RESUMEN

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/genética

RESUMEN

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/instrumentación

RESUMEN

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/trasplante

RESUMEN

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 Biológicos

RESUMEN

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/genética

RESUMEN

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/embriología

RESUMEN

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.

Asunto(s)

Regulación de la Expresión Génica , Miocardio/metabolismo , Troponina I/genética , Células 3T3 , Animales , Secuencia de Bases , ADN , Proteínas de Unión al ADN/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción/metabolismo , Activación Transcripcional

RESUMEN

In striated muscle, chronic increases in workload result in changes in myosin phenotype. The aim of this study was to determine whether such changes occur in the diaphragm of patients with severe chronic obstructive pulmonary disease, a situation characterized by a chronic increase in respiratory load and lung volume. Diaphragm biopsies were obtained from 22 patients who underwent thoracic surgery. Myosin was characterized with electrophoresis in nondenaturing conditions, SDS-glycerol PAGE, and Western blotting with monoclonal antibodies specific for slow and fast myosin heavy chain (MHC) isoforms. Flow volume curves, total lung capacity, and functional residual capacity were measured before surgery in 20 patients. We found that the human diaphragm is composed of at least four myosin isoforms, one slow and three fast, resulting from the combination of three MHC species. Chronic overload was associated with an increase in the slow beta-MHC species at the expense of the fast species (beta-MHC, 78.2 +/- 4.6 and 50.0 +/- 6.5% in emphysematous and control patients, respectively; P < 0.005). Linear correlations were found between beta-MHC percentage and forced expiratory volume in 1 s (r = -0.52; P < 0.02), total lung capacity (r = 0.44; P < 0.05), and functional residual capacity (r = 0.65; P < 0.003). The human adult diaphragm is composed of a balanced proportion of slow and fast myosin isoforms. In patients with chronic obstructive pulmonary disease, the proportion of fast myosins decreases, whereas that of slow myosin increases. This increase appears to be closely related to lung hyperinflation and may reflect an adaptation of the diaphragm to the new functional requirements.

Asunto(s)

Diafragma/fisiopatología , Expresión Génica , Enfermedades Pulmonares/genética , Enfermedades Pulmonares/fisiopatología , Cadenas Pesadas de Miosina/genética , Adulto , Anciano , Western Blotting , Enfermedad Crónica , Diafragma/metabolismo , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares Obstructivas/metabolismo , Masculino , Persona de Mediana Edad , Cadenas Pesadas de Miosina/metabolismo , Respiración/fisiología

RESUMEN

The gene coding for mouse cardiac troponin I (TnI) has been cloned and sequenced. The cardiac TnI gene contains 8 exons and has an exon-intron organization similar to the quail fast skeletal TnI gene except for the region of exons 1-3, which is highly divergent. Comparative analysis suggests that cardiac TnI exon 1 corresponds to fast TnI exons 1 and 2 and that cardiac exon 3, which codes for most of the cardiac-specific amino-terminal extension and has no counterpart in the fast gene, evolved by exon insertion/deletion. The amino acid sequence of cardiac TnI exon 4 shows limited homology (36% identity) with fast TnI exon 4 but is remarkably similar (79% identity) to the corresponding sequence of slow TnI, possibly reflecting an isoform-specific TnC-binding site. The cardiac TnI gene is one of the very few contractile protein genes expressed exclusively in cardiac muscle. To identify the regulatory sequences responsible for the cardiac-specific expression of this gene we transfected cultured cardiac and skeletal muscle cells with fragments up to 4.0 kilobases of the 5'-flanking region linked to a reporter gene. Deletion analysis reveals four major regions in the 5'-flanking sequence, a minimal promoter region, which directs expression at low level in cardiac and skeletal muscle cells, and two upstream cardiac-specific positive regions separated by a negative region.

Asunto(s)

Regulación de la Expresión Génica , Miocardio/metabolismo , Troponina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , ADN , Exones , Ratones , Datos de Secuencia Molecular , Miocardio/citología , Regiones Promotoras Genéticas , Codorniz , Ratas , Secuencias Reguladoras de Ácidos Nucleicos , Homología de Secuencia de Aminoácido , Troponina I

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 Genética

RESUMEN

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 Situ

RESUMEN

Expression of cardiac troponin I (TnIcardiac) and slow skeletal troponin I (TnIslow) genes was analyzed at the mRNA and protein level in the developing rat heart. TnIslow mRNA was detectable by in situ hybridization in the embryonic cardiac tube as early as the 13-somite stage (Embryonic Day 10). In contrast, TnIcardiac transcripts were first detected in the primordial atrium and ventricle of 11-day-old embryos, but were absent in the outflow tract region. TnIslow mRNA levels decreased after birth in atria and later in ventricles but persisted even in adult life in myocytes of the conduction system. TnIslow protein was detected by specific antibodies in atrial myocytes beginning from Embryonic Day 11; in contrast, ventricular myocytes were unreactive until Embryonic Day 18. Western blot analysis of 16-day-old fetal hearts confirmed the expression of TnIcardiac in atrial but not in ventricular myocardium. Slot blot analysis showed that at this stage equivalent amounts of TnIslow and TnIcardiac mRNAs are expressed in atria and ventricles. Similar differences in the expression of TnIslow and TnIcardiac mRNAs and proteins were observed in cultures of embryonic atrial and ventricular myocytes. The results suggest serial rather than simultaneous activation of TnIslow and TnIcardiac genes and they show that different regions of the developing heart differ in their patterns of TnIcardiac expression due to the operation of distinct mechanisms that separately affect the accumulation of TnIcardiac mRNA and protein.

Asunto(s)

Envejecimiento/fisiología , Desarrollo Embrionario y Fetal/fisiología , Corazón/fisiología , Troponina/genética , Animales , Anticuerpos Monoclonales , Células Cultivadas , Femenino , Expresión Génica , Edad Gestacional , Corazón/embriología , Corazón/crecimiento & desarrollo , Inmunohistoquímica , Hibridación in Situ , Masculino , Miocardio/citología , Embarazo , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Mapeo Restrictivo , Troponina/análisis , Troponina I

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 T

RESUMEN

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 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 I

RESUMEN

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 T

RESUMEN

The function and morphology of knee extension/m. vastus lateralis and elbow flexion/m. biceps brachii were studied in young (28 +/- 0.1 years, n = 7) and elderly (68 +/- 0.5 years, n = 8) sedentary subjects and in elderly swimmers (69 +/- 1.9 years, n = 6), runners (70 +/- 0.7 years, n = 5) and strength-trained subjects (68 +/- 0.8 years, n = 7). On average, the training groups had, for the 12-17 years before the measurements were taken, performed their training regimen 3 +/- 0.1 times a week. Compared with the young subjects, the maximal isometric torque of the sedentary elderly subjects was 44% (P less than 0.05) lower in knee extension and 32% (P less than 0.05) lower in elbow flexion, and speed of movement was between 20 and 26% (P less than 0.05) lower in both knee extension and elbow flexion. The cross-sectional area of m. quadriceps femoris and the elbow flexors was also 24% (P less than 0.05) and 20% lower respectively, and the specific tension was 27% (P less than 0.05) lower in m. quadriceps femoris and 14% (P less than 0.05) lower in the elbow flexors. A 27% (P less than 0.05) higher content of myosin heavy chain type I and a 39% (P less than 0.05) higher content of the slow-type myosin light chain--2 was observed in m. vastus lateralis of the sedentary elderly subjects as compared with the young subjects. The same tendency was also seen with m. biceps brachii. Since the histochemical fibre-type distribution was identical and no major co-expression of type I and type II myosin heavy-chain isoforms was observed with immunocytochemistry, the increase in slow myosin isoforms with ageing seems mainly related to a larger relative area of type I fibres, induced by a selective atrophy of type II fibre area. An increased content of the beta-isoform of tropomyosin was also demonstrated with ageing. In contrast to the swimmers and runners, the elderly strength-trained subjects had maximal isometric torques, speed of movements, cross-sectional areas, specific tensions and a content of myosin and tropomyosin isoforms in both muscles studied identical to those of the young controls. These results seem to suggest that strength training can counteract the age-related changes in function and morphology of the ageing human skeletal muscle.

Asunto(s)

Envejecimiento , Músculos/fisiología , Anciano , Articulación del Codo/fisiología , Electroforesis en Gel Bidimensional , Terapia por Ejercicio , Antígenos HLA/biosíntesis , Humanos , Immunoblotting , Articulación de la Rodilla/fisiología , Masculino , Tono Muscular , Músculos/anatomía & histología , Miofibrillas/química , Miosinas/biosíntesis

RESUMEN

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 Endogámicas

RESUMEN

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 Rianodina

RESUMEN

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 I