ResearchPad - muscle-fibers https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Prolyl hydroxylase domain 2 reduction enhances skeletal muscle tissue regeneration after soft tissue trauma in mice]]> https://www.researchpad.co/article/elastic_article_14698 The transcription factor Hypoxia-inducible factor 1 (HIF-1) plays a pivotal role in tissue regeneration. HIF-1 is negatively controlled by O2-dependent prolyl hydroxylases with a predominant role of prolyl hydroxylase 2 isoform (Phd2). Transgenic mice, hypomorphic for this isoform, accumulate more HIF-1 under normoxic conditions. Using these mice, we investigated the influence of Phd2 and HIF-1 on the regenerative capability of skeletal muscle tissue after myotrauma. Phd2-hypomorphic and wild type mice (on C57Bl/6 background) were grouped with regeneration times from 6 to 168 hours after closed mechanic muscle trauma to the hind limb. Tissue samples were analysed by immuno-staining and real-time PCR. Bone marrow derived macrophages of wild type and Phd2-hypomorphic mice were isolated and analysed via flow cytometry and quantitative real-time PCR. Phd2 reduction led to a higher regenerative capability due to enhanced activation of myogenic factors accompanied by induction of genes responsible for glucose and lactate metabolism in Phd2-hypomorphic mice. Macrophage infiltration into the trauma areas in hypomorphic mice started earlier and was more pronounced compared to wild type mice. Phd2-hypomorphic mice also showed higher numbers of macrophages in areas with sustained trauma 72 hours after myotrauma application. In conclusion, we postulate that the HIF-1 pathway is activated secondary to a Phd2 reduction which may lead to i) higher activation of myogenic factors, ii) increased number of positive stem cell proliferation markers, and iii) accelerated macrophage recruitment to areas of trauma, resulting in faster muscle tissue regeneration after myotrauma. With the current development of prolyl hydroxylase domain inhibitors, our findings point towards a potential clinical benefit after myotrauma.

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<![CDATA[Mechanisms underpinning the permanent muscle damage induced by snake venom metalloprotease]]> https://www.researchpad.co/article/5c59febed5eed0c484135375

Snakebite is a major neglected tropical health issue that affects over 5 million people worldwide resulting in around 1.8 million envenomations and 100,000 deaths each year. Snakebite envenomation also causes innumerable morbidities, specifically loss of limbs as a result of excessive tissue/muscle damage. Snake venom metalloproteases (SVMPs) are a predominant component of viper venoms, and are involved in the degradation of basement membrane proteins (particularly collagen) surrounding the tissues around the bite site. Although their collagenolytic properties have been established, the molecular mechanisms through which SVMPs induce permanent muscle damage are poorly understood. Here, we demonstrate the purification and characterisation of an SVMP from a viper (Crotalus atrox) venom. Mass spectrometry analysis confirmed that this protein is most likely to be a group III metalloprotease (showing high similarity to VAP2A) and has been referred to as CAMP (Crotalus atrox metalloprotease). CAMP displays both collagenolytic and fibrinogenolytic activities and inhibits CRP-XL-induced platelet aggregation. To determine its effects on muscle damage, CAMP was administered into the tibialis anterior muscle of mice and its actions were compared with cardiotoxin I (a three-finger toxin) from an elapid snake (Naja pallida) venom. Extensive immunohistochemistry analyses revealed that CAMP significantly damages skeletal muscles by attacking the collagen scaffold and other important basement membrane proteins, and prevents their regeneration through disrupting the functions of satellite cells. In contrast, cardiotoxin I destroys skeletal muscle by damaging the plasma membrane, but does not impact regeneration due to its inability to affect the extracellular matrix. Overall, this study provides novel insights into the mechanisms through which SVMPs induce permanent muscle damage.

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<![CDATA[Nandrolone decanoate administration does not attenuate muscle atrophy during a short period of disuse]]> https://www.researchpad.co/article/5c58d610d5eed0c484031507

Background

A few days of bed rest or immobilization following injury, disease, or surgery can lead to considerable loss of skeletal muscle mass and strength. It has been speculated that such short, successive periods of muscle disuse may be largely responsible for the age-related loss of muscle mass throughout the lifespan.

Objective

To assess whether a single intramuscular injection of nandrolone decanoate prior to immobilization can attenuate the loss of muscle mass and strength in vivo in humans.

Design, setting and participants

Thirty healthy (22 ± 1 years) men were subjected to 7 days of one-legged knee immobilization by means of a full leg cast with (NAD, n = 15) or without (CON, n = 15) prior intramuscular nandrolone decanoate injection (200 mg).

Measures

Before and immediately after immobilization, quadriceps muscle cross-sectional area (CSA) (by means of single-slice computed tomography (CT) scans of the upper leg) and one-legged knee extension strength (one-repetition maximum [1-RM]) were assessed for both legs. Furthermore, muscle biopsies from the immobilized leg were taken before and after immobilization to assess type I and type II muscle fiber cross-sectional area.

Results

Quadriceps muscle CSA decreased during immobilization in both CON and NAD (-6 ± 1% and -6 ± 1%, respectively; main effect of time P<0.01), with no differences between the groups (time × treatment interaction, P = 0.59). Leg muscle strength declined following immobilization (-6 ± 2% in CON and -7 ± 3% in NAD; main effect of time, P<0.05), with no differences between groups (time × treatment interaction, P = 0.55).

Conclusions

This is the first study to report that nandrolone decanoate administration does not preserve skeletal muscle mass and strength during a short period of leg immobilization in vivo in humans.

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<![CDATA[A spasticity model based on feedback from muscle force explains muscle activity during passive stretches and gait in children with cerebral palsy]]> https://www.researchpad.co/article/5c141ebed5eed0c484d2806b

Muscle spasticity is characterized by exaggerated stretch reflexes and affects about 85% of the children with cerebral palsy. However, the mechanisms underlying spasticity and its influence on gait are not well understood. Here, we first aimed to model the response of spastic hamstrings and gastrocnemii in children with cerebral palsy to fast passive stretches. Then, we evaluated how the model applied to gait. We developed three models based on exaggerated proprioceptive feedback. The first model relied on feedback from muscle fiber length and velocity (velocity-related model), the second model relied on feedback from muscle fiber length, velocity, and acceleration (acceleration-related model), and the third model relied on feedback from muscle force and its first time derivative (force-related model). The force-related model better reproduced measured hamstrings and gastrocnemii activity during fast passive stretches (coefficients of determination (R2): 0.73 ± 0.10 and 0.60 ± 0.13, respectively, and root mean square errors (RMSE): 0.034 ± 0.031 and 0.009 ± 0.007, respectively) than the velocity-related model (R2: 0.46 ± 0.15 and 0.07 ± 0.13, and RMSE: 0.053 ± 0.051 and 0.015 ± 0.009), and the acceleration-related model (R2: 0.47 ± 0.15 and 0.09 ± 0.14, and RMSE: 0.052 ± 0.050 and 0.015 ± 0.008). Additionally, the force-related model predicted hamstrings and gastrocnemii activity that better correlated with measured activity during gait (cross correlations: 0.82 ± 0.09 and 0.85 ± 0.06, respectively) than the activity predicted by the velocity-related model (cross correlations: 0.49 ± 0.17 and 0.71 ± 0.22) and the acceleration-related model (cross correlations: 0.51 ± 0.16 and 0.67 ± 0.20). Our results therefore suggest that force encoding in muscle spindles in combination with altered feedback gains and thresholds underlie activity of spastic muscles during passive stretches and gait. Our model of spasticity opens new perspectives for studying movement impairments due to spasticity through simulation.

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<![CDATA[Muscle-Specific Splicing Factors ASD-2 and SUP-12 Cooperatively Switch Alternative Pre-mRNA Processing Patterns of the ADF/Cofilin Gene in Caenorhabditis elegans]]> https://www.researchpad.co/article/5989d9f9ab0ee8fa60b7172a

Pre–mRNAs are often processed in complex patterns in tissue-specific manners to produce a variety of protein isoforms from single genes. However, mechanisms orchestrating the processing of the entire transcript are not well understood. Muscle-specific alternative pre–mRNA processing of the unc-60 gene in Caenorhabditis elegans, encoding two tissue-specific isoforms of ADF/cofilin with distinct biochemical properties in regulating actin organization, provides an excellent in vivo model of complex and tissue-specific pre–mRNA processing; it consists of a single first exon and two separate series of downstream exons. Here we visualize the complex muscle-specific processing pattern of the unc-60 pre–mRNA with asymmetric fluorescence reporter minigenes. By disrupting juxtaposed CUAAC repeats and UGUGUG stretch in intron 1A, we demonstrate that these elements are required for retaining intron 1A, as well as for switching the processing patterns of the entire pre–mRNA from non-muscle-type to muscle-type. Mutations in genes encoding muscle-specific RNA–binding proteins ASD-2 and SUP-12 turned the colour of the unc-60 reporter worms. ASD-2 and SUP-12 proteins specifically and cooperatively bind to CUAAC repeats and UGUGUG stretch in intron 1A, respectively, to form a ternary complex in vitro. Immunohistochemical staining and RT–PCR analyses demonstrate that ASD-2 and SUP-12 are also required for switching the processing patterns of the endogenous unc-60 pre-mRNA from UNC-60A to UNC-60B in muscles. Furthermore, systematic analyses of partially spliced RNAs reveal the actual orders of intron removal for distinct mRNA isoforms. Taken together, our results demonstrate that muscle-specific splicing factors ASD-2 and SUP-12 cooperatively promote muscle-specific processing of the unc-60 gene, and provide insight into the mechanisms of complex pre-mRNA processing; combinatorial regulation of a single splice site by two tissue-specific splicing regulators determines the binary fate of the entire transcript.

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<![CDATA[Musculoskeletal Geometry, Muscle Architecture and Functional Specialisations of the Mouse Hindlimb]]> https://www.researchpad.co/article/5989db3bab0ee8fa60bd4ca7

Mice are one of the most commonly used laboratory animals, with an extensive array of disease models in existence, including for many neuromuscular diseases. The hindlimb is of particular interest due to several close muscle analogues/homologues to humans and other species. A detailed anatomical study describing the adult morphology is lacking, however. This study describes in detail the musculoskeletal geometry and skeletal muscle architecture of the mouse hindlimb and pelvis, determining the extent to which the muscles are adapted for their function, as inferred from their architecture. Using I2KI enhanced microCT scanning and digital segmentation, it was possible to identify 39 distinct muscles of the hindlimb and pelvis belonging to nine functional groups. The architecture of each of these muscles was determined through microdissections, revealing strong architectural specialisations between the functional groups. The hip extensors and hip adductors showed significantly stronger adaptations towards high contraction velocities and joint control relative to the distal functional groups, which exhibited larger physiological cross sectional areas and longer tendons, adaptations for high force output and elastic energy savings. These results suggest that a proximo-distal gradient in muscle architecture exists in the mouse hindlimb. Such a gradient has been purported to function in aiding locomotor stability and efficiency. The data presented here will be especially valuable to any research with a focus on the architecture or gross anatomy of the mouse hindlimb and pelvis musculature, but also of use to anyone interested in the functional significance of muscle design in relation to quadrupedal locomotion.

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<![CDATA[Synergistic Structure in the Speed Dependent Modulation of Muscle Activity in Human Walking]]> https://www.researchpad.co/article/5989dadeab0ee8fa60bbabe3

Recently, a modular organisation has been proposed to simplify control of the large number of muscles involved in human walking. Although previous research indicates that a single set of modular activation patterns can account for muscle activity at different speeds, these studies only provide indirect evidence for the idea that speed regulation in human walking is under modular control. Here, a more direct approach was taken to assess the synergistic structure that underlies speed regulation, by isolating speed effects through the construction of gain functions that represent the linear relation between speed and amplitude for each point in the time-normalized gait cycle. The activity of 13 muscles in 13 participants was measured at 4 speeds (0.69, 1.00, 1.31, and 1.61 ms-1) during treadmill walking. Gain functions were constructed for each of the muscles, and gain functions and the activity patterns at 1.00 ms-1 were both subjected to dimensionality reduction, to obtain modular gain functions and modular basis functions, respectively. The results showed that 4 components captured most of the variance in the gain functions (74.0% ± 1.3%), suggesting that the neuromuscular regulation of speed is under modular control. Correlations between modular gain functions and modular basis functions (range 0.58–0.89) and the associated synergistic muscle weightings (range 0.6–0.95) were generally high, suggesting substantial overlap in the synergistic control of the basic phasing of muscle activity and its modulation through speed. Finally, the combined set of modular functions and associated weightings were well capable of predicting muscle activity patterns obtained at a speed (1.31 ms-1) that was not involved in the initial dimensionality reduction, confirming the robustness of the presently used approach. Taken together, these findings provide direct evidence of synergistic structure in speed regulation, and may inspire further work on flexibility in the modular control of gait.

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<![CDATA[Glutamine Supplementation Stimulates Protein-Synthetic and Inhibits Protein-Degradative Signaling Pathways in Skeletal Muscle of Diabetic Rats]]> https://www.researchpad.co/article/5989da08ab0ee8fa60b7664f

In this study, we investigated the effect of glutamine (Gln) supplementation on the signaling pathways regulating protein synthesis and protein degradation in the skeletal muscle of rats with streptozotocin (STZ)-induced diabetes. The expression levels of key regulatory proteins in the synthetic pathways (Akt, mTOR, GSK3 and 4E-BP1) and the degradation pathways (MuRF-1 and MAFbx) were determined using real-time PCR and Western blotting in four groups of male Wistar rats; 1) control, non-supplemented with glutamine; 2) control, supplemented with glutamine; 3) diabetic, non-supplemented with glutamine; and 4) diabetic, supplemented with glutamine. Diabetes was induced by the intravenous injection of 65 mg/kg bw STZ in citrate buffer (pH 4.2); the non-diabetic controls received only citrate buffer. After 48 hours, diabetes was confirmed in the STZ-treated animals by the determination of blood glucose levels above 200 mg/dL. Starting on that day, a solution of 1 g/kg bw Gln in phosphate buffered saline (PBS) was administered daily via gavage for 15 days to groups 2 and 4. Groups 1 and 3 received only PBS for the same duration. The rats were euthanized, and the soleus muscles were removed and homogenized in extraction buffer for the subsequent measurement of protein and mRNA levels. The results demonstrated a significant decrease in the muscle Gln content in the diabetic rats, and this level increased toward the control value in the diabetic rats receiving Gln. In addition, the diabetic rats exhibited a reduced mRNA expression of regulatory proteins in the protein synthesis pathway and increased expression of those associated with protein degradation. A reduction in the skeletal muscle mass in the diabetic rats was observed and was alleviated partially with Gln supplementation. The data suggest that glutamine supplementation is potentially useful for slowing the progression of muscle atrophy in patients with diabetes.

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<![CDATA[A One-Step Immunostaining Method to Visualize Rodent Muscle Fiber Type within a Single Specimen]]> https://www.researchpad.co/article/5989dafdab0ee8fa60bc5455

In this study, we present a quadruple immunostaining method for rapid muscle fiber typing of mice and rats using antibodies specific to the adult myosin heavy chain (MyHC) isoforms MyHC1, 2A, 2X, and 2B, which are common marker proteins of distinct muscle fiber types. We developed rat monoclonal antibodies specific to each MyHC isoform and conjugated these four antibodies to fluorophores with distinct excitation and emission wavelengths. By mixing the four types of conjugated antibodies, MyHC1, 2A, 2X, and 2B could be distinguished within a single specimen allowing for facile delineation of skeletal muscle fiber types. Furthermore, we could observe hybrid fibers expressing MyHC2X and MyHC2B together in single longitudinal muscle sections from mice and rats, that was not attained in previous techniques. This staining method is expected to be applied to study muscle fiber type transition in response to environmental factors, and to ultimately develop techniques to regulate animal muscle fiber types.

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<![CDATA[Fast-to-Slow Transition of Skeletal Muscle Contractile Function and Corresponding Changes in Myosin Heavy and Light Chain Formation in the R6/2 Mouse Model of Huntington’s Disease]]> https://www.researchpad.co/article/5989da7bab0ee8fa60b98987

Huntington´s disease (HD) is a hereditary neurodegenerative disease resulting from an expanded polyglutamine sequence (poly-Q) in the protein huntingtin (HTT). Various studies report atrophy and metabolic pathology of skeletal muscle in HD and suggest as part of the process a fast-to-slow fiber type transition that may be caused by the pathological changes in central motor control or/and by mutant HTT in the muscle tissue itself. To investigate muscle pathology in HD, we used R6/2 mice, a common animal model for a rapidly progressing variant of the disease expressing exon 1 of the mutant human gene. We investigated alterations in the extensor digitorum longus (EDL), a typical fast-twitch muscle, and the soleus (SOL), a slow-twitch muscle. We focussed on mechanographic measurements of excised muscles using single and repetitive electrical stimulation and on the expression of the various myosin isoforms (heavy and light chains) using dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole muscle and single fiber preparations. In EDL of R6/2, the functional tests showed a left shift of the force-frequency relation and decrease in specific force. Moreover, the estimated relative contribution of the fastest myosin isoform MyHC IIb decreased, whereas the contribution of the slower MyHC IIx isoform increased. An additional change occurred in the alkali MyLC forms showing a decrease in 3f and an increase in 1f level. In SOL, a shift from fast MyHC IIa to the slow isoform I was detectable in male R6/2 mice only, and there was no evidence of isoform interconversion in the MyLC pattern. These alterations point to a partial remodeling of the contractile apparatus of R6/2 mice towards a slower contractile phenotype, predominantly in fast glycolytic fibers.

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<![CDATA[A novel early onset phenotype in a zebrafish model of merosin deficient congenital muscular dystrophy]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdbd14

Merosin deficient congenital muscular dystrophy (MDC1A) is a severe neuromuscular disorder with onset in infancy that is associated with severe morbidities (particularly wheelchair dependence) and early mortality. It is caused by recessive mutations in the LAMA2 gene that encodes a subunit of the extracellular matrix protein laminin 211. At present, there are no treatments for this disabling disease. The zebrafish has emerged as a powerful model system for the identification of novel therapies. However, drug discovery in the zebrafish is largely dependent on the identification of phenotypes suitable for chemical screening. Our goal in this study was to elucidate novel, early onset abnormalities in the candyfloss (caf) zebrafish, a model of MDC1A. We uncovered and characterize abnormalities in spontaneous coiling, the earliest motor movement in the zebrafish, as a fully penetrant change specific to caf mutants that is ideal for future drug testing.

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<![CDATA[Decreased Peak Expiratory Flow Associated with Muscle Fiber-Type Switching in Spinal and Bulbar Muscular Atrophy]]> https://www.researchpad.co/article/5989dafbab0ee8fa60bc4a67

The aim of this study was to characterize the respiratory function profile of subjects with spinal and bulbar muscular atrophy (SBMA), and to explore the underlying pathological mechanism by comparing the clinical and biochemical indices of this disease with those of amyotrophic lateral sclerosis (ALS). We enrolled male subjects with SBMA (n = 40) and ALS (n = 25) along with 15 healthy control subjects, and assessed their respiratory function, motor function, and muscle strength. Predicted values of peak expiratory flow (%PEF) and forced vital capacity were decreased in subjects with SBMA compared with controls. In SBMA, both values were strongly correlated with the trunk subscores of the motor function tests and showed deterioration relative to disease duration. Compared with activities of daily living (ADL)-matched ALS subjects, %PEF, tongue pressure, and grip power were substantially decreased in subjects with SBMA. Both immunofluorescence and RT-PCR demonstrated a selective decrease in the expression levels of the genes encoding the myosin heavy chains specific to fast-twitch fibers in SBMA subjects. The mRNA levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha and peroxisome proliferator-activated receptor delta were up-regulated in SBMA compared with ALS and controls. In conclusion, %PEF is a disease-specific respiratory marker for the severity and progression of SBMA. Explosive muscle strength, including %PEF, was selectively affected in subjects with SBMA and was associated with activation of the mitochondrial biogenesis-related molecular pathway in skeletal muscles.

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<![CDATA[Rac1 Activation Caused by Membrane Translocation of a Guanine Nucleotide Exchange Factor in Akt2-Mediated Insulin Signaling in Mouse Skeletal Muscle]]> https://www.researchpad.co/article/5989da1cab0ee8fa60b7d513

Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4, which is translocated to the plasma membrane following insulin stimulation. Several lines of evidence suggested that the protein kinase Akt2 plays a key role in this insulin action. The small GTPase Rac1 has also been implicated as a regulator of insulin-stimulated GLUT4 translocation, acting downstream of Akt2. However, the mechanisms whereby Akt2 regulates Rac1 activity remain obscure. The guanine nucleotide exchange factor FLJ00068 has been identified as a direct regulator of Rac1 in Akt2-mediated signaling, but its characterization was performed mostly in cultured myoblasts. Here, we provide in vivo evidence that FLJ00068 indeed acts downstream of Akt2 as a Rac1 regulator by using mouse skeletal muscle. Small interfering RNA knockdown of FLJ00068 markedly diminished GLUT4 translocation to the sarcolemma following insulin administration or ectopic expression of a constitutively activated mutant of either phosphoinositide 3-kinase or Akt2. Additionally, insulin and these constitutively activated mutants caused the activation of Rac1 as shown by immunofluorescent microscopy using a polypeptide probe specific to activated Rac1 in isolated gastrocnemius muscle fibers and frozen sections of gastrocnemius muscle. This Rac1 activation was also abrogated by FLJ00068 knockdown. Furthermore, we observed translocation of FLJ00068 to the cell periphery following insulin stimulation in cultured myoblasts. Localization of FLJ00068 in the plasma membrane in insulin-stimulated, but not unstimulated, myoblasts and mouse gastrocnemius muscle was further affirmed by subcellular fractionation and subsequent immunoblotting. Collectively, these results strongly support a critical role of FLJ00068 in Akt2-mediated Rac1 activation in mouse skeletal muscle insulin signaling.

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<![CDATA[Nur77 deletion impairs muscle growth during developmental myogenesis and muscle regeneration in mice]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdb93f

Muscle atrophy is a prevalent condition in illness and aging. Identifying novel pathways that control muscle mass may lead to therapeutic advancement. We previously identified Nur77 as a transcriptional regulator of glycolysis in skeletal muscle. More recently, we showed that Nur77 expression also controls myofiber size in mice. It was unknown, however, whether Nur77’s regulation of muscle size begins during developmental myogenesis or only in adulthood. To determine the importance of Nur77 throughout muscle growth, we examined myofiber size at E18.5, 3 weeks postnatal age, and in young adult mice. Using the global Nur77-/- mice, we showed that Nur77 deficiency reduced myofiber size as early as E18.5. The reduction in myofiber size became more pronounced by 3 weeks of age. We observed comparable reduction in myofiber size in young myofiber-specific Nur77-knockout mice. These findings suggest that Nur77’s effect on muscle growth is intrinsic to its expression in differentiating myofibers, and not dependent on its expression in myogenic stem cells. To determine the importance of Nur77 expression in muscle accretion in mature mice, we generated an inducible-, muscle-specific, Nur77-deficient mouse model. We demonstrated that tamoxifen-induced deletion of Nur77 in 3-month-old mice reduced myofiber size. This change was accompanied by increased activity of Smad2 and FoxO3, two negative regulators of muscle mass. The role of Nur77 in muscle growth was further elaborated in the cardiotoxin-induced muscle regeneration model. Compared to wildtype mice, regenerated myofibers were smaller in Nur77-/- mice. However, when normalized to saline-injected muscle, the recovery of sarcoplasmic area was comparable between Nur77-/- and wildtype mice. These findings suggest that Nur77 deficiency compromises myofiber growth, but not the regenerative capacity of myogenic progenitor cells. Collectively, the findings presented here demonstrate Nur77 as an important regulator of muscle growth both during prenatal and postnatal myogenesis.

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<![CDATA[Quantified Mechanical Properties of the Deltoid Muscle Using the Shear Wave Elastography: Potential Implications for Reverse Shoulder Arthroplasty]]> https://www.researchpad.co/article/5989da60ab0ee8fa60b90dae

The deltoid muscle plays a critical role in the biomechanics of shoulders undergoing reverse shoulder arthroplasty (RSA). However, both pre- and postoperative assessment of the deltoid muscle quality still remains challenging. The purposes of this study were to establish a novel methodology of shear wave elastography (SWE) to quantify the mechanical properties of the deltoid muscle, and to investigate the reliability of this technique using cadaveric shoulders for the purpose of RSA. Eight fresh-frozen cadaveric shoulders were obtained. The deltoid muscles were divided into 5 segments (A1, A2, M, P1 and P2) according to the muscle fiber orientation and SWE values were measured for each segment. Intra- and inter-observer reliability was evaluated using intraclass correlation coefficient (ICC). To measure the response of muscle tension during RSA, the humeral shaft was osteotomized and subsequently elongated by an external fixator (intact to 15 mm elongation). SWE of the deltoid muscle was measured under each stretch condition. Intra- and inter-observer reliability of SWE measurements for all regions showed 0.761–0.963 and 0.718–0.947 for ICC(2,1). Especially, SWE measurements for segments A2 and M presented satisfactory repeatability. Elongated deltoid muscles by the external fixator showed a progressive increase in passive stiffness for all muscular segments. Especially, SWE outcomes of segments A2 and M reliably showed an exponential growth upon stretching (R2 = 0.558 and 0.593). Segmental measurements using SWE could be reliably and feasibly used to quantitatively assess the mechanical properties of the deltoid muscle, especially in the anterior and middle portions. This novel technique based on the anatomical features may provide helpful information of the deltoid muscle properties during treatment of RSA.

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<![CDATA[The Invalidation of HspB1 Gene in Mouse Alters the Ultrastructural Phenotype of Muscles]]> https://www.researchpad.co/article/5989daf0ab0ee8fa60bc10f0

Even though abundance of Hsp27 is the highest in skeletal muscle, the relationships between the expression of HspB1 (encoding Hsp27) and muscle characteristics are not fully understood. In this study, we have analysed the effect of Hsp27 inactivation on mouse development and phenotype. We generated a mouse strain devoid of Hsp27 protein by homologous recombination of the HspB1 gene. The HspB1-/- mouse was viable and fertile, showing neither apparent morphological nor anatomical alterations. We detected a gender dimorphism with marked effects in males, a lower body weight (P < 0.05) with no obvious changes in the growth rate, and a lower plasma lipids profile (cholesterol, HDL and triglycerides, 0.001 < P< 0.05). The muscle structure of the animals was examined by optical microscopy and transmission electron microscopy. Not any differences in the characteristics of muscle fibres (contractile and metabolic type, shape, perimeter, cross-sectional area) were detected except a trend for a higher proportion of small fibres. Different myosin heavy chains electrophoretic profiles were observed in the HspB1-/- mouse especially the presence of an additional isoform. Electron microscopy revealed ultrastructural abnormalities in the myofibrillar structure of the HspB1-/- mouse mutant mice (e.g. destructured myofibrils and higher gaps between myofibrils) especially in the m. Soleus. Combined with our previous data, these findings suggest that Hsp27 could directly impact the organization of muscle cytoskeleton at the molecular and ultrastructural levels.

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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[The SWI/SNF Subunit/Tumor Suppressor BAF47/INI1 Is Essential in Cell Cycle Arrest upon Skeletal Muscle Terminal Differentiation]]> https://www.researchpad.co/article/5989dadbab0ee8fa60bb9c4f

Myogenic terminal differentiation is a well-orchestrated process starting with permanent cell cycle exit followed by muscle-specific genetic program activation. Individual SWI/SNF components have been involved in muscle differentiation. Here, we show that the master myogenic differentiation factor MyoD interacts with more than one SWI/SNF subunit, including the catalytic subunit BRG1, BAF53a and the tumor suppressor BAF47/INI1. Downregulation of each of these SWI/SNF subunits inhibits skeletal muscle terminal differentiation but, interestingly, at different differentiation steps and extents. BAF53a downregulation inhibits myotube formation but not the expression of early muscle-specific genes. BRG1 or BAF47 downregulation disrupt both proliferation and differentiation genetic programs expression. Interestingly, BRG1 and BAF47 are part of the SWI/SNF remodeling complex as well as the N-CoR-1 repressor complex in proliferating myoblasts. However, our data show that, upon myogenic differentiation, BAF47 shifts in favor of N-CoR-1 complex. Finally, BRG1 and BAF47 are well-known tumor suppressors but, strikingly, only BAF47 seems essential in the myoblasts irreversible cell cycle exit. Together, our data unravel differential roles for SWI/SNF subunits in muscle differentiation, with BAF47 playing a dual role both in the permanent cell cycle exit and in the regulation of muscle-specific genes.

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<![CDATA[Oxidative Stress (Glutathionylation) and Na,K-ATPase Activity in Rat Skeletal Muscle]]> https://www.researchpad.co/article/5989db11ab0ee8fa60bcc27b

Background

Changes in ion distribution across skeletal muscle membranes during muscle activity affect excitability and may impair force development. These changes are counteracted by the Na,K-ATPase. Regulation of the Na,K-ATPase is therefore important for skeletal muscle function. The present study investigated the presence of oxidative stress (glutathionylation) on the Na,K-ATPase in rat skeletal muscle membranes.

Results

Immunoprecipitation with an anti-glutathione antibody and subsequent immunodetection of Na,K-ATPase protein subunits demonstrated 9.0±1.3% and 4.1±1.0% glutathionylation of the α isoforms in oxidative and glycolytic skeletal muscle, respectively. In oxidative muscle, 20.0±6.1% of the β1 units were glutathionylated, whereas 14.8±2.8% of the β2-subunits appear to be glutathionylated in glycolytic muscle. Treatment with the reducing agent dithiothreitol (DTT, 1 mM) increased the in vitro maximal Na,K-ATPase activity by 19% (P<0.05) in membranes from glycolytic muscle. Oxidized glutathione (GSSG, 0–10 mM) increased the in vitro glutathionylation level detected with antibodies, and decreased the in vitro maximal Na,K-ATPase activity in a dose-dependent manner, and with a larger effect in oxidative compared to glycolytic skeletal muscle.

Conclusion

This study demonstrates the existence of basal glutathionylation of both the α and the β units of rat skeletal muscle Na,K-ATPase. In addition, the study suggests a negative correlation between glutathionylation levels and maximal Na,K-ATPase activity.

Perspective

Glutathionylation likely contributes to the complex regulation of Na,K-ATPase function in skeletal muscle. Especially, glutathionylation induced by oxidative stress may have a role in Na,K-ATPase regulation during prolonged muscle activity.

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<![CDATA[Narciclasine attenuates diet-induced obesity by promoting oxidative metabolism in skeletal muscle]]> https://www.researchpad.co/article/5989db54ab0ee8fa60bdd08a

Obesity develops when caloric intake exceeds metabolic needs. Promoting energy expenditure represents an attractive approach in the prevention of this fast-spreading epidemic. Here, we report a novel pharmacological strategy in which a natural compound, narciclasine (ncls), attenuates diet-induced obesity (DIO) in mice by promoting energy expenditure. Moreover, ncls promotes fat clearance from peripheral metabolic tissues, improves blood metabolic parameters in DIO mice, and protects these mice from the loss of voluntary physical activity. Further investigation suggested that ncls achieves these beneficial effects by promoting a shift from glycolytic to oxidative muscle fibers in the DIO mice thereby enhancing mitochondrial respiration and fatty acid oxidation (FAO) in the skeletal muscle. Moreover, ncls strongly activates AMPK signaling specifically in the skeletal muscle. The beneficial effects of ncls treatment in fat clearance and AMPK activation were faithfully reproduced in vitro in cultured murine and human primary myotubes. Mechanistically, ncls increases cellular cAMP concentration and ADP/ATP ratio, which further lead to the activation of AMPK signaling. Blocking AMPK signaling through a specific inhibitor significantly reduces FAO in myotubes. Finally, ncls also enhances mitochondrial membrane potential and reduces the formation of reactive oxygen species in cultured myotubes.

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