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/metabolismoRESUMO
Cardiomyogenesis is influenced by factors secreted by anterior-lateral and extra-embryonic endoderm. Differentiation of embryonic stem cells in embryoid bodies allows to study the influence of growth factors on cardiomyogenesis. By these means SPARC was identified as a new factor enhancing cardiomyogenesis [M. Stary, W. Pasteiner, A. Summer, A. Hrdina, A. Eger, G. Weitzer, Parietal endoderm secreted SPARC promotes early cardiomyogenesis in vitro, Exp. Cell Res. 310 (2005) 331-341]. Here we report a similar and new function for S100A4, a calcium-binding protein of the EF-hand type. S100A4 is secreted by parietal endoderm and promotes early differentiation and proliferation of cardiomyocytes. Oligomeric S100A4 supports cardiomyogenesis in a concentration-dependent manner, whereas inhibition of autocrine S100A4 severely attenuates cardiomyogenesis. S100A4 specifically influences transcription in differentiating cardiomyocytes, as evident from increased expression of cardiac transcription factor genes nkx2.5 and mef2C. These data suggest that S100A4, like SPARC, plays a supportive role in early in vitro cardiomyogenesis.
Assuntos
Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Endoderma/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Proteínas S100/administração & dosagem , Proteínas S100/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células , Relação Dose-Resposta a Droga , Embrião de Mamíferos/efeitos dos fármacos , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas de Domínio MADS/metabolismo , Fatores de Transcrição MEF2 , Miócitos Cardíacos/efeitos dos fármacos , Fatores de Regulação Miogênica/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100 , Fatores de Transcrição/metabolismoRESUMO
Cardiomyogenesis proceeds in the presence of signals emanating from extra-embryonic lineages emerging before and during early eutherian gastrulation. In embryonic stem cell derived embryoid bodies, primitive endoderm gives rise to visceral and parietal endoderm. Parietal endoderm undergoes an epithelial to mesenchymal transition shortly before first cardiomyocytes start to contract rhythmically. Here, we demonstrate that Secreted Protein, Acidic, Rich in Cysteine, SPARC, predominantly secreted by mesenchymal parietal endoderm specifically promotes early myocardial cell differentiation in embryoid bodies. SPARC enhanced the expression of bmp2 and nkx2.5 in embryoid bodies and fetal cardiomyocytes. Inhibition of either SPARC or Bmp2 attenuated in both cases cardiomyogenesis and downregulated nkx2.5 expression. Thus, SPARC directly affects cardiomyogenesis, modulates Bmp2 signaling, and contributes to a positive autoregulatory loop of Bmp2 and Nkx2.5 in cardiomyocytes.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Endoderma/metabolismo , Coração/embriologia , Osteonectina/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/genética , Endoderma/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mesoderma/citologia , Mesoderma/fisiologia , Camundongos , Desenvolvimento Muscular/genética , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/genética , Regulação para CimaRESUMO
The unique differentiation potential of inner cell mass derived embryonic stem cells together with their outstanding self-renewal capacity makes them a desirable source for somatic cell therapy of human diseases. Somatic cells are gained by in vitro differentiation of embryonic stem cells, however, the differentiation potential of embryonic stem cells varied even between isogenic cell lines. Variable differentiation potentials may either be a consequence of an inherent inhomogeneity of gene expression in the inner cell mass or may have technical reasons. To understand variations in the differentiation potential, we generated pairs of isogenic, monozygotic twin, and single inner cell mass derived clonal embryonic stem cell lines, and demonstrate that they differentially express the leukaemia inhibitory factor receptor gene. Variations of leukaemia inhibitory factor receptor protein levels are already evident in the inner cell mass and predispose the cardiomyogenic potential of embryonic stem cell lines in a Janus activated kinase dependent manner. Thus, a single inner cell mass may give rise to embryonic stem cell lines with different developmental potentials.