David P. Burns

Evidence of hypoxic tolerance in weak upper airway muscle from young mdx mice

Duchenne muscular dystrophy (DMD) is a genetic disease characterised by deficiency in the protein dystrophin. The respiratory system is weakened and patients suffer from sleep disordered breathing and hypoventilation culminating in periods of hypoxaemia. We examined the effects of an acute (6h) hypoxic stress on sternohyoid muscle function (representative pharyngeal dilator). 8 week old male, wild-type (WT; C57BL/10ScSnJ; n=18) and mdx (C57BL/10ScSn-Dmd(mdx)/J; n=16) mice were exposed to sustained hypoxia (FIO2=0.10) or normoxia. Muscle functional properties were examined ex vivo. Additional WT (n=5) and mdx (n=5) sternohyoid muscle was exposed to an anoxic challenge. Sternohyoid dysfunction was observed in mdx mice with significant reductions in force and power. Following exposure to the acute in vivo hypoxic stress, WT sternohyoid muscle showed evidence of functional impairment (reduced force, work and power). Conversely, mdx sternohyoid showed an apparent tolerance to the acute hypoxic stress. This tolerance was not maintained for mdx following a severe hypoxic stress. A dysfunctional upper airway muscle phenotype is present at 8 weeks of age in the mdx mouse, which may have implications for the control of airway patency in DMD. Hypoxic tolerance in mdx respiratory muscle is suggestive of adaptation to chronic hypoxia, which could be present due to respiratory morbidity. We speculate a role for hypoxia in mdx respiratory muscle morbidity.

Respiratory Control in the mdx Mouse Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a genetic disease caused by defects in the dystrophin gene resulting in loss of the structural protein dystrophin. Patients have reduced diaphragm functional capacity due to progressive muscle weakness. Respiratory morbidity in DMD is further characterised by hypoxaemic periods due to hypoventilation. DMD patients die prematurely due to respiratory and cardiac failure. In this study, we examined respiratory function in young adult male mdx (dystrophin deficient) mice (C57BL/10ScSn-Dmd(mdx)/J; n = 10) and in wild-type controls (WT; C57BL/10ScSnJ; n = 11). Breathing was assessed in unrestrained, unanaesthetised animals by whole-body plethysmography. Ventilatory parameters were recorded during air breathing and during exposure to acute hypoxia (F(i)O(2) = 0.1, 20 min). Data for the two groups of animals were compared using Students t tests. During normoxic breathing, mdx mice had reduced breathing frequency (p = 0.011), tidal volume (p = 0.093) and minute ventilation (p = 0.033) compared to WT. Hypoxia increased minute ventilation in WT and mdx animals. Mdx mice had a significantly increased ventilatory response to hypoxia which manifest as an elevated % change from baseline for minute ventilation (p = 0.0015) compared to WT. We conclude that mdx mice have impaired normoxic ventilation suggestive of hypoventilation. Furthermore, mdx mice have an enhanced hypoxic ventilatory response compared to WT animals which we speculate may be secondary to chronic hypoxaemia. Our results indicate that a significant respiratory phenotype is evident as early as 8 weeks in the mdx mouse model of DMD.

Sensorimotor control of breathing in the mdx mouse model of Duchenne muscular dystrophy

Respiratory failure is a leading cause of mortality in Duchenne muscular dystrophy (DMD), but little is known about the control of breathing in DMD and animal models. We show that young (8 weeks of age) mdx mice hypoventilate during basal breathing due to reduced tidal volume. Basal CO2 production is equivalent in wild‐type and mdx mice. We show that carotid bodies from mdx mice have blunted responses to hyperoxia, revealing hypoactivity in normoxia. However, carotid body, ventilatory and metabolic responses to hypoxia are equivalent in wild‐type and mdx mice. Our study revealed profound muscle weakness and muscle fibre remodelling in young mdx diaphragm, suggesting severe mechanical disadvantage in mdx mice at an early age. Our novel finding of potentiated neural motor drive to breathe in mdx mice during maximal chemoactivation suggests compensatory neuroplasticity enhancing respiratory motor output to the diaphragm and probably other accessory muscles.

Restoration of pharyngeal dilator muscle force in dystrophin‐deficient (mdx) mice following co‐treatment with neutralizing interleukin‐6 receptor antibodies and urocortin 2

What is the central question of this study? We previously reported impaired upper airway dilator muscle function in the mdx mouse model of Duchenne muscular dystrophy (DMD). Our aim was to assess the effect of blocking interleukin‐6 receptor signalling and stimulating corticotrophin‐releasing factor receptor 2 signalling on mdx sternohyoid muscle structure and function. What is the main finding and its importance? The interventional treatment had a positive inotropic effect on sternohyoid muscle force, restoring mechanical work and power to wild‐type values, reduced myofibre central nucleation and preserved the myosin heavy chain type IIb fibre complement of mdx sternohyoid muscle. These data might have implications for development of pharmacotherapies for DMD with relevance to respiratory muscle performance.

Tempol Supplementation Restores Diaphragm Force and Metabolic Enzyme Activities in mdx Mice

Duchenne muscular dystrophy (DMD) is characterized by striated muscle weakness, cardiomyopathy, and respiratory failure. Since oxidative stress is recognized as a secondary pathology in DMD, the efficacy of antioxidant intervention, using the superoxide scavenger tempol, was examined on functional and biochemical status of dystrophin-deficient diaphragm muscle. Diaphragm muscle function was assessed, ex vivo, in adult male wild-type and dystrophin-deficient mdx mice, with and without a 14-day antioxidant intervention. The enzymatic activities of muscle citrate synthase, phosphofructokinase, and lactate dehydrogenase were assessed using spectrophotometric assays. Dystrophic diaphragm displayed mechanical dysfunction and altered biochemical status. Chronic tempol supplementation in the drinking water increased diaphragm functional capacity and citrate synthase and lactate dehydrogenase enzymatic activities, restoring all values to wild-type levels. Chronic supplementation with tempol recovers force-generating capacity and metabolic enzyme activity in mdx diaphragm. These findings may have relevance in the search for therapeutic strategies in neuromuscular disease.

Antioxidant therapy for muscular dystrophy: caveat lector!

We read with interest the excellent article by Pinniger et al. (2017) recently published in The Journal of Physiology, accompanied by an insightful perspective (Head, 2017). However, in our view, the cautionary tone espoused in the original article is overstated, and distracts from the commendable work of the authors revealing impressive effects of antioxidant intervention in the dystrophin-deficient mdx mouse model of Duchenne muscular dystrophy. By way of disclosure, our group advocates antioxidant intervention for respiratory muscle dysfunction in respiratory disease, on the basis of observations in several rodent models of hypoxia (reviewed in O’Halloran & Lewis, 2017). Moreover, we have reported that N-acetylcysteine (NAC) is the most effective antioxidant in preventing respiratory muscle weakness and fatigue following exposure to chronic sustained hypoxia (Lewis et al. 2016) and chronic intermittent hypoxia (Shortt et al. 2014). Pinniger et al. (2017) emphasize the considerable suppression of body mass gain in mice supplemented with NAC, observed in C57 wild-type mice and mdx mice in their study. Whereas the authors report that NAC supplementation did not affect measures of somatic growth in wild-type mice, no data are provided in respect of mdx mice (Pinniger et al. 2017). However, Cao & Picklo (2014) reported no change in femur length or diameter in mice supplemented with 1 g kg−1 NAC in the diet. The striking observation of reduced body mass gain using high dose 2% NAC supplementation in Pinniger et al. (2017) was not accompanied by elevations in cysteine levels, which might otherwise have explained putative adverse outcomes due to toxicity. It is suggested that there are no antecedent reports of stunted body mass gain associated with NAC supplementation (Head, 2017; Pinniger et al. 2017), but several studies have reported reduced body mass gain in NAC-supplemented rodents (Kim et al. 2006; Kondratov et al. 2009; Cao & Picklo, 2014), including significant body mass reductions in male and female mice supplemented with 0.5–1% NAC beginning at 7 months of age (Flurkey et al. 2010), beyond the early growth phase emphasized in Pinniger et al. (2017). A point not addressed in the recent articles (Head, 2017; Pinniger et al. 2017) is the clear evidence that NAC supplementation decreases fat mass. Kim et al. (2006) demonstrated that NAC supplementation decreased visceral fat mass. Ma et al. (2016) reported decreases in epididymal and subcutaneous white adipose tissue, as well as decreased adipocyte expansion in brown adipose tissue and evidence of altered thermogenic gene expression suggesting increased energy expenditure following chronic NAC supplementation. Interestingly, however, notwithstanding changes culminating in altered body composition, Ma et al. (2016) reported that NAC supplementation at 2% in the drinking water for 11 weeks beginning at 6 weeks of age did not affect body mass at any time point, whereas Cao & Picklo (2014) observed that 1 g kg−1 NAC in the diet decreased body mass after 5 weeks of supplementation, although the effects were modest after 17 weeks of supplementation. It was also previously reported that NAC supplementation decreased body mass in a dose-dependent manner (Kim et al. 2006). Given that NAC acidifies the drinking water (pH 2), fluid and food intake may have been reduced in the first few days of supplementation in the study by Pinniger et al. (2017), which might explain the considerable initial drop in body mass in NAC-supplemented mice after 1 week of supplementation. Of note, pH adjustment of NAC-supplemented drinking water is performed in some, but not all studies. Pinniger et al. (2017) did not report food and fluid intake in their study. Of interest, decreased water intake in mice supplemented with 0.5–1% NAC in the drinking water has been reported (Farid et al. 2005; Flurkey et al. 2010). In respect of food consumption in NAC-supplemented animals, either no change (Kim et al. 2006; Hurley et al. 2016; Ma et al. 2016) or decreased food intake (Flurkey et al. 2010) is reported. Metabolic studies are required in the future to accurately determine the effects of NAC supplementation on energy intake and expenditure. On balance, however, the issue of reduced body mass gain may be a red herring, and in any event, as argued by Head (2017), might well be considered a beneficial outcome in reducing muscle stress in dystrophic mice and boys. Importantly, chronic antioxidant supplementation elaborated differential outcomes in muscle mass based on genotype in the study by Pinniger et al. (2017), but this is not emphasized in the article. Reductions in mdx muscle mass could reasonably be viewed as a beneficial outcome, given that fibrosis and remodelling typically lead to heavier muscles in mdx compared with wild-type mice. Indeed, in our experience, mdx mice are routinely heavier than age-matched wild-type counterparts (Burns et al. 2017a,b), with evidence of increased muscle and organ mass, whether animals are sourced as young adults from a commercial supplier or born and raised in our facility. We conclude that the primary focus should be on widening understanding of the impressive effects of NAC supplementation on muscle strength, findings which warrant exploration of the mechanistic action of antioxidant supplementation with potential application to muscles of breathing, since NAC has been shown to delay human diaphragm fatigue (Travaline et al. 1997). However, it is important to acknowledge that whereas NAC supplementation prevents skeletal muscle dysfunction in some models (Shortt et al. 2014; Lewis et al. 2016; Pinniger et al. 2017), it does not in others (Farid et al. 2005). We are reminded by the work of Pinniger et al. (2017) of the need to reflect a wider vista than might otherwise be normally considered in interventional studies in animal models. However, we suggest that warnings should come with warnings! We recognize, as others do, the inherent caveats associated with ‘translational’ drug intervention studies in animal models of disease, necessitating cautious extrapolation of findings to the consideration of human disease and treatments. In the round, in our view, the elegant work of Pinniger et al. (2017) provides a rational basis for comprehensive studies exploring the merits and mishaps of NAC, and other antioxidant strategies, in pre-clinical models of muscular dystrophy in the search for strategies that may

Recovery of respiratory function in mdx mice co‐treated with neutralizing interleukin‐6 receptor antibodies and Urocortin‐2

Impaired ventilatory capacity and diaphragm muscle weakness are prominent features of Duchenne muscular dystrophy, with strong evidence of attendant systemic and muscle inflammation. We performed a 2‐week intervention in young wild‐type and mdx mice, consisting of either injection of saline or co‐administration of a neutralizing interleukin‐6 receptor antibody (xIL‐6R) and urocortin‐2 (Ucn2), a corticotrophin releasing factor receptor 2 agonist. We examined breathing and diaphragm muscle form and function. Breathing and diaphragm muscle functional deficits are improved following xIL‐6R and Ucn2 co‐treatment in mdx mice. The functional improvements were associated with a preservation of mdx diaphragm muscle myosin heavy chain IIx fibre complement. The concentration of the pro‐inflammatory cytokine interleukin‐1β was reduced and the concentration of the anti‐inflammatory cytokine interleukin‐10 was increased in mdx diaphragm following drug co‐treatment. Our novel findings may have implications for the development of pharmacotherapies for the dystrophinopathies with relevance for respiratory muscle performance and breathing.

Breathing with neuromuscular disease: Does compensatory plasticity in the motor drive to breathe offer a potential therapeutic target in muscular dystrophy?

Duchenne muscular dystrophy is a fatal neuromuscular disease associated with respiratory-related morbidity and mortality. Herein, we review recent work by our group exploring deficits and compensation in the respiratory control network governing respiratory homeostasis in a pre-clinical model of DMD, the mdx mouse. Deficits at multiple sites of the network provide considerable challenges to respiratory control. However, our work has also revealed evidence of compensatory neuroplasticity in the motor drive to breathe enhancing diaphragm muscle activity during increased chemical drive. The finding may explain the preserved capacity for mdx mice to increase ventilation in response to chemoactivation. Given the profound dysfunction in the primary pump muscle of breathing, we argue that activation of accessory muscles of breathing may be especially important in mdx (and perhaps DMD). Notwithstanding the limitations resulting from respiratory muscle dysfunction, it may be possible to further leverage intrinsic physiological mechanisms serving to compensate for weak muscles in attempts to preserve or restore ventilatory capacity. We discuss current knowledge gaps and the need to better appreciate fundamental aspects of respiratory control in pre-clinical models so as to better inform intervention strategies in human DMD.

No evidence in support of a prodromal respiratory control signature in the TgF344-AD rat model of Alzheimer’s disease

Alzheimers disease (AD) is a progressive neurodegenerative condition disturbing major brain networks, including those pivotal to the motor control of breathing. The aim of this study was to examine respiratory control in the TgF344-AD transgenic rat model of AD. At 8-11 months of age, basal minute ventilation and ventilatory responsiveness to chemostimulation were equivalent in conscious wild-type (WT) and TgF344-AD rats. Under urethane anesthesia, basal diaphragm and genioglossus EMG activities were similar in WT and TgF344-AD rats. The duration of phenylbiguanide-induced apnoea was significantly shorter in TgF344-AD rats compared with WT. Following bilateral cervical vagotomy, diaphragm and genioglossus EMG responsiveness to chemostimulation were intact in TgF344-AD rats. Amyloid precursor protein C-terminal fragments were elevated in the TgF344-AD brainstem, in the absence of amyloid-β accumulation or alterations in tau phosphorylation. Brainstem pro-inflammatory cytokine concentrations were not increased in TgF344-AD rats. We conclude that neural control of breathing is preserved in TgF344-AD rats at this stage of the disease.

Brainstem network pathology and impaired respiratory drive as successive signatures in a rat model of Parkinson's disease

Parkinsons disease (PD) is a devastating neurodegenerative disorder. Progressive destruction of midbrain dopaminergic neurons and axonal projections of the nigrostriatal pathway disrupts circuitry within the basal ganglia controlling movement. This article is protected by copyright. All rights reserved.