Abstract 135: A Novel TGFb Selective Inhibitor Drives Cardiogenesis in Embryonic Stem Cells
The early signals that drive embryonic stem cells (ESC) to cardiomyocytes are well characterized, but little is known about the pathways that control the later stages of differentiation. Genetic studies in animals are however challenging to study these later steps of heart development and are thus incapable of providing useful information for controlling ESC differentiation. Therefore, we have implemented unbiased high throughput screens on mouse ESC (mESC) to discover small molecules that probe these steps of differentiation to unravel novel biological mechanisms that control cardiac fate. From over 17000 unique molecules, screened in a serum assay relying on a cardiac specific Myh6-GFP reporter to monitor cardiogenesis, we identified a dihydropyridine (DHP), which are well-known L-type calcium channel inhibitors. Subsequent pathway analysis in combination with a structure activity relationship study of over 200 DHP analogs pinpointed the activity specifically to the Activin A/TGFb pathway, with high inhibition efficiency for TGFb signals and poor to none for Activin A signals. Further functional characterization of DHP in the TGFb pathway demonstrated that DHP acts upstream of Smad2/3, positioning the activity at the receptor level, where DHP downregulates the amount of TGFb receptors on the cell surface and targets them for degradation. This TGFb selective inhibitor was then applied in mESC to probe the effects of inhibiting TGFb specifically at several stages of differentiation. When given early in differentiation DHP blocked all mesoderm, indicating that, in serum differentiated cultures, TGFb is essential for mesoderm induction. Secondly, when DHP was added during mesoderm patterning, cardiogenesis was enhanced specifically, whereas other cardiovascular lineages such as smooth muscle, blood and endothelial cells were not affected. In summary, we have identified a new probe of TGFb signaling that shows that TGFb signaling governs cardiopoietic fate by inhibiting formation of cardiomyocytes. Additionally, our molecule and its mechanism have important implications for congenital heart disease, since deficient TGFb signaling leads to vascular and cardiac defects, and elevated signaling underlies Loeys-Deitz syndrome.
- © 2012 by American Heart Association, Inc.