Sabah N. A. Hussain

The Caspase-1 Digestome Identifies the Glycolysis Pathway as a Target during Infection and Septic Shock

Caspase-1 is an essential effector of inflammation, pyroptosis, and septic shock. Few caspase-1 substrates have been identified to date, and these substrates do not account for its wide range of actions. To understand the function of caspase-1, we initiated the systematic identification of its cellular substrates. Using the diagonal gel proteomic approach, we identified 41 proteins that are directly cleaved by caspase-1. Among these were chaperones, cytoskeletal and translation machinery proteins, and proteins involved in immunity. A series of unexpected proteins along the glycolysis pathway were also identified, including aldolase, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, α-enolase, and pyruvate kinase. With the exception of the latter, the identified glycolysis enzymes were specifically cleaved in vitro by recombinant caspase-1, but not caspase-3. The enzymatic activity of wild-type glyceraldehyde-3-phosphate dehydrogenase, but not a non-cleavable mutant, was dampened by caspase-1 processing. In vivo, stimuli that fully activated caspase-1, including Salmonella typhimurium infection and septic shock, caused a pronounced processing of these proteins in the macrophage and diaphragm muscle, respectively. Notably, these stimuli inhibited glycolysis in wild-type cells compared with caspase-1-deficient cells. The systematic characterization of caspase-1 substrates identifies the glycolysis pathway as a caspase-1 target and provides new insights into its function during pyroptosis and septic shock.

AMPK Activation Stimulates Autophagy and Ameliorates Muscular Dystrophy in the mdx Mouse Diaphragm

Duchenne muscular dystrophy (DMD) is characterized by myofiber death from apoptosis or necrosis, leading in many patients to fatal respiratory muscle weakness. Among other pathological features, DMD muscles show severely deranged metabolic gene regulation and mitochondrial dysfunction. Defective mitochondria not only cause energetic deficiency, but also play roles in promoting myofiber atrophy and injury via opening of the mitochondrial permeability transition pore. Autophagy is a bulk degradative mechanism that serves to augment energy production and eliminate defective mitochondria (mitophagy). We hypothesized that pharmacological activation of AMP-activated protein kinase (AMPK), a master metabolic sensor in cells and on-switch for the autophagy-mitophagy pathway, would be beneficial in the mdx mouse model of DMD. Treatment of mdx mice for 4 weeks with an established AMPK agonist, AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside), potently triggered autophagy in the mdx diaphragm without inducing muscle fiber atrophy. In AICAR-treated mdx mice, the exaggerated sensitivity of mdx diaphragm mitochondria to calcium-induced permeability transition pore opening was restored to normal levels. There were associated improvements in mdx diaphragm histopathology and in maximal force-generating capacity, which were not linked to increased mitochondrial biogenesis or up-regulated utrophin expression. These findings suggest that agonists of AMPK and other inducers of the autophagy-mitophagy pathway can help to promote the elimination of defective mitochondria and may thus serve as useful therapeutic agents in DMD.

Autophagy and Skeletal Muscles in Sepsis

Background Mitochondrial injury develops in skeletal muscles during the course of severe sepsis. Autophagy is a protein and organelle recycling pathway which functions to degrade or recycle unnecessary, redundant, or inefficient cellular components. No information is available regarding the degree of sepsis-induced mitochondrial injury and autophagy in the ventilatory and locomotor muscles. This study tests the hypotheses that the locomotor muscles are more prone to sepsis-induced mitochondrial injury, depressed biogenesis and autophagy induction compared with the ventilatory muscles. Methodology/Principal Findings Adult male C57/Bl6 mice were injected with i.p. phosphate buffered saline (PBS) or E. coli lipopolysaccharide (LPS, 20 mg/kg) and sacrificed 24 h later. The tibialis anterior (TA), soleus (SOLD) and diaphragm (DIA) muscles were quickly excised and examined for mitochondrial morphological injury, Ca++ retention capacity and biogenesis. Autophagy was detected with electron microscopy, lipidation of Lc3b proteins and by measuring gene expression of several autophagy-related genes. Electron microscopy revealed ultrastructural injuries in the mitochondria of each muscle, however, injuries were more severe in the TA and SOL muscles than they were in the DIA. Gene expressions of nuclear and mitochondrial DNA transcription factors and co-activators (indicators of biogenesis) were significantly depressed in all treated muscles, although to a greater extent in the TA and SOL muscles. Significant autophagosome formation, Lc3b protein lipidation and upregulation of autophagy-related proteins were detected to a greater extent in the TA and SOL muscles and less so in the DIA. Lipidation of Lc3b and the degree of induction of autophagy-related proteins were significantly blunted in mice expressing a muscle-specific IκBα superrepresor. Conclusion/Significance We conclude that locomotor muscles are more prone to sepsis-induced mitochondrial injury, decreased biogenesis and increased autophagy compared with the ventilatory muscles and that autophagy in skeletal muscles during sepsis is regulated in part through the NFκB transcription factor.

Protective role of PARK2/Parkin in sepsis-induced cardiac contractile and mitochondrial dysfunction

Mitochondrial quality control plays a vital role in the maintenance of optimal mitochondrial function. However, its roles and regulation remain ill-defined in cardiac pathophysiology. Here, we tested the hypothesis that PARK2/Parkin, an E3-ligase recently described as being involved in the regulation of cardiac mitophagy, is important for (1) the maintenance of normal cardiac mitochondrial function; and (2) adequate recovery from sepsis, a condition known to induce reversible mitochondrial injury through poorly understood mechanisms. Investigations of mitochondrial and cardiac function were thus performed in wild-type and Park2-deficient mice at baseline and at 2 different times following administration of a sublethal dose of E. coli lipopolysaccharide (LPS). LPS injection induced cardiac and mitochondrial dysfunctions that were followed by complete recovery in wild-type mice. Recovery was associated with morphological and biochemical evidence of mitophagy, suggesting that this process is implicated in cardiac recovery from sepsis. Under baseline conditions, multiple cardiac mitochondrial dysfunctions were observed in Park2-deficient mice. These mild dysfunctions did not result in a visibly distinct cardiac phenotype. Importantly, Park2-deficient mice exhibited impaired recovery of cardiac contractility and constant degradation of mitochondrial metabolic functions. Interestingly, autophagic clearance of damaged mitochondria was still possible in the absence of PARK2 likely through compensatory mechanisms implicating PARK2-independent mitophagy and upregulation of macroautophagy. Together, these results thus provide evidence that in vivo, mitochondrial autophagy is activated during sepsis, and that compensation for a lack of PARK2 is only partial and/or that PARK2 exerts additional protective roles in mitochondria.

Genetic and histologic evidence for autophagy in asthma pathogenesis

To the Editor: n nAsthma affects all age groups and presents itself as a spectrum of severity and symptoms. Reactive oxidative species (ROS) play a pivotal role in asthma pathogenesis. Exhaled levels of mediators associated with ROS positively correlate with asthma severity.(1) Autophagy, the process of cellular waste disposal through lysosomal -dependent pathways, is induced by ROS to remove oxidized proteins or organelles to minimize tissue damage.(2) Although autophagy is augmented in the lungs of COPD patients compared to healthy control subjects (3), evidence for autophagy in asthma, particularly moderate-to-severe asthma, has not been reported. We hypothesize that autophagy is associated with asthma pathogenesis, and sought to detect its presence using both genetic and histological approaches. n nWe conducted a genetic association study to investigate whether single nucleotide polymorphisms (SNPs) in genes of the autophagy pathway are associated with asthma. We selected 5 genes of the autophagy pathway (unc-51-like kinase 1(ULK1), sequestosome 1 (SQSTM1), microtubule-associated protein 1 light chain 3 beta (MAP1LC3B), beclin 1 (BECN1) and autophagy related 5 homolog (ATG5)). (2) We tested for genetic associations in an asthma family-based study from northeastern Quebec, Canada (The Saguenay-Lac-Saint-Jean (SLSJ) asthma Study) using the family based association test (FBAT) statistic and UNPHASED software for an odds ratio estimate.(4, 5) Patient recruitment has previously been described.(6) These SNPs have been genotyped previously in a genome-wide association study.(6) To reduce the likelihood of false positive findings, we reset the statistical significance threshold from p=0.05 to p=0.001 according to the Bonferroni method. To confirm our positive findings, we tested the association in a second family based population; the non-Hispanic Caucasian participants of the Childhood Asthma Management Program (CAMP), and patient recruitment has previously been described. (7) The SLSJ local ethics committee and the Institutional Review Board for CAMP approved the study, and all subjects gave informed consents. n nIn the SLSJ asthma study a total of 1338 individuals (483 nuclear families) with known asthma status were included in the analysis, and 336 individuals were either probands or affected siblings. Of this group, the male:female ratio was 0.83. The mean age was 16.45 (standard deviation (SD) +/− 9.43) years. 77.1% were atopic. The mean (SD) forced expiratory volume in 1 second (FEV1)% predicted was 94.1(20.1)%. A total of 39 SNPs were tested, and after Bonferroni correction, SNP rs12212740 G>A of ATG5 remained statistically significant (Table 1). Allele G with allele frequency of 0.88 is over - transmitted to asthmatic offspring (p=0.0002), (Odds ratio = 1.35, (95% confidence interval = 1.01 and 1.89). SNP rs12212740 was not associated with asthma in CAMP, however, it was associated with pre - bronchodilator FEV1 (pre-FEV1) (adjusted for age, sex and height) (p=0.04). In the SLSJ cohort, rs12212740 was associated with pre-FEV1 (p=0.007). In both populations, allele G was negatively associated with adjusted pre-FEV1. SNP rs12212740 is located in intron 3 of ATG5, 7kb downstream and 8kb upstream of exon 3 and 4, respectively. At present, the functional consequence of SNP rs12212740 is unknown, and it is probable that the association is due to the linkage disequilibrium between SNP rs12212740 and the true causative variant. Nevertheless, the association between a genetic variant of ATG5 and pre-FEV1 in both study populations suggest that autophagy is associated with reduced lung function in asthmatic subjects. n n n nTable 1 n nAssociation of SNPs and asthma in the SLSJ asthma study n n n nTo determine if autophagy is present in the airways of asthmatic individuals, bronchial biopsy stored at the Tissue Bank of the Respiratory Health Network of the Fonds de la Recherche en Sante du Quebec (McGill University Health Centre site) were obtained. Patient recruitment and bronchoscopy have previously been described.(8) Bronchial biopsy tissue from a moderately severe asthma patient and a healthy control were viewed by electron microscopy (EM) for double membrane autophagosomes. n nHere, we demonstrated by EM in a tissue sample from a moderately severe asthma subject evidence of autophagy in asthma pathogenesis. Using EM, double membrane autophagosomes were detected in fibroblasts and epithelial cells. A representative fibroblast from a bronchial biopsy tissue of a moderate asthma subject is depicted in Figures 1A-C, and epithelial cells in figures 2A-C. Corresponding fibroblast and epithelial cells from a healthy control are depicted in figures 1D-F and figures 2D-F, respectively, where fewer or no autophagosome was detected. n n n nFig 1 n nA double membrane autophagosome was detected in a fibroblast from a bronchial biopsy tissue sample of a moderate asthma subject. Tissue was viewed at magnifications of (A) 1480x, (B) 16100x and (C) 62200x. Boxed areas were viewed under higher magnification ... n n n n n nFig 2 n nAutophagosomes were detected in bronchial epithelial cells from a moderate asthma subject. Tissue was viewed at magnifications of (A) 4030x, (B) 9760x and (C) 37000x, respectively. Corresponding epithelial cells of a healthy subject was viewed at magnifications ... n n n nThis is the first report to present genetic and histological evidence of autophagy in asthma pathogenesis. ATG5 is involved in the elongation step of the autophagosome formation. ATG5 forms a complex with ATG12 and ATG16L1 and the complexes are found on the outer membrane of the forming autophagosome.(2) We speculate that the positive association of allele G with asthma and the negative association with pre-FEV1 in asthmatics may be due to the inverse relationship between pre-FEV1 and asthma severity(9). If allele G is a risk factor for low pre-FEV1, given that pre-FEV1 tends to be lower in those with more severe forms of asthma, the allele would also increase the risk of developing moderate-to-severe asthma. The genetic association of ATG with pre-FEV1 in asthmatics and the detection of autophagosomes in fibroblasts and epithelial cells in tissues from a moderately severe asthma patient suggest an association between autophagy and reduced lung function in asthmatic subjects. At present the mechanistic pathway of autophagy in asthma is unclear, but it opens up a new avenue to explore the mechanism of the chronic nature of asthma pathogenesis.

Role of autophagy in COPD skeletal muscle dysfunction

Chronic obstructive pulmonary disease (COPD) is a debilitating disease caused by parenchymal damage and irreversible airflow limitation. In addition to lung dysfunction, patients with COPD develop weight loss, malnutrition, poor exercise performance, and skeletal muscle atrophy. The latter has been attributed to an imbalance between muscle protein synthesis and protein degradation. Several reports have confirmed that enhanced protein degradation and atrophy of limb muscles of COPD patient is mediated in part through activation of the ubiquitin-proteasome pathway and that this activation is triggered by enhanced production of reactive oxygen species. Until recently, the importance of the autophagy-lysosome pathway in protein degradation of skeletal muscles has been largely ignored, however, recent evidence suggests that this pathway is actively involved in recycling of cytosolic proteins, organelles, and protein aggregates in normal skeletal muscles. The protective role of autophagy in the regulation of muscle mass has recently been uncovered in mice with muscle-specific suppression of autophagy. These mice develop severe muscle weakness, atrophy, and decreased muscle contractility. No information is yet available about the involvement of the autophagy in the regulation of skeletal muscle mass in COPD patients. Pilot experiments on vastus lateralis muscle samples suggest that the autophagy-lysosome system is induced in COPD patients compared with control subjects. In this review, we summarize recent progress related to molecular structure, regulation, and roles of the autophagy-lysosome pathway in normal and diseased skeletal muscles. We also speculate about regulation and functional importance of this system in skeletal muscle dysfunction in COPD patients.

Autophagic flux and oxidative capacity of skeletal muscles during acute starvation

Autophagy is an important proteolytic pathway in skeletal muscles. The roles of muscle fiber type composition and oxidative capacity remain unknown in relation to autophagy. The diaphragm (DIA) is a fast-twitch muscle fiber with high oxidative capacity, the tibialis anterior (TA) muscle is a fast-twitch muscle fiber with low oxidative capacity, and the soleus muscle (SOL) is a slow-twitch muscle with high oxidative capacity. We hypothesized that oxidative capacity is a major determinant of autophagy in skeletal muscles. Following acute (24 h) starvation of adult C57/Bl6 mice, each muscle was assessed for autophagy and compared with controls. Autophagy was measured by monitoring autophagic flux following leupeptin (20 mg/kg) or colchicine (0.4 mg/kg/day) injection. Oxidative capacity was measured by monitoring citrate synthase activity. In control mice, autophagic flux values were significantly greater in the TA than in the DIA and SOL. In acutely starved mice, autophagic flux increased, most markedly in the TA, and several key autophagy-related genes were significantly induced. In both control and starved mice, there was a negative linear correlation of autophagic flux with citrate synthase activity. Starvation significantly induced AMPK phosphorylation and inhibited AKT and RPS6KB1 phosphorylation, again most markedly in the TA. Starvation induced Foxo1, Foxo3, and Foxo4 expression and attenuated the phosphorylation of their gene products. We conclude that both basal and starvation-induced autophagic flux are greater in skeletal muscles with low oxidative capacity as compared with those with high oxidative capacity and that this difference is mediated through selective activation of the AMPK pathway and inhibition of the AKT-MTOR pathways.

Mechanical ventilation triggers abnormal mitochondrial dynamics and morphology in the diaphragm

The diaphragm is a unique skeletal muscle designed to be rhythmically active throughout life, such that its sustained inactivation by the medical intervention of mechanical ventilation (MV) represents an unanticipated physiological state in evolutionary terms. Within a short period after initiating MV, the diaphragm develops muscle atrophy, damage, and diminished strength, and many of these features appear to arise from mitochondrial dysfunction. Notably, in response to metabolic perturbations, mitochondria fuse, divide, and interact with neighboring organelles to remodel their shape and functional properties-a process collectively known as mitochondrial dynamics. Using a quantitative electron microscopy approach, here we show that diaphragm contractile inactivity induced by 6 h of MV in mice leads to fragmentation of intermyofibrillar (IMF) but not subsarcolemmal (SS) mitochondria. Furthermore, physical interactions between adjacent organellar membranes were less abundant in IMF mitochondria during MV. The profusion proteins Mfn2 and OPA1 were unchanged, whereas abundance and activation status of the profission protein Drp1 were increased in the diaphragm following MV. Overall, our results suggest that mitochondrial morphological abnormalities characterized by excessive fission-fragmentation represent early events during MV, which could potentially contribute to the rapid onset of mitochondrial dysfunction, maladaptive signaling, and associated contractile dysfunction of the diaphragm.

Failed reinnervation in aging skeletal muscle

BackgroundSkeletal muscle displays a marked accumulation of denervated myofibers at advanced age, which coincides with an acceleration of muscle atrophy.MethodsIn this study, we evaluated the hypothesis that the accumulation of denervated myofibers in advanced age is due to failed reinnervation by examining muscle from young adult (YA) and very old (VO) rats and from a murine model of sporadic denervation secondary to neurotrypsin over-expression (Sarco mouse).ResultsBoth aging rat muscle and Sarco mouse muscle exhibited marked fiber-type grouping, consistent with repeating cycles of denervation and reinnervation, yet in VO muscle, rapsyn at the endplate increased and was associated with only a 10xa0% decline in acetylcholine receptor (AChR) intensity, whereas in Sarco mice, there was a decline in rapsyn and a 25xa0% decrease in AChR intensity. Transcripts of muscle-specific kinase (21-fold), acetylcholine receptor subunits α (68-fold), ε (threefold) and γ (47-fold), neural cell adhesion molecule (66-fold), and runt-related transcription factor 1 (33-fold) were upregulated in VO muscle of the rat, consistent with the marked persistent denervation evidenced by a large proportion of very small fibers (>20xa0%). In the Sarco mice, there were much smaller increases in denervation transcripts (0–3.5-fold) and accumulation of very small fibers (2–6xa0%) compared to the VO rat, suggesting a reduced capacity for reinnervation in aging muscle. Despite the marked persistent denervation in the VO rat muscle, transcripts of neurotrophins involved in promoting axonal sprouting following denervation exhibited no increase, and several miRNAs predicted to suppress neurotrophins were elevated in VO rat.ConclusionsOur results support the hypothesis that the accumulation of denervated fibers with aging is due to failed reinnervation and that this may be affected by a limited neurotrophin response that mediates axonal sprouting following denervation.

Expression and functional roles of angiopoietin-2 in skeletal muscles.

Background Angiopoietin-1 (ANGPT1) and angiopoietin-2 (ANGPT2) are angiogenesis factors that modulate endothelial cell differentiation, survival and stability. Recent studies have suggested that skeletal muscle precursor cells constitutively express ANGPT1 and adhere to recombinant ANGPT1 and ANGPT2 proteins. It remains unclear whether or not they also express ANGPT2, or if ANGPT2 regulates the myogenesis program of muscle precursors. In this study, ANGPT2 regulatory factors and the effects of ANGPT2 on proliferation, migration, differentiation and survival were identified in cultured primary skeletal myoblasts. The cellular networks involved in the actions of ANGPT2 on skeletal muscle cells were also analyzed. Methodology/Principal Findings Primary skeletal myoblasts were isolated from human and mouse muscles. Skeletal myoblast survival, proliferation, migration and differentiation were measured in-vitro in response to recombinant ANGPT2 protein and to enhanced ANGPT2 expression delivered with adenoviruses. Real-time PCR and ELISA measurements revealed the presence of constitutive ANGPT2 expression in these cells. This expression increased significantly during myoblast differentiation into myotubes. In human myoblasts, ANGPT2 expression was induced by H2O2, but not by TNFα, IL1β or IL6. ANGPT2 significantly enhanced myoblast differentiation and survival, but had no influence on proliferation or migration. ANGPT2-induced survival was mediated through activation of the ERK1/2 and PI-3 kinase/AKT pathways. Microarray analysis revealed that ANGPT2 upregulates genes involved in the regulation of cell survival, protein synthesis, glucose uptake and free fatty oxidation. Conclusion/Significance Skeletal muscle precursors constitutively express ANGPT2 and this expression is upregulated during differentiation into myotubes. Reactive oxygen species exert a strong stimulatory influence on muscle ANGPT2 expression while pro-inflammatory cytokines do not. ANGPT2 promotes skeletal myoblast survival and differentiation. These results suggest that muscle-derived ANGPT2 production may play a positive role in skeletal muscle fiber repair.