RETINOIC ACID ACCELERATES EMBRYONIC STEM CELL-DERIVED CARDIAC DIFFERENTIATION AND ENHANCES DEVELOPMENT OF VENTRICULAR CARDIOMYOCYTES

Pluripotent embryonic stem (ES) cells spontaneously differentiate via embryo-like aggregates into cardiomyocytes of pacemaker-, atrium- and ventricle-like type, which can be distinguished by their specific patt erns of action potentials, It has been shown that retinoic acid (RA) t reatment during ES cell differentiation increases the number of cardio myocytes in a time- and concentration-dependent manner. In order to te st the effect of RA on cardiomyocyte differentiation and specializatio n into ventricle-like cardiomyocytes, we studied gene expression of be ta-galactosidase driven by the ventricular myosin light chain-2 (MLC-2 v) promoter as an indicator for ventricular differentiation. Clones co ntaining the stably integrated expression vector pGNA/MLC-2.1 were sel ected, which revealed an increase of beta-galactosidase activity in ca rdiomyocytes of embryoid bodies at day 7+16, RA, both, in the all-tran s and in the 9-cis configuration resulted in a significant acceleratio n of cardiomyocyte differentiation and a transient increase of beta-ga lactosidase activity. To test whether this acceleration of cardiac dif ferentiation and RA-induced increase of the MLC-2v promotor/beta-galac tosidase activity reflects an increase of cardiac- and ventricle-speci fic gene expression, a semi-quantitative RT-PCR analysis was performed for alpha-cardiac myosin heavy chain (alpha-MHC) and MLC-2v genes, It was shown that both 10(-8) M and 10(-9) M RA resulted in an increased level of alpha-cardiac MMC and MLC-2v mRNA in embryoid bodies in earl y, but not in terminal developmental stages, This led us to the conclu sion that the RA-induced accelerated expression of cardiac-specific ge nes results in an enhanced development of ventricular cardiomyocytes. An increased number of ventricle-like cells after RA treatment was als o found by patch-clamp analysis, The number of cardiomyocytes with Pur kinje- and ventricle-like properties was shown to be increased by RA, whereas the number of pacemaker- and atrium-like cells was reduced and early pacemaker cells were not quantitatively affected. (C) 1997 Acad emic Press Limited.