RESUMEN
Microgravity has a dramatic impact on human physiology, illustrated in particular, with skeletal muscle impairment. A thorough understanding of the mechanisms leading to loss of muscle mass and structural disorders is necessary for defining efficient clinical and spaceflight countermeasures. We investigated the effects of long-term bed rest on the transcriptome of soleus (SOL) and vastus lateralis (VL) muscles in healthy women (BRC group, n = 8), and the potential beneficial impact of protein supplementation (BRN group, n = 8) and of a combined resistance and aerobic training (BRE group, n = 8). Gene expression profiles were obtained using a customized microarray containing 6,681 muscles-relevant genes. A two-class statistical analysis was applied on 2,103 genes with consolidated expression in BRC, BRN, and BRE groups. We identified 472 and 207 mRNAs whose expression was modified in SOL and VL from BRC group, respectively. Further clustering analysis, identifying relevant biological mechanisms and pathways, reported five main subclusters. Three are composed of upregulated mRNAs involved mainly in nucleic acid and protein metabolism, and two made up of downregulated transcripts encoding components involved in energy metabolism. Exercise countermeasure demonstrated drastic compensatory effects, decreasing the number of differentially expressed mRNAs by 89 and 96% in SOL and VL, respectively. In contrast, nutrition countermeasure had moderate effects and decreased the number of differentially-expressed transcripts by 40 and 25% in SOL and VL. Together, these data present a systematic, global and comprehensive view of the adaptive response of female muscle to long-term atrophy.
Asunto(s)
Reposo en Cama , Proteínas en la Dieta/administración & dosificación , Ejercicio Físico , Músculo Esquelético/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Adulto , Análisis por Conglomerados , Suplementos Dietéticos , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
Global and/or dynamic analysis of the cardiac transcriptome may improve our understanding of the adaptation of cardiac tissue or cells to different physiological or pathological conditions. The achievement of sequencing projects on mammalian genomes and the development of DNA chip technology have dramatically extended the scale of gene expression studies from a candidate gene approach to a system approach. In current DNA chip experiments, expression levels of thousands of genes can be determined simultaneously. Obviously, the huge quantities of objects and information generated by these experiments require a computational management of the expression data with adequate mathematical (mostly statistical) algorithms. Here, we will discuss the principle and experimental key points of DNA chips. Four examples will be cited to illustrate applications in the cardiovascular system.
Asunto(s)
Miocardio/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Animales , Fenómenos Fisiológicos Cardiovasculares , Perfilación de la Expresión Génica , Humanos , Transcripción GenéticaRESUMEN
Heart failure is a multifactorial disease that may result from different initiating events. To contribute to an improved comprehension of normal cardiac function and the molecular events leading to heart failure, we performed large-scale gene expression analysis of failing and nonfailing human ventricle. Our aim was to define and compare expression profiles of 4 specific pathophysiological cardiac situations: 1) left ventricle (LV) from nonfailing heart; 2) LV from failing hearts affected by dilated cardiomyopathy (DCM); 3) LV from failing hearts affected by ischemic CM (ICM); 4) right ventricle (RV) from failing hearts affected by DCM or ICM. We used oligonucleotide arrays representing approximately 12,000 human genes. After stringent numerical analyses using several statistical tests, we identified 1,306 genes with a similar expression profile in all 4 cardiac situations, therefore representative of part of the human cardiac expression profile. A total of 95 genes displayed differential expression between failing and nonfailing heart samples, reflecting a reversal to developmental gene expression, dedifferentiation of failing cardiomyocytes, and involvement of apoptosis. Twenty genes were differentially expressed between failing LV and failing RV, identifying possible candidates for different functioning of both ventricles. Finally, no genes were found to be significantly differentially expressed between failing DCM and failing ICM LV, emphasizing that transcriptomal analysis of explanted hearts results mainly in identification of expression profiles of end-stage heart failure and less in determination of expression profiles of the underlying etiology. Taken together, our data resulted in identification of putative transcriptomal landmarks for normal and disturbed cardiac function.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Insuficiencia Cardíaca/genética , Miocardio/química , Miocardio/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Transcripción Genética/genética , Adolescente , Adulto , Anciano , Cardiomiopatías/genética , Cardiomiopatías/fisiopatología , Fibrosis Quística/genética , Femenino , Insuficiencia Cardíaca/fisiopatología , Trasplante de Corazón-Pulmón , Humanos , Masculino , Persona de Mediana Edad , Donantes de Tejidos , Disfunción Ventricular Izquierda/genética , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
The present study involved a global analysis of genes whose expression was modified in rat soleus muscle atrophied after hindlimb suspension (HS). HS muscle unloading is a common model for muscle disuse that especially affects antigravity slow-twitch muscles such as the soleus muscle. A cDNA cloning strategy, based on suppression subtractive hybridization technology, led to the construction of two normalized soleus muscle cDNA libraries that were subtracted in opposite directions, i.e., atrophied soleus muscle cDNAs subtracted by control cDNAs and vice versa. Differential screening of the two libraries revealed 34 genes with altered expression in HS soleus muscle, including 11 novel cDNAs, in addition to the 2X and 2B myosin heavy chain genes expressed only in soleus muscles after HS. Gene up- and down-regulations were quantified by reverse Northern blot and classical Northern blot analysis. The 25 genes with known functions fell into seven important functional categories. The homogeneity of gene alterations within each category gave several clues for unraveling the interplay of cellular events implied in the muscle atrophy phenotype. In particular, our results indicate that modulations in slow- and fast-twitch-muscle component balance, the protein synthesis/secretion pathway, and the extracellular matrix/cytoskeleton axis are likely to be key molecular mechanisms of muscle atrophy. In addition, the cloning of novel cDNAs underlined the efficiency of the chosen technical approach and gave novel possibilities to further decipher the molecular mechanisms of muscle atrophy.
Asunto(s)
Músculo Esquelético/metabolismo , Factores de Edad , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Atrofia/patología , Secuencia de Bases , Northern Blotting , Calcio/metabolismo , Clonación Molecular , Colágeno Tipo III/metabolismo , ADN Complementario/metabolismo , Regulación hacia Abajo , Matriz Extracelular/metabolismo , Femenino , Biblioteca de Genes , Datos de Secuencia Molecular , Cadenas Pesadas de Miosina/biosíntesis , Plásmidos/metabolismo , Poli A/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Análisis de Secuencia de ADN , Factores de Tiempo , Distribución Tisular , Regulación hacia ArribaRESUMEN
Mutations in the dystrophin gene lead to dystrophin deficiency, which is the cause of Duchenne muscular dystrophy (DMD). This important discovery more than 10 years ago opened a new field for very productive investigations. However, the exact functions of dystrophin are still not fully understood and the complex process leading to subsequent muscle fiber necrosis has not been clearly described; hence there has not yet been any marked improvement in patient treatment. To decipher the molecular mechanisms induced by a lack of dystrophin, we started identifying genes whose expression is altered in DMD skeletal muscles. The approach was based on differential screening of a human muscle cDNA array. Nine genes were found to be up- or downregulated. Our results indicate expression alterations in mitochondrial genes, titin, a muscle transcription factor and three novel genes. First characterizations of these novel genes indicated that two of them have striated muscle tissue specificity.
Asunto(s)
Distrofina/deficiencia , Distrofina/genética , Regulación de la Expresión Génica/genética , Genes Reguladores/genética , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Adolescente , Secuencia de Aminoácidos , Niño , Conectina , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , ADN Mitocondrial/genética , Humanos , Masculino , Proteínas de Microfilamentos , Datos de Secuencia Molecular , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Quinasas/genética , ARN Mensajero/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
In rats, changes in myosin expression are induced by the chronic elimination of weight-bearing activity, particularly in the postural muscles. This occurs during spaceflight and hindlimb suspension. Myosin heavy chain (MHC) changes affect fast and slow fiber types differently depending on muscle function. An increase in co-expression of different MHC within the same fiber will signal early changes in muscle fibers. In the rat soleus muscles, the spaceflight-induced increase in fast MHC expression appears to be essentially due to the enhanced or de novo synthesis of IID or IIX MHC. In response to microgravity, the expression of slow-type myosin decreases, while that of fast-type increases. There is scarce information concerning the effect of microgravity on rhesus monkeys (Macaca mulatta), especially on their upper limbs. We investigated the expression of MHC using an immunocytochemical approach to determine the nature and magnitude of the changes in biceps and triceps muscles of rhesus monkeys during the Bion 11 14-day mission.
Asunto(s)
Músculo Esquelético/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Vuelo Espacial , Ingravidez , Adaptación Fisiológica , Animales , Inmunohistoquímica , Macaca mulatta , Masculino , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Lenta/metabolismoRESUMEN
The mdx mouse is an animal model for Duchenne muscular dystrophy (DMD), which is caused by the absence of dystrophin. Mdx limb muscles substantially compensate for the lack of dystrophin while the diaphragm is affected like DMD skeletal muscles. To understand better the complex cascade of molecular events leading to muscle degeneration and compensatory processes in mdx muscles, we analyzed alterations of gene expression in mdx hindlimb and diaphragm muscles as compared to their normal counterparts. The strategy was based on suppression subtractive hybridization followed by reverse Northern quantitative hybridization. Four subtracted/normalized libraries, containing cDNA clones up- or downregulated in mdx hindlimb muscles or diaphragm, were constructed and a total of 1536 cDNA clones were analyzed. Ninety-three cDNAs were found to be differentially expressed in mdx hindlimb muscles and/or diaphragm. They corresponded to 54 known genes and 39 novel cDNAs. The potential role of the known genes is discussed in the context of the mdx phenotype.
Asunto(s)
Diafragma/metabolismo , Regulación de la Expresión Génica , Miembro Posterior , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/genética , Animales , Northern Blotting , ADN Complementario/genética , Regulación hacia Abajo/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Hibridación de Ácido Nucleico , Fenotipo , ARN Mensajero/biosíntesis , Regulación hacia Arriba/genéticaRESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder for which no candidate gene has yet been identified. The gene corresponding to one of the novel human cDNAs that we cloned on the basis of a muscle restricted expression pattern [Piétu G, Alibert O, Guichard B, et al. Genome Res 1996;6:492-503] was mapped in the region of the FSHD1A genetic locus, i.e. one of the loci involved in this muscular dystrophy. The corresponding encoded protein contains a PDZ and a LIM domain, two protein-protein interaction domains, and was very recently shown to bind alpha-actinin-2 and was named ALP (actinin-associated LIM protein) [Xia H, Winokur S, Kuo W, Altherr M, Bredt D. J Cell Biol 1997;139:507-515]. We raised a specific polyclonal anti-ALP serum against an ALP recombinant polypeptide to evaluate the size, level of expression and subcellular localization of ALP in three patients, clearly diagnosed with FSHD disease. Quantitative or qualitative alterations of ALP expression have not been detected in any of them, thus prompting us to exclude ALP as a FSHD gene candidate.
Asunto(s)
Actinina/genética , ADN/genética , Proteínas de Microfilamentos/genética , Distrofias Musculares/genética , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Mapeo Cromosómico , Clonación Molecular , Humanos , Proteínas con Dominio LIM , Persona de Mediana Edad , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/ultraestructura , Distrofias Musculares/metabolismo , Mapeo Restrictivo , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructura , Secuencias Repetidas en Tándem/genéticaRESUMEN
Muscle disuse induces substantial alterations in the highly plastic skeletal muscle tissues, which occur especially in antigravity slow muscles. We differentially screened a muscle cDNA array to identify modifications in gene profile expression induced in slow rat soleus muscle mechanically unloaded by hindlimb suspension as a model for muscle disuse. This study focused on muscle creatine kinase mRNA and protein and glyceraldehyde-3-phosphate dehydrogenase mRNA, which were found to be upregulated in unweighted muscles. These upregulations were analyzed over a 4-wk time course of hindlimb suspension and compared with variations in myosin heavy chain (MHC) isoforms while specifically focusing on type IIx MHC mRNA and protein. The two metabolic marker upregulations clearly preceded IIx MHC contractile protein upregulation. Muscle creatine kinase upregulation was shown to be an excellent, and the earliest, marker of muscle disuse at mRNA and protein levels.
Asunto(s)
Creatina Quinasa/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Músculo Esquelético/metabolismo , Animales , Biomarcadores , Creatina Quinasa/genética , Femenino , Regulación de la Expresión Génica/fisiología , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Suspensión Trasera , Isoenzimas , Músculo Esquelético/patología , Atrofia Muscular/etiología , Atrofia Muscular/patología , Cadenas Pesadas de Miosina/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Systematic analysis of gene transcript repertoires prepared from libraries made with various specific human tissues permitted isolation of many partially sequenced cDNA clones. A few of these represented novel genes with limited or no similarity to known genes from humans or other species. The present study set out to isolate and sequence the full-length cDNA corresponding to one of these novel human transcripts, and identify the corresponding protein product at the subcellular level. Current sequence analyses have revealed that the protein contains a hydrophobic N-terminal segment and an internal leucine-zipper motif. Numerous sites of putative post-translational modifications, such as N-linked glycosylation, myristoylation and phosphorylation sites, were also identified. Using one monoclonal antibody raised against a recombinant fragment, two different 41-43 kDa proteins were detected in human skeletal muscle, heart and placenta homogenates at various ratios. Both immunodetected protein products of the novel human gene were distributed in the transverse tubules and/or near the junctional sarcoplasmic reticulum within skeletal muscle cells. Both proteins had physical properties believed to be attributable to integral membrane components. Finally, the GENX-3414 gene was chromosomally localized at position 4q24-q25.
Asunto(s)
Cromosomas Humanos Par 4 , Proteínas de la Membrana/genética , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Transcripción Genética , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN Complementario , Humanos , Leucina Zippers , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/química , Persona de Mediana Edad , Datos de Secuencia Molecular , Peso Molecular , Proteínas Musculares/biosíntesis , Proteínas Musculares/química , Músculo Esquelético/ultraestructura , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/químicaRESUMEN
One of the most obvious effects of hypokinesia and hypodynamia is muscular atrophy. Changes in myosin expression are induced by the chronic elimination of the weight-bearing activity, particularly in the postural muscles, which occur during spaceflight and hindlimb suspension. Other morphological changes, such as the remodeling of myotendinous junction, are also induced by this reduction in mechanical stress. Moreover, the transversal interface between the cytoskeleton and the extracellular matrix of the muscle fiber can also be modified by the functional demand imposed on muscle. Dystrophin and its associated proteins appear to be essential for the stability of this interface and the deletion of one of these proteins results in a pathological phenotype. Changes in their expression appears to be induced by muscle disuse. We have quantified the changes in the expression of dystrophin and associated proteins induced by a 14-day spaceflight.
Asunto(s)
Distrofina/metabolismo , Músculo Esquelético/metabolismo , Vuelo Espacial , Ingravidez/efectos adversos , Animales , Masculino , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiología , Atrofia Muscular/etiología , Atrofia Muscular/metabolismo , Atrofia Muscular/fisiopatología , Cadenas Pesadas de Miosina/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Unlike the random distribution of fiber types seen in skeletal muscles of most mammals, pig muscle exhibits a rosette pattern consisting of islets of slow fibers surrounded by concentric circles of type IIA and IIB fibers. Within each islet of slow fibers, one of the central fibers is a primary myofiber, whereas all others are secondary fibers. The present study demonstrates that a subpopulation of the slow secondary fibers transiently expresses alpha-myosin heavy chain (MHC). Two cDNA libraries were made from longissimus dorsi skeletal muscle of 14-day-old piglet and adult pig atrium; the latter muscle is mainly composed of alpha-MHC. Screening of the libraries with a human anti-alpha-MHC mAb (F8812F8) demonstrated the presence of positive MHC clones in both libraries; the nucleotide sequence of the 3'-untranslated region (3'-UTR) was identical in both libraries. As this MHC 3'-UTR had 75% homology with the human alpha-MHC, it was identified as pig alpha-MHC. Using specific cRNA probes and mAbs against pig alpha-cardiac and beta/slow/type I MHC, we studied the expression of these MHCs in developing pig semitendinosus muscle by combining in situ hybridization and immunocytochemistry on serial sections at 90 days of gestation, and at 1, 6, 35 days and 6 months of age. The results showed that a subpopulation of secondary fibers that directly abut primary fibers, transiently produced alpha-MHC, both at the levels of the protein and its transcript. Subsequently, these fibres expressed beta-MHC. At 1 day, immunocytochemistry showed that 16% of the secondary fibers expressed alpha-MHC, among which 20% did not yet express beta-MHC. At 6 days, alpha- and beta-MHCs were mostly present in the same fibers, i.e., 23% of the secondary fibers. Thereafter, the proportion of secondary fibers reacting with alpha-MHC mAb decreased to 10% at 5 weeks and 0% at 6 months, whereas beta-MHC was still accumulating in about 38% of the secondary fibers. During the period studied, the distribution of alpha- and beta-MHC transcripts closely matched that of the corresponding proteins. Expression of alpha-MHC was not detected in primary type I muscle fibers and slow type I secondary fibers at the periphery of the rosettes of slow fibers. This study is the first unequivocal demonstration of a transitory expression of alpha-MHC in a subpopulation of secondary fibers in a limb skeletal muscle during mammalian development.
Asunto(s)
Miosinas Cardíacas , Fibras Musculares Esqueléticas/metabolismo , Cadenas Pesadas de Miosina/biosíntesis , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Humanos , Inmunohistoquímica , Hibridación in Situ , Modelos Moleculares , Datos de Secuencia Molecular , Desarrollo de Músculos , Fibras Musculares Esqueléticas/química , Músculo Esquelético/crecimiento & desarrollo , Miosinas/inmunología , Biblioteca de Péptidos , ARN Mensajero/metabolismo , Porcinos , Tendones/crecimiento & desarrolloRESUMEN
The human mineralocorticoid receptor of the steroid receptor family contains a modular structure with domain E which is considered to be a hormone binding domain. Recombinant protein approaches enabled us to clearly determine that this domain is also able to interact with F-actin (Kd about 2 microM) and G-actin. Moreover, it was revealed that this mineralocorticoid receptor domain/actin interaction was modulated by specific mineralocorticoid ligands. Agonist (aldosterone) steroid binding almost totally (91%) abolished the interaction with F-actin, while antagonist (progesterone) binding allowed more than 30% of this binding. Steroid modulation of the interaction between domain E and actin indicated that this actin binding is specific and could be essential for cellular mineralocorticoid receptor activity.
Asunto(s)
Actinas/metabolismo , Mineralocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Aldosterona/metabolismo , Sitios de Unión , Proteínas de Choque Térmico/metabolismo , Humanos , Ligandos , Fragmentos de Péptidos/metabolismo , Progesterona/metabolismo , Unión Proteica , Receptores de Mineralocorticoides/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
In our search for genes up- or down-regulated genes in the mdx mouse model for Duchenne muscular dystrophy, we isolated a down-regulated mitochondrial DNA clone. In addition to this clone, all protein-coding mitochondrial genes tested had tissue-specific and age independent down-regulated expression. This implied mechanisms at the RNA level since no change in the mitochondrial DNA contents were detected. Cytochrome c oxidase activity showed the same range of down-regulated expression. These data provide a molecular basis for energetic metabolism modifications in mdx mice.
Asunto(s)
Envejecimiento/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Mitocondrias Musculares/metabolismo , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , ARN Mensajero/biosíntesis , ARN/biosíntesis , Animales , Secuencia de Bases , Northern Blotting , Cartilla de ADN , Complejo IV de Transporte de Electrones/biosíntesis , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Datos de Secuencia Molecular , Desarrollo de Músculos , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , ARN Mitocondrial , Valores de ReferenciaRESUMEN
Partial deletions of the dystrophin gene are the predominant genetic lesions in Duchenne (DMD) and Becker (BMD) muscular dystrophies. According to the reading frame hypothesis [1], any deletion disrupting the translational reading frame of the mRNA cannot result in expression of the dystrophin molecule and should lead to severe phenotypes of DMD. In contrast, deletions which maintain the reading frame across the deleted exons may give rise to truncated, semifunctional proteins and milder courses of the disease (i.e. BMD). Among the notable exceptions of this hypothesis are very large "in-frame" deletions by which functionally indispensable domains of the dystrophin molecule have been removed. Here, we report on two DMD patients with large intragenic in-frame deletions. Grossly truncated, but stable dystrophin molecules with preserved C-terminal domains were detected at the sarcolemma on cryosections in both patients. However, dystrophin organization on single-teased muscle fibers revealed disarrangement of the costameric pattern, if compared to normal skeletal muscle fibers. Compared to dystrophin-deficient DMD muscle, expression of chromosome-6-encoded dystrophin-related protein (DRP) was greatly diminished in skeletal muscle of both patients. We show, that loss of more than 50% of dystrophin seems to be deleterious for the protein's function and therefore, the extent of the deletions may have an impact on construction of dystrophin mini genes. Moreover, these findings shed new light on the functional significance of the C-terminal domain of dystrophin. They also suggest a negative correlation between sarcolemmal expression of the dystrophin C-terminus and DRP expression at the sarcolemma.
Asunto(s)
Proteínas del Citoesqueleto/genética , Distrofina/genética , Eliminación de Gen , Regulación de la Expresión Génica/genética , Proteínas de la Membrana , Sarcolema/genética , Western Blotting , Niño , Preescolar , ADN/análisis , Exones/genética , Humanos , Inmunohistoquímica , Masculino , Distrofias Musculares/genética , Sistemas de Lectura Abierta/genética , Fenotipo , UtrofinaRESUMEN
Our aim was to investigate the potential functional consequences of myosin heavy chain (MHC) mutations identified in patients with familial hypertrophic cardiomyopathy. We observed the presence of a mutated beta-MHC mRNA in a formalin-fixed paraffin-embedded myocardial tissue of a proband from family A, which Geisterfer-Lowrance et al. [Geisterfer-Lowrance, Kass, Tanigawa, Vosberg, McKenna, Seidman and Seidman (1990) Cell 62, 999-1006] identified as carrying the Arg-403 to Gln mutation. Recombinant DNA methods were then used to obtain size-limited, soluble and undenatured fragments of mutated myosin subfragment 1 focused around the 403 mutation. The present analysis indicated that the 403 mutation did not quantitatively alter the actin- or ATP-binding capacities of two 246-residue or 524-residue-long recombinant MHC fragments containing this mutation. The absence of any apparent impact of the 403 mutation in the recombinant MHC fragments on interactions between actin and ATP is discussed in relation to numerous biochemical and structural reports which demonstrate the crucial role of the central MHC segment, where the 403 mutation occurs, in myosin functions.
Asunto(s)
Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Cardiomiopatía Hipertrófica/genética , Mutación , Miocardio/metabolismo , Miosinas/genética , Arginina , Secuencia de Bases , Sitios de Unión , Expresión Génica , Glutamina , Ventrículos Cardíacos/química , Humanos , Datos de Secuencia Molecular , Miosinas/química , Miosinas/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
Nine monoclonal antibodies were used to test calcium and EDTA effects on the molecular conformation of ventricular VLC2 within myosin. Antibody epitopes were located in six domains of VLC2 using recombinant proteins. The apparent association constants of these antibodies were measured in solution in the presence of calcium or EDTA. An immunofluorescence study was performed to establish whether the observed effects would occur in more integrated systems, as compared to isolated proteins in solution. Our results showed (1) a slight effect of calcium on isolated VLC2, located in the aa 29-45 domain, (2) a clear-cut effect of calcium on VLC2 within myosin, only in the aa 45-59 domain, and (3) in the presence of EDTA, antibody affinities for VLC2 within myosin similar to the affinities for isolated VLC2. These results are discussed in terms of spatial arrangements and binding mechanisms between HC and VLC2. They suggest that there are two processes for stabilizing HC/VLC2 complex formation: one binding via calcium chelation and another involving hydrophobic interactions.
Asunto(s)
Miocardio/química , Miosinas/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Calcio/química , Bovinos , Ácido Edético/química , Mapeo Epitopo , Técnica del Anticuerpo Fluorescente , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Miofibrillas/química , Conformación ProteicaRESUMEN
We correlated utrophin expression with the physiopathological course in mdx mice. Evolution of the pathology was assessed by monitoring expression of developmental MHC in mdx mice versus control. Utrophin expression is detected by dystrophin/utrophin cross-reacting antibodies and can only be evaluated in mdx mouse muscles (in absence of dystrophin). This protein was expressed at the periphery of all myotubes and myofibers during the first postnatal week. It began declining in fast muscles before the third week and disappeared from the soleus between the 3rd and the 4th week. The decrease was concomitant with a sudden degenerative/regenerative process affecting slow muscle earlier and more massively than fast muscles. The pathological process became stable in all muscle types (except the diaphragm), with greater utrophin expression in the soleus. These results in mdx mice along with observed utrophin expression in severely affected DMD patients suggest that overexpression of utrophin is not enough to explain the stability of regenerated fibers in mdx mice.
Asunto(s)
Proteínas del Citoesqueleto/fisiología , Distrofina/deficiencia , Proteínas de la Membrana , Ratones Endogámicos mdx/metabolismo , Proteínas Musculares/biosíntesis , Distrofia Muscular Animal/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Proteínas del Citoesqueleto/biosíntesis , Proteínas del Citoesqueleto/inmunología , Distrofina/genética , Distrofina/inmunología , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx/genética , Músculos/química , Músculos/ultraestructura , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patología , Miosinas/inmunología , UtrofinaRESUMEN
Recombinant DNA methods were used to obtain soluble, undenatured fragments of the heavy chain of myosin subfragment 1 (S-1). These fragments were of preselected lengths and could include protease-sensitive segments that are destroyed when other preparation methods are used. Actin binding by each of the three contiguous segments (residues 1-248, 249-524, and 518-722, essentially spanning the entire S-1 heavy chain) was demonstrated. ATP binding, comparable to that of native S-1, was obtained only with a segment consisting of residues 1-524. Competition among the various fragments for actin was also studied. The data are discussed in relation to the recently reported resolved structure of S-1 [Rayment, I., Rypnieski, R. W., Schmidt-Bäse, K., Smith, R., Tomchick, D. R., Benning, M. M., Winkelmann, D. A., Wesenberg, G. & Holden, H. M. (1993) Science 261, 50-58].