Diaphragm Muscle Weakness Research Articles

Role of myofiber-specific FoxP1 in pancreatic cancer-induced muscle wasting.

Cancer cachexia affects up to 80% of cancer patients and results in reduced quality of life and survival. We previously demonstrated that the transcriptional repressor Forkhead box P1 (FoxP1) is upregulated in skeletal muscle of cachectic mice and people with cancer, and when overexpressed in skeletal muscle is sufficient to induce pathological features characteristic of cachexia. However, the role of myofiber-derived FoxP1 in both normal muscle physiology and cancer-induced muscle wasting remains largely unexplored. To address this gap, we generated a conditional mouse line with myofiber-specific ablation of FoxP1 (FoxP1SkmKO) and found that in cancer-free mice, deletion of FoxP1 in skeletal myofibers resulted in increased myofiber size in both males and females, with a significant increase in muscle mass in males. In response to murine KPC pancreatic tumor burden, we found that myofiber-derived FoxP1 is required for cancer-induced muscle wasting and diaphragm muscle weakness in male mice. In summary, our findings identify myofiber-specific FoxP1 as a negative regulator of skeletal muscle with sex-specific differences in the context of cancer.

Poor correlation between diaphragm ultrasound and invasive gold standard technique derived respiratory muscle strength assessment in patients after hospitalization for COVID-19.

Individuals who survive acute coronavirus disease 2019 (COVID-19) might experience diaphragm muscle weakness. Diaphragm ultrasound may be an easy-to-obtain bedside tool for determining diaphragm function. However, twitch transdiaphragmatic pressure (twPdi) following magnetic stimulation (MS) of the phrenic nerves is the gold standard for non-volitional assessment of diaphragm strength. This study investigated whether diaphragm thickening ratio (DTR) measured on diaphragm ultrasound reflects diaphragm strength as measured by twPdi following MS of the phrenic nerves or other (volitional) invasively obtained pressure values and could therefore be used to accurately diagnose diaphragm weakness. One year after discharge, 50 individuals (14 female, age 58±12 years) who had been hospitalised and treated for moderate-severe COVID-19 underwent standard spirometry and diaphragm ultrasound. TwPdi following cervical MS of the phrenic nerve and volitional inspiratory manoeuvres (Sniff and Mueller manoeuvre) were measured using oesophageal and gastric balloon catheters after transnasal placement. At follow-up, no clinically meaningful restrictive lung function impairment was evident on spirometry. On diaphragm ultrasound, diaphragm dysfunction, i. e. an impaired diaphragm thickening ratio was detected in 24% (12/50) of participants. An objective diagnosis of diaphragm dysfunction, defined as twPdi <16 cmH2O, was made in 60% (30/50) of participants. The measurement results of the two methods did not agree, given that there were many false negative, but also false positive results, so diaphragm ultrasound diagnosed in parts other patients with diaphragm dysfunction than TwPdi. Diaphragm ultrasound had a sensitivity of 26.67% and a specificity of 80.0% in the detection of diaphragm dysfunction (Positive predictive value 66.67%, negative predictive value 42.10%). Diagnosis of diaphragm weakness in individuals who have recovered from COVID-19 cannot be made accurately on diaphragm ultrasound (via DTR), but requires twPdi as the gold standard for assessment of diaphragm strength.

Potential Diaphragm Muscle Weakness-related Dyspnea Persists 2 Years after COVID-19 and Could Be Improved by Inspiratory Muscle Training: Results of an Observational and an Interventional Clinical Trial.

Rationale: Diaphragm muscle weakness might underlie persistent exertional dyspnea, despite normal lung and cardiac function in individuals who were previously hospitalized for acute coronavirus disease (COVID-19) illness. Objectives: The authors sought, first, to determine the persistence and pathophysiological nature of diaphragm muscle weakness and its association with exertional dyspnea 2 years after hospitalization for COVID-19 and, second, to investigate the impact of inspiratory muscle training (IMT) on diaphragm and inspiratory muscle weakness and exertional dyspnea in individuals with long COVID. Methods: Approximately 2 years after hospitalization for COVID-19, 30 individuals (11 women, 19 men; median age, 58 years; interquartile range [IQR] = 51-63) underwent comprehensive (invasive) respiratory muscle assessment and evaluation of dyspnea. Eighteen with persistent diaphragm muscle weakness and exertional dyspnea were randomized to 6 weeks of IMT or sham training; assessments were repeated immediately after and 6 weeks after IMT completion. The primary endpoint was change in inspiratory muscle fatiguability immediately after IMT. Measurements and Main Results: At a median of 31 months (IQR = 23-32) after hospitalization, 21 of 30 individuals reported relevant persistent exertional dyspnea. Diaphragm muscle weakness on exertion and reduced diaphragm cortical activation were potentially related to exertional dyspnea. Compared with sham control, IMT improved diaphragm and inspiratory muscle function (sniff transdiaphragmatic pressure, 83 cm H2O [IQR = 75-91] vs. 100 cm H2O [IQR = 81-113], P = 0.02), inspiratory muscle fatiguability (time to task failure, 365 s [IQR = 284-701] vs. 983 s [IQR = 551-1,494], P = 0.05), diaphragm voluntary activation index (79% [IQR = 63-92] vs. 89% [IQR = 75-94], P = 0.03), and dyspnea (Borg score, 7 [IQR = 5.5-8] vs. 6 [IQR = 4-7], P = 0.03). Improvements persisted for 6 weeks after IMT completion. Conclusions: To the best of the authors' knowledge, this study is the first to identify a potential treatment for persisting exertional dyspnea in long COVID and provide a possible pathophysiological explanation for the treatment benefit. Clinical trial registered with www.clinicaltrials.gov (NCT04854863, NCT05582642).

Postnatal diaphragm muscle and phrenic motor neuron development is impaired in Spastic mice

In rats, there is a strong association between the emergence of mature diaphragm muscle (DIAm) myosin heavy chain isoform expression and the expansion of phrenic motor neuron (PhMN) size (from ~21-28 days postnatal (P)), alongside the manifestation of mature morphological and functional properties. In the Spastic ( Spa) mutant mouse, mutated glycine receptors lead to a spasticity phenotype emerging between P14-28, concomitant with DIAm and PhMN development. In adult Spa mice, DIAm weakness, fiber atrophy and loss of larger PhMNs is evident. We aim to characterize whether these DIAm and PhMN deficits occur contemporaneously to spasticity. Using wildtype (WT) and Spa mice at P14, P21, P28, and P90 (maturity) we evaluated DIAm and PhMN physiologic properties. Ex vivo DIAm strips were used for functional assessments of maximum specific force (Po) and fatigue. DIAm strips were also fresh-frozen, and 8 μm cross-sections were cut and stained with H&amp;E to evaluate cross-sectional area (CSA) of individual DIAm fibers. PhMNs were labelled via rhodamine phrenic nerve dip, processed for longitudinal cryosectioning (70 μm) and prepared for confocal imaging and PhMN assessment. In WT mice, DIAm specific force increased at each age (P14, P21, P28 and P90), whilst in Spa mice, DIAm specific force was reduced from P21 compared to WT. Similarly, DIAm fiber CSA was reduced in Spa compared to WT mice at P21, P28 and P90. The number of PhMNs was similar between genotypes at P21, with a reduction in the number of PhMNs in Spa mice compared to WT evident at P28 continuing to P90. In Spa mice, PhMN size was smaller compared to WT at P21, unchanged at P28 and reduced at P90. The changes in Spa DIAm and PhMNs during the first postnatal month are consistent with the emergence of spasticity from P14 to P28. As most changes occur at ~P21, we are optimistic that interventions prior to weaning may promote the normal growth of DIAm and survival of PhMNs and may stave off later life DIAm behavioral deficits. HL166204 and HL146114. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exercise Rehabilitation Preserves Cardiorespiratory Muscle Function Following Doxorubicin Treatment

Doxorubicin (DOX) is a highly effective chemotherapeutic agent. Unfortunately, cancer patients who have completed DOX treatment often experience exercise intolerance, reduced cardiac function, dyspnea and unfavorable changes to body composition, ultimately, leading to increased incidence of heart failure. Exercise has been shown to reduce the risk of developing chemotherapy-induced cardiorespiratory dysfunction; however, the ability of cancer patients to exercise during treatment can be limited by cancer type, stage and fatigue. Therefore, these experiments utilized a chronic DOX-induced cardiotoxicity model to determine the effects of rehabilitative exercise on cardiorespiratory muscle function. Female Sprague-Dawley rats were treated with DOX once every three weeks for four total cycles via intravenous infusion (i.e., cumulative dose of ~160mg/m2). One-week following the completion of DOX treatment, rats remained sedentary (Sed-DOX) or were prescribed 10-weeks of moderate intensity treadmill running at 30m/min, 0% grade, 1-hour/day, and 3x/week (Mod-DOX). Body composition, exercise tolerance and echocardiography were assessed at three timepoints: 1) pre-treatment; 2) post-treatment and; 3) post-rehabilitation. Diaphragm muscle specific force production was measured post-rehabilitation. From pre- to post-treatment each group gained weight and no differences existed between groups. However, from post-treatment to post-rehabilitation body weight of the DOX-treated groups did not change and was significantly reduced compared to sedentary saline-treated (Sed-Sal) rats from week 15 onwards. Although mean body weight did not differ between the DOX-treated groups, lean mass increased in Mod-DOX rats between each timepoint with no change in fat mass, while fat mass increased in Sed-DOX rats and an initial increase in lean mass was attenuated from post-treatment to post-rehabilitation. Exercise time to fatigue did not change between timepoints within each group; however, exercise run time from post-treatment to post-rehabilitation was significantly longer in Mod-DOX rats compared to Sed-DOX and Sed-Sal. Cardiac fractional shortening percentage (FS%) was reduced in Sed-DOX rats at post-treatment and post-rehabilitation compared to pre-treatment. Additionally, posterior wall shortening velocity (PWSV) was reduced in Sed-DOX rats at post-treatment compared to pre-treatment. No differences in FS% or PWSV were seen between any timepoint in the Mod-DOX group. Finally, ex vivo diaphragm muscle function showed significant diaphragm muscle weakness in Sed-DOX rats at 15-60Hz stimulations compared to Sed-Sal. Diaphragm force production did not differ in Mod-DOX rats compared to Sed-Sal at any stimulation frequency. These results demonstrate a beneficial effect of moderate intensity rehabilitation exercise for the maintenance of body composition, cardiac function and diaphragm muscle function following DOX treatment compared to sedentary rats. NIH R01 HL144858, NIH R01 HL146443, NIH HL134621. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chronic Intermittent Hypoxia-Induced Diaphragm Muscle Weakness Is NADPH Oxidase-2 Dependent.

Chronic intermittent hypoxia (CIH)-induced redox alterations underlie diaphragm muscle dysfunction. We sought to establish if NADPH oxidase 2 (NOX2)-derived reactive oxygen species (ROS) underpin CIH-induced changes in diaphragm muscle, which manifest as impaired muscle performance. Adult male mice (C57BL/6J) were assigned to one of three groups: normoxic controls (sham); chronic intermittent hypoxia-exposed (CIH, 12 cycles/hour, 8 h/day for 14 days); and CIH + apocynin (NOX2 inhibitor, 2 mM) administered in the drinking water throughout exposure to CIH. In separate studies, we examined sham and CIH-exposed NOX2-null mice (B6.129S-CybbTM1Din/J). Apocynin co-treatment or NOX2 deletion proved efficacious in entirely preventing diaphragm muscle dysfunction following exposure to CIH. Exposure to CIH had no effect on NOX2 expression. However, NOX4 mRNA expression was increased following exposure to CIH in wild-type and NOX2 null mice. There was no evidence of overt CIH-induced oxidative stress. A NOX2-dependent increase in genes related to muscle regeneration, antioxidant capacity, and autophagy and atrophy was evident following exposure to CIH. We suggest that NOX-dependent CIH-induced diaphragm muscle weakness has the potential to affect ventilatory and non-ventilatory performance of the respiratory system. Therapeutic strategies employing NOX2 blockade may function as an adjunct therapy to improve diaphragm muscle performance and reduce disease burden in diseases characterised by exposure to CIH, such as obstructive sleep apnoea.

Diaphragm muscle function in a mouse model of early-onset spasticity.

Spasticity is a common symptom in many developmental motor disorders, including spastic cerebral palsy (sCP). In sCP, respiratory dysfunction is a major contributor to morbidity and mortality, yet it is unknown how spasticity influences respiratory physiology or diaphragm muscle (DIAm) function. To investigate the influence of spasticity on DIAm function, we assessed in vivo transdiaphragmatic pressure (Pdi - measured using intraesophageal and intragastric pressure catheters under conditions of eupnea, hypoxia/hypercapnia and occlusion) including maximum Pdi (Pdimax via bilateral phrenic nerve stimulation), ex vivo DIAm-specific force and fatigue (using muscle strips stimulated with platinum plate electrodes), and type-specific characteristics of DIAm fiber cross sections (using immunoreactivity against myosin heavy chain slow and 2A) in spa and wildtype mice. Spa mice show reduced Pdimax, reduced DIAm specific force, and altered fatigability and atrophy of type IIx/IIb fibers. These findings suggest marked DIAm dysfunction may underlie the respiratory phenotype of sCP.NEW & NOTEWORTHY Developmental motor control dysfunctions, including spastic cerebral palsy (sCP) often have respiratory components. Spa mutant mice exhibit a spastic phenotype closely resembling sCP symptoms. Using the spa mouse model of spastic cerebral palsy (sCP), we quantified transdiaphragmatic pressure deficits, diaphragm muscle weakness, and fiber type-specific atrophy, improving our understanding of respiratory dysfunctions in sCP.

Accessory Muscle Support of Breathing in Young Dystrophin‐deficient mdx Mice

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder that occurs due to the absence of the structural protein dystrophin and secondary consequences. DMD is characterized by severe and progressive skeletal muscle weakness, which extends to the respiratory muscles. In young dystrophin‐deficient mdx mice, peak inspiratory pressure‐generating capacity is preserved despite diaphragm muscle weakness and reduced electromyogram (EMG) activitya. Our overarching hypothesis is that accessory muscle compensation limits ventilatory deficit in early dystrophic disease. Four‐month‐old male wild‐type (n=39) and mdx (n=41) mice were studied. Breathing was assessed in conscious mice using whole body plethysmography during normoxia and chemochallenge (10% O2/6% CO2). Thoracic oesophageal pressure and obligatory (diaphragm and external intercostal) respiratory muscle EMG activities were recorded in urethane (1.7g/kg i.p.) anaesthetised spontaneously breathing mice during baseline and sustained tracheal occlusion (maximal muscle activation). Diaphragm and scalene (accessory respiratory muscle) force output were examined ex vivo. Data were statistically compared using unpaired Student’s t test or two‐way ANOVA with Bonferroni post hoc test. Peak diaphragm force ex vivo (P &lt; 0.0001) and baseline diaphragm EMG activity (P = 0.01) were significantly lower in mdx compared with wild‐type. Minute ventilation was equivalent in age‐matched wild‐type and mdx mice during baseline and chemochallenge (P = 0.1). Peak diaphragm (P &lt; 0.0001) and external intercostal (P = 0.01) EMG activities during obstruction were significantly lower in mdx mice compared to wild‐type, while peak inspiratory pressure was equivalent (P = 0.4). Interestingly, peak scalene muscle force ex vivo was preserved in 4‐month‐old mdx mice (P = 0.8). Despite a decline in diaphragm muscle performance (decreased EMG activity and mechanical weakness) at baseline, ventilatory capacity is preserved in mdx mice. Furthermore, despite lower peak electrical activation of the obligatory muscles of breathing and diaphragm muscle functional deficit, peak inspiratory pressure is preserved in 4‐month‐old mdx mice, confirming our previous findings in 2‐month‐old micea. Our finding of preserved scalene muscle force suggests that compensation may be provided by accessory muscles to support ventilatory and non‐ventilatory behaviours in early dystrophic disease. Current studies are examining the EMG activity of accessory respiratory muscles (e.g. scalene, sternomastoid, cleidomastoid and trapezius) in early and advanced dystrophic disease in mdx mice.References a Burns DP, Murphy KH, Lucking EF, O'Halloran KD. Inspiratory pressure‐generating capacity is preserved during ventilatory and non‐ventilatory behaviours in young dystrophic mdx mice despite profound diaphragm muscle weakness. J. Physiol. 2019;597(3):831‐848.

Hyperbaric Oxygen Therapy Following Cervical Spinal Cord Injury Increases BDNF/TrkB Signaling in Diaphragm Muscle

Respiratory insufficiency is the leading cause of morbidity and mortality following cervical spinal cord injury (SCI). Diaphragm atrophy following SCI occurs as a result of damage to respiratory neural circuitry, which leads to impaired muscle contractility. Disruption of redox balance occurs after SCI and increased mitochondrial reactive oxygen species production is an upstream trigger for diaphragm muscle atrophy and weakness. Recently, we demonstrated that increased antioxidant capacity in response to hyperbaric oxygen (HBO) therapy may play a role in preserving mitochondrial redox homeostasis following SCI. However, a precise understanding of the mechanisms by which HBO therapy can prevent diaphragm dysfunction is lacking. In this regard, evidence indicates that the secretion of neurotrophic factors, such as brain derived neurotrophic factor (BDNF), may also contribute to enhanced functional recovery when HBO therapy is initiated acutely following cervical SCI. Through activation of the 5′ adenosine monophosphate‐activated protein kinase (AMPK) and serine‐threonine protein kinase (AKT) signaling via the phosphorylation of its receptor tropomyosin receptor kinase B (TrKb), BDNF expression has been shown to modulate mitochondrial biogenesis and skeletal muscle proteolysis. Therefore, we tested the hypothesis that 10 days of HBO therapy initiated in the acute phase following cervical SCI can increase diaphragm mitochondrial biogenesis and prevent diaphragm proteolytic signaling by activating BDNF/TrkB signaling. To test this, adult male Sprague‐Dawley rats were separated into three groups (n=8/group): (1) non‐injured, room air exposure (CON); (2) lateral‐cervical spinal cord contusion, room air exposure (SCI); and (3) lateral‐cervical spinal cord contusion, HBO therapy (SCI+HBO). Animals in the SCI and SCI+HBO groups were anesthetized, and following laminectomy at the C3–C4 level, a lateral‐cervical spinal contusion was made using a mechanical impactor. HBO therapy consisted of 10 days of exposure starting on the day of SCI, using a 40 L chamber flushed with 100% O2 and pressurized to 3 ATA. Our results indicate that HBO therapy delivered in the acute phase of SCI positively affected BDNF/TrkB signaling. Specifically, diaphragm protein expression of BDNF, p‐AKT/AKT and p‐AMPK/AMPK were increased after 10 days of HBO therapy compared to CON animals (p&lt;0.05). Additionally, HBO therapy prevented the SCI‐induced reduction in diaphragm peroxisome proliferator‐activated receptor‐gamma coactivator (PGC1‐α) protein expression (p&lt;0.05), as well as the caspase‐3‐specific cleavage of α‐II‐spectrin observed in the SCI group (p&lt;0.05). In summary, these findings demonstrate that HBO therapy can induce BDNF signaling and highlight an additional mechanism for the protective effects of HBO therapy to preserve diaphragm muscle function following SCI.

α-tocopherol, a slow-binding inhibitor of acetylcholinesterase

Acetylcholinesterase (AChE) is reversibly inhibited by α-tocopherol (α-T). Steady state kinetic analysis shows that α-T is a mixed slow-binding inhibitor of type A of human enzyme (Kci = 0.49 μM; Kui = 1.6 μM) with a residence time of 2 min on target. Molecular dynamics (MD) simulations support this mechanism, and indicate that α-T first forms multiple non-specific interactions with AChE surface near the gorge entrance, then binds to the peripheral side with alkylene chain slowly sliding down the gorge, inducing no significant conformational change.α-T slightly modulates the progressive inhibition of AChE by the cyclic organophosphorus, cresyl saligenylphosphate, accelerating the fast pseudo-first order process of phosphorylation. A moderate accelerating effect of α-T on phosphorylation by paraoxon was also observed after pre-incubation of AChE in the presence of α-T. This accelerating effect of α-T on ex vivo paraoxon-induced diaphragm muscle weakness was also observed. The effect of α-T on AChE phosphylation was interpreted in light of molecular modeling results. From all results it is clear that α-T does not protect AChE against phosphylation by organophosphorus.

Muscle-specific deletion of the vitamin D receptor in mice is associated with diaphragm muscle weakness.

Diseases or conditions where diaphragm muscle (DIAm) function is impaired, including chronic obstructive pulmonary disease, cachexia, asthma, and aging, are associated with an increased risk of pulmonary symptoms, longer duration of hospitalizations, and increasing requirements for mechanical ventilation. Vitamin D deficiency is associated with proximal muscle weakness that resolves following therapy with vitamin D3. Skeletal muscle expresses the vitamin D receptor (VDR), which responds to the active form of vitamin D, 1,25-dihydroxyvitamin D3 by altering gene expression in target cells. In knockout mice without skeletal muscle VDRs, there is marked atrophy of muscle fibers and a change in skeletal muscle biochemistry. We used a tamoxifen-inducible skeletal muscle Cre recombinase in Vdrfl/fl mice (Vdrfl/fl actin.iCre+) to assess the role of muscle-specific VDR signaling on DIAm-specific force, fatigability, and fiber type-dependent morphology. Vdrfl/fl actin.iCre+ mice treated with vehicle and Vdrfl/fl mice treated with tamoxifen served as controls. Seven days following the final treatment, mice were euthanized, the DIAm was removed, and isometric force and fatigue were assessed in DIAm strips using direct muscle stimulation. The proportion and cross-sectional areas of DIAm fiber types were evaluated by immunolabeling with myosin heavy chain antibodies differentiating type I, IIa and IIx, and/or IIb fibers. We show that in mice with skeletal muscle-specific VDR deletion, maximum specific force and residual force following fatigue are impaired, along with a selective atrophy of type IIx and/or IIb fibers. These results show that the VDR has a significant biological effect on DIAm function independent of systemic effects on mineral metabolism.NEW & NOTEWORTHY Vitamin D deficiency and vitamin D receptor (VDR) polymorphisms are associated with adverse pulmonary and diaphragm muscle (DIAm)-associated respiratory outcomes. We used a skeletal muscle-specific tamoxifen-inducible VDR knockout to investigate DIAm dysfunction following reduced VDR signaling. Marked DIAm weakness and atrophy of type IIx and/or IIb fibers are present in muscle-specific tamoxifen-induced VDR knockout mice compared with controls. These results show that the VDR has a significant biological effect on DIAm function independent of systemic effects on mineral metabolism.

Contribution of extra‐diaphragmatic inspiratory muscles to peak inspiratory pressure in wild‐type and dystrophic ( mdx ) mice

Peak inspiratory pressure-generating capacity is preserved in mdx dystrophic mice, despite profound diaphragm muscle weakness and a reduction in peak diaphragm electromyogram activity1. Our data reveal a capacity for compensation afforded by accessory muscles of breathing, adequate to sustain peak respiratory system performance, at least in early dystrophic disease1,2. We hypothesise that there is a greater contribution by extra-diaphragmatic inspiratory muscles to peak respiratory system performance in mdx mice, a compensation that is ultimately lost with advancing disease. In anesthetized 4-month old wild-type and mdx mice, inspiratory pressure and respiratory electromyogram activities were measured under baseline conditions and following unilateral and bilateral phrenic nerve section, confirmed by bilateral loss of phasic electrical activity in costal electromyogram recordings. Parasternal intercostal electromyogram was recorded in phrenicotomized mice during sustained tracheal occlusion to generate maximal respiratory muscle activation and peak inspiratory pressure. In separate 4-month-old mice with intact phrenic nerves, peak inspiratory pressure was -74±13 cmH20 (mean±SD) in wild-type control mice (n=12) and -90±21 cmH20 in mdx mice (n=13), confirming our previous observation of a retained capacity to achieve normal peak inspiratory pressure in mdx mice, despite diaphragm weakness (50% reduction in peak diaphragm force)1. In 4-month-old phrenicotomized mice, peak inspiratory pressure was -26±9 cmH20 in wild-type mice (n=8) and -55±10 cmH20 in mdx mice (n=8); p<0.05. Peak parasternal EMG activity coinciding with peak pressure generation during airway obstruction was greater in wild-type 34±22 mV.s compared to mdx 23±11 mV.s. Our results confirm the greater reliance on extra-diaphragmatic inspiratory muscles in support of peak inspiratory performance in mdx mice. Despite a 50% reduction in peak diaphragm force, mdx mice generate peak inspiratory pressures that match or exceed those of wild-type mice. Following acute paralysis of the diaphragm, mdx mice generate peak inspiratory pressures that are double that of wild-type mice. These findings suggest plasticity in extra-diaphragmatic motor pathways of breathing. Since there was no evidence of greater recruitment of the obligatory inspiratory parasternal intercostal muscles, we hypothesize a greater contribution by accessory motor pathways in support of peak inspiratory performance in dystrophic mice. We reason that this compensatory plasticity is progressively lost in advancing dystrophic disease, culminating in respiratory morbidity and ultimately failure.

Impact of congenital diaphragmatic hernia on diaphragm muscle function in neonatal rats.

Congenital diaphragmatic hernia (CDH) is characterized by incomplete partitioning of the thoracic and abdominal cavities by the diaphragm muscle (DIAm). The resulting in utero invasion of the abdominal viscera into the thoracic cavity leads to impaired fetal breathing movements, severe pulmonary hypoplasia, and pulmonary hypertension. We hypothesized that in a well-established rodent model of Nitrofen-induced CDH, DIAm isometric force generation, and DIAm fiber cross-sectional areas would be reduced compared with nonlesioned littermate and Control pups. In CDH and nonlesioned pups at embryonic day 21 or birth, DIAm isometric force responses to supramaximal field stimulation (200 mA, 0.5 ms duration pulses in 1-s duration trains at rates ranging from 10 to 100 Hz) was measured ex vivo. Further, DIAm fatigue was determined in response to 120 s of repetitive stimulation at 40 Hz in 330-ms duration trains repeated each second. The DIAm was then stretched to Lo, frozen, and fiber cross-sectional areas were measured in 10 μm transverse sections. In CDH pups, there was a marked reduction in DIAm-specific force and force following 120 s of fatiguing contraction. The cross-sectional area of DIAm fibers was also reduced in CDH pups compared with nonlesioned littermates and Control pups. These results show that CDH is associated with a dramatic weakening of the DIAm, which may contribute to poor survival despite various surgical efforts to repair the hernia and improve lung development.NEW & NOTEWORTHY There are notable respiratory deficits related to congenital diaphragmatic hernia (CDH), yet the contribution, if any, of frank diaphragm muscle weakness to CDH is unexplored. Here, we use the well-established Nitrofen teratogen model to induce CDH in rat pups, followed by diaphragm muscle contractility and morphological assessments. Our results show diaphragm muscle weakness in conjunction with reduced muscle fiber density and size, contributing to CDH morbidity.

NADPH oxidase 2 knockout prevents chronic intermittent hypoxia‐induced diaphragm muscle weakness but not increased propensity for apnoea in a mouse model of obstructive sleep apnoea syndrome

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 diaphragm muscle function and aberrant respiratory plasticity which manifests as destabilised breathing during sleep. We sought to investigate the putative role of the superoxide‐generating NADPH oxidase 2 (NOX2) enzyme in CIH‐induced diaphragm 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 during light hours for 2 weeks. Adult male (C57BL/6J) mice were assigned to one of 5 groups: normoxic controls, CIH‐exposed and CIH+apocynin (NADPH oxidase 2 inhibitor, 2mM) given in the drinking water throughout the CIH exposure, NOX2 null (B6.129S‐Cybbtm1Din/J) assigned to a sham or CIH exposure. On day 15, whole body plethysmography was used to measure breathing parameters on a breath‐by‐breath basis in room air. An apnoea was defined as ≥ 2 missed breaths. Diaphragm muscle contractile function was examined ex vivo. Gene expression was examined by qRT‐PCR. Markers of oxidative stress were determined using spectrophotometric assays. Values are expressed as mean ± SD and data were statistically compared by unpaired Student t‐test.Exposure to CIH significantly decreased diaphragm muscle peak specific force by ~45% compared with sham exposure. Apocynin and NOX2 gene knockout completely prevented CIH‐induced diaphragm muscle weakness. CIH resulted in no overt oxidative stress in diaphragm evidenced by unaltered thiobarbituric acid reactive substances (TBARS) and citrate synthase activity. CIH increased mRNA expression of NRF2, MuRF1, BNIP3, LC3B, GABARAPL1, PARK2, Myostatin, Myogenin, MEF2C, MyoD. Apocynin and NOX2 gene knockout prevented these CIH‐induced increases. Basal minute ventilation was unchanged following 2 weeks of CIH exposure however the number of apnoeas per hour was significantly increased compared with sham exposed mice. Apocynin intervention significantly 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 observed in wild‐type mice.Mice show signs of profound diaphragm muscle dysfunction and increased mRNA expression of genes relating to muscle atrophy, mitophagy, autophagy and regeneration following 2 weeks of CIH. Studies in NOX2 null mice reveal that NOX2 is necessary for CIH‐induced diaphragm 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 OSA). The reduction in apnoea frequency following treatment with apocynin implicates ROS (that are not NOX2‐derived) in the manifestation of CIH‐induced respiratory disturbances. Our results have implications for human OSAS and point to antioxidant intervention as a potential therapeutic strategy.

Diaphragm protection: what should we target?

Diaphragm weakness can impact survival and increases comorbidities in ventilated patients. Mechanical ventilation is linked to diaphragm dysfunction through several mechanisms of injury, referred to as myotrauma. By monitoring diaphragm activity and titrating ventilator settings, the critical care clinician can have a direct impact on diaphragm injury. Both the absence of diaphragm activity and excessive inspiratory effort can result in diaphragm muscle weakness, and recent evidence demonstrates that a moderate level of diaphragm activity during mechanical ventilation improves ICU outcome. This supports the hypothesis that by avoiding ventilator overassistance and underassistance, the clinician can implement a diaphragm-protective ventilation strategy. Furthermore, eccentric diaphragm contractions and end-expiratory shortening could impact diaphragm strength as well. This review describes these potential targets for diaphragm protective ventilation. A ventilator strategy that results in appropriate levels of diaphragm activity has the potential to be diaphragm-protective and improve clinical outcome. Monitoring respiratory effort during mechanical ventilation is becoming increasingly important.

Skeletal Muscle-specific Nox2 Knockout Increases Survival and Partially Protects Against Diaphragm Muscle Weakness in Heart Failure with Reduced Ejection Fraction

Skeletal Muscle-specific Nox2 Knockout Increases Survival and Partially Protects Against Diaphragm Muscle Weakness in Heart Failure with Reduced Ejection Fraction

Hemorrhagic Shock Sensitized the Diaphragm to Ventilator-Induced Dysfunction through the Activation of IL-6/JAK/STAT Signaling-Mediated Autophagy in Rats.

Mechanical ventilation (MV) is a major life support technique for the management of trauma-associated hemorrhagic shock (HS). Ventilator-induced diaphragm dysfunction (VIDD), one of the most common complications of MV, has been well demonstrated in animal and human studies. However, few data are available concerning the effects of MV on diaphragm function in HS victims. In the present study, we found diaphragm muscle atrophy and weakness in HS but not in healthy animals after 4 hours of MV. The inhibition of autophagy resulted in reduced muscle fiber atrophy and improved forces. In addition, we observed diaphragmatic interleukin- (IL-) 6 overexpression and activation of its downstream signaling JAK/STAT in HS animals after MV, and either the neutralization of IL-6 or the inhibition of the JAK/STAT pathway attenuated autophagy, diaphragm atrophy, and weakness. Importantly, treatment with nonselective antioxidant exerted no protective effects against VIDD in HS animals. In addition, in vitro study showed that exogenous IL-6 was able to induce activation of JAK/STAT signaling and to increase autophagy in C2C12 cells. Moreover, the inhibition of JAK/STAT signaling abolished IL-6-induced cell autophagy. Together, our results suggested that HS sensitized the diaphragm to ventilator-induced atrophy and weakness through the activation of IL-6/JAK/STAT signaling-mediated autophagy in rats.

Western Diet-Induced Obesity Does Not Cause Diaphragm Muscle Abnormalities

PURPOSE: Obese individuals demonstrate ventilatory abnormalities at rest and during exercise, which may reflect inspiratory (diaphragm) muscle weakness. Several studies have shown diaphragm abnormalities in genetically obese animal models. However, the effects of an obesogenic diet on the diaphragm are not well defined. The purpose of this study was to determine if a Western diet (high-saturated fat, high-sugar: HFHS) causes diaphragm muscle pathology, perhaps related to oxidative stress. METHODS: Adult male Wistar rats were randomly allocated to one of two ad libitum diets for 24 weeks: healthy control (n=8) or HFHS diet (n=8). Diaphragm muscle then underwent i) in vitro contractile function assessment, ii) histology and immunohistochemistry for determination of fibrosis and fiber cross-sectional area (CSA) and iii) analysis of mitochondrial reactive oxygen species (ROS) emission. Data are mean ± SE. RESULTS: We previously reported that HFHS diet did not impair diaphragm maximal specific force or peak power. Alongside this maintenance of contractile function, HFHS-fed rats did not differ in diaphragm muscle interstitial fibrosis (in %: lean 3.4±0.8, HFHS 2.7±1.7), type I fiber CSA (in μm2: lean 1954±121, HFHS 2174±103), type IIa fiber CSA (in μm2: lean 2062±329, HFHS 2412±155), or type IIb/x fiber CSA (in μm2: lean 4165±1232, HFHS 4523±526). Additionally, no shifts in fiber type occurred. Interestingly, HFHS diaphragm demonstrated a trend toward a lower succinate-induced increase in ROS emission (in pmol/min: lean 1.313 ± 0.2, HFHS 0.73 ± 0.2, p = 0.09). Analyses of glutathione and antioxidant enzymes are ongoing. CONCLUSION: A high-saturated fat, high-sugar diet did not induce diaphragm muscle dysfunction or morphological changes. These results, combined with our previous findings of normal contractile function, suggest that intrinsic diaphragm muscle abnormalities do not contribute to breathing difficulties in obesity.

Endoplasmic reticulum stress and unfolded protein response in diaphragm muscle dysfunction of patients with stable chronic obstructive pulmonary disease.

Respiratory muscle dysfunction is common in patients with chronic obstructive pulmonary disease (COPD). Chronic contractile activity induces endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in animals (animals and humans). We hypothesized that the respiratory muscle dysfunction associated with COPD may upregulate ER stress and UPR expression in diaphragm of stable patients with different degrees of airway obstruction and normal body composition. In diaphragm muscle specimens of patients with mild and moderate-to-severe COPD with preserved body composition and non-COPD controls (thoracotomy because of lung localized neoplasms), expression of protein misfolding (ER stress) and UPR markers, proteolysis and apoptosis (qRT-PCR and immunoblotting), and protein aggregates (lipofuscin, histology) were quantified. All patients and non-COPD controls were also clinically evaluated: lung and muscle functions and exercise capacity. Compared with non-COPD controls, patients exhibited mild and moderate-to-severe airflow limitation and diffusion capacity and impaired exercise tolerance and diaphragm strength. Moreover, compared with the controls, in the diaphragm of the COPD patients, slow-twitch fiber proportions increased, gene expression but not protein levels of protein disulfide isomerase family A member 3 and phosphatidylinositol 3-kinase catalytic subunit type 3 were upregulated, and no significant differences were found in markers of UPR transmembrane receptor pathways (activating transcription factor-6, inositol-requiring enzyme-1α, and protein kinase-like ER kinase), lipofuscin aggregates, proteolysis, or apoptosis. In stable COPD patients with a wide range of disease severity, reduced diaphragm force of contraction, and normal body composition, ER stress and UPR signaling were not induced in the main respiratory muscle. These findings imply that ER stress and UPR are probably not involved in the documented diaphragm muscle dysfunction (reduced strength) observed in all the study patients, even in those with severe airflow limitation. Hence, in stable COPD patients with normal body composition, therapeutic strategies targeted to treat diaphragm muscle dysfunction should not include UPR modulators, even in those with a more advanced disease. NEW & NOTEWORTHY In stable chronic obstructive pulmonary disease patients with a wide range of disease severity, diaphragm muscle weakness, and normal body composition, endoplasmic reticulum stress and unfolded protein response (UPR) signaling were not induced in the main respiratory muscle. These findings imply that endoplasmic reticulum stress and UPR are not involved in the documented diaphragm muscle dysfunction observed in the study patients, even in those with severe airflow limitation. In stable chronic obstructive pulmonary disease patients with normal body composition, therapeutic strategies should not include UPR modulators.

Role of NADPH oxidase in chronic intermittent hypoxia‐induced respiratory dysfunction: Insights from pharmacological and transgenic approaches

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.