The role of NADPH oxidase-2 in chronic intermittent hypoxia-induced respiratory system dysfunction

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Drummond, Sarah E.
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University College Cork
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Obstructive sleep apnoea syndrome (OSAS) is characterised by exposure to chronic intermittent hypoxia (CIH) as a consequence of repetitive occlusions of the upper airway in patients during sleep. Exposure to CIH evokes redox changes which culminate in impaired upper airway and diaphragm muscle function. Excessive reactive oxygen species (ROS) are also associated with aberrant respiratory plasticity, which manifests as destabilised breathing during sleep. There is a paucity of information regarding the molecular mechanisms underlying these effects. We sought to investigate the putative role of the superoxide-generating NADPH oxidase-2 (NOX2) enzyme in CIH-induced respiratory muscle dysfunction and respiratory mal-adaptation. 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 for 2 weeks. Adult male (C57BL/6J) mice were assigned to one of 5 groups: normoxic controls, CIH-exposed, CIH + apocynin (NOX2 inhibitor, 2mM) given in the drinking water throughout the exposure to CIH, and NOX2 null (B6.129S-Cybbtm1Din/J) assigned to a sham or CIH exposure. On day 15, whole body plethysmography (WBP) was used to measure breathing parameters on a breath-by-breath basis in room air and in response to chemostimulation. An apnoea was defined as ≥ 2 missed breaths. Sternohyoid and diaphragm muscle contractile function was examined ex vivo. Gene expression was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Western blot was used to measure protein expression. NOX enzyme activity and indices of oxidative stress were determined using spectrophotometric assays. Basal minute ventilation (V̇I) was unchanged following 2 weeks of exposure to CIH, 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-exposed group. Apnoea index was increased in NOX2 null mice exposed to CIH, reminiscent of that observed in wild‐type mice. Exposure to CIH resulted in severe sternohyoid and diaphragm muscle weakness, evidenced by a ~45% reduction in the force-generating capacity of these respiratory muscles compared with sham. Exposure to CIH increased NOX enzyme activity in the sternohyoid, with no alteration to the gene or protein expression of NOX subunits. In contrast, exposure to CIH increased the protein and mRNA expression of NOX4 in the diaphragm, while NOX activity and expression of all other NOX subunits remained unchanged. No indication of overt oxidative stress was observed in the diaphragm or sternohyoid muscle following exposure to CIH. Apocynin treatment and NOX2 gene knock-out completely prevented CIH‐induced diaphragm and sternohyoid muscle weakness. Exposure to CIH increased the mRNA expression of genes relating to autophagy, atrophy and muscle differentiation in the diaphragm in a NOX2-dependent manner; these CIH-induced responses were not observed in the sternohyoid. Mice show signs of profound respiratory muscle dysfunction following exposure to a mild-to-moderate paradigm of CIH. The putative NOX inhibitor, apocynin, prevents CIH-induced respiratory muscle weakness. Moreover, studies in NOX2 null mice reveal that NOX2 is obligatory for CIH-induced respiratory muscle weakness. The mechanisms underpinning CIH-induced muscle weakness likely differ between sternohyoid and diaphragm muscle, evidenced by varied molecular responses to CIH, which may relate to differences in fibre-type expression. CIH-induced respiratory muscle weakness may contribute to upper airway obstruction and impaired swallow and cough, relevant to OSAS. The reduction in apnoea frequency following treatment with apocynin, but not NOX2 knock-out, implicates ROS (that are not NOX2-derived) in the manifestation of CIH-induced respiratory disturbances. CIH-induced increase in the propensity for apnoea may be of clinical relevance as it may underpin progression in the severity of OSAS pathology (i.e. mild-to-moderate-to-severe OSAS). Our results have implications for human OSAS and point to antioxidant intervention, potentially targeting NOX2 blockade, as a potential therapeutic strategy.
Obstructive sleep apnoea , Chronic intermittent hypoxia , NADPH oxidase , Respiratory control system
Drummond, S. E. 2020. The role of NADPH oxidase-2 in chronic intermittent hypoxia-induced respiratory system dysfunction. PhD Thesis, University College Cork.