Single rabbit stomach smooth muscle cell myosin heavy chain SMB expression and shortening velocity

Isolated single smoothmuscle cells (SMCs) from different regions of the rabbit stomach wereused to determine a possible correlation between unloaded shorteningvelocity and smooth muscle (SM) myosin heavy chain (MHC) S1 headisoform composition (SMA, no head insert; SMB, with head insert).-Toxin-permeabilized isolated single cells were maximally activatedto measure unloaded shortening velocity and subsequently used in anRT-PCR reaction to determine the SMA/SMB content of the same cell. SMMHC SMA and SMB isoforms are uniquely distributed in the stomach with cells from the fundic region expressing little SMB (38.1 ± 7.3% SMB; n = 16); cells from the antrum express primarilySMB (94.9 ± 1.0% SMB; n = 16). Mean fundic cellunloaded shortening velocity was 0.014 ± 0.002 cell lengths/scompared with 0.036 ± 0.002 for the antrum cells. Unloadedshortening velocity in these cells was significantly correlated withtheir percent SMB expression (r² = 0.58).Resting cell length does not correlate with the percent SMB expression(n = 32 cells). Previously published assays of purifiedor expressed SMA and SMB heavy meromyosin show a twofold difference inactin filament sliding speed in in vitro motility assays. Extrapolationof our data to 0-100% SMB would give a 10-fold range ofshortening velocity, which is closer to the ~20-fold range reportedfrom various SM tissues. This suggests that mechanisms in addition tothe MHC S1 head isoforms regulate shortening velocity.

     

Expression of smooth muscle and nonmuscle myosin heavy chains in cultured vascular smooth muscle cells

We explored the hypothesis that discrepancies in the literature concerning the nature of myosin expression in cultured smooth muscle cells are due to the appearance of a new form of myosin heavy chain (MHC) in vitro. Previously, we used a very porous sodium dodecyl sulfate gel electrophoresis system to detect two MHCs in intact smooth muscles (SM1 and SM2) which differ by less than 2% in molecular weight (Rovner, A. S., Thompson, M. M., and Murphy, R. A. (1986) Am. J. Physiol. 250, C861-C870). Myosin-containing homogenates of rat aorta cells in primary culture were electrophoresed on this gel system, and Western blots were performed using smooth muscle-specific and nonmuscle-specific myosin antibodies. Subconfluent, rapidly proliferating cultures contained a form of heavy chain not found in rat aorta cells in vivo (NM) with electrophoretic mobility and antigenicity identical to the single unique heavy chain seen in nonmuscle cells. Moreover, these cultures expressed almost none of the smooth muscle heavy chains. In contrast, postconfluent growth-arrested cultures expressed increased levels of the two smooth muscle heavy chains, along with large amounts of NM. Analysis of cultures pulsed with [35S] methionine indicated that subconfluent cells were synthesizing almost exclusively NM, whereas postconfluent cells synthesized SM1 and SM2 as well as larger amounts of NM. Similar patterns of MHC content and synthesis were found in subconfluent and postconfluent passaged cells. These results show that cultured vascular smooth muscle cells undergo differential expression of smooth muscle- and nonmuscle-specific MHC forms with changes in their growth state, which appear to parallel changes in expression of the smooth muscle and nonmuscle forms of actin (Owens, G. K., Loeb, A., Gordon, D., and Thompson, M. M. (1986) J. Cell Biol. 102, 343-352). The reappearance of the smooth muscle MHCs in postconfluent cells suggests that density-related growth arrest promotes cytodifferentiation, but the continued expression of the nonmuscle MHC form in these smooth muscle cells indicates that other factors are required to induce the fully differentiated state while in culture.     

Shortening velocity and myosin heavy- and light-chain isoform mRNA in rabbit arterial smooth muscle cells

In smooth muscle cells (SMCs)isolated from rabbit carotid, femoral, and saphenous arteries, relativemyosin isoform mRNA levels were measured in RT-PCR to test forcorrelations between myosin isoform expression and unloaded shorteningvelocity. Unloaded shortening velocity and percent smooth muscle myosinheavy chain 2 (SM2) and myosin light chain 17b(MLC_17b) mRNA levels were not significantly different insingle SMCs isolated from the luminal and adluminal regions of thecarotid media. Saphenous artery SMCs shortened significantly faster(P < 0.05) than femoral SMCs and had more SM2 mRNA(P < 0.05) than carotid SMCs and lessMLC_17b mRNA (P < 0.001) and higher tissuelevels of SMB mRNA (P < 0.05) than carotid and femoralSMCs. No correlations were found between percent SM2 and percentMLC_17b mRNA levels and unloaded shortening velocity in SMCsfrom these arteries. We have previously shown that myosin heavy chain(MHC) SM1/SM2 and SMA/SMB and MLC_17a/MLC_17b isoform mRNA levels correlate with protein expression for these isoforms in rabbit smooth muscle tissues. Thus we interpret these results to suggest that 1) SMC myosin isoform expression andunloaded shortening velocity do not vary with distance from the lumenof the carotid artery but do vary in arteries located longitudinally within the arterial tree, 2) MHC SM1/SM2 and/orMLC_17a/MLC_17b isoform expression does notcorrelate with unloaded shortening velocity, and 3)intracellular expression of the MHC SM1/SM2 and MLC_17a/MLC_17b isoforms is not coregulated.

     

Pro-enkephalin gene derived peptides in the porcine stomach: cellular distribution and molecular forms

Antisera to the enkephalin variants Met-enk Arg6Phe7 and Met-enk Arg6Gly7Leu8 have been used in immunohistochemistry and radioimmunoassay studies of hog stomach. In the mucosa of the antrum, but not fundus, there was identified a population of immunoreactive endocrine-like cells. When extracts of antral mucosa were fractionated by gel filtration on Sephadex G50 the predominant immunoreactive forms of Met-enk Arg6Phe7 and Met-enk Arg6Gly7Leu8 were found to elute before the standards, and were compatible with N-terminally extended variants. In the muscle layers of both antrum and fundus, immunoreactive nerve fibers were found, these were especially numerous in the myenteric plexus. In extracts of the antral muscle, 50-60% of both Met-enk Arg6Phe7 and Met-enk Arg6Gly7Leu8 immunoreactivity eluted in the position of the standards and the remainder had the properties of N-terminally extended variants. In the fundus muscle the variants accounted for 70-80% of total activity. The results indicate that the proenkephalin gene is expressed in neurones and endocrine cells of the hog stomach. The different patterns of molecular forms found in different regions of the stomach suggest that the precursor is processed by different pathways in different populations of endocrine cells and neurones.     

Nonmuscle and smooth muscle myosin isoforms in bovine endothelial cells

A panel of monoclonal antibodies, specific for human platelet (NM-A9, NM-F6, and NM-G2) and for bovine smooth muscle (SM-E7) myosin heavy chains (MHC), were used to study the composition and the distribution of myosin isoforms in bovine endothelial cells (EC), in vivo and in vitro. Using indirect and double immunofluorescence techniques, we have found that in the intact aortic endothelium there is expression of nonmuscle MHC (NM-MHC), exclusively. By contrast, hepatic sinusoidal endothelium as well as cultured bovine aortic EC (BAEC) in the subconfluent phase of growth show coexistence of NM- and smooth muscle MHC (SM-MHC) isoforms. SM myosin immunoreactivity disappears when cultured BAEC become confluent. In this phase of cell growth, NM-MHC isoforms are localized differently within the cells, i.e., in the cytoplasm around the nucleus or in the cortical, submembranous region of EC cytoplasm. A third type of intracellular distribution of NM-MHC immunoreactivity was evident in the cell periphery of binucleated, confluent BAEC. These data indicate that (1) several myosin isoforms are differently distributed in bovine endothelia; and (2) SM myosin expression and the specific subcellular localization of NM myosin isoforms within EC might be regulated by cell-cell interactions.     

Induction of endogenous myosin light chain 1 and cardiac alpha-actin expression in L6E9 cells by MyoD1.

Rat L6E9 muscle cells commit to terminal differentiation by forming a large muscle syncitia complete with the expression of a large number of muscle-specific contractile protein genes. To determine whether these cells, which fail to synthesize MLC (myosin light chain) 1 and cardiac alpha-actin, exhibit a deficiency in the expression of muscle determination genes, we measured expression of MyoD1, myogenin, Myf-5, and MRF-4. Results show these cells do not synthesize MyoD1, yet express the other myogenic determination genes. Transient expression of exogenous MyoD1 in these cells is sufficient to activate endogenous MLC 1 and cardiac alpha-actin mRNA synthesis during muscle differentiation. Previously undetected myosin heavy chain (MHC) isoforms (beta-MHC and perinatal MHC) are also transcribed at low levels in L6E9 muscle cells, and in MyoD1-transfected L6E9 cells no change occurs in their expression. Furthermore, treatment with the demethylating agent 5-azacytidine activates expression of the endogenous MyoD1 gene in L6E9 cells and, subsequently, rescues deficiencies in their myogenic biochemical program. These results demonstrate that the endogenous MyoD1 gene in L6E9 cells is not defective and can be functionally activated. Also, the MyoD1 protein plays an essential role, which cannot be compensated by other known muscle determination proteins, in the induction of MLC 1 and cardiac alpha-actin expression.     

Unusual intrafusal fibres in human muscle spindles

We have studied the morphology and pattern of expression of myosin heavy chain (MHC) isoforms of intrafusal fibres in a human first lumbrical muscle. Each intrafusal fibre type, namely nuclear bag1, nuclear bag2 and nuclear chain fibres, had a distinct MHC composition and distribution of different MHC isoforms along the whole length of intrafusal fibres. However, most muscle spindles analyzed also contained one or several intrafusal fibres exhibiting an extrafusal or mixed pattern of immunoreactivity which did not correspond to any of the described intrafusal fibre types. We conclude that the latter fibres do not represent new intrafusal fibre types, but their morphology and expression of MHC merely reflects the differences in their innervation owing to their unusual localization at the edge or outside the axial bundle of intrafusal fibres.     

Temporal and tissue-specific expression of myosin heavy chain isoforms in developing and adult avian muscle

We have raised monoclonal antibodies (Mabs) to myosin heavy chain isoforms (MHCs) that have specific patterns of temporal expression during the development of quail pectoral muscle and that are expressed in very restricted, tissue-specific patterns in adult birds. We find that an early embryonic, a perinatal, and an adult-specific, fast myosin heavy chain are co-expressed at different levels in the pectoral muscle of 8-12 day quail embryos. The early embryonic MHC disappears from the pectoral muscle at approximately 14 days in ovo, whereas the perinatal MHC persists until 26 days post-hatching. The adult-specific MHC accumulates preferentially and eventually completely replaces the other isoforms. These Mabs cross-react with the homologous isoforms of the chick and detect a similar pattern of MHC expression in the pectoral muscle of developing chicks. Although the early embryonic and perinatal MHC isoforms recognized by our Mabs are expressed in the pectoral muscle only during distinct developmental stages, our Mabs also recognize MHC isoforms present in the heart and extraocular muscle of adult quail. Immunofingerprinting using Staphylococcus aureus protease V8 suggests that the early embryonic and perinatal MHC isoforms that we see are strongly homologous with the adult ventricular and extraocular muscle isoforms, respectively. These observations suggest that at least three distinct MHC isoforms, which are normally expressed in adult muscles, are co-expressed during the early development of the pectoral muscle in birds. In this respect, the pattern of expression of the MHCs recognized by our Mabs in developing, fast muscle is very similar to the patterns described for other muscle contractile proteins.     

Immunochemical analysis of myosin heavy chains in the developing chicken heart

Monoclonal antibodies (McAb) against myosin from the pectoralis muscle of the adult chicken have been generated and shown to react specifically with the myosin heavy chain (MHC). The reactivities of two such McAbs with myosin from adult chicken atrial and ventricular myocardium were further analysed by immunoautoradiography, radioimmunoassay, and immunofluorescence microscopy. Monoclonal antibody MF 20 was found to bind both atrial and ventricular MHC and stain all striated muscle cells of the adult chicken heart. In contrast, McAb B1 bound specifically to atrial myocytes in immunofluorescence studies, while immunoautoradiography and radioimmunoassay demonstrated the specificity of this antibody for the atrial MHC. Upon reacting these McAbs with myosin isolated from embryonic hearts where definitive atria and ventricles were present, the same specificity of antibody binding was observed. Immunofluorescence studies demonstrated that all striated muscle cells of the embryonic heart contained MHCs recognized by MF 20, while only atrial muscle cells were bound by B1. When extracts of presumptive atrial and ventricular tissue were reacted with MF 20 and B1, significant reactivity of MF 20 was first observed at stage 10 in the presumptive ventricle and thereafter this McAb reacted with all regions of the developing myocardium. Binding of B1 was detected approximately 1 day later at stage 15 and was confined to atrial-forming tissues. These data demonstrate antigenic similarity between adult and embryonic MHC isolated from atrial myocardium and suggest the expression of an atrial-specific MHC early in the regional differentiation of the heart.     

Expression of smooth muscle MyHC B in blood vessels of hypertrophied heart in experimentally hypertensive rats

We demonstrated recently a significantly lower fraction of cardiac precapillary arterioles that expressed smooth muscle myosin heavy chain (MyHC) B (SMB) in spontaneously hypertensive rats. To clarify whether this reduction of SMB expression is of genetic origin, we investigated SMB expression in cardiac precapillary arterioles of normotensive and experimentally hypertensive rats (one clip, one kidney or ANG II minipump). We observed similar SMB expression patterns in precapillary arterioles of experimentally hypertensive rats compared with normotensive controls. These observations suggest that the downregulation of SMB in spontaneously hypertensive rats is of genetic origin rather than an adaptive response to chronically enhanced blood pressure and cardiac hypertrophy.     

Induction of zygote formation by ethylene during the sexual development of the cellular slime mold Dictyostelium mucoroides

Immunochemical studies have identified a distinct myosin heavy chain (MHC) in the chicken embryonic skeletal muscle that was undetectable in this muscle in the posthatch period by both immunocytochemical and the immunoblotting procedures. This embryonic isoform, identified by antibody 96J, which also recognises the cardiac and SM1 myosin heavy chains, differs from the embryonic myosin heavy chain belonging to the fast class described previously. Although the fast embryonic isoform is a major species present in the leg and pectoral embryonic muscles, slow embryonic isoform was present in significant amounts during early embryonic development. Immunocytochemical studies using another monoclonal antibody designated 9812, which is specific for SM1 MHC, showed this isoform to be restricted to only presumptive slow muscle cells. From these studies and those reported on the changes in SM2 MHC, it is proposed that as is the case for the fast class, there also exists a slow class of myosin heavy chains composed of slow embryonic, SM1 and SM2 isoforms. The differentiation of a muscle cell involves transitions in a series of myosin isozymes in both presumptive fast and slow skeletal muscle cells.     

Segregated assembly of muscle myosin expressed in nonmuscle cells.

Skeletal muscle myosin cDNAs were expressed in a simian kidney cell line (COS) and a mouse myogenic cell line to investigate the mechanisms controlling early stages of myosin filament assembly. An embryonic chicken muscle myosin heavy chain (MHC) cDNA was linked to constitutive promoters from adenovirus or SV40 and transiently expressed in COS cells. These cells accumulate hybrid myosin molecules composed of muscle MHCs and endogenous, nonmuscle, myosin light chains. The muscle myosin is found associated with a Triton insoluble fraction from extracts of the COS cells by immunoprecipitation and is detected in 2.4 +/- 0.8-micron-long filamentous structures distributed throughout the cytoplasm by immunofluorescence microscopy. These structures are shown by immunoelectron microscopy to correspond to loosely organized bundles of 12-16-nm-diameter myosin filaments. The muscle and nonmuscle MHCs are segregated in the transfected cells; the endogenous nonmuscle myosin displays a normal distribution pattern along stress fibers and does not colocalize with the muscle myosin filament bundles. A similar assembly pattern and distribution are observed for expression of the muscle MHC in a myogenic cell line. The myosin assembles into filament bundles, 1.5 +/- 0.6 micron in length, that are distributed throughout the cytoplasm of the undifferentiated myoblasts and segregated from the endogenous nonmuscle myosin. In both cell lines, formation of the myosin filament bundles is dependent on the accumulation of the protein. In contrast to these results, the expression of a truncated MHC that lacks much of the rod domain produces an assembly deficient molecule. The truncated MHC is diffusely distributed throughout the cytoplasm and not associated with cellular stress fibers. These results establish that the information necessary for the segregation of myosin isotypes into distinct cellular structures is contained within the primary structure of the MHC and that other factors are not required to establish this distribution.     

Detection of a ventricular-specific myosin heavy chain in adult and developing chicken heart

In the present study, a monoclonal antibody (McAb), ALD19, generated against myosin of slow tonic muscle, was shown to react with the heavy chain of ventricular myosin in the adult chicken heart. With this antibody, it was possible to detect a ventricular-specific myosin during myocardial differentiation and to show that the epitope recognized by ALD19 was present from the earliest stages of ventricular differentiation and maintained throughout development only in the ventricle. A second McAb, specific for atrial myosin heavy chain (MHC) (Gonzalez-Sanchez, A., and D. Bader, 1984, Dev. Biol., 103:151-158), was used as a control to detect an atrial-specific myosin in the caudal portion of the developing heart at Hamburger-Hamilton stage 15. It was found that the appearance of ventricular MHC predated the expression of atrial MHC by approximately 1 d in ovo and that specific MHCs were always differentially distributed. While a common primordial MHC may be present in the early heart, this study showed the tissue-specific expression of a ventricular MHC during the initial stages of heart development and its differential accumulation throughout development.     

Myosin heavy chain composition of single cells from avian slow skeletal muscle is strongly correlated with velocity of shortening during development

We have determined the myosin heavy chain (MHC) composition (using a sensitive sodium dodecyl sulfate-polyacrylamide gel electrophoresis system) and the maximal velocity of shortening (Vmax) of single cells from neonatal and adult chicken anterior latissimus dorsi (ALD) muscles. In addition, the MHC, myosin light chain, and regulatory protein (i.e., troponin and tropomyosin subunits) compositions of bundles of ALD fibers were determined at late embryonic, neonatal, and adult ages. At young ages, there are two MHCs in ALD muscle, SM1 and SM2, with SM1 decreasing in relative amount with increasing age, as shown previously by others. The mean Vmax of single fibers also decreases from neonatal to adult ages. A strong quantitative correlation is demonstrated between the specific MHC composition and Vmax among individual cells of the ALD muscle at several ages. Since virtually no changes occur in the regulatory protein and myosin light chain compositions of the ALD muscle between late embryonic and adult ages, it appears that the MHC composition of an individual cell in this muscle is the primary determinant of the maximal shortening velocity. These results are the first to illustrate the functional significance of the developmental transition in myosin heavy chain composition of an avian slow skeletal muscle, consistent with our previous findings on mammalian muscle.     

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.     

Developmental consequences of the lack of myosin heavy chain in Dictyostelium discoideum

Two different Dictyostelium discoideum cell lines that lack myosin heavy chain protein (MHC A) have been previously described. One cell line (mhcA) was created by antisense RNA inactivation of the endogenous mRNA and the other (HMM) by insertional mutagenesis of the endogenous myosin gene. The two cell lines show similar developmental defects; they are delayed in aggregation and become arrested at the mound stage. However, when cells that lack myosin heavy chain are mixed with wild-type cells, some of the mutant cells are capable of completing development to form mature spores. The pattern of expression of a number of developmentally regulated genes has been examined in both mutant cell lines. Although morphogenesis becomes aberrant before aggregation is completed, all of the markers that we have examined are expressed normally. These include genes expressed prior to aggregation as well as prespore genes expressed later in development. It appears that the signals necessary for cell-type differentiation are generated in the aborted structures formed by cells lacking MHC A. The mhcA cells have negligible amounts of MHC A protein while the HMM cells express normal amounts of a fragment of the myosin heavy chain protein similar to heavy meromyosin (HMM). The expression of myosin light chain was examined in these two cell lines. HMM cells accumulate normal amounts of the 18,000-D light chain, while the amount of light chain in mhcA cells is dramatically reduced. It is likely that the light chains assemble normally with the HMM fragment in HMM cells, while in cells lacking myosin heavy chain (mhcA) the light chains are unstable.     

Human cardiac myosin heavy chain genes and their linkage in the genome.

Human myosin heavy chains are encoded by a multigene family consisting of at least 10 members. A gene-specific oligonucleotide has been used to isolate the human beta myosin heavy chain gene from a group of twelve nonoverlapping genomic clones. We have shown that this gene (which is expressed in both cardiac and skeletal muscle) is located 3.6kb upstream of the alpha cardiac myosin gene. We find that DNA sequences located upstream of rat and human alpha cardiac myosin heavy chain genes are very homologous over a 300bp region. Analogous regions of two other myosin genes expressed in different muscles (cardiac and skeletal) show no such homology to each other. While a human skeletal muscle myosin heavy chain gene cluster is located on chromosome 17, we show that the beta and alpha human cardiac myosin heavy chain genes are located on chromosome 14.