Morphology of perimysial and endomysial connective tissue in skeletal muscle

The fibrous components of the endomysium and perimysium of various muscles from four animal species were examined by scanning electron microscopy. All muscles were qualitatively similar. Endomysium consisted of a dense feltwork of collagen fibrils completely covering individual muscle fibre surfaces. Perimysium consisted of three fibrous components, (1) coarse, crimped fibres laid down in a well-ordered criss-cross pattern, (2) a loose feltwork of non-crimped fibrils, (3) fine noncrimped bundles of fibrils with no directional organization. The perimysium showed gradation from dense sheets of collagen down to the most delicate of sheets found on every muscle fibre surface overlying the endomysium.     

Indices of skeletal muscle damage and connective tissue breakdown following eccentric muscle contractions

 Indirect indices of exercise-induced human skeletal muscle damage and connective tissue breakdown were studied following a single bout of voluntary eccentric muscle contractions. Subjects (six female, two male), mean (SD) age 22 (2) years performed a bout of 50 maximum voluntary eccentric contractions of the knee extensors of a single leg. The eccentric exercise protocol induced muscle soreness (P < 0.05 Wilcoxon test), chronic force loss, and a decline in the 20:100 Hz percutaneous electrical myostimulation force ratio [P < 0.01, repeated measures analysis of variance (ANOVA)]. Serum creatine kinase (CK) and lactate dehydrogenase (LDH) activities were elevated (P < 0.01, repeated measures ANOVA) following the bout. The mean (SD) CK and LDH levels recorded 3 days post-exercise were 2815 (4144) IU · l^–1 and 375 (198) IU · l^–1, respectively. Serum alkaline phosphatase activity showed no changes throughout the study, and a non-significant increase (P = 0.058, repeated measures ANOVA) in pyridinoline was recorded following the bout. Urinary hydroxyproline (HP) and hydroxylysine (HL) excretion, expressed in terms of creatinine (Cr) concentration, increased after exercise (P < 0.05 and P < 0.01, respectively, repeated measures ANOVA). An increased HP:Cr was recorded 2 days post-exercise and HL:Cr was increased above baseline on days 2, 5, and 9 post-exercise. This indirect evidence of exercise-induced muscle damage suggests that myofibre disruption was caused by the eccentric muscle contractions. Elevated urine concentrations of indirect indices of collagen breakdown following eccentric muscle contractions suggests an increased breakdown of connective tissue, possibly due to a localised inflammatory response. Accepted: 9 October 1996     

An immunological study of glycosaminoglycans in the connective tissue of bovine and cod skeletal muscle

The presence of sulfated glycosaminoglycans (GAGs) was demonstrated in the connective tissue of bovine and cod skeletal muscle by histochemical staining using Alcian blue added MgCl₂ (0.06 M and 0.4 M, respectively). For further identification of the sulfated GAGs, a panel of monoclonal antibodies, 1B5, 2B6, 3B3 and 5D4 was used that recognizes epitopes in chondroitin-0-sulfate (C0S), chondroitin-4-sulfate/dermatan sulfate (C4S/DS), chondroitin-6-sulfate (C6S) and keratan sulfate (KS), respectively. Light microscopy and Western blotting techniques showed that in bovine and cod muscle C0S and C6S were primarily localized pericellularly, whereas cod exhibited a more intermittent staining. C4S was expressed around the separate cells and also in the perimysium and myocommata. In contrast to bovine muscle, which hardly expressed highly sulfated KS, cod exhibited a very strong and consistent staining. Western blotting showed that C0S and C6S were mainly associated with proteoglycans (PGs) of high molecular sizes in both species. Contrary to bovine muscle, C4S in cod was associated with molecules of various sizes. Both cod and bovine muscle contained KSPGs of similar sizes as C4S. KSPGs of different sizes and buoyant densities, sensitive to keratanase I and II were found expressed in cod.     

No effect of menstrual cycle on myofibrillar and connective tissue protein synthesis in contracting skeletal muscle

We tested the hypothesis that acute exercise would stimulate synthesis of myofibrillar protein and intramuscular collagen in women and that the phase of the menstrual cycle at which the exercise took place would influence the extent of the change. Fifteen young, healthy female subjects were studied in the follicular (FP, n=8) or the luteal phase (LP, n=7, n=1 out of phase) 24 h after an acute bout of one-legged exercise (60 min of kicking at 67% W(max)), samples being taken from the vastus lateralis in both the exercised and resting legs. Rates of synthesis of myofibrillar and muscle collagen proteins were measured by incorporation of [(13)C]leucine. Myofibrillar protein synthesis (means+/-SD; rest FP: 0.053+/-0.009%/h, LP: 0.055+/-0.013%/h) was increased at 24-h postexercise (FP: 0.131+/-0.018%/h, P<0.05, LP: 0.134+/-0.018%/h, P< 0.05) with no differences between phases. Similarly, muscle collagen synthesis (rest FP: 0.024+/- 0.017%/h, LP: 0.021+/- 0.006%/h) was elevated at 24-h postexercise (FP: 0.073+/- 0.016%/h, P<0.05, LP: 0.072+/- 0.015%/h, P<0.05), but the responses did not differ between menstrual phases. Therefore, there is no effect of menstrual cycle phase, at rest or in response to an acute bout of exercise, on myofibrillar protein synthesis and muscle collagen synthesis in women.     

Engineering vascularized skeletal muscle tissue

One of the major obstacles in engineering thick, complex tissues such as muscle is the need to vascularize the tissue in vitro. Vascularization in vitro could maintain cell viability during tissue growth, induce structural organization and promote vascularization upon implantation. Here we describe the induction of endothelial vessel networks in engineered skeletal muscle tissue constructs using a three-dimensional multiculture system consisting of myoblasts, embryonic fibroblasts and endothelial cells coseeded on highly porous, biodegradable polymer scaffolds. Analysis of the conditions for induction and stabilization of the vessels in vitro showed that addition of embryonic fibroblasts increased the levels of vascular endothelial growth factor expression in the construct and promoted formation and stabilization of the endothelial vessels. We studied the survival and vascularization of the engineered muscle implants in vivo in three different models. Prevascularization improved the vascularization, blood perfusion and survival of the muscle tissue constructs after transplantation.     

The synthesis of connective tissue protein in smooth muscle cells

The synthesis of elastin by smooth muscle cells was clearly demonstrated by amino acid analyses and the presence of lysine-derived crosslinks. The values obtained were compatible with those found in amorphous elastin isolated from rabbit aortic tissue. Collagen synthesis by these same cells was monitored by the appearance of [^{1 4}C] hydroxyproline when the cells were grown in the presence of [^{1 4}C] proline. When the cells were pulsed with [^{1 4}C] lysine, one could detect [^{1 4}C] hydroxylysine and [^{1 4}C] glucosylgalactosylhydroxylysine. Further evidence for the synthesis of elastin and collagen was the finding of radiolabelled ϵ-hydroxynorleucine and the reduced aldol condensate of two residues of allysine after reduction of [^{1 4}C] lysine pulsed cells with NaBH₄.     

Biomaterials for skeletal muscle tissue engineering

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Ultrastructural changes in the connective tissue of skeletal muscles following denervation]

The ultrastructural changes found in the endomysium of rats after denervation of the diaphragm and m. plantaris were studied. Within the first week after crossing the peripheral nerve in the nedomysium there appeared an increased amount of neutrophils and monocytes as well as phagocytic material in the cytoplasm of histocytes. Activization of the cytoplasm of fibroblasts which manifested itself in the appearance of numerous vesicles and multiple free and bound ribosomes was detected by the end of the second and the beginning of the third weeks after denervation. At the same period eosinophils invaded the endomysium and became closely surrounded by numerous collagenic fibres. After reparation of neuromuscular synapses these changes disappeared. On the basis of these results and others founded in previous studies of denervated and reinnervated skeletal muscles the authors consider these changes in the endomysium appearing under the above experimental conditions to be manifestations of metabolic interrelations between the endomysium connective tissue and muscle fibres.     

Alanine metabolism in skeletal muscle in tissue culture

Alanine production by skeletal muscle in tissue culture was studied using an established myogenic line (L₆) of rat skeletal muscle cells. Correlation analyses were performed on rates of metabolism of alanine, glucose, lactate and pyruvate over incubation periods up to 96 h. Alanine production did not correlate significantly with glucose utilization (r = 0.24, P < 0.20). Alanine production, however, did correlate with lactate production (r = 0.72, P < 0.0005) as well as medium (r = 0.50, P < 0.025) and intracellular (r = 0.85, P < 0.0005) pyruvate concentrations. The intercepts of the latter two correlation analyses indicated that when medium or cell pyruvate fell below 0.28 mM or 1 nmol/mg protein, respectively, net alanine consumption occurred. Alanine synthesis also correlated (r = 0.71, P < 0.0005) with the percent change in the cell mass action ratio for the sum of the alanine and aspartate aminotransferase reactions, i.e., [alanine] [malate]/[aspartate] [lactate]. These results suggest that alanine production is not necessarily linked to the rate of glucose utilization but rather to pyruvate overflow above a critical intracellular level; under conditions of pyruvate overflow, alanine synthesis is driven by the tendency to establish equilibrium between metabolites of the linked amino acid transaminases in skeletal muscle.     

Synaptic activity and connective tissue remodeling in denervated frog muscle

Denervation of skeletal muscle results in dramatic remodeling of the cellular and molecular composition of the muscle connective tissue. This remodeling is concentrated in muscle near neuromuscular junctions and involves the accumulation of interstitial cells and several extracellular matrix molecules. Given the role of extracellular matrix in neurite outgrowth and synaptogenesis, we predict that this remodeling of the junctional connective tissue directly influences the regeneration of the neuromuscular junction. As one step toward understanding the role of this denervation-induced remodeling in synapse formation, we have begun to look for the signals that are involved in initiating the junctional accumulations of interstitial cells and matrix molecules. Here, the role of muscle inactivity as a signal was examined. The distributions of interstitial cells, fibronectin, and tenascin were determined in muscles inactivated by presynaptic blockade of muscle activity with tetrodotoxin. We found that blockade of muscle activity for up to 4 wk produced neither the junctional accumulation of interstitial cells nor the junctional concentrations of tenascin and fibronectin normally present in denervated frog muscle. In contrast, the muscle inactivity induced the extrajunctional appearance of two synapse-specific molecules, the acetylcholine receptor and a muscle fiber antigen, mAb 3B6. These results demonstrate that the remodeling of the junctional connective tissue in response to nerve injury is a unique response of muscle to denervation in that it is initiated by a mechanism that is independent of muscle activity. Thus connective tissue remodeling in denervated skeletal muscle may be induced by signals released from or associated with the nerve other than the evoked release of neurotransmitter.     

The efficiency of in vitro isolation and myogenic differentiation of MSCs derived from adipose connective tissue,bone marrow,and skeletal muscle tissue

The objective of the study is to evaluate efficiency of in vitro isolation and myogenic differentiation of mesenchymal stem cells (MSCs) derived from adipose connective tissue (AD-MSCs), bone marrow (BM-MSCs), and skeletal muscle tissue (MC-MSCs). MSCs were isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue of two adult 6-wk-old rats. Cultured MSCs were treated with 5-azacytidine (AZA) to induce myogenic differentiation. Isolated MSCs and differentiated cells were evaluated by immunocytochemistry (ICC), fluorescence-activated cell sorting (FACS), PCR, and RT-PCR. AD-MSCs showed the highest proliferation rate while BM-MSCs had the lowest one. In ICC, isolated MSCs had strong CD90- and CD44-positive expression and negative expression of CD45, CD31, and CD34, while AZA-treated MSCs had strong positive desmin expression. In FACS analysis, AD-MSCs had the highest percentage of CD90- and CD44-positive-expressing cells (99% and 96%) followed by BM-MSCs (97% and 94%) and MC-MSCs (92% and 91%).At 1 wk after incubation with AZA treatment, the peak of myogenin expression reached 93% in differentiated MC-MSCs, 83.3% in BM-MSCs, and 77% in AD-MSCs. MSCs isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue have the same morphology and phenotype, but AD-MSCs were the most easily accessible and had the highest rate of growth on cultivation and the highest percentage of stem cell marker expression. Moreover, although MC-MSCs showed the highest rate of myogenic differentiation potential and expression of myoblast markers, AD-MSCs and BM-MSCs still can be valuable alternatives. The differentiated myoblastic cells could be an available new choice for myoblastic auto-transplantation in regeneration medicine.     

The synthesis of connective tissue protein in smooth muscle cells.

The synthesis of elastin by smooth muscle cells was clearly demonstrated by amino acid analyses and the presence of lysine-derived crosslinks. The values obtained were compatible with those found in amorphous elastin isolated from rabbit aortic tissue. Collagen synthesis by these same cells was monitored by the appearance of [14C] hydroxyproline when the cells were grown in the presence of [14C] proline. When the cells were pulsed with [14C] lysine, one could detect [14C] hydroxylysine and [14C] glucosylgalactosylhydroxylysine. Further evidence for the synthesis of elastin and collagen was the finding of radiolabelled epsilon-hydroxynorleucine and the reduced aldol condensate of two residues of allysine after reduction of [14C] lysine pulsed cells with NaBH4.     

Antigen-induced antigen endocytosis in primitive reticular cells of connective tissue of innervated and denervated skeletal muscle from the allergized guinea pig

Electron microscope studies demonstrated that soluble proteins (ferritin and horseradish peroxidase) administered in vitro to strips of guinea pig diaphragmatic muscle under physiological conditions were internalized by vesicles of primitive reticular cells (PRCs) found in the connective tissue. This endocytosis effect was enhanced when the muscle preparations were obtained from animals actively allergized to the protein. Denervation increased the occurrence of peroxidase-positive vesicles in the peroxidase-antiperoxidase system. These results suggest that the antigen-antibody interaction induces endocytosis directed to the specific antigen.     

Alanine metabolism in skeletal muscle in tissue culture.

Alanine production by skeletal muscle in tissue culture was studied using an established myogenic line (L6) of rat skeletal muscle cells. Correlation analyses were performed on rates of metabolism of alanine, glucose, lactate and pyruvate over incubation periods up to 96 h. Alanine production did not correlate significantly with glucose utilization (r = 0.24, P less than 0.20). Alanine production, however, did correlate with lactate production (r = 0.72, P less than 0.0005) as well as medium (r = 0.50, P less than 0.025) and intracellular (r = 0.85, P less than 0.0005) pyruvate concentrations. The intercepts of the latter two correlation analyses indicated that when medium or cell pyruvate fell below 0.28 mM or 1 nmol/mg protein, respectively, net alanine consumption occurred. Alanine synthesis also correlated (r = 0.71, P less than 0.0005) with the percent change in the cell mass action ratio for the sum of the alanine and aspartate aminotransferase reactions, i.e., [alanine] [malate]/[aspartate] [lactate]. These results suggest that alanine production is not necessarily linked to the rate of glucose utilization but rater to pyruvate overflow above a critical intracellular level; under conditions of pyruvate overflow, alanine synthesis is driven by the tendency to establish equilibrium between metabolites of the linked amino acid transaminases in skeletal muscle.     

Determination of lipoprotein-lipase activity in human skeletal muscle tissue.

An in vitro assay system was developed for the determination of lipoprotein-lipase activity in 10--30-mg specimens of human skeletal muscle tissue. The reaction medium of the assay was based on a glycine buffer of pH 8.3 (at 37 degrees C) with a heparin concentration of 1.5 g/l (about 180 IU/ml). The enzyme activity was measured as the release of [3H]oleic acid from a serum-activated, triglyceride emulsion, in which [3H]trioleate was used as trace substance. The enzyme activity studied had the characteristic properties of lipoprotein-lipase activity, i.e. it was activated by the addition of serum or apolipoprotein C-II and inhibited in the presence of high ionic strength, protamine sulphate or apolipoprotein C-III. A mean Km of 0.40 +/- 0.13 (S.D.) mmol/l for triglyceride substrate was found in tissue samples that had very different concentrations of lipoprotein-lipase activity. This Km was similar to the low fasting concentrations of very low density lipoprotein triglycerides often found in healthy individuals. The lipoprotein-lipase activity was not decreased freezing and storing the tissue specimens in liquid nitrogen. The within-day variation of the method was 16 percent and the between-day variation 8 percent. Muscle tissue from the vastus lateralis muscle had, on the average, a 60 percent higher concentration of lipoprotein-lipase activity than the rectus abdominis muscle in the same subject.     

A note on adhesion of bacteria to chicken muscle connective tissue

Laboratory cultures of bacteria were tested for the ability to attach to collagen fibres of intact chicken muscle connective tissue. All salmonellas, fimbriate strains of Escherichia coli and a strain of Campylobacter coli were able to attach to tissue only when suspended in distilled water. Prior immersion of tissue in sterile water for 20 min or extended immersion in these bacterial suspensions was a prerequisite for adhesion. Attachment could be prevented by the addition of physiological levels of sodium chloride to the attachment medium. Furthermore, attached bacteria could be removed by rinsing tissues in saline media.