V2a neurons restore diaphragm function in mice following spinal cord injury.

The specific roles that different types of neurons play in recovery from injury is poorly understood. Here, we show that increasing the excitability of ipsilaterally projecting, excitatory V2a neurons using designer receptors exclusively activated by designer drugs (DREADDs) restores rhythmic bursting activity to a previously paralyzed diaphragm within hours, days, or weeks following a C2 hemisection injury. Further, decreasing the excitability of V2a neurons impairs tonic diaphragm activity after injury as well as activation of inspiratory activity by chemosensory stimulation, but does not impact breathing at rest in healthy animals. By examining the patterns of muscle activity produced by modulating the excitability of V2a neurons, we provide evidence that V2a neurons supply tonic drive to phrenic circuits rather than increase rhythmic inspiratory drive at the level of the brainstem. Our results demonstrate that the V2a class of neurons contribute to recovery of respiratory function following injury. We propose that altering V2a excitability is a potential strategy to prevent respiratory motor failure and promote recovery of breathing following spinal cord injury.

An acytokinetic cell division creates PIP2-enriched membrane asymmetries leading to slit diaphragm assembly in Drosophila nephrocytes.

Vertebrate podocytes and Drosophila nephrocytes display slit diaphragms, specialised cell junctions that are essential for the execution of the basic excretory function of ultrafiltration. To elucidate the mechanisms of slit diaphragm assembly we have studied their formation in Drosophila embryonic garland nephrocytes. These cells of mesenchymal origin lack overt apical-basal polarity. We find that their initial membrane symmetry is broken by an acytokinetic cell division that generates PIP2-enriched domains at their equator. The PIP2-enriched equatorial cortex becomes a favourable domain for hosting slit diaphragm proteins and the assembly of the first slit diaphragms. Indeed, when this division is either prevented or forced to complete cytokinesis, the formation of diaphragms is delayed to larval stages. Furthermore, although apical polarity determinants also accumulate at the equatorial cortex, they do not appear to participate in the recruitment of slit diaphragm proteins. The mechanisms we describe allow the acquisition of functional nephrocytes in embryos, which may confer on them a biological advantage similar to the formation of the first vertebrate kidney, the pronephros.

Structural insights into plasmalemma vesicle-associated protein (PLVAP): Implications for vascular endothelial diaphragms and fenestrae.

In many organs, small openings across capillary endothelial cells (ECs) allow the diffusion of low-molecular weight compounds and small proteins between the blood and tissue spaces. These openings contain a diaphragm composed of radially arranged fibers, and current evidence suggests that a single-span type II transmembrane protein, plasmalemma vesicle-associated protein-1 (PLVAP), constitutes these fibers. Here, we present the three-dimensional crystal structure of an 89-amino acid segment of the PLVAP extracellular domain (ECD) and show that it adopts a parallel dimeric alpha-helical coiled-coil configuration with five interchain disulfide bonds. The structure was solved using single-wavelength anomalous diffraction from sulfur-containing residues (sulfur SAD) to generate phase information. Biochemical and circular dichroism (CD) experiments show that a second PLVAP ECD segment also has a parallel dimeric alpha-helical configuration-presumably a coiled coil-held together with interchain disulfide bonds. Overall, ~2/3 of the ~390 amino acids within the PLVAP ECD adopt a helical configuration, as determined by CD. We also determined the sequence and epitope of MECA-32, an anti-PLVAP antibody. Taken together, these data lend strong support to the model of capillary diaphragms formulated by Tse and Stan in which approximately ten PLVAP dimers are arranged within each 60- to 80-nm-diameter opening like the spokes of a bicycle wheel. Passage of molecules through the wedge-shaped pores is presumably determined both by the length of PLVAP-i.e., the long dimension of the pore-and by the chemical properties of amino acid side chains and N-linked glycans on the solvent-accessible faces of PLVAP.

Thoracoabdominal Wall Motion-Guided Biofeedback Treatment of Abdominal Distention: A Randomized Placebo-Controlled Trial.

BACKGROUND & AIMS: Abdominal distention results from abdominophrenic dyssynergia (ie, diaphragmatic contraction and abdominal wall relaxation) in patients with disorders of gut-brain interaction. This study aimed to validate a simple biofeedback procedure, guided by abdominothoracic wall motion, for treating abdominal distention. METHODS: In this randomized, parallel, placebo-controlled trial, 42 consecutive patients (36 women and 6 men; ages 17-64 years) with meal-triggered visible abdominal distention were recruited. Recordings of abdominal and thoracic wall motion were obtained using inductance plethysmography via adaptable belts. The signal was shown to patients in the biofeedback group, who were taught to mobilize the diaphragm. In contrast, the signal was not shown to the patients in the placebo group, who were given a placebo capsule. Three sessions were performed over a 4-week intervention period, with instructions to perform exercises (biofeedback group) or to take placebo 3 times per day (control group) at home. Outcomes were assessed through response to an offending meal (changes in abdominothoracic electromyographic activity and girth) and clinical symptoms measured using daily scales for 7 days. RESULTS: Patients in the biofeedback group (n = 19) learned to correct abdominophrenic dyssynergia triggered by the offending meal (intercostal activity decreased by a mean ± SE of 82% ± 10%, anterior wall activity increased by a mean ± SE of 97% ± 6%, and increase in girth was a mean ± SE of 108% ± 4% smaller) and experienced improved clinical symptoms (abdominal distention scores decreased by a mean ± SE of 66% ± 5%). These effects were not observed in the placebo group (all, P < .002). CONCLUSIONS: Abdominothoracic wall movements serve as an effective biofeedback signal for correcting abdominophrenic dyssynergia and abdominal distention in patients with disorders of gut-brain interaction. ClincialTrials.gov, Number: NCT04043208.

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).

The slow-release adiponectin analog ALY688-SR modifies early-stage disease development in the D2.mdx mouse model of Duchenne muscular dystrophy.

Fibrosis is associated with respiratory and limb muscle atrophy in Duchenne muscular dystrophy (DMD). Current standard of care partially delays the progression of this myopathy but there remains an unmet need to develop additional therapies. Adiponectin receptor agonism has emerged as a possible therapeutic target to lower inflammation and improve metabolism in mdx mouse models of DMD but the degree to which fibrosis and atrophy are prevented remain unknown. Here, we demonstrate that the recently developed slow-release peptidomimetic adiponectin analog, ALY688-SR, remodels the diaphragm of murine model of DMD on DBA background (D2.mdx) mice treated from days 7-28 of age during early stages of disease. ALY688-SR also lowered interleukin-6 (IL-6) mRNA but increased IL-6 and transforming growth factor-ß1 (TGF-ß1) protein contents in diaphragm, suggesting dynamic inflammatory remodeling. ALY688-SR alleviated mitochondrial redox stress by decreasing complex I-stimulated H2O2 emission. Treatment also attenuated fibrosis, fiber type-specific atrophy, and in vitro diaphragm force production in diaphragm suggesting a complex relationship between adiponectin receptor activity, muscle remodeling, and force-generating properties during the very early stages of disease progression in murine model of DMD on DBA background (D2.mdx) mice. In tibialis anterior, the modest fibrosis at this young age was not altered by treatment, and atrophy was not apparent at this young age. These results demonstrate that short-term treatment of ALY688-SR in young D2.mdx mice partially prevents fibrosis and fiber type-specific atrophy and lowers force production in the more disease-apparent diaphragm in relation to lower mitochondrial redox stress and heterogeneous responses in certain inflammatory markers. These diverse muscle responses to adiponectin receptor agonism in early stages of DMD serve as a foundation for further mechanistic investigations.NEW & NOTEWORTHY There are limited therapies for the treatment of Duchenne muscular dystrophy. As fibrosis involves an accumulation of collagen that replaces muscle fibers, antifibrotics may help preserve muscle function. We report that the novel adiponectin receptor agonist ALY688-SR prevents fibrosis in the diaphragm of D2.mdx mice with short-term treatment early in disease progression. These responses were related to altered inflammation and mitochondrial functions and serve as a foundation for the development of this class of therapy.

Irisin attenuates ventilator-induced diaphragmatic dysfunction by inhibiting endoplasmic reticulum stress through activation of AMPK.

Mechanical ventilation (MV) is an essential life-saving technique, but prolonged MV can cause significant diaphragmatic dysfunction due to atrophy and decreased contractility of the diaphragm fibres, called ventilator-induced diaphragmatic dysfunction (VIDD). It is not clear about the mechanism of occurrence and prevention measures of VIDD. Irisin is a newly discovered muscle factor that regulates energy metabolism. Studies have shown that irisin can exhibit protective effects by downregulating endoplasmic reticulum (ER) stress in a variety of diseases; whether irisin plays a protective role in VIDD has not been reported. Sprague-Dawley rats were mechanically ventilated to construct a VIDD model, and intervention was performed by intravenous administration of irisin. Diaphragm contractility, degree of atrophy, cross-sectional areas (CSAs), ER stress markers, AMPK protein expression, oxidative stress indicators and apoptotic cell levels were measured at the end of the experiment.Our findings showed that as the duration of ventilation increased, the more severe the VIDD was, the degree of ER stress increased, and the expression of irisin decreased.ER stress may be one of the causes of VIDD. Intervention with irisin ameliorated VIDD by reducing the degree of ER stress, attenuating oxidative stress, and decreasing the apoptotic index. MV decreases the expression of phosphorylated AMPK in the diaphragm, whereas the use of irisin increases the expression of phosphorylated AMPK. Irisin may exert its protective effect by activating the phosphorylated AMPK pathway.

Ventilator-induced diaphragm dysfunction: phenomenology and mechanism(s) of pathogenesis.

Mechanical ventilation (MV) is used to support ventilation and pulmonary gas exchange in patients during critical illness and surgery. Although MV is a life-saving intervention for patients in respiratory failure, an unintended side-effect of MV is the rapid development of diaphragmatic atrophy and contractile dysfunction. This MV-induced diaphragmatic weakness is labelled as 'ventilator-induced diaphragm dysfunction' (VIDD). VIDD is an important clinical problem because diaphragmatic weakness is a risk factor for the failure to wean patients from MV. Indeed, the inability to remove patients from ventilator support results in prolonged hospitalization and increased morbidity and mortality. The pathogenesis of VIDD has been extensively investigated, revealing that increased mitochondrial production of reactive oxygen species within diaphragm muscle fibres promotes a cascade of redox-regulated signalling events leading to both accelerated proteolysis and depressed protein synthesis. Together, these events promote the rapid development of diaphragmatic atrophy and contractile dysfunction. This review highlights the MV-induced changes in the structure/function of diaphragm muscle and discusses the cell-signalling mechanisms responsible for the pathogenesis of VIDD. This report concludes with a discussion of potential therapeutic opportunities to prevent VIDD and suggestions for future research in this exciting field.

Conserved mammalian muscle mechanics during eccentric contractions.

Skeletal muscle has a broad range of biomechanical functions, including power generation and energy absorption. These roles are underpinned by the force-velocity relationship, which comprises two distinct components: a concentric and an eccentric force-velocity relationship. The concentric component has been extensively studied across a wide range of muscles with different muscle properties. However, to date, little progress has been made in accurately characterising the eccentric force-velocity relationship in mammalian muscle with varying muscle properties. Consequently, mathematical models of this muscle behaviour are based on a poorly understood phenomenon. Here, we present a comprehensive assessment of the concentric force-velocity and eccentric force-velocity relationships of four mammalian muscles (soleus, extensor digitorum longus, diaphragm and digastric) with varying biomechanical functions, spanning three orders of magnitude in body mass (mouse, rat and rabbits). The force-velocity relationship was characterised using a hyperbolic-linear equation for the concentric component a hyperbolic equation for the eccentric component, at the same time as measuring the rate of force development in the two phases of force development in relation to eccentric lengthening velocity. We demonstrate that, despite differences in the curvature and plateau height of the eccentric force-velocity relationship, the rates of relative force development were consistent for the two phases of the force-time response during isovelocity lengthening ramps, in relation to lengthening velocity, in the four muscles studied. Our data support the hypothesis that this relationship depends on cross-bridge and titin activation. Hill-type musculoskeletal models of the eccentric force-velocity relationship for mammalian muscles should incorporate this biphasic force response. KEY POINTS: The capacity of skeletal muscle to generate mechanical work and absorb energy is underpinned by the force-velocity relationship. Despite identification of the lengthening (eccentric) force-velocity relationship over 80 years ago, no comprehensive study has been undertaken to characterise this relationship in skeletal muscle. We show that the biphasic force response seen during active muscle lengthening is conserved over three orders of magnitude of mammalian skeletal muscle mass. Using mice with a small deletion in titin, we show that part of this biphasic force profile in response to muscle lengthening is reliant on normal titin activation. The rate of force development during muscle stretch may be a more reliable way to describe the forces experienced during eccentric muscle contractions compared to the traditional hyperbolic curve fitting, and functions as a novel predictor of force-velocity characteristics that may be used to better inform hill-type musculoskeletal models and assess pathophysiological remodelling.

Mucopolysaccharidosis (MPS IIIA) mice have increased lung compliance and airway resistance, decreased diaphragm strength, and no change in alveolar structure.

Mucopolysaccharidosis type IIIA (MPS IIIA) is characterized by neurological and skeletal pathologies caused by reduced activity of the lysosomal hydrolase, sulfamidase, and the subsequent primary accumulation of undegraded heparan sulfate (HS). Respiratory pathology is considered secondary in MPS IIIA and the mechanisms are not well understood. Changes in the amount, metabolism, and function of pulmonary surfactant, the substance that regulates alveolar interfacial surface tension and modulates lung compliance and elastance, have been reported in MPS IIIA mice. Here we investigated changes in lung function in 20-wk-old control and MPS IIIA mice with a closed and open thoracic cage, diaphragm contractile properties, and potential parenchymal remodeling. MPS IIIA mice had increased compliance and airway resistance and reduced tissue damping and elastance compared with control mice. The chest wall impacted lung function as observed by an increase in airway resistance and a decrease in peripheral energy dissipation in the open compared with the closed thoracic cage state in MPS IIIA mice. Diaphragm contractile forces showed a decrease in peak twitch force, maximum specific force, and the force-frequency relationship but no change in muscle fiber cross-sectional area in MPS IIIA mice compared with control mice. Design-based stereology did not reveal any parenchymal remodeling or destruction of alveolar septa in the MPS IIIA mouse lung. In conclusion, the increased storage of HS which leads to biochemical and biophysical changes in pulmonary surfactant also affects lung and diaphragm function, but has no impact on lung or diaphragm structure at this stage of the disease.NEW & NOTEWORTHY Heparan sulfate storage in the lungs of mucopolysaccharidosis type IIIA (MPS IIIA) mice leads to changes in lung function consistent with those of an obstructive lung disease and includes an increase in lung compliance and airway resistance and a decrease in tissue elastance. In addition, diaphragm muscle contractile strength is reduced, potentially further contributing to lung function impairment. However, no changes in parenchymal lung structure were observed in mice at 20 wk of age.

Small molecule inhibition of glycogen synthase I reduces muscle glycogen content and improves biomarkers in a mouse model of Pompe disease.

Pompe disease is a rare genetic disorder caused by a deficiency of the enzyme acid alpha-glucosidase (GAA). This enzyme is responsible for breaking down glycogen, leading to the abnormal accumulation of glycogen, which results in progressive muscle weakness and metabolic dysregulation. In this study, we investigated the hypothesis that the small molecule inhibition of glycogen synthase I (GYS1) may reduce muscle glycogen content and improve metabolic dysregulation in a mouse model of Pompe disease. To address this hypothesis, we studied four groups of male mice: a control group of wild-type (WT) B6129SF1/J mice fed either regular chow or a GYS1 inhibitor (MZ-101) diet (WT-GYS1), and Pompe model mice B6;129-Gaatm1Rabn/J fed either regular chow (GAA-KO) or MZ-101 diet (GAA-GYS1) for 7 days. Our findings revealed that GAA-KO mice exhibited abnormal glycogen accumulation in the gastrocnemius, heart, and diaphragm. In contrast, inhibiting GYS1 reduced glycogen levels in all tissues compared with GAA-KO mice. Furthermore, GAA-KO mice displayed reduced spontaneous activity during the dark cycle compared with WT mice, whereas GYS1 inhibition counteracted this effect. Compared with GAA-KO mice, GAA-GYS1 mice exhibited improved glucose tolerance and whole body insulin sensitivity. These improvements in insulin sensitivity could be attributed to increased AMP-activated protein kinase phosphorylation in the gastrocnemius of WT-GYS1 and GAA-GYS1 mice. Additionally, the GYS1 inhibitor led to a reduction in the phosphorylation of GSS641 and the LC3 autophagy marker. Together, our results suggest that targeting GYS1 could serve as a potential strategy for treating glycogen storage disorders and metabolic dysregulation.NEW & NOTEWORTHY We investigated the effects of small molecule inhibition of glycogen synthase I (GYS1) on glucose metabolism in a mouse model of Pompe disease. GYS1 inhibition reduces abnormal glycogen accumulation and molecular biomarkers associated with Pompe disease while also improving glucose intolerance. Our results collectively demonstrate that the GYS1 inhibitor represents a novel approach to substrate reduction therapy for Pompe disease.

Identification of eupneic breathing using machine learning.

The diaphragm muscle (DIAm) is the primary inspiratory muscle in mammals. In awake animals, considerable heterogeneity in the electromyographic (EMG) activity of the DIAm reflects varied ventilatory and nonventilatory behaviors. Experiments in awake animals are an essential component to understanding the neuromotor control of breathing, which has especially begun to be appreciated within the last decade. However, insofar as the intent is to study the control of breathing, it is paramount to identify DIAm EMG activity that in fact reflects breathing. Current strategies for doing so in a reproducible, reliable, and efficient fashion are lacking. In the present article, we evaluated DIAm EMG from awake animals using hierarchical clustering across four-dimensional feature space to classify eupneic breathing. Our model, which can be implemented with automated threshold of the clustering dendrogram, successfully identified eupneic breathing with high F1 score (0.92), specificity (0.70), and accuracy (0.88), suggesting that it is a robust and reliable tool for investigating the neural control of breathing.NEW & NOTEWORTHY The heterogeneity of diaphragm muscle (DIAm) activity in awake animals reflects real motor behavior diversity but makes assessments of eupneic breathing challenging. The present article uses an unsupervised machine learning model to identify eupneic breathing amidst a deluge of different DIAm electromyography (EMG) burst patterns in awake rats. This technique offers a scalable and reliable tool that improves efficiency of DIAm EMG analysis and minimizes potential sources of bias.

Functional involvement of the sternohyoid muscle during breathing and swallowing in rats.

The sternohyoid muscle depresses the hyoid bone, but it is unclear whether the muscle contributes to respiratory and swallowing mechanisms. This study aimed to clarify whether the sternohyoid muscle participates in the respiration and swallowing reflex and how the activity is modulated in two conditions: with airway stenosis and with a fixed sternohyoid muscle length. Electromyographic activity in the sternohyoid, digastric, thyrohyoid, and diaphragm muscles was recorded in anesthetized rats. The sternohyoid muscle activity was observed in the inspiratory phase and during swallowing, and was well coordinated with digastric and thyrohyoid muscle activity. With airway stenosis, the respiratory activity per respiratory cycle was facilitated in all assessed muscles but the facilitation of activity per second occurred only in the digastric, thyrohyoid, and sternohyoid muscles. With airway stenosis, the swallowing activity was facilitated only in the digastric muscle but not in the thyrohyoid and sternohyoid muscles. Swallowing activity was not observed in the sternohyoid muscle in the condition with the sternohyoid muscle length fixed, although increased inspiratory activity remained. The current results suggest that 1) the sternohyoid muscle is slightly activated in the inspiratory phase, 2) the effect of airway stenosis on respiratory function may differ between the upper airway muscles and diaphragm, and 3) swallowing activity in the sternohyoid muscle is not dominantly controlled by the swallowing central pattern generator but instead occurs as a myotatic reflex.NEW & NOTEWORTHY We found that the sternohyoid muscle was activated in the inspiratory phase. However, increased airway resistance had different effects on the extrathoracic muscles than on the diaphragm. The swallowing activity of the sternohyoid disappeared when the muscle length was fixed. These findings suggest that the sternohyoid muscle may be activated not by the swallowing central pattern generator but as a myotatic reflex.

Definition of diaphragmatic sleep disordered breathing and clinical meaning in Duchenne muscular dystrophy.

BACKGROUND: Diaphragmatic sleep disordered breathing (dSDB) has been recently identified as sleep dysfunction secondary to diaphragmatic weakness in Duchenne muscular dystrophy (DMD). However, scoring criteria for the identification of dSDB are missing.This study aimed to define and validate dSDB scoring criteria and to evaluate whether dSDB severity correlates with respiratory progression in DMD. METHODS: Scoring criteria for diaphragmatic apnoea (dA) and hypopnoeas (dH) have been defined by the authors considering the pattern observed on cardiorespiratory polygraphy (CR) and the dSDB pathophysiology.10 sleep professionals (physiologists, consultants) blinded to each other were involved in a two-round Delphi survey to rate each item of the proposed dSDB criteria (Likert scale 1-5) and to recognise dSDB among other SDB. The scorers' accuracy was tested against the authors' panel.Finally, CR previously conducted in DMD in clinical setting were rescored and diaphragmatic Apnoea-Hypopnoea Index (dAHI) was derived. Pulmonary function (forced vital capacity per cent of predicted, FVC%pred), overnight oxygen saturation (SpO2) and transcutaneous carbon dioxide (tcCO2) were correlated with dAHI. RESULTS: After the second round of Delphi, raters deemed each item of dA and dH criteria as relevant as 4 or 5. The agreement with the panel in recognising dSDB was 81%, kappa 0.71, sensitivity 77% and specificity 85%.32 CRs from DMD patients were reviewed. dSDB was previously scored as obstructive. The dAHI negatively correlated with FVC%pred (r=-0.4; p<0.05). The total number of dA correlated with mean overnight tcCO2 (r 0.4; p<0.05). CONCLUSIONS: dSDB is a newly defined sleep disorder that correlates with DMD progression. A prospective study to evaluate dSDB as a respiratory measure for DMD in clinical and research settings is planned.

Improvements of the shape and strength of the diaphragm after endoscopic lung volume reduction.

RATIONALE: Endoscopic lung volume reduction improves lung function, quality of life and exercise capacity in severe emphysema patients. However, its effect on the diaphragm function is not well understood. We hypothesised that endoscopic lung volume reduction increases its strength by modifying its shape. OBJECTIVES: To investigate changes in both diaphragm shape and strength induced by the insertion of endobronchial valves. METHODS: In 19 patients, both the diaphragm shape and strength were investigated respectively by 3D Slicer software applied on CT scans acquired at functional residual capacity and by transdiaphragmatic pressure measurements by bilateral magnetic stimulation of the phrenic nerves before and 3 months after unilateral valves insertion. MEASUREMENTS AND MAIN RESULTS: After lung volume reduction (median (IQR), 434 mL (-597 to -156], p<0.0001), diaphragm strength increased (transdiaphragmatic pressure: 3 cmH2O (2.3 to 4.2), p<0.0001). On the treated side, this increase was associated with an increase in the coronal (16 mm (13 to 24), p<0.0001) and sagittal (26 mm (21 to 30), p<0.0001) lengths as well as in the area of the zone of apposition (62 cm2 (3 to 100), p<0.0001) with a decrease in the coronal (8 mm (-12 to -4), p<0.0001) and sagittal (9 mm (-18 to -2), p=0.0029) radii of curvature. CONCLUSIONS: Endoscopic lung volume reduction modifies the diaphragm shape by increasing its length and its zone of apposition and by decreasing its radius of curvature on the treated side, resulting in an increase in its strength. TRIAL REGISTRATION NUMBER: NCT05799352.

Endocytosis mediated by an atypical CUBAM complex modulates slit diaphragm dynamics in nephrocytes.

The vertebrate endocytic receptor CUBAM, consisting of three cubilin monomers complexed with a single amnionless molecule, plays a major role in protein reabsorption in the renal proximal tubule. Here, we show that Drosophila CUBAM is a tripartite complex composed of Amnionless and two cubilin paralogues, Cubilin and Cubilin2, and that it is required for nephrocyte slit diaphragm (SD) dynamics. Loss of CUBAM-mediated endocytosis induces dramatic morphological changes in nephrocytes and promotes enlarged ingressions of the external membrane and SD mislocalisation. These phenotypes result in part from an imbalance between endocytosis, which is strongly impaired in CUBAM mutants, and exocytosis in these highly active cells. Of note, rescuing receptor-mediated endocytosis by Megalin/LRP2 or Rab5 expression only partially restores SD positioning in CUBAM mutants, suggesting a specific requirement of CUBAM in SD degradation and/or recycling. This finding and the reported expression of CUBAM in podocytes suggest a possible unexpected conserved role for this endocytic receptor in vertebrate SD remodelling.

Defective mesothelium and limited physical space are drivers of dysregulated lung development in a genetic model of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a developmental disorder associated with diaphragm defects and lung hypoplasia. The etiology of CDH is complex and its clinical presentation is variable. We investigated the role of the pulmonary mesothelium in dysregulated lung growth noted in the Wt1 knockout mouse model of CDH. Loss of WT1 leads to intrafetal effusions, altered lung growth, and branching defects prior to normal closure of the diaphragm. We found significant differences in key genes; however, when Wt1 null lungs were cultured ex vivo, growth and branching were indistinguishable from wild-type littermates. Micro-CT imaging of embryos in situ within the uterus revealed a near absence of space in the dorsal chest cavity, but no difference in total chest cavity volume in Wt1 null embryos, indicating a redistribution of pleural space. The altered space and normal ex vivo growth suggest that physical constraints are contributing to the CDH lung phenotype observed in this mouse model. These studies emphasize the importance of examining the mesothelium and chest cavity as a whole, rather than focusing on single organs in isolation to understand early CDH etiology.

Influence of an inspiratory muscle fatigue protocol on healthy youths on respiratory muscle strength, vertical jump performance and muscle oxygen saturation: a randomized controlled trial.

BACKGROUND: Inspiratory muscle fatigue has been shown to have effects on limbs blood flow and physical performance. This study aimed to evaluate the influence of an inspiratory muscle fatigue protocol on respiratory muscle strength, vertical jump performance and muscle oxygen saturation in healthy youths. METHODS: A randomized and double-blinded controlled clinical trial, was conducted. Twenty-four participants aged 18-45 years, non-smokers and engaged in sports activity at least three times a week for a minimum of one year were enrolled in this investigation. Participants were randomly assigned to three groups: Inspiratory Muscle Fatigue (IMFG), Activation, and Control. Measurements of vertical jump, diaphragmatic ultrasound, muscle oxygen saturation, and maximum inspiratory pressure were taken at two stages: before the intervention (T1) and immediately after treatment (T2). RESULTS: The IMFG showed lower scores in muscle oxygen saturation and cardiorespiratory variables after undergoing the diaphragmatic fatigue intervention compared to the activation and control groups (p < 0.05). For the vertical jump variables, intragroup differences were found (p < 0.01), but no differences were shown between the three groups (p > 0.05). CONCLUSIONS: Inspiratory muscle fatigue appears to negatively impact vertical jump performance, muscle oxygen saturation and inspiratory muscle strength in healthy youths. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT06271876. Date of registration 02/21/2024. https://clinicaltrials.gov/study/NCT06271876 .

Reduction of ringing artifacts induced by diaphragm drifting in free-breathing dynamic pulmonary MRI using 3D koosh-ball acquisition.

PURPOSE: To reduce the ringing artifacts of the motion-resolved images in free-breathing dynamic pulmonary MRI. METHODS: A golden-step based interleaving (GSI) technique was proposed to reduce ringing artifacts induced by diaphragm drifting. The pulmonary MRI data were acquired using a superior-inferior navigated 3D radial UTE sequence in an interleaved manner during free breathing. Successive interleaves were acquired in an incoherent fashion along the polar direction. Four-dimensional images were reconstructed from the motion-resolved k-space data obtained by retrospectively binning. The reconstruction algorithms included standard nonuniform fast Fourier transform (NUFFT), Voronoi-density-compensated NUFFT, extra-dimensional UTE, and motion-state weighted motion-compensation reconstruction. The proposed interleaving technique was compared with a conventional sequential interleaving (SeqI) technique on a phantom and eight subjects. RESULTS: The quantified ringing artifacts level in the motion-resolved image is positively correlated with the quantified nonuniformity level of the corresponding k-space. The nonuniformity levels of the end-expiratory and end-inspiratory k-space binned from GSI data (0.34 ± 0.07, 0.33 ± 0.05) are significantly lower with statistical significance (p < 0.05) than that binned from SeqI data (0.44 ± 0.11, 0.42 ± 0.12). Ringing artifacts are substantially reduced in the dynamic images of eight subjects acquired using the proposed technique in comparison with that acquired using the conventional SeqI technique. CONCLUSION: Ringing artifacts in the motion-resolved images induced by diaphragm drifting can be reduced using the proposed GSI technique for free-breathing dynamic pulmonary MRI. This technique has the potential to reduce ringing artifacts in free-breathing liver and kidney MRI based on full-echo interleaved 3D radial acquisition.

Effects of aging on diaphragm hyperemia and blood flow distribution in male and female Fischer 344 rats.

Aging is associated with inspiratory muscle dysfunction; however, the impact of aging on diaphragm blood flow (BF) regulation, and whether sex differences exist, is unknown. We tested the hypotheses in young animals that diaphragm BF and vascular conductance (VC) would be greater in females and that aging would decrease the diaphragm's ability to increase BF with contractions. Young (4-6 mo) and old (22-24 mo) Fischer 344 rats were divided into four groups: young female (YF, n = 7), young male (YM, n = 8), old female (OF, n = 9), and old male (OM, n = 9). Diaphragm BF (mL/min/100 g) and VC (mL/mmHg/min/100 g) were determined, via fluorescent microspheres, at rest and during 1 Hz contractions. In YF versus OF, aging blunted the increase in medial costal diaphragm BF (44 ± 5% vs. 16 ± 12%; P < 0.05) and VC (43 ± 7% vs. 21 ± 12%; P < 0.05). Similarly, in YM versus OM, aging blunted the increase in medial costal diaphragm BF (43 ± 6% vs. 24 ± 12%; P < 0.05) and VC (50 ± 6% vs. 34 ± 10%; P < 0.05). In female rats, age increased dorsal costal diaphragm BF, whereas in male rats, age increased crural diaphragm BF (P < 0.05). Compared with age-matched females, dorsal costal diaphragm BF was lower in YM and OM (P < 0.05). In conclusion, aging results in an inability to augment medial costal diaphragm BF and alters regional diaphragm BF distribution in response to muscular contractions. Furthermore, sex differences in regional diaphragm BF are present in young and old animals.NEW & NOTEWORTHY This is the first study, to our knowledge, to demonstrate that old age impairs the hyperemic response and alters blood flow distribution in the diaphragm of both female and male rats. In addition, this investigation provides novel evidence of sex differences in regional diaphragm blood flow distribution with contractions. The data presented herein suggest that aging compromises diaphragm vascular function and provides a potential mechanism for the diaphragm contractile dysfunction associated with old age.