A multimodal exercise countermeasure prevents the negative impact of head-down tilt bed rest on muscle volume and mitochondrial health in older adults.

Mitochondrial dysfunctions are thought to contribute to muscle atrophy and weakness that develop during ageing and mechanical unloading caused by immobilization, bed rest and microgravity. Older adults are at greater risk of developing muscle and mitochondrial dysfunctions in response to unloading. Although exercise is well known to promote muscle and mitochondrial health, its protective effect during mechanical unloading in older adults remains largely unexplored. Here, we investigated the impact of 14 days of head-down tilt bed rest (HDBR) with and without a multimodal exercise countermeasure in older men and women (55-65 years). Leg muscle volume was assessed using magnetic resonance imaging. Biopsies of the vastus lateralis were performed to assess markers of mitochondrial content, respiration, reactive oxygen species (ROS) production and calcium retention capacity (mCRC). Indices of mitochondrial quality control (MQC), including markers of fusion (MFN1 and 2), fission (Drp1), mitophagy (Parkin) and autophagy (p62 and LC3I and II) were measured using immunoblots. Muscle cross-sections were stained for neural cell adhesion molecule (NCAM, a marker of denervation). HDBR triggered muscle atrophy, decreased mitochondrial content and respiration and increased mitochondrial ROS production. HDBR had no impact on mCRC or MQC markers but increased markers of autophagy and denervation. Exercise prevented the deleterious effects of HDBR on leg muscle volume, mitochondrial ROS production and markers of autophagy and denervation. Exercise also increased mitochondrial content and respiration without altering mCRC and MQC markers. Collectively, our results indicate that an exercise countermeasure that can be performed in bed is effective in protecting muscle and mitochondrial health during HDBR in older adults. KEY POINTS: Conditions associated with muscle unloading, such as immobilization, bed rest or microgravity, result in muscle atrophy and weakness, particularly in older adults. Mitochondrial dysfunctions are thought to contribute to muscle atrophy caused by unloading and ageing. However, whether exercise can counteract the deleterious effects of unloading in older adults remains largely unexplored. Here, we report that older adults exposed to 14 days of head-down tilt bed rest (HDBR) displayed upper leg muscle atrophy, a decrease in mitochondrial content and respiration, an increase in H2O2 emission, and an increase in autophagy and denervation markers. No impact of HDBR on mitochondrial quality control was observed. A multimodal exercise countermeasure prevented the deleterious effects of HDBR on upper leg muscle volume, mitochondrial reactive oxygen species emission, and markers of autophagy and denervation and increased mitochondrial content and respiration. These findings highlight the effectiveness of exercise in promoting muscle and mitochondrial health in older adults undergoing bed rest.

The impact of high-fat feeding and parkin overexpression on skeletal muscle mass, mitochondrial respiration, and H2O2 emission.

Obesity is a major risk factor for developing various health problems, including insulin resistance and type 2 diabetes. Although controversial, accumulation of mitochondrial dysfunction, and notably an increase in mitochondrial reactive oxygen species (ROS) production, was proposed as a key contributor leading to obesity-induced insulin resistance. Here, our goal was to investigate whether Parkin overexpression, a key regulator of the removal of dysfunctional mitochondria through mitophagy, could confer protection against obesity-induced mitochondrial dysfunction. To this end, intramuscular injections of adeno-associated viruses (AAVs) were performed to overexpress Parkin in limb muscle of 6-mo-old mice fed a control diet (CD) or a high-fat diet (HFD) for 12 wk. An AAV-expressing the green fluorescent protein (GFP) was used as control. HFD increased fat mass, altered glycemia, and resulted in insulin resistance. Parkin overexpression resulted in an increase in muscle mass in both CD and HFD mice. In CD mice, Parkin overexpression increased maximal mitochondrial respiration and lowered H2O2 emission. HFD increased mitochondrial respiration and, surprisingly, also lowered H2O2 emission. Parkin overexpression did not significantly impact mitochondrial function in HFD mice. Taken altogether, our results indicate that Parkin overexpression positively impacts muscle and mitochondrial health under basal conditions and challenges the notion that intrinsic mitochondrial dysfunction is involved in the development of insulin resistance caused by high-fat feeding.

Angiotensin-converting enzyme 2 expression in COPD and IPF fibroblasts: the forgotten cell in COVID-19.

The COVID-19 pandemic is associated with severe pneumonia and acute respiratory distress syndrome leading to death in susceptible individuals. For those who recover, post-COVID-19 complications may include development of pulmonary fibrosis. Factors contributing to disease severity or development of complications are not known. Using computational analysis with experimental data, we report that idiopathic pulmonary fibrosis (IPF)- and chronic obstructive pulmonary disease (COPD)-derived lung fibroblasts express higher levels of angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 entry and part of the renin-angiotensin system that is antifibrotic and anti-inflammatory. In preclinical models, we found that chronic exposure to cigarette smoke, a risk factor for both COPD and IPF and potentially for SARS-CoV-2 infection, significantly increased pulmonary ACE2 protein expression. Further studies are needed to understand the functional implications of ACE2 on lung fibroblasts, a cell type that thus far has received relatively little attention in the context of COVID-19.

Surrogate Humane Endpoints in Small Animal Models of Acute Lung Injury: A Modified Delphi Consensus Study of Researchers and Laboratory Animal Veterinarians.

OBJECTIVES: In many jurisdictions, ethical concerns require surrogate humane endpoints to replace death in small animal models of acute lung injury. Heterogenous selection and reporting of surrogate endpoints render interpretation and generalizability of findings between studies difficult. We aimed to establish expert-guided consensus among preclinical scientists and laboratory animal veterinarians on selection and reporting of surrogate endpoints, monitoring of these models, and the use of analgesia. DESIGN: A three-round consensus process, using modified Delphi methodology, with researchers who use small animal models of acute lung injury and laboratory animal veterinarians who provide care for these animals. Statements on the selection and reporting of surrogate endpoints, monitoring, and analgesia were generated through a systematic search of MEDLINE and Embase. Participants were asked to suggest any additional potential statements for evaluation. SETTING: A web-based survey of participants representing the two stakeholder groups (researchers, laboratory animal veterinarians). Statements were rated on level of evidence and strength of support by participants. A final face-to-face meeting was then held to discuss results. SUBJECTS: None. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Forty-two statements were evaluated, and 29 were rated as important, with varying strength of evidence. The majority of evidence was based on rodent models of acute lung injury. Endpoints with strong support and evidence included temperature changes and body weight loss. Behavioral signs and respiratory distress also received support but were associated with lower levels of evidence. Participants strongly agreed that analgesia affects outcomes in these models and that none may be necessary following nonsurgical induction of acute lung injury. Finally, participants strongly supported transparent reporting of surrogate endpoints. A prototype composite score was also developed based on participant feedback. CONCLUSIONS: We provide a preliminary framework that researchers and animal welfare committees may adapt for their needs. We have identified knowledge gaps that future research should address.

Mitochondrial Dynamics and Mitophagy in Skeletal Muscle Health and Aging.

The maintenance of mitochondrial integrity is critical for muscle health. Mitochondria, indeed, play vital roles in a wide range of cellular processes, including energy supply, Ca2+ homeostasis, retrograde signaling, cell death, and many others. All mitochondria-containing cells, including skeletal muscle cells, dispose of several pathways to maintain mitochondrial health, including mitochondrial biogenesis, mitochondrial-derived vesicles, mitochondrial dynamics (fusion and fission process shaping mitochondrial morphology), and mitophagy-the process in charge of the removal of mitochondria though autophagy. The loss of skeletal muscle mass (atrophy) is a major health problem worldwide, especially in older people. Currently, there is no treatment to counteract the progressive decline in skeletal muscle mass and strength that occurs with aging, a process termed sarcopenia. There is increasing data, including our own, suggesting that accumulation of dysfunctional mitochondria contributes to the development of sarcopenia. Impairments in mitochondrial dynamics and mitophagy were recently proposed to contribute to sarcopenia. This review summarizes the current state of knowledge on the role played by mitochondrial dynamics and mitophagy in skeletal muscle health and in the development of sarcopenia. We also highlight recent studies showing that enhancing mitophagy in skeletal muscle is a promising therapeutic target to prevent or even treat skeletal muscle dysfunction in the elderly.

Negative regulation of angiogenesis by novel micro RNAs.

Angiopoietin-1 (Ang-1) is a ligand of Tie-2 receptors that promotes survival, migration, and differentiation of endothelial cells (ECs). Recent studies have identified several microRNA (miRNA) families that either promote or inhibit angiogenesis. To date, the nature and functional importance of miRNAs in Ang-1-induced angiogenesis are unknown. Microarray screening of known miRNAs in human umbilical vein endothelial cells (HUVECs) revealed that the expressions of miR-103b, miR-330-5p, miR-557, miR-575, miR-1287-5p, and miR-1468-5p significantly decrease following exposure to Ang-1 for 24 h. Exposure to the angiogenesis factors angiopoietin-2 (Ang-2), vascular endothelial growth factor, fibroblast growth factor 2, and transforming growth factor ß also inhibits miR-103b expression, but exerts varying effects on the other miRNAs. By overexpressing miR-103b, miR-330-5p, miR-557, miR-575, miR-1287-5p, and miR-1468-5p with selective mimics, we demonstrated that the pro-survival effects of Ang-1 are eliminated, Caspase-3 activity increases, and cell migration, proliferation, and capillary-like tube formation decreases. Conversely, transfection with selective miRNA inhibitors increases cell survival, inhibits Caspase-3 activity, and stimulates migration, proliferation and tube formation. miRNet miRNA-target gene network analyses revealed that miR-103, miR-330-5p, miR-557, miR-575, miR-1287-5p, and miR-1468-5p directly interact with 47, 95, 165, 108, 49, and 16 gene targets, respectively. Since many of these genes are positive regulators of angiogenic processes, we conclude that these miRNAs function as anti-angiogenic miRNAs and that their downregulation may be essential for Ang-1-induced angiogenesis to occur.

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.

Protective role of Parkin in skeletal muscle contractile and mitochondrial function.

KEY POINTS: Parkin, an E3 ubiquitin ligase encoded by the Park2 gene, has been implicated in the regulation of mitophagy, a quality control process in which defective mitochondria are degraded. The exact physiological significance of Parkin in regulating mitochondrial function and contractility in skeletal muscle remains largely unexplored. Using Park2-/- mice, we show that Parkin ablation causes a decrease in muscle specific force, a severe decrease in mitochondrial respiration, mitochondrial uncoupling and an increased susceptibility to opening of the permeability transition pore. These results demonstrate that Parkin plays a protective role in the maintenance of normal mitochondrial and contractile functions in skeletal muscles. ABSTRACT: Parkin is an E3 ubiquitin ligase encoded by the Park2 gene. Parkin has been implicated in the regulation of mitophagy, a quality control process in which defective mitochondria are sequestered in autophagosomes and delivered to lysosomes for degradation. Although Parkin has been mainly studied for its implication in neuronal degeneration in Parkinson disease, its role in other tissues remains largely unknown. In the present study, we investigated the skeletal muscles of Park2 knockout (Park2-/- ) mice to test the hypothesis that Parkin plays a physiological role in mitochondrial quality control in normal skeletal muscle, a tissue highly reliant on mitochondrial content and function. We first show that the tibialis anterior (TA) of Park2-/- mice display a slight but significant decrease in its specific force. Park2-/- muscles also show a trend for type IIB fibre hypertrophy without alteration in muscle fibre type proportion. Compared to Park2+/+ muscles, the mitochondrial function of Park2-/- skeletal muscles was significantly impaired, as indicated by the significant decrease in ADP-stimulated mitochondrial respiratory rates, uncoupling, reduced activities of respiratory chain complexes containing mitochondrial DNA (mtDNA)-encoded subunits and increased susceptibility to opening of the permeability transition pore. Muscles of Park2-/- mice also displayed a decrease in the content of the mitochondrial pro-fusion protein Mfn2 and an increase in the pro-fission protein Drp1 suggesting an increase in mitochondrial fragmentation. Finally, Park2 ablation resulted in an increase in basal autophagic flux in skeletal muscles. Overall, the results of the present study demonstrate that Parkin plays a protective role in the maintenance of normal mitochondrial and contractile functions in normal skeletal muscles.

Differential Regulation of the Autophagy and Proteasome Pathways in Skeletal Muscles in Sepsis.

OBJECTIVES: Skeletal muscle fiber atrophy develops in response to severe sepsis, but it is unclear as to how the proteolytic pathways that are involved in its development are differentially regulated. We investigated the link between sepsis-induced fiber atrophy and activation of the proteasome and autophagy pathways and whether the degree of activation is more severe and sustained in limb muscles than it is in the diaphragm. DESIGN: Randomized controlled experiment. SETTING: Animal research laboratory. SUBJECTS: Adult male C57/BL6 mice. INTERVENTIONS: Two groups of animals were studied. The sepsis group was subjected to a cecal ligation and perforation technique, whereas the control (sham) group was subjected to abdominal surgery without cecal ligation and perforation. Measurements for both groups were performed 24, 48, and 96 hours after the surgical procedure. MEASUREMENTS AND MAIN RESULTS: Atrophy was quantified in the diaphragm and tibialis anterior by measuring fiber diameter. Autophagy was evaluated using electron microscopic detection of autophagosomes and by measuring LC3B protein lipidation and autophagy-related protein expressions. Proteasomal degradation was quantified by measuring chymotrypsin-like activity of the 26S proteasome and messenger RNA expressions of muscle-specific E3 ligases. Sepsis triggered transient fiber atrophy in the diaphragm that lasted for 24 hours and prolonged atrophy in the tibialis anterior that persisted for 96 hours. The autophagy and proteasome pathways were activated in both muscles at varying intensities over the time course of sepsis. Activation was more pronounced in the tibialis anterior than in the diaphragm. Sepsis inhibited the V-Akt thymoma viral oncogene homolog 1 and complex 1 of the mammalian target of rapamycin pathways and stimulated the AMP-activated protein kinase pathway in both muscles. CONCLUSIONS: Sepsis triggers more severe and sustained muscle fiber atrophy in limb muscles when compared with respiratory muscle. This response is associated with enhanced proteasomal and autophagic proteolytic pathway activities and is triggered by inhibition of the AKT and complex 1 of the mammalian target of rapamycin pathways and activation of the AMPK pathway.

Mechanisms of Chronic Muscle Wasting and Dysfunction after an Intensive Care Unit Stay. A Pilot Study.

RATIONALE: Critical illness survivors often experience permanent functional disability due to intensive care unit (ICU)-acquired weakness. The mechanisms responsible for long-term weakness persistence versus resolution are unknown. OBJECTIVES: To delineate cellular mechanisms underlying long-term weakness persistence in ICU survivors. METHODS: We conducted a nested, prospective study of critically ill patients mechanically ventilated for 7 days or longer. The patients were recruited from the RECOVER program and serially assessed over 6 months after ICU discharge. Twenty-seven of 82 patients consented to participate; 15 and 11 patients were assessed at 7 days and 6 months after ICU discharge, respectively. MEASUREMENTS AND MAIN RESULTS: We assessed motor functional capacity, quadriceps size, strength, and voluntary contractile capacity and performed electromyography, nerve conduction studies, and vastus lateralis biopsies for histologic, cellular, and molecular analyses. Strength and quadriceps cross-sectional areas were decreased 7 days after ICU discharge. Weakness persisted to 6 months and correlated with decreased function. Quadriceps atrophy resolved in 27% patients at 6 months. Muscle mass reconstitution did not correlate with resolution of weakness, owing to persistent impaired voluntary contractile capacity. Compared with Day 7, increased ubiquitin-proteasome system-mediated muscle proteolysis, inflammation, and decreased mitochondrial content all normalized at 6 months. Autophagy markers were normal at 6 months. Patients with sustained atrophy had decreased muscle progenitor (satellite) cell content. CONCLUSIONS: Long-term weakness in ICU survivors results from heterogeneous muscle pathophysiology with variable combinations of muscle atrophy and impaired contractile capacity. These findings are not explained by ongoing muscle proteolysis, inflammation, or diminished mitochondrial content. Sustained muscle atrophy is associated with decreased satellite cell content and compromised muscle regrowth, suggesting impaired regenerative capacity.

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.

The JAK-STAT pathway is critical in ventilator-induced diaphragm dysfunction.

Mechanical ventilation (MV) is one of the lynchpins of modern intensive-care medicine and is life saving in many critically ill patients. Continuous ventilator support, however, results in ventilation-induced diaphragm dysfunction (VIDD) that likely prolongs patients' need for MV and thereby leads to major associated complications and avoidable intensive care unit (ICU) deaths. Oxidative stress is a key pathogenic event in the development of VIDD, but its regulation remains largely undefined. We report here that the JAK-STAT pathway is activated in MV in the human diaphragm, as evidenced by significantly increased phosphorylation of JAK and STAT. Blockage of the JAK-STAT pathway by a JAK inhibitor in a rat MV model prevents diaphragm muscle contractile dysfunction (by ~85%, p < 0.01). We further demonstrate that activated STAT3 compromises mitochondrial function and induces oxidative stress in vivo, and, interestingly, that oxidative stress also activates JAK-STAT. Inhibition of JAK-STAT prevents oxidative stress-induced protein oxidation and polyubiquitination and recovers mitochondrial function in cultured muscle cells. Therefore, in ventilated diaphragm muscle, activation of JAK-STAT is critical in regulating oxidative stress and is thereby central to the downstream pathogenesis of clinical VIDD. These findings establish the molecular basis for the therapeutic promise of JAK-STAT inhibitors in ventilated ICU patients.

Angiopoietin-1 enhances skeletal muscle regeneration in mice.

Activation of muscle progenitor cell myogenesis and endothelial cell angiogenesis is critical for the recovery of skeletal muscle from injury. Angiopoietin-1 (Ang-1), a ligand of Tie-2 receptors, enhances angiogenesis and skeletal muscle satellite cell survival; however, its role in skeletal muscle regeneration after injury is unknown. We assessed the effects of Ang-1 on fiber regeneration, myogenesis, and angiogenesis in injured skeletal muscle (tibialis anterior, TA) in mice. We also assessed endogenous Ang-1 levels and localization in intact and injured TA muscles. TA fiber injury was triggered by cardiotoxin injection. Endogenous Ang-1 mRNA levels immediately decreased in response to cardiotoxin then increased during the 2 wk. Ang-1 protein was expressed in satellite cells, both in noninjured and recovering TA muscles. Positive Ang-1 staining was present in blood vessels but not in nerve fibers. Four days after the initiation of injury, injection of adenoviral Ang-1 into injured muscles resulted in significant increases in in situ TA muscle contractility, muscle fiber regeneration, and capillary density. In cultured human skeletal myoblasts, recombinant Ang-1 protein increased survival, proliferation, migration, and differentiation into myotubes. The latter effect was associated with significant upregulation of the expression of the myogenic regulatory factors MyoD and Myogenin and certain genes involved in cell cycle regulation. We conclude that Ang-1 strongly enhances skeletal muscle regeneration in response to fiber injury and that this effect is mediated through induction of the myogenesis program in muscle progenitor cells and the angiogenesis program in endothelial cells.

An official American Thoracic Society/European Respiratory Society statement: update on limb muscle dysfunction in chronic obstructive pulmonary disease.

BACKGROUND: Limb muscle dysfunction is prevalent in chronic obstructive pulmonary disease (COPD) and it has important clinical implications, such as reduced exercise tolerance, quality of life, and even survival. Since the previous American Thoracic Society/European Respiratory Society (ATS/ERS) statement on limb muscle dysfunction, important progress has been made on the characterization of this problem and on our understanding of its pathophysiology and clinical implications. PURPOSE: The purpose of this document is to update the 1999 ATS/ERS statement on limb muscle dysfunction in COPD. METHODS: An interdisciplinary committee of experts from the ATS and ERS Pulmonary Rehabilitation and Clinical Problems assemblies determined that the scope of this document should be limited to limb muscles. Committee members conducted focused reviews of the literature on several topics. A librarian also performed a literature search. An ATS methodologist provided advice to the committee, ensuring that the methodological approach was consistent with ATS standards. RESULTS: We identified important advances in our understanding of the extent and nature of the structural alterations in limb muscles in patients with COPD. Since the last update, landmark studies were published on the mechanisms of development of limb muscle dysfunction in COPD and on the treatment of this condition. We now have a better understanding of the clinical implications of limb muscle dysfunction. Although exercise training is the most potent intervention to address this condition, other therapies, such as neuromuscular electrical stimulation, are emerging. Assessment of limb muscle function can identify patients who are at increased risk of poor clinical outcomes, such as exercise intolerance and premature mortality. CONCLUSIONS: Limb muscle dysfunction is a key systemic consequence of COPD. However, there are still important gaps in our knowledge about the mechanisms of development of this problem. Strategies for early detection and specific treatments for this condition are also needed.

Autophagy in locomotor muscles of patients with chronic obstructive pulmonary disease.

RATIONALE: Locomotor muscle atrophy develops in patients with chronic obstructive pulmonary disease (COPD) partly because of increased protein degradation by the ubiquitin-proteasome system. It is not known if autophagy also contributes to protein degradation. OBJECTIVES: To investigate whether autophagy is enhanced in locomotor muscles of stable patients with COPD, to quantify autophagy-related gene expression in these muscles, and to identify mechanisms of autophagy induction. METHODS: Muscle biopsies were obtained from two cohorts of control subjects and patients with COPD and the numbers of autophagosomes in the vastus lateralis and tibialis anterior muscles, the levels of LC3B protein lipidation, and the expression of autophagy-related genes were measured in the vastus lateralis muscle. To investigate potential pathways that might induce the activation of autophagy, measures were taken of protein kinase B (AKT), mTORC1, and AMPK pathway activation, transcription factor regulation, proteasome activation, and oxidative stress. MEASUREMENTS AND MAIN RESULTS: Autophagy is enhanced in the locomotor muscles of patients with COPD as shown by significantly higher numbers of autophagosomes in affected muscles as compared with control subjects. Autophagosome number inversely correlates with FEV1. In the vastus lateralis, LC3B protein lipidation is increased by COPD and the expression of autophagy-related gene expressions is up-regulated. LC3B lipidation inversely correlates with thigh cross-sectional area, FEV1, and FEV1/FVC ratio. Enhanced autophagy is associated with activation of the AMPK pathway and FOXO transcription factors, inhibition of the mTORC1 and AKT pathways, and the development of oxidative stress. CONCLUSIONS: Autophagy is significantly enhanced in locomotor muscles of stable patients with COPD. The degree of autophagy correlates with severity of muscle atrophy and lung function impairment.

MFN2 overexpression in skeletal muscles of young and old mice causes a mild hypertrophy without altering mitochondrial respiration and H2O2 emission.

AIM: Sarcopenia, the aging-related loss of muscle mass and function, is a debilitating process negatively impacting the quality of life of affected individuals. Although the mechanisms underlying sarcopenia are incompletely understood, impairments in mitochondrial dynamics, including mitochondrial fusion, have been proposed as a contributing factor. However, the potential of upregulating mitochondrial fusion proteins to alleviate the effects of aging on skeletal muscles remains unexplored. We therefore hypothesized that overexpressing Mitofusin 2 (MFN2) in skeletal muscle in vivo would mitigate the effects of aging on muscle mass and improve mitochondrial function. METHODS: MFN2 was overexpressed in young (7 mo) and old (24 mo) male mice for 4 months through intramuscular injections of an adeno-associated viruses. The impacts of MFN2 overexpression on muscle mass and fiber size (histology), mitochondrial respiration, and H2O2 emission (Oroboros fluororespirometry), and various signaling pathways (qPCR and western blotting) were investigated. RESULTS: MFN2 overexpression increased muscle mass and fiber size in both young and old mice. No sign of fibrosis, necrosis, or inflammation was found upon MFN2 overexpression, indicating that the hypertrophy triggered by MFN2 overexpression was not pathological. MFN2 overexpression even reduced the proportion of fibers with central nuclei in old muscles. Importantly, MFN2 overexpression had no impact on muscle mitochondrial respiration and H2O2 emission in both young and old mice. MFN2 overexpression attenuated the increase in markers of impaired autophagy in old muscles. CONCLUSION: MFN2 overexpression may be a viable approach to mitigate aging-related muscle atrophy and may have applications for other muscle disorders.

Angiopoietin-1 and vascular endothelial growth factor regulation of leukocyte adhesion to endothelial cells: role of nuclear receptor-77.

OBJECTIVE: Vascular endothelial growth factor (VEGF) promotes leukocyte adhesion to endothelial cells (ECs). Angiopoietin-1 (Ang-1) inhibits this response. Nuclear receptor-77 (Nur77) is a proangiogenic nuclear receptor. In the present study, we assessed the influence of Ang-1 and VEGF on Nur77 expression in ECs, and evaluated its role in Ang-1/VEGF-mediated leukocyte adhesion. METHODS AND RESULTS: Expression of Nur77 was evaluated with real-time polymerase chain reaction and immunoblotting. Adhesion of leukocytes to ECs was monitored with inverted microscopy. Nur77 expression or activity was inhibited using adenoviruses expressing dominant-negative form of Nur77, retroviruses expressing Nur77 in the antisense direction, and small interfering RNA oligos. Both Ang-1 and VEGF induce Nur77 expression, by >5- and 30-fold, respectively. When combined, Ang-1 potentiates VEGF-induced Nur77 expression. Ang-1 induces Nur77 through the phosphoinositide 3-kinase and extracellular signal-regulated protein kinase 1/2 pathways. VEGF induces Nur77 expression through the protein kinase D/histone deacetylase 7/myocyte enhancer factor 2 and extracellular signal-regulated protein kinase 1/2 pathways. VEGF induces nuclear factor-kappaB transcription factor, vascular cell adhesion molecule-1, and E-selectin expressions, and promotes leukocyte adhesion to ECs. Ang-1 inhibits these responses. This inhibitory effect of Ang-1 disappears when Nur77 expression is disrupted, restoring the inductive effects of VEGF on adhesion molecule expression, and increased leukocyte adhesion to ECs. CONCLUSIONS: Nur77 promotes anti-inflammatory effects of Ang-1, and functions as a negative feedback inhibitor of VEGF-induced EC activation.

Autophagy ablation in skeletal muscles worsens sepsis-induced muscle wasting, impairs whole-body metabolism, and decreases survival.

Septic patients frequently develop skeletal muscle wasting and weakness, resulting in severe clinical consequences and adverse outcomes. Sepsis triggers sustained induction of autophagy, a key cellular degradative pathway, in skeletal muscles. However, the impact of enhanced autophagy on sepsis-induced muscle dysfunction remains unclear. Using an inducible and muscle-specific Atg7 knockout mouse model (Atg7iSkM-KO), we investigated the functional importance of skeletal muscle autophagy in sepsis using the cecal ligation and puncture model. Atg7iSkM-KO mice exhibited a more severe phenotype in response to sepsis, marked by severe muscle wasting, hypoglycemia, higher ketone levels, and a decreased in survival as compared to mice with intact Atg7. Sepsis and Atg7 deletion resulted in the accumulation of mitochondrial dysfunction, although sepsis did not further worsen mitochondrial dysfunction in Atg7iSkM-KO mice. Overall, our study demonstrates that autophagy inactivation in skeletal muscles triggers significant worsening of sepsis-induced muscle and metabolic dysfunctions and negatively impacts survival.