RESUMEN
Skeletal muscle is able to respond to a range of stimuli, including stretch and increased load, by increasing in diameter and length in the absence of myofiber division. This type of cellular growth (hypertrophy) is a highly complex process involving division of muscle precursor cells (myoblasts) and their fusion to existing muscle fibers as well as increased protein synthesis and decreased protein degradation. Underlying the alterations in protein levels are increases in a range of specific mRNAs including those coding for structural proteins and proteins that regulate the hypertrophic process. Seven days of passive stretch in vivo of tibialis anterior (TA) muscle has been shown to elicit muscle hypertrophy. We have identified a cDNA corresponding to an mRNA that exhibits increased expression in response to 7 days of passive stretch imposed on TA muscles in vivo. This 944-bp novel murine transcript is expressed primarily in cardiac and skeletal muscle and to a lesser extent in brain. Translation of the transcript revealed an open reading frame of 85 amino acids encoding a nuclear localization signal and two overlapping casein kinase II phosphorylation sites. This gene has been called "small muscle protein (X chromosome)" (Smpx; HGMW-approved human gene symbol SMPX) and we hypothesize that it plays a role in skeletal muscle hypertrophy.
Asunto(s)
Proteínas Musculares , Músculo Esquelético/metabolismo , Porinas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Diferenciación Celular/genética , Mapeo Cromosómico , ADN Complementario/química , ADN Complementario/genética , Embrión de Mamíferos/metabolismo , Exones , Regulación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Intrones , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Músculo Esquelético/citología , Porinas/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Organismos Libres de Patógenos Específicos , Estrés Mecánico , Distribución Tisular , Cromosoma X/genéticaRESUMEN
In response to extended periods of stretch, skeletal muscle typically exhibits cell hypertrophy associated with sustained increases in mRNA and protein synthesis. Several soluble hypertrophic agonists have been identified, yet relatively little is known as to how mechanical load is converted into intracellular signals regulating gene expression or how increased cell size is maintained. In skeletal muscle, hypertrophy is generally regarded as a beneficial adaptive response to increased workload. In some cases, however, hypertrophy can be detrimental as seen in long-term cardiac hypertrophy. Skeletal muscle wasting (atrophy) is a feature of both inherited and acquired muscle disease and normal aging. Elucidating the molecular regulation of cell size is a fundamental step toward comprehending the complex molecular systems underlying muscle hypertrophy and atrophy. Subtractive hybridization between passively stretched and control murine skeletal muscle tissue identified an mRNA that undergoes increased expression in response to passive stretch. Encoded within the mRNA is an open reading frame of 311 amino acids containing a highly conserved type 1 peroxisomal targeting signal and a serine lipase active center. The sequence shows identity to a family of serine hydrolases and thus is named serine hydrolase-like (Serhl). The predicted three-dimensional structure displays a core alpha/beta-hydrolase fold and catalytic triad characteristic of several hydrolytic enzymes. Endogenous Serhl protein immunolocalizes to perinuclear vesicles as does Serhl-FLAG fusion protein transiently expressed in muscle cells in vitro. Overexpression of Serhl-FLAG has no effect on muscle cell phenotype in vitro. Serhl's expression patterns and its response to passive stretch suggest that it may play a role in normal peroxisome function and skeletal muscle growth in response to mechanical stimuli.
Asunto(s)
Proteínas Musculares/genética , Músculo Esquelético/enzimología , Serina Endopeptidasas/análisis , Serina Endopeptidasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Técnicas de Cultivo de Célula , Clonación Molecular , ADN , Inducción Enzimática , Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Contracción Muscular , Proteínas Musculares/biosíntesis , Proteínas Musculares/metabolismo , Músculo Esquelético/citología , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/metabolismoRESUMEN
OBJECTIVE: The mechanisms by which Coxsackie B viruses cause myocarditis or dilated cardiomyopathy are not well understood. This study examined changes in the expression of cardiac genes resulting from Coxsackievirus B3 (CVB3) infection of mice. METHODS: Mice (five per group) were experimentally infected with CVB3 or mock-infected with diluent. Altered expression of genes was initially identified by cDNA array, and confirmed by semiquantitative RT-PCR, western blot and immunohistochemistry. RESULTS: Forty-two up-regulated or down-regulated genes were observed in cDNA arrays carrying 588 known mouse genes. Among these, one down-regulated gene, Bag-1, known to be involved in inhibition of apoptosis and modulation of chaperone activity, was investigated further. Semiquantitative RT-PCR showed that Bag-1 expression was down-regulated by up to 30% in virus-infected mouse heart on day 7 compared to the mock-infected. Cell fractionation and western blot analysis confirmed that Bag-1 isoform p32 was predominant in the cytoplasm of mouse myocardium and down-regulated at 4 days or 7 days after CVB3 infection. In contrast, Bag-1 isoform p50 appeared to increase in the nuclear fraction of mouse heart at 7 days after infection. Down regulated expression and distribution of Bag-1 protein or evidence of apoptosis in the infected mouse heart was demonstrated by immunostaining or histochemistry (TUNEL assay), respectively. CONCLUSION: CVB3 infection induced differential expression of Bag-1 in cytoplasmic and nuclear fractions of mouse heart and apoptosis. This may be important in the pathogenesis of enterovirus heart muscle disease.
Asunto(s)
Proteínas Portadoras/metabolismo , Infecciones por Coxsackievirus/metabolismo , Enterovirus Humano B , Miocarditis/metabolismo , Animales , Apoptosis , Proteínas Portadoras/genética , Infecciones por Coxsackievirus/genética , Infecciones por Coxsackievirus/patología , ADN Complementario/genética , Proteínas de Unión al ADN , Regulación hacia Abajo , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos , Miocarditis/genética , Miocarditis/patología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TranscripciónRESUMEN
Mechanically induced hypertrophy of skeletal muscles involves shifts in gene expression leading to increases in the synthesis of specific proteins. Full characterization of the regulation of muscle hypertrophy is a prerequisite for the development of novel therapies aimed at treating muscle wasting (atrophy) in human aging and disease. Using suppression subtractive hybridization, cDNAs corresponding to mRNAs that increase in relative abundance in response to mechanical stretch of mouse skeletal muscles in vivo were identified. A novel 1100-bp transcript was detected exclusively in skeletal muscle. This exhibited a fourfold increase in expression after 7 days of stretch. The transcript had an open reading frame of 328 amino acids encoding an ATP/GTP binding domain, a nuclear localization signal, two PEST protein-destabilization motifs, and a 132-amino-acid ankyrin-repeat region. We have named this gene ankyrin-repeat domain 2 (stretch-responsive muscle) (Ankrd2). We hypothesize that Ankrd2 plays an important role in skeletal muscle hypertrophy.
Asunto(s)
Repetición de Anquirina/genética , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Secuencias de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular , Células Cultivadas , Clonación Molecular , ADN Complementario/genética , Exones , Expresión Génica , Biblioteca Genómica , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas Musculares/biosíntesis , Proteínas Musculares/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/embriología , Proteínas Nucleares , Especificidad de Órganos/genética , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Proteínas Represoras , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Estrés MecánicoRESUMEN
An approach has been developed for the screening of allelic variation of minisatellite DNA loci that substantially reduces the time and hazards involved. Primers were designed for a minisatellite region isolated from a gadoid fish species (Merlangius merlangus L.), enabling amplification by polymerase chain reaction, so that differences in the number of minisatellite repeat units (allelic variability) were detectable by ethidium bromide fluorescence (over UV light) following separation by agarose gel electrophoresis. This amplifiable minisatellite variable number tandem repeat region, the first non-primate marker of its kind can be used successfully with DNA extracted by a rapid Chelex protocol. From a sample of 97 individuals, 24 alleles were resolved (750-2200 kb) and heterozygosity was estimated at 0.94.