Gassanov Natig, Er Fikret, Zagidullin Naufal, Jankowski Marek, Gutkowska Jolanta, Hoppe Uta C
Department of Internal Medicine III, University of Cologne, Cologne, Germany.
Differentiation. 2008 Nov;76(9):971-80. doi: 10.1111/j.1432-0436.2008.00283.x. Epub 2008 Jun 17.
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 10(-5), 10(-7), and 10(-9) M RA at early (days 1-5 [d1-5]) and late (d6-10) 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 10(-5) M RA on EB development by inducing marked apoptosis. Morphologic and expression analysis demonstrated that 10(-7) M RA applied at d1-5 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 d1-5 or d6-10 displayed unchanged I(Ca,L) and I(to) channel expression compared with untreated cells. However, patch clamp studies revealed a significant increase of I(Ca,L) and I(to) current densities associated with increased levels of the underlying channel subunits in 6-7-day-old cardiomyocytes upon early RA exposure. In contrast, I(f) 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 I(Ca,L) and I(to) ion channels without affecting differentiation of pacemaker cells.
虽然维甲酸（RA）信号通路与胚胎心脏发育有关，但其在特定心脏亚型分化中的意义仍不清楚。在本研究中，我们利用胚胎干细胞（ES细胞）中利钠肽（ANP）的谱系特异性表达，研究RA对心房和起搏器样表型分化的影响。在早期（第1 - 5天[d1 - 5]）和晚期（第6 - 10天）发育阶段，将胚状体（EB）暴露于10^(-5)、10^(-7)和10^(-9) M的RA中，并检测RA对谱系特异性心脏标志物和离子通道表达的影响。我们的初步实验表明，10^(-5) M的RA通过诱导明显的凋亡对EB发育产生有害影响。形态学和表达分析表明，在d1 - 5时应用10^(-7) M的RA最有效地诱导心房亚谱系。RA不影响起搏器样细胞的分化，与RA浓度和应用时间无关。相反，在发育早期暴露于RA会抑制心室特异性MLC - 2v基因的表达。晚期给予RA在心肌分化方面没有显著变化。与未处理的细胞相比，在d1 - 5或d6 - 10时暴露于RA的终末分化心肌细胞显示I(Ca,L)和I(to)通道表达没有变化。然而，膜片钳研究显示，早期RA暴露后，6 - 7日龄心肌细胞中I(Ca,L)和I(to)电流密度显著增加，同时相关通道亚基水平升高。相比之下，I(f)电流密度和HCN4表达在很大程度上不受RA影响。我们的结果表明，RA以时间和浓度依赖性方式诱导表达ANP的EB向心房表型分化，并加速I(Ca,L)和I(to)离子通道的表达，而不影响起搏器细胞的分化。
