Metzger J M, Lin W I, Samuelson L C
Department of Physiology, School of Medicine, University of Michigan, Ann Arbor 48109-0622.
J Cell Biol. 1994 Aug;126(3):701-11. doi: 10.1083/jcb.126.3.701.
Mouse embryonic stem (ES) cells differentiate in vitro into a variety of cell types including spontaneously contracting cardiac myocytes. We have utilized the ES cell differentiation culture system to study the development of the cardiac contractile apparatus in vitro. Difficulties associated with the cellular and developmental heterogeneity of this system have been overcome by establishing attached cultures of differentiating ES cells, and by the micro-dissection of the contracting cardiac myocytes from culture. The time of onset and duration of continuous contractile activity of the individual contracting myocytes was determined by daily visual inspection of the cultures. A functional assay was used to directly measure force production in ES cell-derived cardiac myocyte preparations. The forces produced during spontaneous contractions in the membrane intact preparation, and during activation by Ca2+ subsequent to chemical permeabilization of the surface membranes were determined in the same preparation. Results showed a transition in contractile sensitivity to Ca2+ in ES cell-derived cardiac myocytes during development in vitro. Cardiac preparations isolated from culture following the initiation of spontaneous contractile activity showed marked sensitivity of the contractile apparatus to activation by Ca2+. However, the Ca2+ sensitivity of tension development was significantly decreased in preparations isolated from culture following prolonged continuous contractile activity in vitro. The alteration in Ca2+ sensitivity obtained in vitro paralleled that observed during murine cardiac myocyte development in vivo. This provides functional evidence that ES cell-derived cardiac myocytes recapitulate cardiogenesis in vitro. Alterations in Ca2+ sensitivity could be important in optimizing the cardiac contractile response to variations in the myoplasmic Ca2+ transient during embryogenesis. The potential to stably transfect ES cells with cardiac regulatory genes, together with the availability of a functional assay using control and genetically modified ES cell-derived cardiac myocytes, will permit determination of the functional significance of altered cardiac gene expression during cardiogenesis in vitro.
小鼠胚胎干细胞（ES细胞）在体外可分化为多种细胞类型，包括自发收缩的心肌细胞。我们利用ES细胞分化培养系统在体外研究心脏收缩装置的发育。通过建立分化ES细胞的贴壁培养，并从培养物中显微解剖收缩的心肌细胞，克服了该系统细胞和发育异质性带来的困难。通过每天目视检查培养物来确定单个收缩心肌细胞持续收缩活动的起始时间和持续时间。使用功能测定法直接测量ES细胞来源的心肌细胞制剂中的力产生。在同一制剂中测定完整膜制剂自发收缩期间以及表面膜化学通透后Ca2+激活期间产生的力。结果显示，在体外发育过程中，ES细胞来源的心肌细胞对Ca2+的收缩敏感性发生了转变。自发收缩活动开始后从培养物中分离的心脏制剂显示收缩装置对Ca2+激活具有明显的敏感性。然而，在体外长时间持续收缩活动后从培养物中分离的制剂中，张力发展的Ca2+敏感性显著降低。体外获得的Ca2+敏感性变化与在小鼠体内心肌细胞发育过程中观察到的变化相似。这提供了功能证据，表明ES细胞来源的心肌细胞在体外重现了心脏发生过程。Ca2+敏感性的改变可能对优化胚胎发育过程中肌质Ca2+瞬变变化时的心脏收缩反应很重要。用心脏调节基因稳定转染ES细胞的潜力，以及使用对照和基因修饰的ES细胞来源的心肌细胞进行功能测定的可用性，将有助于确定体外心脏发生过程中改变的心脏基因表达的功能意义。
