Human Muscle Samples Research Articles

Interleukin-17 increases the effects of IL-1β on muscle cells: arguments for the role of T cells in the pathogenesis of myositis

We studied the production of interleukin (IL)-6 and CCL20/macrophage inflammatory protein-3α (MIP-3α) by human myoblasts and muscle samples in response to IL-17 alone or in combination with IL-1β. Both IL-17 and IL-1β induced IL-6 production by normal myoblasts and muscle samples. IL-17 had no effect on CCL20 production by myoblasts. Combination of IL-17 and IL-1β further increased IL-6 and CCL20 production by muscle samples but not that of CK. IL-17 induced also HLA class I, C-Fos, nuclear factor κB (NF-κB) and C-Jun expression by myoblasts but not that of HLA class II, CD40, vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1). Finally, immunostaining of dermatomyositis (DM) and polymyositis (PM) muscle biopsies showed IL-17 and CCL20 expression. Our study shows that low levels of cytokines produced by T cells (IL-17) and monocytes (IL-1β) can act in combination on skeletal myoblasts and muscle tissue.

Discovering orphan receptor function using human in vitro pharmacology.

mRNA encoding several recently discovered orphan receptors and their putative ligands has been identified in human tissues, suggesting the existence of new transmitter systems that may be important in the regulation of human cardiac and vascular function. Data obtained from in vitro pharmacological studies using healthy and pathological surgical samples of human arteries, veins and heart muscle are proving advantageous in the identification and characterisation of orphan receptor systems, such as urotensin-II, ghrelin, apelin and relaxin, which are most likely to provide novel targets for drugs to treat human cardiovascular disease.

Two-dimensional protein map of human vastus lateralis muscle.

Protein samples of human vastus lateralis muscle were analyzed by two-dimensional gel electrophoresis, using immobilized pH gradients encompassing several pH regions in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. More than 500 protein spots on each gel were detected by silver staining, of which 150 were excised, digested in-gel with trypsin and characterized by matrix assisted laser desorptioin/ionization-mass spectrometry and tandem electrospray mass spectrometry. Using these techniques, 124 spots including contractile proteins and metabolic enzymes were identified. This database should provide a valuable resource for the definition of the functional properties of muscle fibres and for assessment of the influence of e.g., hypoxia, ischaemia and ageing on protein level.

Current aspects of lactate exchange: lactate/H+ transport in human skeletal muscle.

Skeletal muscle is capable of producing and releasing large amounts of lactate and at the same time taking up lactate and using it as a respiratory fuel. The release and uptake of lactate both involve transmembrane transport, which is mediated mainly by a membrane protein called the monocarboxylate transporter (MCT). MCTs mediate membrane transport with an obligatory 1:1 coupling between lactate and H+ fluxes, and is therefore of great importance for pH regulation, especially during intense muscle activity. The total lactate and H+ transport capacity is higher in membranes from oxidative fibers than in membranes from more glycolytic fibers. There are two isoforms of MCT present in skeletal muscle, MCT1 and MCT4. In human muscle samples, there is a positive correlation between the proportion of type I fibers and MCT1 density. In contrast, the MCT4 density in human muscle is independent of fiber type and displays a large interindividual variation. Although the two isoforms have identical transport kinetics (Km), they may have different roles in muscle. MCT1 and MCT4 respond differently to a high-intensity training session, which suggests that these two isoforms are regulated differently.

AmpFℓSTR® Profiler Plus™ and AmpFℓSTR® COfiler™ Analysis of Tissues Stored in GenoFix™, a New Tissue Preservation Solution for Mass Disaster DNA Identification

A preliminary study was conducted to assess the capability of a new alcohol-based tissue fixative, GenoFix, to preserve DNA from biopsy tissues stored at room temperature and/or -20 degrees C in a freezer, for subsequent short tandem repeat (STR) DNA typing analysis. Fresh human smooth muscle samples were stored at room temperature in GenoFix for one month and up to one year and seven months before being processed using the megaplex STR systems, AmpFlSTR Profiler Plus and AmpFlSTR COfiler. Alternatively, muscle tissues in GenoFix were placed at -20 degrees C in a freezer for up to 3 1/2 years following two to three months in the fixative at room temperature. DNA analysis was also carried out on tissues stored in GenoFix for one month at room temperature and subsequently paraffin-embedded and stored at room temperature for four years. The AmpFlSTR Profiler Plus and AmpFlSTR COfiler STR profiles produced, using DNA extracted from all fixed tissue samples, were of very good quality. The fluorescent signals were well balanced across the nine STR loci or six loci comprised in the megaplexes surveyed and profiles showed no differences with those observed for the control blood of the respective donor patients. Continuous exposure to GenoFix at room temperature (up to one year and seven months) did not compromise the STR typing analysis of the fixed tissues. No adverse effects were noted on the STR typeability of tissues fixed with GenoFix and stored at -20 degrees C in a freezer for up to 3 1/2 years. STR profiles generated from the paraffin-embedded tissues fixed in GenoFix were of excellent quality. This preliminary study suggests that GenoFix can be used to store tissue samples at room temperature for up to one year and seven months or at -20 degrees C in a freezer for longer storage (up to 3 1/2 years). This new and odorless tissue fixative promotes tissue and DNA preservation in a very effective manner and as such may prove useful in criminal investigations or mass disaster identifications carried out in remote locations and in which a small or large number of tissue samples are collected for further analyses.

Age-dependent changes of antioxidant activities and markers of free radical damage in human skeletal muscle

This study was conducted in order to provide evidence for the role of reactive oxygen species (ROS) in human skeletal muscle aging. We used human muscle samples obtained from hospitalized patients in an open study with matched pairs of individuals of different ages. The subjects, ranging in age from 17 to 91 years, were grouped as follows: 17–25-, 26–35-, 36–45-, 46–55-, 56–65-, 66–75-, 76–85-, and 86–91-year-old groups. To investigate the relationship between muscle aging and oxidative damage we measured total and Mn-dependent superoxide dismutase (total SOD, MnSOD), glutathione peroxidase (GSHPx), and catalase (CAT) activities; total reduced and oxidized glutathione (GSHtot, GSH, and GSSG) levels; lipid peroxidation (LPO), and protein carbonyl content (PrC). Total SOD activity decreases significantly with age in the 66–75-year-old group, although MnSOD activity increases significantly in the 76–85-year-old group. The activity of the two H 2O 2 detoxifying enzymes (GSHPx and CAT) did not change with age, as do GSHtot and GSH levels. GSSG levels increased significantly (76–85- and 86–91-year-old groups) with age. We observed a significant increase in LPO levels (66–75- and 76–85-year-old groups), although the PrC content shows a trend of increase without gaining the statistical significance. These results support the idea that ROS play an important role in the human muscle aging process.

Mitochondrial function in human skeletal muscle is not impaired by high intensity exercise.

The hypothesis that high-intensity (HI) intermittent exercise impairs mitochondrial function was investigated with different microtechniques in human muscle samples. Ten male students performed three bouts of cycling at 130% of peak O2 consumption (V.O2,peak). Muscle biopsies were taken from the vastus lateralis muscle at rest, at fatigue and after 110 min recovery. Mitochondrial function was measured both in isolated mitochondria and in muscle fibre bundles made permeable with saponin (skinned fibres). In isolated mitochondria there was no change in maximal respiration, rate of adenosine 5'-triphosphate (ATP) production (measured with bioluminescence) and respiratory control index after exercise or after recovery. The ATP production per consumed oxygen (P/O ratio) also remained unchanged at fatigue but decreased by 4% (P<0.05) after recovery. In skinned fibres, maximal adenosine 5'-diphosphate (ADP)-stimulated respiration increased by 23% from rest to exhaustion (P<0.05) and remained elevated after recovery, whereas the respiratory rates in the absence of ADP and at 0.1 mM ADP (submaximal respiration) were unchanged. The ratio between respiration at 0.1 and 1 mM ADP (ADP sensitivity index) decreased at fatigue (P<0.05) but after the recovery period was not significantly different from that at rest. It is concluded that mitochondrial oxidative potential is maintained or improved during exhaustive HI exercise. The finding that the sensitivity of mitochondrial respiration to ADP is reversibly decreased after strenuous exercise may indicate that the control of mitochondrial respiration is altered.

HEAT SHOCK PROTEIN 27 DOES NOT APPEAR IN HUMAN SERUM FOLLOWING EXERCISE-INDUCED MUSCLE DAMAGE 297

Serum samples were purified to remove serum albumin and IgG so as to detect the presence of HSP 27 following eccentric exercise. Untrained subjects (4 males, 2 females; ages 20-42 yrs) performed 2 sets of 25 maximal eccentric contractions of the elbow flexors. Indirect indicators of muscle damage were assessed pre-, immediately post-, 24, 48, 72, 96, and 120 h post-exercise. Blood samples were collected pre-exercise, 2, 6, 12 hours post-exercise and daily for 5 days, and serum creatine kinase activity (CK) was measured. Serum albumin was removed by blue sepharose affinity chromatography (BS); Protein A affinity chromatography (PA) eliminated IgG after BS. HSP 27 abundance was analyzed by Western blotting and serum Transferrin (Tf) was analyzed as a control. Eccentric exercise caused a 52% decrease in MVC 24 hours post-exercise. Serum CK activity peaked 96 hrs following the exercise (3679 IU/L, range 84-9752 IU/L). Arm circumference, range of motion and muscle soreness changes were indicative of exercise-induced muscle damage. Tf abundance did not appear to change during the course of the investigation, and was also unaffected by the purification procedures. Following BS, 22% of the serum proteins were recovered. PA recovered 11% of the total protein found in whole serum. Following exercise-induced muscle damage HSP 27 was not detected at any timepoint, in either whole serum or PA flow through. Samples doped with purified HSP 27 demonstrated that the protein bound to the blue sepharose column and was eluted only by changes in pH. Although HSP 27 has been documented in human muscle samples after damage inducing exercise, it apparently does not efflux from damaged tissue to a significant extent; this may be due to the large oligomeric structure of HSP 27 in vivo.

Contractile properties of rat, rhesus monkey, and human type I muscle fibers.

It is well known that skeletal muscle intrinsic maximal shortening velocity is inversely related to species body mass. However, there is uncertainty regarding the relationship between the contractile properties of muscle fibers obtained from commonly studied laboratory animals and those obtained from humans. In this study we determined the contractile properties of single chemically skinned fibers prepared from rat, rhesus monkey, and human soleus and gastrocnemius muscle samples under identical experimental conditions. All fibers used for analysis expressed type I myosin heavy chain as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Allometric coefficients for type I fibers from each muscle indicated that there was little change in peak tension (force/fiber cross-sectional area) across species. In contrast, both soleus and gastrocnemius type I fiber maximal unloaded shortening velocity (Vo), the y-intercept of the force-velocity relationship (Vmax), peak power per unit fiber length, and peak power normalized for fiber length and cross-sectional area were all inversely related to species body mass. The present allometric coefficients for soleus fiber Vo (-0.18) and Vmax (-0.11) are in good agreement with published values for soleus fibers obtained from common laboratory and domesticated mammals. Taken together, these observations suggest that the Vo of slow fibers from quadrupeds and humans scale similarly and can be described by the same quantitative relationships. These findings have implications in the design and interpretation of experiments, especially those that use small laboratory mammals as a model of human muscle function.

Calcium Content and Respiratory Control Index of Isolated Skeletal Muscle Mitochondria: Effects of Different Isolation Media

Calcium (Ca) content measured with graphite furnace atomic absorption spectrometry and polarographical measurement of respiratory control index (RCI) were performed on isolated skeletal muscle mitochondria. Four different isolation media were used in order to develop a method for isolating physiologically intact mitochondria in small samples with an endogenous Ca content as close as possible toin vivoconditions. With ruthenium red in the isolation medium, the mean Ca content decreased fourfold to 19.3 ± 6.8 nmol/mg protein and RCI increased about 25% to 6.9 ± 1.1 compared with a standard medium. With EGTA or EGTA plus ruthenium red, the mean Ca content decreased further to 5.8 ± 1.0 and 5.1 ± 0.9 nmol/mg protein and the mean RCI values increased to 8.4 ± 1.2 and 8.8 ± 1.3, respectively. Isolated mitochondria from human muscle samples using EGTA and ruthenium red in the isolation medium had mean Ca values of 11.0 ± 0.9 nmol/mg protein and mean RCI values of 12.2 ± 1.6. The data indicate that the Ca concentration in the skeletal muscle mitochondria is inversely proportional to RCI, and the mitochondrial functional property is significantly improved upon lowering the intramitochondrial Ca accumulation by the incorporation of a chelating agent (EGTA) and a potent Ca antagonist, ruthenium red, in the isolation media.

Distribution of Radioactive Caesium in the Population of Northern Sweden 1988-1993

During the period from May 1988 until June 1993 the 137 Cs concentration was measured in 751 samples of psoas muscle from selected medico-legal autopsy cases in the northern half of Sweden. In this area the deposition level of 137 Cs from the Chernobyl accident varied from negligible to 100 kBq.m -2 . Northern Sweden is characterised by large boreal forest areas and a sparse population. The rural population often has a high level of subsistence through meat from reindeer, moose and other game, fresh water fish, forest wild berries, and mushrooms. From a multiple linear regression performed on the 137 Cs concentration in the 751 measured samples of human muscle, the effective half-time of caesium whole-body content in the population could be assessed as 3.7 years. A slight increase in 137 Cs concentration was observed with the age of the individual and a significant difference between the sexes, the level for men exceeded that for women by 23%. The dose commitment to this population of approximately 900,000 inhabitants from internal radiation due to the Chernobyl debris could, by this model, be estimated at 220 man.Sv which, with the current ICRP lifetime risk estimates, would cause an addition of ten fatal cancer cases.

Use of double labeling and photo CD for morphometric analysis of injured skeletal muscle.

We used computer-assisted analysis of myofiber cross-sectional areas to measure skeletal muscle responses to injury and disease. We developed a simple, inexpensive method for measuring myofiber size in human muscle samples using Kodak photo compact discs (CDs) as the image source. The photo CD serves as a permanent image storage medium and provides a high-resolution image that can be used to detect small myofibers. The use of double labeling for dystrophin and desmin allowed positive identification of both degenerating and regenerating fibers in a single biopsy specimen.

Assay of Acyl-CoA Dehydrogenase Activity in Frozen Muscle Biopsies: Application to Medium-Chain Acyl-CoA Dehydrogenase Deficiency

The acyl-CoA dehydrogenases (ACDs) are mitochondrial enzymes that dehydrogenate acyl-coenzyme A esters of different chain lengths. Inherited deficiencies of these dehydrogenases are commonly associated with muscle weakness and lipid storage. Numerous assays including spectrophotometric, fluorometric, chemical, and radiochemical procedures have been used, but there is need for a rapid, reproducible assay for the different acyl-CoA dehydrogenases in small frozen samples of human muscle biopsies. We describe a comparative study of dye-linked spectrophotometric assays of the long, medium, and short chain acyl-CoA dehydrogenases in frozen rat and human muscle samples. An optimal procedure is described confirming the value of glass-glass homogenization and assay of a 600g supernatant. Higher activities for all acyl-CoA dehydrogenases, citrate synthase, and cytochrome c oxidase were obtained in rat in contrast to human. The substrate-linked dye reduction method was found superior to the ferricenium or electron transfer flavoprotein acceptor systems. Application of the phenazine ethosulfate-DCPIP-linked method to medium-chain acyl-CoA dehydrogenase (MCAD) was studied in detail and the effect of immunoprecipitation of MCAD allowed for the determination of substrate specificity and the degree of crossover between long-, medium-, and short-chain ACD activity following immunoprecipitation. Finally, a comparison of the specificity and validity of the assay in a patient with MCAD deficiency was performed.

The effects of the method of death and lapsed time on proton relaxation time T1 in autopsied muscle samples.

The variation of measured magnetic resonance T1 relaxation times of autopsied human muscle samples is confusing. Hence, the authors studied rats' muscles to evaluate the effect of fiber type, the relative area of nonmyofiber space, fat and water content, cell death, and the mechanism of death on proton T1. Rats were studied on a 0.1 T magnetic resonance device. We studied how death by cervical dislocation, pentobarbital injection, or a combination of these methods, as well as the amount of time lapsed after death, variably affected T1. Death itself did not affect T1, but the mechanism of death did: rats killed by cervical dislocation after ether anesthesia had longer T1 than those killed with an overdose of pentobarbital. T1 was significantly shorter 1 day after death than at 4 hours after and returned to baseline levels within 4 days after death. Repeated warming caused variation in T1 and obscured other possible changes. Investigation methods should be strictly controlled and standardized before measurements of the relaxation time, T1, of muscle tissue will provide consistent results.

Purification of human muscle satellite cells by flow cytometry

To purify satellite cells directly from human muscle biopsies, we have developed a method based on size separation of dissociated cells by flow cytometry. Immediately after tryptic dissociation of human muscle biopsies and elimination of erythrocytes, microscopic observation and flow cytometry analysis of cell suspensions revealed two populations of cells differing in size and nucleocytoplasmic ratio. Clonal cultures of these two cell types with a manual procedure demonstrated that only the small cells were myogenic satellite cells. Flow cytometry-sorting and analysis of the small cell population showed that (1) all sorted cells contained desmin immediately after dissociation and plating; (2) more than 98% of the cells expressed the 5.1.H11 epitope after 2 weeks of proliferation in culture; and (3) 90% of the sorted cells were able to form myotubes when cultivated at low density or in clonal cultures. Thus, human muscle satellite cells can be directly purified from human muscle samples using flow cytometry.

A Rapid Fluorometric Method for the Determination of Carnitine Palmitoyltransferase

A rapid and sensitive fluorometric assay for the measurement of carnitine palmitoyltransferase (CPT) activity is described. In this assay the coenzyme A (CoA) liberated from palmitoyl-CoA by CPT reacts with N-(9-acridinyl)-maleimide to form a fluorescent product. By comparing the fluorescence intensities of external CoA standards, CPT activity can be determined. The coefficients of variation within-run and between-run of the method were 6.9 and 10%, respectively. The CPT activity of human muscle samples measured with this assay is comparable to those obtained with a commonly used radiochemical assay and CPT deficiency in skeletal muscle from a patient was detected by this new fluorometric assay.

MR Relaxation Times and Fiber Type Predominance of the Psoas and Multifidus Muscle

MR relaxation times, fiber composition, nonmyofiber space, water content, and fat content of human psoas and multifidus muscle samples of 10 male cadavers were studied in vitro. The T1 and T2 relaxation times of multifidus muscle were significantly longer than those of the psoas muscle. On average, type 1 fibers (slow fibers with a small cross-sectional diameter) predominated in both muscles. There was no correlation between the relative mass of type 1 or 2 fibers (fast fibers with a large cross-sectional diameter) or nonmyofiber space and the relaxation times. The quantity of fat in the muscle did not correlate with the relaxation times either.

Mr Relaxation Times and Fiber Type Predominance of the Psoas and Multifidus Muscle

MR relaxation times, fiber composition, nonmyofiber space, water content, and fat content of human psoas and multifidus muscle samples of 10 male cadavers were studied in vitro. The T1 and T2 relaxation times of multifidus muscle were significantly longer than those of the psoas muscle. On average, type 1 fibers (slow fibers with a small cross-sectional diameter) predominated in both muscles. There was no correlation between the relative mass of type 1 or 2 fibers (fast fibers with a large cross-sectional diameter) or nonmyofiber space and the relaxation times. The quantity of fat in the muscle did not correlate with the relaxation times either.

A technique for studies of the contractile apparatus in single human muscle fibre segments obtained by percutaneous biopsy.

Human muscle samples were obtained with the percutaneous biopsy technique. The samples were membrane-hyperpermeabilized (skinned) using a chemical or freeze-drying technique. Short single fibre segments were dissected from the sample, transferred to an experimental chamber, connected to a force transducer and manipulator, and exposed to temperature-controlled solutions. The force generating-capacity, the sensitivity of the contractile apparatus to calcium and the caffeine threshold for calcium release from the sarcoplasmic reticulum could be studied in the short muscle fibre segments obtained from man with the percutaneous muscle biopsy technique. The average length of the fibre segments between the connectors was 0.44 +/- 0.21 mm. Thus, detailed studies of the contractile machinery can be made on human skinned muscle fibres with only minimal discomfort to the patient or subject during biopsy, which should be useful in studies of neuromuscular disease, muscle plasticity or in applied physiology.

Immunologic study of vinculin in Duchenne muscular dystrophy.

Using immunologic techniques, we studied vinculin, a cytoskeletal protein associated with the membrane-skeleton of the muscle fiber. We examined muscle biopsies from five patients with Duchenne muscular dystrophy (DMD), two with Becker's muscular dystrophy (BMD), three normal human muscle samples, and four biopsies from disease control patients. All DMD patients showed patchy and low-intensity immunostain at the sarcolemma of most fibers and, by immunoblot analysis, the content of vinculin was 42 to 61% of control values. There was no significant vinculin deficiency in samples from patients with BMD and other disease controls. The data suggest that vinculin content is reduced only in muscle where dystrophin is absent or sparse.