Muscle Ring Finger Research Articles

Attenuation of ventilation-induced diaphragm dysfunction through toll-like receptor 4 and nuclear factor-κB in a murine endotoxemia model

Mechanical ventilation (MV) is often used to maintain life in patients with sepsis and sepsis-related acute lung injury. However, controlled MV may cause diaphragm weakness due to muscle injury and atrophy, an effect termed ventilator-induced diaphragm dysfunction (VIDD). Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) signaling pathways may elicit sepsis-related acute inflammatory responses and muscle protein degradation and mediate the pathogenic mechanisms of VIDD. However, the mechanisms regulating the interactions between VIDD and endotoxemia are unclear. We hypothesized that mechanical stretch with or without endotoxin treatment would augment diaphragmatic structural damage, the production of free radicals, muscle proteolysis, mitochondrial dysfunction, and autophagy of the diaphragm via the TLR4/NF-κB pathway. Male C57BL/6 mice, either wild-type or TLR4-deficient, aged between 6 and 8 weeks were exposed to MV (6 mL/kg or 10 mL/kg) with or without endotoxemia for 8 h. Nonventilated mice were used as controls. MV with endotoxemia aggravated VIDD, as demonstrated by the increases in the expression levels of TLR4, caspase-3, atrogin-1, muscle ring finger-1, and microtubule-associated protein light chain 3-II. In addition, increased NF-κB phosphorylation and oxidative loads, disorganized myofibrils, disrupted mitochondria, autophagy, and myonuclear apoptosis were also observed. Furthermore, MV with endotoxemia reduced P62 levels and diaphragm muscle fiber size (P < 0.05). Endotoxin-exacerbated VIDD was attenuated by pharmacologic inhibition with a NF-κB inhibitor or in TLR4-deficient mice (P < 0.05). Our data indicate that endotoxin-augmented MV-induced diaphragmatic injury occurs through the activation of the TLR4/NF-κB signaling pathway.Mechanical ventilation may cause diaphragm weakness due to muscle injury and atrophy. This study shows that endotoxin-augmented mechanical ventilation-induced diaphragmatic injury occurs through the activation of toll-like receptor 4 and nuclear factor-κB signaling pathways. These findings may aid in the development of treatments for patients with sepsis.

Avenanthramides attenuate inflammation and atrophy in muscle cells

BackgroundChronic inflammation is an important etiologic mechanism for muscle atrophy. Oat-derived phytochemical avenanthramides (AVAs) have been shown to suppress inflammatory responses in human clinical studies and in several cell lines in vitro, but their role in skeletal muscle is unclear. The aim of this study was to investigate whether AVA treatment can prevent tumor necrosis factor (TNF)-α-induced muscle fiber atrophy in C2C12 cells. MethodsWe treated 70% confluent cells for 24 h with AVA. Then, TNF-α was added to cell-cultured medium. Subsequently, cells were harvested at different time points. The cells were examined using various biochemical techniques for measuring protein, messenger RNA levels, nuclear binding activity, and viability. Fluorescence microscope was used for analysis of the myotube morphology. ResultsCells treated with TNF-α significantly increased nuclear factor κB activation, indicated by a marked decrease of IκB (p < 0.05) and a 6.6-fold increase in p65-DNA binding (p < 0.01); however, 30 μmol of AVA-A, -B, and -C treatment reduced the binding by 33%, 18%, and 19% (p < 0.01), respectively, compared with cells treated with TNF-α without AVA. The interleukin-6 level increased by 2.5 fold (p < 0.01) with TNF-α, but decreased by 24%, 32%, and 28% (p < 0.01), respectively, with AVA-A, -B, and -C. The interleukin-1β level also showed a 47% increase with TNF-α (p < 0.01), whereas this increment was abolished in all AVA-treated cells. Reactive oxygen species production was 1.3-fold higher in the TNF-α-treated group (p < 0.01) but not in the TNF-α + AVAs groups. Messenger RNA levels of muscle-specific E3 ubiquitin ligase atrogin-1 increased 23% in TNF-α vs. control (p < 0.05) but was decreased by 46%, 34%, and 53% (p < 0.01), respectively, with treatment of AVA-A, -B, and -C. Moreover, TNF-α treatment increased the muscle RING finger 1 messenger RNA level by 76% (p < 0.01); this change was abolished by AVAs. Cells treated with TNF-α demonstrated a reduced proliferation compared with control cells (p < 0.01), but this effect was not seen in TNF-α + AVAs cells. The diameter of the C2C12 myotube decreased by 28% (p < 0.01) with TNF-α, whereas it showed no change when AVAs were included in the cell media. ConclusionThese results indicated that AVAs can reduce proinflammatory cytokine and reactive oxygen species production and ameliorate TNF-α-induced myotube atrophy in muscle cells.

Impact of Very Early Physical Therapy During Septic Shock on Skeletal Muscle: A Randomized Controlled Trial.

As the catabolic state induced by septic shock together with the physical inactivity of patients lead to the rapid loss of muscle mass and impaired function, the purpose of this study was to test whether an early physical therapy during the onset of septic shock regulates catabolic signals and preserves skeletal muscle mass. Randomized controlled trial. Tertiary mixed ICU. Adult patients admitted for septic shock within the first 72 hours. Patients were assigned randomly into two groups. The control group benefited from manual mobilization once a day. The intervention group had twice daily sessions of both manual mobilization and 30-minute passive/active cycling therapy. Skeletal muscle biopsies and electrophysiology testing were performed at day 1 and day 7. Muscle biopsies were analyzed for histology and molecular components of signaling pathways regulating protein synthesis and degradation as well as inflammation markers. Hemodynamic values and patient perception were collected during each session. Twenty-one patients were included. Three died before the second muscle biopsy. Ten patients in the control and eight in the intervention group were analyzed. Markers of the catabolic ubiquitin-proteasome pathway, muscle atrophy F-box and muscle ring finger-1 messenger RNA, were reduced at day 7 only in the intervention group, but without difference between groups (muscle atrophy F-box: -7.3% ± 138.4% in control vs -56.4% ± 37.4% in intervention group; p = 0.23 and muscle ring finger-1: -30.8% ± 66.9% in control vs -62.7% ± 45.5% in intervention group; p = 0.15). Muscle fiber cross-sectional area (µm) was preserved by exercise (-25.8% ± 21.6% in control vs 12.4% ± 22.5% in intervention group; p = 0.005). Molecular regulations suggest that the excessive activation of autophagy due to septic shock was lower in the intervention group, without being suppressed. Markers of anabolism and inflammation were not modified by the intervention, which was well tolerated by the patients. Early physical therapy during the first week of septic shock is safe and preserves muscle fiber cross-sectional area.

Experimental Spinal Cord Injury Causes Left-Ventricular Atrophy and Is Associated with an Upregulation of Proteolytic Pathways.

Spinal cord injury (SCI) causes autonomic dysfunction, altered neurohumoral control, profound hemodynamic changes, and an increased risk of heart disease. In this prospective study, we investigated the cardiac consequences of chronic experimental SCI in rats by combining cutting edge in vivo techniques (magnetic resonance imaging [MRI] and left-ventricular [LV] pressure-volume catheterization) with histological and molecular assessments. Twelve weeks post-SCI, MRI-derived structural indices and in vivo LV catheterization-derived functional indices indicated the presence of LV atrophy (LV mass in Control vs. SCI = 525 ± 38.8 vs. 413 ± 28.6 mg, respectively; p = 0.0009), reduced ventricular volumes (left-ventricular end-diastolic volume in Control vs. SCI = 364 ± 44 vs. 221 ± 35 μL, respectively; p = 0.0004), and contractile dysfunction (end-systolic pressure-volume relationship in Control vs. SCI = 1.31 ± 0.31 vs. 0.76 ± 0.11 mm Hg/μL, respectively; p = 0.0045). Cardiac atrophy and contractile dysfunction in SCI were accompanied by significantly lower blood pressure, reduced circulatory norepinephrine, and increased angiotensin II. At the cellular level, we found the presence of reduced cardiomyocyte size and increased expression of angiotensin II type 1 receptors and transforming growth factor-beta receptors (TGF-β receptor 1 and 2) post-SCI. Importantly, we found more than a two-fold increase in muscle ring finger-1 and Beclin-1 protein level following SCI, indicating the upregulation of the ubiquitin-proteasome system and autophagy-lysosomal machinery. Our data provide novel evidence that SCI-induced cardiomyocyte atrophy and systolic cardiac dysfunction are accompanied by an upregulation of proteolytic pathways, the activation of which is likely due to loss of trophic support from the sympathetic nervous system, neuromechanical unloading, and altered neurohumoral pathways.

Electrical Stimulation Prevents Preferential Skeletal Muscle Myosin Loss in Steroid-Denervation Rats.

Severe muscle weakness concomitant with preferential depletion of myosin has been observed in several pathological conditions. Here, we used the steroid-denervation (S-D) rat model, which shows dramatic decrease in myosin content and force production, to test whether electrical stimulation (ES) treatment can prevent these deleterious changes. S-D was induced by cutting the sciatic nerve and subsequent daily injection of dexamethasone for 7 days. For ES treatment, plantarflexor muscles were electrically stimulated to produce four sets of five isometric contractions each day. Plantarflexor in situ isometric torque, muscle weight, skinned muscle fiber force, and protein and mRNA expression were measured after the intervention period. ES treatment partly prevented the S-D-induced decreases in plantarflexor in situ isometric torque and muscle weight. ES treatment fully prevented S-D-induced decreases in skinned fiber force and ratio of myosin heavy chain (MyHC) to actin, as well as increases in the reactive oxygen/nitrogen species-generating enzymes NADPH oxidase (NOX) 2 and 4, phosphorylation of p38 MAPK, mRNA expression of the muscle-specific ubiquitin ligases muscle ring finger-1 (MuRF-1) and atrogin-1, and autolyzed active calpain-1. Thus, ES treatment is an effective way to prevent muscle impairments associated with loss of myosin.

Formoterol treatment prevents the effects of endotoxin on muscle TNF/NF-kB, Akt/mTOR, and proteolytic pathways in a rat model. Role of IGF-I and miRNA 29b

Inflammatory diseases are associated with muscle wasting as a result of an increase in proteolysis. The purpose of this study was to elucidate whether administration of a β2 adrenergic agonist, formoterol, was able to prevent the acute effects of sepsis induced by liposaccharide (LPS) injection on rat gastrocnemius muscle and to evaluate the possible roles of corticosterone, IGF-I, miR-23a, and miR-29b. For this purpose, male Wistar rats were injected with LPS and/or formoterol. Formoterol treatment decreased LPS-induced increase in serum corticosterone, TNFα upregulation, and NF-κB(p65) and Forkhead box protein O1 activation in the gastrocnemius. Atrogin-1, muscle RING-finger protein-1, microtubule-associated protein-1 light chain 3b (LC3b), and the lipidation of LC3b-I to LC3b-II were increased by LPS, and formoterol blocked these effects. Serum IGF-I and its mRNA levels in the gastrocnemius were decreased, whereas mecano growth factor and IGF binding protein 3 mRNA levels were increased in the rats injected with LPS but not in the rats that received LPS and formoterol. Similarly, LPS decreased Akt and mammalian target of rapamycin phosphorylation, and formoterol blocked these decreases. Finally, miR-29b expression in the gastrocnemius was upregulated by endotoxin injection, whereas miR-23a was not significantly different. Formoterol treatment did not significantly modify LPS-induced increase in muscle miR-29b. Furthermore, in control rats formoterol increased the expression of this miRNA. We conclude that formoterol decreases endotoxin-induced inflammation and proteolysis in rat skeletal muscle. Those responses can be a direct effect of β2 adrenergic receptor stimulation or/and of blocking the effects of LPS on corticosterone and IGF-I. Muscle miR-23a and -29b do not seem to play an important role in those responses.

High-intensity eccentric training ameliorates muscle wasting in colon 26 tumor-bearing mice.

Eccentric (ECC) contractions are used to maintain skeletal muscle mass and strength in healthy subjects and patients. Here we investigated the effects of ECC training induced by electrical stimulation (ES) on muscle wasting in colon 26 (C-26) tumor-bearing mice. Mice were divided into four groups: control (CNT), CNT + ECC, C-26, and C-26 + ECC. Cancer cachexia was induced by a subcutaneous injection of C-26 cells and developed for four weeks. In experiment 1, muscle protein synthesis rate and mammalian target of rapamycin complex (mTORC) 1 signaling were investigated six hours after one bout of ECC-ES (2 s contraction given every 6 s, 20°/s, 4 sets of 5 contractions). In experiment 2, ECC-ES training, a total of 14 sessions, was performed every other day starting one day after C-26 injection. Compared to the CNT mice, the gastrocnemius muscle weight was significantly decreased in the tumor-bearing mice. This change was accompanied by a reduction in protein synthesis rate and a marked increase in the expression levels of genes including regulated in development and DNA damage responses (REDD) 1, forkhead box protein O1 (FoxO1), muscle-specific E3 ubiquitin ligases atrogin-1, and muscle ring finger 1 (MuRF-1) mRNA. ECC-ES increased the protein synthesis rate and the phosphorylation levels of p70S6K (Thr389) and rpS6 (Ser240/244), markers for mTORC1 signaling, and reversed an upregulation of MuRF-1 mRNA in muscles from C-26 mice. Our findings suggest that ECC-ES training reduces skeletal muscle atrophy in C-26 tumor-bearing mice through activation of mTORC1 signaling and the inhibition of ubiquitin-proteasome pathway. Thus, ECC-ES training might be used to effectively ameliorate muscle wasting in patients with cancer cachexia.

Muscle RING-finger protein-1 (MuRF1) functions and cellular localization are regulated by SUMO1 post-translational modification.

The muscle RING-finger protein-1 (MuRF1) is an E3 ubiquitin ligase expressed in skeletal and cardiac muscle tissues and it plays important roles in muscle remodeling. Upregulation of MuRF1 gene transcription participates in skeletal muscle atrophy, on contrary downregulation of protein expression leads to cardiac hypertrophy. MuRF1 gene point mutations have been found to generate protein aggregate myopathies defined as muscle disorder characterized by protein accumulation in muscle fibers. We have discovered that MuRF1 turned out to be also a target for a new post-translational modification arbitrated by conjugation of SUMO1 and it is mediated by the SUMO ligases E2 UBC9 and the E3 PIASγ/4. SUMOylation takes place at lysine 238 localized at the second coiled-coil protein domain that is required for efficient substrate interaction for polyubiquitination. We provided evidence that SUMOylation is essential for MuRF1 nuclear translocation and its mitochondria accumulation is enhanced in hyperglycemic conditions delivering a stabilization of the overall SUMOylated proteins in cultured myocytes. Thus, our findings add this SUMO1 post-translational modification as a new concept to understand muscle disorders related to the defect in MuRF1 activity.

Pyrroloquinoline quinone attenuates cachexia-induced muscle atrophy via suppression of reactive oxygen species.

Cachexia, a wasting syndrome, is most commonly observed in individuals with advanced cancer including lung cancer, esophageal cancer, liver cancer, etc. The characteristic sign of cachexia is muscle atrophy. To date, effective countermeasures have been still deficiency to alleviate muscle atrophy. Reactive oxygen species (ROS) are important regulators of muscle atrophy. Therefore, the effects of a naturally antioxidant, pyrroloquinoline quinone (PQQ), were explored on muscle atrophy induced by cachexia in the present study. Tumor necrosis factor-α (TNF-α) induced C2C12 myotubes atrophy model was constructed. The atrophied C2C12 myotubes were dealt with the presence or absence of N-acetyl-L-cysteine (NAC, an antioxidant for ROS abolition) (5 mM) or PQQ (80 µM) for 24 hours. ROS content was determined by dichlorodihydrofluorescein diacetate (DCFH-DA) staining. The diameter of myotubes was analyzed by myosin heavy chain (MHC) staining. The protein levels of MHC, muscle atrophy F-box (MAFbx) and muscle RING finger-1 (MuRF-1) in each group were observed by Western blotting. First, ROS generation was enhanced in C2C12 myotubes treated with TNF-α. NAC treatments significantly avoided the reduction in the diameter of C2C12 myotubes, and concomitantly increased MHC levels, and decreased ROS contents, MuRF1 and MAFbx levels. These data suggested that the increased ROS induced by TNF-α might play a central role in muscle wasting. PQQ (a naturally occurring antioxidant) administration inhibited C2C12 myotubes atrophy induced by TNF-α, as evidenced by the increase of the diameter of C2C12 myotubes, together with increased MHC levels and decreased ROS, MAFbx and MuRF-1 levels. PQQ resists atrophic effect dependent on, at least in part, decreased ROS in skeletal muscle treated with TNF-α.

The phosphodiesterase-4 inhibitor roflumilast reverts proteolysis in skeletal muscle cells of patients with COPD cachexia.

Peripheral muscle weakness and mass loss are characteristic features in severe chronic obstructive pulmonary disease (COPD). We hypothesized that the phosphodiesterase (PDE)-4 inhibitor roflumilast-induced cAMP may ameliorate proteolysis and metabolism in skeletal muscles of COPD patients with severe muscle wasting. In myogenic precursor cells (isolated from muscle biopsies and cultured up to obtain differentiated myotubes) from 10 severe COPD patients and 10 healthy controls, which were treated with 1 μM roflumilast N-oxide (RNO) for three time cohorts (1, 6, and 24 h), genes of antioxidant defense and oxidative stress marker, myogenesis and muscle metabolism, proteolysis (tyrosine release assay) and ubiquitin-proteasome system markers, autophagy, and myosin isoforms were analyzed using RT-PCR and immunoblotting. In COPD patients at 6 h RNO treatment, myotube tyrosine release, total protein ubiquitination, and tripartite motif-containing protein 32 levels were significantly lower than healthy controls, whereas at 24 h RNO treatment, myotube myosin heavy chain ( MyHC) -I and MyHC-IIx expression levels were upregulated in both patients and controls. In the 6-h RNO cohort, in patients and controls, myotube expression of nuclear factor (erythroid-derived 2)-like 2 ( NRF2) and its downstream antioxidants sirtuin-1, FGF-inducible 14, and insulin-like growth factor-1 was upregulated, whereas that of myocyte-specific enhancer factor 2C, myogenic differentiation, myogenin, myostatin, atrogin-1, and muscle RING-finger protein-1 was downregulated. In myotubes of severe COPD patients with cachexia, roflumilast-induced cAMP signaling exerts beneficial effects by targeting muscle protein breakdown (tyrosine release), along with reduced expression of proteolytic markers of the ubiquitin-proteasome system and that of myostatin. In both patients and controls, roflumilast also favored antioxidant defense through upregulation of the NRF2 pathway and that of the histone deacetylase sirtuin-1, whereas it improved the expression of slow- and fast-twitch myosin isoforms. These findings show that muscle dysfunction and wasting may be targeted by roflumilast-induced cAMP signaling in COPD. These results have potential therapeutic implications, as this PDE-4 inhibitor is currently available for the treatment of systemic inflammation and exacerbations in patients with severe COPD. NEW & NOTEWORTHY In myotubes of cachectic chronic obstructive pulmonary disease (COPD) patients, cAMP signaling exerted beneficial effects by targeting muscle proteolysis and reducing gene expression of proteolytic markers of the ubiquitin-proteasome system and that of myostatin. In myotubes of patients and controls, roflumilast also favored antioxidant defense through upregulation of the nuclear factor (erythroid-derived 2)-like 2 pathway, of sirtuin-1, and of gene expression of slow- and fast-twitch isoforms. These findings have potential clinical implications for the treatment of muscle wasting in patients with COPD and cachexia.

Possible Role of NADPH Oxidase 4 in Angiotensin II-Induced Muscle Wasting in Mice.

Background: Muscle wasting is a debilitating phenotype associated with chronic heart failure (CHF). We have previously demonstrated that angiotensin II (AII) directly induces muscle wasting in mice through the activation of NADPH oxidase (Nox). In this study, we tested the hypothesis that deficiency of NADPH oxidase 4 (Nox4), a major source of oxidative stress, ameliorates AII-induced muscle wasting through the regulation of redox balance.Methods and Results: Nox4 knockout (KO) and wild-type (WT) mice were used. At baseline, there were no differences in physical characteristics between the WT and KO mice. Saline (vehicle, V) or AII was infused via osmotic minipumps for 4 weeks, after which, the WT + AII mice showed significant increases in Nox activity and NOX4 protein compared with the WT + V mice, as well as decreases in body weight, gastrocnemius muscle weight, and myocyte cross-sectional area. These changes were significantly attenuated in the KO + AII mice (27 ± 1 vs. 31 ± 1 g, 385 ± 3 vs. 438 ± 13 mg, and 1,330 ± 30 vs. 2281 ± 150 μm2, respectively, all P < 0.05). The expression levels of phospho-Akt decreased, whereas those of muscle RING Finger-1 (MuRF-1) and MAFbx/atrogin-1 significantly increased in the WT + AII mice compared with the WT + V mice. Furthermore, nuclear factor erythroid-derived 2-like 2 (Nrf2) and the expression levels of Nrf2-regulated genes significantly decreased in the WT + AII mice compared with the WT + V mice. These changes were significantly attenuated in the KO + AII mice (P < 0.05).Conclusion: Nox4 deficiency attenuated AII-induced muscle wasting, partially through the regulation of Nrf2. The Nox4–Nrf2 axis may play an important role in the development of AII-induced muscle wasting.

Muscle‐specific regulation of right ventricular transcriptional responses to chronic hypoxia induced heart failure by the Muscle Ring Finger‐1 (MuRF1) ubiquitin ligase in vivo

BackgroundWe recently identified a role for the muscle‐specific ubiquitin ligase MuRF1 (Muscle Ring Finger‐1) in right‐sided heart failure secondary to pulmonary hypertension induced by chronic hypoxia (CH). MuRF1−/− challenged to chronic hypoxia for three weeks were resistant to CH induced changes in right ventricle (RV) weight, ejection fraction, and volume (systolic and diastolic) and provided significantly increased perfusion to skeletal muscle. In contrast, MuRF1 Tg+ mice exhibited significant decreases in RV weight and ejection fraction indicative of a maladaptive dilated phenotype (heart failure), consistent with the significant decreases in skeletal muscle perfusion observed. The present study was undertaken to understand the underlying transcriptional alterations in the right ventricle of MuRF1−/− and MuRF1 Tg+ mice using microarray analysis.ResultsAfter three weeks of chronic hypoxia (15% O2) exposure, right ventricular total RNA was isolated, labeled, and hybridized to microarray chips. Microarray analysis of the MuRF1−/− right ventricle identified 37,095 defined genes with signals, with 590 found to be significantly different (p&lt;0.01) from WT controls. Analysis of the 70 top differentially expressed genes (&gt;2 fold or &lt;−2 fold) revealed their function in 1) transcriptional regulation; and 2) oxidoreductase function. Analysis of these differentially expressed genes for common transcription factor promoter regions identified homeobox (HOX) D12 and HOXC13 transcription factors, along with RREB‐1. Microarray analysis of the MuRF1 Tg+ right ventricle identified 37,095 defined genes with signals, with 150 found to be significantly different (p&lt;0.01) from WT controls. Analysis of the 46 top differentially expressed genes (&gt;3 fold or &lt;−3 fold) revealed their function in 1) oxidoreductase‐metabolic regulation, 2) glycoprotein‐transmembrane integral protein structure, and 3) alternative splicing / splice variants.ConclusionsThe differentially expressed genes in MuRF1−/− and MuRF1 Tg+ RV after CH exposure have common functional annotations related to oxidoreductase (including antioxidant) and transmembrane component functions. Moreover, the functionally‐enhanced MuRF1−/− hearts regulate genes related to transcription, homeobox proteins, and kinases/phosphorylation. These studies also reveal potential indirect effects MuRF1 may have with known substrates such as SFR and PPARalpha by regulating RREB‐1 and reveal for the first time mechanisms by which MuRF1 may transcriptionally regulate anti‐oxidant systems in the face of right heart failure.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The muscle‐specific ubiquitin ligase MuRF1 regulates autophagy via FOXO1/3 ubiquitination to inhibit NF‐κB signaling and protect against cardiac inflammation in vivo

The muscle‐specific ubiquitin ligase MuRF1 (muscle ring finger‐1) has recently been shown to regulate signal transduction by modulating transcription factor activity through ubiquitin‐mediated post‐translational modification. Therefore, we investigated the ability of MuRF1 to regulate autophagy. MuRF1−/− hearts exhibited decreased autophagic flux (decrease in LC3II and VPS34 protein levels), while MuRF1Tg+ hearts exhibited increased autophagic flux (increase in last phase autophagosomes and VPS34 protein levels). Interestingly, the FOXO1/3a transcription factors, known supporters of autophagy, correlated with MuRF1 expression. MuRF1−/− hearts expressed significantly less of the FOXO‐regulated P62, while MuRF1Tg+ expressed significantly more P62 by immunoblot analysis. Since P62 supports NF‐κB signaling by IKKβ phosphorylation, MuRF1−/− and MuRF1Tg+ mice were challenged with LPS to determine resistance to endotoxin‐induced cardiac dysfunction to identify the role of MuRF1‐P62 in inhibiting cardiac NF‐κB signaling. MuRF1−/− mice were significantly protected against LPS‐induced systolic dysfunction, while MuRF1 Tg+ mice were more susceptible to LPS‐induced heart failure. NF‐κB activity was significantly inhibited in MuRF1−/− mice at the level of the IKK complex, consistent with decreased FOXO‐regulated P62 to reduce downstream NF‐κB signaling. These studies identify the first ubiquitin ligase to regulate autophagy in the heart and describe novel therapeutic targets that could be used to block myocyte‐specific inflammationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of the Regulation and Function of Tetratricopeptide 39c (Ttc39c) in Skeletal Muscle

Ttc39c has been identified as a novel gene in skeletal muscle that is differentially regulated in response to neurogenic atrophy in wild‐type (WT) mice compared to Muscle RING Finger 1 (MuRF1) knockout (KO) mice. MuRF1 is a ubiquitin E3 ligase that is known to be an important mediator of muscle wasting; however, more recent research suggests that MuRF1 may also function as a regulator of atrophy‐induced gene expression. Quantitative PCR and Western blot analysis determined that Ttc39c is expressed in both proliferating and differentiated muscle cells. Furthermore, comparison of Ttc39c expression in undifferentiated and differentiated C2C12 cells demonstrated that Ttc39c levels are elevated in proliferating myoblasts, but decrease in differentiated myotubes. The transcriptional regulation of two Ttc39c isoforms with alternative transcriptional start sites were examined by cloning the unique promoter regions of each transcript. The Ttc39c reporter gene constructs were transfected into muscle cells and confirmed to have significant transcriptional activity in cultured muscle cells and are transcriptionally responsive to ectopic expression of myogenic regulatory factors (MRF). Furthermore, conserved E‐box elements in the proximal promoter regions of both Ttc39c transcripts were identified, mutated, and analyzed for their role in the transcriptional regulation of Ttc39c expression. The mutation of the conserved E‐box sequences rendered the Ttc39c reporter genes inactive, suggesting that these elements are potentially necessary for Ttc39c expression. Finally, immunoprecipitation (IP) of Ttc39c from C2C12 cells resulted in the co‐IP of numerous binding partners, which are currently being identified by proteomic analysis in the hope of elucidating a potential function for Ttc39c in skeletal muscle based on identified binding partners. Interestingly, ectopic expression of Ttc39c in C2C12 mouse myoblasts resulted in delayed cell fusion and reduction in myotube formation. The identification and characterization of novel genes that are activated during neurogenic atrophy helps improve our understanding of the molecular genetic events that result in muscle wasting and could eventually lead to new therapeutic targets in the future.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants to D.W.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of chronic dexamethasone administration on hyperglycemia and insulin release in goats

BackgroundDexamethasone (Dex), a synthetic glucocorticoid, is among the most commonly used drugs worldwide in animals and humans as an anti-inflammatory and immunosuppressive agent. GC has profound effects on plasma glucose level and other metabolic conditions. However, the effect of prolonged use of Dex on glucose metabolism in ruminants is still unclear.ResultsTen goats were randomly assigned to two groups: the control goats were injected with saline, and the Dex-treated goats were intramuscularly injected daily for 21 d with 0.2 mg/kg Dex. The results showed that plasma glucose and insulin concentrations were significantly increased after Dex administration (P < 0.05). Additionally, the content of hepatic glycogen was also markedly increased in Dex-treated goats (P < 0.01), while the content of glycogen in dorsal longissimus was unchanged by Dex (P > 0.05). The expression of several key genes, involved in blood glucose regulation, was detected by real-time PCR in the small intestine, skeletal muscle and liver. The expression of glucose transporter type 2 (GLUT2), sodium-glucose transporter 1 (SGLT1) and sodium-potassium ATPase (Na-K/ATPase) in the small intestine were generally increased by Dex, and GLUT2 mRNA expression was significantly up-regulated (P < 0.05). In liver, the expression of genes involved in gluconeogenesis including glucose-6-phosphatase catalytic subunit (G6PC), cytosolic form of phosphoenolpyruvate carboxykinase (PCK1) and pyruvate carboxylase (PC), were significantly down-regulated by Dex. However, the protein expression levels of PCK1 & PCK2 were significantly increased by Dex, suggesting a post-transcriptional regulation. In dorsal longissimus, the mRNA expression of genes associated with gluconeogenesis and the insulin signaling pathway were generally up-regulated by Dex, but the mRNA expression of two markers of muscle atrophy, namely F-box protein 32 (FBXO32/Atrogin1) and muscle RING-finger protein 1 (MuRF1), was not altered by Dex.ConclusionsTaken together, these results indicate that chronic administration of a low dosage of Dex induces hyperglycemia mainly through gluconeogenesis activation in the goat liver.

Improvement of flesh quality in rainbow trout (Oncorhynchus mykiss ) fed supranutritional dietary selenium yeast is associated with the inhibited muscle protein degradation

Supranutritional dietary selenium (Se) has been demonstrated beneficial for fish health, however, its effects on fish flesh quality remain unclear. This study investigated the effects of supranutritional dietary Se on the flesh quality of rainbow trout (Oncorhynchus mykiss) and their potential mechanism. Fish were fed a basal diet supplemented with or without graded Se yeast for 10 weeks. Results showed that Se supplementation significantly increased fillet crude protein and enhanced fillet water-holding capacity as well as fillet firmness. Supplementing with both 2 and 4 mg/kg Se significantly downregulated the expressions of two autophagy–lysosome-related genes (autophagy-related 12-like and gamma-aminobutyric acid type A receptor-associated protein-like 1) in fish muscle, while supplementing with 4 mg/kg Se also significantly downregulated the expression of two ubiquitin–proteasome-related genes (muscle RING finger 2 and F-box protein 25). Correlation analysis indicated that the improved fillet quality parameters were closely correlated with the expressions of these differentially expressed genes. This study revealed that dietary Se was effective for the improvement of rainbow trout flesh quality, and the improved fish flesh quality was associated with the inhibited protein degradation in fish muscle.

Resistance training prevents muscle fibrosis and atrophy via down-regulation of C1q-induced Wnt signaling in senescent mice.

Increased complement component 1q (C1q) secretion with aging leads to muscle fibrosis and atrophy whereas resistance training attenuates circulating C1q levels. This study aimed to clarify whether resistance exercise-induced reduction of C1q secretion contributes to the inhibition of fibrosis and atrophy in aged muscles. Young (13-wk-old) and aged (38-wk-old) senescence-accelerated mouse prone 1 mice were randomly assigned to one of 4 groups: a young or aged sedentary control group, or a young or aged resistance training (climbing a ladder 3 d/wk for 12 wk) group. We found that resistance training ameliorated muscle fibrosis and atrophy in aged mice, concomitant with decreased circulating and muscle C1q levels and attenuated activation of muscle Wnt signaling (glycogen synthase kinase β/β-catenin), including β-catenin in satellite (Pax7+/DAPI+) and fibroblast (vimentin+/DAPI+) cells. Furthermore, during muscle regeneration after mice were injured by cardiotoxin injection, we observed a reduction in circulating C1q levels, the inhibition of muscle fibrosis and repair, and decreased in the activation of muscle cytoplasmic and nuclear β-catenin in aged mice from the resistance training group, but these effects were cancelled by a single preadministration of exogenous recombinant C1q. In addition, resistance training attenuated aging-related muscle loss concomitant with decreased expression of both muscle ring-finger protein 1 and muscle atrophy F-box in the muscle. Thus, resistance training-induced changes in circulating C1q levels may contribute to the prevention of muscle fibrosis and atrophy via muscle Wnt signaling in senescent mice.-Horii, N., Uchida, M., Hasegawa, N., Fujie, S., Oyanagi, E., Yano, H., Hashimoto, T., Iemitsu, M. Resistance training prevents muscle fibrosis and atrophy via down-regulation of C1q-induced Wnt signaling in senescent mice.

Inhibition of interleukin-6 decreases atrogene expression and ameliorates tail suspension-induced skeletal muscle atrophy.

BackgroundInterleukin-6 (IL-6) is an inflammatory cytokine. Whether systemic IL-6 affects atrogene expression and disuse-induced skeletal muscle atrophy is unclear.MethodsTail-suspended mice were used as a disuse-induced muscle atrophy model. We administered anti-mouse IL-6 receptor antibody, beta-hydroxy-beta-methylbutyrate (HMB) and vitamin D to the mice and examined the effects on atrogene expression and muscle atrophy.ResultsSerum IL-6 levels were elevated in the mice. Inhibition of IL-6 receptor suppressed muscle RING finger 1 (MuRF1) expression and prevented muscle atrophy. HMB and vitamin D inhibited the serum IL-6 surge, downregulated the expression of MuRF1 and atrogin-1 in the soleus muscle, and ameliorated atrophy in the mice.ConclusionSystemic IL-6 affects MuRF1 expression and disuse-induced muscle atrophy.

The E3 ubiquitin ligase MuRF2 attenuates LPS-induced macrophage activation by inhibiting production of inflammatory cytokines and migration.

Muscle RING‐finger (MuRF) proteins are E3 ubiquitin ligases that are expressed in striated muscle. MuRF2 is an important member of this family, but whether it is expressed in tissues other than striated muscle has not been thoroughly elucidated to date. In this study, we determined that MuRF2 is also expressed in other vital organs, including liver, lung, brain, spleen and kidney. Moreover, we show that the level of MuRF2 expression is significantly decreased in hepatic mononuclear cells of mice with lipopolysaccharide (LPS)/d‐galactosamine‐induced hepatitis and negatively correlated with the serum levels of alanine aminotransferase and aspartate aminotransferase in these mice. Furthermore, the expression of MuRF2 was down‐regulated in RAW264.7 cells activated with LPS but not in cells treated with polyinosinic‐polycytidylic acid (Poly(I:C)) or with lipidosome plus Poly(I:C). We also found that MuRF2 was able to translocate from the cytoplasm to the nucleus in RAW264.7 cells activated with LPS but not in cells treated with Poly(I:C). In addition, we demonstrated that interleukin 6 and tumour necrosis factor α production and macrophage migration were inhibited after MuRF2 was overexpressed in RAW264.7 cells. We further verified that nuclear factor‐κB p65 subunit level was greatly reduced in RAW264.7 macrophage nuclei by gain of function. Taken together, these findings indicate that MuRF2 may rescue LPS‐induced macrophage activation by suppressing the production of proinflammatory cytokines and cell migration. We also identify a novel function of MuRF2 in non‐muscle tissues and cells.

Effects of a Single Bout of Aerobic Exercise on Skeletal Muscle Protein Turnover in Mice

Background and Objective Aerobic exercise has a strong effect on skeletal muscle metabolism, in both males and females of all ages. However, the effect of a single bout of aerobic exercise on the regulation of protein balance remains unknown. In the present study, we investigated the effect of a single bout of treadmill-based exercise on the levels of various protein synthesis-related eukaryotic proteins (initiation factor 2a, eukaryotic initiation factor 4E, eukaryotic initiation factor 4E-binding protein 1, and eukaryotic elongation factor 2), breakdown-related proteins (microtubule-associated protein 1 light chain 3 alpha (LC3), autophagy-related 7, and muscle RING-finger protein 1 (MuRF1)), and polyubiquitination in 3-month-old male ICR mice. Material and Methods Twenty-four male mice were randomized into four time-point groups; each group of mice was run on a rodent treadmill for 10 min at 10 m/min at a slope of 5° between 7:00 p.m. and 8:00 p.m. for 2 days during the adaptation period. On the third day, exercise was performed for 50 min at a speed of 12.3 m/min; the control mice did not perform any exercise. Gastrocnemius muscles were collected immediately after the mice were sacrificed by cervical dislocation at 0, 3, 6, or 12 hours post-exercise. Results Levels of synthesis-related proteins were significantly reduced at 3 and 6 hours into the recovery period, whereas levels of breakdown-related proteins, including that of the autophagy marker LC3, increased immediately after exercise but not during the recovery period. MuRF1 level was determined in the gastrocnemius muscle to identify the factors involved in this increase. We found that increased MuRF1 levels were associated with an increase in polyubiquitination during the recovery period. Conclusion Our results suggest a potential role of optimal time points in muscle protein metabolism during recovery from a single bout of treadmill exercise.