Myosin heavy-chain isoforms in adult and developing rabbit vascular smooth muscle

Two monoclonal antibodies specific for smooth muscle myosin (designated SM-E7 and SM-A9) and one monoclonal anti-(human platelet myosin) antibody (designated NM-G2) have been used to study myosin heavy chain composition of smooth muscle cells in adult and in developing rabbit aorta. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Western blotting experiments revealed that adult aortic muscle consisted of two myosin heavy chains (MCH) of smooth muscle type, named MHC-1 (205 kDa), and MHC-2 (200 kDa). In the fetal/neonatal stage of development, vascular smooth muscle was found to contain only MHC-1 but not MHC-2. Non-muscle myosin heavy chain, which showed the same electrophoretic mobility as the slower migrating MHC, was expressed in an inverse manner with respect to MHC-2, i.e. it was detectable only in the early stages of development. The distinct pattern of smooth and non-muscle myosin isoform expression during development may be related to the different functional properties of smooth muscle cells during vascular myogenesis.     

The heavy-chain stoichiometry of smooth muscle myosin is a characteristic of smooth muscle tissues

The stoichiometry of the two heavy chains of myosin in smooth muscle was determined by electrophoresing extracts of native myosin and of dissociated myosin on sodium dodecyl sulfate (SDS) 4%-polyacrylamide gels. The slower migrating heavy chain was 3.6 times more abundant in toad stomach, 2.3 in rabbit myometrium, 2.0 in rat femoral artery, 1.3 in guinea pig ileum, 0.93 in pig trachea and 0.69 in human bronchus, than the more rapidly migrating chain. Both heavy chains were identified as smooth muscle myosin by immunoblotting using antibodies to smooth muscle and non-muscle myosin. The unequal proportion of heavy chains suggested the possibility of native isoforms of myosin comprised of heavy-chain homodimers. To test this, native myosin extracts wer electrophoresed on non-dissociating (pyrophosphate) gels. When each band was individually analysed on SDS-polyacrylamide gel the slowest was found to be filamin and the other bands were myosin in which the relative proportion of the heavy chains was unchanged from that found in the original tissue extracts. Since this is incompatible with either a heterodimeric or a homodimeric arrangement it suggests that pyrophosphate gel electrophoresis is incapable of separating putative isoforms of native myosin.     

Characterization of myosin heavy chains in cultured aorta smooth muscle cells. A comparative study

Myosin heavy chains (MHCs) from rat aorta smooth muscle cells were analyzed prior to and after these cells were placed into cell culture using sodium dodecyl sulfate-5% polyacrylamide gels, immunoblots, and two-dimensional peptide maps of tryptic digests. Rat aorta smooth muscle cells prior to culture were found to contain two MHCs (mass = 204 and 200 kDa) which cross-reacted with antibodies raised to smooth muscle myosin, but not with antibodies raised to platelet myosin. Tryptic peptide maps of these two MHCs showed no major differences when compared to each other and to maps of vas deferens and uterus smooth muscle MHCs. When rat aorta smooth muscle cells were placed into culture, the MHCs isolated from the cell extracts differed, depending on whether the cells were rapidly growing or postconfluent. Extracts from log-phase cultures contained predominantly MHCs that migrated more rapidly than smooth muscle myosin in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (mass = 196 kDa) and cross-reacted with antibodies raised to platelet myosin, but not to smooth muscle myosin. Tryptic peptide maps of this MHC were very similar to those obtained with MHCs from non-muscle sources such as platelets and fibroblasts. In contrast, extracts from postconfluent rat aorta cell cultures contained three MHCs (mass = 204, 200, and 196 kDa). Using immunoblots and peptide maps, the fastest migrating MHC was found to be identical to the 196-kDa non-muscle MHC, while the two slower migrating MHCs had the same properties as aorta smooth muscle MHCs prior to culture. These results suggest that smooth muscle cells grown in primary culture contain predominantly (greater than 80%) non-muscle myosin while actively growing, but at a postconfluent stage, contain more equivalent amounts of smooth muscle and non-muscle myosins.     

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.     

Tissue-specific and non-tissue-specific heavy-chain isoforms of myosin in the brain as revealed by monoclonal antibodies.

Four types of monoclonal antibody (BM-1, BM-2, BM-3 and BM-4) each having distinctive tissue specificity were obtained by immunizing mice with purified bovine cerebrum myosin. Both BM-1 and BM-2 reacted most efficiently with cerebrum myosin and less efficiently with myosins from other limited nonmuscle tissues, the tissue specificity of BM-1 being much narrower than that of BM-2. BM-3 reacted more efficiently with several other nonmuscle myosins than with cerebellar or cerebral myosin. BM-4 recognized various nonmuscle and smooth muscle myosins with a nearly equal efficiency. Cerebral myosin as well a cerebellar myosin contained two or more electrophoretic variants of the heavy chains. BM-1 and BM-3 as well as BM-2 and BM-3 were found to recognize selectively these distinct heavy-chain isoforms. The antigenic sites of the three tissue-specific antibodies (BM-1, BM-2 and BM-3) were all localized near the head/tail junction of the myosin molecules, while that of non-tissue-specific antibody BM-4 was near the center of the tail. These and additional results indicate that mammalian brain tissues as well as several other nonmuscle tissues contain multiple heavy-chain isoforms of myosin, the levels of which differed considerably from one tissue to another.     

Evidence for new forms of cardiac myosin heavy chains in mechanical heart overloading and in ageing

Heavy chains of myosin rods and subfragment 1 were isolated from normal hearts and from mechanically overloaded hearts of young and older rats. These myosin heavy-chain fragments were cleaved by cyanogen bromide or partially proteolysed by pronase and by chymotrypsin after denaturation with sodium dodecyl sulfate. The peptides, analyzed by electrophoresis on a one-dimensional polyacrylamide slab gel, varied depending on the origin of the cardiac myosin heavy chains. Some bands present in the peptide patterns of the normal heart of young rats were missing from the pattern of greatly hypertrophied hearts and vice versa. We conclude that mechanical overloading of the heart stimulates the synthesis of cardiac myosin 'isozyme' with a heavy-chain primary structure which is different from that observed in the normal heart of young rat. The patterns from myosin heavy-chain peptides from the hearts of older rats were different from those for peptides from young rat hearts; these results also indicate the presence of a new myosin heavy chain specific to ageing. No difference was detected between the peptide patterns of heavy chains isolated from hypertrophied hearts of young and older rats, and those isolated from normal hearts of older rats.     

Diazepam induces relaxation of chick embryo muscle fibers in vitro and inhibits myosin synthesis

Diazepam (Valium/Roche) causes an immediate cessation of spontaneous contraction in chick embryo skeletal muscle fibers growing in vitro. Between 24–48 h later in the presence of 100 μM diazepam the relaxed muscle fibers no longer accumulate myosin as measured by the total amount of myosin heavy-chain peptide extracted from the cell cultures and identified by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The myosin heavy chain assay procedure was standardized by quantitative precipitation of myosin with antibody to column purified chicken skeletal muscle myosin. Failure to accumulate myosin is related to a progressive inhibition of myosin synthesis. Diazepam-treated cultures showed an 80% inhibition of myosin heavy-chain synthesis over a period of 4 days. At the same time the rate of myosin heavy-chain degradation increases in diazepam-treated cultures relative to matched control cultures. Total protein synthesis was only marginally affected suggesting that diazepam may differentially inhibit myofibrillar protein synthesis. All of the observed effects of diazepam were reversible if drug exposure was limited to 48 h. The apparent specificity and reversibility of diazepam suggests that the drug will be useful in probing the mechanisms of terminal skeletal muscle cell differentiation and the hypotrophic relationship between chronic relaxation and inhibition of accumulation of myosin and perhaps other myofibrillar proteins.     

Embryonic chicken gizzard: smooth muscle and non-muscle myosin isoforms

Antibodies to smooth muscle and non-muscle myosin allow the development of smooth muscle and its capillary system in the embryonic chicken gizzard to be followed by immunofluorescent techniques. Although smooth muscle development proceeds in a serosal to luminal direction, angiogenetic cell clusters develop independently at the luminal side close to the epithelial layer, and the presumptive capillaries invade the developing muscle in a luminal to serosal direction. The smooth muscle and non-muscle myosin heavy chains in this avian system cannot be separated by SDS polyacrylamide gel electrophoresis and do not show isoform specificity in immunoblotting, unlike the system found in mammals. Only two myosin heavy chains with M_r of 200 and 196 kDa were separable and considerable immunological cross-reactivity was found between the denatured myosin isoform heavy chains.     

A chicken embryo-specific myosin light chain (L23) appears to be identical with one of brain myosin light chains

A novel embryo-specific myosin light chain of 23 kDa molecular weight (L23) was found previously in embryonic chicken skeletal, cardiac, and smooth muscles (Takano-Ohmuro et al. (1985) J. Cell Biol. 100, 2025-2030). When we examined myosin in embryonic and adult brain by two-dimensional electrophoresis, 23 kDa myosin light chain present in brain (Burridge & Bray (1975) J. Mol. Biol. 99, 1-14) comigrated with L23. Two monoclonal antibodies, EL-64 and MT-185d, were applied to clarify the identity of the brain 23 kDa myosin light chain and the chicken embryonic muscle L23. The two antibodies recognize different antigenic determinants in the L23 molecule; the former antibody is specific for L23, whereas the latter recognizes the sequence common to fast skeletal muscle myosin light chains 1 and 3, and also L23. The immunoblots combined with two-dimensional gel electrophoresis showed that both EL-64 and MT-185d can bind to the brain 23 kDa myosin light chain as well as the chicken embryonic muscle L23. These results indicate that chicken brain and chicken embryonic muscles contain a common myosin light chain of 23 kDa molecular weight.     

Myosin at the apical pole of ciliated epithelial cells as revealed by a monoclonal antibody

A monoclonal antibody (CC-212), obtained in a fusion experiment in which basal bodies from quail oviduct were used as immunogen, has been shown to label the apical pole of ciliated cells and to react with a 200-kD protein. This monoclonal antibody was demonstrated to be an anti-myosin from smooth muscle or from nonmuscular cells using the following criteria: On Western blots it reacted with the myosin heavy chains from gizzard and platelet extracts and from cultured cell line extracts, but did not react with striated muscle myosin heavy chains. By immunofluorescence it decorated the stress fibers of well-spread cells with a characteristic striated pattern, while it did not react with myotubes containing organized myofibrils. On native ciliated cells as well as on Triton-extracted ciliated cortices from quail oviduct, this monoclonal antibody decorated the apical pole with a stronger labeling of the periphery of the apical area. Ultrastructural localization was attempted using the immunogold technique on the same preparation. Myosin was associated with a filamentous material present between striated rootlets and the proximal extremities of the basal bodies. No labeling of the basal body itself or of axoneme was observed.     

Selective purification of the 20,000-Da light chains of smooth muscle myosin

The 20,000-Da light chains of gizzard smooth muscle myosin have been purified to homogeneity. Actomyosin, prepared by MgATP extraction of myofibrils, was denatured in 8 M urea, 1 M guanidine HCl, and 0.05% sodium dodecyl sulfate. Myosin heavy chains were precipitated with ethanol and the light chain enriched fraction was dialyzed and subjected to chromatography on DEAE-Sephacel. Fractions containing the 20,000-Da light chains were further purified by hydrophobic chromatography on phenyl-Sepharose. The 20,000-Da light chains eluted at low ionic strength from the phenyl-Sepharose column were judged to be greater than 95% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained only 0.04 mol of phosphate/mol of light chain. The yield of light chains was calculated to be 219 +/- 17 mg/kg of starting gizzard smooth muscle. This method may be useful for preparation of homogeneous 20,000-Da smooth muscle myosin light chains in the quantities necessary for study of contractile systems.     

NFATc1 and slow-to-fast shift of myosin heavy chain isoforms under functional unloading of the rat m. soleus

It was determined whether the content of NFATc1 (nuclear factor of activation of T cells) in the nuclear and cytoplasmic extracts is related to an increase in the content of fibers containing type IIa myosin heavy chains under gravitational unloading of m. soleus. It was found that three isoforms of NFATc1 with molecular masses of 140, 110, and 86 kDa are present in m. soleus. Under unloading, the translocation of 140 kDa NFATc1 into the nucleus, a decrease in the content of 110 kDa NFATc1 in the cytoplasmic extract of m. soleus, and an increase in the content of 86 kDa NFATc1 in the nuclear extract of m. soleus take place. The content of fibers containing type IIa myosin heavy chains under gravitational unloading increases. The increase in the level of 140 and 86 kDa NFATc1 in the nucleus is accompanied by a decrease in the percentage of fibers containing type I myosin heavy chains and an increase in the percentage of muscle fibers containing type IIa myosin heavy chains.     

Two smooth muscle myosin heavy chains differ in their light meromyosin fragment

Smooth muscle myosin heavy chains [SM1, approximately 205 kilodaltons (kDa), and SM2, approximately 200 kDa] were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Peptide maps of the two heavy chains showed unique patterns. Limited proteolytic cleavage of purified swine stomach myosin was performed by using a variety of proteases to produce the major myosin fragments which were resolved on SDS gels. A single band was obtained for heavy meromyosin in the soluble fraction following chymotrypsin digestion. However, a variable number of bands were observed for light meromyosin fragments in the insoluble fraction after chymotrypsin digestion. Peptide mapping indicated that the two bands observed after short digestion times with chymotrypsin had relative mobility and solubility properties consistent with approximately 100- and 95-kDa light meromyosin (LMM) fragments. These results indicate that the region of difference between SM1 and SM2 lies in the LMM fragment.     

Embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain

It has been demonstrated that embryonic chicken gizzard smooth muscle contains a unique embryonic myosin light chain of 23,000 mol wt, called L23 (Katoh, N., and S. Kubo, 1978, Biochem. Biophys. Acta, 535:401-411; Takano-Ohmuro, H., T. Obinata, T. Mikawa, and T. Masaki, 1983, J. Biochem. (Tokyo), 93:903-908). When we examined myosins in developing chicken ventricular and pectoralis muscles by two-dimensional gel electrophoresis, the myosin light chain (Le) that completely comigrates with L23 was detected in both striated muscles at early developmental stages. Two monoclonal antibodies, MT-53f and MT-185d, were applied to characterize the embryonic light chain Le of striated muscles. Both monoclonal antibodies were raised to fast skeletal muscle myosin light chains; the former antibody is specific to fast muscle myosin light chains 1 and 3, whereas the latter recognizes not only fast muscle myosin light chains but also the embryonic smooth muscle light chain L23. The immunoblots combined with both one- and two-dimensional gel electrophoresis showed that Le reacts with MT-185d but not with MT-53f. These results strongly indicate that Le is identical to L23 and that embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.     

NFATc1 and slow-to-fast transition of myosin heavy chain isoforms in gravitational unloading of the rat soleus

An attempt was made to determine whether or not the concentration of NFATc1 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1) in nuclear and cytoplasmic extracts is related to an increase in the concentration of fibers containing type IIa myosin heavy chains under modeled gravitational unloading of m. soleus. Experiments were carried out on Wistar rats using the Morey-Holton tail suspension model. It was found that the soleus contains three isoforms of NFATc1 (140, 110, and 86 kDa). Under unloading, the 140-kDa isoform is translocated into the nucleus, the concentration of the 110-kDa isoform in the cytoplasmic extract decreases, and the concentration of the 86-kDa isoform in the nuclear extract increases. Under gravitational unloading of the muscle, the concentration of fibers containing type IIa myosin heavy chains increases. The increase in the concentration of the 140-and 86-kDa NFATc1 isoforms in the nucleus is accompanied by a decrease in the fraction of muscle fibers containing type I myosin heavy chains and an increase in the fraction containing type IIa chains.     

Detection and distribution of myosin isozymes in vertebrate smooth muscle

Crude extracts of taenia coli (guinea-pig), gizzard (chicken), stomach, colon, ureter, bladder, mesenteric vein, mesenteric artery, uterus and vas deferens (dog) were electrophoresed under conditions which do not denature myosin (pyrophosphate gels). Two isozymes (G1 and G2) were observed in all cases. Their mobilities are the same in all organs, but there are some variations in their relative proportions. They have an ATPase activity. Based on electrophoretic mobility the light chains (L20 and L17) seem to be the same for both isozymes whilst the heavy chains are different. Isozyme G2 contains one type of heavy chain of an apparent molecular mass of 230 kDa, whilst isozyme G1 contains two types of heavy chains: one of apparent molecular mass of 230 kDa, and the other of apparent molecular mass of 200 kDa.     

Myosin isoenzymes and their subunits in urodelan amphibian fast skeletal muscle. Coexistence of larval and adult heavy chains in neotenic individuals

The distributions of native myosin isoforms were examined by electrophoresis under non-dissociating conditions, in the fast twitch dorsal skeletal muscle of young larvae, neotenic adults and metamorphosed adults of urodelan amphibians. Both heavy and light chains of myosin isoenzymes were analysed. In pyrophosphate acrylamide gel electrophoresis three isoenzymes were demonstrated in larval myosin; other isoforms of lower electrophoretic mobility were observed in metamorphosed adults myosin. Larval and adult isoenzymes were shown to coexist in myosin from neotenic adults. Analysis of heavy chains in denaturing conditions and proteolytic digestion revealed the sequential occurrence during development of two types of heavy chains, one larval and one adult, that coexist in the myosin of neotenic adults only. Analysis of light chain patterns under denaturing conditions revealed the existence of three fast light chains which displayed no modification during the course of development. The neotenic urodelan amphibian species model represents actually the only model in which the coexistence of larval (or neonatal) and adult heavy chains is maintained throughout life in adults.     

Characterization of monoclonal antibodies to human platelet myosin that recognize highly conserved epitopes within the 50 kDa fragment of myosin subfragment-1.

Three monoclonal antibodies directed against human platelet myosin heavy chains (MCH) that recognize homologous sequences contained within the functionally active subfragment-1, in platelet and rabbit skeletal muscle myosin were studied. These antibodies are distinguished by their affinities to different myosins and their differential effect on various ATPase activities. Epitope mapping was accomplished by analyzing antibody binding to proteolytic peptides of myosin head subfragment-1 under various experimental conditions. The epitopes recognized by these anti-human platelet MHC monoclonal antibodies reside within a small region of the 50 kDa fragment, beginning 9 kDa from its C-terminus and extending a stretch of 6 kDa towards the N-terminus. These epitopes lie between residues 535-586, and are contained within a highly conserved area of myosin heavy chain.     

Contraction kinetics and myosin isoform composition in smooth muscle from hypertrophied rat urinary bladder

Mechanical properties and isoform composition of myosin heavy and light chains were studied in hypertrophying rat urinary bladders. Growth of the bladder was induced by partial ligation of the urethra. Preparations were obtained after 10 days. In maximally activated skinned preparations from the hypertrophying tissue, the maximal shortening velocity and the rate of force development following photolytic release of ATP were reduced by about 20 and 25%, respectively. Stiffness was unchanged. The relative content of the basic isoform of the essential 17 kDa myosin light chain was doubled in the hypertrophied tissue. The expression of myosin heavy chain with a 7 amino acid insert at the 25K/50K region was determined using a peptide-derived antibody against the insert sequence. The relative amount of heavy chain with insert was decreased to 50%, in the hypertrophic tissue. The kinetics of the cross-bridge turn-over in the newly formed myosin in the hypertrophic smooth muscle is reduced, which might be related to altered expression of myosin heavy or light chain isoforms. © 1996 Wiley-Liss, Inc.