Abstract Whereas retinoid acid (RA) signaling has been implicated in embryonic heart development, its significance in differentiation of specific cardiac subtypes remains largely unknown. In the present study, we took advantage of lineage-specific expression of atrial natriuretic peptide (ANP) in embryonic stem (ES) cells to study RA-induced effects on differentiation of atrial- and pacemaker-like phenotypes. Embryoid bodies (EB) were exposed to 1022125, 1022127, and 1022129†M RA at early (days120135 [d120135]) and late (d6201310) developmental stages, and RA effects on expression of lineage-specific cardiac markers and ion channels were examined. Our initial experiments revealed a detrimental effect of 1022125†M RA on EB development by inducing marked apoptosis. Morphologic and expression analysis demonstrated that 1022127†M RA applied at d120135 was most effective to induce the atrial sublineage. RA did not affect differentiation of pacemaker-like cells, independent of RA concentration and application time. Conversely, RA exposure at an early developmental stage inhibited ventricular-specific MLC-2v gene expression. Late-stage RA administration exhibited no significant alterations in cardiomyogenic differentiation. Terminally differentiated cardiomyocytes exposed to RA at d120135 or d6201310 displayed unchanged ICa,L and Ito channel expression compared with untreated cells. However, patch clamp studies revealed a significant increase of ICa,L and Ito current densities associated with increased levels of the underlying channel subunits in 620137-day-old cardiomyocytes upon early RA exposure. In contrast, If current density and HCN4 expression remained largely unaffected by RA. Our results imply that RA induces differentiation of ANP-expressing EBs toward an atrial phenotype in a time- and concentration-dependent manner and accelerates expression of ICa,L and Ito ion channels without affecting differentiation of pacemaker cells.
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