOX2 gene knockout prevented these CIH‐induced increases. Basal minute ventilation was unchanged following 2 weeks of CIH exposure however the number of apnoeas per hour was significantly increased compared with sham exposed mice. Apocynin intervention significantly reduced the frequency of apnoeas compared with the CIH group. Apnoea index was increased in NOX2 null mice exposed to CIH compared with NOX2 null sham mice reminiscent of that observed in wild‐type mice.

Mice show signs of profound diaphragm muscle dysfunction and increased mRNA expression of genes relating to muscle atrophy, mitophagy, autophagy and regeneration following 2 weeks of CIH. Studies in NOX2 null mice reveal that NOX2 is necessary for CIH‐induced diaphragm muscle weakness. CIH‐induced increase in the propensity for apnoea may be of biological relevance as it may underpin progression in the severity of OSAS pathology (i.e. mild to moderate OSA). The reduction in apnoea frequency following treatment with apocynin implicates ROS (that are not NOX2‐derived) in the manifestation of CIH‐induced respiratory disturbances. Our results have implications for human OSAS and point to antioxidant intervention as a potential therapeutic strategy.