Myocardin overexpression is sufficient for promoting the development of a mature smooth muscle cell-like phenotype from human embryonic stem cells.

BACKGROUND: Myocardin 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/PRINCIPAL: Findings 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/SIGNIFICANCE: Myocardin 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

Myocardin promotes calcium influx in response to carbachol.

<p>Embryoid bodies were treated with Ad-LacZ or Ad-Myo from day 10 to day 28 and then dispersed into a single cell suspension. Cells were loaded with Fluo-4, a calcium sensitive fluorophore, and intracellular [Ca²⁺] was measured by flow cytometry before and after the addition of the muscarinic agonist, carbachol in arbitrary units (AU). Representative data from a single experiment (A) and means of three studies (B) show significant increases in fluorescence following addition of carbachol. Error bars represent s.e.m., ns = not significant, * = p<0.05, C = carbachol.</p

Tbx20 Is an Essential Regulator of Embryonic Heart Growth in Zebrafish.

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

Contractile phenotype is promoted by myocardin overexpression.

<p>Embryoid bodies were treated with Ad-LacZ or Ad-Myo from day 10 to day 28, dispersed into a single cell suspension and seeded into collagen gels in 24 well plates (A). Gel contraction in response to carbachol was significantly increased in the Ad-myo group (B). Data points represent the means (±s.e.m.) of three experiments. SMCs derived from human ESCs using a 2-dimensional culture protocol were individually examined for contraction following transduction with Ad-LacZ or Ad-Myo using time lapse microscopy. Percentage of contractile cells increased from 29% with Ad-LacZ to 53% with Ad-Myo (C). Results represent the mean values (±s.e.m.) from 10 randomly chosen optical fields. **p<0.01.</p

Myocardin overexpression increases number of SMC-like cells.

<p>Embryoid bodies were enzymatically dispersed into single cells at day 17 or day 28 and flow cytometric assessment for SMC markers was carried out. Groups that had been treated with no virus, Ad-LacZ or Ad-Myo from day 10 onwards were used to quantify the proportion of SMαA⁺ cells (A & B) and SMMHC⁺ cells (C & D). Both FL1 and FL2 channels were measured for all samples to distinguish specific signal for SMαA (FL1 in A) and SMMHC (FL2 in B) due to the high levels of autofluorescence in embryoid body-derived cells. In the no virus group, SMαA staining was quantified as median SMαA⁺ signal/median SMαA⁻ signal at both day 17 and day 28 (E). Data presented in A and C are representative flow cytometric plots from a single study with the means from three independent experiments specified in the gated regions and as bar charts ± s.e.m. (B, D & E). **p<0.01.</p

Timing of viral transduction reveals an early role in SMC induction for myocardin.

<p>Embryoid bodies were transduced with Ad-LacZ or Ad-Myo early (day 10 alone or days 10 & 14), late (days 18 & 23) or throughout differentiation (days 10, 14, 18 &13). Late delivery of Ad-Myo reduced number of SMαA⁺ cells (A) (*p<0.05 by ANOVA and Tukey HSD) but had no significant effect on SMMHC⁺ cell numbers (B). Values represent mean cell proportions from three independent experiments (± s.e.m.). *p<0.05.</p

Selective upregulation of CArG-dependent genes by myocardin.

<p>Embryoid bodies were treated with no virus, Ad-LacZ or Ad-Myo from day 10 to day 28 and then harvested for RNA. SMC marker expression for a range of CArG-dependent and non-dependent genes was measured using real time RT-PCR and is normalised by three housekeeping genes and then presented relative to no virus controls. RT-PCR data represent means from at least three independent experiments. Error bars represent s.e.m. SMαA and SMMHC expression levels in response to myocardin were significantly higher than the other genes and thus the precise levels are depicted by numbers above the black bars (± s.e.m.).</p

Development of SMCs in human embryoid bodies.

<p>(A) Low power dark field image of day 28 human embryoid body grown in 20% FBS showing outgrowth of cells (white arrowheads) from the central embryoid body mass. (B and C) Expression of SMC specific genes in embryoid bodies by real time RT-PCR at days 0, 15 and 28 is normalised by three housekeeping genes (GAPDH, UBC, 18S) and then presented relative to undifferentiated human ESCs. RT-PCR data represent means from three independent experiments. Bars represent s.e.m. Cells that stain for SMαA (D) and SMMHC (E) clearly seen at day 28 within embryoid bodies by immunofluorescence. Nuclei counterstained (blue) with DAPI. Bar in A = 1000 µm, bars in D and E = 20 µm.</p

Primer sequences used for real time RT-PCR.

<p>Primer sequences used for real time RT-PCR.</p