Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA080

Poster Communications

NADPH oxidase 2 is necessary for chronic intermittent hypoxia-induced respiratory muscle weakness in adult male mice

S. E. Drummond1, D. P. Burns1, V. Healy1, K. D. O'Halloran1

1. Department of Physiology, University College Cork, Cork, Ireland.


Obstructive sleep apnoea syndrome (OSAS), the most prevalent form of sleep-disordered breathing (SDB), 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 upper airway and diaphragm muscle function. 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 enzyme in CIH-induced respiratory muscle dysfunction. 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. 10-11 week old male (C57BL/6J) mice were assigned to one of 3 groups: normoxic controls (sham; n=24), CIH-exposed (n=24) and CIH+apocynin (NADPH oxidase 2 inhibitor, 2mM; n=24) given in the drinking water throughout the CIH exposure. In separate studies, 10-11 week old NOX2 null (B6.129S-Cybbtm1Din/J) male mice were assigned to a sham (n=12) or CIH (n=12) exposure. Sternohyoid and diaphragm muscle contractile function was examined ex vivo. Gene expression was examined by qRT-PCR. Western blot was used to measure protein expression. NOX enzyme activity was determined using a spectrophotometric assay. All values are expressed as mean ± SD and data were statistically compared by unpaired Student t-test. Exposure to CIH significantly decreased sternohyoid (9.4 ± 1.6 vs. 5.2 ± 1.4 N/cm2; p<0.001) and diaphragm (22.6 ± 5.6 vs. 12.5 ± 2.3 N/cm2; p<0.01) muscle peak specific force by ~45% compared with sham exposure. CIH significantly increased NOX enzyme activity in the sternohyoid (0.6 ± 0.1 vs. 0.8 ± 0.1 slope/mg/hr; p<0.01), with no significant alterations in gene or protein expression of NOX subunits compared with sham. CIH decreased the expression of p22phox (1.1 ± 0.4 vs. 0.6 ± 0.4 mRNA fold change relative to control; p<0.05) and rac (1.2 ± 0.6 vs. 0.7 ± 0.3 mRNA fold change relative to control; p<0.05) genes and increased protein expression of NOX4 (0.5 ± 0.2 vs. 1.0 ± 0.2 O.D./Ponceau S (a.u.); p<0.01) in the diaphragm, while NOX activity remained unchanged compared with sham. Administration of apocynin and NOX2 gene knockout completely prevented CIH-induced sternohyoid and diaphragm muscle weakness. Mice show signs of profound respiratory muscle dysfunction following exposure to 2 weeks of CIH. The putative NOX inhibitor, apocynin, prevents CIH-induced respiratory muscle weakness. Studies in NOX2 null mice reveal that NOX2 is necessary for CIH-induced sternohyoid and diaphragm muscle weakness. Our results have implications for human OSAS.

Where applicable, experiments conform with Society ethical requirements