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

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

Primer sequences used for real time RT-PCR.

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

Myocardin has a dominant instructive role on development of SMC-like cells in the embryoid body.

<p>Embryoid bodies were treated with Ad-LacZ or Ad-Myo from day 10 to day 28 then dispersed and fixed for flow cytometry. (A) The subset of cells transduced with the Ad-Myo virus was identified by flow cytometric detection of the 3′ FLAG tag fused to the myocardin transgene. (B) The effect of Ad-Myo treatment on %SMMHC⁺ cells was analysed by flow cytometry. The majority (90%) of FLAG⁺ Ad-Myo transduced cells demonstrated a SMC-like phenotype. Data are representative of three independent experiments.</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

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

Ectopic expression of wild-type Tbx20 rescues <i>whz</i> mutant embryos.

<p><b>(A, B)</b> Lateral view of <i>whz</i> mutant embryos control-injected with KCl <b>(A)</b> and wild-type zebrafish <i>tbx20</i> mRNA <b>(B)</b>, respectively. <b>(C)</b> Ectopic expression of wild-type zebrafish <i>tbx20</i> mRNA can rescue the heart phenotype of 73% of homozygous <i>whz</i> mutant embryos, whereas injection of KCl has no effect. <b>(D, E)</b> Dissected <i>whz</i> hearts injected with KCl <b>(D)</b> and wild-type <i>tbx20</i> mRNA <b>(E)</b> are stained against MEF-2 (red) after incorporation of 5-ethynyl-2'-deoxyuridine (EdU; green) to visualize cardiomyocyte proliferation. <b>(F)</b> <i>whz</i> mutant hearts injected with wild-type <i>tbx20</i> mRNA show significantly increased numbers of ventricular cardiomyocytes at 72 hpf <b>(E)</b> compared to control-injected <i>whz</i> mutants <b>(D)</b> (<i>whz+tbx20</i> mRNA: 142.5±10 SD, <i>whz</i> + KCl: 87.82±10 SD, n = 10; p = 0.0001). <b>(G)</b> Ventricular cardiomyocyte proliferation in <i>whz</i> mutant embryos injected with <i>tbx20</i> mRNA is significantly enhanced compared to control injected mutants at 72 hpf (<i>whz</i>+<i>tbx20</i> mRNA: 7±3% SD, <i>whz</i>+KCl: 1±2% SD, n = 10; p = 0.0001).</p

Effects of the <i>whz</i> mutation on embryonic heart morphology and growth.

<p><b>(A-D)</b> Lateral view of wild-type (wt; <b>A, C</b>) and <i>whz</i> mutant (<b>B, D</b>) embryos at 72 hours post fertilization (hpf). <i>Whz</i> mutants show pericardial edema, blood congestion at the cardiac inflow tract and stretched heart chambers. <b>(E, F)</b> Hematoxylin and Eosin staining of sagittal histological sections of wt <b>(E)</b> and <i>whz</i> mutant <b>(F)</b> hearts at 72 hpf. Similar to wild-types, in <i>whz</i> atria and ventricles, myocardial (myo) and endocardial (endo) cell layers are clearly defined and separated by an atrio-ventricular canal (AVC). In contrast to wild-type hearts, <i>whz</i> mutant ventricles appear small and the myocardium monolayered. <b>(G-I)</b> Dissected wt <b>(G)</b> and <i>whz</i> mutant <b>(H)</b> hearts at 72 hpf, stained with a cardiomyocyte-specific MEF-2 antibody (nuclei; red) and co-stained with S46, exclusively marking atrial cardiomyocytes (green)<b>. (I)</b> <i>whz</i> mutant hearts show significantly reduced ventricular cardiomyocytes at 72 hpf (sib: 144.2±10 SD and <i>whz</i>: 94.9±10 SD, n = 10; p<0.0001), whereas cardiomyocyte numbers are comparable between wt and <i>whz</i> ventricles at 48 hpf (wt: 93.4±10 SD and <i>whz</i>: 88.2±10 SD, n = 10; p>0.05).</p