Davidson Mercy M, Nesti Claudia, Palenzuela Lluis, Walker Winsome F, Hernandez Evelyn, Protas Lev, Hirano Michio, Isaac Nithila D
Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
J Mol Cell Cardiol. 2005 Jul;39(1):133-47. doi: 10.1016/j.yjmcc.2005.03.003.
Background. - We have established proliferating human cardiomyocyte cell lines derived from non-proliferating primary cultures of adult ventricular heart tissue, using a novel method that may be applicable to many post-mitotic primary cultures. Methods and results. - Primary cells from human ventricular tissue, were fused with SV40 transformed, uridine auxotroph human fibroblasts, devoid of mitochondrial DNA. This was followed by selection in uridine-free medium to eliminate unfused fibroblasts. The fused cells were subcloned and screened for cell type-specific markers. Four clones (AC1, AC10, AC12, AC16) that express markers characteristic of cardiomyocytes were studied. Clones were homogeneous morphologically, and expressed transcription factors (GATA4, MYCD, NFATc4), contractile proteins such as alpha- and beta-myosin heavy chain, alpha-cardiac actin, troponin I, desmoplakin, alpha actinin, the muscle-specific intermediate filament protein, desmin, the cardiomyocyte-specific peptide hormones, BNP, the L-type calcium channel alpha1C subunit and gap junction proteins, connexin-43 and connexin-40. Furthermore, dye-coupling studies confirmed the presence of functional gap junctions. EM ultra structural analysis revealed the presence of myofibrils in the subsarcolemmal region, indicating a precontractile developmental stage. When grown in mitogen-depleted medium, the AC cells stopped proliferating and formed a multinucleated syncytium. When the SV40 oncogene was silenced using the RNAi technique, AC16 cells switched from a proliferating to a more differentiated quiescent state, with the formation of multinucleated syncyntium. Concurrently, the cells expressed BMP2, an important signaling molecule for induction of cardiac-specific markers, that was not expressed by the proliferating cells. The presence of the combination of transcription factors in addition to muscle-specific markers is a good indication for the presence of a cardiac transcription program in these cells. CONCLUSIONS. - Based on the expression of myogenic markers and a fully functional respiratory chain, the AC cells have retained the nuclear DNA and the mitochondrial DNA of the primary cardiomyocytes. They can be frozen and thawed repeatedly and can differentiate when grown in mitogen-free medium. These cell lines are potentially useful in vitro models to study developmental regulation of cardiomyocytes in normal and pathological states.
背景。——我们利用一种可能适用于许多有丝分裂后原代培养物的新方法，从成人心室心脏组织的非增殖原代培养物中建立了增殖性人类心肌细胞系。
方法与结果。——将人类心室组织的原代细胞与经SV40转化的、缺乏线粒体DNA的尿苷营养缺陷型人类成纤维细胞融合。随后在无尿苷培养基中进行筛选，以去除未融合的成纤维细胞。对融合细胞进行亚克隆，并筛选细胞类型特异性标记物。研究了四个表达心肌细胞特征性标记物的克隆（AC1、AC10、AC12、AC16）。这些克隆在形态上是均匀一致的，表达转录因子（GATA4、MYCD、NFATc4）、收缩蛋白，如α和β肌球蛋白重链、α心肌肌动蛋白、肌钙蛋白I、桥粒斑蛋白、α辅肌动蛋白、肌肉特异性中间丝蛋白结蛋白、心肌细胞特异性肽激素脑钠肽、L型钙通道α1C亚基以及缝隙连接蛋白连接蛋白43和连接蛋白40。此外，染料偶联研究证实了功能性缝隙连接的存在。电子显微镜超微结构分析显示在肌膜下区域存在肌原纤维，表明处于收缩前的发育阶段。当在无有丝分裂原的培养基中生长时，AC细胞停止增殖并形成多核合胞体。当使用RNAi技术使SV40癌基因沉默时，AC16细胞从增殖状态转变为更分化的静止状态，形成多核合胞体。同时，这些细胞表达骨形态发生蛋白2（BMP2），这是一种诱导心脏特异性标记物的重要信号分子，增殖细胞不表达该分子。除肌肉特异性标记物外，转录因子的组合存在很好地表明这些细胞中存在心脏转录程序。
结论。——基于肌源性标记物的表达和完全功能性的呼吸链，AC细胞保留了原代心肌细胞的核DNA和线粒体DNA。它们可以反复冻融，在无有丝分裂原的培养基中生长时可以分化。这些细胞系是研究正常和病理状态下心肌细胞发育调控的潜在有用体外模型。
