Lack of compensatory mitophagy in skeletal muscles during sepsis.

Skeletal muscle dysfunction is a major problem in critically ill patients suffering from sepsis. This condition is associated with mitochondrial dysfunction and increased autophagy in skeletal muscles. Autophagy is a proteolytic mechanism involved in eliminating dysfunctional cellular components, including mitochondria. The latter process, referred to as mitophagy, is essential for maintaining mitochondrial quality and skeletal muscle health. Recently, a fluorescent reporter system called mito-QC (i.e. mitochondrial quality control) was developed to specifically quantify mitophagy levels. In the present study, we used mito-QC transgenic mice and confocal microscopy to morphologically monitor mitophagy levels during sepsis. To induce sepsis, Mito-QC mice received Escherichia coli lipopolysaccharide (10 mg kg-1 i.p.) or phosphate-buffered saline and skeletal muscles (hindlimb and diaphragm) were excised 48 h later. In control groups, there was a negative correlation between the basal mitophagy level and overall muscle mitochondrial content. Sepsis increased general autophagy in both limb muscles and diaphragm but had no effect on mitophagy levels. Sepsis was associated with a downregulation of certain mitophagy receptors (Fundc1, Bcl2L13, Fkbp8 and Phbb2). The present study suggests that general autophagy and mitophagy can be dissociated from one another, and that the characteristic accumulation of damaged mitochondria in skeletal muscles under the condition of sepsis may reflect a failure of adequate compensatory mitophagy. KEY POINTS: There was a negative correlation between the basal level of skeletal muscle mitophagy and the mitochondrial content of individual muscles. Mitophagy levels in limb muscles and the diaphragm were unaffected by lipopolysaccharide (LPS)-induced sepsis. With the exception of BNIP3 in sepsis, LPS administration induced either no change or a downregulation of mitophagy receptors in skeletal muscles.

Dynamic equilibrium of skeletal muscle macrophage ontogeny in the diaphragm during homeostasis, injury, and recovery.

The diaphragm is a unique skeletal muscle due to its continuous activation pattern during the act of breathing. The ontogeny of macrophages, pivotal cells for skeletal muscle maintenance and regeneration, is primarily based on two distinct origins: postnatal bone marrow-derived monocytes and prenatal embryonic progenitors. Here we employed chimeric mice to study the dynamics of these two macrophage populations under different conditions. Traditional chimeric mice generated through whole body irradiation showed virtually complete elimination of the original tissue-resident macrophage pool. We then developed a novel method which employs lead shielding to protect the diaphragm tissue niche from irradiation. This allowed us to determine that up to almost half of tissue-resident macrophages in the diaphragm can be maintained independently from bone marrow-derived monocytes under steady-state conditions. These findings were confirmed by long-term (5 months) parabiosis experiments. Acute diaphragm injury shifted the macrophage balance toward an overwhelming predominance of bone marrow (monocyte)-derived macrophages. However, there was a remarkable reversion to the pre-injury ontological landscape after diaphragm muscle recovery. This diaphragm shielding method permits analysis of the dynamics of macrophage origin and corresponding function under different physiological and pathological conditions. It may be especially useful for studying diseases which are characterized by acute or chronic injury of the diaphragm and accompanying inflammation.

Computed tomography reveals hypertrophic remodelling of the diaphragm in cystic fibrosis but not in COPD.

Background: Computed tomography (CT) is increasingly used for assessing skeletal muscle characteristics. In cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), reduced limb muscle mass predicts poor clinical outcomes. However, the degree to which quantity or quality of respiratory and nonrespiratory muscles is affected by these diseases remains controversial. Methods: Thoracic CT images of 29 CF, 21 COPD and 20 normal spirometry control subjects were analysed to measure indices of muscle quantity (volume or cross-sectional area) and quality (radiodensity) in respiratory (diaphragm, abdominal) and nonrespiratory (pectoralis, lumbar paraspinal) muscles. Multivariable linear regression assessed relationships of CT measurements with body mass index (BMI), forced expiratory volume in 1 s (FEV1) % pred, inflammation and infection biomarkers, nutritional status and CF genotype. Results: Diaphragm volume in CF was significantly higher than in COPD (by 154%) or controls (by 140%). Abdominal muscle area in CF was also greater than in COPD (by 130%). Nonrespiratory muscles in COPD had more low radiodensity muscle (marker of lipid content) compared to CF and controls. In CF but not COPD, higher BMI and FEV1 % pred were independently associated with higher diaphragm and/or abdominal muscle quantity indices. Serum creatinine also predicted respiratory and nonrespiratory muscle quantity in CF, whereas other biomarkers including genotype correlated poorly with muscle CT parameters. Conclusions: Our data suggest that the CF diaphragm undergoes hypertrophic remodelling, whereas in COPD the nonrespiratory muscles show altered muscle quality consistent with greater lipid content. Thoracic CT can thus identify distinctive respiratory and nonrespiratory muscle remodelling signatures associated with different chronic lung diseases.

Trained immunity as a potential target for therapeutic immunomodulation in Duchenne muscular dystrophy.

Dysregulated inflammation involving innate immune cells, particularly of the monocyte/macrophage lineage, is a key contributor to the pathogenesis of Duchenne muscular dystrophy (DMD). Trained immunity is an evolutionarily ancient protective mechanism against infection, in which epigenetic and metabolic alterations confer non-specific hyperresponsiveness of innate immune cells to various stimuli. Recent work in an animal model of DMD (mdx mice) has shown that macrophages exhibit cardinal features of trained immunity, including the presence of innate immune system "memory". The latter is reflected by epigenetic changes and durable transmissibility of the trained phenotype to healthy non-dystrophic mice by bone marrow transplantation. Mechanistically, it is suggested that a Toll-like receptor (TLR) 4-regulated, memory-like capacity of innate immunity is induced at the level of the bone marrow by factors released from the damaged muscles, leading to exaggerated upregulation of both pro- and anti-inflammatory genes. Here we propose a conceptual framework for the involvement of trained immunity in DMD pathogenesis and its potential to serve as a new therapeutic target.

Characterization of skeletal muscle wasting pathways in diaphragm and limb muscles of cystic fibrosis mice.

Patients with cystic fibrosis (CF) often suffer from skeletal muscle atrophy, most often attributed to physical inactivity and nutritional factors. CF is also characterized by abnormally elevated systemic inflammation. However, it is unknown whether the lack of a functional CF transmembrane conductance regulator (CFTR) gene predisposes to exaggerated inflammation-induced muscle proteolysis. CF mice (CFTR-/-) and their wild-type (WT = CFTR+/+) littermate controls were systemically injected with Pseudomonas-derived lipopolysaccharide (LPS). After 24 h, the diaphragm and limb muscles (fast-twitch tibialis anterior, and slow-twitch soleus) were assessed for induction of inflammatory cytokines (TNFα, IL1ß, and IL6), oxidative stress, canonical muscle proteolysis pathways (Calpain, Ubiquitin-Proteasome, Autophagy), muscle fiber histology, and diaphragm contractile function. At baseline, CF and WT muscles did not differ with respect to indices of inflammation, proteolysis, or contractile function. After LPS exposure, there was significantly greater induction of all proteolysis pathways (calpain activity; ubiquitin-proteasome: MuRF1 and Atrogin1; autophagy: LC3B, Gabarapl-1, and BNIP3) in CF mice for the diaphragm and tibialis anterior, but not the soleus. Proteolysis pathway upregulation and correlations with inflammatory cytokine induction were most prominent in the tibialis anterior. Diaphragm force normalized to muscle cross-sectional area was reduced by LPS to an equivalent degree in CF and WT mice. CF skeletal muscles containing a high proportion of fast-twitch fibers (diaphragm, tibialis anterior) exhibit abnormally exaggerated upregulation of multiple muscle wasting pathways after exposure to an acute inflammatory stimulus, but not under basal conditions.

TLR4 is a regulator of trained immunity in a murine model of Duchenne muscular dystrophy.

Dysregulation of the balance between pro-inflammatory and anti-inflammatory macrophages has a key function in the pathogenesis of Duchenne muscular dystrophy (DMD), a fatal genetic disease. We postulate that an evolutionarily ancient protective mechanism against infection, known as trained immunity, drives pathological inflammation in DMD. Here we show that bone marrow-derived macrophages from a murine model of DMD (mdx) exhibit cardinal features of trained immunity, consisting of transcriptional hyperresponsiveness associated with metabolic and epigenetic remodeling. The hyperresponsive phenotype is transmissible by bone marrow transplantation to previously healthy mice and persists for up to 11 weeks post-transplant. Mechanistically, training is induced by muscle extract in vitro. The functional and epigenetic changes in bone marrow-derived macrophages from dystrophic mice are TLR4-dependent. Adoptive transfer experiments further support the TLR4-dependence of trained macrophages homing to damaged muscles from the bone marrow. Collectively, this suggests that a TLR4-regulated, memory-like capacity of innate immunity induced at the level of the bone marrow promotes dysregulated inflammation in DMD.

Macrophage plasticity in Duchenne muscular dystrophy: a nexus of pathological remodelling with therapeutic implications.

Duchenne muscular dystrophy (DMD) is characterized by chronic skeletal muscle necrosis, leading to muscle regeneration failure and fibrosis. Although macrophages (MPs) are normally essential for muscle regeneration, dysregulated MP function promotes pathological muscle remodelling. Infiltrating MPs can be predominantly pro-inflammatory (M1 biased), anti-inflammatory (M2 biased) or of a mixed phenotype and can originate from the adult bone marrow (monocyte dependent) or embryonic precursors (monocyte independent). In mdx mice (genetic model of DMD) lacking either Toll-like receptor (Tlr) 2 or Tlr4, it is found that MP infiltration of dystrophic muscle is significantly reduced and that the MP phenotype is shifted toward a more anti-inflammatory profile. This is accompanied by significant improvements in muscle histology and force production. Lack of the chemokine receptor CCR2, which impedes monocyte release from the bone marrow, leads to similar beneficial effects in mdx mice. Evidence was also found for Tlr4-regulated induction of trained innate immunity in MPs cultured from the bone marrow of mdx mice before their entry into the muscle. These MPs exhibit epigenetic and metabolic alterations, accompanied by non-specific hyper-responsiveness to multiple stimuli, which is manifested by potentiated upregulation of both pro- and anti-inflammatory genes. In summary, exaggerated recruitment of monocyte-derived MPs and signs of trained innate immunity at the level of the bone marrow are features of the immunophenotype associated with dystrophic muscle disease. These phenomena are regulated by Toll-like receptors that bind endogenous damage-associated molecular pattern (DAMP) molecules, suggesting that DAMP release from dystrophic muscles modulates MP plasticity at the bone marrow level through Toll-like receptor-driven mechanisms.

Evolution of inspiratory muscle function in children during mechanical ventilation.

BACKGROUND: There is no universally accepted method to assess the pressure-generating capacity of inspiratory muscles in children on mechanical ventilation (MV), and no study describing its evolution over time in this population. METHODS: In this prospective observational study, we have assessed the function of the inspiratory muscles in children on various modes of MV. During brief airway occlusion maneuvers, we simultaneously recorded airway pressure depression at the endotracheal tube (ΔPaw, force generation) and electrical activity of the diaphragm (EAdi, central respiratory drive) over five consecutive inspiratory efforts. The neuro-mechanical efficiency ratio (NME, ΔPaw/EAdimax) was also computed. The evolution over time of these indices in a group of children in the pediatric intensive care unit (PICU) was primarily described. As a secondary objective, we compared these values to those measured in a group of children in the operating room (OR). RESULTS: In the PICU group, although median NMEoccl decreased over time during MV (regression coefficient - 0.016, p = 0.03), maximum ΔPawmax remained unchanged (regression coefficient 0.109, p = 0.50). Median NMEoccl at the first measurement in the PICU group (after 21 h of MV) was significantly lower than at the only measurement in the OR group (1.8 cmH2O/µV, Q1-Q3 1.3-2.4 vs. 3.7 cmH2O/µV, Q1-Q3 3.5-4.2; p = 0.015). Maximum ΔPawmax in the PICU group was, however, not significantly different from the OR group (35.1 cmH2O, Q1-Q3 21-58 vs. 31.3 cmH2O, Q1-Q3 28.5-35.5; p = 0.982). CONCLUSIONS: The function of inspiratory muscles can be monitored at the bedside of children on MV using brief airway occlusions. Inspiratory muscle efficiency was significantly lower in critically ill children than in children undergoing elective surgery, and it decreased over time during MV in critically ill children. This suggests that both critical illness and MV may have an impact on inspiratory muscle efficiency.

Validation of home portable monitoring for the diagnosis of sleep-disordered breathing in adolescents and adults with neuromuscular disorders.

STUDY OBJECTIVES: Sleep-disordered breathing (SDB) is common in patients with neuromuscular disorders (NMD), developing before chronic hypercapnia appears. Polysomnography (PSG) is the diagnostic gold standard but is often impractical and poorly accessible for individuals with NMD. We sought to determine the diagnostic accuracy, feasibility, and patient preference of home sleep apnea testing (HSAT) compared with PSG for the detection of SDB in NMD. METHODS: Participants with NMD at risk for SDB aged ≥ 13 years underwent HSAT followed by overnight PSG with concomitant laboratory sleep apnea testing (same device as HSAT). Sensitivity and specificity were calculated for standard apnea-hypopnea index cutoffs for mild (≥ 5 events/h), moderate (≥ 15 events/h), and severe SDB (≥ 30 events/h) and for an oxygen desaturation index ≥ 5 events/h. Receiver operating characteristic curves were built. A questionnaire assessed patient preference. RESULTS: Of 38 participants, 73% had moderate to severe SDB and 79% had technically acceptable HSAT. For an apnea-hypopnea index ≥ 15 events/h, HSAT sensitivity and specificity were 50% and 88%, respectively. For an oxygen desaturation index ≥ 5 events/h, HSAT sensitivity and specificity were 95% and 78%, respectively. The area under the receiver operating characteristic curve for an apnea-hypopnea index ≥ 15 events/h was 0.88 (95% confidence interval, 0.69-1.00) for HSAT. The HSAT underestimated the apnea-hypopnea index from PSG (bias, -10.7 ± 15.9 events/h). HSAT was preferred to PSG by 61% of participants. CONCLUSIONS: HSAT is feasible, preferred by patients, and reliable for detecting SDB in most patients, although it cannot definitively rule out SDB. Therefore, HSAT is a viable diagnostic approach for SDB in NMD when PSG is not feasible, recognizing that it does not accurately distinguish between upper-airway obstruction and hypoventilation. Additional work is needed to further optimize home sleep testing in NMD. CITATION: Westenberg JN, Petrof BJ, Noel F, et al. Validation of home portable monitoring for the diagnosis of sleep-disordered breathing in adolescents and adults with neuromuscular disorders. J Clin Sleep Med. 2021;17(8):1579-1590.

Diaphragm Morphology Assessed by Computed Tomography in Chronic Obstructive Pulmonary Disease.

Rationale: Chronic obstructive pulmonary disease (COPD) is associated with abnormal skeletal muscle morphology and function. Objectives: To test the hypothesis that in vivo diaphragm muscle morphology assessed by computed tomography (CT) imaging would be associated with COPD severity, exacerbations, health status, and exercise capacity. Methods: The COPD Morphometry Study is a cross-sectional study that enrolled a clinical sample of smokers with COPD. Spirometry was performed and COPD severity was defined according to guidelines. Three-dimensional left hemidiaphragm morphology was segmented from contiguous axial CT images acquired at maximal inspiration, yielding quantitative measures of diaphragm CT density in Hounsfield units, dome height, and muscle volume. Exacerbations prompting pharmacotherapy or hospitalization in the preceding 12 months and St. George's Respiratory Questionnaire for COPD were assessed. Incremental symptom-limited cycle ergometry quantified peak oxygen uptake ([Formula: see text]o2Peak). Associations were adjusted for age, sex, body height, body mass index, and smoking status. Results: Among 65 smokers with COPD (75% male; [mean ± standard deviation (SD)] 56 ± 26 pack-years; forced expiratory volume in 1 second [FEV1] percentage predicted 55 ± 23%), mean diaphragm CT density was 3.1 ± 10 Hounsfield units, dome height was 5.2 ± 1.3 cm, and muscle volume was 57 ± 24 cm3. A 1-SD decrement in the diaphragm CT density was associated with 8.3% lower FEV1, 3.27-fold higher odds of exacerbation history, 9.7-point higher score on the St. George's Respiratory Questionnaire for COPD, and 2.5 ml/kg/min lower [Formula: see text]o2Peak. A 1-SD decrement in dome height was associated with 11% lower FEV1 and 1.3 ml/kg/min lower [Formula: see text]o2Peak. There were no associations with diaphragm volume observed. Conclusions: CT-assessed diaphragm morphology was associated with COPD severity, exacerbations, impaired health status, and exercise intolerance. The mechanisms and functional impact of lower diaphragm CT density merit investigation.

Age-Dependent Progression in Lung Pathophysiology can be Prevented by Restoring Fatty Acid and Ceramide Imbalance in Cystic Fibrosis.

PURPOSE: Cystic fibrosis (CF) is a progressive disease which causes a continuous decline in lung capacity with age. Our study aimed to investigate the age-dependent deterioration in lung function and the effects of treatment with Fenretinide formulation (LAU-7b) in Cftr knockout (KO) mice. METHODS: Non-invasive whole-body plethysmography (WBP) was done to measure the baseline lung functions of KO and wild-type (WT) mice at the ages of 2 and 4 months. Mice were then treated for 21 days with PBS or 10 mg/kg/day LAU-7b initiated at 4 and 7 months. Standard airway resistance measurements, haematoxylin and eosin staining, and analysis of lipids, and markers of oxidation were performed. RESULTS: The 4- and 7-month-old KO mice had significantly higher lung enhanced pause (Penh) and resistance values than age-matched WT mice and 2-month-old KO mice. Likewise, analysis of ceramides showed that PBS-treated mice had higher levels of long-chain ceramides (LCCs; C14-C18) and lower levels of very-long-chain ceramides (VLCCs; C24-C26) compared to LAU-7b-treated mice. Cftr KO mice displayed markedly greater inflammatory cell infiltration and epithelial hyperplasia at the ages of 2, 4, and 7 months compared to WT. LAU-7b treatment significantly diminished this cellular infiltration and epithelial hyperplasia compared to PBS-treated mice. CONCLUSION: Our results demonstrate a progressive age-dependent decline in lung function in Cftr KO mice. Treatment with LAU-7b corrects the lipid imbalance observed in the aging KO and WT mice and, more importantly, inhibits the age-dependent deterioration in lung physiology and histopathology.

Fenretinide favorably affects mucins (MUC5AC/MUC5B) and fatty acid imbalance in a manner mimicking CFTR-induced correction.

Cystic fibrosis (CF) is the most common genetic disease in Caucasians. CF is manifested by abnormal accumulation of mucus in the lungs, which serves as fertile ground for the growth of microorganisms leading to recurrent infections and ultimately, lung failure. Mucus in CF patients consists of DNA from dead neutrophils as well as mucins produced by goblet cells. MUC5AC mucin leads to pathological plugging of the airways whereas MUC5B has a protective role against bacterial infection. Therefore, decreasing the level of MUC5AC while maintaining MUC5B intact would in principle be a desirable mucoregulatory treatment outcome. Fenretinide prevented the lipopolysaccharide-induced increase of MUC5AC gene expression, without affecting the level of MUC5B, in a lung goblet cell line. Additionally, fenretinide treatment reversed the pro-inflammatory imbalance of fatty acids by increasing docosahexaenoic acid and decreasing the levels of arachidonic acid in a lung epithelial cell line and primary leukocytes derived from CF patients. Furthermore, for the first time we also demonstrate the effect of fenretinide on multiple unsaturated fatty acids, as well as differential effects on the levels of long- compared to very-long-chain saturated fatty acids which are important substrates of complex phospholipids. Finally, we demonstrate that pre-treating mice with fenretinide in a chronic model of P. aeruginosa lung infection efficiently decreases the accumulation of mucus. These findings suggest that fenretinide may offer a new approach to therapeutic modulation of pathological mucus production in CF.

Differential regulation of myofibrillar proteins in skeletal muscles of septic mice.

Sepsis elicits skeletal muscle atrophy as a result of decreased total protein synthesis and/or increased total protein degradation. It is unknown how and whether sepsis differentially affects the expression of specific myofibrillar proteins in respiratory and limb muscles. In this study, we measured the effects of sepsis myofibrillar mRNAs and their corresponding protein levels in the diaphragm (DIA) and tibialis anterior (TA) muscles in a murine cecal ligation and perforation (CLP) model of sepsis. Male mice (C57/BL6j) underwent CLP-induced sepsis. Sham-operated mice were subjected to the same surgical procedures, except for CLP. Mice were euthanized 24, 48, or 96 h postsurgery. Transcript and protein levels of autophagy-related genes, ubiquitin E3 ligases, and several myofibrillar genes were quantified. Sepsis elicited transient fiber atrophy in the DIA and prolonged atrophy in the TA. Atrophy was coincident with increased autophagy and ubiquitin E3 ligase expression. Myosin heavy chain isoforms decreased at 24 h in the DIA and across the time-course in the TA, myosin light chain isoforms decreased across the time-course in both muscles, and troponins T and C as well as tropomyosin decreased after 24 and 48 h in both the DIA and TA. α-Actin and troponin I were unaffected by sepsis. Sepsis-induced decreases in myofibrillar protein levels coincided with decreased mRNA expressions of these proteins, suggesting that transcriptional inhibition is involved. We hypothesize that sepsis-induced muscle atrophy is mediated by decreased transcription and enhanced degradation of specific myofibrillar proteins, including myosin heavy and light chains, troponin C, troponin T, and tropomyosin.

Oxidants Regulated Diaphragm Proteolysis during Mechanical Ventilation in Rats.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: Diaphragm dysfunction and atrophy develop during controlled mechanical ventilation. Although oxidative stress injures muscle during controlled mechanical ventilation, it is unclear whether it causes autophagy or fiber atrophy. WHAT THIS ARTICLE TELLS US THAT IS NEW: Pretreatment of rats undergoing 24 h of mechanical ventilation with N-acetylcysteine prevents decreases in diaphragm contractility, inhibits the autophagy and proteasome pathways, but has no influence on the development of diaphragm fiber atrophy. BACKGROUND: Diaphragm dysfunction and atrophy develop during prolonged controlled mechanical ventilation. Fiber atrophy has been attributed to activation of the proteasome and autophagy proteolytic pathways. Oxidative stress activates the proteasome during controlled mechanical ventilation, but it is unclear whether it also activates autophagy. This study investigated whether pretreatment with the antioxidant N-acetylcysteine affects controlled mechanical ventilation-induced diaphragm contractile dysfunction, fiber atrophy, and proteasomal and autophagic pathway activation. The study also explored whether proteolytic pathway activity during controlled mechanical ventilation is mediated by microRNAs that negatively regulate ubiquitin E3 ligases and autophagy-related genes. METHODS: Three groups of adult male rats were studied (n = 10 per group). The animals in the first group were anesthetized and allowed to spontaneously breathe. Animals in the second group were pretreated with saline before undergoing controlled mechanical ventilation for 24 h. The animals in the third group were pretreated with N-acetylcysteine (150 mg/kg) before undergoing controlled mechanical ventilation for 24 h. Diaphragm contractility and activation of the proteasome and autophagy pathways were measured. Expressions of microRNAs that negatively regulate ubiquitin E3 ligases and autophagy-related genes were measured with quantitative polymerase chain reaction. RESULTS: Controlled mechanical ventilation decreased diaphragm twitch force from 428 ± 104 g/cm (mean ± SD) to 313 ± 50 g/cm and tetanic force from 2,491 ± 411 g/cm to 1,618 ± 177 g/cm. Controlled mechanical ventilation also decreased diaphragm fiber size, increased expression of several autophagy genes, and augmented Atrogin-1, MuRF1, and Nedd4 expressions by 36-, 41-, and 8-fold, respectively. Controlled mechanical ventilation decreased the expressions of six microRNAs (miR-20a, miR-106b, miR-376, miR-101a, miR-204, and miR-93) that regulate autophagy genes. Pretreatment with N-acetylcysteine prevented diaphragm contractile dysfunction, attenuated protein ubiquitination, and downregulated E3 ligase and autophagy gene expression. It also reversed controlled mechanical ventilation-induced microRNA expression decreases. N-Acetylcysteine pretreatment had no affect on fiber atrophy. CONCLUSIONS: Prolonged controlled mechanical ventilation activates the proteasome and autophagy pathways in the diaphragm through oxidative stress. Pathway activation is accomplished, in part, through inhibition of microRNAs that negatively regulate autophagy-related genes.

Mechanical ventilation causes diaphragm dysfunction in newborn lambs.

BACKGROUND: Diaphragm weakness occurs rapidly in adult animals treated with mechanical ventilation (MV), but the effects of MV on the neonatal diaphragm have not been determined. Furthermore, it is unknown whether co-existent lung disease exacerbates ventilator-induced diaphragmatic dysfunction (VIDD). We investigated the impact of MV (mean duration = 7.65 h), either with or without co-existent respiratory failure caused by surfactant deficiency, on the development of VIDD in newborn lambs. METHODS: Newborn lambs (1-4 days) were assigned to control (CTL, non-ventilated), mechanically ventilated (MV), and MV + experimentally induced surfactant deficiency (MV+SD) groups. Immunoblotting and quantitative PCR assessed inflammatory signaling, the ubiquitin-proteasome system, autophagy, and oxidative stress. Immunostaining for myosin heavy chain (MyHC) isoforms and quantitative morphometry evaluated diaphragm atrophy. Contractile function of the diaphragm was determined in isolated myofibrils ex vivo. RESULTS: Equal decreases (25-30%) in myofibrillar force generation were found in MV and MV+SD diaphragms compared to CTL. In comparison to CTL, both MV and MV+SD diaphragms also demonstrated increased STAT3 transcription factor phosphorylation. Ubiquitin-proteasome system (Atrogin1 and MuRF1) transcripts and autophagy indices (Gabarapl1 transcripts and the ratio of LC3B-II/LC3B-I protein) were greater in MV+SD relative to MV alone, but fiber type atrophy was not observed in any group. Protein carbonylation and 4-hydroxynonenal levels (indices of oxidative stress) also did not differ among groups. CONCLUSIONS: In newborn lambs undergoing controlled MV, there is a rapid onset of diaphragm dysfunction consistent with VIDD. Superimposed lung injury caused by surfactant deficiency did not influence the severity of early diaphragm weakness.

AMPK Activation Regulates LTBP4-Dependent TGF-ß1 Secretion by Pro-inflammatory Macrophages and Controls Fibrosis in Duchenne Muscular Dystrophy.

Chronic inflammation and fibrosis characterize Duchenne muscular dystrophy (DMD). We show that pro-inflammatory macrophages are associated with fibrosis in mouse and human DMD muscle. DMD-derived Ly6Cpos macrophages exhibit a profibrotic activity by sustaining fibroblast production of collagen I. This is mediated by the high production of latent-TGF-ß1 due to the higher expression of LTBP4, for which polymorphisms are associated with the progression of fibrosis in DMD patients. Skewing macrophage phenotype via AMPK activation decreases ltbp4 expression by Ly6Cpos macrophages, blunts the production of latent-TGF-ß1, and eventually reduces fibrosis and improves DMD muscle force. Moreover, fibro-adipogenic progenitors are the main providers of TGF-ß-activating enzymes in mouse and human DMD, leading to collagen production by fibroblasts. In vivo pharmacological inhibition of TGF-ß-activating enzymes improves the dystrophic phenotype. Thus, an AMPK-LTBP4 axis in inflammatory macrophages controls the production of TGF-ß1, which is further activated by and acts on fibroblastic cells, leading to fibrosis in DMD.

Diaphragm Weakness in the Critically Ill: Basic Mechanisms Reveal Therapeutic Opportunities.

The diaphragm is the primary muscle of inspiration. Its capacity to respond to the load imposed by pulmonary disease is a major determining factor both in the onset of ventilatory failure and in the ability to successfully separate patients from ventilator support. It has recently been established that a very large proportion of critically ill patients exhibit major weakness of the diaphragm, which is associated with poor clinical outcomes. The two greatest risk factors for the development of diaphragm weakness in critical illness are the use of mechanical ventilation and the presence of sepsis. Loss of force production by the diaphragm under these conditions is caused by a combination of defective contractility and reduced diaphragm muscle mass. Importantly, many of the same molecular mechanisms are implicated in the diaphragm dysfunction associated with both mechanical ventilation and sepsis. This review outlines the primary cellular mechanisms identified thus far at the nexus of diaphragm dysfunction associated with mechanical ventilation and/or sepsis, and explores the potential for treatment or prevention of diaphragm weakness in critically ill patients through therapeutic manipulation of these final common pathway targets.

Subcellular origin of mitochondrial DNA deletions in human skeletal muscle.

OBJECTIVE: In patients with mitochondrial DNA (mtDNA) maintenance disorders and with aging, mtDNA deletions sporadically form and clonally expand within individual muscle fibers, causing respiratory chain deficiency. This study aimed to identify the sub-cellular origin and potential mechanisms underlying this process. METHODS: Serial skeletal muscle cryosections from patients with multiple mtDNA deletions were subjected to subcellular immunofluorescent, histochemical, and genetic analysis. RESULTS: We report respiratory chain-deficient perinuclear foci containing mtDNA deletions, which show local elevations of both mitochondrial mass and mtDNA copy number. These subcellular foci of respiratory chain deficiency are associated with a local increase in mitochondrial biogenesis and unfolded protein response signaling pathways. We also find that the commonly reported segmental pattern of mitochondrial deficiency is consistent with the three-dimensional organization of the human skeletal muscle mitochondrial network. INTERPRETATION: We propose that mtDNA deletions first exceed the biochemical threshold causing biochemical deficiency in focal regions adjacent to the myonuclei, and induce mitochondrial biogenesis before spreading across the muscle fiber. These subcellular resolution data provide new insights into the possible origin of mitochondrial respiratory chain deficiency in mitochondrial myopathy. Ann Neurol 2018;84:289-301.

Pulmonary and diaphragmatic pathology in collagen type I α1 mutant mice with osteogenesis imperfecta.

BackgroundOsteogenesis imperfecta (OI) is most often caused by mutations in type I collagen genes. Respiratory complications have been largely attributed to spine and ribcage deformities. We hypothesized that direct involvement of the pulmonary parenchyma and/or diaphragm by the disease may occur.MethodsIn Col1a1Jrt/+ mice, a model of severe dominant OI, mean linear intercept length (Lm) was used to assess the distal airspace size. Cross-sectional area (CSA) and myosin heavy chain (MyHC) phenotype of the diaphragm muscle fibers, as well as contractile properties, were determined. OI mice were also treated with neutralizing antibodies against transforming growth factor-ß (TGF-ß).ResultsDistal airspace enlargement occurred in OI mice (Lm +27%). Diaphragmatic thickness and fiber number were reduced, with increases in fast-twitch type IIx/IIb MyHC fibers. Ex vivo force generation (normalized for CSA) of the diaphragm was also significantly reduced. The increased Lm values found in OI mice were not prevented by anti-TGF-ß antibody treatment.ConclusionsThe Col1a1Jrt/+ mouse model of OI demonstrates: (1) pulmonary airspace enlargement not driven by TGF-ß; and (2) reduced muscle mass and intrinsic contractile weakness of the diaphragm. These results suggest a complex and multifaceted basis for respiratory complications in OI that cannot be solely attributed to bone manifestations.