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.

Microbes, metabolites and muscle: Is the gut-muscle axis a plausible therapeutic target in Duchenne muscular dystrophy?

NEW FINDINGS: What is the topic of this review? The contribution of gut microbial signalling to skeletal muscle maintenance and development and identification of potential therapeutic targets in progressive muscle degenerative diseases such as Duchenne muscular dystrophy. What advances does it highlight? Gut microbe-derived metabolites are multifaceted signalling molecules key to muscle function, modifying pathways contributing to skeletal muscle wasting, making them a plausible target for adjunctive therapy in muscular dystrophy. ABSTRACT: Skeletal muscle is the largest metabolic organ making up ∼50% of body mass. Because skeletal muscle has both metabolic and endocrine properties, it can manipulate the microbial populations within the gut. In return, microbes exert considerable influence on skeletal muscle via numerous signalling pathways. Gut bacteria produce metabolites (i.e., short chain fatty acids, secondary bile acids and neurotransmitter substrates) that act as fuel sources and modulators of inflammation, influencing host muscle development, growth and maintenance. The reciprocal interactions between microbes, metabolites and muscle establish a bidirectional gut-muscle axis. The muscular dystrophies constitute a broad range of disorders with varying disabilities. In the profoundly debilitating monogenic disorder Duchenne muscular dystrophy (DMD), skeletal muscle undergoes a reduction in muscle regenerative capacity leading to progressive muscle wasting, resulting in fibrotic remodelling and adipose infiltration. The loss of respiratory muscle in DMD culminates in respiratory insufficiency and eventually premature death. The pathways contributing to aberrant muscle remodelling are potentially modulated by gut microbial metabolites, thus making them plausible targets for pre- and probiotic supplementation. Prednisone, the gold standard therapy for DMD, drives gut dysbiosis, inducing a pro-inflammatory phenotype and leaky gut barrier contributing to several of the well-known side effects associated with chronic glucocorticoid treatment. Several studies have observed that gut microbial supplementation or transplantation exerts positive effects on muscle, including mitigating the side effects of prednisone. There is growing evidence in support of the potential for an adjunctive microbiota-directed regimen designed to optimise gut-muscle axis signalling, which could alleviate muscle wasting in DMD.

Three Days of Chronic Intermittent Hypoxia Induce ß1-Adrenoceptor Dependent Increases in Left Ventricular Contractility.

Sleep apnea is characterized by bouts of chronic intermittent hypoxia (CIH) that elicit sympathetic hyperactivity resulting in residual hypertension. We previously demonstrated that exposure to CIH increases cardiac output and sought to determine if enhanced cardiac contractility manifests prior to hypertension.Male Wistar rats were exposed to cyclical bouts of hypoxia (FiO2 = 0.05 nadir; 90 s) and normoxia (FiO2 = 0.21; 210 s) 8 h/day for 3 days (CIH; n = 6). Control animals (n = 7) were exposed to room air. Data are presented as mean ± SD and were analyzed using unpaired Student t-tests.Three-day exposure to CIH did not elicit changes in heart rate and blood pressure (p > 0.05). However, baseline left ventricular contractility (dP/dtMAX) was significantly increased in CIH-exposed animals compared with control (15300 ± 2002 vs. 12320 ± 2725 mmHg/s; p = 0.025), despite no difference in catecholamine concentrations. Acute ß1-adrenoceptor inhibition reduced contractility in CIH-exposed animals (-7604 ± 1298 vs. -4747 ± 2080 mmHg/s; p = 0.014), to levels equivalent to control, while preserving cardiovascular parameters. Sympathetic ganglion blockade (hexamethonium 25 mg/kg; i.v.) produced equivalent cardiovascular responses suggesting similar global sympathetic activity between groups. Interestingly, gene expression of the ß1-adrenoceptor pathway in cardiac tissue was unchanged.Our results suggest that CIH increases cardiac contractility via ß1-adrenoceptor dependent mechanisms prior to development of global sympathetic hyperactivity suggesting that positive cardiac inotropy contributes to the development of hypertension in CIH-exposed rats.

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.

Cerebrovascular and blood pressure responses during voluntary apneas are larger than rebreathing.

Both voluntary rebreathing (RB) of expired air and voluntary apneas (VA) elicit changes in arterial carbon dioxide and oxygen (CO2 and O2) chemostimuli. These chemostimuli elicit synergistic increases in cerebral blood flow (CBF) and sympathetic nervous system activation, with the latter increasing systemic blood pressure. The extent that simultaneous and inverse changes in arterial CO2 and O2 and associated increases in blood pressure affect the CBF responses during RB versus VAs are unclear. We instrumented 21 healthy participants with a finometer (beat-by-beat mean arterial blood pressure; MAP), transcranial Doppler ultrasound (middle and posterior cerebral artery velocity; MCAv, PCAv) and a mouthpiece with sample line attached to a dual gas analyzer to assess pressure of end-tidal (PET)CO2 and PETO2. Participants performed two protocols: RB and a maximal end-inspiratory VA. A second-by-second stimulus index (SI) was calculated as PETCO2/PETO2 during RB. For VA, where PETCO2 and PETO2 could not be measured throughout, SI values were calculated using interpolated end-tidal gas values before and at the end of the apneas. MAP reactivity (MAPR) was calculated as the slope of the MAP/SI, and cerebrovascular reactivity (CVR) was calculated as the slope of MCAv or PCAv/SI. We found that compared to RB, VA elicited ~ fourfold increases in MAPR slope (P < 0.001), translating to larger anterior and posterior CVR (P ≤ 0.01). However, cerebrovascular conductance (MCAv or PCAv/MAP) was unchanged between interventions (P ≥ 0.2). MAP responses during VAs are larger than those during RB across similar chemostimuli, and differential CVR may be driven by increases in perfusion pressure.

Cosmic kidney disease: an integrated pan-omic, physiological and morphological study into spaceflight-induced renal dysfunction.

Missions into Deep Space are planned this decade. Yet the health consequences of exposure to microgravity and galactic cosmic radiation (GCR) over years-long missions on indispensable visceral organs such as the kidney are largely unexplored. We performed biomolecular (epigenomic, transcriptomic, proteomic, epiproteomic, metabolomic, metagenomic), clinical chemistry (electrolytes, endocrinology, biochemistry) and morphometry (histology, 3D imaging, miRNA-ISH, tissue weights) analyses using samples and datasets available from 11 spaceflight-exposed mouse and 5 human, 1 simulated microgravity rat and 4 simulated GCR-exposed mouse missions. We found that spaceflight induces: 1) renal transporter dephosphorylation which may indicate astronauts' increased risk of nephrolithiasis is in part a primary renal phenomenon rather than solely a secondary consequence of bone loss; 2) remodelling of the nephron that results in expansion of distal convoluted tubule size but loss of overall tubule density; 3) renal damage and dysfunction when exposed to a Mars roundtrip dose-equivalent of simulated GCR.

Comparison of mild physiological effects of surgical masks and N95 respirators over 60 min at rest.

Dead-space-associated rebreathing of expired air and heat trapping with use of surgical masks and N95 respirators may underlie anecdotal reports of adverse symptoms associated with medical face barriers. Limited data exist directly comparing the physiological effects of masks and respirators at rest. We assessed the short-term physiological effects of both barrier types over 60 min at rest, including face microclimate temperature, end-tidal gases, and venous blood acid-base variables. We recruited 34 participants into two trials: surgical masks (n = 17) and N95 respirators (n = 17). In a seated position, participants underwent a 10-min baseline without a barrier and then wore a standardized surgical mask or dome-shaped N95 respirator for 60 min, followed by a 10-min washout. We instrumented healthy human participants with a peripheral pulse oximeter ([Formula: see text]) and a nasal cannula connected to a dual gas analyzer for measurement of the pressure of end-tidal [Formula: see text] and [Formula: see text], with an associated temperature probe for face microclimate temperature. Venous (v) blood samples were obtained at baseline and following 60-min mask/respirator wearing to assess [Formula: see text], [HCO3-]v and pHv. Compared with baseline during/following 60 min, temperature, [Formula: see text], [Formula: see text], and [HCO3-]v were mildly but significantly higher, and [Formula: see text] and [Formula: see text] were significantly lower, but [Formula: see text] was unaffected. The magnitude of effects was similar between barrier types. Temperature and [Formula: see text] returned to baseline levels within 1-2 min following removal of the barrier. These mild physiological effects may underlie reports of qualitative symptoms while wearing masks or respirators. However, the magnitudes were mild, not physiologically relevant and reversed immediately with the removal of the barrier.NEW & NOTEWORTHY Anecdotal reports suggest mild physiological effects of wearing surgical masks and/or N95 respirators, including heat trapping and rebreathing of expired air. There are limited data directly comparing the physiological effects of wearing medical barriers at rest. We found that the time course and magnitude of changes to face microclimate temperature, end-tidal gases, and venous blood gases and acid-base variables were mild in magnitude, not physiologically relevant, equivalent between barrier types, and immediately reversible on removal.

Acute hyperglycemia does not affect central respiratory chemoreflex responsiveness to CO2 in healthy humans.

The central respiratory chemoreceptor complex (CCRC) is comprised of brainstem neurons and surrounding interoceptors, which collectively increase ventilation in response to elevated brainstem tissue CO2/[H+] (i.e., central chemoreflex; CCR). The extent that the CCRC detects/responds to other metabolically related chemostimuli is unknown. We aimed to test the effects of acute oral glucose ingestion on CCR reactivity in heathy human participants (n = 38). We instrumented participants with a pneumotachometer (minute ventilation) and a gas sample line connected to a dual gas analyzer (pressure of end-tidal CO2). Following a baseline (BL) period and capillary blood [glucose] (BG) sample, fasted (F) participants underwent a modified hyperoxic rebreathing test to assess CCR reactivity. Participants then consumed a 75 g standard glucose beverage (glucose loaded; GL). Following 30-min, they underwent a second BL, BG sample and hyperoxic rebreathing test. BG and metabolic rate were higher in GL, confirming the metabolic stimulus. However, the ventilatory recruitment threshold and the CCR responses were unchanged between F and GL states.

Steady-state chemoreflex drive captures ventilatory acclimatization during incremental ascent to high altitude: Effect of acetazolamide.

Ventilatory acclimatization (VA) is important to maintain adequate oxygenation with ascent to high altitude (HA). Transient hypoxic ventilatory response tests lack feasibility and fail to capture the integrated steady-state responses to chronic hypoxic exposure in HA fieldwork. We recently characterized a novel index of steady-state respiratory chemoreflex drive (SSCD), accounting for integrated contributions from central and peripheral respiratory chemoreceptors during steady-state breathing at prevailing chemostimuli. Acetazolamide is often utilized during ascent for prevention or treatment of altitude-related illnesses, eliciting metabolic acidosis and stimulating respiratory chemoreceptors. To determine if SSCD reflects VA during ascent to HA, we characterized SSCD in 25 lowlanders during incremental ascent to 4240 m over 7 days. We subsequently compared two separate subgroups: no acetazolamide (NAz; n = 14) and those taking an oral prophylactic dose of acetazolamide (Az; 125 mg BID; n = 11). At 1130/1400 m (day zero) and 4240 m (day seven), steady-state measurements of resting ventilation (V̇I ; L/min), pressure of end-tidal (PET )CO2 (Torr), and peripheral oxygen saturation (SpO2 ; %) were measured. A stimulus index (SI; PET CO2 /SpO2 ) was calculated, and SSCD was calculated by indexing V̇I against SI. We found that (a) both V̇I and SSCD increased with ascent to 4240 m (day seven; V̇I : +39%, p < 0.0001, Hedges' g = 1.52; SSCD: +56.%, p < 0.0001, Hedges' g = 1.65), (b) and these responses were larger in the Az versus NAz subgroup (V̇I : p = 0.02, Hedges' g = 1.04; SSCD: p = 0.02, Hedges' g = 1.05). The SSCD metric may have utility in assessing VA during prolonged stays at altitude, providing a feasible alternative to transient chemoreflex tests.

The effects of acute incremental hypocapnia on the magnitude of neurovascular coupling in healthy participants.

The high metabolic demand of cerebral tissue requires that local perfusion is tightly coupled with local metabolic rate (neurovascular coupling; NVC). During chronic altitude exposure, where individuals are exposed to the antagonistic cerebrovascular effects of hypoxia and hypocapnia, pH is maintained through renal compensation and NVC remains stable. However, the potential independent effect of acute hypocapnia and respiratory alkalosis on NVC remains to be determined. We hypothesized that acute steady-state hypocapnia via voluntary hyperventilation would attenuate the magnitude of NVC. We recruited 17 healthy participants and insonated the posterior cerebral artery (PCA) with transcranial Doppler ultrasound. NVC was elicited using a standardized strobe light stimulus (6 Hz; 5 × 30 s on/off) where absolute delta responses from baseline (BL) in peak, mean, and total area under the curve (tAUC) were quantified. From a BL end-tidal (PET )CO2  level of 36.7 ± 3.2 Torr, participants were coached to hyperventilate to reach steady-state hypocapnic steps of Δ-5 Torr (31.6 ± 3.9) and Δ-10 Torr (26.0 ± 4.0; p < 0.001), which were maintained during the presentation of the visual stimuli. We observed a small but significant reduction in NVC peak (ΔPCAv) from BL during controlled hypocapnia at both Δ-5 (-1.58 cm/s) and Δ-10 (-1.37 cm/s), but no significant decrease in mean or tAUC NVC response was observed. These data demonstrate that acute respiratory alkalosis attenuates peak NVC magnitude at Δ-5 and Δ-10 Torr PET CO2 , equally. Although peak NVC magnitude was mildly attenuated, our data illustrate that mean and tAUC NVC are remarkably stable during acute respiratory alkalosis, suggesting multiple mechanisms underlying NVC.