A monoclonal antibody to the embryonic myosin heavy chain of rat skeletal muscle

A monoclonal antibody, 2B6, has been prepared against the embryonic myosin heavy chain of rat skeletal muscle. On solid phase radioimmunoassay, 2B6 shows specificity to myosin isozymes known to contain the embryonic myosin heavy chain and on immunoblots of denatured contractile proteins and on competitive radioimmunoassay, it reacts only with the myosin heavy chain of embryonic myosin and not with the myosin heavy chain of neonatal or adult fast and slow myosin isozymes or with other contractile or noncontractile proteins. This specificity is maintained with cat, dog, guinea pig, and human myosins, but not with chicken myosins. 2B6 was used to define which isozymes in the developing animal contained the embryonic myosin heavy chain and to characterize the changes in embryonic myosin heavy chain in fast versus slow muscles during development. Finally, 2B6 was used to demonstrate that thyroid hormone hastens the disappearance of embryonic myosin heavy chain during development, while hypothyroidism retards its decrease. This confirmed our previous conclusion that thyroid hormones orchestrate changes in isozymes during development.     

Plasminogen activator inhibitor-1 detaches cells from extracellular matrices by inactivating integrins

The binding of urokinase plaminogen activator (uPA) to its cell surface receptor (uPAR; CD87) promotes cell adhesion by increasing the affinity of the receptor for both vitronectin (VN) and integrins. We provide evidence that plasminogen activator inhibitor (PAI)-1 can detach cells by disrupting uPAR-VN and integrin-VN interactions and that it does so by binding to the uPA present in uPA-uPAR-integrin complexes on the cell surface. The detached cells cannot reattach to VN unless their surface integrins are first activated by treatment with MnCl2. Immunoprecipitation and subcellular fractionation experiments reveal that PAI-1 treatment triggers deactivation and disengagement of uPA-uPAR-integrin complexes and their endocytic clearance by the low density lipoprotein receptor-related protein. Transfection experiments demonstrate that efficient cell detachment by PAI-1 requires an excess of matrix-engaged uPA-uPAR-integrin complexes over free engaged integrins and that changes in this ratio alter the efficacy of PAI-1. Together, these results suggest a VN-independent, uPA-uPAR-dependent mechanism by which PAI-1 induces cell detachment. This pathway may represent a general mechanism, since PAI-1 also can detach cells from fibronectin and type-1 collagen. This novel "deadhesive" activity of PAI-1 toward a variety of cells growing on different extracellular matrices may begin to explain why high PAI-1 levels often are associated with a poor prognosis in human metastatic disease.     

A monoclonal antibody against cytochrome c oxidase distinguishes cardiac and skeletal muscle mitochondria

The mitochondrial enzyme cytochrome c oxidase (COX) in eukaryotes consists of at least seven subunits, three of which (I-III) are encoded by mitochondrial DNA (mitDNA) and the others (IV-VII) by the nuclear genome. There is increasing evidence that COX in mammals exists in multiple tissue-specific forms, presumably specified by nuclearly encoded subunits. We performed immunologic studies in human cardiac and skeletal muscle, using a monoclonal antibody raised against subunit IV of COX purified from human cardiac muscle. In immunotitration studies, the antibody bound with high affinity to mitochondria from cardiac muscle, but reacted only weakly with mitochondria from skeletal muscle. Similarly, immunocytochemical studies showed prominent mitochondrial staining in frozen sections of heart, but no staining in sections of mature skeletal muscle. Although this antibody did not stain mitochondria in mature skeletal muscle, it clearly stained mitochondria in myoblasts and immature myotubes of human muscle cultures, suggesting that mitochondria in immature muscle cells are different from those in mature muscle, and similar to heart mitochondria. Immunotitration data using either native or denatured COX protein from heart or skeletal muscle showed similar immunoreactivity. These studies indicate that the epitope for recognition by this antibody is exposed in mitochondria from heart and immature muscle cells, but masked in mitochondria from mature skeletal muscle.     

Selective isoactin release from cultured embryonic skeletal muscle cells

The culture medium of embryonic quail myoblasts, labeled for 24 h with [35S]L-methionine, was analyzed by two-dimensional gel autoradiography. The major polypeptide observed had a 43,000 molecular weight and an isoelectric point of 5.4. This polypeptide could be specifically adsorbed to DNAse-I Sepharose. A tryptic peptide map of the [35S]methionine-labeled peptides of intracellular actin and the extracellular major polypeptide were virtually identical. These findings identify the released polypeptide as actin. A comparison of two-dimensional gel patterns of intracellular and extracellular labeled polypeptides showed a large number of differences indicating the actin release did not result from general cellular breakdown. The released actin was not filamentous as judged by its behavior during Bio-Gel A-5m chromatography (Bio-Rad Laboratories, Richmond, Calif.) The released actin did not originate solely from contaminating fibroblasts in the culture because actin was also observed in the medium in clonal myoblast cultures and in purified myotube preparations. Finally, the nonmuscle isoactins, as opposed to muscle alpha-isoactin, were released preferentially. These results indicate that within the developing muscle cell where both muscle and nonmuscle specific isoactins are simultaneously present, the different isoactins may be physically or functionally compartmentalized with the nonmuscle isoactins existing primarily at or near the cell surface.     

A monoclonal antibody against meningococcus group B polysaccharides distinguishes embryonic from adult N-CAM

The neural cell adhesion molecules (N-CAM) occur chiefly in two molecular forms that are selectively expressed at various stages of development. Highly sialylated forms prevalent in embryonic and neonatal brain are gradually replaced by less sialylated forms as development proceeds. Here we describe a monoclonal antibody raised against the capsular polysaccharides of meningococcus group B (Men B) which specifically distinguishes embryonic N-CAM from adult N-CAM. This antibody recognizes alpha 2-8-linked N-acetylneuraminic acid units (NeuAc alpha 2-8). Immunoblot together with immunoprecipitation experiments with cell lines or tissue extracts showed that N-CAM are the major glycoproteins bearing such polysialosyl units. Moreover we could not detect any sialoglycolipid reactive with this antibody in mouse brain or in the neural cell lines examined. By indirect immunofluorescence staining this anti-Men B antibody decorated cells such as AtT20 (D16/16), which expressed the embryonic forms of N-CAM, but not cells that expressed the adult forms. In primary cultures this antibody allowed us to follow the embryonic-to-adult conversion in individual cells. In addition, the existence of cross-reactive polysialosyl structures on Men B and N-CAM in embryonic brain cells for caution in efforts to develop immunotherapy against neonatal meningitis.     

Cytoskeleton of embryonic skeletal muscle cells

Cytoskeletal organization in embryonic skeletal muscle cells has been examined by transmission electron microscopy; the technique involves preparation using the platinum replication method of freezedried samples, with and without cryofracture. The cytoskeletons in developing muscle cells appear to play a role in preserving cell shape as well as in anchoring myofibrils to cell membrane.     

Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody

A novel monoclonal antibody, SM/C-2.6, specific for mouse muscle satellite cells was established. SM/C-2.6 detects mononucleated cells beneath the basal lamina of skeletal muscle, and the cells co-express M-cadherin. Single fiber analyses revealed that M-cadherin+ mononucleated cells attaching to muscle fibers are stained with SM/C-2.6. SM/C-2.6+ cells, which were freshly purified by FACS from mouse skeletal muscle, became MyoD+ in vitro in proliferating medium, and the cells differentiated into desmin+ and nuclear-MyoD+ myofibers in vitro when placed under differentiation conditions. When the sorted cells were injected into mdx mouse muscles, donor cells differentiated into muscle fibers. Flow cytometric analyses of SM/C-2.6+ cells showed that the quiescent satellite cells were c-kit-, Sca-1-, CD34+, and CD45-. More, SM/C-2.6+ cells were barely included in the side population but in the main population of cells in Hoechst dye efflux assay. These results suggest that SM/C-2.6 identifies and enriches quiescent satellite cells from adult mouse muscle, and that the antibody will be useful as a powerful tool for the characterization of cellular and molecular mechanisms of satellite cell activation and proliferation.     

In situ localization of cholesterol in skeletal muscle by use of a monoclonal antibody.

A common perception is that cholesterol, the major structural lipid found in mammalian membranes, is localized nearly exclusively to the plasma membrane of living cells and that it is found in much smaller quantities in internal membranes. This perception is based almost exclusively on cell fractionation studies, in which density gradient centrifugation is used for purification of discrete subcellular membrane fractions. Here we describe a monoclonal antibody, MAb 2C5-6, previously reported to detect purified cholesterol in synthetic membranes (Swartz GM Jr, Gentry MK, Amende LM, Blanchette-Mackie EJ, and Alving CR. Proc Natl Acad Sci USA 85: 1902-1906, 1988), that is capable of detecting cholesterol in situ in the membranes of skeletal muscle sections. Localization of cholesterol, the dihydropyridine receptor of the T tubule, and the Ca(2+)-ATPase of the sarcoplasmic reticulum (SERCA2) by means of double and triple immunostaining protocols clearly demonstrates that cholesterol is primarily localized to the sarcoplasmic reticulum membranes of skeletal muscle rather than the sarcolemmal or T tubule membranes. The availability of this reagent and its ability to spatially localize cholesterol in situ may provide a greater understanding of the relationship between membrane cholesterol content and transmembrane signaling in skeletal muscle.     

A smooth muscle-specific monoclonal antibody recognizes smooth muscle actin isozymes

Injection of chicken gizzard actin into BALB/c mice resulted in the isolation of a smooth muscle-specific monoclonal antibody designated CGA7. When assayed on methanol-Carnoy's fixed, paraffin-embedded tissue, it bound to smooth muscle cells and myoepithelial cells, but failed to decorate striated muscle, endothelium, connective tissue, epithelium, or nerve. CGA7 recognized microfilament bundles in early passage cultures of rat aortic smooth muscle cells and human leiomyosarcoma cells but did not react with human fibroblasts. In Western blot experiments, CGA7 detected actin from chicken gizzard and monkey ileum, but not skeletal muscle or fibroblast actin. Immunoblots performed on two-dimensional gels demonstrated that CGA7 recognizes gamma-actin from chicken gizzard and alpha- and gamma-actin from rat colon muscularis. This antibody was an excellent tissue-specific smooth muscle marker.     

A monoclonal antibody stains myogenic cells in regenerating newt muscle

Monoclonal antibodies have been used to study minced muscle regeneration in the adult newt, Notophthalmus viridescens. The contralateral limb was amputated and the immunostaining patterns in the regenerating blastema were compared with the minced tissue in sectioned material. Staining with a myofibre-specific antibody, called 12/101 (Kintner & Brockes, 1984), showed that myofibre degeneration was complete by 8-10 days after mincing, with myogenesis commencing 2 days later. Another monoclonal antibody, called 22/18, previously shown to label a subset of cells in the regeneration blastema of the newt (Kintner & Brockes, 1984, 1985), was found also to recognize a population of cells in regenerating minced muscle. At 6 days after mincing, the number of 22/18-positive (22/18+) cells was low but by days 12-16, during the period of myogenesis, their number had increased to become a major population within the minced tissue. A small number of the 22/18+ cells could be double labelled with 12/101 at this time. Prior to this, there was a phase in which 12/101 staining had disappeared from the mince. Cells immunoreactive with both antibodies after this phase confirm that at least some of the 22/18+ cells are myogenic. The number of 22/18+ cells decreased as muscle repair and maturation progressed. These results show that 22/18 is not specifically associated with blastemal cells but is a more general marker for regenerating systems in the newt. They further suggest an alternative interpretation of the double-labelled cells used by Kintner & Brockes (1984) as evidence for myofibre dedifferentiation in limb regeneration. Instead, we propose that such cells represent new myogenesis occurring by tissue repair of locally damaged muscle fibres.     

Generation of skeletal muscle from transplanted embryonic stem cells in dystrophic mice

Embryonic stem (ES) cells have great therapeutic potential because of their capacity to proliferate extensively and to form any fully differentiated cell of the body, including skeletal muscle cells. Successful generation of skeletal muscle in vivo, however, requires selective induction of the skeletal muscle lineage in cultures of ES cells and following transplantation, integration of appropriately differentiated skeletal muscle cells with recipient muscle. Duchenne muscular dystrophy (DMD), a severe progressive muscle wasting disease due to a mutation in the dystrophin gene and the mdx mouse, an animal model for DMD, are characterized by the absence of the muscle membrane associated protein, dystrophin. Here, we show that co-culturing mouse ES cells with a preparation from mouse muscle enriched for myogenic stem and precursor cells, followed by injection into mdx mice, results occasionally in the formation of normal, vascularized skeletal muscle derived from the transplanted ES cells. Study of this phenomenon should provide valuable insights into skeletal muscle development in vivo from transplanted ES cells.     

A new 185,000-dalton skeletal muscle protein detected by monoclonal antibodies

The M line, which transverses the center of the thick filament region of skeletal muscle sarcomeres, appears to be a complex array of multiple structural elements. To date, two proteins have definitely been shown to be associated with the M line. They are MM-CK, localized in the M 4,4' substriations, and a 165,000-dalton (164 kd) protein, referred to as both M-protein and myomesin. Here we report the positive identification of a third M-line protein of 185 kd. In the course of making monoclonal antibodies (mAbs) against a 165-kd fraction, we also obtained mAbs that bound to the M line of isolated myofibrils as detected by indirect immunofluorescence, but recognized a protein band of 185 kd in immunoblotting experiments with either the original immunogen or low ionic strength myofibril extracts as antigenic targets. The evidence that the 185- and 165-kd proteins are distinct protein species is based on the separation of the two proteins into discrete peaks by ion exchange chromatography, the distinctive patterns of their degradation products, and non-cross-reactivity of any of seven mAbs. These mAbs recognize three unique antigenic determinants on the 185-kd molecule and at least two and probably four sites on the 165-kd molecule as determined from competitive binding and immunofluorescence experiments. To resolve the problem of multiple nomenclature for the 165-kd protein, the 185-kd protein will be referred to as myomesin and the 165-kd protein as M-protein.     

The extracellular matrix dimension of skeletal muscle development

Cells anchor to substrates by binding to extracellular matrix (ECM). In addition to this anchoring function however, cell–ECM binding is a mechanism for cells to sense their surroundings and to communicate and coordinate behaviour amongst themselves. Several ECM molecules and their receptors play essential roles in muscle development and maintenance. Defects in these proteins are responsible for some of the most severe muscle dystrophies at every stage of life from neonates to adults. However, recent studies have also revealed a role of cell–ECM interactions at much earlier stages of development as skeletal muscle forms. Here we review which ECM molecules are present during the early phases of myogenesis, how myogenic cells interact with the ECM that surrounds them and the potential consequences of those interactions. We conclude that cell–ECM interactions play significant roles during all stages of skeletal muscle development in the embryo and suggest that this “extracellular matrix dimension” should be added to our conceptual network of factors contributing to skeletal myogenesis.     

A ribonuclease from human skeletal muscle

1. A ribonuclease has been prepared from human muscle by ammonium sulphate fractionation, heat treatment and ion-exchange chromatography. 2. The enzyme degrades polycytidylic acid and polyuridylic acid to the nucleoside 3'-phosphates, with nucleoside 2':3'-cyclic phosphates as intermediates. Polyadenylic acid and polyguanylic acid are not attacked. 3. The enzyme has maximal activity at pH8.5. The molecular weight (by gel filtration) is between 11000 and 12000. It is relatively heat-stable. It exhibits optimum activity in a medium of high ionic strength, and is inhibited by several bivalent cations, particularly Zn(2+).     

Embryonic myosin heavy chain as a differentiation marker of developing human skeletal muscle and rhabdomyosarcoma. A monoclonal antibody study

Hybridoma cell lines were obtained from the fusion of NS-O myeloma cells with spleen cells of mice immunized with bovine fetal skeletal myosin. A stable hybridoma clone, BF-G6, produced immunoglobulin G1 k antibodies reacting specifically with embryonic-type myosin heavy chains present in fetal but not in neonatal or adult human skeletal muscle, as determined by enzyme immunoassay and immunoblot analysis. Fetal but not adult skeletal muscle fibers were stained by this monoclonal antibody in indirect immunofluorescence assays; smooth muscle cells and cardiac muscle cells, as well as non-muscle cells were also unreactive. Solid tumors of infants and children were tested for reactivity with BF-G6 by immunofluorescence and immunoperoxidase staining. Embryonic myosin heavy chain was expressed in rhabdomyosarcomas but not in other types of tumor, except for Wilms' tumor. Rhabdomyosarcoma cells isolated from a bone marrow metastasis and grown in vitro for several months were also labelled by BF-G6. Embryonic myosin heavy chain can thus be used as a specific differentiation marker of normal and neoplastic skeletal muscle tissue.     

A monoclonal antibody to the beta subunit of the skeletal muscle dihydropyridine receptor immunoprecipitates the brain omega-conotoxin GVIA receptor.

Antibodies against the subunits of the dihydropyridine-sensitive L-type calcium channel of skeletal muscle were tested for their ability to immunoprecipitate the high affinity (Kd = 0.13 nM) 125I-omega-conotoxin GVIA receptor from rabbit brain membranes. Monoclonal antibody VD2(1) against the beta subunit of the dihydropyridine receptor from skeletal muscle specifically immunoprecipitated up to 86% of the 125I-omega-conotoxin receptor solubilized from brain membranes whereas specific antibodies against the alpha 1, alpha 2, and gamma subunits did not precipitate the brain receptor. Purified skeletal muscle dihydropyridine receptor inhibited the immunoprecipitation of the brain omega-conotoxin receptor by monoclonal antibody VD2(1). The dihydropyridine receptor from rabbit brain membranes was also precipitated by monoclonal antibody VD2(1). However, neither the neuronal ryanodine receptor nor the sodium channel was precipitated by monoclonal antibody VD2(1). The omega-conotoxin receptor immunoprecipitated by monoclonal antibody VD2(1) showed high affinity 125I-omega-conotoxin binding, which was inhibited by unlabeled omega-contoxin and by CaCl2 but not by nitrendipine or by diltiazem. An antibody against the beta subunit of the skeletal muscle dihydropyridine receptor stained 58- and 78-kDa proteins on immunoblot of the omega-conotoxin receptor, partially purified through heparin-agarose chromatography and VD2(1)-Sepharose chromatography. These results suggest that the brain omega-conotoxin-sensitive calcium channel contains a component homologous to the beta subunit of the dihydropyridine-sensitive calcium channel of skeletal muscle and brain.     

A monoclonal anticarbohydrate antibody detecting superfast myosin in the masseter muscle

Adrenal medullary chromaffin cells secrete catecholamines through exocytosis of their intracellular chromaffin granules. Osmotic granule swelling has been implicated to play a role in the generation of membrane stress associated with the fusion of the granule membrane. However, controversy exists as to whether swelling occurs before or after the actual fusion event. Using morphometric methods we have determined the granule diameter distributions in rapidly frozen, freeze-substituted chromaffin cells. Our measurements show that intracellular chromaffin granules increase in size from an average of 234 nm to 274 nm or 277 nm in cells stimulated to secrete with nicotine or high external K⁺, respectively. Granule swelling occurs before the formation of membrane contact. Ammonium chloride, an agent which inhibits stimulated catecholamine secretion by approximately 50% by altering the intragranular pH, also inhibits granule swelling. In addition, ammonium chloridetreated secreting cells show more granule-plasma membrane contacts than untreated secreting cells. Sodium propionate induces granule swelling in the absence of secretagogue and has been shown to enhance nicotine- and high K⁺- induced catecholamine release. These results indicate that in adrenal chromaffin cells granule swelling is an essential step in exocytosis before fusion pore formation, and is related to the pH of the granule environment.