RESUMO
SULF1/SULF2 enzymes regulate cell signalling that impacts the growth and differentiation of many tissues. To determine their possible role in cartilage and bone growth or repair, their expression was examined during development and bone fracture healing using RT-PCR and immunochemical analyses. Examination of epiphyseal growth plates revealed differential, inverse patterns of SULF1 and SULF2 expressions, with the former enriched in quiescent and the latter in hypertrophic chondrocyte zones. Markedly higher levels of both SULFs, however, were expressed in osteoblasts actively forming bone when compared with proliferating pre-osteoblasts in the periosteum or the entombed osteocytes which express the lowest levels. The increased expression of Sulf1 and Sulf2 in differentiating osteoblasts was further confirmed by RT-PCR analysis of mRNA levels in rat calvarial osteoblast cultures. SULF1 and SULF2 were expressed in most foetal articular chondrocytes but down-regulated in a larger subset of cells in the post-natal articular cartilage. Unlike adult articular chondrocytes, SULF1/SULF2 expression varied markedly in post-natal hypertrophic chondrocytes in the growth plate, with very high SULF2 expression compared with SULF1 apparent during neonatal growth in both primary and secondary centres of ossification. Similarly, hypertrophic chondrocytes expressed greatly higher levels of SULF2 but not SULF1 during bone fracture healing. SULF2 expression unlike SULF1 also spread to the calcifying matrix around the hypertrophic chondrocytes indicating its possible ligand inhibiting role through HSPG desulphation. Higher levels of SULF2 in both developing and healing bone closely correlated with parallel increases in hedgehog signalling analysed by ptc1 receptor expression.
Assuntos
Osso e Ossos/metabolismo , Cartilagem Articular/metabolismo , Condrogênese/fisiologia , Consolidação da Fratura/fisiologia , Osteogênese/fisiologia , Sulfotransferases/biossíntese , Animais , Osso e Ossos/lesões , Calcificação Fisiológica/fisiologia , Diferenciação Celular , Células Cultivadas , Condrócitos/metabolismo , Lâmina de Crescimento/fisiologia , Humanos , Masculino , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteócitos/citologia , Osteócitos/metabolismo , Receptores Patched/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Sulfatases , Sulfotransferases/genéticaRESUMO
Sulf1A expression, which is a characteristic of embryonic muscle, is undetectable in mature muscle fibres and quiescent satellite cells, but is re-activated in vivo upon injury and in vitro following activation of satellite cells. Sulf1A is known to enhance canonical Wnt signalling, and its association with Wnt1-induced satellite cell proliferation in vitro in the present study further confirmed this. However, exogenous Wnt6 decreased satellite cell proliferation but promoted the adoption of a hyper-elongated cell morphology in myoblasts on isolated single fibres in culture. Such Wnt6-induced cellular hyper-elongation and inhibition of proliferation was found to be dependent upon Sulf1A, as treatment with Sulf1A neutralising antibodies abolished both these effects. This indicates that Sulf1A can regulate Wnt6 signalling and cellular differentiation in skeletal muscle.
RESUMO
Most myofibrillar proteins, including troponin I and troponin T subunits of troponin complex, undergo developmental stage-specific isoform transitions in vertebrate heart before attaining adult contractile and regulatory characteristics. Only the cardiac/slow skeletal muscle type isoform of troponin C, however, has been shown to be expressed in both adult and developing heart. The changes in troponin C could be functionally important as the TnC isoforms vary in their affinities for Ca(2+). For example, fast troponin C has two Ca(2+) binding sites while slow/cardiac troponin C has a single regulatory site. This study demonstrates the co-expression of both fast and slow transcripts of troponin C in not only quail embryonic skeletal muscle but also embryonic heart using two different analytical techniques of polymerase chain reaction and in situ hybridisation procedure. Fast troponin C expression in the quail heart using in situ hybridisation procedure was first observed at embryonic day 3, with maximum expression at day 5 after which its level in the developing heart was gradually down regulated. In situ hybridisation staining of sections at these developmental stages demonstrated the expression of both fast and slow transcripts of troponin C in all cardiomyocytes.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Desenvolvimento Muscular , Músculo Esquelético/embriologia , Codorniz/embriologia , Troponina C/biossíntese , Animais , Cálcio/metabolismo , Coração/crescimento & desenvolvimento , Hibridização In Situ , Músculo Esquelético/crescimento & desenvolvimento , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Codorniz/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Troponina C/genéticaRESUMO
Id2 cDNA was isolated from a subtractive screen of stage-12 quail caudal somites. In situ hybridisation analysis identified the previously un-described expression of Id2 mRNA in distinct medial and lateral domains of the somitic dermamyotome in both quail and chick embryos. Id2 expression in somites was highly dynamic being first initiated in the lateral domain of the dermamyotome of stage-8-10 embryos, followed by expression in a separate medial domain. Id2 mRNA during subsequent embryonic development could be detected in both medial and lateral domains in the anterior to mid regions while the posterior, recently segmented somites, showed expression only in the lateral domain, which was eventually down regulated in the anterior-most somites. Tissue manipulation studies revealed that Id2 expression in somites required positive signalling from not only axial structures and lateral plate mesoderm but also surface ectoderm. In addition, Id2 expression was also observed in anterior and posterior domains of developing avian limb buds and interdigital tissue.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteína 2 Inibidora de Diferenciação/fisiologia , Animais , Sequência de Bases , Padronização Corporal , Embrião de Galinha , DNA Complementar/metabolismo , Regulação para Baixo , Ectoderma/metabolismo , Perfilação da Expressão Gênica , Biblioteca Gênica , Genes Reguladores , Hibridização In Situ , Proteína 2 Inibidora de Diferenciação/metabolismo , Botões de Extremidades/metabolismo , Mesoderma/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Codorniz , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Somitos/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
The developmental signaling functions of cell surface heparan sulfate proteoglycans (HSPGs) are dependent on their sulfation states. Here, we report the identification of QSulf1, the avian ortholog of an evolutionarily conserved protein family related to heparan-specific N-acetyl glucosamine sulfatases. QSulf1 expression is induced by Sonic hedgehog in myogenic somite progenitors in quail embryos and is required for the activation of MyoD, a Wnt-induced regulator of muscle specification. QSulf1 is localized on the cell surface and regulates heparan-dependent Wnt signaling in C2C12 myogenic progenitor cells through a mechanism that requires its catalytic activity, providing evidence that QSulf1 regulates Wnt signaling through desulfation of cell surface HSPGs.
Assuntos
Padronização Corporal , Proteoglicanas de Heparan Sulfato/metabolismo , Músculos/embriologia , Proteínas Proto-Oncogênicas/metabolismo , Codorniz/embriologia , Transdução de Sinais , Células-Tronco/metabolismo , Sulfatases/metabolismo , Proteínas de Peixe-Zebra , Sequência de Aminoácidos , Animais , Células CHO , Membrana Celular/metabolismo , Células Cultivadas , Clonagem Molecular , Técnicas de Cocultura , Cricetinae , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Proteínas Hedgehog , Heparina/metabolismo , Heparina/farmacologia , Heparitina Sulfato/metabolismo , Dados de Sequência Molecular , Músculos/citologia , Músculos/metabolismo , Mutação , Proteína MyoD/genética , Proteína MyoD/metabolismo , Oligonucleotídeos Antissenso , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Somitos/metabolismo , Sulfatases/química , Sulfatases/genética , Transativadores/genética , Transativadores/metabolismo , Transfecção , Proteínas WntRESUMO
The development of skeletal muscle in mouse oesophagus was investigated by studying the expression of skeletal muscle type myosin heavy chain (MHC), troponin I (TnI) and tropoinin T (TnT) using immunocytochemical and immunoblotting procedures. Both slow and fast muscle fibres were first detected in outer layer muscularis externa of cranial oesophagus at 14 days gestation. The fast MHC was present in all skeletal muscle fibres of oesophagus while the slow MHC was restricted to only a subset of myotubes during foetal development, indicating that slow and fast fibres emerged during early stages of myogenesis. A small number of cells expressed both slow and fast MHCs in the caudal region of adult mouse oesophagus, suggesting that some muscle fibres did not differentiate fully even in the adult. The conversion of some muscle fibre types, from slow to fast, was apparent during postnatal development. This was indicated by a gradual reduction in the number of slow MHC positive fibres during postnatal growth. The complete suppression of slow MHC was observed in cranial oesophagus by 4 weeks of age. However, the persistence of some slow MHC in the caudal oesophagus was apparent even in the adult. The conversion of muscle fibres from slow to fast type was also evidenced by immunoblotting study of fast and slow TnI. The expression level of slow TnI decreased while that of fast TnI increased during neonatal growth period. Compared with the limb skeletal muscles, the onset of the adult fast TnT isoform expression was delayed in mouse oesophagus and its developmental isoforms were not completely suppressed in the adult, although their expression level was reduced.
Assuntos
Esôfago/citologia , Esôfago/crescimento & desenvolvimento , Desenvolvimento Muscular , Fibras Musculares de Contração Rápida/citologia , Fibras Musculares de Contração Lenta/citologia , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Animais , Esôfago/metabolismo , Camundongos , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Isoformas de Proteínas/metabolismo , Troponina I/metabolismo , Troponina T/metabolismoRESUMO
Mouse esophageal muscle is composed of skeletal muscle in the adult, but it has been proposed to be derived from differentiated smooth muscle cells by transdifferentiation during late fetal and early postnatal development (Patapoutian et al. [1995] Science 270:1818-1821). We characterize skeletal muscle precursors in mouse esophagus by investigating the expression of four myogenic regulatory factor transcripts: MyoD, Myf-5, myogenin, and MRF4. Myf-5 was first detected at cranial region of esophageal muscle at 12-13 days of gestation, followed by coexpression of MyoD and MRF4 at 14 days of gestation, and myogenin at embryonic day 15. The expression of these myogenic factors showed outer to inner layer and cranial to caudal progression during fetal and early postnatal development of mouse esophagus. The early appearance of myogenic regulatory factors starting at 12-13 days of gestation indicates that the cells in the mouse esophageal wall are committed to become skeletal muscle-type cells before any differentiated smooth or skeletal muscle cells are observed at 14-15 days of gestation.
Assuntos
Proteínas de Ligação a DNA , Esôfago/embriologia , Proteínas Musculares/genética , Músculo Esquelético/embriologia , Proteína MyoD/genética , Fatores de Regulação Miogênica/genética , Miogenina/genética , Transativadores , Animais , Desenvolvimento Embrionário e Fetal , Esôfago/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Camundongos , Desenvolvimento Muscular , Músculo Esquelético/crescimento & desenvolvimento , Fator Regulador Miogênico 5 , Fatores de TempoRESUMO
Muscularis externa of mouse oesophagus is composed of two skeletal muscle layers in the adult. Unlike rest of skeletal muscle in the body, the oesophageal skeletal muscle in the mouse has been proposed to be derived from fully differentiated smooth muscle cells by transdifferentiation during later foetal and early postnatal development (Patapoutian et al. [1995] Science 270:1818-1821). Here we characterised the nature of cells in muscularis externa of the mouse oesophagus by ultrastructural and immunoctyochemical analyses. The presence of differentiated skeletal muscle cells identified by positive staining for skeletal muscle specific myosin heavy chain became first apparent in the outer layer of cranial oesophagus at 14 days gestation. The transient expression of smooth muscle type alpha-actin in mouse oesophageal muscle was also apparent during foetal development. This isoform, however, was not smooth muscle specific during early development as it was also detected in foetal skeletal muscles. Compared with oesophagus, the suppression of this smooth muscle type alpha-actin during foetal development was faster in non-oesophageal skeletal muscle cells. The development of skeletal muscle in oesophagus showed a cranial to caudal and an outer layer to inner layer progression. During early foetal development, mouse oesophagus is composed of undifferentiated mesenchymal cells that formed cell clusters. Two types of cells with different staining densities could be distinguished within these cell clusters by electron microscopy. The centrally located pale staining cells gave rise to skeletal muscle cells while the peripherally positioned dense staining cells gave rise to smooth muscle cells, indicating the existence of both skeletal and smooth muscle cell precursors in mouse oesophagus during early foetal development. Further development showed an increase in the proportion of skeletal muscle cells and a decrease in size and number of the smooth muscle type cells. Apart from decrease in cell size, some other morphological features of smooth muscle cell degeneration were also observed during later foetal and early neonatal development. No smooth muscle cells undergoing transdifferentiation were observed. Both immunochemical and ultrastructural observations, thus, demonstrated the presence of skeletal muscle cells in early foetal oesophagus. It is concluded that the transient appearance of smooth muscle cells may provide a scaffold for the laying down of skeletal muscle layers in mouse oesophagus, the final disappearance of which may be triggered by lack of smooth muscle innervation.
Assuntos
Esôfago/embriologia , Músculo Esquelético/embriologia , Músculo Liso/embriologia , Actinina/metabolismo , Actinas/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Embrião de Mamíferos/ultraestrutura , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Camundongos , Microscopia Eletrônica , Cadeias Pesadas de Miosina/metabolismo , Fatores de Tempo , Troponina I/metabolismoRESUMO
We have cloned cDNA sequences of both rat and mouse slow troponin T gene. These sequences share a high level of homology with each other and with the human slow troponin T gene although we were unable to detect an alternatively spliced exon present at 3' end of human slow troponin T cDNA in either mouse or rat cDNAs. Northern blot analysis detected a high level expression of slow troponin T in adult mouse Soleus with a lower level expression in mixed postnatal skeletal muscles. Unlike late fetal and postnatal skeletal muscles in which slow troponin T expression is restricted to slow muscle fibre rich regions only, in situ hybridisation analysis detected this isoform to be highly expressed in somitic myotome and all muscle masses at 10-14 days of gestation after which its expression was rapidly downregulated. The unexpected expression of slow troponin T mRNA in fetal heart was apparent by both northern blotting and in situ hybridisation analyses. Slow troponin T mRNA in fetal heart was first detected at 10 day in utero reaching maximum levels of expression at 12-15 days gestation. The slow troponin T in the heart was mainly expressed in the ventral ventricles until day 15 after which low level expression was also observed in both atria. Slow troponin T mRNA in both atrium and ventricle was mainly expressed in outer wall of the myocardium although it was also expressed in interventricular septum. This study therefore shows that in addition to being a cell type specific marker during later fetal and postnatal skeletal muscle development, slow troponin T represented one of the major developmental isoforms expressed in embryonic and fetal skeletal muscle as well as in the cardiac muscle.
Assuntos
Coração/embriologia , Coração/crescimento & desenvolvimento , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/embriologia , Músculo Esquelético/crescimento & desenvolvimento , Miocárdio/metabolismo , Troponina T/genética , Fatores Etários , Processamento Alternativo/genética , Animais , DNA Complementar/análise , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Camundongos , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/citologia , Músculo Esquelético/metabolismo , Miocárdio/citologia , Isoformas de Proteínas/genética , RNA Mensageiro/metabolismo , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
The vertebrate fast skeletal muscle troponin T gene, TnTf, produces a complexity of isoforms through differential mRNA splicing. The mechanisms that regulate splicing and the physiological significance of TnTf isoforms are poorly understood. To investigate these questions, we have determined the complete sequence structure of the quail TnTf gene, and we have characterized the developmental expression of alternatively spliced TnTf mRNAs in quail embryonic muscles. We report the following: 1) the quail TnTf gene is significantly larger than the rat TnTf gene and has 8 non-homologous exons, including a pectoral muscle-specific set of alternatively spliced exons; 2) specific sequences are implicated in regulated exon splicing; 3) a 900-base pair sequence element, composed primarily of intron sequence flanking the pectoral muscle-specific exons, is tandemly repeated 4 times and once partially, providing direct evidence that the pectoral-specific TnT exon domain arose by intragenic duplications; 4) a chicken repeat 1 retrotransposon element resides upstream of this repeated intronic/pectoral exon sequence domain and is implicated in transposition of this element into an ancestral genome; and 5) a large set of novel isoforms, produced by regulated exon splicing, is expressed in quail muscles, providing insights into the developmental regulation, physiological function, and evolution of the vertebrate TnTf isoforms.
Assuntos
Processamento Alternativo/genética , Troponina T/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sequência Conservada , Coturnix , Evolução Molecular , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Fibras Musculares de Contração Rápida/metabolismo , Músculo Esquelético/metabolismo , Isoformas de Proteínas/genética , RNA Mensageiro/genética , Sequências Repetitivas de Ácido Nucleico , Alinhamento de Sequência , Troponina T/químicaRESUMO
Adult skeletal muscles are composed of different fibre types. What initiates the distinctive muscle fibre type-specific specialization in a developing embryo is still controversial. In vitro studies of avian muscles have shown the expression of one of the slow myosin heavy chains, SM2, in only some myotubes. In this report we demonstrate the expression of another slow myosin heavy chain, SM1, restricted to only some chicken myotubes (presumptive slow) in vitro. We also demonstrate that as is the case for avian species, distinct fast and slow myogenic cells are detectable in mammalian species, human and rat, during in vitro development in the absence of innervation. While antibodies to fast myosin heavy chains stained all myotubes dark in these muscle cell cultures, antibodies to slow myosin heavy chains stained only a proportion of the myotubes (presumptive slow). The other myotubes were either unstained or only weakly stained with slow myosin heavy chain antibodies. The muscle cell cultures prepared from different developmental stages of rat skeletal muscles showed a reduction in the number of slow myosin heavy chain-positive myotubes with advancing foetal growth. It is concluded that embryonic myogenic cells that are likely to form distinct fast or slow muscle fibre types are intrinsically heterogeneous, not only in avian but also in mammalian species, although extrinsic factors reinforce and modify such commitment throughout subsequent development.
Assuntos
Aves/embriologia , Mamíferos/embriologia , Fibras Musculares de Contração Rápida/citologia , Fibras Musculares de Contração Lenta/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Embrião de Galinha , Humanos , Morfogênese , Ratos , Especificidade da EspécieRESUMO
Three non-identical, full length troponin-I (Tn-I) clones were isolated from an Atlantic salmon myotomal (trunk) muscle cDNA library. The primary structures, which are predicted to range from 172 to 180 amino acids in length, exhibit similar percent identity scores when compared with fast, slow and cardiac specific Tn-Is from higher vertebrates. When the sequence data are considered along with the results of Western blotting it is evident that Tn-I is more heterogeneous in Atlantic salmon than has been previously shown in higher vertebrates.
Assuntos
Salmão , Troponina I/química , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Oceano Atlântico , Clonagem Molecular , Dados de Sequência Molecular , Proteínas Musculares/química , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
To analyse the myogenic cell lineages in human foetal skeletal muscle, muscle cell cultures were prepared from different foetal stages of development. The in vitro muscle cell phenotype was defined by staining the myotubes with antibodies to fast and slow skeletal muscle type myosin heavy chains using immunoperoxidase or double immunofluorescence procedures. The antibodies to fast skeletal muscle myosin heavy chains stained nearly all myotubes dark in cell cultures prepared from quadriceps muscles at 10-18 weeks of gestation. The antibodies to slow skeletal muscle myosin heavy chains, in contrast, stained only 10-40% of the myotubes very dark. The remaining myotubes were further subdivided into two populations, one of which was unstained while the other stained with variable intensity for slow myosin heavy chain. The slow myosin heavy chain staining was not influenced by the nature of the substratum used to culture these cells, although the growth of muscle cell cultures was greatly improved on matrigel-coated dishes. The presence of both slow and fast myosin heavy chains was detected even when myotubes were grown on uncoated petri dishes. The myotube diversity was further investigated by analysing the clonal populations of human foetal skeletal muscle cells in vitro. When cultured at clonal densities, two types of myogenic clones were identified by their differential staining with antibodies to slow myosin heavy chain. As was the case with the high density muscle cell cultures, virtually all myotubes in both groups of clones stained with antibodies to fast myosin heavy chains. Antibodies to slow myosin heavy chains stained nearly all myotubes dark in one group of myogenic clones, but only a subset of the myotubes stained dark for slow myosin heavy chain in the second group of clones. The proportion of slow myosin heavy chain positive myotubes in this group varied in different clones. The myogenic diversity was thus apparent in both high density and clonal human muscle cell cultures, and myogenic cells retained their ability to modify their muscle cell phenotype.
Assuntos
Fibras Musculares de Contração Rápida/citologia , Fibras Musculares de Contração Lenta/citologia , Músculo Esquelético/embriologia , Células-Tronco/citologia , Adulto , Fatores Etários , Materiais Biocompatíveis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Clonais , Colágeno/farmacologia , Combinação de Medicamentos , Feto/citologia , Gelatina/farmacologia , Humanos , Laminina/farmacologia , Fibras Musculares de Contração Rápida/química , Fibras Musculares de Contração Lenta/química , Músculo Esquelético/química , Músculo Esquelético/citologia , Cadeias Pesadas de Miosina/análise , Cadeias Pesadas de Miosina/genética , Fenótipo , Proteoglicanas/farmacologiaRESUMO
The process of skeletal muscle repair was investigated by immunocytochemical evaluation of chicken leg muscles injured by a localized crush or superficial cut. Only the damaged parts of the muscle fibers, approximately 400-500 microm across, along the longitudinal axis, expressed ventricular myosin heavy chain. The level of this myosin heavy chain along the fiber length further decreased with time. Unlike the newly generated independent regenerating myotubes, even the injured parts of original mature muscle fibers positive for ventricular myosin heavy chain in the immediate vicinity of injury did not show changes in the expression of slow or fast myosin heavy chains in these regions. It is concluded that muscle fibers injured by superficial cut or crush methods used in this study despite being multinucleated were rapidly repaired by localized changes without affecting the major gene expression in the uninjured parts of the fibers.
Assuntos
Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/biossíntese , Regeneração , Envelhecimento/metabolismo , Animais , Galinhas , Técnicas Imunoenzimáticas , Fibras Musculares de Contração Rápida/fisiologia , Fibras Musculares de Contração Lenta/fisiologia , Músculo Esquelético/lesões , Músculo Esquelético/fisiologia , Cadeias Pesadas de Miosina/imunologiaRESUMO
To investigate the role of innervation and to determine if the process of muscle differentiation is preprogrammed, the expression of insulin-like growth factors (IGF-I and IGF-II), troponin I and troponin T mRNAs was studied in regenerating transplants of rat Extensor digitorum longus muscle in the presence and absence of nerve. The role of innervation was further investigated by denervating some adult fast (Gastrocnemius and Plantaris) and slow (Soleus) skeletal muscles. In normal adult skeletal muscles, IGF-I, IGF-II and developmental fast troponin T mRNA containing exon y, are undetectable or present at very low levels. Induction of all these mRNAs was observed in regenerating muscles in both the presence and absence of nerve as well as following denervation of adult fast and slow skeletal muscles. Their low level expression was maintained in adult denervated skeletal muscles but gradually suppressed in both innervated and noninnervated regenerating extensor digitorum longus muscle transplants after 2 months. Fast troponin T mRNA was synthesized in both innervated and noninnervated EDL transplants although the level of this transcript changed markedly in response to denervation of both adult fast and slow skeletal muscles. The fast troponin T mRNA containing exon 17 was also initially expressed in both regenerating muscles but its level was reduced with time in both transplants and in all adult denervated skeletal muscles. Fast and slow troponin I mRNAs were synthesised during EDL muscle regeneration in both the presence and absence of nerve but the slow troponin I expression was not maintained in noninnervated transplants. The level of fast troponin I mRNA decreased in denervated fast skeletal muscles but markedly increased in denervated Soleus. The level of slow troponin I mRNA was slightly increased in denervated fast skeletal muscles but considerably reduced in denervated Soleus.
Assuntos
Denervação Muscular , Músculo Esquelético/fisiologia , RNA Mensageiro/metabolismo , Somatomedinas/genética , Troponina/genética , Animais , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Ratos , Ratos Wistar , Regeneração , Troponina/metabolismo , Troponina I/genética , Troponina I/metabolismo , Troponina TRESUMO
We have examined four models of experimental cardiac hypertrophy and heart failure for alterations in troponin isoform expression, particularly in the re-expression of the fetal isoforms. Cardiac protein extracts from experimental and sham-operated control rats were analyzed using one dimensional gel electrophoresis, followed by Western blotting and detection with antibodies specific for troponin I and T. No alteration in protein profile was observed for these proteins between control, hypertrophied and failing heart samples. The data demonstrate that reversion to the fetal pattern of troponin expression is not a feature of experimental cardiac hypertrophy and heart failure in the rat.
Assuntos
Cardiomegalia/metabolismo , Insuficiência Cardíaca/metabolismo , Troponina/metabolismo , Animais , Cardiomegalia/induzido quimicamente , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Insuficiência Cardíaca/induzido quimicamente , Masculino , Contração Miocárdica/fisiologia , Ratos , Ratos Wistar , Troponina I , Troponina TRESUMO
A monoclonal antibody has been prepared to slow skeletal muscle type myosin heavy chain which in the rat distinguishes fast and slow myocytes within the somitic myotome at 11.5 days in utero. The distribution of slow myotubes identified by this antibody in developing limb buds is also restricted to presumptive slow muscle cell type regions only. No slow myoblasts in hindlimb buds, however, were detected at 14.5 day in utero when a small number of fast muscle cells were already present. The presence of slow muscle cell population detected by this specific antibody became apparent a day later. This study thus demonstrated the diversification into different muscle cell types during both early embryonic and foetal development.
Assuntos
Mamíferos/embriologia , Mesoderma/citologia , Fibras Musculares de Contração Rápida/embriologia , Fibras Musculares de Contração Lenta/embriologia , Animais , Anticorpos Monoclonais/imunologia , Biomarcadores , Extremidades/embriologia , Coração Fetal/citologia , Idade Gestacional , Técnicas Imunoenzimáticas , Miosinas/análise , Miosinas/imunologia , Ratos , Ratos WistarRESUMO
In theory, the rat fast skeletal muscle troponin T gene can generate 64 different isoforms. Here we report the identification of a novel alternative exon (exon y) that increases the potential isoform variation to 128. The inclusion of exon y in fast skeletal muscle troponin T mRNA occurs in perinatal, but not adult, skeletal muscle. Exon y is located between exons 8 and 9. This is the first time that a developmentally regulated exon located amongst a set of alternatively spliced exons has been described. Exon y is included in two mRNA isoforms. The proteins that these mRNAs would encode have molecular masses greater than that of the largest fast skeletal muscle troponin T isoform lacking exon y. These two proteins correlate well in both size and pattern of expression with the two fast skeletal muscle troponin T isoforms expressed in perinatal skeletal muscle. These results indicate that there is coordinated regulation of the splicing of exon y with other alternative exons.
Assuntos
Éxons , Regulação da Expressão Gênica , Desenvolvimento Muscular , Troponina/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , DNA , Dados de Sequência Molecular , Músculos/metabolismo , Ratos , Homologia de Sequência de Aminoácidos , Troponina/biossíntese , Troponina TRESUMO
Recent reports have demonstrated the presence of two isoforms of troponin I in the human fetal heart, namely, cardiac troponin I and slow skeletal muscle troponin I. Structural and physiological considerations indicate that these isoforms would confer differing contractile properties on the myocardium, particularly on the phosphorylation-mediated regulation of contractility by adrenergic agonists. We have investigated the developmental expression of these isoforms in the human heart from 9 weeks of gestation to 9 months of postnatal life, using Western blots revealed with troponin I antibodies to detect troponin protein isoforms and Northern blots to detect the corresponding mRNAs. The results show the following: 1) Slow skeletal muscle troponin I is the predominant isoform throughout fetal life. 2) After birth, the slow skeletal isoform is lost, with cardiac troponin I being the only isoform detectable by 9 months of postnatal development. 3) The protein isoforms and their corresponding mRNAs follow the same pattern of accumulation, suggesting that the transition in troponin expression is regulated at the level of gene transcription. The developmental transition in troponin I isoform content has implications for contractility of the fetal and postnatal myocardium. We further analyzed right and left ventricular muscle samples from 17 hearts in end-stage heart failure resulting from pulmonary hypertension, ischemic heart disease, or dilated cardiomyopathy. Cardiac troponin I mRNA remained abundant in each case, and slow skeletal muscle troponin I mRNA was not detectable in any of sample. We conclude that alterations in troponin I isoform content do not therefore contribute to the altered contractile characteristics of the adult failing ventricle.