Abstract 15633: Canonical Wnt Signaling Promotes Pacemaker Cell Specification of Cardiac Mesodermal Cells
Cardiac differentiation of embryonic stem cells (ESCs) gives rise to both chamber (atrial/ventricular) cardiomyocytes and pacemaker cells. Recent findings point to canonical Wnt signaling as a mediator of cardiac sinoatrial node development in chick embryos. We sought to test if early regulation of this pathway may influence cardiac progenitors to bifurcate to either chamber cardiomyocytes or pacemaker cells. The prevailing model of cardiac differentiation of ESCs relies on maintaining a saturating level of Dkk1, added exogenously to inhibit canonical Wnt signaling. We hypothesized that canonical Wnt signaling may lead the progenitor cells to commit to cardiac pacemaker cell fate while its suppression leads differentiation to chamber cardiomyocytes. To this end, Flk-1+/PdgfR-α+ mesodermal progenitor cells were derived from mouse ESCs by treatment with activin-A and BMP-4. Then, the progenitors were subjected to either cardiac differentiation medium (CDM) containing 150 ng/mL of recombinant Dkk1 or the same medium without the exogenous Wnt inhibitor (Wnt-intact medium, WM). At week 3, spontaneous beating rates were significantly higher in freshly-isolated single cardiomyocytes that had been derived in WM compared to those in CDM (248±20 vs. 122±16 bpm, n>21, p5-fold higher transcript levels of pacemaker cell-specific genes (Hcn4, Tbx18, Shox2), and exhibited 3-fold higher number of Hcn4+/Tbx3+ cells compared to those in CDM. Spontaneously-beating cells in WM fired rhythmic action potentials with steeper phase-4 depolarization (84±9 vs. 37±10 mV/s, n>21, p9, p<0.01) compared to those grown in CDM. Treating the WM progenitors with a small molecule inhibitor of Wnt/β-catenin pathway, IWR-1, suppressed pacemaker lineage (Tbx18 and Shox2) gene expression. Conversely, treatment with Wnt3a, an activator of the Wnt/β-catenin pathway, heightened Tbx18/Shox2 expression. Taken together, our data identify the Wnt/β-catenin pathway as a critical determinant of cardiac myocyte subtype commitment during ESC differentiation: intact Wnt signaling favors the pacemaker lineage, while its suppression favors the chamber cardiomyocyte lineage.
Author Disclosures: W. Liang: None. E.H. Kim: None. J. Mak: None. R. Zhang: None. A.G. Torrente: None. J.I. Goldhaber: None. E. Marbán: None. H. Cho: None.
- © 2014 by American Heart Association, Inc.