Skeletal Muscle Extracellular Matrix Research Articles

Integrin-associated ILK and PINCH1 protein content are reduced in skeletal muscle of maintenance haemodialysis patients.

Patients with renal failure undergoing maintenance haemodialysis are associated with insulin resistance and protein metabolism dysfunction. Novel research suggests that disruption to the transmembrane protein linkage between the cytoskeleton and the extracellular matrix in skeletal muscle may contribute to reduced amino acid metabolism and insulin resistance in haemodialysis. ILK, PINCH1 and pFAKTyr397 were significantly decreased in haemodialysis compared to controls, whereas Rac1 and Akt2showed no different between groups. Rac1 deletion in the Rac1 knockout model did not alter the expression of integrin-associated proteins. Phenylalanine kinetics were reduced in the haemodialysis group at 30 and 60min post meal ingestion compared to controls; both groups showed similar levels of insulin sensitivity and β-cell function. Key proteins in the integrin-cytoskeleton linkage are reduced in haemodialysis patients, suggesting for the first time that integrin-associated proteins dysfunction may contribute to reduced phenylalanine flux without affecting insulin resistance in haemodialysis patients. Muscle atrophy, insulin resistance and reduced muscle phosphoinositide 3-kinase-Akt signalling are common characteristics of patients undergoing maintenance haemodialysis (MHD). Disruption to the transmembrane protein linkage between the cytoskeleton and the extracellular matrix in skeletal muscle may contribute to reduced amino acid metabolism and insulin resistance in MHD patients. Eight MHD patients (age: 56±5years: body mass index: 32±2kgm-2 ) and non-diseased controls (age: 50±2years: body mass index: 31±1kgm-2 ) received primed continuous l-[ring-2 H5 ]phenylalanine before consuming a mixed meal. Phenylalanine metabolism was determined using two-compartment modelling. Muscle biopsies were collected prior to the meal and at 300min postprandially. In a separate experiment, skeletal muscle tissue from muscle-specific Rac1 knockout (Rac1mKO) was harvested to investigate whether Rac1 depletion disrupted the cytoskeleton-integrin linkage, allowing for cross-model examination of proteins of interest. ILK, PINCH1 and pFAKTyr397 were significantly lower in MHD (P<0.01). Rac1 and Akt showed no difference between groups for the human trial. Rac1 deletion in the Rac1mKO model did not alter the expression of integrin-associated proteins. Phenylalanine rates of appearance and disappearance, as well as metabolic clearance rates, were lower in the MHD group at 30 and 60min post meal ingestion compared to controls (P<0.05). Both groups showed similar levels of insulin sensitivity and β-cell function. Key proteins in the integrin-cytoskeleton linkage are reduced in MHD patients, suggesting for the first time that integrin-associated proteins dysfunction may contribute to reduced phenylalanine flux without affecting insulin resistance in haemodialysis patients.

Remodeling the Skeletal Muscle Extracellular Matrix in Older Age-Effects of Acute Exercise Stimuli on Gene Expression.

With advancing age, the skeletal muscle extracellular matrix (ECM) undergoes fibrotic changes that may lead to increased muscle stiffness, injury susceptibility and strength loss. This study tested the potential of different exercises to counter these changes by stimulating the activity of genes associated with ECM remodeling. Twenty-six healthy men (66.9 ± 3.9 years) were stratified to two of four groups, performing unilateral (i) conventional resistance exercise, (ii) conventional resistance exercise followed by self-myofascial release (CEBR), (iii) eccentric-only exercise (ECC) or (iv) plyometric jumps (PLY). The non-trained leg served as control. Six hours post-exercise, vastus lateralis muscle biopsy samples were analyzed for the expression of genes associated with ECM collagen synthesis (COL1A1), matrix metallopeptidases (collagen degradation; MMPs) and peptidase inhibitors (TIMP1). Significant between-group differences were found for MMP3, MMP15 and TIMP1, with the greatest responses in MMP3 and TIMP1 seen in CEBR and in MMP15 in ECC. MMP9 (3.24–3.81-fold change) and COL1A1 (1.47–2.40-fold change) were increased in CEBR and PLY, although between-group differences were non-significant. The expression of ECM-related genes is exercise-specific, with CEBR and PLY triggering either earlier or stronger remodeling than other stimuli. Training studies will test whether execution of such exercises may help counter age-associated muscle fibrosis.

Insulin Resistance, Skeletal Muscle Extracellular Matrix Remodeling, And The Effect Of Exercise

Extracellular matrix (ECM) remodeling in skeletal muscle is a potential mechanism linking obesity with metabolic dysfunction. It is also a constructive feature of skeletal muscle adaptation to exercise training. PURPOSE: To test the hypothesis that skeletal muscle ECM remodeling associated with insulin resistance can be minimized by exercise training. METHODS: Six-week-old male C57/BL6J mice (n=48) were divided into two groups, high-fat (60% calories from fat) diet (HF, n=36) and normal chow-fed control (C, n=12) group. After 12 weeks of feeding, HF mice developed insulin resistance, as confirmed by insulin and glucose tolerance tests (ITT and GTT). HF mice were then randomly assigned to three groups: high-fat diet only group (HFS, n=12), high-fat diet + aerobic exercise group (HF+AE, n=12), high-fat diet + resistance training group (HF+RT, n=12). The HF+AE and HF+RT groups were subject to aerobic (treadmill running) and resistive (vertical ladder climbing) training, for 12 weeks. After training, gastrocnemius muscle was harvested and analyzed for ECM factors using immunohistochemistry, ECM PCR array, and western blotting. ANOVA was performed to test the significance of group differences at p<0.05. RESULTS: High-fat feeding induced higher deposition of collagens (COLI, III and IV) in the skeletal muscle of HFS group, and increased gene and protein expression of MMP3, CDH1, ITGAL and SELL and decreased the expression of TIMP3 in HFS group, as compared to group C. These changes were minimized even reversed by either aerobic or resistance exercise training (mRNA fold change relative to C in HF+AE and HF+RT vs. HFS: MMP3, 1.1 and 1.6 vs. 2.6; CDH1, 1.7 and 1.1 vs. 2.0; ITGAL, 1.9 and -1.0 vs. 2.0; SELL, 1.4 and 1.0 vs. 2.1; TIMP3, 1.2 and -1.0 vs. -1.2; p<0.05). These effects were accompanied by a significant improvement in insulin sensitivity (GTT AUC glucose in mmol/l x 120 min: C, 27.2±3.0; HFS, 39.7 ± 7.0; HF+AE, 32.4± 7.5; HF+RT, 30.3± 6.1; p<0.05). CONCLUSION: Both aerobic and resistive exercise training can minimize changes in skeletal muscle ECM associated with insulin resistance. Skeletal muscle ECM remodeling may play a significant role in mediating the metabolic benefits of exercise training. This study was supported by National Nature Science Foundation of China (31470060).

Decellularized skeletal muscles display neurotrophic effects in three-dimensional organotypic cultures.

Skeletal muscle decellularization allows the generation of natural scaffolds that retain the extracellular matrix (ECM) mechanical integrity, biological activity, and three‐dimensional (3D) architecture of the native tissue. Recent reports showed that in vivo implantation of decellularized muscles supports muscle regeneration in volumetric muscle loss models, including nervous system and neuromuscular junctional homing. Since the nervous system plays pivotal roles during skeletal muscle regeneration and in tissue homeostasis, support of reinnervation is a crucial aspect to be considered. However, the effect of decellularized muscles on reinnervation and on neuronal axon growth has been poorly investigated. Here, we characterized residual protein composition of decellularized muscles by mass spectrometry and we show that scaffolds preserve structural proteins of the ECM of both skeletal muscle and peripheral nervous system. To investigate whether decellularized scaffolds could per se attract neural axons, organotypic sections of spinal cord were cultured three dimensionally in vitro, in presence or in absence of decellularized muscles. We found that neural axons extended from the spinal cord are attracted by the decellularized muscles and penetrate inside the scaffolds upon 3D coculture. These results demonstrate that decellularized scaffolds possess intrinsic neurotrophic properties, supporting their potential use for the treatment of clinical cases where extensive functional regeneration of the muscle is required.

Implications of Skeletal Muscle Extracellular Matrix Remodeling in Metabolic Disorders: Diabetes Perspective.

The extracellular matrix (ECM) provides a scaffold for cells, controlling biological processes and providing structural as well as mechanical support to surrounding cells. Disruption of ECM homeostasis results in several pathological conditions. Skeletal muscle ECM is a complex network comprising collagens, proteoglycans, glycoproteins, and elastin. Recent therapeutic approaches targeting ECM remodeling have been extensively deliberated. Various ECM components are typically found to be augmented in the skeletal muscle of obese and/or diabetic humans. Skeletal muscle ECM remodeling is thought to be a feature of the pathogenic milieu allied with metabolic dysregulation, obesity, and eventual diabetes. This narrative review explores the current understanding of key components of skeletal muscle ECM and their specific roles in the regulation of metabolic diseases. Additionally, we discuss muscle-specific integrins and their role in the regulation of insulin sensitivity. A better understanding of the importance of skeletal muscle ECM remodeling, integrin signaling, and other factors that regulate insulin activity may help in the development of novel therapeutics for managing diabetes and other metabolic disorders.

Exercise-Induced Increases in Insulin Sensitivity After Bariatric Surgery Are Mediated By Muscle Extracellular Matrix Remodeling.

Exercise seems to enhance the beneficial effect of bariatric (Roux-en-Y gastric bypass [RYGB]) surgery on insulin resistance. We hypothesized that skeletal muscle extracellular matrix (ECM) remodeling may underlie these benefits. Women were randomized to either a combined aerobic and resistance exercise training program following RYGB (RYGB + ET) or standard of care (RYGB). Insulin sensitivity was assessed by oral glucose tolerance test. Muscle biopsy specimens were obtained at baseline and 3 and 9 months after surgery and subjected to comprehensive phenotyping, transcriptome profiling, molecular pathway identification, and validation in vitro. Exercise training improved insulin sensitivity beyond surgery alone (e.g., Matsuda index: RYGB 123% vs. RYGB + ET 325%; P ≤ 0.0001). ECM remodeling was reduced by surgery alone, with an additive benefit of surgery and exercise training (e.g., collagen I: RYGB -41% vs. RYGB + ET -76%; P ≤ 0.0001). Exercise and RYGB had an additive effect on enhancing insulin sensitivity, but surgery alone did not resolve insulin resistance and ECM remodeling. We identified candidates modulated by exercise training that may become therapeutic targets for treating insulin resistance, in particular, the transforming growth factor-β1/SMAD 2/3 pathway and its antagonist follistatin. Exercise-induced increases in insulin sensitivity after bariatric surgery are at least partially mediated by muscle ECM remodeling.

3D myotube guidance on hierarchically organized anisotropic and conductive fibers for skeletal muscle tissue engineering.

Tissue engineering represents a promising approach for the functional restoration of large volumetric skeletal muscle loss. However, design and fabrication of 3D hierarchically organized scaffolds that closely mimic the natural micro-/nanostructures of skeletal muscle extracellular matrix (ECM) is still an ongoing challenge. Here, we constructed a hierarchically organized, anisotropic and conductive scaffold with microscale melt electrowriting (MEW) grooves parallel aligned on the top of unidirectionally oriented nanofibrous mesh to guide myoblast cell alignment, elongation and differentiation into myotubes. A 7-days study of H9c2 myoblast cells cultured on the scaffolds indicated that the combination of nanoscale and microscale anisotropic surface topography enhanced myogenesis. Specifically, the parallel patterned scaffold effectively enhanced both the elongation and maturation of myotubes, as indicated by the increased myotube length (>600μm), higher heavy myosin chain (MHC) surface coverage and maturation index on the parallel patterned scaffold compared to their pure (2.4-fold MHC surface coverage, 1.6-fold maturation index) and perpendicular patterned counterparts (1.7-fold MHC surface coverage, 1.5-fold maturation index). Furthermore, a gold nanolayer coating on the aligned nanofibrous mesh surface provided electroactive cues to enhance the formation of myotubes. With the increase of Au coating thickness, improved myoblasts alignment (74.6%, 80.5%, 85.6%, 94.9% for Au 0, 10, 40, 70nm respectively) and higher MHC-positive expression (57.5% for Au 0nm, 90.3%, 92.5%, 95.0% for Au10, 40, 70nm respectively) were observed. Finally, the formation and morphology of myotubes were also found dependent on the spacing between microgrooves (100, 200, 300μm), where myotubes formed on 200μm spacing scaffold showed the highest alignment (within 10° along nanofibers) and elongation (>850μm). The hierarchically organized, anisotropic and conductive fibers hold great potential for regeneration of skeletal muscle tissue.

Neonatal transplantation of iPSC-derived MSCs affects systemic collagen vi restoration in ullrich congenital muscular dystrophy mice

Background & Aim Collagen VI is ubiquitously expressed extracellular matrix of skeletal muscle, which is supplemented mainly by interstitial fibroblasts. Mutations in Col6 gene cause a spectrum of COL6-related myopathies, with Ullrich congenital muscular dystrophy (UCMD) regarded at the severe end. As collagen VI is systemically defective, patients with UCMD present early-onset muscle weakness, proximal joint contracture and distal hyperextensibility. The skeletal muscle of col6a1−/− mice have a subpopulation of very small-sized myofibers, representing the characteristic pathology in which newly regenerating myofibers fail to go through the normal maturation process and remain immature and small. We hypothesized that MSC transplantation could restore collagen VI in skeletal muscles with additional therapeutic effects and performed a transplantation study using human iPSC-derived MSCs (iMSCs) on neonatal Col6a1−/−/NSG mice. Methods, Results & Conclusion We injected 5 × 106 human iMSCs with a luciferase expressing piggy-bac vector into the abdominal cavity of Col6a1−/−/NSG mice on postnatal day 2 and analyzed them at 4 weeks. Mice that received a boosting ip transplantation at 4 weeks were analyzed at 8 weeks. They were compared with non-treated Col6a1−/−/NSG mice or WT/NSG mice. Intraperitoneally transplanted iMSCs were distributed to all major organs but the brain. Both the engraftment of donor cells and resulting collagen VI restoration were confirmed at the quadriceps and diaphragm. The mice showed increased muscle weight, with enlarged diameters as well as increased total number of myofibers. Functional tests at 8 weeks showed better performance in the treated mice. Further analysis revealed that continuous muscle regeneration was maintained in the transplanted Col6a1−/−/NSG mice, with increased number of MyoD+ cells and MYH3+ myofibers compared to mice without intervention. Therefore, collagen VI supplementation by iMSC transplantation may compensate the natural course of muscle atrophic changes observed in Col6a1−/−/NSG mice by facilitating healthy muscle maturation as well as maintaining continuous regeneration. This study demonstrated the histological and functional therapeutic effects of iMSC transplantation in Col6a1−/−/NSG mice while also revealing its mechanisms. These promising results are a proof of concept for systemic MSC transplantation as a therapy for UCMD.

Skeletal Muscle Extracellular Matrix - What Do We Know About Its Composition, Regulation, and Physiological Roles? A Narrative Review.

Skeletal muscle represents the largest body-composition component in humans. In addition to its primary function in the maintenance of upright posture and the production of movement, it also plays important roles in many other physiological processes, including thermogenesis, metabolism and the secretion of peptides for communication with other tissues. Research attempting to unveil these processes has traditionally focused on muscle fibers, i.e., the contractile muscle cells. However, it is a frequently overlooked fact that muscle fibers reside in a three-dimensional scaffolding that consists of various collagens, glycoproteins, proteoglycans, and elastin, and is commonly referred to as extracellular matrix (ECM). While initially believed to be relatively inert, current research reveals the involvement of ECM cells in numerous important physiological processes. In interaction with other cells, such as fibroblasts or cells of the immune system, the ECM regulates muscle development, growth and repair and is essential for effective muscle contraction and force transmission. Since muscle ECM is highly malleable, its texture and, consequently, physiological roles may be affected by physical training and disuse, aging or various diseases, such as diabetes. With the aim to stimulate increased efforts to study this still poorly understood tissue, this narrative review summarizes the current body of knowledge on (i) the composition and structure of the ECM, (ii) molecular pathways involved in ECM remodeling, (iii) the physiological roles of muscle ECM, (iv) dysregulations of ECM with aging and disease as well as (v) the adaptations of muscle ECM to training and disuse.

Influence of the integrin alpha-1 subunit and its relationship with high-fat diet upon extracellular matrix synthesis in skeletal muscle and tendon.

Integrins are important for mechanosensation in tissue and play, together with nutrition, a role in regulating extracellular matrix (ECM) in skeletal muscle and tendon. Integrin receptors are dimers that consist of an α and β subunit and bridge extracellular and intracellular signals. The present study investigates whether the deletion of the integrin receptor α1 subunit influences collagen and other matrix proteins in the musculotendinous tissue and whether it causes any compensatory changes in other integrin subunits in C57BL/6J mice. In addition, we study whether a high-fat diet (HFD) influences these responses in muscle or tendon. Mice on a HFD had a higher number of non-enzymatic cross-links in skeletal muscle ECM and increased gene expression of collagen and other extracellular matrix proteins. In contrast to gene expression, total collagen protein content was decreased by HFD in the muscle with no change in tendon. Integrin α1 subunit knockout resulted in a decrease of collagen type I and III, TGF-β1 and IGF-1 gene expression in muscle of HFD mice but did not affect total collagen protein compared with wild-type (WT) littermates in either muscle or tendon. There was no compensatory increase in the genes that express other integrin subunits. In conclusion, HFD induced a significant increase in expression of ECM genes in muscle. On the protein level, HFD resulted in a lower collagen content in muscle. Tendons were unaffected by the diet. Deletion of the integrin α1 subunit did not affect collagen protein or gene expression in muscle or tendon.

Dose optimization of decellularized skeletal muscle extracellular matrix hydrogels for improving perfusion and subsequent validation in an aged hindlimb ischemia model.

Peripheral artery disease (PAD) affects more than 27 million individuals in North America and Europe, and current treatment strategies mainly aim to restore blood perfusion. However, many patients are ineligible for existing procedures, and these therapies are often ineffective. Previous studies have demonstrated success of an injectable decellularized skeletal muscle extracellular matrix (ECM) hydrogel in a young rat hindlimb ischemia model of PAD, but further pre-clinical studies are necessary prior to clinical translation. In this study, varying concentrations of a skeletal muscle ECM hydrogel were investigated for material properties and in vivo effects on restoring blood perfusion. Rheological measurements indicated an increase in viscosity and mechanical strength with the higher concentrations of the ECM hydrogels. When injecting dye-labelled ECM hydrogels into a healthy rat, differences were also observed for the spreading and degradation rate of the various concentrations. The three concentrations for the ECM hydrogel were then further examined in a young rat hindlimb ischemia model. The efficacy of the optimal ECM hydrogel concentration was then further confirmed in an aged mouse hindlimb ischemia model. These results further validate the use of decellularized skeletal muscle ECM hydrogels for improving blood perfusion in small animal models of PAD.

Diversity of extracellular matrix morphology in vertebrate skeletal muscle.

Existing data suggest the extracellular matrix (ECM) of vertebrate skeletal muscle consists of several morphologically distinct layers: an endomysium, perimysium, and epimysium surrounding muscle fibers, fascicles, and whole muscles, respectively. These ECM layers are hypothesized to serve important functional roles within muscle, influencing passive mechanics, providing avenues for force transmission, and influencing dynamic shape changes during contraction. The morphology of the skeletal muscle ECM is well described in mammals and birds; however, ECM morphology in other vertebrate groups including amphibians, fish, and reptiles remains largely unexamined. It remains unclear whether a multilayered ECM is a common feature of vertebrate skeletal muscle, and whether functional roles attributed to the ECM should be considered in mechanical analyses of non-mammalian and non-avian muscle. To explore the prevalence of a multilayered ECM, we used a cell maceration and scanning electron microscopy technique to visualize the organization of ECM collagen in muscle from six vertebrates: bullfrogs (Lithobates catesbeianus), turkeys (Meleagris gallopavo), alligators (Alligator mississippiensis), cane toads (Rhinella marina), laboratory mice (Mus musculus), and carp (Cyprinus carpio). All muscles studied contained a collagen-reinforced ECM with multiple morphologically distinct layers. An endomysium surrounding muscle fibers was apparent in all samples. A perimysium surrounding groups of muscle fibers was apparent in all but carp epaxial muscle; a muscle anatomically, functionally, and phylogenetically distinct from the others studied. An epimysium was apparent in all samples taken at the muscle periphery. These findings show that a multilayered ECM is a common feature of vertebrate muscle and suggest that a functionally relevant ECM should be considered in mechanical models of vertebrate muscle generally. It remains unclear whether cross-species variations in ECM architecture are the result of phylogenetic, anatomical, or functional differences, but understanding the influence of such variation on muscle mechanics may prove a fruitful area for future research.

Spatio-temporal expression and distribution of collagen VI during zebrafish development

Collagen VI (ColVI) is an extracellular matrix (ECM) protein involved in a range of physiological and pathological conditions. Zebrafish (Danio rerio) is a powerful model organism for studying vertebrate development and for in vivo analysis of tissue patterning. Here, we performed a thorough characterization of ColVI gene and protein expression in zebrafish during development and adult life. Bioinformatics analyses confirmed that zebrafish genome contains single genes encoding for α1(VI), α2(VI) and α3(VI) ColVI chains and duplicated genes encoding for α4(VI) chains. At 1 day post-fertilization (dpf) ColVI transcripts are expressed in myotomes, pectoral fin buds and developing epidermis, while from 2 dpf abundant transcript levels are present in myosepta, pectoral fins, axial vasculature, gut and craniofacial cartilage elements. Using newly generated polyclonal antibodies against zebrafish α1(VI) protein, we found that ColVI deposition in adult fish delineates distinct domains in the ECM of several organs, including cartilage, eye, skin, spleen and skeletal muscle. Altogether, these data provide the first detailed characterization of ColVI expression and ECM deposition in zebrafish, thus paving the way for further functional studies in this species.

Unraveling the pathophysiology of Bethlem Myopathy using a unique zebrafish model for the disease

Bethlem myopathy (BM) is a neuromuscular disease characterized by joint contractures and muscle weakness. BM is caused by mutations in one of the genes encoding one of the three α-chains of collagen VI (COLVI), a component of the skeletal muscle extracellular matrix. Nowadays, an unresolved question is to understand how alteration of COLVI located outside the muscle cells leads to functional modifications in muscle fibers. The zebrafish model col6a1Δex14 is currently the unique animal model of the disease since it is the only model to reproduce a mutation that is the most frequently found in BM patients. In patient and col6a1Δex14 zebrafish muscles, the structure of the sarcoplasmic reticulum has been found to be altered, thus suggesting dysfunction in intracellular Ca2+ handling and/or in ion channels that are known to control Ca2+ homeostasis and to play pivotal roles in muscle function and pathogenesis. Therefore, our project aims at exploring the properties of ion channels and intracellular Ca2+ regulation using electrophysiological approaches and intracellular Ca2+ measurement at rest and during activity in isolated muscle fibers from col6a1Δex14 zebrafish. On one hand, this project should contribute to decipher how alteration in an extracellular matrix component transduces pathogenic signals within muscle fiber and should possibly lead to identify therapeutic targets for this currently incurable disease. On the other hand, because functional studies on zebrafish muscle cells are scarce, this project will provide a sound database on the electrophysiological properties of this cell model.

Manufacturing considerations for producing and assessing decellularized extracellular matrix hydrogels.

Although several decellularized extracellular matrix (ECM) sheets or patches have been commercialized for use in the clinic, only one injectable decellularized ECM hydrogel, a decellularized myocardial matrix, has reached clinical trials. Consequently, very little information is available for established manufacturing standards or assessments of these materials. Here we present detailed methodology for investigating three parameters related to manufacturing optimization for a porcine derived skeletal muscle ECM hydrogel - animal-to-animal variability, bioburden reduction, and harvesting conditions. Results from characterization assays, including residual dsDNA content and sulfated glycosaminoglycan content, did not yield noteworthy differences amongst individual animals or following the addition of a bioburden reducing agent. However, the tissue collected under different harvesting conditions contained varying amounts of fat, and the protein compositions of the decellularized products differed, which could ultimately impact subsequent efficacy in vitro or in vivo. As decellularized ECM hydrogels continue to be evaluated for various applications, the differences between laboratory-scale and manufacturing-scale material batches should be thoroughly considered to avoid costly and timely optimization during scale-up.

Extracellular matrix composition of connective tissues: a systematic review and meta-analysis

The function of connective tissues depends on the physical and biochemical properties of their extracellular matrix (ECM), which are in turn dictated by ECM protein composition. With the primary objective of obtaining quantitative estimates for absolute and relative amounts of ECM proteins, we performed a systematic review of papers reporting protein composition of human connective tissues. Articles were included in meta-analysis if they contained absolute or relative quantification of proteins found in the ECM of human bone, adipose tissue, tendon, ligament, cartilage and skeletal muscle. We generated absolute quantitative estimates for collagen in articular cartilage, intervertebral disk (IVD), skeletal muscle, tendon, and adipose tissue. In addition, sulfated glycosaminoglycans were quantified in articular cartilage, tendon and skeletal muscle; total proteoglycans in IVD and articular cartilage, fibronectin in tendon, ligament and articular cartilage, and elastin in tendon and IVD cartilage. We identified significant increases in collagen content in the annulus fibrosus of degenerating IVD and osteoarthritic articular cartilage, and in elastin content in degenerating disc. In contrast, collagen content was decreased in the scoliotic IVD. Finally, we built quantitative whole-tissue component breakdowns. Quantitative estimates improve our understanding of composition of human connective tissues, providing insights into their function in physiology and pathology.

The Development of Cancer Cachexia Negatively Impacts Skeletal Muscle Extracellular Matrix Remodeling

The Development of Cancer Cachexia Negatively Impacts Skeletal Muscle Extracellular Matrix Remodeling

Inhibition of TLR9 attenuates skeletal muscle fibrosis in aged sarcopenic mice via the p53/SIRT1 pathway.

Sarcopenia is an age-related syndrome characterized by a gradual loss of muscle mass and function, but its pathophysiological mechanism remains unclear. Skeletal muscle extracellular matrix (ECM) remodeling is an important pathological change in sarcopenia, and fibrosis is the most obvious manifestation of this change. We found that the expression of the immunoreceptor Toll-like receptor 9 (TLR9) is significantly increased in skeletal muscle in aged mice and is positively related to muscle fibrosis. Moreover, in previous reports, the longevity gene Sirt1 was reported to attenuate ECM deposition and improve muscle function. In this study, we hypothesized that TLR9 modulated skeletal muscle fibrosis via Sirt1. We used TLR9 knockout (TLR9 KO) mice and C57 mice, and grip strength and body composition were compared at different ages. We found that TLR9 knockout significantly attenuated skeletal muscle fibrosis and improved muscle function in aged mice. Furthermore, silent information regulator 1 (Sirt1) activity in mice was inhibited by Ex527, which is a specific inhibitor of Sirt1. Negative Sirt1 regulation via the activation of TLR9-related signaling pathways participated in skeletal muscle fibrosis in the sarcopenic mice, and this process might mediated by the Sirt1/Smad signaling pathway. Our findings revealed that fibrosis changes in the gastrocnemius muscle in sarcopenic mice are closely related to TLR9 activation, and TLR9 modulation could be a therapeutic strategy for combating sarcopenia during aging.

Analysis of α-Dystroglycan/LG Domain Binding Modes: Investigating Protein Motifs That Regulate the Affinity of Isolated LG Domains.

Dystroglycan (DG) is an adhesion complex that links the cytoskeleton to the surrounding extracellular matrix in skeletal muscle and a wide variety of other tissues. It is composed of a highly glycosylated extracellular α-DG associated noncovalently with a transmembrane β-DG whose cytodomain interacts with dystrophin and its isoforms. Alpha-dystroglycan (α-DG) binds tightly and in a calcium-dependent fashion to multiple extracellular proteins and proteoglycans, each of which harbors at least one, or, more frequently, tandem arrays of laminin-globular (LG) domains. Considerable biochemical and structural work has accumulated on the α-DG-binding LG domains, highlighting a significant heterogeneity in ligand-binding properties of domains from different proteins as well as between single and multiple LG domains within the same protein. Here we review biochemical, structural, and functional information on the LG domains reported to bind α-dystroglycan. In addition, we have incorporated bioinformatics and modeling to explore whether specific motifs responsible for α-dystroglycan recognition can be identified within isolated LG domains. In particular, we analyzed the LG domains of slits and agrin as well as those of paradigmatic α-DG non-binders such as laminin-α3. While some stretches of basic residues may be important, no universally conserved motifs could be identified. However, the data confirm that the coordinated calcium atom within the LG domain is needed to establish an interaction with the sugars of α-DG, although it appears that this alone is insufficient to mediate significant α-DG binding. We develop a scenario involving different binding modes of a single LG domain unit, or tandemly repeated units, with α-DG. A variability of binding modes might be important to generate a range of affinities to allow physiological regulation of this interaction, reflecting its crucial biological importance.

Age-specific response of skeletal muscle extracellular matrix to acute resistance exercise: A pilot study

The extracellular matrix (ECM) plays an essential role in the development, growth and repair of skeletal muscles and serves to transmit contractile force. However, its regulation is poorly understood. This study investigates the age-specificity of the effects of acute resistance exercise on ECM gene expression. To this purpose, five young (YM, 23.8 ± 2.2 yrs.) and 5 elderly (EM, 66.8 ± 4.1 yrs.) men performed one session of unilateral leg press and leg extension exercises. Six hours post-exercise, biopsies were taken from the vastus lateralis muscles of both legs. A PCR array was used to profile the expression of 84 ECM-related genes, of which 6 were validated by qPCR. The PCR array found 9 and 4 ECM-associated genes to be selectively altered (>1.5-fold change) in YM or EM only. Four further genes were upregulated in YM but downregulated in EM. Of the 6 genes validated on individual samples MMP9 expression increased in YM (9.7-fold) and decreased (0.2-fold) in EM. MMP15 was downregulated in EM only (0.6-fold). A significant correlation between leg extension 1 RM and changes in COL7A1 expression (ρ = 0.71) suggests a potential influence of fitness levels. In conclusion, acute resistance exercise affects ECM gene expression at least partly in an age-specific manner. The altered expression of genes encoding matrix metalloproteinases (MMP3, MMP9, MMP15) highlights the role of remodelling processes in the response to an acute bout of resistance exercise. Larger studies are required to verify the age-associated differences in gene expression profiles and establish their functional implications.