Differentiation of cardiomyocytes requires functional serine residues within the amino-terminal domain of desmin.

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.

Desmin stimulates differentiation of cardiomyocytes and up-regulation of brachyury and nkx2.5.

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.

Parietal endoderm secreted S100A4 promotes early cardiomyogenesis in embryoid bodies.

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.

Parietal endoderm secreted SPARC promotes early cardiomyogenesis in vitro.

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.

Single inner cell masses yield embryonic stem cell lines differing in lifr expression and their developmental potential.

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.