Westfall M V, Samuelson L C, Metzger J M
Department of Physiology, University of Michigan, Ann Arbor, 48109-0622, USA.
Dev Dyn. 1996 May;206(1):24-38. doi: 10.1002/(SICI)1097-0177(199605)206:1<24::AID-AJA3>3.0.CO;2-2.
We studied troponin I (TnI) isoform expression in the mouse embryonic stem (ES) cell model of cardiogenesis as an essential first step to understanding the relationship between TnI isoform transitions and myofibrillar function. Cultures of differentiating ES cells were grown on coverslips to permit microscopic inspection of foci of spontaneously contracting cardiac myocytes developing in culture. TnI expression was followed over time to test whether the cardiac myocytes undergo the developmental pattern of expression characteristic of vertebrate cardiogenesis, in which slow skeletal TnI (ssTnI) is expressed initially, followed by induction of cardiac (cTnI) isoform expression. Cardiac TnI expression was examined using the cardiac-specific, monoclonal TI-1 antibody (Ab) while all striated muscle ThI isoforms were detected using the monoclonal TI-4 Ab. Cardiac-specific TnI expression was detected in only 8% (8/96) of foci contracting less than 5 days while TI-4 positive staining was present in 95% (71/73) of foci. These results indicate that other striated muscle TnI isoforms were being expressed in most of the TI-4 positive staining foci. The proportion of contracting foci expressing the cardiac isoform increased steadily over time, such that 100% of foci contracting more than 20 days (13/13) stained positive with the TI-1 Ab. Dual labeling experiments with both TI-1 and TI-4 anti-TnI Abs in the same culture confirmed that within each foci, the area expressing cTnI increased with the days of spontaneous contraction. Western blot analysis of micro-dissected ES cell derived cardiac myocytes confirmed that TI-4 immunostaining at early developmental time points represented ssTnI, and not the fast skeletal TnI isoform. We conclude that ES cell-derived cardiac myocytes display the developmental induction of cardiac TnI expression characteristic of vertebrate cardiac development. Thus, this model should be useful for studying the regulation and functional significance of TnI isoform expression during in vitro cardiogenesis.
我们研究了肌钙蛋白I（TnI）同工型在小鼠胚胎干细胞（ES）心脏发生模型中的表达，这是理解TnI同工型转变与肌原纤维功能之间关系的重要第一步。将分化中的ES细胞培养在盖玻片上，以便在显微镜下观察培养物中自发收缩的心肌细胞灶。随着时间推移跟踪TnI表达，以测试心肌细胞是否经历脊椎动物心脏发生特有的表达发育模式，即最初表达慢骨骼肌TnI（ssTnI），随后诱导心脏（cTnI）同工型表达。使用心脏特异性单克隆TI-1抗体（Ab）检测心脏TnI表达，而使用单克隆TI-4 Ab检测所有横纹肌TnI同工型。在收缩少于5天的灶中，仅8%（8/96）检测到心脏特异性TnI表达，而在73个灶中的95%（71/73）存在TI-4阳性染色。这些结果表明，在大多数TI-4阳性染色灶中表达的是其他横纹肌TnI同工型。表达心脏同工型的收缩灶比例随时间稳步增加，以至于收缩超过20天的灶（13/13）中有100%用TI-1 Ab染色呈阳性。在同一培养物中用TI-1和TI-4抗TnI Abs进行双重标记实验证实，在每个灶内，表达cTnI的面积随自发收缩天数增加。对显微解剖的ES细胞来源的心肌细胞进行蛋白质印迹分析证实，在早期发育时间点的TI-4免疫染色代表ssTnI，而不是快骨骼肌TnI同工型。我们得出结论，ES细胞来源的心肌细胞表现出脊椎动物心脏发育特有的心脏TnI表达的发育诱导。因此，该模型对于研究体外心脏发生过程中TnI同工型表达的调控及其功能意义应该是有用的。
