RESUMO
Desmin contributes to the stability of the myocardium and its amino-terminal domain influences intermediate filament formation and interacts with a variety of proteins and DNAs. Specific serine residues located in this domain are reversibly phosphorylated in a cell cycle and developmental stage-dependent manner as has been demonstrated also for other cytoplasmic type III intermediate filament proteins. Although absence of desmin apparently does not affect cardiomyogenesis, homozygous deletion of the amino-terminal domain of desmin severely inhibited in vitro cardiomyogenesis. To demonstrate the significance of phosphorylation of this domain in cardiomyogenic commitment and differentiation, we inhibited phosphorylation of serine residues 6, 7, and 8 by mutation to alanine, and investigated early cardiomyogenesis in heterozygous embryoid bodies. As control, serine residues 31 and 32, which are not phosphorylated by kinases mutating serine residues 6, 7, and 8, were mutated to alanine in a second set. Desmin(S6,7,8A) interfered with cardiomyogenesis and myofibrillogenesis in a dominant negative fashion, whereas desmin(S31,32A) produced only a mild phenotype. Desmin(S6,7,8A) led to the down-regulation of the transcription factor genes brachyury, goosecoid, nkx2.5, and mef2C and increased apoptosis of presumptive mesoderm and differentiating cardiomyocytes. Surviving cardiomyocytes which were few in number had no myofibrils. Demonstration that some but not any mutant desmin interfered with the very beginning of cardiomyogenesis suggests an important function of temporarily phosphorylated serine residues 6, 7, and 8 in the amino-terminal domain of desmin in cardiomyogenic commitment and differentiation.
Assuntos
Diferenciação Celular/fisiologia , Desmina/genética , Miócitos Cardíacos/citologia , Fragmentos de Peptídeos/fisiologia , Serina/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células , Desmina/fisiologia , Humanos , Desenvolvimento Muscular/genética , Fragmentos de Peptídeos/genética , Estrutura Terciária de Proteína/genética , Serina/fisiologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
Desmin contributes to structural integrity and function of the myocardium but its function seems to be redundant in early cardiomyogenesis in the desmin null mouse model. To test the hypothesis that desmin also plays a supportive role in cardiomyogenic commitment and early differentiation of cardiomyocytes we investigated cardiomyogenesis in embryoid bodies expressing different desmin alleles. Constitutive expression of desmin and increased synthesis during mesoderm formation led to the up-regulation of brachyury and nkx2.5 genes, accelerated early cardiomyogenesis and resulted in the development of large, proliferating, highly interconnected, and synchronously beating cardiomyocyte clusters, whereas desmin null cardiomyocytes featured an opposite phenotype. In contrast, constitutive expression of amino-terminally truncated desmin(Delta1-48) interfered with the beginning of cardiomyogenesis, caused down-regulation of mesodermal and myocardial transcription factors, and hampered myofibrillogenesis and survival of cardiomyocytes. These results provide first evidence that a type III intermediate filament protein takes part in regulating the differentiation of mesoderm to cardiomyocytes at the very beginning of cardiomyogenesis.
Assuntos
Diferenciação Celular/fisiologia , Desmina/fisiologia , Proteínas Fetais/biossíntese , Proteínas Fetais/genética , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Miócitos Cardíacos/citologia , Proteínas com Domínio T/biossíntese , Proteínas com Domínio T/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Regulação para Cima/genética , Animais , Agregação Celular/genética , Diferenciação Celular/genética , Linhagem Celular , Desmina/biossíntese , Desmina/deficiência , Desmina/genética , Proteína Homeobox Nkx-2.5 , Mesoderma/citologia , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismoAssuntos
Genes myc/genética , Linfoma de Células B/patologia , Linfoma Folicular/patologia , Linfoma Difuso de Grandes Células B/patologia , Deleção Cromossômica , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 18 , Cromossomos Humanos Par 8 , Deleção de Genes , Humanos , Linfoma de Células B/genética , Linfoma Folicular/genética , Linfoma Difuso de Grandes Células B/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-6/genética , Translocação GenéticaRESUMO
Desmin fulfils important functions in maintenance of muscle cells and mutations in the desmin gene have been linked to a variety of myopathies. To ascertain the role of desmin's amino-terminal domain in muscle cells we generated embryonic stem cells constitutively expressing desmin(Delta1-48) in a null background and investigated muscle cell development in vitro. Desmin(Delta1-48) lacking the first 48 amino acid residues promotes fusion of myoblasts, rescues myogenesis and down-regulates vimentin expression in embryoid bodies, but hampers cardiomyogenesis and blocks smooth muscle development. These results demonstrate that desmin's amino-terminus has different roles in skeletal, cardiac, and smooth muscle cell development and function.
