Myosin heavy-chain isoforms in human smooth muscle

The myosin heavy-chain composition of human smooth muscle has been investigated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, enzyme immunoassay, and enzyme-immunoblotting procedures. A polyclonal and a monoclonal antibody specific for smooth muscle myosin heavy chains were used in this study. The two antibodies were unreactive with sarcomeric myosin heavy chains and with platelet myosin heavy chain on enzyme immunoassay and immunoblots, and stained smooth muscle cells but not non-muscle cells in cryosections and cultures processed for indirect immunofluorescence. Two myosin heavy-chain isoforms, designated MHC-1 and MHC-2 (205 kDa and 200 kDa, respectively) were reactive with both antibodies on immunoblots of pyrophosphate extracts from different smooth muscles (arteries, veins, intestinal wall, myometrium) electrophoresed in 4% polyacrylamide gels. In the pulmonary artery, a third myosin heavy-chain isoform (MHC-3, 190 kDa) electrophoretically and antigenically distinguishable from human platelet myosin heavy chain, was specifically recognized by the monoclonal antibody. Analysis of muscle samples, directly solubilized in a sodium dodecyl sulfate solution, and degradation experiments performed on pyrophosphate extracts ruled out the possibility that MHC-3 is a proteolytic artefact. Polypeptides of identical electrophoretic mobility were also present in the other smooth muscle preparations, but were unreactive with this antibody. The presence of three myosin heavy-chain isoforms in the pulmonary artery may be related to the unique physiological properties displayed by the smooth muscle of this artery.     

Myosin heavy chain isoforms in white, red and ventricle muscles of barbel (Barbus barbus L.).

1. Actomyosin extracts of trunk, heart, and head muscles from barbel (Barbus barbus L.) were analyzed by SDS-polyacrylamide gel electrophoresis to study their myosin heavy chain composition. 2. Four heavy chain isoforms were found: trunk white, trunk red, and ventricle muscles yielded one heavy chain typical of the muscle type; head muscles devoid of red fibers displayed two heavy chain isoforms, the slow migrating one corresponding to the trunk white muscle type. 3. The electrophoretic mobility of red and ventricle myosin heavy chains related to that of white isoforms appeared highly modified by the glycerol content of the gels.     

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.     

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.     

Changes in myosin isozymes during development of chicken gizzard muscle

The distribution of myosin isozymes in embryonic and adult chicken gizzard muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, there were three isozyme components in embryonic gizzard myosin, but only one isozyme in adult gizzard myosin. The mobility of the fastest migrating embryonic isozyme was similar to that of the adult isozyme. The three embryonic isozymes differ from each other in the light chain distribution. Two of them contain an embryo-specific myosin light chain, which is characterized by its molecular weight and isoelectric point, whereas the other embryonic myosin isozyme contained the same light chains as the adult myosin. The pattern of peptide fragments of embryonic heavy chain produced by digestion with alpha-chymotrypsin in the presence of SDS was not distinguishable from that of adult myosin heavy chain. Thus there are myosin isozymes specific to embryonic gizzard muscle which exhibit embryo-specific light chain compositions, but are similar to adult gizzard myosin in their heavy chain structure.     

Localization of a light-chain binding site on smooth muscle myosin revealed by light-chain overlay of sodium dodecyl sulfate-polyacrylamide electrophoretic gels

The interactions of smooth muscle myosin and its light chains have been examined by incubating sodium dodecyl sulfate-polyacrylamide gels of myosin with radioactively labeled regulatory or essential light chains. The technique involves sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fixation with methanol and acetic acid followed by an extensive series of washes. The gel is incubated overnight with labeled light chains in the presence of bovine serum albumin and then washed extensively to remove unbound protein. Following staining and destaining, the gel is autoradiographed to reveal which protein bands have bound light chain. The myosin heavy chain was able to rebind labeled regulatory or essential light chains despite the harsh procedure described above. By fragmenting the myosin heavy chain proteolytically, we were able to determine the binding site for both types of light chains to be within the 26,000-Da COOH-terminal segment of smooth muscle subfragment 1 (S-1) or the 20,000-Da COOH-terminal segment of skeletal muscle S-1. The extent of binding was 0.1-0.4 mol of light chain/mol of S-1 heavy chain. No binding was observed to portions of the myosin molecule which do not contain this segment such as myosin rod, light meromyosin, S-2, or the NH2-terminal 75,000-Da segment of S-1.     

Analysis of myosin light and heavy chain types in single human skeletal muscle fibers

In this study, myosin types in human skeletal muscle fibers were investigated with electrophoretic techniques. Single fibers were dissected out of lyophilized surgical biopsies and typed by staining for myofibrillar ATPase after preincubation in acid or alkaline buffers. After 14C-labelling of the fiber proteins in vitro by reductive methylation, the myosin light chain pattern was analysed on two-dimensional gels and the myosin heavy chains were investigated by one-dimensional peptide mapping. Surprisingly, human type I fibers, which contained only the slow heavy chain, were found to contain variable amounts of fast myosin light chains in addition to the two slow light chains LC1s and LC2s. The majority of the type I fibers in normal human muscle showed the pattern LC1s, LC2s and LC1f. Further evidence for the existence in human muscle of a hybrid myosin composed of a slow heavy chain with fast and slow light chains comes from the analysis of purified human myosin in the native state by pyrophosphate gel electrophoresis. With this method, a single band corresponding to slow myosin was obtained; this slow myosin had the light chain composition LC1s, LC2s and LC1f. Type IIA and IIB fibers, on the other hand, revealed identical light chain patterns consisting of only the fast light chains LC1f, LC2f and LC3f but were found to have different myosin havy chains. On the basis of the results presented, we suggest that the histochemical ATPase normally used for fibre typing is determined by the myosin heavy chain type (and not by the light chains). Thus, in normal human muscle a number of 'hybrid' myosins were found to occur, namely two extreme forms of fast myosins which have the same light chains but different heavy chains (IIA and IIB) and a continuum of slow forms consisting of the same heavy chain and slow light chains with a variable fast light chain composition. This is consistent with the different physiological roles these fibers are thought to have in muscle contraction.     

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.     

Changes in myosin isozymes during development of chicken breast muscle

The patterns of myosin isozymes in embryonic and adult chicken pectoralis muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light chains and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, the predominant isozyme component in embryonic pectoralis myosin could be clearly distinguished from adult myosin isozymes. SDS-polyacrylamide gel electrophoresis indicated that the light chain composition of embryonic myosin was also different from that of adult myosin. The pattern of peptide fragments produced by myosin digestion with a-chymotrypsin differed significantly between embryonic and adult skeletal myosin. These results suggest that myosin in the embryonic pectoralis muscle is different in both light and heavy chain composition from myosin in the same adult tissue.     

Synthesis and assembly of native myosin on muscle polyribosomes

We have used the overload-induced growth of avian muscles to study the assembly of the newly synthesized myosins which were separated by non-denaturing pyrophosphate-polyacrylamide gel electrophoresis. Using this model, we have observed the appearance of fast-like isomyosins in polyribosomes prepared from slow anterior latissimus dorsi muscle after 72 h of overload. These new isoforms comigrating with native myosin from fast posterior latissimus dorsi muscle were not yet present in cellular extracts from the same muscle. The in vitro translation system utilizing muscle specific polyribosomes directs the synthesis of the corresponding myosin isoforms. Under denaturing conditions, myosin heavy chains and light chains dissociate to the expected subunit composition of each specific isoform. The synthesis and assembly of native myosin on polyribosomes indicate that myosin exists as a single mature protein prior to the incorporation in the thick filament.     

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.     

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.     

The 204-kDa smooth muscle myosin heavy chain is phosphorylated in intact cells by casein kinase II on a serine near the carboxyl terminus

The heavy chain of smooth muscle myosin was found to be phosphorylated following immunoprecipitation from cultured bovine aortic smooth muscle cells. Of a variety of serine/threonine kinases assayed, only casein kinase II and calcium/calmodulin-dependent protein kinase II phosphorylated the smooth muscle myosin heavy chain to a significant extent in vitro. Two-dimensional maps of tryptic peptides derived from heavy chains phosphorylated in cultured cells revealed one major and one minor phosphopeptide. Identical tryptic peptide maps were obtained from heavy chains phosphorylated in vitro with casein kinase II but not with calcium/calmodulin-dependent protein kinase II. Of note, the 204-kDa smooth muscle myosin heavy chain but not the 200-kDa heavy chain isoform was phosphorylated by casein kinase II. Partial sequence of the tryptic phosphopeptides generated following phosphorylation by casein kinase II yielded Val-Ile-Glu-Asn-Ala-Asp-Gly-Ser*-Glu-Glu-Glu-Val. The Ser* represents the Ser(PO4) which is in an acidic environment, as is typical for casein kinase II phosphorylation sites. By comparison with the deduced amino acid sequence for rabbit uterine smooth muscle myosin (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737), we have localized the phosphorylated serine residue to the non-helical tail of the 204-kDa isoform of the smooth muscle myosin heavy chain. The ability of the 204-kDa isoform, but not the 200-kDa isoform, to serve as a substrate for casein kinase II suggests that these two isoforms can be regulated differentially.     

Analysis of smooth muscle myosin phosphorylation with native pyrophosphate gels and its application to studies on myosin phosphorylation in contracting smooth muscle

Gizzard smooth muscle myosin, the 20,000 Mr light chain (L20) of which had been phosphorylated in vitro with a calmodulin-myosin light chain kinase system, was separated into 5 isolated bands in a pyrophosphate polyacrylamide gel. Their mobilities were in the following order: myosin with 2 unphosphorylated L20 (GM) less than myosin with 1 unphosphorylated and 1 mono-phosphorylated L20 (GMP1) less than myosin with 2 mono-phosphorylated L20 (GMP2) less than myosin with 1 mono-phosphorylated and 1 di-phosphorylated L20 (GMP3) less than myosin with 2 di-phosphorylated L20 (GMP4). We used this pyrophosphate polyacrylamide gel electrophoresis to analyze the phosphorylated state of taenia coli smooth muscle during K+-induced contraction. During the initial 2 min contraction, phosphorylated forms corresponding to GMP1 and GMP2 were detected in addition to the unphosphorylated form.     

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.     

The human muscle proteome in aging

The aim of the present study was to assess age-dependent changes of proteins in the vastus lateralis muscle of physically active elderly and young subjects by a combination of two-dimensional difference gel electrophoresis, SDS-PAGE and ESI-MS/MS. The differences observed in the elderly group included down-regulation of regulatory myosin light chains, particularly the phosphorylated isoforms, a higher proportion of myosin heavy chain isoforms 1 and 2A, and enhanced oxidative and reduced glycolytic capacity.     

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.     

Expression of myosin heavy chain isoforms in regenerating myotubes of innervated and denervated chicken pectoral muscle

Monoclonal antibodies were prepared to stage-specific chicken pectoral muscle myosin heavy chain isoforms. From comparison of serial sections reacted with these antibodies, the myosin heavy chain isoform composition of individual myofibers was determined in denervated pectoral muscle and in regenerating myotubes that developed following cold injury of normal and denervated muscle. It was found that the neonatal myosin heavy chain reappeared in most myofibers following denervation of the pectoral muscle. Regenerating myotubes in both innervated and denervated muscle expressed all of the myosin heavy chain isoforms which have thus far been characterized in developing pectoral muscle. However, the neonatal and adult myosin heavy chains appeared more rapidly in regenerating myotubes compared to myofibers in developing muscle. While the initial expression of these isoforms in the regenerating areas was similar in innervated and denervated muscles, the neonatal myosin heavy chain did not disappear from noninnervated regenerating fibers. These results indicate that innervation is not required for the appearance of fast myosin heavy chain isoforms, but that the nerve plays some role in the repression of the neonatal myosin heavy chain.