Horackova M, Byczko Z
Department of Physiology and Biophysics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada.
Exp Cell Res. 1997 Nov 25;237(1):158-75. doi: 10.1006/excr.1997.3775.
In this study, we used laser confocal scanning microscopy and immunofluorescent markers to describe the establishment of long-term (1-5 week) cultures of adult guinea pig cardiomyocytes and adult rat cardiomyocytes. Providing that the preparation of freshly isolated guinea pig cardiomyocytes consists mostly (> 80%) of rod-shaped, Ca(2+)-tolerant, and quiescent cells and these are plated under optimal conditions and density (10(5) cells/cm2), these myocytes have the following characteristics: (1) they remain elongated with regular ultrastructural characteristics and quiescent for 1 week; (2) within 10-14 days, they reestablish intercellular contacts and resume contractile activity, which becomes synchronous all through the confluent layer; (3) their myofibrillar striations remain regular all through the adaptation to culture conditions without any sign of dedifferentiation or redifferentiation; (4) they form adherence junctions (as indicated by their immunoreactivity to an antibody against N-cadherin) over the entire cellular surface; (5) they appear to retain their ability to express atrial natriuretic peptide (ANP), as indicated by immunoreactivity to anti-ANP antibody; (6) this activity seems to be directly related to the surface area of the myocytes in contact with the substrate. In contrast, rat cardiomyocytes cultured under very similar and optimal conditions exhibit very different characteristics during their adaptation in long-term cultures: (1) although 85-90% of freshly isolated cells are also rod-shaped and Ca2+ tolerant they exhibit slow spontaneous contractions; (2) they round up during the first few days, and during the first week they dedifferentiate, losing their regular striated appearance; (3) they spread, becoming irregularly shaped, and, unlike the guinea pig cardiomyocytes, they do not form confluent layers, no matter what the plating density is; (4) they atrophy at a very early stage in the cultures, so that by the fourth week, they have lost most of their myofibrils; (5) the initial rounding up is largely eliminated by exposure for 24 h to 1 microM ryanodine or 20 mM butanedione monoxime, compounds that suppress the spontaneous contractions. In conclusion, our studies demonstrate that adult guinea pig cardiomyocytes adapt and survive in long-term (1-5 week) cultures much better than do adult rat cardiomyocytes, indicating that the long-term cultures of adult guinea pig ventricular myocytes provide a valuable experimental model which opens new possibilities for studying the cellular and molecular regulation of myocardial function under the acute or chronic influence of various intrinsic and/or extrinsic factors.
在本研究中，我们使用激光共聚焦扫描显微镜和免疫荧光标记物来描述成年豚鼠心肌细胞和成年大鼠心肌细胞长期（1 - 5周）培养物的建立。倘若新鲜分离的豚鼠心肌细胞制备物大多（>80%）由杆状、耐钙且静止的细胞组成，并且将这些细胞在最佳条件和密度（10⁵个细胞/cm²）下接种，这些心肌细胞具有以下特征：（1）它们保持细长形态，具有规则的超微结构特征，并且静止1周；（2）在10 - 14天内，它们重新建立细胞间连接并恢复收缩活性，在汇合层中收缩活性变得同步；（3）在适应培养条件的整个过程中，它们的肌原纤维条纹保持规则，没有任何去分化或再分化的迹象；（4）它们在整个细胞表面形成黏附连接（通过它们对N - 钙黏蛋白抗体的免疫反应性表明）；（5）它们似乎保留了表达心房钠尿肽（ANP）的能力，通过对抗ANP抗体的免疫反应性表明；（6）这种活性似乎与与底物接触的心肌细胞表面积直接相关。相比之下，在非常相似的最佳条件下培养的大鼠心肌细胞在长期培养适应过程中表现出非常不同的特征：（1）尽管85 - 90%的新鲜分离细胞也是杆状且耐钙的，但它们表现出缓慢的自发收缩；（2）在最初几天它们变圆，并且在第一周它们去分化，失去其规则的条纹外观；（3）它们铺展，变得形状不规则，并且与豚鼠心肌细胞不同，无论接种密度如何，它们都不会形成汇合层；（4）它们在培养的早期阶段就萎缩，以至于到第四周时，它们已经失去了大部分肌原纤维；（5）通过暴露于1 μM 兰尼碱或20 mM丁二酮单肟24小时，可很大程度上消除最初的变圆现象，这两种化合物可抑制自发收缩。总之，我们的研究表明成年豚鼠心肌细胞在长期（1 - 5周）培养中比成年大鼠心肌细胞适应和存活得更好，这表明成年豚鼠心室肌细胞的长期培养提供了一个有价值的实验模型，为研究各种内在和/或外在因素的急性或慢性影响下心肌功能的细胞和分子调节开辟了新的可能性。
