ResearchPad - muscle-biochemistry https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Ontogenetic changes in energetic reserves, digestive enzymes, amino acid and energy content of <i>Lithodes santolla</i> (Anomura: Lithodidae): Baseline for culture]]> https://www.researchpad.co/article/elastic_article_14500 The southern king crab (SKC) Lithodes santolla is an important commercial species in southern South America. Fishing pressure has caused the deterioration of its stocks. Currently, culture techniques are being developed for producing SKC juveniles to enhance the natural population and to recover the fishing stock. Therefore, it is necessary to know about physiology, energetic and nutritional requirements for SKC maintenance in hatchery. Thus, this study aims to evaluate the biochemical and physiological changes in the midgut gland, muscle and hemolymph of juveniles, pre-adults and adults of wild SKC. The energetic reserves, digestive enzymes activity, amino acid profile and energy were quantified in twelve juveniles, ten pre-adult, and ten adult crabs. Juveniles showed high glycogen and low lipids in the midgut gland, and low proteins and low lactate in muscle. In the hemolymph, juveniles had high lipids. Pre-adults had high glycogen and lipids in the midgut gland, and both high protein and lactate in muscle. In the hemolymph, pre-adults had high lipids. Adults had low glycogen and high lipids in midgut gland, and both high proteins and high lactate in muscle. In hemolymph, adults had high glucose and lactate. Juveniles and pre-adults had high proteinase activity, whereas adults had high lipase activity. Major essential amino acids of SKC were arginine, methionine, and tryptophan, and the non-essential amino acids were glycine, aspartic acid and glutamic acid. On another hand, SKC had similar energy in the midgut gland and muscle, regardless of the ontogenetic stage. Moreover, we demonstrated that the biochemical energy calculation underestimates the actual measured values by a calorimeter. Thus, our results help to understand the physiological changes, energetic and nutritional requirements of L. santolla, and this study is a baseline for research on diet formulation for maintaining this species under culture conditions.

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<![CDATA[Computational model for the patella onset]]> https://www.researchpad.co/article/5c1966c6d5eed0c484b52d75

The patella is a sesamoid bone embedded within the quadriceps tendon and the patellar tendon that articulates with the femur. However, how is it formed is still unknown. Therefore, here we have evaluated, computationally, how three theories explain, independently, the patella onset. The first theory was proposed recently, in 2015. This theory suggested that the patella is initially formed as a bone eminence, attached to the anterodistal surface of the femur, while the quadriceps tendon is forming. Thereafter, a joint develops between the eminence and the femur, regulated by mechanical load. We evaluated this theory by simulating the biochemical environment that surrounds the tendon development. As a result, we obtained a patella-like structure embedded within the tendon, especially for larger flexion angles. The second and third theories are the most accepted until now. They state that the patella develops within tendons in response to the mechanical environment provided by the attaching muscles. The second theory analyzed the mechanical conditions (high hydrostatic stress) that (according to previous Carter theories) lead to the differentiation from tendon to fibrocartilage, and then, to bone. The last theory was evaluated using the self-optimizing capability of biological tissue. It was considered that the development of the patella, due to tissue topological optimization of the developing quadriceps tendon, is a feasible explanation of the patella appearance. For both theories, a patella onset was obtained as a structure embedded within the tendon. This model provided information about the relationship between the flexion angle and the patella size and shape. In conclusion, the computational models used to evaluate and analyze the selected theories allow determining that the patella onset may be the result of a combination of biochemical and mechanical factors that surround the patellar tendon development.

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<![CDATA[Polarization-resolved microscopy reveals a muscle myosin motor-independent mechanism of molecular actin ordering during sarcomere maturation]]> https://www.researchpad.co/article/5af0b562463d7e1d4f6521e7

Sarcomeres are stereotyped force-producing mini-machines of striated muscles. Each sarcomere contains a pseudocrystalline order of bipolar actin and myosin filaments, which are linked by titin filaments. During muscle development, these three filament types need to assemble into long periodic chains of sarcomeres called myofibrils. Initially, myofibrils contain immature sarcomeres, which gradually mature into their pseudocrystalline order. Despite the general importance, our understanding of myofibril assembly and sarcomere maturation in vivo is limited, in large part because determining the molecular order of protein components during muscle development remains challenging. Here, we applied polarization-resolved microscopy to determine the molecular order of actin during myofibrillogenesis in vivo. This method revealed that, concomitantly with mechanical tension buildup in the myotube, molecular actin order increases, preceding the formation of immature sarcomeres. Mechanistically, both muscle and nonmuscle myosin contribute to this actin order gain during early stages of myofibril assembly. Actin order continues to increase while myofibrils and sarcomeres mature. Muscle myosin motor activity is required for the regular and coordinated assembly of long myofibrils but not for the high actin order buildup during sarcomere maturation. This suggests that, in muscle, other actin-binding proteins are sufficient to locally bundle or cross-link actin into highly regular arrays.

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<![CDATA[The Order of Exercise during Concurrent Training for Rehabilitation Does Not Alter Acute Genetic Expression, Mitochondrial Enzyme Activity or Improvements in Muscle Function]]> https://www.researchpad.co/article/5989dab8ab0ee8fa60bada01

Concurrent exercise combines different modes of exercise (e.g., aerobic and resistance) into one training protocol, providing stimuli meant to increase muscle strength, aerobic capacity and mass. As disuse is associated with decrements in strength, aerobic capacity and muscle size concurrent training is an attractive modality for rehabilitation. However, interference between the signaling pathways may result in preferential improvements for one of the exercise modes. We recruited 18 young adults (10 ♂, 8 ♀) to determine if order of exercise mode during concurrent training would differentially affect gene expression, protein content and measures of strength and aerobic capacity after 2 weeks of knee-brace induced disuse. Concurrent exercise sessions were performed 3x/week for 6 weeks at gradually increasing intensities either with endurance exercise preceding (END>RES) or following (RES>END) resistance exercise. Biopsies were collected from the vastus lateralis before, 3 h after the first exercise bout and 48 h after the end of training. Concurrent exercise altered the expression of genes involved in mitochondrial biogenesis (PGC-1α, PRC, PPARγ), hypertrophy (PGC-1α4, REDD2, Rheb) and atrophy (MuRF-1, Runx1), increased electron transport chain complex protein content, citrate synthase and mitochondrial cytochrome c oxidase enzyme activity, muscle mass, maximum isometric strength and VO2peak. However, the order in which exercise was completed (END>RES or RES>END) only affected the protein content of mitochondrial complex II subunit. In conclusion, concurrent exercise training is an effective modality for the rehabilitation of the loss of skeletal muscle mass, maximum strength, and peak aerobic capacity resulting from disuse, regardless of the order in which the modes of exercise are performed.

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<![CDATA[Activated Protein Synthesis and Suppressed Protein Breakdown Signaling in Skeletal Muscle of Critically Ill Patients]]> https://www.researchpad.co/article/5989da31ab0ee8fa60b84780

Background

Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3β (GSK3β) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU) patients compared with healthy controls.

Methodology/Principal Findings

ICU patients were systemically inflamed, moderately hyperglycemic, received insulin therapy, and showed a tendency to lower plasma branched chain amino acids compared with controls. Using Western blotting we measured Akt, GSK3β, mTOR, ribosomal protein S6 kinase (S6k), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), and muscle ring finger protein 1 (MuRF1); and by RT-PCR we determined mRNA expression of, among others, insulin-like growth factor 1 (IGF-1), FoxO 1, 3 and 4, atrogin1, MuRF1, interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) and myostatin. Unexpectedly, in critically ill ICU patients Akt-mTOR-S6k signaling was substantially higher compared with controls. FoxO1 mRNA was higher in patients, whereas FoxO3, atrogin1 and myostatin mRNAs and MuRF1 protein were lower compared with controls. A moderate correlation (r2 = 0.36, p<0.05) between insulin infusion dose and phosphorylated Akt was demonstrated.

Conclusions/Significance

We present for the first time muscle protein turnover signaling in critically ill ICU patients, and we show signaling pathway activity towards a stimulation of muscle protein synthesis and a somewhat inhibited proteolysis.

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<![CDATA[Deregulated MicroRNAs in Myotonic Dystrophy Type 2]]> https://www.researchpad.co/article/5989d9ddab0ee8fa60b686c0

Myotonic Dystrophy Type-2 (DM2) is an autosomal dominant disease caused by the expansion of a CCTG tetraplet repeat. It is a multisystemic disorder, affecting skeletal muscles, the heart, the eye, the central nervous system and the endocrine system. Since microRNA (miRNA) expression is disrupted in Myotonic Dystrophy Type-1 and many other myopathies, miRNAs deregulation was studied in skeletal muscle biopsies of 13 DM2 patients and 13 controls. Eleven miRNAs were deregulated: 9 displayed higher levels compared to controls (miR-34a-5p, miR-34b-3p, miR-34c-5p, miR-146b-5p, miR-208a, miR-221-3p and miR-381), while 4 were decreased (miR-125b-5p, miR-193a-3p, miR-193b-3p and miR-378a-3p). To explore the relevance of DM2 miRNA deregulation, the predicted interactions between miRNA and mRNA were investigated. Global gene expression was analyzed in DM2 and controls and bioinformatic analysis identified more than 1,000 miRNA/mRNA interactions. Pathway and function analysis highlighted the involvement of the miRNA-deregulated mRNAs in multiple aspects of DM2 pathophysiology. In conclusion, the observed miRNA dysregulations may contribute to DM2 pathogenetic mechanisms.

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<![CDATA[Increasing O-GlcNAcylation Level on Organ Culture of Soleus Modulates the Calcium Activation Parameters of Muscle Fibers]]> https://www.researchpad.co/article/5989da3cab0ee8fa60b88232

O-N-acetylglucosaminylation is a reversible post-translational modification which presents a dynamic and highly regulated interplay with phosphorylation. New insights suggest that O-GlcNAcylation might be involved in striated muscle physiology, in particular in contractile properties such as the calcium activation parameters. By the inhibition of O-GlcNAcase, we investigated the effect of the increase of soleus O-GlcNAcylation level on the contractile properties by establishing T/pCa relationships. We increased the O-GlcNAcylation level on soleus biopsies performing an organ culture of soleus treated or not with PUGNAc or Thiamet-G, two O-GlcNAcase inhibitors. The enhancement of O-GlcNAcylation pattern was associated with an increase of calcium affinity on slow soleus skinned fibers. Analysis of the glycoproteins pattern showed that this effect is solely due to O-GlcNAcylation of proteins extracted from skinned biopsies. We also characterized the O-GlcNAcylated contractile proteins using a proteomic approach, and identified among others troponin T and I as being O-GlcNAc modified. We quantified the variation of O-GlcNAc level on all these identified proteins, and showed that several regulatory contractile proteins, predominantly fast isoforms, presented a drastic increase in their O-GlcNAc level. Since the only slow isoform of contractile protein presenting an increase of O-GlcNAc level was MLC2, the effect of enhanced O-GlcNAcylation pattern on calcium activation parameters could involve the O-GlcNAcylation of sMLC2, without excluding that an unidentified O-GlcNAc proteins, such as TnC, could be potentially involved in this mechanism. All these data strongly linked O-GlcNAcylation to the modulation of contractile activity of skeletal muscle.

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<![CDATA[Involvement of the FoxO1/MuRF1/Atrogin-1 Signaling Pathway in the Oxidative Stress-Induced Atrophy of Cultured Chronic Obstructive Pulmonary Disease Myotubes]]> https://www.researchpad.co/article/5989da6aab0ee8fa60b92b01

Oxidative stress is thought to be one of the most important mechanisms implicated in the muscle wasting of chronic obstructive pulmonary disease (COPD) patients, but its role has never been demonstrated. We therefore assessed the effects of both pro-oxidant and antioxidant treatments on the oxidative stress levels and atrophic signaling pathway of cultured COPD myotubes. Treatment of cultured COPD myotubes with the pro-oxidant molecule H2O2 resulted in increased ROS production (P = 0.002) and protein carbonylation (P = 0.050), in association with a more pronounced atrophy of the myotubes, as reflected by a reduced diameter (P = 0.003), and the activated expression of atrophic markers MuRF1 and FoxO1 (P = 0.022 and P = 0.030, respectively). Conversely, the antioxidant molecule ascorbic acid induced a reduction in ROS production (P<0.001) and protein carbonylation (P = 0.019), and an increase in the myotube diameter (P<0.001) to a level similar to the diameter of healthy subject myotubes, in association with decreased expression levels of MuRF1, atrogin-1 and FoxO1 (P<0.001, P = 0.002 and P = 0.042, respectively). A significant negative correlation was observed between the variations in myotube diameter and the variations in the expression of MuRF1 after antioxidant treatment (P = 0.047). Moreover, ascorbic acid was able to prevent the H2O2-induced atrophy of COPD myotubes. Last, the proteasome inhibitor MG132 restored the basal atrophy level of the COPD myotubes and also suppressed the H2O2-induced myotube atrophy. These findings demonstrate for the first time the involvement of oxidative stress in the atrophy of COPD peripheral muscle cells in vitro, via the FoxO1/MuRF1/atrogin-1 signaling pathway of the ubiquitin/proteasome system.

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<![CDATA[Necdin Enhances Myoblasts Survival by Facilitating the Degradation of the Mediator of Apoptosis CCAR1/CARP1]]> https://www.researchpad.co/article/5989db4aab0ee8fa60bd9e5f

Regeneration of muscle fibers, lost during pathological muscle degeneration or after injuries, is sustained by the production of new myofibers by means of the satellite cells. Survival of the satellite cells is a critical requirement for efficient muscle reconstitution. Necdin, a member of the MAGE proteins family, is expressed in satellite cell-derived myogenic precursors during perinatal growth and in the adult upon activation during muscle regeneration, where it plays an important role both in myoblast differentiation and survival. We show here that necdin exerts its pro-survival activity by counteracting the action of the pro-apoptotic protein Cell Cycle Apoptosis Regulatory Protein (CCAR1/CARP1) that we have identified as a new molecular interactor of necdin by two-hybrid screening. Necdin is responsible for the maintenance of CCAR1 protein levels, by implementing its ubiquitination and degradation through the proteasome. Taken together, these data shed new light on the molecular mechanism of necdin anti-apoptotic activity in myogenesis.

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<![CDATA[Energy Metabolism during Repeated Sets of Leg Press Exercise Leading to Failure or Not]]> https://www.researchpad.co/article/5989d9fbab0ee8fa60b722ba

This investigation examined the influence of the number of repetitions per set on power output and muscle metabolism during leg press exercise. Six trained men (age 34±6 yr) randomly performed either 5 sets of 10 repetitions (10REP), or 10 sets of 5 repetitions (5REP) of bilateral leg press exercise, with the same initial load and rest intervals between sets. Muscle biopsies (vastus lateralis) were taken before the first set, and after the first and the final sets. Compared with 5REP, 10REP resulted in a markedly greater decrease (P<0.05) of the power output, muscle PCr and ATP content, and markedly higher (P<0.05) levels of muscle lactate and IMP. Significant correlations (P<0.01) were observed between changes in muscle PCr and muscle lactate (R2 = 0.46), between changes in muscle PCr and IMP (R2 = 0.44) as well as between changes in power output and changes in muscle ATP (R2 = 0.59) and lactate (R2 = 0.64) levels. Reducing the number of repetitions per set by 50% causes a lower disruption to the energy balance in the muscle. The correlations suggest that the changes in PCr and muscle lactate mainly occur simultaneously during exercise, whereas IMP only accumulates when PCr levels are low. The decrease in ATP stores may contribute to fatigue.

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<![CDATA[Ablation of the Cardiac-Specific Gene Leucine-Rich Repeat Containing 10 (Lrrc10) Results in Dilated Cardiomyopathy]]> https://www.researchpad.co/article/5989da9eab0ee8fa60ba4b73

Leucine-rich repeat containing 10 (LRRC10) is a cardiac-specific protein exclusively expressed in embryonic and adult cardiomyocytes. However, the role of LRRC10 in mammalian cardiac physiology remains unknown. To determine if LRRC10 is critical for cardiac function, Lrrc10-null (Lrrc10−/−) mice were analyzed. Lrrc10/ mice exhibit prenatal systolic dysfunction and dilated cardiomyopathy in postnatal life. Importantly, Lrrc10−/− mice have diminished cardiac performance in utero, prior to ventricular dilation observed in young adults. We demonstrate that LRRC10 endogenously interacts with α-actinin and α-actin in the heart and all actin isoforms in vitro. Gene expression profiling of embryonic Lrrc10−/− hearts identified pathways and transcripts involved in regulation of the actin cytoskeleton to be significantly upregulated, implicating dysregulation of the actin cytoskeleton as an early defective molecular signal in the absence of LRRC10. In contrast, microarray analyses of adult Lrrc10−/− hearts identified upregulation of oxidative phosphorylation and cardiac muscle contraction pathways during the progression of dilated cardiomyopathy. Analyses of hypertrophic signal transduction pathways indicate increased active forms of Akt and PKCε in adult Lrrc10−/− hearts. Taken together, our data demonstrate that LRRC10 is essential for proper mammalian cardiac function. We identify Lrrc10 as a novel dilated cardiomyopathy candidate gene and the Lrrc10−/− mouse model as a unique system to investigate pediatric cardiomyopathy.

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<![CDATA[Tissue-Specific Strategies of the Very-Long Chain Acyl-CoA Dehydrogenase-Deficient (VLCAD−/−) Mouse to Compensate a Defective Fatty Acid β-Oxidation]]> https://www.researchpad.co/article/5989daa3ab0ee8fa60ba6916

Very long-chain acyl-CoA dehydrogenase (VLCAD)-deficiency is the most common long-chain fatty acid oxidation disorder presenting with heterogeneous phenotypes. Similar to many patients with VLCADD, VLCAD-deficient mice (VLCAD−/−) remain asymptomatic over a long period of time. In order to identify the involved compensatory mechanisms, wild-type and VLCAD−/− mice were fed one year either with a normal diet or with a diet in which medium-chain triglycerides (MCT) replaced long-chain triglycerides, as approved intervention in VLCADD. The expression of the mitochondrial long-chain acyl-CoA dehydrogenase (LCAD) and medium-chain acyl-CoA dehydrogenase (MCAD) was quantified at mRNA and protein level in heart, liver and skeletal muscle. The oxidation capacity of the different tissues was measured by LC-MS/MS using acyl-CoA substrates with a chain length of 8 to 20 carbons. Moreover, in white skeletal muscle the role of glycolysis and concomitant muscle fibre adaptation was investigated. In one year old VLCAD−/− mice MCAD and LCAD play an important role in order to compensate deficiency of VLCAD especially in the heart and in the liver. However, the white gastrocnemius muscle develops alternative compensatory mechanism based on a different substrate selection and increased glucose oxidation. Finally, the application of an MCT diet over one year has no effects on LCAD or MCAD expression. MCT results in the VLCAD−/− mice only in a very modest improvement of medium-chain acyl-CoA oxidation capacity restricted to cardiac tissue. In conclusion, VLCAD−/− mice develop tissue-specific strategies to compensate deficiency of VLCAD either by induction of other mitochondrial acyl-CoA dehydrogenases or by enhancement of glucose oxidation. In the muscle, there is evidence of a muscle fibre type adaptation with a predominance of glycolytic muscle fibres. Dietary modification as represented by an MCT-diet does not improve these strategies long-term.

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<![CDATA[Skeletal Muscle 11beta-HSD1 Controls Glucocorticoid-Induced Proteolysis and Expression of E3 Ubiquitin Ligases Atrogin-1 and MuRF-1]]> https://www.researchpad.co/article/5989daa9ab0ee8fa60ba8cdd

Recent studies demonstrated expression and activity of the intracellular cortisone-cortisol shuttle 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in skeletal muscle and inhibition of 11beta-HSD1 in muscle cells improved insulin sensitivity. Glucocorticoids induce muscle atrophy via increased expression of the E3 ubiquitin ligases Atrogin-1 (Muscle Atrophy F-box (MAFbx)) and MuRF-1 (Muscle RING-Finger-1). We hypothesized that 11beta-HSD1 controls glucocorticoid-induced expression of atrophy E3 ubiquitin ligases in skeletal muscle. Primary human myoblasts were generated from healthy volunteers. 11beta-HSD1-dependent protein degradation was analyzed by [3H]-tyrosine release assay. RT-PCR was used to determine mRNA expression of E3 ubiquitin ligases and 11beta-HSD1 activity was measured by conversion of radioactively labeled [3H]-cortisone to [3H]-cortisol separated by thin-layer chromatography. We here demonstrate that 11beta-HSD1 is expressed and biologically active in interconverting cortisone to active cortisol in murine skeletal muscle cells (C2C12) as well as in primary human myotubes. 11beta-HSD1 expression increased during differentiation from myoblasts to mature myotubes (p<0.01), suggesting a role of 11beta-HSD1 in skeletal muscle growth and differentiation. Treatment with cortisone increased protein degradation by about 20% (p<0.001), which was paralleled by an elevation of Atrogin-1 and MuRF-1 mRNA expression (p<0.01, respectively). Notably, pre-treatment with the 11beta-HSD1 inhibitor carbenoxolone (Cbx) completely abolished the effect of cortisone on protein degradation as well as on Atrogin-1 and MuRF-1 expression. In summary, our data suggest that 11beta-HSD1 controls glucocorticoid-induced protein degradation in human and murine skeletal muscle via regulation of the E3 ubiquitin ligases Atrogin-1 and MuRF-1.

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<![CDATA[Two Weeks of Metformin Treatment Enhances Mitochondrial Respiration in Skeletal Muscle of AMPK Kinase Dead but Not Wild Type Mice]]> https://www.researchpad.co/article/5989daddab0ee8fa60bba726

Metformin is used as an anti-diabetic drug. Metformin ameliorates insulin resistance by improving insulin sensitivity in liver and skeletal muscle. Reduced mitochondrial content has been reported in type 2 diabetic muscles and it may contribute to decreased insulin sensitivity characteristic for diabetic muscles. The molecular mechanism behind the effect of metformin is not fully clarified but inhibition of complex I in the mitochondria and also activation of the 5′AMP activated protein kinase (AMPK) has been reported in muscle. Furthermore, both AMPK activation and metformin treatment have been associated with stimulation of mitochondrial function and biogenesis. However, a causal relationship in skeletal muscle has not been investigated. We hypothesized that potential effects of in vivo metformin treatment on mitochondrial function and protein expressions in skeletal muscle are dependent upon AMPK signaling. We investigated this by two weeks of oral metformin treatment of muscle specific kinase dead α2 (KD) AMPK mice and wild type (WT) littermates. We measured mitochondrial respiration and protein activity and expressions of key enzymes involved in mitochondrial carbohydrate and fat metabolism and oxidative phosphorylation. Mitochondrial respiration, HAD and CS activity, PDH and complex I-V and cytochrome c protein expression were all reduced in AMPK KD compared to WT tibialis anterior muscles. Surprisingly, metformin treatment only enhanced respiration in AMPK KD mice and thereby rescued the respiration defect compared to the WT mice. Metformin did not influence protein activities or expressions in either WT or AMPK KD mice.

We conclude that two weeks of in vivo metformin treatment enhances mitochondrial respiration in the mitochondrial deficient AMPK KD but not WT mice. The improvement seems to be unrelated to AMPK, and does not involve changes in key mitochondrial proteins.

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<![CDATA[Skeletal Muscle NADPH Oxidase Is Increased and Triggers Stretch-Induced Damage in the mdx Mouse]]> https://www.researchpad.co/article/5989db3aab0ee8fa60bd490f

Recent studies have shown that oxidative stress contributes to the pathogenesis of muscle damage in dystrophic (mdx) mice. In this study we have investigated the role of NADPH oxidase as a source of the oxidative stress in these mice. The NADPH oxidase subunits gp91phox, p67phox and rac 1 were increased 2–3 fold in tibilais anterior muscles from mdx mice compared to wild type. Importantly, this increase occurred in 19 day old mice, before the onset of muscle necrosis and inflammation, suggesting that NADPH oxidase is an important source of oxidative stress in mdx muscle. In muscles from 9 week old mdx mice, gp91phox and p67phox were increased 3–4 fold and NADPH oxidase superoxide production was 2 times greater than wild type. In single fibers from mdx muscle NADPH oxidase subunits were all located on or near the sarcolemma, except for p67phox,which was expressed in the cytosol. Pharmacological inhibition of NADPH oxidase significantly reduced the intracellular Ca2+ rise following stretched contractions in mdx single fibers, and also attenuated the loss of muscle force. These results suggest that NADPH oxidase is a major source of reactive oxygen species in dystrophic muscle and its enhanced activity has a stimulatory effect on stretch-induced Ca2+ entry, a key mechanism for muscle damage and functional impairment.

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<![CDATA[TGF-β Small Molecule Inhibitor SB431542 Reduces Rotator Cuff Muscle Fibrosis and Fatty Infiltration By Promoting Fibro/Adipogenic Progenitor Apoptosis]]> https://www.researchpad.co/article/5989dab7ab0ee8fa60bad4ed

Rotator cuff tears represent a large burden of muscle-tendon injuries in our aging population. While small tears can be repaired surgically with good outcomes, critical size tears are marked by muscle atrophy, fibrosis, and fatty infiltration, which can lead to failed repair, frequent re-injury, and chronic disability. Previous animal studies have indicated that Transforming Growth Factor-β (TGF-β) signaling may play an important role in the development of these muscle pathologies after injury. Here, we demonstrated that inhibition of TGF-β1 signaling with the small molecule inhibitor SB431542 in a mouse model of massive rotator cuff tear results in decreased fibrosis, fatty infiltration, and muscle weight loss. These observed phenotypic changes were accompanied by decreased fibrotic, adipogenic, and atrophy-related gene expression in the injured muscle of mice treated with SB431542. We further demonstrated that treatment with SB431542 reduces the number of fibro/adipogenic progenitor (FAP) cells—an important cellular origin of rotator cuff muscle fibrosis and fatty infiltration, in injured muscle by promoting apoptosis of FAPs. Together, these data indicate that the TGF-β pathway is a critical regulator of the degenerative muscle changes seen after massive rotator cuff tears. TGF-β promotes rotator cuff muscle fibrosis and fatty infiltration by preventing FAP apoptosis. TGF-β regulated FAP apoptosis may serve as an important target pathway in the future development of novel therapeutics to improve muscle outcomes following rotator cuff tear.

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<![CDATA[Skeletal Muscle Myofibrillar and Sarcoplasmic Protein Synthesis Rates Are Affected Differently by Altitude-Induced Hypoxia in Native Lowlanders]]> https://www.researchpad.co/article/5989da42ab0ee8fa60b8a412

As a consequence to hypobaric hypoxic exposure skeletal muscle atrophy is often reported. The underlying mechanism has been suggested to involve a decrease in protein synthesis in order to conserve O2. With the aim to challenge this hypothesis, we applied a primed, constant infusion of 1-13C-leucine in nine healthy male subjects at sea level and subsequently at high-altitude (4559 m) after 7–9 days of acclimatization. Physical activity levels and food and energy intake were controlled prior to the two experimental conditions with the aim to standardize these confounding factors. Blood samples and expired breath samples were collected hourly during the 4 hour trial and vastus lateralis muscle biopsies obtained at 1 and 4 hours after tracer priming in the overnight fasted state. Myofibrillar protein synthesis rate was doubled; 0.041±0.018 at sea-level to 0.080±0.018%⋅hr−1 (p<0.05) when acclimatized to high altitude. The sarcoplasmic protein synthesis rate was in contrast unaffected by altitude exposure; 0.052±0.019 at sea-level to 0.059±0.010%⋅hr−1 (p>0.05). Trends to increments in whole body protein kinetics were seen: Degradation rate elevated from 2.51±0.21 at sea level to 2.73±0.13 µmol⋅kg−1⋅min−1 (p = 0.05) at high altitude and synthesis rate similar; 2.24±0.20 at sea level and 2.43±0.13 µmol⋅kg−1⋅min−1 (p>0.05) at altitude. We conclude that whole body amino acid flux is increased due to an elevated protein turnover rate. Resting skeletal muscle myocontractile protein synthesis rate was concomitantly elevated by high-altitude induced hypoxia, whereas the sarcoplasmic protein synthesis rate was unaffected by hypoxia. These changed responses may lead to divergent adaptation over the course of prolonged exposure.

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<![CDATA[Glucagon Like Peptide-1-Induced Glucose Metabolism in Differentiated Human Muscle Satellite Cells Is Attenuated by Hyperglycemia]]> https://www.researchpad.co/article/5989da7aab0ee8fa60b981d8

Background

Glucagon like peptide-1 (GLP-1) stimulates insulin secretion from the pancreas but also has extra-pancreatic effects. GLP-1 may stimulate glucose uptake in cultured muscle cells but the mechanism is not clearly defined. Furthermore, while the pancreatic effects of GLP-1 are glucose-dependent, the glucose-dependency of its extra-pancreatic effects has not been examined.

Methods

Skeletal muscle satellite cells isolated from young (22.5±0.97 yr), lean (BMI 22.5±0.6 kg/m2), healthy males were differentiated in media containing either 22.5 mM (high) or 5 mM (normal) glucose for 7 days in the absence or presence of insulin and/or various GLP-1 concentrations. Myocellular effects of GLP-1, insulin and glucose were assessed by western-blot, glucose uptake and glycogen synthesis.

Results

We firstly show that the GLP-1 receptor protein is expressed in differentiated human muscle satellite cells (myocytes). Secondly, we show that in 5 mM glucose media, exposure of myocytes to GLP-1 results in a dose dependent increase in glucose uptake, GLUT4 amount and subsequently glycogen synthesis in a PI3K dependent manner, independent of the insulin signaling cascade. Importantly, we provide evidence that differentiation of human satellite cells in hyperglycemic (22.5 mM glucose) conditions increases GLUT1 expression, and renders the cells insulin resistant and interestingly GLP-1 resistant in terms of glucose uptake and glycogen synthesis. Hyperglycemic conditions did not affect the ability of insulin to phosphorylate downstream targets, PKB or GSK3. Interestingly we show that at 5 mM glucose, GLP-1 increases GLUT4 protein levels and that this effect is abolished by hyperglycemia.

Conclusions

GLP-1 increases glucose uptake and glycogen synthesis into fully-differentiated human satellite cells in a PI3-K dependent mechanism potentially through increased GLUT4 protein levels. The latter occurs independently of the insulin signaling pathway. Attenuation of both GLP-1 and insulin-induced glucose metabolism by hyperglycemia is likely to occur downstream of PI3K.

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<![CDATA[Myocardin Overexpression Is Sufficient for Promoting the Development of a Mature Smooth Muscle Cell-Like Phenotype from Human Embryonic Stem Cells]]> https://www.researchpad.co/article/5989db1cab0ee8fa60bce431

Background

Myocardin is thought to have a key role in smooth muscle cell (SMC) development by acting on CArG-dependent genes. However, it is unclear whether myocardin-induced SMC maturation and increases in agonist-induced calcium signalling are also associated with increases in the expression of non-CArG-dependent SMC-specific genes. Moreover, it is unknown whether myocardin promotes SMC development from human embryonic stem cells.

Methodology/Principal

Findings The effects of adenoviral-mediated myocardin overexpression on SMC development in human ESC-derived embryoid bodies were investigated using immunofluorescence, flow cytometry and real time RT-PCR. Myocardin overexpression from day 10 to day 28 of embryoid body differentiation increased the number of smooth muscle α-actin+ and smooth muscle myosin heavy chain+ SMC-like cells and increased carbachol-induced contractile function. However, myocardin was found to selectively regulate only CArG-dependent SMC-specific genes. Nevertheless, myocardin expression appeared to be sufficient to specify the SMC lineage.

Conclusions/Significance

Myocardin increases the development and maturation of SMC-like cells from human embryonic stem cells despite not activating the full repertoire of SMC genes. These findings have implications for vascular tissue engineering and other applications requiring large numbers of functional SMCs.

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<![CDATA[Delayed Recovery of Skeletal Muscle Mass following Hindlimb Immobilization in mTOR Heterozygous Mice]]> https://www.researchpad.co/article/5989da89ab0ee8fa60b9d790

The present study addressed the hypothesis that reducing mTOR, as seen in mTOR heterozygous (+/−) mice, would exaggerate the changes in protein synthesis and degradation observed during hindlimb immobilization as well as impair normal muscle regrowth during the recovery period. Atrophy was produced by unilateral hindlimb immobilization and data compared to the contralateral gastrocnemius. In wild-type (WT) mice, the gradual loss of muscle mass plateaued by day 7. This response was associated with a reduction in basal protein synthesis and development of leucine resistance. Proteasome activity was consistently elevated, but atrogin-1 and MuRF1 mRNAs were only transiently increased returning to basal values by day 7. When assessed 7 days after immobilization, the decreased muscle mass and protein synthesis and increased proteasome activity did not differ between WT and mTOR+/− mice. Moreover, the muscle inflammatory cytokine response did not differ between groups. After 10 days of recovery, WT mice showed no decrement in muscle mass, and this accretion resulted from a sustained increase in protein synthesis and a normalization of proteasome activity. In contrast, mTOR+/− mice failed to fully replete muscle mass at this time, a defect caused by the lack of a compensatory increase in protein synthesis. The delayed muscle regrowth of the previously immobilized muscle in the mTOR+/− mice was associated with a decreased raptor•4EBP1 and increased raptor•Deptor binding. Slowed regrowth was also associated with a sustained inflammatory response (e.g., increased TNFα and CD45 mRNA) during the recovery period and a failure of IGF-I to increase as in WT mice. These data suggest mTOR is relatively more important in regulating the accretion of muscle mass during recovery than the loss of muscle during the atrophy phase, and that protein synthesis is more sensitive than degradation to the reduction in mTOR during muscle regrowth.

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