Intracompartmental sorting of essential myosin light chains: molecular dissection and in vivo monitoring by epitope tagging.

The isoprotein-specific intracompartmental sorting of the three essential myosin light chains (LCs), the skeletal muscle LC-1f and LC-3f and the nonmuscle LC-3nm, was investigated. Epitope tagging was used to monitor the intracellular localization to different cytoskeletal structures of the exogenously introduced constructs in adult rat cardiomyocytes (ARCs), which exhibit both stress fibers and regenerating myofibrils. LC-1f and LC-3f bind almost exclusively to the sarcomeric myosin heavy chain (MHC) with high affinity, while the LC-3nm interacts with stress fibers and sarcomeres equally well. Sorting appears to be directed by a hierarchical order of different affinities. Domain mapping by deletion and by construction of a LC-1f/3nm chimera suggests that the LCs are composed of three functionally distinct domains: a basal MHC binding site in the C-terminus; the central part, modulating the preferential interaction with MHC isoforms; and the isoprotein-specific N-terminus of the essential LC, which is probably not involved in the sorting process.     

Myosin heavy-chain-based isomyosins in developing, adult fast-twitch and slow-twitch muscles.

A modified method of electrophoresis under nondenaturing conditions made it possible to separate rat muscle extracts of defined myosin heavy chain (HC) and light chain (LC) composition into subsets of developmental, fast and slow myosin heavy-chain-based isomyosins. The fastest migrating isomyosins were the neonatal isomyosins (nM1, nM2, nM3), followed by the slightly slower migrating embryonic isomyosins (eM1, eM2, eM3, eM4). Of the nine adult fast isomyosins, the HCIIb-based isomyosins (FM1b, FM2b, FM3b) were the fastest migrating. These were followed by the HCIId-based isomyosins (FM1d, FM2d, FM3d). The HCIIa-based isomyosins (FM1a, FM2a, FM3a) were the slowest. Our results suggest that FM3a is identical with the so-called intermediate isomyosin (IM) described in the literature. The slow myosin heavy-chain-based isomyosins (SM1, SM2, SM3) migrated far behind the fast isomyosins. Whereas the gross electrophoretic mobilities of each of these isomyosin triplets is determined by the specific heavy chain complement, the different mobilities of the bands within each triplet result from different alkali light chain combinations. Thus, the fastest triplet bands of the neonatal (nM1) and adult fast isomyosins (FM1b, FM1d, FM1a) represent the LC3f homodimers, the slowest (nM3, FM3b, FM3d, FM3a) the LC1f homodimers, and the intermediate bands (nM2, FM2b, FM2d, FM2a) the LC1f/LC3f heterodimers. Different proportions of the adult fast isomyosin triplet bands indicate that the affinity for LC3f decreases in the order HCIIb, HCIId, HCIIa. The three slow isomyosins represent LC1sa (SM1) and LC1sb (SM3) homodimers and a LC1sa/LC1sb heterodimer (SM2). Circumstantial evidence suggests an inverse order in rabbit muscle where SM1 and SM3 most likely represent LC1sb and LC1sa homodimers, respectively.     

Nascent muscle fiber appearance in overloaded chicken slow-tonic muscle

The application of a weight overload to the humerus of chickens induces a hypertrophy of anterior latissimus dorsi (ALD) muscle fibers. This growth is accompanied by a rapid and almost complete replacement of one slow-tonic myosin isoform, SM-1, by another slow-tonic isoform, SM-2. In addition, a population of small fibers appears mainly in extrafascicular spaces and, concurrently, three additional myosin bands are detected by gel electrophoresis. Five antibodies against myosin heavy chain (MHC) isoforms were selected as immunocytochemical probes to determine the cellular location and nature of these myosins. The antibodies react with ventricular, fast skeletal muscle and either SM-1 or SM-2, or both the slow-tonic MHCs. The antifast and antiventricular antibodies react with myosin present in the 10-day embryonic ALD muscle but do not react with myosin in posthatch ALD muscle. The small fibers in overloaded muscle contain a myosin isoform characteristically expressed during the embryonic stage of ALD muscle development and therefore are named nascent myofibers. Some of the nascent myofibers do not react with the antibody to both slow-tonic MHCs, indicating the lack of the normal adult slow-tonic myosins which are expressed in 10-day embryos. In order to explore the origin of the nascent fibers, an electron microscopic study was performed. Stereological analysis of the existing fibers shows a stimulation of numbers and sizes of satellite cells. In addition, the volume occupied by nonmuscle and undifferentiated cells increases dramatically. Myotube formation with incipient myofibrils is seen in extrafascicular spaces. These data suggest that new muscle fiber formation accompanies hypertrophy in overloaded chicken ALD muscle and the process may involve satellite cell migration.     

Immunolocalization of muscle and nonmuscle isoforms of actin in myogenic cells and adult skeletal muscle

In vertebrate skeletal muscle, the proliferating myoblasts synthesize nonmuscle isoforms of actin, and the cells begin to express muscle-specific actin isoforms during their myogenic differentiation. To study the distributions of the actin isoforms in myogenic cells and fully differentiated skeletal muscle, we prepared a peptide antibody specific for the skeletal alpha isoform of actin and used this antibody along with an antibody specifically reactive with nonmuscle gamma actin to stain cultured myotubes and adult skeletal myofibrils by double-indirect immunofluorescence. At this level of resolution, no differences in isoform localization were seen: Both muscle and nonmuscle actins were detected in the myotubes and in the striations of mature myofibrils. Myotubes were also double-stained using immunogold electron microscopy, and the isoform distributions were determined quantitatively by counting the two sizes of gold particles that corresponded to labeling with each antibody. A quantitative analysis of immunoreactivity revealed that, although both forms were present in all actin-containing structures, nonmuscle actin was relatively more prevalent along the edges (cortical microfilaments) of the myotubes, whereas the muscle isoform predominated in the interior regions (containing forming myofibrils). Thus, we have found evidence of a heterogeneous distribution of muscle and nonmuscle actin isoforms in differentiating myogenic cells, and we have demonstrated that a nonmuscle actin isoform is a component of the muscle contractile apparatus.     

The second EF-hand is responsible for the isoform-specific sorting of myosin essential light chain.

It has been known that isoforms of myosin essential light chain (LC) exhibit the isoform-specific sorting within cardiac myocytes and fibroblasts. In order to analyze which domain of LC is responsible for the sorting, various chimeric cDNA constructs between human nonmuscle isoform (LC3nm) and chicken fast skeletal muscle isoform (LC3f) were generated and expressed in cultured chicken cardiac myocytes. If chimeras contained LC3f sequence at the place that was restricted by BssHII and PstI, they were preferentially sorted to sarcomeres and precisely localized at A-bands, and their incorporation levels into the A-bands were identical with that of the wild type LC3f. However, other chimeras were distributed throughout the cytoplasm like the wild type LC3nm. Comparison of amino acid sequences revealed that 12 amino acids are different between chicken LC3f and human LC3nm in the BssHII-PstI fragment, and these amino acids are located within the second EF-hand of LC. These results indicated that the second EF-hand is responsible for the isoform-specific sorting of LC. Although the second EF-hand is not included in the key contacts with myosin heavy chain, it is supposed that this domain is important for the relative disposition of neighboring domains. Thus, the 12 amino acids in the second EF-hand might play a key role for modulation of overall configuration of LC, thereby influencing the precise association of the key contacts.     

Subunit composition of rodent isomyosins and their distribution in hindlimb skeletal muscles

Three adult skeletal muscle sarcomeric myosin heavy chain (MHC) genes have been identified in the rat, suggesting that the expressed native myosin isoforms can be differentiated, in part, on the basis of their MHC composition. This study was undertaken to ascertain whether the five major native isomyosins [3 fast (Fm1, Fm2, Fm3), 1 slow (Sm), and 1 intermediate (Im)], typically expressed in the spectrum of adult rat skeletal muscles comprising the hindlimb, could be further differentiated on the basis of their MHC profiles in addition to their light chain composition. Results show that in muscles comprised exclusively of fast-twitch glycolytic (FG) fibers and consisting of Fm1, Fm2, and Fm3, such as the tensor fasciae latae, only one MHC, designated as fast type IIb, could be resolved. In soleus muscle, comprised of both slow-twitch oxidative and fast-twitch oxidative-glycolytic fibers and expressing Sm and Im, two MHC bands were resolved and designated as slow/cardiac beta-MHC and fast type IIa MHC. In muscles expressing a mixture of all three fiber types and a full complement of isomyosins, as seen in the plantaris, the MHC could be resolved into three bands. Light chain profiles were characterized for each muscle type, as well as for the purified isomyosins. These data suggest that Im (IIa) consists of a mixture of fast and slow light chains, whereas Fm (IIb) and Sm (beta) isoforms consist solely of fast- and slow-type light chains, respectively. Polypeptide mapping of denatured myosin extracted from muscles expressing contrasting isoform phenotypes suggests differences in the MHC primary structure between slow, intermediate, and fast myosin isotypes. These findings demonstrate that 1) Fm, Im, and Sm isoforms are differentiated on the bases of both their heavy and light chain components and 2) each isomyosin is distributed in a characteristic fashion among rat hindlimb skeletal muscles. Furthermore, these data suggest that the ratio of isomyosins in a given muscle or muscle region is of physiological importance to the function of that muscle during muscular activity.     

Myofibrillogenesis in the first cardiomyocytes formed from isolated quail precardiac mesoderm

De novo assembly of myofibrils was investigated in explants of precardiac mesoderm from quail embryos to address a controversy about different models of myofibrillogenesis. The sequential expression of sarcomeric components was visualized in double- and triple-stained explants before, during, and just after the first cardiomyocytes began to beat. In explants from stage 6 embryos, cultured for 10 h, ectoderm, endoderm, and the precardiac mesoderm displayed arrays of stress fibers with alternating bands of the nonmuscle isoforms of alpha-actinin and myosin IIB. With increasing time in culture, mesoderm cells contained fibrils composed of actin, nonmuscle myosin IIB, and sarcomeric alpha-actinin. Several hours later, before beating occurred, both nonmuscle and muscle myosin II localized in some of the fibrils in the cells. Concentrations of muscle myosin began as thin bundles, dispersed in the cytoplasm, often overlapping one another, and progressed to small, aligned A-band-sized aggregates. The amount of nonmuscle myosin decreased dramatically when Z-bands formed, the muscle myosin became organized into A-bands, and the cells began beating. The sequential changes in protein composition of the fibrils in the developing muscle cells supports the model of myofibrillogenesis in which assembly begins with premyofibrils and progresses through nascent myofibrils to mature myofibrils.     

Monoclonal antibodies to the muscle isoform of alpha-actinin--a marker for the study of the differentiation of skeletal and cardiac muscles

A battery of monoclonals to the rabbit skeletal muscle alpha-actinin has been produced. The majority of monoclonals proved to be species-specific by indirect immunofluorescence on the isolated rabbit skeletal myofibrils and on the differentiating cultures of chicken and rat skeletal muscles. One monoclonal, EA-53, reacts with the skeletal muscle alpha-actinin of various species (rat, rabbit, chicken) in immunofluorescence and immunoblotting. The monoclonal EA-53 recognizes also heart muscle alpha-actinin in cultured cardiomyocytes of human, rat and mouse origin. EA-53 does not stain alpha-actinin in myoblasts, fibroblasts, and endothelial cells. The monoclonal antibody EA-53 discriminating muscle and nonmuscle alpha-actinin isoforms could be used as a tool to study the mechanisms of skeletal and cardiac myogenesis.     

Two distinct nonmuscle myosin-heavy-chain mRNAs are differentially expressed in various chicken tissues. Identification of a novel gene family of vertebrate non-sarcomeric myosin heavy chains

Two distinct cDNA clones for nonmuscle myosin heavy chain (MHC) were isolated from a chicken fibroblast cDNA library by cross-hydridization under a moderate stringency with chicken gizzard smooth muscle MHC cDNA. These two fibroblast MHC and the gizzard MHC are each encoded in different genes in the chicken genome. Northern blot analysis showed that both of the nonmuscle MHC mRNAs were expressed not only in fibroblasts but also in a variety of tissues including brain, lung, kidney, spleen, and skeletal, cardiac and smooth muscles. However, the relative contents of the two nonmuscle MHC mRNAs varied greatly among tissues. The encoded amino acid sequences of the nonmuscle MHCs were highly similar to each other (81% identity) and to the smooth muscle MHC (81-84%), but much less similar to vertebrate skeletal muscle MHCs (38-41%) or to protista nonmuscle MHCs (35-36%). A phylogenic tree of MHC isoforms was constructed by calculating the similarity scores between these MHC sequences. An examination of the tree showed that the vertebrate sarcomeric (skeletal and cardiac) MHC isoforms are encoded in a very closely related multigene family, and that the vertebrate non-sarcomeric (smooth muscle and nonmuscle) MHC isoforms define a distinct, less conserved MHC gene family.     

Expression of myofibrillar proteins and parvalbumin isoforms in white muscle of dorada during development

Several polyacrylamide gel electrophoresis techniques were used to study developmental changes in myofibrillar protein composition and parvalbumin distribution in the myotomal muscle of Brycon moorei . Two myosin LC2 chains and two troponin I isoforms were successively detected. Up to four troponin T isoforms were synthesized. Slow red-muscle myofibrils from adult fish showed no common component (except actin) with larval, juvenile or adult fast white-muscle myofibrils. During growth of B. moorei , two classes of parvalbumin isoforms were sequentially expressed: larval PA I, PA IIa, and PA IIb and adult PA III. In adult fish, the content in Tn T-2 isoform decreased from the anterior to the posterior myomeres, in favour of Tn T-1 and Tn T-4. The parvalbumin content also diminished from the rostral to the caudal muscle. The fast rate of transition from larval to adult isoforms appeared to parallel the extremely fast growth of B. moorei . Sequential expression of these isoforms presumably reflected variations in the contractile properties of the muscle fibres, required by changes in physiological demands of the propulsive musculature.     

α-Smooth muscle actin is transiently expressed in embryonic rat cardiac and skeletal muscles

Actin isoform expression may change during development, and in certain physiological, experimental and pathological situations. It is accepted that during sarcomeric (skeletal and cardiac) muscle development, the alpha-skeletal and alpha-cardiac isoforms of actin accumulate rapidly at the onset of muscle fibre formation, while there is a rapid fall in the expression of nonmuscle (beta and gamma) actin isoforms. Here we show that, before birth, both skeletal and myocardial cells express significant amounts of alpha-smooth muscle actin mRNA and protein. This expression is transient and disappears over the 1-7 days following birth. Our findings show that the program regulating actin isoform expression in sarcomeric muscle development is complex and that alpha-smooth muscle actin participates in this process.     

Roles of troponin isoforms in pH dependence of contraction in rabbit fast and slow skeletal and cardiac muscles.

Skinned fibers prepared from rabbit fast and slow skeletal and cardiac muscles showed acidotic depression of the Ca2+ sensitivity of force generation, in which the magnitude depends on muscle type in the order of cardiac>fast skeletal>slow skeletal. Using a method that displaces whole troponin-complex in myofibrils with excess troponin T, the roles of Tn subunits in the differential pH dependence of the Ca2+ sensitivity of striated muscle were investigated by exchanging endogenous troponin I and troponin C in rabbit skinned cardiac muscle fibres with all possible combinations of the corresponding isoforms expressed in rabbit fast and slow skeletal and cardiac muscles. In fibers exchanged with fast skeletal or cardiac troponin I, cardiac troponin C confers a higher sensitivity to acidic pH on the Ca2+ sensitive force generation than fast skeletal troponin C independently of the isoform of troponin I present. On the other hand, fibres exchanged with slow skeletal troponin I exhibit the highest resistance to acidic pH in combination with either isoform of troponin C. These results indicate that troponin C is a determinant of the differential pH sensitivity of fast skeletal and cardiac muscles, while troponin I is a determinant of the pH sensitivity of slow skeletal muscle.     

Effects of resistance training on myosin function studied by the in vitro motility assay in young and older men.

It is generally believed that the maximum shortening velocity (V(o)) of a skeletal muscle fiber type does not vary unless a change in myosin heavy chain (MHC) isoform composition occurs. However, recent findings have shown that V(o) of a given fiber type can change after training, suggesting the hypothesis that the function of myosin can vary without a change in isoform. The present study addressed the latter hypothesis by studying the function of isolated myosin isoforms by the use of the in vitro motility assay (IVMA) technique. Four young (age 23-29 yr, YO) and four elderly men (age 68-82 yr, EL) underwent a 12-wk progressive resistance training program of the knee extensor muscles and to one pre- and one posttraining biopsy of the vastus lateralis muscle. The significant increase in one-repetition maximum posttraining in both YO and EL indicated that training was effective. After training, MHC isoform composition showed a shift from MHC(2X) toward MHC(2A) in YO and no shift in EL. The velocity of sliding (V(f)) of actin filaments on pure myosin isoforms extracted from single fibers was studied in IVMA. One hundred sixty IVMA samples were prepared from 480 single fibers, and at least 50 filaments were analyzed in each experiment. Whereas no training-induced change was observed in V(f) of myosin isoform 1 either in YO or in EL, a significant increase in V(f) of myosin isoform 2A after training was observed in both YO (18%) and EL (19%). The results indicate that resistance training can change the velocity of the myosin molecule.     

The Superfast Human Extraocular Myosin Is Kinetically Distinct from the Fast Skeletal IIa,IIb, and IId Isoforms

Humans express five distinct myosin isoforms in the sarcomeres of adult striated muscle (fast IIa, IId, the slow/cardiac isoform I/β, the cardiac specific isoform α, and the specialized extraocular muscle isoform). An additional isoform, IIb, is present in the genome but is not normally expressed in healthy human muscles. Muscle fibers expressing each isoform have distinct characteristics including shortening velocity. Defining the properties of the isoforms in detail has been limited by the availability of pure samples of the individual proteins. Here we study purified recombinant human myosin motor domains expressed in mouse C₂C₁₂ muscle cells. The results of kinetic analysis show that among the closely related adult skeletal isoforms, the affinity of ADP for actin·myosin (K_AD) is the characteristic that most readily distinguishes the isoforms. The three fast muscle myosins have K_AD values of 118, 80, and 55 μm for IId, IIa, and IIb, respectively, which follows the speed in motility assays from fastest to slowest. Extraocular muscle is unusually fast with a far weaker K_AD = 352 μm. Sequence comparisons and homology modeling of the structures identify a few key areas of sequence that may define the differences between the isoforms, including a region of the upper 50-kDa domain important in signaling between the nucleotide pocket and the actin-binding site.     

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.     

Skeletal muscle calcineurin: influence of phenotype adaptation and atrophy

Calcineurin (CaN) has been implicated as a signaling molecule that can transduce physiological stimuli (e.g., contractile activity) into molecular signals that initiate slow-fiber phenotypic gene expression and muscle growth. To determine the influence of muscle phenotype and atrophy on CaN levels in muscle, the levels of soluble CaN in rat muscles of varying phenotype, as assessed by myosin heavy chain (MHC)-isoform proportions, were determined by Western blotting. CaN levels were significantly greater in the plantaris muscle containing predominantly fast (IIx and IIb) MHC isoforms, compared with the soleus (predominantly type I MHC) or vastus intermedius (VI, contains all 4 adult MHC isoforms). Three months after a complete spinal cord transection (ST), the CaN levels in the VI muscle were significantly reduced, despite a significant increase in fast MHC isoforms. Surprisingly, the levels of CaN in the VI were highly correlated with muscle mass but not MHC isoform proportions in ST and control rats. These data demonstrate that CaN levels in skeletal muscle are highly correlated to muscle mass and that the normal relationship with phenotype is lost after ST.