The mdx mouse model of Duchenne muscular dystrophy shows evidence of hypoventilation and pronounced diaphragm dysfunction. Six-week-old male mdx (n = 32) and wild-type (WT; n = 32) mice received either saline (0.9% w/v) or a co-administration of neutralizing interleukin-6 receptor antibodies (xIL-6R; 0.2 mg kg ) and corticotrophin-releasing factor receptor 2 agonist (urocortin-2; 30 μg kg ) subcutaneously over 2 weeks. Breathing and diaphragm muscle contractile function (ex vivo) were examined. Diaphragm structure was assessed using histology and immunofluorescence. Muscle cytokine concentration was determined using a multiplex assay. Minute ventilation and diaphragm muscle peak force at 100 Hz were significantly depressed in mdx compared with WT. Drug treatment completely restored ventilation in mdx mice during normoxia and significantly increased mdx diaphragm force- and power-generating capacity. The number of centrally nucleated muscle fibres and the areal density of infiltrates and collagen content were significantly increased in mdx diaphragm; all indices were unaffected by drug co-treatment. The abundance of myosin heavy chain (MyHC) type IIx fibres was significantly decreased in mdx diaphragm; drug co-treatment preserved MyHC type IIx complement in mdx muscle. Drug co-treatment increased the cross-sectional area of MyHC type I and IIx fibres in mdx diaphragm. The cytokines IL-1β, IL-6, KC/GRO and TNF-α were significantly increased in mdx diaphragm compared with WT. Drug co-treatment significantly decreased IL-1β and increased IL-10 in mdx diaphragm. Drug co-treatment had no significant effect on WT diaphragm muscle structure, cytokine concentrations or function. Recovery of breathing and diaphragm force in mdx mice was impressive in our studies, with implication for human dystrophinopathies.

Chronic kidney disease (CKD) occurs in more than 50% of patients with obstructive sleep apnea (OSA). However, the impact of intermittent hypoxia (IH) on renal function and oxygen homeostasis is unclear. Male Sprague-Dawley rats were exposed to IH (270 s at 21% O2; 90 s hypoxia, 6.5% O2 at nadir) for 4 h [acute IH (AIH)] or to chronic IH (CIH) for 8 h/day for 2 wk. Animals were anesthetized and surgically prepared for the measurement of mean arterial pressure (MAP), and left renal excretory function, renal blood flow (RBF), and renal oxygen tension (Po2). AIH had no effect on MAP (123 ± 14 vs. 129 ± 14 mmHg, means ± SE, sham vs. IH). The CIH group was hypertensive (122 ± 9 vs. 144 ± 15 mmHg, P < 0.05). Glomerular filtration rate (GFR) (0.92 ± 0.27 vs. 1.33 ± 0.33 ml/min), RBF (3.8 ± 1.5 vs. 7.2 ± 2.4 ml/min), and transported sodium (TNa) (132 ± 39 vs. 201 ± 47 μmol/min) were increased in the AIH group (all P < 0.05). In the CIH group, GFR (1.25 ± 0.28 vs. 0.86 ± 0.28 ml/min, P < 0.05) and TNa (160 ± 39 vs. 120 ± 40 μmol/min, P < 0.05) were decreased, while RBF (4.13 ± 1.5 vs. 3.08 ± 1.5 ml/min) was not significantly different. Oxygen consumption (QO2) was increased in the AIH group (6.76 ± 2.60 vs. 13.60 ± 7.77 μmol/min, P < 0.05), but it was not significantly altered in the CIH group (3.97 ± 2.63 vs. 6.82 ± 3.29 μmol/min). Cortical Po2 was not significantly different in the AIH group (46 ± 4 vs. 46 ± 3 mmHg), but it was decreased in the CIH group (44 ± 5 mmHg vs. 38 ± 2 mmHg, P < 0.05). For AIH, renal oxygen homeostasis was preserved through a maintained balance between O2 supply (RBF) and consumption (GFR). For CIH, mismatched TNa and QO2 reflect inefficient O2 utilization and, thereby, sustained decrease in cortical Po2.

Respiratory muscle weakness occurs due to dystrophin deficiency in Duchenne muscular dystrophy (DMD). The mdx mouse model of DMD shows evidence of impaired respiratory muscle performance with attendant inflammation and oxidative stress. We examined the effects of N-acetylcysteine (NAC) supplementation on respiratory system performance in mdx mice. Eight-week-old male wild type (n = 10) and mdx (n = 20) mice were studied; a subset of mdx (n = 10) received 1% NAC in the drinking water for 14 days. We assessed breathing, diaphragm, and external intercostal electromyogram (EMG) activities and inspiratory pressure during ventilatory and non-ventilatory behaviours. Diaphragm muscle structure and function, cytokine concentrations, glutathione status, and mRNA expression were determined. Diaphragm force-generating capacity was impaired in mdx compared with wild type. Diaphragm muscle remodelling was observed in mdx, characterized by increased muscle fibrosis, immune cell infiltration, and central myonucleation. NAC supplementation rescued mdx diaphragm function. Collagen content and immune cell infiltration were decreased in mdx + NAC compared with mdx diaphragms. The cytokines IL-1β, IL-6 and KC/GRO were increased in mdx plasma and diaphragm compared with wild type; NAC decreased systemic IL-1β and KC/GRO concentrations in mdx mice. We reveal that NAC treatment improved mdx diaphragm force-generating capacity associated with beneficial anti-inflammatory and anti-fibrotic effects. These data support the potential use of NAC as an adjunctive therapy in human dystrophinopathies.

Oxygen deficit (hypoxia) is a major feature of cardiorespiratory diseases characterized by diaphragm dysfunction, yet the putative role of hypoxic stress as a driver of diaphragm dysfunction is understudied. We explored the cellular and functional consequences of sustained hypoxic stress in a mouse model. Adult male mice were exposed to 8 hours of normoxia, or hypoxia (FiO2 = 0.10) with or without antioxidant pretreatment (N-acetyl cysteine, 200 mg/kg i.p.). Ventilation and metabolism were measured. Diaphragm muscle contractile function, myofibre size and distribution, gene expression, protein signalling cascades, and oxidative stress (TBARS) were determined. Hypoxia caused pronounced diaphragm muscle weakness, unrelated to increased respiratory muscle work. Hypoxia increased diaphragm HIF-1α protein content and activated MAPK, mTOR, Akt, and FoxO3a signalling pathways, largely favouring protein synthesis. Hypoxia increased diaphragm lipid peroxidation, indicative of oxidative stress. FoxO3 and MuRF-1 gene expression were increased. Diaphragm 20S proteasome activity and muscle fibre size and distribution were unaffected by acute hypoxia. Pretreatment with N-acetyl cysteine substantially enhanced cell survival signalling, prevented hypoxia-induced diaphragm oxidative stress, and prevented hypoxia-induced diaphragm dysfunction. Hypoxia is a potent driver of diaphragm weakness, causing myofibre dysfunction without attendant atrophy. N-acetyl cysteine protects the hypoxic diaphragm and may have application as a potential adjunctive therapy.

No abstract

We report the case of a 61‐year‐old woman presenting with a subacute eruption of a florid, circinate eruption with peripheral pustules. Histological findings included a neutrophilic intraepidermal pustule with associated acantholysis, and a mixed inflammatory cell infiltrate within the dermis.

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 upper airway and diaphragm muscle function. Excessive ROS are also associated with impaired respiratory control, 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 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 for 2 weeks. Adult male (C57BL/6J) mice were assigned to one of 5 groups: normoxic controls (sham), CIH‐exposed, CIH+apocynin (NOX2 inhibitor, 2mM) given in the drinking water throughout the CIH and NOX2 null (B6.129S‐Cybbtm1Din/J) sham or CIH exposed. On day 15, whole body plethysmography was used to measure breathing parameters in unrestrained, unaesthetized mice in room air. Flow signals recorded were analysed offline and an apnoea was defined as ≥ 2 missed breaths. 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.CIH decreased sternohyoid and diaphragm muscle peak specific force by ~45% compared with sham exposure. CIH increased NOX enzyme activity in the sternohyoid, with no alterations in gene or protein expression of NOX subunits compared with sham. CIH decreased the expression of p22phox and rac genes and increased protein expression of NOX4 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. Basal minute ventilation remained unchanged following CIH exposure however the number of apnoeas per hour was increased compared with sham. Apocynin intervention 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 in wild‐type mice.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 this CIH‐induced respiratory 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 OSAS). The reduction in apnoea frequency following treatment with apocynin implicates ROS in the manifestation of CIH‐induced respiratory disturbances in a non‐NOX2 dependant manner. Our results have implications for human OSAS and point to antioxidant intervention as a potential therapeutic strategy.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Obstructive sleep apnoea syndrome (OSAS) is the most prevalent form of sleep disordered breathing (SDB) which affects 1 in 5 people worldwide. Chronic intermittent hypoxia (CIH) is a hallmark feature of SDB as a consequence of repetitive upper airway occlusions in patients during sleep. These recurring hypoxia‐reoxygenation cycles result in the overproduction of reactive oxygen species (ROS), ultimately yielding a state of oxidative stress. Excessive levels of ROS have been associated with aberrant plasticity at multiple levels of the respiratory system including impaired upper airway muscle function. However, there is a paucity of information regarding the molecular mechanisms underlying these effects. The NADPH oxidase (NOX) family of enzymes have been linked to a multitude of CIH induced morbidities in a range of organs and systems with NOX subunits also having been identified in skeletal muscle.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) for 90 seconds 8 hr/day for 2 weeks during light hours. 10–11 week old male mice (C57BL/6J) were randomly assigned to one of 3 groups: Normoxic controls (sham; n=24), CIH exposed (n=24), and CIH + Apocynin (2 mM; n=24) treated throughout the gas exposure. 10–11 week old NOX2 null (B6.129S‐Cybbtm1Din/J) male mice were also assigned to a sham (n=12) or CIH (n=12) group. Following this, sternohyoid muscle contractile function was examined ex vivo. Gene expression of the entire family of NOX enzymes was examined by RT‐PCR. Western blot was used to quantify NOX2 and NOX4 protein expression. NOX enzyme activity was determined using a NOX activity assay. Data were expressed as mean±SD and were statistically compared by unpaired Student t‐test or two‐way ANOVA with Bonferroni post‐hoc test.2 weeks of exposure to CIH resulted in a significant decrease of ~45% in the peak specific force (Fmax) of the sternohyoid muscle when compared to normoxic controls (sham). CIH resulted in no significant alteration to the gene expression of any members of the NOX family of enzymes when compared to sham controls. There was no significant difference in NOX2 or NOX4 protein expression between CIH and sham groups. However, CIH exposure resulted in a significant increase in sternohyoid NOX enzyme activity compared to shams. Treatment with Apocynin (2 mM) throughout the 2 week CIH exposure prevented sternohyoid muscle weakness, restoring force to levels equivalent to controls. Furthermore, NOX2 knockout prevented CIH induced sternohyoid muscle weakness.Adult male C57BL/6J mice show signs of profound sternohyoid muscle dysfunction following exposure to 2 weeks of CIH when compared with sham animals. This occurs without alteration to the gene or protein expression of various NOX subunits. Increased NOX activity suggests a CIH induced increase in NOX enzyme assembly rather than abundance. Administration of the putative NOX inhibitor, Apocynin, prevents sternohyoid muscle weakness. NOX2 knockout also entirely prevents sternohyoid muscle weakness confirming its specific role in CIH induced muscle dysfunction. The striking muscle phenotype in the C57 mouse and its prevention in the absence of NOX2 provides a novel and robust platform for the study of mechanisms of hypoxia‐related muscle dysfunction, which may have relevance to respiratory conditions characterised by CIH, such as sleep apnoea.Support or Funding InformationDepartment of Physiology, University College Cork, Cork, IrelandThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.