Wobus A M, Kaomei G, Shan J, Wellner M C, Rohwedel J, Fleischmann B, Katus H A, Hescheler J, Franz W M
Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany.
J Mol Cell Cardiol. 1997 Jun;29(6):1525-39. doi: 10.1006/jmcc.1997.0433.
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 patterns of action potentials. It has been shown that retinoic acid (RA) treatment during ES cell differentiation increases the number of cardiomyocytes in a time- and concentration-dependent manner. In order to test the effect of RA on cardiomyocyte differentiation and specialization into ventricle-like cardiomyocytes, we studied gene expression of beta-galactosidase driven by the ventricular myosin light chain-2 (MLC-2v) promoter as an indicator for ventricular differentiation. Clones containing the stably integrated expression vector pGNA/MLC-2.1 were selected, which revealed an increase of beta-galactosidase activity in cardiomyocytes of embryoid bodies at day 7 + 16. RA, both, in the all-trans and in the 9-cis configuration resulted in a significant acceleration of cardiomyocyte differentiation and a transient increase of beta-galactosidase activity. To test whether this acceleration of cardiac differentiation and RA-induced increase of the MLC-2v promotor/beta-galactosidase activity reflects an increase of cardiac- and ventricle-specific 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 MHC and MLC-2v mRNA in embryoid bodies in early, but not in terminal developmental stages. This led us to the conclusion that the RA-induced accelerated expression of cardiac-specific genes results in an enhanced development of ventricular cardiomyocytes. An increased number of ventricle-like cells after RA treatment was also found by patch-clamp analysis. The number of cardiomyocytes with Purkinje- 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.
多能胚胎干细胞通过类似胚胎的聚集体自发分化为起搏器样、心房样和心室样类型的心肌细胞，这些细胞可通过其特定的动作电位模式加以区分。研究表明，在胚胎干细胞分化过程中进行视黄酸（RA）处理，可使心肌细胞数量呈时间和浓度依赖性增加。为了测试RA对心肌细胞分化以及向心室样心肌细胞特化的影响，我们研究了由心室肌球蛋白轻链-2（MLC-2v）启动子驱动的β-半乳糖苷酶的基因表达，以此作为心室分化的指标。筛选出含有稳定整合表达载体pGNA/MLC-2.1的克隆，这些克隆显示在第7 + 16天时，胚状体心肌细胞中β-半乳糖苷酶活性增加。全反式和9-顺式构型的RA均导致心肌细胞分化显著加速，且β-半乳糖苷酶活性短暂增加。为了测试这种心脏分化的加速以及RA诱导的MLC-2v启动子/β-半乳糖苷酶活性增加是否反映了心脏和心室特异性基因表达的增加，我们对α-心肌肌球蛋白重链（α-MHC）和MLC-2v基因进行了半定量逆转录聚合酶链反应（RT-PCR）分析。结果表明，10^(-8) M和10^(-9) M的RA均导致胚状体在早期而非末期发育阶段α-心肌MHC和MLC-2v mRNA水平升高。这使我们得出结论，RA诱导的心脏特异性基因加速表达导致心室心肌细胞发育增强。通过膜片钳分析还发现，RA处理后心室样细胞数量增加。具有浦肯野样和心室样特性的心肌细胞数量显示因RA而增加，而起搏器样和心房样细胞数量减少，早期起搏器细胞数量未受定量影响。
