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The role of extracellular matrix composition in structure and function of bioengineered skeletal muscle.

ABSTRACT: One of the obstacles to the potential clinical utility of bioengineered skeletal muscle is its limited force generation capacity. Since engineered muscle, unlike most native muscle tissue, is composed of relatively short myofibers, we hypothesized that, its force production and transmission would be profoundly influenced by cell-matrix interactions. To test this hypothesis, we systematically varied the matrix protein type (collagen I/fibrin/Matrigel) and concentration in engineered, hydrogel-based neonatal rat skeletal muscle bundles and assessed the resulting tissue structure, generation of contractile force, and intracellular Ca(2+) handling. After two weeks of culture, the muscle bundles consisted of highly aligned and cross-striated myofibers and exhibited standard force-length and force-frequency relationships achieving tetanus at 40 Hz. The use of 2 mg/ml fibrin (control) yielded isometric tetanus amplitude of 1.4 ± 0.3 mN as compared to 0.9 ± 0.4 mN measured in collagen I-based bundles. Higher fibrin and Matrigel concentrations synergistically yielded further increase in active force generation to 2.8 ± 0.5 mN without significantly affecting passive mechanical properties, tetanus-to-twitch ratio, and twitch kinetics. Optimized matrix composition yielded significant cellular hypertrophy (protein/DNA ratio = 11.4 ± 4.1 vs. 6.5 ± 1.9 ?g/?g in control) and a prolonged Ca(2+) transient half-width (Ca(50) = 232.8 ± 33.3 vs. 101.7 ± 19.8 ms). The use of growth-factor-reduced Matrigel, instead of standard Matrigel did not alter the obtained results suggesting enhanced cell-matrix interactions rather than growth factor supplementation as an underlying cause for the measured increase in contractile force. In summary, biomaterial-based manipulation of cell-matrix interactions represents an important target for improving contractile force generation in engineered skeletal muscle.

SUBMITTER: Hinds S

PROVIDER: S-EPMC3057410 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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The role of extracellular matrix composition in structure and function of bioengineered skeletal muscle.

Hinds Sara S Bian Weining W Dennis Robert G RG Bursac Nenad N

Biomaterials 20110213 14

One of the obstacles to the potential clinical utility of bioengineered skeletal muscle is its limited force generation capacity. Since engineered muscle, unlike most native muscle tissue, is composed of relatively short myofibers, we hypothesized that, its force production and transmission would be profoundly influenced by cell-matrix interactions. To test this hypothesis, we systematically varied the matrix protein type (collagen I/fibrin/Matrigel) and concentration in engineered, hydrogel-bas ...[more]

PMID: 21324402

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Project description:ObjectiveSkeletal muscle plasticity and remodeling are critical for adapting tissue function to use, disuse, and regeneration. The aim of this study was to identify genes and molecular pathways that regulate the transition from atrophy to compensatory hypertrophy or recovery from injury. Here, we have used a mouse model of hindlimb unloading and reloading, which causes skeletal muscle atrophy, and compensatory regeneration and hypertrophy, respectively.MethodsWe analyzed mouse skeletal muscle at the transition from hindlimb unloading to reloading for changes in transcriptome and extracellular fluid proteome. We then used qRT-PCR, immunohistochemistry, and bulk and single-cell RNA sequencing data to determine Mustn1 gene and protein expression, including changes in gene expression in mouse and human skeletal muscle with different challenges such as exercise and muscle injury. We generated Mustn1-deficient genetic mouse models and characterized them in vivo and ex vivo with regard to muscle function and whole-body metabolism. We isolated smooth muscle cells and functionally characterized them, and performed transcriptomics and proteomics analysis of skeletal muscle and aorta of Mustn1-deficient mice.ResultsWe show that Mustn1 (Musculoskeletal embryonic nuclear protein 1, also known as Mustang) is highly expressed in skeletal muscle during the early stages of hindlimb reloading. Mustn1 expression is transiently elevated in mouse and human skeletal muscle in response to intense exercise, resistance exercise, or injury. We find that Mustn1 expression is highest in smooth muscle-rich tissues, followed by skeletal muscle fibers. Muscle from heterozygous Mustn1-deficient mice exhibit differences in gene expression related to extracellular matrix and cell adhesion, compared to wild-type littermates. Mustn1-deficient mice have normal muscle and aorta function and whole-body glucose metabolism. We show that Mustn1 is secreted from smooth muscle cells, and that it is present in arterioles of the muscle microvasculature and in muscle extracellular fluid, particularly during the hindlimb reloading phase. Proteomics analysis of muscle from Mustn1-deficient mice confirms differences in extracellular matrix composition, and female mice display higher collagen content after chemically induced muscle injury compared to wild-type littermates.ConclusionsWe show that, in addition to its previously reported intracellular localization, Mustn1 is a microprotein secreted from smooth muscle cells into the muscle extracellular space. We explore its role in muscle ECM deposition and remodeling in homeostasis and upon muscle injury. The role of Mustn1 in fibrosis and immune infiltration upon muscle injury and dystrophies remains to be investigated, as does its potential for therapeutic interventions.

Project description: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.

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The role of extracellular matrix composition in structure and function of bioengineered skeletal muscle.

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The role of extracellular matrix composition in structure and function of bioengineered skeletal muscle.

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