Figure 1.
The small molecule BMP inhibitor, dorsomorphin, induces cardiomyogenesis in mouse ES cells.
(A) Chemical structures of dorsomorphin (DM), a selective BMP inhibitor, and 676489, a DM analog which inhibits VEGF/VEGFR2, but not BMP signaling. (B) ES cells treated with dorsomorphin (DM) from day −3 to 2 formed large areas of contracting cardiomyocytes that expressed DsRed-Nuc under the α-MHC promoter by day 12 of differentiation (right), but DMSO-treated cells did not (left). Upper panels depict representative red fluorescence images. Lower panels show the corresponding bright-field images. (C, D) Dorsomorphin treatment resulted in strong increases in expression of cardiac markers Nkx2.5 (*p = 0.021, **p = 0.020, #p = 0.0013), and Myh6 (*p = 0.046, **p = 0.026). Q-PCR results represent relative expression normalized to that of DMSO-treated cells at Day 0. Measurements were from at least three independent experiments for each time-point.
Figure 2.
Quantitative assessment of cardiomyocyte induction by dorsomorphin in a 96-well format.
(A) Dorsomorphin (DM) treatment in a 96-well microtiter plate format reproducibly induced formation of beating embryoid bodies in CGR8 ES cells (94.4% of EBs, *p<0.0001, results from at least three independent experiments involving over 300 EBs per condition). (B) DM treatment reproducibly induced cardiomyogenesis in R1 ES cells (91.3% of EBs, results from at least 92 EBs). All results are compared to DMSO-vehicle treatment as negative control. Red bars, dorsomorphin-treated. Black bars, DMSO-treated. Error bars represent standard error. (C) Time window for cardiomyocyte induction by dorsomorphin. DM treatments from Day −3 to 2, Day −2 to 2, Day −1 to 2, Day 0 to 2, and Day 0 to 1, represented by red bars, were nearly equivalent in promoting formation of beating cardiomyocytes in mouse ES cells at day 12 of differentiation. Increases in the frequencies of beating EBs with the above DM treatment protocols were all highly statistically significant in comparison to DMSO treatment over same time periods (P<0.0001 for each condition). Differences between the above DM treatments were not statistically significant. In contrast, DM treatment from Days −3 to 0 did not result in significant increase in cardiomyocyte formation compared to DMSO control. Based on these results, the minimal temporal requirement for cardiac induction by DM can be narrowed down to the first 24 hours of ES cell differentiation (shown between two dotted lines). Frequencies of DM-treated EBs that contract spontaneously by day 12 were obtained from at least 200 EBs for each time point on three or more separate days. Ave. denotes average percentage of EBs that contract spontaneously. By contrast, Noggin treatment (300 ng/mL), represented by orange bars, during the first 24 hours of differentiation did not efficiently induce the formation of beating EBs. However, in agreement with the prior report by Yuasa et al, Noggin treatment from Days −3 to 2 (orange bars) efficiently induced the formation of beating EBs. For Noggin treatment, results were obtained from 96 EBs per condition. (D) Dose-response curve for cardiomyocyte induction by DM treatment from Day 0 to 1. ES cells were treated with various concentrations of DM at day 0 to 1 of differentiation, and percentages of EBs that contract spontaneously at day 12 of differentiation were determined. Results were then used to create a dose-response curve. Results were obtained from at least 52 EBs per condition.
Figure 3.
Dorsomorphin treatment during the initial 24 hours of ES cell differentiation robustly induces cardiomyogenesis.
(A, B) Dorsomorphin (DM) treatment from day 0 to 1 of differentiation resulted in strong increases in expression of cardiac markers Nkx2.5 (*p = 0.003, **p = 0.0019, #p = 0.0001), and Myh6 (*p = 0.023, **p = 0.029, #p = 0.0089). All results are compared to DMSO-vehicle treatment as negative control. Red bars, DM-treated. Black bars, DMSO-treated. Error bars represent standard error. Q-PCR results represent relative expression normalized to that of DMSO-treated cells at day 0. Measurements were obtained from at least three independent experiments for each time-point. (C) Western blot showing markedly higher levels of the cardiac α-Myosin Heavy Chain (α-MHC) protein in DM-treated ES cells on day 10 in comparison to DMSO-treated controls. Antibody against α-Tubulin was used as loading control. (D) DM treatment greatly increased the number of fluorescent nuclei that expressed DsRed as a percentage of total cells (DAPI+) at day 12 (error bars, standard error; p<0.0001 vs. DMSO control). Results from trypsin dissociated cells from 11 DM-treated EBs and 14 DMSO-treated controls. (E) Representative FACS analysis showing an approximately 30-fold increase in the fraction of α-actinin+ cells following DM treatment vs. DMSO controls.
Figure 4.
Dorsomorphin treatment promotes the formation of large areas comprised of cardiomyocytes.
(A) Dorsomorphin-treated ES cells formed larger areas of contracting cardiomyocytes that immunostained for the sarcomeric proteins cardiac Troponin-T (c-TnT), α-Actinin and α-cardiac myosin heavy chain (α-MHC) at day 10 (Green, left panels). The areas that immunostained for sarcomeric proteins also immunostained for the cardiac-specific transcription factor Nkx2.5 (Red, middle panels), confirming that these regions are comprised mainly of cardiomyocytes. Merged images are on the right. (B) Merged 40× confocal images showing the details of sarcomeric protein and Nkx2.5 immunostaining (right, α-actinin/Nkx2.5; middle, c-troponin-T/Nkx2.5; right, α-cardiac MHC/Nkx2.5). DM-treated cells showed many cardiomyocytes with organized sarcomeric structures. (C) 10× images of DMSO-treated cells. In control conditions, cells that immunostain for sarcomeric proteins and Nkx2.5 are rare and typically form much smaller foci without discernable sarcomeric organization. The panels depict representative immunofluorescence images.
Figure 5.
Dorsomorphin treatment promotes cardiomyogenesis at the expense of other mesodermal lineages.
(A) Dorsomorphin treatment during the first 24 hours of ES cell differentiation (from day 0 to 1) blunted the induction of BryT expression at day 3 (*p = 0.0016), but resulted in higher BryT expression at day 4, in comparison to controls (**p = 0.017). (B) Dorsomorphin treatment caused similar, yet even more striking, changes in Mesp1 expression (*p<0.0001, **p = 0.0107). In addition, dorsomorphin treatment resulted in significant decreases in (C) Gata1 expression at days 4 to 8 (*p = 0.0382, **p = 0.0044, and #p = 0.0019), in (D) MyH11 expression at days 8 and 10 (*p = 0.0032 and **p = 0.0040), in (E) Flk-1 expression at day 3 to 6 (*p = 0.0002, **p = 0.0092, and #p = 0.0021), and in (F) VE-Cadherin (VE-cad; vascular endothelium-cadherin) expression at day 4 to 8 (*P = 0.0076, **p = 0.0364, and #P = 0.0153). All results are compared to DMSO control. Red bars, dorsomorphin-treated. Black bars, DMSO-treated. Q-PCR results were obtained from at least three independent experiments. Error bars, standard error.