Chronic N-acetyl cysteine treatment does not improve respiratory system performance in the mdx mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is characterised by respiratory muscle injury, inflammation, fibrosis and weakness, ultimately culminating in respiratory failure. The dystrophin-deficient mouse model of DMD (mdx) shows evidence of respiratory muscle remodelling and dysfunction contributing to impaired respiratory system performance. The antioxidant N-acetylcysteine (NAC) has been shown to exert anti-inflammatory and anti-fibrotic effects leading to improved respiratory muscle performance in a range of animal models of muscle dysfunction, including mdx mice, following short-term administration (2 weeks). We sought to build on previous work by exploring the effects of chronic NAC administration (3 months) on respiratory system performance in mdx mice. One-month-old male mdx mice were randomised to receive normal drinking water (n = 30) or 1% NAC in the drinking water (n = 30) for 3 months. At 4 months of age, we assessed breathing in conscious mice by plethysmography followed by ex vivo assessment of diaphragm force-generating capacity. Additionally, diaphragm histology was performed. In separate studies, in anaesthetised mice, respiratory electromyogram (EMG) activity and inspiratory pressure across a range of behaviours were determined, including assessment of peak inspiratory pressure-generating capacity. NAC treatment did not affect force-generating capacity of the mdx diaphragm. Collagen content and immune cell infiltration were unchanged in mdx + NAC compared with mdx diaphragms. Additionally, there was no significant effect of NAC on breathing, ventilatory responsiveness, inspiratory EMG activity or inspiratory pressure across the range of behaviours from basal conditions to peak system performance. We conclude that chronic NAC treatment has no apparent beneficial effects on respiratory system performance in the mdx mouse model of DMD suggesting limited potential of NAC treatment alone for human DMD.

Loss of compensation afforded by accessory muscles of breathing leads to respiratory system compromise in the mdx mouse model of Duchenne muscular dystrophy.

Despite profound diaphragm weakness, peak inspiratory pressure-generating capacity is preserved in young mdx mice revealing adequate compensation by extra-diaphragmatic muscles of breathing in early dystrophic disease. We hypothesised that loss of compensation gives rise to respiratory system compromise in advanced dystrophic disease. Studies were performed in male wild-type (n = 196) and dystrophin-deficient mdx mice (n = 188) at 1, 4, 8, 12 and 16 months of age. In anaesthetised mice, inspiratory pressure and obligatory and accessory respiratory EMG activities were recorded during baseline and sustained tracheal occlusion for up to 30-40 s to evoke peak system activation to task failure. Obligatory inspiratory EMG activities were lower in mdx mice across the ventilatory range to peak activity, emerging in early dystrophic disease. Early compensation protecting peak inspiratory pressure-generating capacity in mdx mice, which appears to relate to transforming growth factor-ß1-dependent fibrotic remodelling of the diaphragm and preserved accessory muscle function, was lost at 12 and 16 months of age. Denervation and surgical lesion of muscles of breathing in 4-month-old mice revealed a greater dependency on diaphragm for peak inspiratory performance in wild-type mice, whereas mdx mice were heavily dependent upon accessory muscles (including abdominal muscles) for peak performance. Accessory EMG activities were generally preserved or enhanced in young mdx mice, but peak EMG activities were lower than wild-type by 12 months of age. In general, ventilation was reasonably well protected in mdx mice until 16 months of age. Despite the early emergence of impairments in the principal obligatory muscles of breathing, peak inspiratory performance is compensated in early dystrophic disease due to diaphragm remodelling and facilitated contribution by accessory muscles of breathing. Loss of compensation afforded by accessory muscles underpins the emergence of respiratory system morbidity in advanced dystrophic disease. KEY POINTS: Despite diaphragm weakness, peak inspiratory performance is preserved in young dystrophin-deficient mdx mice revealing adequate compensation by extra-diaphragmatic muscles. Peak obligatory muscle (diaphragm, external intercostal, and parasternal intercostal) EMG activities are lower in mdx mice, emerging early in dystrophic disease, before the temporal decline in peak performance. Peak EMG activities of some accessory muscles are lower, whereas others are preserved. There is greater recruitment of the trapezius muscle in mdx mice during peak system activation. In phrenicotomised mice with confirmed diaphragm paralysis, there is a greater contribution made by extra-diaphragmatic muscles to peak inspiratory pressure in mdx compared with wild-type mice. Surgical lesion of accessory (including abdominal) muscles adversely affects peak pressure generation in mdx mice. Diaphragm remodelling leading to stiffening provides a mechanical advantage to peak pressure generation via the facilitated action of extra-diaphragmatic muscles in early dystrophic disease. Peak accessory EMG activities are lower in 12-month-old mdx compared to wild-type mice. Peak inspiratory pressure declines in mdx mice with advanced disease. We conclude that compensation afforded by accessory muscles of breathing declines in advanced dystrophic disease precipitating the emergence of respiratory system dysfunction.

Ventilatory Effects of Acute Intermittent Hypoxia in Conscious Dystrophic Mice.

Exposure to acute intermittent hypoxia (AIH) elicits a form of respiratory plasticity known as long-term facilitation (LTF). Interest has grown in developing AIH interventions to treat ventilatory insufficiency, with promising results in spinal cord injury and amyotrophic lateral sclerosis. Therapeutic AIH may have application in neuromuscular disorders including muscular dystrophies. We sought to establish hypoxic ventilatory responsiveness and the expression of ventilatory LTF in X-linked muscular dystrophy (mdx) mice.Experiments were performed in 15 male wild-type (BL10) and 15 male mdx mice at 4 months of age. Ventilation was assessed using whole-body plethysmography. Baseline measures of ventilation and metabolism were established. Mice were exposed to 10 successive bouts of hypoxia, each lasting 5 min, interspersed with 5-min bouts of normoxia. Measurements were taken for 60 min following termination of AIH.In mdx mice, ventilation was significantly increased 60 min post-AIH compared to baseline. However, metabolic CO2 production was also increased. Therefore, ventilatory equivalent was unaffected by AIH exposure, i.e., no ventilatory LTF manifestation. In wild-type mice, ventilation and metabolism were not affected by AIH.Eliciting ventilatory LTF is dependent on many factors and may require concomitant isocapnia or hypercapnia during AIH exposures and/or repeated daily AIH exposures, which is worthy of further pursuit.