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.

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.

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.

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.

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.

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.

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.

Leaky ryanodine receptors contribute to diaphragmatic weakness during mechanical ventilation.

Ventilator-induced diaphragmatic dysfunction (VIDD) refers to the diaphragm muscle weakness that occurs following prolonged controlled mechanical ventilation (MV). The presence of VIDD impedes recovery from respiratory failure. However, the pathophysiological mechanisms accounting for VIDD are still not fully understood. Here, we show in human subjects and a mouse model of VIDD that MV is associated with rapid remodeling of the sarcoplasmic reticulum (SR) Ca(2+) release channel/ryanodine receptor (RyR1) in the diaphragm. The RyR1 macromolecular complex was oxidized, S-nitrosylated, Ser-2844 phosphorylated, and depleted of the stabilizing subunit calstabin1, following MV. These posttranslational modifications of RyR1 were mediated by both oxidative stress mediated by MV and stimulation of adrenergic signaling resulting from the anesthesia. We demonstrate in the murine model that such abnormal resting SR Ca(2+) leak resulted in reduced contractile function and muscle fiber atrophy for longer duration of MV. Treatment with ß-adrenergic antagonists or with S107, a small molecule drug that stabilizes the RyR1-calstabin1 interaction, prevented VIDD. Diaphragmatic dysfunction is common in MV patients and is a major cause of failure to wean patients from ventilator support. This study provides the first evidence to our knowledge of RyR1 alterations as a proximal mechanism underlying VIDD (i.e., loss of function, muscle atrophy) and identifies RyR1 as a potential target for therapeutic intervention.

Ventilator-induced diaphragmatic dysfunction in MDX mice.

INTRODUCTION: Patients with Duchenne muscular dystrophy (DMD) frequently undergo mechanical ventilation (MV) for treatment of hypoventilation, but the susceptibility of the dystrophic diaphragm to ventilator-induced diaphragmatic dysfunction (VIDD) has not been examined. METHODS: Dystrophic mice (mdx-genetic homolog of DMD) were assigned to non-ventilated control (CTL) and MV (for 6 hours) groups. Biochemical markers of oxidative/cellular stress, metabolism, and proteolysis were compared along with ex-vivo diaphragmatic force production. RESULTS: MV significantly depressed maximal diaphragmatic force production compared with baseline values. In addition, MV triggered oxidative stress responses, STAT3 phosphorylation, and an upregulation of cellular pathways associated with muscle proteolysis and/or wasting (autophagy, E3 ubiquitin ligases, and myostatin). DISCUSSION: Short-term MV induces rapid diaphragmatic force loss and biochemical changes consistent with VIDD in mdx mice. This may have implications for the optimal use of intermittent MV in DMD patients. Muscle Nerve 57: 442-448, 2018.

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.

Toll-like receptor 4 ablation in mdx mice reveals innate immunity as a therapeutic target in Duchenne muscular dystrophy.

Toll-like receptor 4 (TLR4) recognizes specific structural motifs associated with microbial pathogens and also responds to certain endogenous host molecules associated with tissue damage. In Duchenne muscular dystrophy (DMD), inflammation plays an important role in determining the ultimate fate of dystrophic muscle fibers. In this study, we used TLR4-deficient dystrophic mdx mice to assess the role of TLR4 in the pathogenesis of DMD. TLR4 expression was increased and showed enhanced activation following agonist stimulation in mdx diaphragm muscle. Genetic ablation of TLR4 led to significantly increased muscle force generation in dystrophic diaphragm muscle, which was associated with improved histopathology including decreased fibrosis, as well as reduced pro-inflammatory gene expression and macrophage infiltration. TLR4 ablation in mdx mice also altered the phenotype of muscle macrophages by inducing a shift toward a more anti-inflammatory (iNOS(neg) CD206(pos)) profile. In vitro experiments confirmed that lack of TLR4 is sufficient to influence macrophage activation status in response to classical polarizing stimuli such as IFN-gamma and IL-4. Finally, treatment of dystrophic mice with glycyrrhizin, an inhibitor of the endogenous TLR4 ligand, high mobility group box (HMGB1), also pointed to involvement of the HMGB1-TLR4 axis in promoting dystrophic diaphragm pathology. Taken together, our findings reveal TLR4 and the innate immune system as important players in the pathophysiology of DMD. Accordingly, targeting either TLR4 or its endogenous ligands may provide a new therapeutic strategy to slow disease progression.

Divergent impact of Toll-like receptor 2 deficiency on repair mechanisms in healthy muscle versus Duchenne muscular dystrophy.

Injury to skeletal muscle, whether acute or chronic, triggers macrophage-mediated innate immunity in a manner which can be either beneficial or harmful for subsequent repair. Endogenous ligands for Toll-like receptor 2 (TLR2) are released by damaged tissues and might play an important role in activating the innate immune system following muscle injury. To test this hypothesis, we compared macrophage behaviour and muscle repair mechanisms in mice lacking TLR2 under conditions of either acute (cardiotoxin-induced) or chronic (mdx mouse genetic model of Duchenne muscular dystrophy; DMD) muscle damage. In previously healthy muscle subjected to acute damage, TLR2 deficiency reduced macrophage numbers in the muscle post-injury but did not alter the expression pattern of the prototypical macrophage polarization markers iNOS and CD206. In addition, there was abnormal persistence of necrotic fibres and impaired regeneration in TLR2-/- muscles after acute injury. In contrast, TLR2 ablation in chronically diseased muscles of mdx mice not only resulted in significantly reduced macrophage numbers but additionally modified their phenotype by shifting from inflammatory (iNOS(pos) CD206(neg) ) to more anti-inflammatory (iNOS(neg) CD206(pos) ) characteristics. This decrease in macrophage-mediated inflammation was associated with ameliorated muscle histopathology and improved force-generating capacity of the dystrophic muscle. Our results suggest that the role of TLR2 in macrophage function and skeletal muscle repair depends greatly upon the muscle injury context, and raise the possibility that inhibition of TLR2 could serve as a useful therapeutic measure in DMD.

Prolonged controlled mechanical ventilation in humans triggers myofibrillar contractile dysfunction and myofilament protein loss in the diaphragm.

BACKGROUND: Prolonged controlled mechanical ventilation (CMV) in humans and experimental animals results in diaphragm fibre atrophy and injury. In animals, prolonged CMV also triggers significant declines in diaphragm myofibril contractility. In humans, the impact of prolonged CMV on myofibril contractility remains unknown. The objective of this study was to evaluate the effects of prolonged CMV on active and passive human diaphragm myofibrillar force generation and myofilament protein levels. METHODS AND RESULTS: Diaphragm biopsies were obtained from 13 subjects undergoing cardiac surgery (control group) and 12 brain-dead organ donors (CMV group). Subjects in each group had been mechanically ventilated for 2-4 and 12-74 h, respectively. Specific force generation of diaphragm myofibrils was measured with atomic force cantilevers. Rates of force development (Kact), force redevelopment after a shortening protocol (Ktr) and relaxation (Krel) in fully activated myofibrils (pCa(2+)=4.5) were calculated to assess myosin cross-bridge kinetics. Myofilament protein levels were measured with immunoblotting and specific antibodies. Prolonged CMV significantly decreased active and passive diaphragm myofibrillar force generation, Kact, Ktr and Krel. Myosin heavy chain (slow), troponin-C, troponin-I, troponin-T, tropomyosin and titin protein levels significantly decreased in response to prolonged CMV, but no effects on α-actin, α-actinin or nebulin levels were observed. CONCLUSIONS: Prolonged CMV in humans triggers significant decreases in active and passive diaphragm myofibrillar force generation. This response is mediated, in part, by impaired myosin cross-bridge kinetics and decreased myofibrillar protein levels.

Ventilator-induced diaphragmatic dysfunction: what have we learned?

PURPOSE OF REVIEW: The purpose of the review is to summarize and discuss recent research regarding the role of mechanical ventilation in producing weakness and atrophy of the diaphragm in critically ill patients, an entity termed ventilator-induced diaphragmatic dysfunction (VIDD). RECENT FINDINGS: Severe weakness of the diaphragm is frequent in mechanically ventilated patients, in whom it contributes to poor outcomes including increased mortality. Significant progress has been made in identifying the molecular mechanisms responsible for VIDD in animal models, and there is accumulating evidence for occurrence of the same cellular processes in the diaphragms of human patients undergoing prolonged mechanical ventilation. SUMMARY: Recent research is pointing the way to novel pharmacologic therapies as well as nonpharmacologic methods for preventing VIDD. The next major challenge in the field will be to move these findings from the bench to the bedside in critically ill patients.

Pompe Disease: Diagnosis and Management. Evidence-Based Guidelines from a Canadian Expert Panel.

Pompe disease is a lysosomal storage disorder caused by a deficiency of the enzyme acid alpha-glucosidase. Patients have skeletal muscle and respiratory weakness with or without cardiomyopathy. The objective of our review was to systematically evaluate the quality of evidence from the literature to formulate evidence-based guidelines for the diagnosis and management of patients with Pompe disease. The literature review was conducted using published literature, clinical trials, cohort studies and systematic reviews. Cardinal treatment decisions produced seven management guidelines and were assigned a GRADE classification based on the quality of evidence in the published literature. In addition, six recommendations were made based on best clinical practices but with insufficient data to form a guideline. Studying outcomes in rare diseases is challenging due to the small number of patients, but this is in particular the reason why we believe that informed treatment decisions need to consider the quality of the evidence.

Relationship between Autophagy and Ventilator-induced Diaphragmatic Dysfunction.

BACKGROUND: Mechanical ventilation (MV) is associated with atrophy and weakness of the diaphragm muscle, a condition termed ventilator-induced diaphragmatic dysfunction (VIDD). Autophagy is a lysosomally mediated proteolytic process that can be activated by oxidative stress, which has the potential to either mitigate or exacerbate VIDD. The primary goals of this study were to (1) determine the effects of MV on autophagy in the diaphragm and (2) evaluate the impact of antioxidant therapy on autophagy induction and MV-induced diaphragmatic weakness. METHODS: Mice were assigned to control (CTRL), MV (for 6 h), MV + N-acetylcysteine, MV + rapamycin, and prolonged (48 h) fasting groups. Autophagy was monitored by quantifying (1) autophagic vesicles by transmission electron microscopy, (2) messenger RNA levels of autophagy-related genes, and (3) the autophagosome marker protein LC3B-II, with and without administration of colchicine to calculate the indices of relative autophagosome formation and degradation. Force production by mouse diaphragms was determined ex vivo. RESULTS: Diaphragms exhibited a 2.2-fold (95% CI, 1.8 to 2.5) increase in autophagic vesicles visualized by transmission electron microscopy relative to CTRL after 6 h of MV (n = 5 per group). The autophagosome formation index increased in the diaphragm alone (1.5-fold; 95% CI, 1.3 to 1.8; n = 8 per group) during MV, whereas prolonged fasting induced autophagosome formation in both the diaphragm (2.5-fold; 95% CI, 2.2 to 2.8) and the limb muscle (4.1-fold; 95% CI, 1.8 to 6.5). The antioxidant N-acetylcysteine further augmented the autophagosome formation in the diaphragm during MV (1.4-fold; 95% CI, 1.2 to 1.5; n = 8 per group) and prevented MV-induced diaphragmatic weakness. Treatment with the autophagy-inducing agent rapamycin also largely prevented the diaphragmatic force loss associated with MV (n = 6 per group). CONCLUSIONS: In this model of VIDD, autophagy is induced by MV but is not responsible for diaphragmatic weakness. The authors propose that autophagy may instead be a beneficial adaptive response that can potentially be exploited for therapy of VIDD.

Bcl-2-associated autophagy regulator Naf-1 required for maintenance of skeletal muscle.

Nutrient-deprivation autophagy factor-1 (NAF-1) was identified as an endoplasmic reticulum (ER) BCL-2-interacting protein, which functions to mediate the ability of ER BCL-2 to antagonize Beclin 1-dependent autophagy and depress ER calcium stores. In humans, a point mutation in Naf-1 (synonyms: Cisd2, Eris, Miner1 and Noxp70) is responsible for the neurodegenerative disorder Wolfram Syndrome 2. Here, we describe the generation and characterization of the Naf-1 gene deletion in mice. Naf-1 null mice display discernable clinical signs of degeneration at 2-3 months of age, with early evidence of significant defects in the structure and performance of skeletal muscle. Skeletal muscles from Naf-1 knockout mice demonstrate a significant shift towards slow-twitch (type I) fibers and greater resistance to muscle fatigue. Force-generating capacity is dramatically reduced in Naf-1(-/-) muscle. Consistent with its role in ER BCL-2-mediated regulation of autophagy and calcium flux, these physiological deficiencies were accompanied by augmented autophagy and dysregulated calcium homeostasis. In contrast, this also included adaptive enlargement of mitochondria with extensive cristae structures. Thus, NAF-1, a BCL-2-associated autophagy regulator, is required for homeostatic maintenance of skeletal muscle. Our findings uncover a novel pathway that is required for normal muscle maintenance, which may ultimately provide a novel therapeutic target for treating certain muscle pathologies.

Muscle-specific inhibition of the classical nuclear factor-κB pathway is protective against diaphragmatic weakness in murine endotoxemia.

OBJECTIVE: Diaphragmatic weakness and acute respiratory failure are common in sepsis. Nuclear factor-κB acts as a general coordinator of the systemic inflammatory response, but its role within the diaphragm itself during sepsis is unknown. We investigated the potential protective effect upon the diaphragm of inhibiting nuclear factor-κB only within muscle fibers during acute endotoxemia. DESIGN: Prospective study in experimental animals. SETTING: University research laboratory. INTERVENTIONS: Wild-type and transgenic (muscle-specific IκBα super-repressor) mice with skeletal muscle-specific inhibition of the classical nuclear factor-κB pathway were subjected to acute endotoxemia. Muscle-specific ubiquitin ligases (muscle RING-finger protein 1 and atrogin-1), caspase-3 activity, inhibitor of apoptosis proteins, proinflammatory cytokines (interleukin-1ß, monocyte chemoattractant protein-1, and tumor necrosis factor-α), and diaphragmatic contractility were evaluated after 24 hours. MEASUREMENTS AND MAIN RESULTS: In wild-type mice, endotoxemia significantly increased proinflammatory cytokines (fold-change messenger RNA: interleukin-1ß = 7.6, monocyte chemoattractant protein-1 = 15.3, and tumor necrosis factor-α = 2.2) and proteolysis effectors (fold-change messenger RNA: muscle RING-finger protein 1 = 5.7, atrogin-1 = 2.8; caspase-3 activity elevated by 28%) in the diaphragm, while reducing its force-generating capacity by 38%. In nonendotoxemic muscle-specific IκBα super-repressor diaphragms, caspase-3 activity was unexpectedly increased by 40% above basal wild-type levels and inhibitors of apoptosis proteins were down-regulated, but force production remained normal. In muscle-specific IκBα super-repressor mice subjected to endotoxemia, proinflammatory cytokines, muscle RING-finger protein 1, and atrogin-1 were not significantly increased above their basal levels, and diaphragmatic weakness and further increases in caspase-3 activity were completely prevented. CONCLUSIONS: These results suggest that nuclear factor-κB signaling within skeletal muscle fibers is a key pathway leading to diaphragmatic weakness during acute endotoxemia, most likely via effects on multiple inflammatory mediators. In addition, inhibition of nuclear factor-κB signaling within diaphragm muscle fibers has complex effects on caspase-3 activation, which could have implications for the treatment of sepsis-induced diaphragmatic dysfunction.