Canonical WNT Pathway Enriches Cardiac Pacemaker Cell Population During Embryonic Stem Cell Differentiation

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Background: Embryonic stem cells (ESCs) can be guided to differentiate into cardiomyocytes by blocking canonical Wnt pathway (e.g., with Dkk-1) during cardiac specification stage. We hypothesized that canonical Wnt signaling may be an important negotiator during cardiac progenitors' commitment towards pacemaker or atrial/ventricular lineages. Methods: Mouse ESCs were treated with activin-A/BMP-4 for 40 hours in a defined medium to initiate cardiac differentiation. Flk-1+/PdgfR-α+ cardiac progenitors are FACS-purified and seeded as monolayers with Dkk-1 (day-0). Results: At day-4, ∼65% of cells are positive for cTnT, a pan-cardiomyocyte marker. Some cTnT-positive cells express one or more of pacemaker-lineage markers, Shox2/Tbx18/Tbx3/Hcn4. Spontaneously-beating areas are observed starting day-2, and some single cells exhibit spontaneous, rhythmic action potentials with hallmark pacemaker electrophysiology such as phase-4 depolarization and depolarized maximal diastolic potential. Still, the monolayers beat in syncytium, resembling the passive contractions of atrial/ventricular myocardium. Removal of Dkk-1 significantly increases pacemaker gene transcript levels, Tbx18 and Shox2 by 5-fold (p<0.05, n=4), Hcn1 and Hcn4 by 2-fold (p<0.05, n=4) compared to the cells cultured with Dkk-1. Conversely, ventricular/atrial lineage markers, Nkx2.5 and Scn5a were suppressed by 4- and 8-fold, respectively, compared to control (p<0.05, n=4). In contrast to the syncytial contractions of the monolayers cultured with Dkk-1, intact canonical Wnt signaling (no Dkk-1) induces formation of discrete, node-like structures which beat autonomously. The beating rates of cells cultured without Dkk-1 are ∼3x faster than that of cells cultured with Dkk-1 (161.5±11.5 vs. 48.0±2.9 bpm, p<0.01, n=4) at week-2. Single spontaneously-beating cells isolated from no-Dkk-1 group are frequently spindle-shaped replicating the morphology of genuine sinoatrial node pacemaker cells. Conclusions: Endogenous, canonical Wnt pathway promotes differentiation of mouse cardiac progenitor cells into pacemaker cells rather than to normally-quiescent cardiomyocytes.