BackgroundMyocardin is thought to have a key role in smooth muscle cell (SMC) development by acting on CArG-dependent genes. However, it is unclear whether myocardin-induced SMC maturation and increases in agonist-induced calcium signalling are also associated with increases in the expression of non-CArG-dependent SMC-specific genes. Moreover, it is unknown whether myocardin promotes SMC development from human embryonic stem cells.Methodology/PrincipalFindings The effects of adenoviral-mediated myocardin overexpression on SMC development in human ESC-derived embryoid bodies were investigated using immunofluorescence, flow cytometry and real time RT-PCR. Myocardin overexpression from day 10 to day 28 of embryoid body differentiation increased the number of smooth muscle α-actin+ and smooth muscle myosin heavy chain+ SMC-like cells and increased carbachol-induced contractile function. However, myocardin was found to selectively regulate only CArG-dependent SMC-specific genes. Nevertheless, myocardin expression appeared to be sufficient to specify the SMC lineage.Conclusions/SignificanceMyocardin increases the development and maturation of SMC-like cells from human embryonic stem cells despite not activating the full repertoire of SMC genes. These findings have implications for vascular tissue engineering and other applications requiring large numbers of functional SMCs.

The molecular mechanisms that regulate cardiomyocyte proliferation during embryonic heart growth are not completely deciphered yet. In a forward genetic N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we identified the recessive embryonic-lethal zebrafish mutant line weiches herz (whz). Homozygous mutant whz embryos display impaired heart growth due to diminished embryonic cardiomyocyte proliferation resulting in cardiac hypoplasia and weak cardiac contraction. By positional cloning, we found in whz mutant zebrafish a missense mutation within the T-box 20 (Tbx20) transcription factor gene leading to destabilization of Tbx20 protein. Morpholino-mediated knock-down of Tbx20 in wild-type zebrafish embryos phenocopies whz, indicating that the whz phenotype is due to loss of Tbx20 function, thereby leading to significantly reduced cardiomyocyte numbers by impaired proliferation of heart muscle cells. Ectopic overexpression of wild-type Tbx20 in whz mutant embryos restored cardiomyocyte proliferation and heart growth. Interestingly, ectopic overexpression of Tbx20 in wild-type zebrafish embryos resulted, similar to the situation in the embryonic mouse heart, in significantly reduced proliferation rates of ventricular cardiomyocytes, suggesting that Tbx20 activity needs to be tightly fine-tuned to guarantee regular cardiomyocyte proliferation and embryonic heart growth in vivo.

BackgroundMyocardin is a cardiac-specific and smooth muscle cell (SMC)-specific transcription factor with a key role in development. It regulates a wide variety of SMC-specific contractile markers by acting as an accessory protein for serum response factor, binding to its DNA binding sites (CArG boxes), and so has a major effect on SMC phenotype. However, its precise role in SMC development remains unclear. Moreover, there are no data on its requirement in human SMC development.MethodologyWe investigated whether myocardin was required for SMC development from human embryonic stem cells in an embryoid body model and whether we could promote SMC development at high efficiency by overexpressing myocardin.Results & conclusionsEmbryoid bodies from human embryonic stem cells were found to express increasing quantities of SMC markers up to 60 days of development with the appearance of visibly contractile SMC patches as a late phenomenon. Overexpression of myocardin using an adenovirus vector increased differentiation of pluripotent cells into the SMC lineage, although only a subset of cells was susceptible to the overexpressed transcription factor. Loss of function studies using a truncated myocardin dominant negative construct resulted in only minor or no reduction in SMC differentiation, suggesting that redundant pathways exist during embryonic development in human cells.