Viero Cedric, Kraushaar Udo, Ruppenthal Sandra, Kaestner Lars, Lipp Peter
Institute for Molecular Cell Biology, Medical Faculty, Saarland University, Building 61, 66421 Homburg/Saar, Germany.
Cell Calcium. 2008 Jan;43(1):59-71. doi: 10.1016/j.ceca.2007.04.001. Epub 2007 Sep 5.
For studying heart pathologies on the cellular level, cultured adult cardiac myocytes represent an important approach. We aimed to explore a novel adult rat ventricular myocyte culture system with minimised dedifferentiation allowing extended experimental manipulation of the cells such as expression of exogenous proteins. Various culture conditions were investigated including medium supplement, substrate coating and electrical pacing for one week. Adult myocytes were probed for (i) viability, (ii) morphology, (iii) frequency dependence of contractions, (iv) Ca(2+) transients, and (v) their tolerance towards adenovirus-mediated expression of the Ca(2+) sensor "inverse pericam". Conventionally, in either serum supplemented or serum-free medium, myocytes dedifferentiated into flat cells within 3 days or cell physiology and morphology were impaired, respectively. In contrast, myocytes cultured in medium supplemented with an insulin-transferrin-selenite mixture on substrates coated with extracellular matrix proteins showed an increased cell attachment and a conserved cross-striation. Moreover, these myocytes displayed optimised preservation of their contractile behaviour and Ca(2+) signalling even under conditions of continuous electrical pacing. Sustained expression of inverse pericam did not alter myocyte function and allowed long lasting high speed Ca(2+) imaging of electrically driven adult myocytes. Our single-cell model thus provides a new advance for high-content screening of these highly specialised cells.
为了在细胞水平上研究心脏病理，培养的成年心肌细胞是一种重要的方法。我们旨在探索一种新型的成年大鼠心室肌细胞培养系统，该系统能将去分化降至最低，从而允许对细胞进行扩展的实验操作，如外源蛋白的表达。我们研究了各种培养条件，包括培养基补充物、底物包被和为期一周的电刺激。对成年心肌细胞进行了以下检测：（i）活力，（ii）形态，（iii）收缩的频率依赖性，（iv）Ca(2+)瞬变，以及（v）它们对腺病毒介导的Ca(2+)传感器“反向 pericam”表达的耐受性。传统上，在补充血清或无血清培养基中，心肌细胞分别在3天内去分化为扁平细胞或细胞生理和形态受到损害。相比之下，在补充有胰岛素-转铁蛋白-亚硒酸盐混合物的培养基中培养于包被有细胞外基质蛋白的底物上的心肌细胞，显示出细胞附着增加和保留的横纹。此外，即使在持续电刺激的条件下，这些心肌细胞也表现出其收缩行为和Ca(2+)信号的优化保存。反向 pericam 的持续表达不会改变心肌细胞功能，并允许对电驱动的成年心肌细胞进行持久的高速Ca(2+)成像。因此，我们的单细胞模型为这些高度特化细胞的高内涵筛选提供了新的进展。
