RESUMEN
We have previously reported the identification of a distinct myosin heavy chain (MyHC) isoform in a major subpopulation of rat skeletal muscle fibers, referred to as 2X fibers (Schiaffino, S., L. Gorza, S. Sartore, L. Saggin, M. Vianello, K. Gundersen, and T. Lømo. 1989. J. Muscle Res. Cell Motil. 10:197-205). However, it was not known whether 2X-MyHC is the product of posttranslational modification of other MyHCs or is coded by a distinct mRNA. We report here the isolation and characterization of cDNAs coding a MyHC isoform that is expressed in type 2X skeletal muscle fibers. 2X-MyHC transcripts differ from other MyHC transcripts in their restriction map and 3' end sequence and are thus derived from a distinct gene. In situ hybridization analyses show that 2X-MyHC transcripts are expressed at high levels in the diaphragm and fast hindlimb muscles and can be coexpressed either with 2B- or 2A-MyHC transcripts in a number of fibers. At the single fiber level the distribution of each MyHC mRNA closely matches that of the corresponding protein, determined by specific antibodies on serial sections. In hindlimb muscles 2X-, 2A-, and 2B-MyHC transcripts are first detected by postnatal day 2-5 and display from the earliest stages a distinct pattern of distribution in different muscles and different fibers. The emergence of type 2 MyHC isoforms thus defines a distinct neonatal phase of fiber type differentiation during muscle development. The functional significance of MyHC isoforms is discussed with particular reference to the velocity of shortening of skeletal muscle fibers.
Asunto(s)
Regulación de la Expresión Génica , Músculos/metabolismo , Miosinas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Diferenciación Celular , ADN , Estimulación Eléctrica , Hibridación in Situ , Datos de Secuencia Molecular , Desarrollo de Músculos , Miosinas/metabolismo , Ratas , Mapeo Restrictivo , Hormonas Tiroideas/fisiología , Transcripción GenéticaRESUMEN
Human embryonic stem cells (hESCs) may become important for cardiac repair due to their potentially unlimited ability to generate cardiomyocytes (CMCs). Moreover, genetic manipulation of hESC-derived CMCs would be a very promising technique for curing myocardial disorders. At the present time, however, inducing the differentiation of hESCs into CMCs is extremely difficult and, therefore, an easy and standardizable technique is needed to evaluate differentiation strategies. Vectors driving cardiac-specific expression may represent an important tool not only for monitoring new cardiac-differentiation strategies, but also for the manipulation of cardiac differentiation of ESCs. To this aim, we generated cardiac-specific lentiviral vectors (LVVs) in which expression is driven by a short fragment of the cardiac troponin-I proximal promoter (TNNI3) with a human cardiac alpha-actin enhancer, and tested its suitability in inducing tissue-specific gene expression and ability to track the CMC lineage during differentiation of ESCs. We determined that (1) TNNI3-LVVs efficiently drive cardiac-specific gene expression and mark the cardiomyogenic lineage in human and mouse ESC differentiation systems (2) the cardiac alpha-actin enhancer confers a further increase in gene-expression specificity of TNNI3-LVVs in hESCs. Although this technique may not be useful in tracking small numbers of cells, data suggested that TNNI3-based LVVs are a powerful tool for manipulating human ESCs and modifying hESC-derived CMCs.
Asunto(s)
Células Madre Embrionarias/citología , Terapia Genética/métodos , Insuficiencia Cardíaca/terapia , Miocitos Cardíacos/citología , Actinas/genética , Animales , Diferenciación Celular , Línea Celular , Elementos de Facilitación Genéticos , Citometría de Flujo , Ingeniería Genética , Vectores Genéticos/farmacología , Humanos , Lentivirus/genética , Ratones , Regiones Promotoras Genéticas , Transducción Genética/métodos , Troponina I/genéticaRESUMEN
The adventitial layer surrounding the blood vessels has long been exclusively considered a supporting tissue the main function of which is to provide adequate nourishment to the muscle layers of tunica media. Although functionally interconnected, the adventitial and medial layers are structurally interfaced at the external elastic lamina level, clearly distinguishable at the maturational phase of vascular morphogenesis. Over the last few years the "passive" role that the adventitia seemed to play in experimental and spontaneous vascular pathologies involving proliferation, migration, differentiation, and apoptosis of vascular smooth muscle cells (VSMCs) has been questioned. It has been demonstrated that fibroblasts from the adventitia display an important partnership with the resident medial VSMCs in terms of phenotypic conversion, proliferation, apoptotic, and migratory properties the result of which is neointima formation and vascular remodeling. This article is an attempt at reviewing the major themes and more recent findings dealing with the phenotypic conversion process that leads adventitial "passive" (static) fibroblasts to become "activated" (mobile) myofibroblasts. This event shows some facets in common with vascular morphogenesis, ie, the process of recruitment, incorporation, and phenotypic conversion of cells surrounding the primitive endothelial tube in the definitive vessel wall. We hypothesize that during the response to vascular injuries in the adult, "activation" of adventitial fibroblasts is, at least in part, reminiscent of a developmental program that also invests, although with distinct spatiotemporal features, medial VSMCs.
Asunto(s)
Arteriopatías Oclusivas/patología , Arterias/patología , Fibroblastos/patología , Músculo Liso Vascular/patología , Túnica Íntima/patología , Animales , Antígenos de Diferenciación/metabolismo , Arteriopatías Oclusivas/metabolismo , Arterias/embriología , Arterias/metabolismo , Arterias/cirugía , Diferenciación Celular , División Celular , Movimiento Celular , Progresión de la Enfermedad , Fibroblastos/metabolismo , Humanos , Morfogénesis , Músculo Liso Vascular/embriología , Músculo Liso Vascular/metabolismo , Venas/trasplanteRESUMEN
Multiple cell populations with different embryological histories are involved in the morphogenesis of the cardiac arterial and venous poles as well as in the correct alignment and connection of the developing vessels with the cardiac chambers. Formation of the aorta and the pulmonary trunk is a complicated process orchestrated via a specific sequence of highly integrated spatiotemporal events of cell proliferation, migration, differentiation, and apoptosis. The peculiar susceptibility of this intricate cell network to be altered explains the frequency of congenital cardiovascular diseases of the arterial and venous poles. We review this topic from the "vascular point of view," putting major emphasis on (1) the existence of different cell lineages from which smooth muscle cells of the aorticopulmonary trunk can be derived, (2) the establishment of cell/tissue boundaries in the cardiovascular connecting regions, and (3) the animal models that can mimic human congenital defects of the arterial and venous poles of the heart.
Asunto(s)
Sistema Cardiovascular/embriología , Linaje de la Célula , Animales , Anomalías Cardiovasculares/embriología , Anomalías Cardiovasculares/fisiopatología , Sistema Cardiovascular/citología , Circulación Coronaria , Modelos Animales de Enfermedad , Corazón/embriología , Corazón/fisiología , Corazón/fisiopatología , Humanos , Modelos BiológicosRESUMEN
Reverse transcription polymerase chain reaction (RT-PCR) of cytokeratin-19 (CK-19) has been widely used to detect small numbers of circulating malignant epithelial cells in the bone marrow or the peripheral blood of patients with breast cancer. However, a high percentage of false positive results has been recorded and conflicting reports question the clinical relevance of this technical approach. We demonstrate that the use of a new nested primer pair for CK-19 RT-PCR avoids false positive results without affecting the sensitivity of the assay. Our experiments were carried out using MCF-7 cells alone or mixed with peripheral blood mononuclear cells (PBMNC) of healthy donors. The results were also validated in a large series of healthy donors and in a preliminary study on a limited number of patients with breast cancer, thus suggesting that our assay is feasible for application in the clinical evaluation of occult malignant epithelial cells.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Cartilla de ADN/genética , Queratinas/genética , Neoplasias Primarias Desconocidas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Adulto , Anciano , Secuencia de Bases , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Humanos , Persona de Mediana Edad , Datos de Secuencia Molecular , Neoplasias Primarias Desconocidas/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/instrumentaciónRESUMEN
The subunits of the troponin complex-troponin C, troponin T, and troponin I-are responsible for the Ca(2+)-dependent regulation of contractile activity in heart and skeletal muscle. Distinct troponin T and I isoforms, generated by different genes or by alternative splicing from the same gene, are expressed during cardiac development. Troponin switching affects the Ca(2+) sensitivity of the contractile system and may account for the greater resistance to hypoxia and acidosis, and the impaired responsiveness to adrenergic stimulation of the fetal and neonatal heart.
RESUMEN
We examined the myosin heavy-chain (MHC), troponin T (TnT), and troponin I (TnI) isoform composition in the rat soleus muscle after 21 days of hindlimb suspension using electrophoretic and immunoblotting analysis with specific monoclonal antibodies. The suspended soleus showed a shift in the MHC isoform distribution with a marked increase (from 1.0 to 33%) in the relative amount of type IIa and IIx MHC and a corresponding decrease in type I MHC. However, type IIb MHC, which represents a major component in fast-twitch muscles, was not detected in suspended soleus muscles. TnT and TnI isoform composition was also changed with the appearance of fast-type TnI and TnT bands. However, a high-mobility TnT band, which represents a major component in fast-twitch muscles, was not expressed in suspended soleus. These isoform transitions may be related to the increased maximal velocity of shortening and higher calcium sensitivity previously reported in the rat soleus after hindlimb suspension.
Asunto(s)
Miembro Posterior/fisiología , Músculos/metabolismo , Miosinas/metabolismo , Troponina/metabolismo , Ingravidez/efectos adversos , Animales , Anticuerpos Monoclonales/inmunología , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Isomerismo , Ratas , Ratas Wistar , Troponina I , Troponina TRESUMEN
During early cardiac development the atrial myocardium is continuous with the ventricular myocardium throughout the atrioventricular canal. The atrioventricular canal undergoes complex remodelling involving septation, formation of atrioventricular valves and insulation between atria and ventricles except at the level of the atrioventricular node. Understanding of these processes has been hampered by the lack of markers specific for this heart region. We have generated transgenic mice expressing beta-galactosidase under the control of the cardiac troponin I gene that show transgene expression mainly confined to the atrioventricular canal myocardium during early embryonic development. With further development beta-galactosidase positive cells are observed in the atrioventricular node and in the lower rim of both right and left atria, supporting the view that atrioventricular canal myocardium contributes to the atrioventricular node and is in part incorporated into the lower rim of the atria. These results identify the atrioventricular canal myocardium as a distinct transcriptional domain.
Asunto(s)
Nodo Atrioventricular/embriología , Corazón/embriología , Troponina I/genética , Animales , Nodo Atrioventricular/química , Expresión Génica , Atrios Cardíacos/embriología , Ventrículos Cardíacos/embriología , Ratones , Ratones Transgénicos , Miocardio/química , Cadenas Pesadas de Miosina/análisis , beta-Galactosidasa/análisis , beta-Galactosidasa/genéticaRESUMEN
The effect of ageing on human skeletal muscle was investigated using needle biopsies from young and aged subjects and from aged subjects trained with different activity patterns. Histochemical staining for myofibrillar ATPase of ageing m. vastus lateralis demonstrated an unchanged fibre type distribution but a selective atrophy of type IIa and type IIb fibres. Analysis of myosin heavy chain (MHC) composition showed that type I MHC increased with ageing (P less than 0.05). The relative content of the MHC isoforms correlated with the relative area of the respective fibre types. Sarcoplasmic reticulum (SR) proteins were investigated in muscle extracts by electrophoretic and immunoblotting techniques. When compared to a young control group (28 +/- 0.1 years old, n = 7) blots of post-myofibrillar supernatant proteins probed with polyclonal antibodies to the rabbit fast SR Ca-ATPase, a marker of extrajunctional SR, showed that the content of Ca-ATPase was significantly lower (P less than 0.05) in the old control group (68 +/- 0.5 years old, n = 8). On the other hand the content of calsequestrin (CS), the major intraluminal protein of SR terminal cisternae (TC), and of the 350-kDa ryanodine-binding protein, which is localized in the junctional regions of TC, did not show a concomitant decrease. These results suggest that ageing differentially affects extrajunctional and junctional SR of human skeletal muscle. These age-related changes were not observed within a group of old strength-trained subjects.
Asunto(s)
Envejecimiento , Ejercicio Físico , Músculos/metabolismo , Retículo Sarcoplasmático/metabolismo , Anciano , ATPasas Transportadoras de Calcio/metabolismo , Calsecuestrina/metabolismo , Electroforesis en Gel Bidimensional , Histocitoquímica , Humanos , Masculino , Persona de Mediana Edad , Contracción Muscular , Proteínas Musculares/metabolismo , Receptores Colinérgicos/metabolismo , Canal Liberador de Calcio Receptor de RianodinaRESUMEN
In the bovine nodal conduction tissue we have described the existence of a novel cardiac myosin isoform, immunologically related to the myosin types expressed during skeletal muscle development. Using different monoclonal antibodies specific for the embryonic and the neonatal skeletal myosin heavy chain types we investigated the myosin composition of the rat sino-atrial and atrio-ventricular nodes. We find that nodal conduction tissue fibers of the rat heart contain a distinct cardiac myosin isoform antigenically similar to the skeletal embryonic myosin heavy chain. The expression of this myosin isoform in nodal tissue appears to be developmentally regulated and partially controlled by thyroid hormone. Reactive cardiac fibers were detected in the nodal regions only during fetal development and a few days after birth, whereas very rare labelled fibers could be observed in the adult nodes. This myosin type does not represent a primordial cardiac myosin isoform since it was not detected in the embryonic heart before 13.5 days of gestation. When congenital hypothyroidism was induced in rats, the post-natal disappearance of reactive fibers in the nodal regions was delayed. On the other hand, hypothyroidism induced in the adult rats did not change the number of the reactive nodal fibers with respect to the euthyroid hearts.
Asunto(s)
Sistema de Conducción Cardíaco/análisis , Hipotiroidismo/inmunología , Miocardio/análisis , Miosinas/análisis , Animales , Hipotiroidismo Congénito , Corazón/embriología , Sistema de Conducción Cardíaco/embriología , Sistema de Conducción Cardíaco/inmunología , Masculino , Miosinas/inmunología , Ratas , Ratas EndogámicasRESUMEN
Fetal rat skeletal muscles express a troponin T (TnT) isoform similar to the TnT isoform expressed in the embryonic heart with respect to electrophoretic mobility and immunoreactivity with cardiac TnT-specific monoclonal antibodies. Immunoblotting analyses reveal that both the embryonic and the adult isoforms of cardiac TnT are transiently expressed during the neonatal stages. In addition, other TnT species, different from both cardiac TnTs and from the TnT isoforms expressed in adult muscles, are present in skeletal muscles during the first two postnatal weeks. By immunocytochemistry, cardiac TnT is detectable at the somitic stage and throughout embryonic and fetal development, and disappears during the first weeks after birth, persisting exclusively in the bag fibers of the muscle spindles. Cardiac TnT is re-expressed in regenerating muscle fibers following a cold injury and in mature muscle fibers after denervation. Developmental regulation of this TnT variant is not coordinated with that of the embryonic myosin heavy chain with respect to timing of disappearance and cellular distribution. No obligatory correlation between the two proteins is likewise found in regenerating and denervated muscles.
Asunto(s)
Músculos/embriología , Miocardio/metabolismo , Troponina/análisis , Animales , Animales Recién Nacidos , Diferenciación Celular , Desarrollo Embrionario y Fetal , Técnicas para Inmunoenzimas , Desnervación Muscular , Músculos/inervación , Músculos/metabolismo , Subfragmentos de Miosina/análisis , Ratas , Ratas Endogámicas , Regeneración , Troponina TRESUMEN
Monoclonal antibodies identify two distinct isoforms of troponin I in rat cardiac muscle, one predominant in the embryonic and fetal heart and one predominant in the adult heart. The two isoforms can be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with apparent molecular weights of 27,000 and 31,500, respectively. The adult isoform is specifically recognized by a monoclonal antibody that is unreactive with the embryonic variant, while two other monoclonal antibodies recognize both isoforms. A monoclonal antibody to cardiac troponin T was used to isolate by affinity chromatography the troponin complex from adult and neonatal rat heart. Affinity purified troponin from neonatal heart was found to contain both the embryonic and adult isoforms of troponin I. Comparative immunoblotting analysis with different muscle tissues shows that embryonic troponin I is identical with respect to electrophoretic mobility and pattern of immunoreactivity to the major troponin I isoform found in adult slow skeletal muscle. Troponin I switching may be implicated in developmental changes involving Ca2+ and pH sensitivity of the contractile system and response to beta-adrenergic stimulation.
Asunto(s)
Corazón/crecimiento & desarrollo , Troponina/análisis , Envejecimiento , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales , Bovinos , Feto , Atrios Cardíacos/crecimiento & desarrollo , Ventrículos Cardíacos/crecimiento & desarrollo , Immunoblotting , Ratas , Troponina IRESUMEN
We have isolated and sequenced a full-length cDNA clone of rat cardiac troponin I (TnI). The amino acid sequence of rat cardiac TnI is highly similar to that of other mammalian species in the portion of the molecule (residues 33-210) that is also homologous to skeletal muscle TnI isoforms. In contrast, a lower degree of similarity is present in the cardiac TnI-specific amino terminal extension (residues 1-32). This region contains a conserved serine residue that has been shown to be selectively phosphorylated by cAMP-dependent protein kinase. Cardiac TnI mRNA is weakly expressed in the 18-day fetal heart and accumulates in neonatal and postnatal stages. No difference can be demonstrated between TnI mRNAs present in fetal and postnatal heart by RNAase protection assays. The fetal and neonatal, but not the adult heart, contain significant amounts of slow skeletal TnI transcripts, detected by oligonucleotide probes specific for the 5'- and 3'-untranslated regions of slow skeletal TnI mRNA. In situ hybridization studies show that cardiac and slow skeletal TnI mRNAs are coexpressed in the rat heart from embryonic day 11 throughout fetal and perinatal stages. Changes in troponin isoform expression during development may be responsible for the difference in calcium sensitivity and in the response to beta-adrenergic stimulation between fetal and adult heart.
Asunto(s)
Expresión Génica/fisiología , Corazón/crecimiento & desarrollo , ARN Mensajero/genética , Troponina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Bovinos , ADN Circular/análisis , Técnicas Genéticas , Corazón/embriología , Corazón/fisiología , Humanos , Datos de Secuencia Molecular , Conejos , Ratas , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Troponina IRESUMEN
Using immunofluorescence procedures with specific polyclonal and monoclonal antimyosin antibodies we have found that embryonic and neonatal myosin heavy chains (MHCs), which in rat skeletal muscle disappear during the first weeks after birth, are reexpressed in adult muscle after denervation. Reactivity for embryonic and neonatal MHCs was detected in some fibers as early as 3 days after denervation, became more evident by 7 days, and occurred exclusively in the type 2A fiber population. Paralysis of innervated muscles by tetrodotoxin block of the sciatic nerve also resulted in the reappearance of embryonic and neonatal MHCs in type 2A fibers. Significant variation in the degree of immunoreactivity was observed in different segments of the same muscle fiber, suggesting that coordination of muscle fiber nuclei in the control of myosin heavy chain gene expression is partially lost following denervation.
Asunto(s)
Animales Recién Nacidos/metabolismo , Feto/metabolismo , Desnervación Muscular , Músculos/metabolismo , Miosinas/metabolismo , Parálisis/metabolismo , Envejecimiento/metabolismo , Animales , Técnica del Anticuerpo Fluorescente , Músculos/embriología , Músculos/inervación , Parálisis/inducido químicamente , Ratas , Ratas Endogámicas , Nervio Ciático/efectos de los fármacos , Tetrodotoxina/farmacologíaRESUMEN
Intracellular rapidly exchanging Ca2+ stores are identified and defined in terms of intralumenal low-affinity, high-capacity Ca(2+)-binding proteins, of which calsequestrin (CS) is the prototype in striated muscles. In chicken striated muscles, there is a single gene for CS [Choi and Clegg (1990) Dev. Biol. 142, 169-177]. In the chicken brain, the gene for CS was found to be selectively expressed in Purkinje neurons, as judged by Northern blotting, in situ hybridization and immunocytochemistry. The synthetic machinery for CS was found to be restricted to the cell body, i.e. excluded from dendrites and axon.
Asunto(s)
Calsecuestrina/genética , Expresión Génica , Células de Purkinje/metabolismo , Animales , Northern Blotting , Pollos/genética , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Hibridación in SituRESUMEN
A monoclonal antibody specific for cardiac troponin T has been used to investigate troponin changes during development in the rat heart. Specificity of the antibody was determined by immunoblot analysis with purified bovine cardiac troponin. In the rat heart, immunoblot analysis shows that anticardiac troponin T reacts with a 42.5-kDa band in fetal ventricles and with a 41-kDa band in adult ventricles. The faster migrating troponin T is present in traces in the fetal heart and increases markedly during the first 2 weeks after birth, concomitantly with the progressive decrease of the slower migrating form that is no longer detectable in the adult. The pattern of reactivity of the monoclonal antibody is not modified by alkaline phosphatase pretreatment, suggesting that the antibody is not specific for a phosphorylated epitope. Conditions known to affect cardiac myosin composition, such as hypothyroidism and hypertrophy secondary to systemic hypertension, do not change the troponin T isoform profile of adult rat ventricles. The expression and accumulation of the adult isoforms of troponin T are not suppressed by propylthiouracil treatment of pregnant and nursing rats.
Asunto(s)
Corazón/crecimiento & desarrollo , Troponina/metabolismo , Envejecimiento , Animales , Cardiomegalia/metabolismo , Corazón/embriología , Hipertensión/metabolismo , Hipotiroidismo/metabolismo , Músculos/metabolismo , Miocardio/metabolismo , Ratas , Ratas Endogámicas , Valores de Referencia , Troponina TRESUMEN
The expression of 4 myosin heavy chain (MHC) isoforms was analyzed in the rat soleus (SOL) and extensor digitorum longus (EDL) muscles after denervation and chronic electric stimulation. The stimulation frequencies used were 20 and 150 Hz and the amount of stimulation was either large (20 Hz), intermediate (150 Hz), or small (150 Hz). These patterns resemble some features of normal motor unit activity in SOL and EDL of freely moving rats (Hennig and Lømo, 1985). The relative expression of each MHC isoform depended strongly on the stimulation pattern. Furthermore, for any particular stimulation pattern, fibers in SOL and EDL expressed different MHCs. Coexistence of different MHC types in the same fiber was frequently observed in stimulated muscles. 20-Hz stimulation preserved normal expression of type 1-MHC in SOL but failed to induce type 1-MHC in type 2 fibers of the EDL, where type 2A- and 2X-MHC expression dominated and type 2B-MHC expression was completely suppressed. 150-Hz low-amount stimulation preserved nearly normal 2B-MHC expression in many type 2 fibers of the EDL but failed to induce type 2B-MHC expression in the SOL, where 2X-MHC became predominant. 150-Hz high-amount stimulation differed from 150-Hz small amount stimulation by suppressing almost all type 2B-MHC expression in EDL and by inducing considerable type 2A-MHC expression in the SOL. Scattered fibers in EDL that were probably the original type 1 fibers responded differently from both type 2 fibers in the EDL and from type 1 fibers in the SOL to stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Músculos/metabolismo , Miosinas/metabolismo , Animales , Desnervación , Estimulación Eléctrica , Técnicas para Inmunoenzimas , Isomerismo , Masculino , Músculos/fisiología , Ratas , Ratas EndogámicasRESUMEN
We report on the existence of a myosin heavy chain (MHC) isoform with unique structural properties in extraocular (EO) muscles. Differences in MHC composition are apparent using a polyclonal antibody prepared against myosin isolated from bovine EO muscle myosin. In enzyme immunoassays and western blotting experiments, this anti-EO myosin antibody reacted specifically with the heavy chains of EO muscle myosin and not with the heavy chains of other myosins. The distribution of this new MHC isoform in the globe rotating muscles from different mammalian species was analysed using a panel of specific anti-myosin antibodies and comparing the histochemical myosin ATPase profile of muscle fibres with their isomyosin content. Most fibres which display a type 2 ATPase reaction pattern were selectively labelled by anti-EO antibodies. A few type 2 fibres were found to react with both anti-EO and anti-2A myosin antibodies and others, located almost exclusively in the orbital layers, reacted with anti-foetals as well as anti-EO antibodies. The presence of a distinct form of myosin in EO muscle fibres is probably related to the particular functional characteristics of these muscles, which are known to be exceptionally fast-contracting but display a very low tension output.
Asunto(s)
Miosinas/análisis , Músculos Oculomotores/citología , Fragmentos de Péptidos/análisis , Animales , Complejo Antígeno-Anticuerpo , Bovinos , Reacciones Cruzadas , Sueros Inmunes , Subfragmentos de Miosina , Miosinas/inmunología , Fragmentos de Péptidos/inmunologíaRESUMEN
Previous studies have demonstrated that the primordial pulmonary veins originate as an outgrowth of the atrial cells and anastomosis with the pulmonary venous plexus. As a consequence of this embryologic origin the tunica media of these vessels is composed of cardiac cells that express atrial specific markers (Lyons et al. [1990] J Cell Biol 111:2427-2436; Jones et al. [1994] Dev Dyn 200:117-128). We used transgenic mice for the cardiac troponin I (cTNI) gene and smooth muscle (SM) myosin heavy chain as differentiation markers, to analyze how cardiac and SM cells contribute to the formation and structural remodeling of the pulmonary veins during development. We show here that the tunica media of the adult mouse pulmonary veins contains an outer layer of cardiac cells and an intermediate SM cell compartment lining down on the inner endothelium. This structural organization is well expressed in the intrapulmonary veins from the beginning of vasculogenesis, with cardiac cells accumulating over preexisting roots of endothelial and SM cells and extending to the third bifurcation of the pulmonary branches without reaching the more distal tips of the vessels. On the other hand, SM cells, which are widely distributed in the intrapulmonary veins from the embryonic stage E16, accumulate also in the extrapulmonary branches and reach the posterior wall of the left atrium, including the orifices of the pulmonary veins. This event takes place around birth when the pulmonary blood flow starts to function properly. A model for the development of the pulmonary veins is presented, based upon our analysis.
Asunto(s)
Músculo Liso Vascular/citología , Miocardio/citología , Venas Pulmonares/embriología , Actinas/análisis , Actinas/inmunología , Animales , Desarrollo Embrionario y Fetal/fisiología , Femenino , Expresión Génica , Genes Reporteros , Corazón/embriología , Técnicas Inmunológicas , Hibridación in Situ , Pulmón/irrigación sanguínea , Pulmón/embriología , Masculino , Ratones , Ratones Transgénicos , Microscopía Confocal , Modelos Biológicos , Músculo Liso Vascular/embriología , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/metabolismo , Miocardio/inmunología , Miocardio/metabolismo , Miosinas/análisis , Miosinas/inmunología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/inmunología , Venas Pulmonares/citología , Venas Pulmonares/crecimiento & desarrollo , Venas Pulmonares/metabolismo , Troponina I/análisis , Troponina I/genética , Troponina I/inmunología , Túnica Media/citología , Túnica Media/embriologíaRESUMEN
The cardiac troponin I gene is one of the few sarcomeric protein genes exclusively expressed in cardiac muscle. We show here that this specificity is controlled by a proximal promoter (-230/+16) in transfected cardiac cells in culture, in the adult hearts, and in transgenic animals. Functional analysis indicates that MEF2/Oct-1, Sp1, and GATA regulatory elements are required for optimal gene activation because selective mutations produce weak or inactive promoters. MEF2 and Oct-1 transcription factors bind to the same A/T-rich element. A mutation that blocks this binding markedly reduces gene activation in vivo and in vitro, and overexpression of MEF2A, MEF2C, and MEF2D in noncardiac cells transactivates the cardiac troponin I promoter. Disruption of these elements inactivates the cardiac troponin I promoter in cultured cardiac cells but has a less important role in transfected adult heart. Moreover, nuclear extracts from an almost pure population of adult cardiac cells contain much lower levels of GATA binding activity compared with fetal cardiac cells. These findings point to a differential role of GATA factors in the maintenance of gene expression in the adult heart as compared with the activation of cardiac genes in fetal cardiomyocytes. Overexpression of GATA family members transactivates the cardiac troponin I promoter, and GATA-5 and GATA-6 are stronger transactivators than GATA-4, a property apparently unique to the cardiac troponin I promoter. Transgenic mice carrying the -230/+126 base pair promoter express beta-galactosidase reporter gene in the heart both at early stages of cardiogenesis and in the adult animals. These results indicate that the ability of the cardiac troponin I proximal promoter to target expression of a downstream gene in the heart is also maintained when the transgene is integrated into the genome.