Prolonged environment-induced hyperthermia alters autophagy in oxidative skeletal muscle in Sus scrofa.

Prolonged heat stress represents a continuing threat to human health and agricultural production. Despite the broad, negative impact of prolonged hyperthermia little is known about underlying pathological mechanisms leading to negative health outcomes, which has limited the development of etiological interventions and left clinicians and producers with only cooling and rehydration strategies. The purpose of this investigation was to determine the extent to which prolonged environment-induced hyperthermia altered autophagy in oxidative skeletal muscle in a large animal model, serving the dual purpose of accurately modeling human physiology as well as agricultural production. We hypothesized that prolonged hyperthermia would induce autophagy in skeletal muscle, independent of the accompanying caloric restriction. To test this hypothesis pigs were treated as follows: thermoneutral (20 °C), heat stress (35 °C), or were held under thermoneutral conditions but pair-fed to the heat stress group for seven days. Upon euthanasia the red portion of the semitendinosus was collected. We found that prolonged hyperthermic exposure increased oxidative stress without a corresponding change in antioxidant enzyme activities. Hyperthermia prevented initiation of autophagy despite increased markers of nucleation, elongation and autophagosome formation. However, p62 relative protein abundance, which is inversely correlated with autophagic degradation, was strongly increased suggesting suppressed degradation of autophagosomes. Markers of mitophagy and mitochondrial abundance were largely similar between groups. These data indicate that faulty autophagy plays a key role in hyperthermic muscle dysfunction.

Chronic kidney disease induces autophagy leading to dysfunction of mitochondria in skeletal muscle.

Chronic kidney disease (CKD) causes loss of lean body mass by multiple mechanisms. This study examines whether autophagy-mediated proteolysis contributes to CKD-induced muscle wasting. We tested autophagy in the muscle of CKD mice with plantaris muscle overloading to mimic resistance exercise or with acupuncture plus low-frequency electrical stimulation (Acu/LFES) treatment. In CKD muscle, Bnip3, Beclin-1, and LC3II mRNAs and proteins were increased compared with those in control muscle, indicating autophagosome-lysosome formation induction. Acu/LFES suppressed the CKD-induced upregulation of autophagy. However, overloading increased autophagy-related proteins in normal and CKD muscle. Serum from uremic mice induces autophagy formation but did not increase the myosin degradation or actin break down in cultured muscle satellite cells. We examined mitochondrial biogenesis, copy number, and ATP production in cultured myotubes, and found all three aspects to be decreased by uremic serum. Inhibition of autophagy partially reversed this decline in cultured myotubes. In CKD mice, the mitochondrial copy number, biogenesis marker peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), mitochondrial transcription factor A (TFAM), and mitochondrial fusion marker Mitofusin-2 (Mfn2) are decreased. Both muscle overloading and Acu/LFES increased mitochondrial copy number, and reversed the CKD-induced decreases in PGC-1α, TFAM, and Mfn2. We conclude that the autophagy is activated in the muscle of CKD mice. However, myofibrillar protein is not directly broken down through autophagy. Instead, CKD-induced upregulation of autophagy leads to dysfunction of mitochondria and decrease of ATP production.

miR-182 attenuates atrophy-related gene expression by targeting FoxO3 in skeletal muscle.

Skeletal muscle atrophy occurs in response to a variety of conditions including chronic kidney disease, diabetes, cancer, and elevated glucocorticoids. MicroRNAs (miR) may play a role in the wasting process. Activation of the forkhead box O3 (FoxO3) transcription factor causes skeletal muscle atrophy in patients, animals, and cultured cells by increasing the expression of components of the ubiquitin-proteasome and autophagy-lysosome proteolytic systems. To identify microRNAs that potentially modulate the atrophy process, an in silico target analysis was performed and miR-182 was predicted to target FoxO3 mRNA. Using a combination of immunoblot analysis, quantitative real-time RT-PCR, and FoxO3 3'-UTR luciferase reporter genes, miR-182 was confirmed to regulate FoxO3 expression in C2C12 myotubes. Transfection of miR-182 into muscle cells decreased FoxO3 mRNA 30% and FoxO3 protein 67% (P < 0.05) and also prevented a glucocorticoid-induced upregulation of multiple FoxO3 gene targets including MAFbx/atrogin-1, autophagy-related protein 12 (ATG12), cathepsin L, and microtubule-associated protein light chain 3 (LC3). Treatment of C2C12 myotubes with dexamethasone (Dex) (1 µM, 6 h) to induce muscle atrophy decreased miR-182 expression by 63% (P < 0.05). Similarly, miR-182 was decreased 44% (P < 0.05) in the gastrocnemius muscle of rats injected with streptozotocin to induce diabetes compared with controls. Finally, miR-182 was present in exosomes isolated from the media of C2C12 myotubes and Dex increased its abundance. These data identify miR-182 as an important regulator of FoxO3 expression that participates in the control of atrophy-inducing genes during catabolic diseases.

miR-23a is decreased during muscle atrophy by a mechanism that includes calcineurin signaling and exosome-mediated export.

Skeletal muscle atrophy is prevalent in chronic diseases, and microRNAs (miRs) may play a key role in the wasting process. miR-23a was previously shown to inhibit the expression of atrogin-1 and muscle RING-finger protein-1 (MuRF1) in muscle. It also was reported to be regulated by cytoplasmic nuclear factor of activated T cells 3 (NFATc3) in cardiomyocytes. The objective of this study was to determine if miR-23a is regulated during muscle atrophy and to evaluate the relationship between calcineurin (Cn)/NFAT signaling and miR-23a expression in skeletal muscle cells during atrophy. miR-23a was decreased in the gastrocnemius of rats with acute streptozotocin-induced diabetes, a condition known to increase atrogin-1 and MuRF1 expression and cause atrophy. Treatment of C2C12 myotubes with dexamethasone (Dex) for 48 h also reduced miR-23a as well as RCAN1.4 mRNA, which is transcriptionally regulated by NFAT. NFATc3 nuclear localization and the amount of miR-23a decreased rapidly within 1 h of Dex administration, suggesting a link between Cn signaling and miR-23a. The level of miR-23a was lower in primary myotubes from mice lacking the α- or ß-isoform of the CnA catalytic subunit than wild-type mice. Dex did not further suppress miR-23a in myotubes from Cn-deficient mice. Overexpression of CnAß in C2C12 myotubes prevented Dex-induced suppression of miR-23a. Finally, miR-23a was present in exosomes isolated from the media of C2C12 myotubes, and Dex increased its exosomal abundance. Dex did not alter the number of exosomes released into the media. We conclude that atrophy-inducing conditions downregulate miR-23a in muscle by mechanisms involving attenuated Cn/NFAT signaling and selective packaging into exosomes.

Inhibition of the ubiquitin-proteasome pathway does not protect against ventilator-induced accelerated proteolysis or atrophy in the diaphragm.

BACKGROUND: Mechanical ventilation (MV) is a life-saving intervention in patients with acute respiratory failure. However, prolonged MV results in ventilator-induced diaphragm dysfunction (VIDD), a condition characterized by both diaphragm fiber atrophy and contractile dysfunction. Previous work has shown that calpain, caspase-3, and the ubiquitin-proteasome pathway (UPP) are all activated in the diaphragm during prolonged MV. However, although it is established that both calpain and caspase-3 are important contributors to VIDD, the role that the UPP plays in the development of VIDD remains unknown. These experiments tested the hypothesis that inhibition of the UPP will protect the diaphragm against VIDD. METHODS: The authors tested this prediction in an established animal model of MV using a highly specific UPP inhibitor, epoxomicin, to prevent MV-induced activation of the proteasome in the diaphragm (n = 8 per group). RESULTS: The results of this study reveal that inhibition of the UPP did not prevent ventilator-induced diaphragm muscle fiber atrophy and contractile dysfunction during 12 h of MV. Also, inhibition of the UPP does not affect MV-induced increases in calpain and caspase-3 activity in the diaphragm. Finally, administration of the proteasome inhibitor did not protect against the MV-induced increases in the expression of the E3 ligases, muscle ring finger-1 (MuRF1), and atrogin-1/MaFbx. CONCLUSION: Collectively, these results indicate that proteasome activation does not play a required role in VIDD development during the first 12 h of MV.

Negative pressure ventilation and positive pressure ventilation promote comparable levels of ventilator-induced diaphragmatic dysfunction in rats.

BACKGROUND: Mechanical ventilation is a life-saving intervention for patients with respiratory failure. Unfortunately, a major complication associated with prolonged mechanical ventilation is ventilator-induced diaphragmatic atrophy and contractile dysfunction, termed ventilator-induced diaphragmatic dysfunction (VIDD). Emerging evidence suggests that positive pressure ventilation (PPV) promotes lung damage (ventilator-induced lung injury [VILI]), resulting in the release of signaling molecules that foster atrophic signaling in the diaphragm and the resultant VIDD. Although a recent report suggests that negative pressure ventilation (NPV) results in less VILI than PPV, it is unknown whether NPV can protect against VIDD. Therefore, the authors tested the hypothesis that compared with PPV, NPV will result in a lower level of VIDD. METHODS: Adult rats were randomly assigned to one of three experimental groups (n = 8 each): (1) acutely anesthetized control (CON), (2) 12 h of PPV, and (3) 12 h of NPV. Dependent measures included indices of VILI, diaphragmatic muscle fiber cross-sectional area, diaphragm contractile properties, and the activity of key proteases in the diaphragm. RESULTS: Our results reveal that no differences existed in the degree of VILI between PPV and NPV animals as evidenced by VILI histological scores (CON = 0.082 ± 0.001; PPV = 0.22 ± 0.04; NPV = 0.25 ± 0.02; mean ± SEM). Both PPV and NPV resulted in VIDD. Importantly, no differences existed between PPV and NPV animals in diaphragmatic fiber cross-sectional area, contractile properties, and the activation of proteases. CONCLUSION: These results demonstrate that NPV and PPV result in similar levels of VILI and that NPV and PPV promote comparable levels of VIDD in rats.

Cross-talk between the calpain and caspase-3 proteolytic systems in the diaphragm during prolonged mechanical ventilation.

OBJECTIVE: Diaphragmatic weakness, due to both atrophy and contractile dysfunction, is a well-documented response following prolonged mechanical ventilation. Evidence indicates that activation of the proteases calpain and caspase-3 is essential for mechanical ventilation-induced diaphragmatic weakness to occur. We tested the hypothesis that a regulatory cross-talk exists between calpain and caspase-3 in the diaphragm during prolonged mechanical ventilation. To test this prediction, we determined whether selective pharmacological inhibition of calpain would prevent activation of caspase-3 and conversely whether selective inhibition of caspase-3 would abate calpain activation. DESIGN: Animal study. SETTING: University Research Laboratory. SUBJECTS: Female Sprague-Dawley rats. INTERVENTIONS: Animals were randomly divided into control or one of three 12-hr mechanical ventilation groups that were treated with/without a selective pharmacological protease inhibitor: 1) control, 2) mechanical ventilation, 3) mechanical ventilation with a selective caspase-3 inhibitor, and 4) mechanical ventilation with a selective calpain inhibitor. MEASUREMENTS AND MAIN RESULTS: Compared to control, mechanical ventilation resulted in calpain and caspase-3 activation in the diaphragm accompanied by atrophy of type I, type IIa, and type IIx/IIb fibers. Independent inhibition of either calpain or caspase-3 prevented this mechanical ventilation-induced atrophy. Pharmacological inhibition of calpain prevented mechanical ventilation-induced activation of diaphragmatic caspase-3 and inhibition of caspase-3 prevented activation of diaphragmatic calpain. Furthermore, calpain inhibition also prevented the activation of caspase-9 and caspase-12, along with the cleavage of Bid to tBid, all upstream signals for caspase-3 activation. Lastly, caspase-3 inhibition prevented the mechanical ventilation-induced degradation of the endogenous calpain inhibitor, calpastatin. CONCLUSIONS: Collectively, these results indicate that mechanical ventilation-induced diaphragmatic atrophy is dependent on the activation of both calpain and caspase-3. Importantly, these findings provide the first experimental evidence in diaphragm muscle that calpain inhibition prevents the activation of caspase-3 and vice versa and caspase-3 inhibition prevents the activation of calpain. These findings support our hypothesis that a regulatory calpain/caspase-3 cross-talk exists whereby calpain can promote caspase-3 activation and active caspase-3 can enhance calpain activity in diaphragm muscle during prolonged mechanical ventilation.

Nuclear factor-κB signaling contributes to mechanical ventilation-induced diaphragm weakness*.

OBJECTIVES: Although mechanical ventilation is a life-saving measure for patients in respiratory failure, prolonged mechanical ventilation results in diaphragmatic weakness attributable to fiber atrophy and contractile dysfunction. Therefore, identifying the signaling pathways responsible for mechanical ventilation-induced diaphragmatic weakness is important. In this context, it is established that oxidative stress is required for mechanical ventilation-induced diaphragmatic weakness to occur. Numerous redox-sensitive signaling pathways exist in muscle including the transcription factor nuclear factor-κB. Although it has been suggested that nuclear factor-κB contributes to proteolytic signaling in inactivity-induced atrophy in locomotor muscles, the role that nuclear factor-κB plays in mechanical ventilation-induced diaphragmatic weakness is unknown. We tested the hypothesis that nuclear factor-κB activation plays a key signaling role in mechanical ventilation-induced diaphragmatic weakness and that oxidative stress is required for nuclear factor-κB activation. DESIGN: Cause and effect was determined by independently treating mechanically ventilated animals with either a specific nuclear factor-κB inhibitor (SN50) or a clinically relevant antioxidant (curcumin). MEASUREMENTS AND MAIN RESULTS: Inhibition of nuclear factor-κB activity partially attenuated both mechanical ventilation-induced diaphragmatic atrophy and contractile dysfunction. Further, treatment with the antioxidant curcumin prevented mechanical ventilation-induced activation of nuclear factor-κB in the diaphragm and rescued the diaphragm from both mechanical ventilation-induced atrophy and contractile dysfunction. CONCLUSIONS: Collectively, these findings support the hypothesis that nuclear factor-κB activation plays a significant signaling role in mechanical ventilation-induced diaphragmatic weakness and that oxidative stress is an upstream activator of nuclear factor-κB. Finally, our results suggest that prevention of mechanical ventilation-induced oxidative stress in the diaphragm could be a useful clinical strategy to prevent or delay mechanical ventilation-induced diaphragmatic weakness.

Both high level pressure support ventilation and controlled mechanical ventilation induce diaphragm dysfunction and atrophy.

OBJECTIVES: Previous workers have demonstrated that controlled mechanical ventilation results in diaphragm inactivity and elicits a rapid development of diaphragm weakness as a result of both contractile dysfunction and fiber atrophy. Limited data exist regarding the impact of pressure support ventilation, a commonly used mode of mechanical ventilation-that permits partial mechanical activity of the diaphragm-on diaphragm structure and function. We carried out the present study to test the hypothesis that high-level pressure support ventilation decreases the diaphragm pathology associated with CMV. METHODS: Sprague-Dawley rats were randomly assigned to one of the following five groups:1) control (no mechanical ventilation); 2) 12 hrs of controlled mechanical ventilation (12CMV); 3) 18 hrs of controlled mechanical ventilation (18CMV); 4) 12 hrs of pressure support ventilation (12PSV); or 5) 18 hrs of pressure support ventilation (18PSV). MEASUREMENTS AND MAIN RESULTS: We carried out the following measurements on diaphragm specimens: 4-hydroxynonenal-a marker of oxidative stress, active caspase-3 (casp-3), active calpain-1 (calp-1), fiber type cross-sectional area, and specific force (sp F). Compared with the control, both 12PSV and 18PSV promoted a significant decrement in diaphragmatic specific force production, but to a lesser degree than 12CMV and 18CMV. Furthermore, 12CMV, 18PSV, and 18CMV resulted in significant atrophy in all diaphragm fiber types as well as significant increases in a biomarker of oxidative stress (4-hydroxynonenal) and increased proteolytic activity (20S proteasome, calpain-1, and caspase-3). Furthermore, although no inspiratory effort occurs during controlled mechanical ventilation, it was observed that pressure support ventilation resulted in large decrement, approximately 96%, in inspiratory effort compared with spontaneously breathing animals. CONCLUSIONS: High levels of prolonged pressure support ventilation promote diaphragmatic atrophy and contractile dysfunction. Furthermore, similar to controlled mechanical ventilation, pressure support ventilation-induced diaphragmatic atrophy and weakness are associated with both diaphragmatic oxidative stress and protease activation.

Mitochondria-targeted antioxidants protect against mechanical ventilation-induced diaphragm weakness.

BACKGROUND: Mechanical ventilation is a life-saving intervention used to provide adequate pulmonary ventilation in patients suffering from respiratory failure. However, prolonged mechanical ventilation is associated with significant diaphragmatic weakness resulting from both myofiber atrophy and contractile dysfunction. Although several signaling pathways contribute to diaphragm weakness during mechanical ventilation, it is established that oxidative stress is required for diaphragmatic weakness to occur. Therefore, identifying the site(s) of mechanical ventilation- induced reactive oxygen species production in the diaphragm is important. OBJECTIVE: These experiments tested the hypothesis that elevated mitochondrial reactive oxygen species emission is required for mechanical ventilation-induced oxidative stress, atrophy, and contractile dysfunction in the diaphragm. DESIGN: Cause and effect was determined by preventing mechanical ventilation-induced mitochondrial reactive oxygen species emission in the diaphragm of rats using a novel mitochondria-targeted antioxidant (SS-31). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Compared to mechanically ventilated animals treated with saline, animals treated with SS-31 were protected against mechanical ventilation-induced mitochondrial dysfunction, oxidative stress, and protease activation in the diaphragm. Importantly, treatment of animals with the mitochondrial antioxidant also protected the diaphragm against mechanical ventilation-induced myofiber atrophy and contractile dysfunction. CONCLUSIONS: These results reveal that prevention of mechanical ventilation-induced increases in diaphragmatic mitochondrial reactive oxygen species emission protects the diaphragm from mechanical ventilation-induced diaphragmatic weakness. This important new finding indicates that mitochondria are a primary source of reactive oxygen species production in the diaphragm during prolonged mechanical ventilation. These results could lead to the development of a therapeutic intervention to impede mechanical ventilation-induced diaphragmatic weakness.

Indices of Defective Autophagy in Whole Muscle and Lysosome Enriched Fractions From Aged D2-mdx Mice.

Duchenne muscular dystrophy (DMD) is a fatal, progressive muscle disease caused by the absence of functional dystrophin protein. Previous studies in mdx mice, a common DMD model, identified impaired autophagy with lysosomal insufficiency and impaired autophagosomal degradation as consequences of dystrophin deficiency. Thus, we hypothesized that lysosomal abundance would be decreased and degradation of autophagosomes would be impaired in muscles of D2-mdx mice. To test this hypothesis, diaphragm and gastrocnemius muscles from 11 month-old D2-mdx and DBA/2J (healthy) mice were collected. Whole muscle protein from diaphragm and gastrocnemius muscles, and protein from a cytosolic fraction (CF) and a lysosome-enriched fraction (LEF) from gastrocnemius muscles, were isolated and used for western blotting. Initiation of autophagy was not robustly activated in whole muscle protein from diaphragm and gastrocnemius, however, autophagosome formation markers were elevated in dystrophic muscles. Autophagosome degradation was impaired in D2-mdx diaphragms but appeared to be maintained in gastrocnemius muscles. To better understand this muscle-specific distinction, we investigated autophagic signaling in CFs and LEFs from gastrocnemius muscles. Within the LEF we discovered that the degradation of autophagosomes was similar between groups. Further, our data suggest an expanded, though impaired, lysosomal pool in dystrophic muscle. Notably, these data indicate a degree of muscle specificity as well as model specificity with regard to autophagic dysfunction in dystrophic muscles. Stimulation of autophagy in dystrophic muscles may hold promise for DMD patients as a potential therapeutic, however, it will be critical to choose the appropriate model and muscles that most closely recapitulate findings from human patients to further develop these therapeutics.

Effects of purposeful soccer heading on circulating small extracellular vesicle concentration and cargo.

BACKGROUND: Considering the potential cumulative effects of repetitive head impact (HI) exposure, we need sensitive biomarkers to track short- and long-term effects. Circulating small extracellular vesicles (sEVs) (<200 nm) traffic biological molecules throughout the body and may have diagnostic value as biomarkers for disease. The purpose of this study was to identify the microRNA (miRNA) profile in circulating sEVs derived from human plasma following repetitive HI exposure. METHODS: Healthy adult (aged 18-35 years) soccer players were randomly assigned to one of 3 groups: the HI group performed 10 standing headers, the leg impact group performed 10 soccer ball trapping maneuvers over 10 min, and the control group did not participate in any soccer drills. Plasma was collected before testing and 24 h afterward, and sEVs were isolated and characterized via nanoparticle tracking analysis. Next-generation sequencing was utilized to identify candidate miRNAs isolated from sEVs, and candidate microRNAs were analyzed via quantitative polymerase chain reaction. In silico target prediction was performed using TargetScan (Version 7.0; targetscan.org) and miRWalk (http://mirwalk.umm.uni-heidelberg.de/) programs, and target validation was performed using luciferase reporter vectors with a miR-7844-5p mimic in human embryonic kidney (HEK) 293T/17 cells. RESULTS: Plasma sEV concentration and size were not affected across time and group following repetitive HI exposure. After 24 h, the HI read count from next-generation sequencing showed a 4-fold or greater increase in miR-92b-5p, miR-423-5p, and miR-24-3p and a 3-fold or greater decrease in miR-7844-5p, miR-144-5p, miR-221-5p, and miR-22-3p. Analysis of quantitative polymerase chain reaction revealed that leg impact did not alter the candidate miRNA levels. To our knowledge, miR-7844-5p is a previously unknown miRNA. We identified 8 miR-7844-5p mRNA targets: protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B), LIM and senescent cell antigen-like domains 1 (LIMS1), autophagy-related 12 (ATG12), microtubule-associated protein 1 light chain 3 beta (MAP1LC3B), integrin subunit alpha-1 (ITGA1), mitogen-activated protein kinase 1 (MAPK1), glycogen synthase kinase 3ß (GSK3ß), and mitogen-activated protein kinase 8 (MAPK8). CONCLUSION: Collectively, these data indicate repetitive HI exposure alters plasma sEV miRNA content, but not sEV size or number. Furthermore, for the first time we demonstrate that previously unknown miR-7844-5p targets mRNAs known to be involved in mitochondrial apoptosis, autophagy regulation, mood disorders, and neurodegenerative disease.

PGC-1α overexpression increases transcription factor EB nuclear localization and lysosome abundance in dystrophin-deficient skeletal muscle.

Duchenne muscular dystrophy (DMD) is caused by the absence of functional dystrophin protein and results in progressive muscle wasting. Dystrophin deficiency leads to a host of dysfunctional cellular processes including impaired autophagy. Autophagic dysfunction appears to be due, at least in part, to decreased lysosomal abundance mediated by decreased nuclear localization of transcription factor EB (TFEB), a transcription factor responsible for lysosomal biogenesis. PGC-1α overexpression decreased disease severity in dystrophin-deficient skeletal muscle and increased PGC-1α has been linked to TFEB activation in healthy muscle. The purpose of this study was to determine the extent to which PGC-1α overexpression increased nuclear TFEB localization, increased lysosome abundance, and increased autophagosome degradation. We hypothesized that overexpression of PGC-1α would drive TFEB nuclear translocation, increase lysosome biogenesis, and improve autophagosome degradation. To address this hypothesis, we delivered PGC-1α via adeno-associated virus (AAV) vector injected into the right limb of 3-week-old mdx mice and the contralateral limbs received a sham injection. At 6 weeks of age, this approach increased PGC-1α transcript by 60-fold and increased TFEB nuclear localization in gastrocnemii from PGC-1α treated limbs by twofold compared to contralateral controls. Furthermore, lamp2, a marker of lysosome abundance, was significantly elevated in muscles from limbs overexpressing PGC-1α. Lastly, increased LC3II and similar p62 in PGC-1α overexpressing-limbs compared to contralateral limbs are supportive of increased degradation of autophagosomes. These data provide mechanistic insight into PGC-1α-mediated benefits to dystrophin-deficient muscle, such that increased TFEB nuclear localization in dystrophin-deficient muscle leads to increased lysosome biogenesis and autophagy.

Muscle Stem Cell-Derived Extracellular Vesicles Reverse Hydrogen Peroxide-Induced Mitochondrial Dysfunction in Mouse Myotubes.

Muscle stem cells (MuSCs) hold great potential as a regenerative therapeutic but have met numerous challenges in treating systemic muscle diseases. Muscle stem cell-derived extracellular vesicles (MuSC-EVs) may overcome these limitations. We assessed the number and size distribution of extracellular vesicles (EVs) released by MuSCs ex vivo, determined the extent to which MuSC-EVs deliver molecular cargo to myotubes in vitro, and quantified MuSC-EV-mediated restoration of mitochondrial function following oxidative injury. MuSCs released an abundance of EVs in culture. MuSC-EVs delivered protein cargo into myotubes within 2 h of incubation. Fluorescent labeling of intracellular mitochondria showed co-localization of delivered protein and mitochondria. Oxidatively injured myotubes demonstrated a significant decline in maximal oxygen consumption rate and spare respiratory capacity relative to untreated myotubes. Remarkably, subsequent treatment with MuSC-EVs significantly improved maximal oxygen consumption rate and spare respiratory capacity relative to the myotubes that were damaged but received no subsequent treatment. Surprisingly, MuSC-EVs did not affect mitochondrial function in undamaged myotubes, suggesting the cargo delivered is able to repair but does not expand the existing mitochondrial network. These data demonstrate that MuSC-EVs rapidly deliver proteins into myotubes, a portion of which co-localizes with mitochondria, and reverses mitochondria dysfunction in oxidatively-damaged myotubes.

A comparison of exosome purification methods using serum of Marek's disease virus (MDV)-vaccinated and -tumor-bearing chickens.

Marek's disease (MD) is an alphaherpesvirus (Marek's disease virus, MDV)-induced pathology of chickens associated with paralysis, immunosuppression, neurological signs, and T-cell lymphomas. MD is controlled in poultry production via live attenuated vaccines. The purpose of the current study was to compare methods for precipitating exosomes from vaccinated and protected chicken sera (VEX) and tumor-bearing chicken sera (TEX) for biomarker analysis of vaccine-induced protection and MD lymphomas respectively. A standard polyethylene glycol (PEG, 8%) method was compared to a commercial reagent (total exosome isolation reagent, TEI) for exosome yield and RNA content. Although exosomes purified by PEG or TEI were comparable in size and morphology, TEI-reagent yielded 3-4-fold greater concentration. Relative expression of 8 out of 10 G. gallus- and MDV1-encoded miRNAs examined displayed significant difference depending upon the precipitation method used. Standard PEG yields comparable, albeit lower amounts of exosomes than the TEI-reagent and a distinctive miRNA composition.

Xanthine oxidase contributes to mechanical ventilation-induced diaphragmatic oxidative stress and contractile dysfunction.

Respiratory muscle weakness resulting from both diaphragmatic contractile dysfunction and atrophy has been hypothesized to contribute to the weaning difficulties associated with prolonged mechanical ventilation (MV). While it is clear that oxidative injury contributes to MV-induced diaphragmatic weakness, the source(s) of oxidants in the diaphragm during MV remain unknown. These experiments tested the hypothesis that xanthine oxidase (XO) contributes to MV-induced oxidant production in the rat diaphragm and that oxypurinol, a XO inhibitor, would attenuate MV-induced diaphragmatic oxidative stress, contractile dysfunction, and atrophy. Adult female Sprague-Dawley rats were randomly assigned to one of six experimental groups: 1) control, 2) control with oxypurinol, 3) 12 h of MV, 4) 12 h of MV with oxypurinol, 5) 18 h of MV, or 6) 18 h of MV with oxypurinol. XO activity was significantly elevated in the diaphragm after MV, and oxypurinol administration inhibited this activity and provided protection against MV-induced oxidative stress and contractile dysfunction. Specifically, oxypurinol treatment partially attenuated both protein oxidation and lipid peroxidation in the diaphragm during MV. Further, XO inhibition retarded MV-induced diaphragmatic contractile dysfunction at stimulation frequencies >60 Hz. Collectively, these results suggest that oxidant production by XO contributes to MV-induced oxidative injury and contractile dysfunction in the diaphragm. Nonetheless, the failure of XO inhibition to completely prevent MV-induced diaphragmatic oxidative damage suggests that other sources of oxidant production are active in the diaphragm during prolonged MV.

Acute hormonal and neuromuscular responses to hypertrophy, strength and power type resistance exercise.

The purpose of the current study was to determine the acute neuroendocrine response to hypertrophy (H), strength (S), and power (P) type resistance exercise (RE) equated for total volume. Ten male subjects completed three RE protocols and a rest day (R) using a randomized cross-over design. The protocols included (1) H: 4 sets of 10 repetitions in the squat at 75% of 1RM (90 s rest periods); (2) S: 11 sets of three repetitions at 90% of 1RM (5 min rest periods); and (3) P: 8 sets of 6 repetitions of jump squats at 0% of 1RM (3 min rest periods). Total testosterone (T), cortisol (C), and sex hormone binding globulin (SHBG) were determined prior to (PRE), immediately post (IP), 60 min post, 24 h post, and 48 h post exercise bout. Peak force, rate of force development, and muscle activity from the vastus medialis (VM) and biceps femoris (BF) were determined during a maximal isometric squat test. A unique pattern of response was observed in T, C, and SHBG for each RE protocol. The percent change in T, C, and SHBG from PRE to IP was significantly (p

Comparison of exosomes purified via ultracentrifugation (UC) and Total Exosome Isolation (TEI) reagent from the serum of Marek's disease virus (MDV)-vaccinated and tumor-bearing chickens.

Extracellular vesicles (EVs) is a collective term used to refer microparticles, exosomes, and apoptotic bodies produced by a variety of cells and released into interstitial spaces and bodily fluids. Serum exosomes can serve as invaluable biomarkers, containing m/miRNAs, lipids, and proteins, indicative of various conditions. There are currently limited studies on the characterization and mutual consensus of biomarker profiles of serum exosomes purified by different methods. Here we compared the advantages and disadvantages of two commonly used serum exosome purification procedures including ultracentrifugation (UC) and Total Exosome Isolation (TEI) reagent, by analyzing exosome size distribution, concentration, morphology and miRNA expression profiles. Serum was obtained from Marek's disease virus (MDV)-infected chickens that were either vaccinated against Marek's disease (MD), and thus protected, or unvaccinated and bearing MDV-induced tumors. Nanoparticle tracking analysis (NTA) and Transmission Electron Microscopy (TEM) were performed to evaluate particle size, concentration, and morphological integrity, respectively. Our results indicate that the size distribution of particles purified by either procedure is consistent with that of exosomes (30-150 nm). TEI reagent generated higher yields and co-isolated additional EV populations that are slightly larger (∼180 nm). Based on the miRNA expression profiles from a previous high throughput sequencing experiment of exosome small RNAs, we selected six cellular and four MDV1 miRNAs, to validate their expression in UC- and TEI-purified exosomes. miRNA expression profiles displayed relative correlation between the two procedures, but distinctive differences were observed in abundance with TEI-purified exosomes showing higher miRNA expression consistent with higher yield than those purified by UC. TEI-purified exosomes from vaccinated chickens exhibited greater expression of tumor suppressor miRNA, gga-mir-146b and least expression of oncomiR, gga-mir-21 compared to those obtained from tumor-bearing chickens. We propose that gga-mir-146 and -21 can serve as serum exosome biomarkers for vaccine-induced protection and MD tumors respectively.

The effect of resistance exercise on humoral markers of oxidative stress.

UNLABELLED: Previous research attempts to identify an oxidative stress response to acute resistance exercise have yielded mixed results. Inconsistencies in the current literature base probably reflect study-to-study variance in resistance exercise protocols; where high volume and short recovery elicit the most identifiable oxidative stress response. PURPOSE: This study examined the effect of resistance exercise intensity on blood oxidative stress. METHODS: To elicit a blood oxidative stress, 10 subjects undertook two different back squat protocols: 1) a hypertrophy protocol of four sets, 10 repetitions with 90 s of rest at 75% one-repetition max (1RM); and 2) a strength protocol of 11 sets, three repetitions with 5 min of rest at 90% 1RM. The resistance exercise protocols were standardized for total volume and completed in a randomized crossover fashion with 1 wk between trials. Blood drawn before (PRE), immediately following exercise (IP), and 60 min following exercise (60POST) was analyzed for markers of oxidative stress and damage. RESULTS: In response to both hypertrophy and strength exercise protein carbonyls were significantly elevated IP and 60POST while plasma lipid hydroperoxides were not. Following the hypertrophy protocol, trolox equivalent antioxidant capacity was elevated IP while urate lower than baseline. At the 60POST time point plasma ferric reducing ability of plasma was elevated following the hypertrophy protocol. Based on protein carbonyl data, a similar oxidative stress was incurred following both hypertrophy and strength protocols. CONCLUSION: Normalization for time of blood draw following the two protocols indicates that the magnitude of blood oxidative protein damage was identical between the protocols. These findings demonstrate that both resistance exercise protocols elicited a blood oxidative stress in a time-dependent fashion.

Crosstalk between autophagy and oxidative stress regulates proteolysis in the diaphragm during mechanical ventilation.

Mechanical ventilation (MV) results in the rapid development of ventilator-induced diaphragm dysfunction (VIDD). While the mechanisms responsible for VIDD are not fully understood, recent data reveal that prolonged MV activates autophagy in the diaphragm, which may occur as a result of increased cellular reactive oxygen species (ROS) production. Therefore, we tested the hypothesis that (1) accelerated autophagy is a key contributor to VIDD; and that (2) oxidative stress is required to increase the expression of autophagy genes in the diaphragm. Our findings reveal that targeted inhibition of autophagy in the rat diaphragm prevented MV-induced muscle atrophy and contractile dysfunction. Attenuation of VIDD in these animals occurred as a result of increased diaphragm concentration of the antioxidant catalase and reduced mitochondrial ROS emission, which corresponded to reductions in the activity of calpain and caspase-3. To determine if increased ROS production is required for the upregulation of autophagy biomarkers in the diaphragm, rats that were administered the mitochondrial-targeted peptide SS-31 during MV. Results from this study demonstrated that mitochondrial ROS production in the diaphragm during MV is required for the increased expression of key autophagy genes (i.e. LC3, Atg7, Atg12, Beclin1 and p62), as well as for increased activity of cathepsin L. Together, these data reveal that autophagy is required for VIDD, and that autophagy inhibition reduces MV-induced diaphragm ROS production and prevents a positive feedback loop whereby increased autophagy is stimulated by oxidative stress, resulting in further increases in ROS and autophagy.