Adult Slow Myosin Research Articles

Developmental expression of dystrophin, dystrophin-associated glycoproteins and other membrane cytoskeletal proteins in human skeletal and heart muscle

Dystrophin, utrophin and the dystrophin-associated glycoproteins, β-dystroglycan and adhalin, were analyzed, together with the membrane cytoskeletal proteins β-spectrin, vinculin and talin, and adult and fetal myosin heavy chains, in 25 normal human fetuses from 8 to 24 weeks of gestation. Dystrophin was present in heart and skeletal muscle from 8 weeks although in the latter was mainly in the cytoplasm at this stage. Utrophin expression increased until around gestational weeks 19/21, but by 24 weeks immunostaining and immunoblot band intensities had reduced. Beta-dystroglycan was scarce in skeletal muscle at 8 weeks, increased with maturation and was more abundant in heart of the same age. Adhalin appeared later than β-dystroglycan on skeletal muscle fiber surfaces, positivity became more intense as the fibers matured. In heart adhalin was detectable only in groups of cells at 12–16 weeks. From 8 weeks all fetal myotubes expressed β-spectrin on their surfaces, while vinculin and talin positivity was mainly at the periphery of the fascicles, increasing with age. Adult slow myosin was seen in most myotubes at 10 weeks. Secondary myotubes then formed which increasingly expressed adult fast myosin, while still retaining fetal myosin. By 24 weeks most fibers expressing adult slow myosin had lost fetal myosin and were more mature in the expression of most membrane proteins. Muscle membrane organization during human fetal development is a complex process and takes place earlier in heart than skeletal muscle.

Effects of chronic electrical stimulation on myosin heavy chain expression in satellite cell cultures derived from rat muscles of different fiber-type composition.

Myotube cultures were established from satellite cells of three rat muscles of different fiber-type composition, slow-twitch soleus, diaphragm, and fast-twitch tibialis anterior (TA). Effects of chronic electrical stimulation were studied by exposing these cultures for up to 13 days to a stimulus pattern consisting of 250 ms impulse trains of 40 Hz, repeated every 4 s. Changes in myosin expression were assessed at the mRNA level by Northern blotting and in situ hybridization. Expression of slow myosin at the protein level was analysed by immunoblotting and immunohistochemistry with two antibodies, one specific to adult slow myosin, the other reacting with developmental and adult slow myosin heavy chain (MHCI) isoforms. In all three myotube cultures stimulation enhanced the mRNA and protein expression of a developmental isoform of slow myosin (MHCI). However, the three myotube cultures differed in the extent of the increase in MHCI. It was greatest in soleus-derived myotubes, least in TA-derived myotubes, and intermediate in diaphragm-derived myotubes. In addition to the increase in slow myosin, long-term stimulation led to an isoform switch, as indicated by an increase in myotubes reacting with the antibody specific for the adult MHCI. Our results suggest that enhanced contractile activity promotes the expression of the slow phenotype predetermined in satellite cells of slow-twitch, type I fibers. The different extents of increased slow myosin expression may thus be explained as reflecting different percentages of type I fibers and consequently of slow-type satellite cells in the corresponding donor muscles.

Expression of myosin isoforms during notexin-induced regeneration of rat soleus muscles

Myosin isozymes and their fiber distribution were studied during regeneration of the soleus muscle of young adult (4–6 week old) rats. Muscle degeneration and regeneration were induced by a single subcutaneous injection of a snake toxin, notexin. If reinnervation of the regenerating muscle was allowed to occur (functional innervation nearly complete by 7 days), then fiber diameters continued to increase and by 28 days after toxin treatment they attained the same values as fibers in the contralateral soleus. If the muscles were denervated at the time of toxin injection, the early phases of regeneration still took place but the fibers failed to continue to increase in size. Electrophoresis of native myosin showed multiple bands between 3 and 21 days of regeneration which could be interpreted as indicating the presence of embryonic, neonatal, fast and slow myosins in the innervated muscles. Adult slow myosin became the exclusive form in innervated regenerates. In contrast, adult fast myosin became the predominant form in denervated regenerating muscles. Immunocytochemical localization of myosin isozymes demonstrated that in innervated muscles the slow form began to appear in a heterogeneous fashion at about 7 days, and became the major form in all fibers by 21–28 days. Thus, the regenerated muscle was almost entirely composed of slow fibers, in clear contrast to the contralateral muscle which was still substantially mixed. In denervated regenerating muscles, slow myosin was not detected biochemically or immunocytochemically whereas fast myosin was detected in all denervated fibers by 21–28 days. The regenerating soleus muscle therefore is clearly different from the developing soleus muscle in that the former is composed of a uniform fiber population with respect to myosin transitions. Moreover the satellite cells which account for the regeneration process in the soleus muscle do not appear to be predetermined with respect to myosin heavy chain expression, since the fibers they form can express either slow or fast isoforms. The induction of the slow myosin phenotype is entirely dependent on a positive, extrinsic influence of the nerve.

Myonuclear birthdates distinguish the origins of primary and secondary myotubes in embryonic mammalian skeletal muscles.

Myotubes were isolated from enzymically disaggregated embryonic muscles and examined with light microscopy. Primary myotubes were seen as classic myotubes with chains of central nuclei within a tube of myofilaments, whereas secondary myotubes had a smaller diameter and more widely spaced nuclei. Primary myotubes could also be distinguished from secondary myotubes by their specific reaction with two monoclonal antibodies (MAbs) against adult slow myosin heavy chain (MHC). Myonuclei were birth dated with [3H]thymidine autoradiography or with 2-bromo-5'-deoxyuridine (BrdU) detected with a commercial monoclonal antibody. After a single pulse of label during the 1-2 day period when primary myotubes were forming, some primary myotubes had many myonuclei labelled, usually in adjacent groups, while in others no nuclei were labelled. If a pulse of label was administered after this time labelled myonuclei appeared in most secondary myotubes, while primary myotubes received few new nuclei. Labelled and unlabelled myonuclei were not grouped in the secondary myotubes, but were randomly interspersed. We conclude that primary myotubes form by a nearly synchronous fusion of myoblasts with similar birthdates. In contrast, secondary myotubes form in a progressive fashion, myoblasts with asynchronous birthdates fusing laterally with secondary myotubes at random positions along their length. These later-differentiating myoblasts do not fuse with primary myotubes, despite being closely apposed to their surface. Furthermore, they do not generally fuse with each other, as secondary myotube formation is initiated only in the region of the primary myotube endplate.

Coexpression of myosin isoforms in muscle of patients with neurogenic disease.

Three well-characterized antimyosin heavy chain monoclonal antibodies (McAbs) were used as immunocytochemical reagents to study myosin isoform expression in relationship to adenosine triphosphatase (ATPase) defined fiber types in human muscle. The biopsy specimens were from patients with neurogenic muscle disease whose muscle exhibited fiber type grouping and group atrophy. The use of McAbs revealed heretofore unrecognized coexpression of multiple myosin isoforms in selected fibers in the pathologic samples which was not apparent with ATPase reactions and not present in normal muscle. The fibers containing multiple myosin isoforms were probably undergoing neurally directed fiber type transformation. Furthermore, a small population of fibers in neurogenic specimens expressed a "prenatal" myosin signifying the presence of regenerating fibers. We also demonstrated immunocytochemical evidence of the persistence of adult slow myosin in denervated mature human skeletal muscle despite the reputed necessity of innervation for maintenance of expression of this myosin isoform proffered by others.

Fast muscle fibers are preferentially affected in Duchenne muscular dystrophy

We show that Duchenne muscular dystrophy (DMD) selectively affects a subset of skeletal muscle fibers specialized for fast contraction. Muscle fiber types were characterized immunohistochemically with monoclonal antibodies that distinguish isoforms of fetal and adult-fast or adult-slow myosin heavy chain present in the same fiber. Fetal myosin expression increased with patient age and was not due to arrested development but rather to de novo synthesis, which served as a sensitive indicator of muscle regeneration. A subset of fast fibers were the first to degenerate (type Ilb). Extensive fast fiber regeneration occurred before slow fibers were affected. These results suggest that the DMD gene product has a specific function in a subpopulation of muscle fibers specialized to respond to the highest frequency of neuronal stimulation with maximal rates of contraction.

Immunocytochemical analysis of myosin heavy chains in human fetal skeletal muscles

Quadriceps muscle samples from human fetuses (10 weeks of gestation to term) were studied using immunocytochemical methods with monoclonal antibodies against fetal, adult-slow and adult-fast B myosin heavy chains. The monoclonal antibodies were selected for their virtually exclusive specificity for a particular isomyosin and used to investigate the expression of different myosin heavy chains during fetal development of muscle fibres. Concomitant studies of the myofibrillary ATPase pattern of muscle fibres were carried out. A fetal-specific myosin was persistently expressed during fetal life but at a continuously decreasing rate. Adult-slow myosin was observed in a small pool of muscle fibres, histochemically undifferentiated, in fetuses of 14–16 weeks of gestation. However, adult isomyosins appeared intensively only in the late fetal period, progressively replacing fetal myosin. The genes coding for adult-slow myosin are expressed earlier that those coding for adult-fast myosin. Myosin heavy chains specific for the neonatal period were not demonstrated with the antibodies used in this study. The contribution, provided by the present study, to the knowledge of the sequence of events in the expression of myosin heavy chains during normal muscle development, may allow a better understanding of eventual myosin changes which may occur in genetic muscle disorders.

Myosin isozyme transitions occurring during the postnatal development of the rat soleus muscle

The myosin isozymes present in the developing rat soleus muscle from 1 week to 6 weeks after birth were investigated using biochemical and immunological methods. Electrophoresis of native myosin reveals that adult slow myosin is present in the soleus as early as 1 week after birth. At this time, embryonic and neonatal myosin can also be demonstrated. Using an immunotransfer technique, the presence of slow myosin heavy chain can be demonstrated at all time points examined whereas neonatal myosin heavy chain diminishes in quantity between 2 and 3 weeks, and is undetectable in the adult soleus. Specific polyclonal antibodies were prepared to embryonic, neonatal, and adult fast and slow myosins. Immunocytochemistry reveals a cellular heterogeneity at all stages examined. Different combinations of myosin isozymes can be found in the soleus fibers depending on the stage of development; these results suggest therefore that myosin isozyme transitions are occurring. Approximately half the fibers contain embryonic and slow myosin at 1 week after birth; these fibers subsequently contain only slow myosin. A second group of fibers contains embryonic and neonatal myosin at 1 week and most of them subsequently accumulate adult fast myosin. A portion of this latter group begins to acquire slow myosin from 4 weeks of age. These data are interpreted to suggest that a preprogrammed sequence of myosin isozymes is embryonic → neonatal → adult fast. At any time during development of an individual fiber, induction of slow myosin accumulation and repression of other types can occur.

Myosin transitions in developing fast and slow muscles of the rat hindlimb

Myosin isozymes from the slow soleus and fast EDL muscles of the rat hindlimb were analyzed by pyrophosphate gel electrophoresis, by peptide mapping of heavy chains, and by antibody staining. At the earliest stage examined, 20 days gestation, distinctions between the developing fast and slow muscles were seen by all these criteria; all fibers in the distal hindlimb reacted strongly with antibody to adult fast myosin. Some fibers also reacted with antibody to adult slow myosin; these fibers had a precise, axial distribution in the hindlimb. This pattern of staining which includes the entire soleus, foreshadows the adult distribution of slow fibers and may indicate that the specific pattern of innervation of the limb is already determined. In the early developing soleus there are four fetal and neonatal isozymes plus two isozymes present in equal proportions in the ‘slow’ area of the pyrophosphate gel. The mobility of these two slow isozymes decreases with maturity and the slowest moving isozyme gradually becomes the dominant species. Thus early diversity between the soleus and EDL is expressed by myosins which are distinct from the mature isozymes. The relative proportion of slow isozymes significantly increases with development and as this occurs the fetal and neonatal isozymes are progressively eliminated. Transiently at least one mature fast isozyme appears in the soleus. This is present at 15 days postpartum and probably correlates with the population of fast, type II fibers, which comprise 50% of this muscle cell population at 15 days. The EDL contained three fetal and neonatal isozymes and only one slow isozyme which does not change in mobility with age. Slow isozymes in the soleus and EDL are thus not identical. Each muscle underwent a unique series of changes until the adult pattern of isozymes and heavy chains was reached about one month postpartum.

Effects of hypothyroidism on myosin isozyme transitions in developing rat muscle

Hypothyroidism was induced in young rats by methylthiouracil treatment of pregnant mothers from 18 days of gestation to 4 weeks after birth. Electrophoretic analysis of native myosin isozymes revealed a persistence of neonatal and embryonic myosin in developing fast and slow muscles up to at least 28 days after birth. The appearance of adult fast myosin was inhibited in 28-day old animals, however adult slow myosin was found in the soleus muscle. Immunocytochemical results on the soleus demonstrate a cellular heterogeneity in the response to hypothyroidism. About half fibers have a normal complement of slow myosin and do not contain neonatal myosin. Only the remaining fibers contain the large amounts of neonatal myosin demonstrated by electrophoresis.

Myosin types during the development of embryonic chicken fast and slow muscles.

We have studied the myosin types present in developing fast and slow muscles of the chicken embryo. Myosin light chains were characterized by their mobility on sodium dodecyl sulfate/polyacrylamide gels; myosin heavy chains were identified by their reaction with antibodies specific for adult fast or adult slow myosin heavy chains. During development, the pectoralis muscle, a fast muscle in the adult, contains heavy chains and two of the three light chains characteristic of adult fast muscle myosin. However, the anterior latissimus dorsi muscle, a slow muscle in the adult, also contains fast myosin light and heavy chains during early development. Only after the time of innervation does this muscle begin synthesizing predominantly the slow myosin heavy and light chains. We hypothesize that the synthesis of fast myosin in both early fast and slow muscles is the result of the endogenous program for muscle development; initiation of the synthesis of slow myosin, however, is dependent upon exogenous factors.