Co-expression of multiple myosin heavy chain genes, in addition to a tissue-specific one, in extraocular musculature

We have investigated the developmental transitions of myosin heavy chain (MHC) gene expression in the rat extraocular musculature (EOM) at the mRNA level using S1-nuclease mapping techniques and at the protein level by polypeptide mapping and immunochemistry. We have isolated a genomic clone, designated lambda 10B3, corresponding to an MHC gene which is expressed in the EOM fibers (recti and oblique muscles) of the adult rat but not in hind limb muscles. Using cDNA and genomic probes for MHC genes expressed in skeletal (embryonic, neonatal, fast oxidative, fast glycolytic, and slow/cardiac beta-MHC), cardiac (alpha-MHC), and EOM (lambda 10B3) muscles, we demonstrate the concomitant expression at the mRNA level of at least six different MHC genes in adult EOM. Protein and immunochemical analyses confirm the presence of at least four different MHC types in EOM. Immunocytochemistry demonstrates that different myosin isozymes tend to segregate into individual myofibers, although some fibers seem to contain more than one MHC type. The results also show that the EOM fibers exhibit multiple patterns of MHC gene regulation. One set of fibers undergoes a sequence of isoform transitions similar to the one described for limb skeletal muscles, whereas other EOM myofiber populations arrest the MHC transition at the embryonic, neonatal/adult, or adult EOM-specific stage. Thus, the MHC gene family is not under the control of a strict developmental clock, but the individual genes can modify their expression by tissue-specific and/or environmental factors.     

Eyestalk ablation has little effect on actin and myosin heavy chain gene expression in adult lobster skeletal muscles

The organization of skeletal muscles in decapod crustaceans is significantly altered during molting and development. Prior to molting, the claw muscles atrophy dramatically, facilitating their removal from the base of the claw. During development, lobster claw muscles exhibit fiber switching over several molt cycles. Such processes may be influenced by the secretion of steroid molting hormones, known collectively as ecdysteroids. To assay the effects of these hormones, we used eyestalk ablation to trigger an elevation of circulating ecdysteroids and then quantified myofibrillar mRNA levels with real-time PCR and myofibrillar protein levels by SDS-PAGE. Levels of myosin heavy chain (MHC) and actin proteins and the mRNA encoding them were largely unaffected by eyestalk ablation, but in muscles from intact animals, myofibrillar gene expression was modestly elevated in premolt and postmolt animals. In contrast, polyubiquitin mRNA was significantly elevated (about 2-fold) in claw muscles from eyestalk-ablated animals with elevated circulating ecdysteroids. Moreover, patterns of MHC and actin gene expression are significantly different among slow and fast claw muscles. Consistent with these patterns, the three muscle types differed in the relative amounts of myosin heavy chain and actin proteins. All three muscles also co-expressed fast and slow myosin isoforms, even in fibers that are generally regarded as exclusively fast or slow. These results are consistent with other recent data demonstrating co-expression of myosin isoforms in lobster muscles.     

Single-fiber myosin heavy chain polymorphism during postnatal development: modulation by hypothyroidism

The primary objective of this study was to follow the developmental time course of myosin heavy chain (MHC) isoform transitions in single fibers of the rodent plantaris muscle. Hypothyroidism was used in conjunction with single-fiber analyses to better describe a possible linkage between the neonatal and fast type IIB MHC isoforms during development. In contrast to the general concept that developmental MHC isoform transitions give rise to muscle fibers that express only a single MHC isoform, the single-fiber analyses revealed a very high degree of MHC polymorphism throughout postnatal development. In the adult state, MHC polymorphism was so pervasive that the rodent plantaris muscles contained approximately 12-15 different pools of fibers (i.e., fiber types). The degree of polymorphism observed at the single-fiber level made it difficult to determine specific developmental schemes analogous to those observed previously for the rodent soleus muscle. However, hypothyroidism was useful in that it confirmed a possible link between the developmental regulation of the neonatal and fast type IIB MHC isoforms.     

Myofibrillar gene expression in differentiating lobster claw muscles

Lobster claw muscles undergo a process of fiber switching during development, where isomorphic muscles containing a mixture of both fast and slow fibers, become specialized into predominantly fast, or exclusively slow, muscles. Although this process has been described using histochemical methods, we lack an understanding of the shifts in gene expression that take place. In this study, we used several complementary techniques to follow changes in the expression of a number of myofibrillar genes in differentiating juvenile lobster claw muscles. RNA probes complementary to fast and slow myosin heavy chain (MHC) mRNA were used to label sections of 7th stage (approximately 3 months old) juvenile claw muscles from different stages of the molt cycle. Recently molted animals (1-5 days postmolt) had muscles with distinct regions of fast and slow gene expression, whereas muscles from later in the molt cycle (7-37 days postmolt) had regions of fast and slow MHC expression that were co-mingled and indistinct. Real-time PCR was used to quantify several myofibrillar genes in 9th and 10th stages (approximately 6 months old) juvenile claws and showed that these genes were expressed at significantly higher levels in the postmolt claws, as compared with the intermolt and premolt claws. Finally, Western blot analyses of muscle fibers from juvenile lobsters approximately 3 to 30 months in age showed a shift in troponin-I (TnI) isoform expression as the fibers differentiated into the adult phenotypes, with expression of the adult fast fiber TnI pattern lagging behind the adult slow fiber TnI pattern. Collectively, these data show that juvenile and adult fibers differ both qualitatively and quantitative in the expression of myofibrillar proteins and it may take as much as 2 years for juvenile fibers to achieve the adult phenotype.     

Diversity of fast myosin heavy chain expression during development of gastrocnemius, bicep brachii, and posterior latissimus dorsi muscles in normal and dystrophic chickens

The expression of fast myosin heavy chain (MHC) isoforms was examined in developing bicep brachii, lateral gastrocnemius, and posterior latissimus dorsi (PLD) muscles of inbred normal White Leghorn chickens (Line 03) and genetically related inbred dystrophic White Leghorn chickens (Line 433). Utilizing a highly characterized monoclonal antibody library we employed ELISA, Western blot, immunocytochemical, and MHC epitope mapping techniques to determine which MHCs were present in the fibers of these muscles at different stages of development. The developmental pattern of MHC expression in the normal bicep brachii was uniform with all fibers initially accumulating embryonic MHC similar to that of the pectoralis muscle. At hatching the neonatal isoform was expressed in all fibers; however, unlike in the pectoralis muscle the embryonic MHC isoform did not disappear. With increasing age the neonatal MHC was repressed leaving the embryonic MHC as the only detectable isoform present in the adult bicep brachii muscle. While initially expressing embryonic MHC in ovo, the post-hatch normal gastrocnemius expressed both embryonic and neonatal MHCs. However, unlike the bicep brachii muscle, this pattern of expression continued in the adult muscle. The adult normal gastrocnemius stained heterogeneously with anti-embryonic and anti-neonatal antibodies indicating that mature fibers could contain either isoform or both. Neither the bicep brachii muscle nor the lateral gastrocnemius muscle reacted with the adult specific antibody at any stage of development. In the developing posterior latissimus dorsi muscle (PLD), embryonic, neonatal, and adult isoforms sequentially appeared; however, expression of the embryonic isoform continued throughout development. In the adult PLD, both embryonic and adult MHCs were expressed, with most fibers expressing both isoforms. In dystrophic neonates and adults virtually all fibers of the bicep brachii, gastrocnemius, and PLD muscles were identical and contained embryonic and neonatal MHCs. These results corroborate previous observations that there are alternative programs of fast MHC expression to that found in the pectoralis muscle of the chicken (M.T. Crow and F.E. Stockdale, 1986, Dev. Biol. 118, 333-342), and that diversification into fibers containing specific MHCs fails to occur in the fast muscle fibers of the dystrophic chicken. These results are consistent with the hypothesis that avian muscular dystrophy is a developmental disorder that is associated with alterations in isoform switching during muscle maturation.     

Possible Influence of B Chromosomes on Genes Included in Immune Response and Parasite Burden in Apodemus flavicollis

Genetic background underlying wild populations immune response to different parasites is still not well understood. We studied immune response to multiple infections and to competition between different parasite species at different developmental stages in population of yellow-necked mouse, Apodemus flavicollis. Quantitative real-time PCR was used to investigate associations of MHC II-DRB, IL-10 and Tgf-β genes expressions with presence of intestinal parasites at different developmental stages. Furthermore, we were interested whether the host related characteristics (sex, age, body condition, presence of B chromosomes or expression of other genes) or characteristics of present parasites (number of adult parasites of each identified species, egg count of each parasite genus, total number of nematode individuals) affect differential expression of the studied genes. A significant invert association between the expression of MHC II-DRB and Tgf-β gene was found, which together with absence of IL-10 association confirmed modified Th2 as the main type of immune response to nematode infections. Effect of recorded parasites and parasite life-cycle stage on expression levels of MHC II-DRB gene was detected only through interactions with host-related characteristics such as sex, age, and the presence of B chromosomes. The presence of B chromosomes is associated with lower expression level of Tgf-β gene. Although the influence of host genetic background on parasite infection has already been well documented, this is the first study in mammals that gave presence of B chromosomes on immune response full consideration.     

Developmentally regulated expression of three slow isoforms of myosin heavy chain: diversity among the first fibers to form in avian muscle.

At least three slow myosin heavy chain (MHC) isoforms were expressed in skeletal muscles of the developing chicken hindlimb, and differential expression of these slow MHC isoforms produced distinct fiber types from the outset of skeletal muscle myogenesis. Immunohistochemistry with isoform-specific monoclonal antibodies demonstrated differences in MHC content among the fibers of the dorsal and ventral premuscle masses and distinctions among fibers before splitting of the premuscle masses into individual muscles (Hamburger and Hamilton Stage 25). Immunoblot analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin extracted from the hindlimb demonstrated the presence throughout development of different mobility classes of MHCs with epitopes associated with slow MHC isoforms. Immunopeptide mapping showed that one of the MHCs expressed in the embryonic limb was the same slow MHC isoform, slow MHC1 (SMHC1), that is expressed in adult slow muscles. SMHC1 was expressed in the dorsal and ventral premuscle masses, embryonic, fetal, and some neonatal and adult hindlimb muscles. In the embryo and fetus SMHC1 was expressed in future fast, as well as future slow muscles, whereas in the adult only the slow muscles retained expression of SMHC1. Those embryonic muscles destined in the adult to contain slow fibers or mixed fast/slow fibers not only expressed SMHC1, but also an additional slow MHC not previously described, designated as slow MHC3 (SMHC3). Slow MHC3 was shown by immunopeptide mapping to contain a slow MHC epitope (reactive with mAb S58) and to be structurally similar to a MHC expressed in the atria of the adult chicken heart. SMHC3 was designated as a slow MHC isoform because (i) it was expressed only in those muscles destined to be of the slow type in the adult, (ii) it was expressed only in primary fibers of muscles that subsequently are of the slow type, and (iii) it had an epitope demonstrated to be present on other slow, but not fast, isoforms of avian MHC. This study demonstrates that a difference in phenotype between fibers is established very early in the chicken embryo and is based on the fiber type-specific expression of three slow MHC isoforms.     

Emergence of the mature myosin phenotype in the rat diaphragm muscle

Immunohistochemical analysis of myosin heavy chain (MHC) isoform expression in perinatal and adult rat diaphragm muscles was performed with antibodies which permitted the identification of all known MHC isoforms found in typical rat muscles. Isoform switching, leading to the emergence of the adult phenotype, was more complex than had been previously described. As many as four isoforms could be coexpressed in a single myofiber. Elimination of developmental isoforms did not usually result in the myofiber immediately achieving its adult phenotype. Activation of genes for specific adult isoforms might be delayed to puberty. For example, two of the three fast MHCs, MHC2X and MHC2A appeared perinatally, while MHC2B did not appear until 30 days postnatal. By Day 60 this isoform was present in approximately 27% of the myofibers, but in most myofibers expression of this isoform was transient (i.e., at Day greater than or equal to 115, less than 4% of the myofibers expressed MHC2B). Fibers which contained MHC beta/slow during the late fetal and early neonatal period coexpressed MHCemb. A marked increase in the frequency of fibers containing MHC beta/slow occurred between 4 and 21 days postnatal. These slow fibers arose from a population of myofibers which expressed MHCemb and MHCneo during their development, and they accounted for the majority of slow fibers found in the adult diaphragm. The adult myosin phenotype of the diaphragm myofibers (as determined with immunocytochemistry, and 5% SDS-PAGE) was not achieved until the rat was greater than or equal to 115 days old.     

The developmental program of fast myosin heavy chain expression in avian skeletal muscles

We have examined the types of fast myosin heavy chains (MHCs) expressed in a number of different developing chicken skeletal muscles by combining peptide mapping and immunoblotting to identify fast MHC-specific peptides among the total mixture of MHC digestion products. Using this technique, we have identified three different fast MHC patterns among the different fast and mixed (i.e., fast and slow) fiber type muscles of the adult. While the different muscles all underwent sequential changes in fast MHC isoform expression during their development, the exact sequence of these changes and the isoform patterns expressed varied from muscle to muscle. During late embryonic or fetal development, all muscles expressed a similar fast MHC pattern (designated here as the fetal pattern) which was replaced shortly after hatching with a different fast MHC pattern (the neonatal pattern). During the transition from the neonatal to the adult state that occurred sometime in the first year after hatching, many of the muscles underwent additional changes in fast MHC isoform expression. In muscles such as the pectoralis major and pectoralis minor, a new fast MHC isoform pattern was seen in the adult so that the developmental program of isoform switching in these muscles involved the sequential appearance of distinct fetal, neonatal, and adult fast MHCs. Other muscles, such as the sartorius and posterior latissimus dorsi, underwent a qualitatively different program of isoform switching and expressed as an adult a fast MHC pattern that was indistinguishable from that expressed during fetal development. Finally, in some muscles, such as the superficial biceps, no change in isoform pattern was detected during the neonatal to adult transition--in these muscles, expression of the neonatal MHC isoform pattern apparently persisted into the adult state. These data indicate that no single scheme or program of fast MHC isoform switching can describe all the developmental changes that occur in fast MHC isoform expression in the chicken and that at least three different programs of isoform switching and expression can be identified.     

Effects of spaceflight and thyroid deficiency on rat hindlimb development. II. Expression of MHC isoforms.

Both slow-twitch and fast-twitch muscles are undifferentiated after birth as to their contractile protein phenotype. Thus we examined the separate and combined effects of spaceflight (SF) and thyroid deficiency (TD) on myosin heavy chain (MHC) gene expression (protein and mRNA) in muscles of neonatal rats (7 and 14 days of age at launch) exposed to SF for 16 days. Spaceflight markedly reduced expression of the slow, type I MHC gene by approximately 55%, whereas it augmented expression of the fast IIx and IIb MHCs in antigravity skeletal muscles. In fast muscles, SF caused subtle increases in the fast IIb MHC relative to the other adult MHCs. In contrast, TD prevented the normal expression of the fast MHC phenotype, particularly the IIb MHC, whereas TD maintained expression of the embryonic/neonatal MHC isoforms; this response occurred independently of gravity. Collectively, these results suggest that normal expression of the type I MHC gene requires signals associated with weight-bearing activity, whereas normal expression of the IIb MHC requires an intact thyroid state acting independently of the weight-bearing activities typically encountered during neonatal development of laboratory rodents. Finally, MHC expression in developing muscles is chiefly regulated by pretranslational processes based on the tight relationship between the MHC protein and mRNA data.     

Disuse and passive stretch cause rapid alterations in expression of developmental and adult contractile protein genes in skeletal muscle

Contractile proteins exist as a number of isoforms that show a developmental and tissue-specific pattern of expression. Using gene-specific cDNA probes, the expression of the sarcomeric myosin heavy chain (MHC) multi-gene family and of cardiac (foetal) alpha-actin was analysed in three different rat hindlimb muscles immobilised for 5 days in either the shortened or lengthened positions. For each of the MHC genes normally expressed in adult muscle (slow, IIA and IIB), the effect of disuse alone (immobilisation in the shortened position) upon expression was markedly different to that of passive stretch (immobilisation in the lengthened position) in each of the three muscles. However, the same adult sarcomeric myosin heavy chain gene can be affected in a different, or even opposite, manner by either disuse or passive stretch depending on the muscle in which it is being expressed. The fast IIB MHC gene, for example, exhibits a rapid induction in the slow postural soleus muscle, in response to disuse but no such induction occurs in the faster plantaris and gastrocnemius muscles. Furthermore, the induction of this gene in the soleus was prevented by passive stretch. The MHC gene, normally only expressed in embryonic skeletal muscle, showed a similar response in all three muscles and was reinduced in adult muscle in response to passive stretch but not by disuse alone. In contrast, the isoform of alpha-actin which is normally only present in significant quantities in embryonic skeletal muscle and which is reduced postnatally, is not reinduced by passive stretch but is reduced still further by immobilisation in the shortened position.     

Expression of class I histocompatibility antigens in neuroectodermal tumors is independent of the expression of a transfected neuroblastoma myc gene

We have evaluated the relationship between the neuronal myc gene (NMYC) and class I major histocompatibility complex (MHC) expression in human neuroblastoma (NB) tumor cell lines. Class I MHC surface Ag expression in NB cell lines varied from nearly undetectable to levels nearly as high as in a lymphoblastoid cell line. Class I MHC mRNA levels in NMYC-amplified NB cell lines were lower than levels observed in single copy NMYC NB cell lines. However, considerable variation in class I MHC surface Ag and mRNA expression was evident in NMYC-amplified cell lines. To determine directly whether NMYC might modulate class I MHC expression in NB, we transfected a plasmid containing a recombinant NMYC gene into two tumor cell lines derived from a NB and a related neuroepithelioma tumor. Constitutive overexpression of the recombinant NMYC gene produced no consistent change in class I MHC surface Ag or mRNA levels. To determine whether class I MHC expression might be developmentally regulated in adrenal medullary cells, the precursor cells of adrenal NB tumors, beta 2-microglobulin expression was measured in fetal and adult adrenal glands. beta 2-Microglobulin expression was not evident in the neuroblasts of a 24-wk-old fetal adrenal gland, whereas beta 2-microglobulin expression was present in the adult adrenal medulla. These data suggest that variation in class I MHC expression among NB cells may reflect the developmental stage at which neuroblasts were arrested during tumorigenesis.     

Proteolytic mRNA expression in response to acute resistance exercise in human single skeletal muscle fibers.

The purpose of this study was to characterize changes in mRNA expression of select proteolytic markers in human slow-twitch [myosin heavy chain (MHC) I] and fast-twitch (MHC IIa) single skeletal muscle fibers following a bout of resistance exercise (RE). Muscle biopsies were obtained from the vastus lateralis of eight young healthy sedentary men [23 +/- 2 yr (mean +/- SD), 93 +/- 17 kg, 183 +/- 6 cm] before and 4 and 24 h after 3 x 10 repetitions of bilateral knee extensions at 65% of one repetition maximum. The mRNA levels of TNF-alpha, calpains 1 and 2, muscle RING (really interesting novel gene) finger-1 (MuRF-1), atrogin-1, caspase-3, B-cell leukemia/lymphoma (Bcl)-2, and Bcl-2-associated X protein (Bax) were quantified using real-time RT-PCR. Generally, MHC I fibers had higher (1.6- to 5.0-fold, P < 0.05) mRNA expression pre- and post-RE. One exception was a higher (1.6- to 3.9-fold, P < 0.05) Bax-to-Bcl-2 mRNA ratio in MHC IIa fibers pre- and post-RE. RE increased (1.4- to 4.8-fold, P < 0.05) MuRF-1 and caspase-3 mRNA levels 4-24 h post-RE in both fiber types, whereas Bax-to-Bcl-2 mRNA ratio increased 2.2-fold (P < 0.05) at 4 h post-RE only in MHC I fibers. These results suggest that MHC I fibers have a greater proteolytic mRNA expression pre- and post-RE compared with MHC IIa fibers. The greatest mRNA induction following RE was in MuRF-1 and caspase-3 in both fiber types. This altered and specific proteolytic mRNA expression among slow- and fast-twitch muscle fibers indicates that the ubiquitin/proteasomal and caspase pathways may play an important role in muscle remodeling with RE.     

Primary structure and developmental acidic to basic transition of 13 alternatively spliced mouse fast skeletal muscle troponin T isoforms

Large samples of original cDNAs encoding neonatal and adult mouse fast skeletal muscle troponin T (fTnT) have been isolated and characterized. The results demonstrate expression relationships of 8 alternatively spliced exons of the fTnT gene and reveal the primary structure of as many as 13 fTnT isoforms that diverge into acidic and basic classes due to differential mRNA splicing in the N-terminal variable region. In the C-terminal variable region encoded by the mutually exclusive exons 16 and 17, the splicing pathway and structure of exon 16 appears to be adult fTnT-specific, suggesting an adaptation to the functional demands of mature fast skeletal muscle. The cloned cDNAs were expressed in E. coli as standards to identify a high M_r to low M_r, acidic to basic fTnT isoform transition in postnatal developing skeletal muscles. Different from the developmental cardiac TnT switch generated by alternative splicing of a single exon, the fTnT isoform transition is an additive effect of alternative splicing of multiple N-terminal-coding exons, especially exons 4, 8 and fetal that are expressed at higher frequencies in the neonatal than in the adult muscle. The developmental fTnT isoform primary structure transition in both N- and C-terminal variable regions suggest a physiological importance of the apparently complex TnT isoform expression.     

Characterization of cDNA and genomic sequences corresponding to an embryonic myosin heavy chain

We report here the isolation and characterization of cDNA and genomic sequences corresponding to a rat embryonic myosin heavy chain (MHC) protein. This gene, which is present as a single copy in the rat genome, comprises about 25 kilobase pairs of DNA and contains approximately 80% intronic sequences. The embryonic MHC gene belongs to a highly conserved multigene family, and exhibits a high degree of nucleotide and amino acid sequence conservation with other sarcomeric MHC genes from nematode to man. S1 nuclease mapping experiments using cDNA and genomic probes show that this MHC gene is transiently expressed during skeletal muscle development. Its mRNA is detected in fetal skeletal muscle during early development and persists up to 2 weeks after birth with the overlapping expression of neonatal and adult skeletal MHC mRNAs. However, this MHC is not expressed in the adult skeletal muscle with the exception of extraocular muscle fibers. The transient expression during muscle development of the isoform produced by this gene and its sequential replacement by other MHCs raises interesting questions about the mechanism controlling MHC isozyme transitions and the physiological significance of the individual MHCs in muscle fibers.     

Developmentally regulated expression of vascular smooth muscle myosin heavy chain isoforms

Two types of smooth muscle myosin heavy chain (MHC) isoforms, SM1 and SM2, were recently identified to have different carboxyl termini (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). SM1 and SM2 are considered to be generated from a single gene through alternative RNA splicing. In this study we investigated expression of vascular MHC isoforms during development in rabbits at the mRNA, protein, and histological levels. In adults, all smooth muscle cells reacted with both anti-SM1 and anti-SM2 antibodies on immunofluorescence, suggesting the coexpression of SM1 and SM2 in a single cell. In fetal and perinatal rabbits, however, only anti-SM1 antibody consistently reacted with smooth muscles. Reactivity with anti-SM2 antibody was negative in the fetal and neonatal blood vessels and gradually increased during 30 days after birth. These developmental changes in SM1 and SM2 expression at the histological level coincided with mRNA expression of each MHC isoform as determined by S1 nuclease mapping, indicating that expression of SM1 and SM2 is controlled at the level of RNA splicing. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin from fetal and perinatal aortas revealed the presence of large amount of SM2. Interestingly, fetal SM2 did not cross-react with our anti-SM2 antibody on immunoblotting. We conclude that expression of SM1 and SM2 are differentially regulated during development and that a third type of MHC isoform may exist in embryonic and perinatal vascular smooth muscles.