Weisser-Thomas J, Dieterich E, Janssen P M L, Schmidt-Schweda S, Maier L S, Sumbilla C, Pieske B
Georg-August-Universität Göttingen, Abteilung Kardiologie und Pneumologie, Zentrum Innere Medizin, Robert-Koch-Str. 40, 37075 Göttingen, Germany.
J Pharmacol Toxicol Methods. 2005 Mar-Apr;51(2):91-103. doi: 10.1016/j.vascn.2004.10.005. Epub 2004 Dec 10.
Adenovirus-mediated gene transfer into cardiomyocytes has emerged as an interesting tool to study functional effects of single proteins. However, the functional consequences of cell isolation, cell culture per se and adenovirus-mediated transfer of the LacZ or SERCA1 gene in failing human cardiomyocytes warrant further investigation.
Primary cell culture was performed without or after adenovirus-mediated gene transfer of LacZ or SERCA1. Functional behavior of myocytes was assessed under basal conditions (field stimulation, 0.5 Hz, 37 degrees C), and during inotropic stimulation with isoproterenol (ISO; 10(-9)-10(-5) M), Ca(2+) (1.5-15 mM) or increasing stimulation rates (0.25-2.5 Hz). Results were compared to trabeculae from the same hearts.
Freshly isolated myocytes showed full inotropic competence as compared to multicellular preparations. The response to stimulation with ISO and Ca(2+), as well as changes in stimulation rate resulted in a maximal increase in fractional cell shortening (FS) to 215+/-24% and 291+/-34%, and a frequency-dependent decline in FS to 46+/-5% of the basal value, respectively. After 48 h of cell culture, basal FS did not change significantly compared to fresh cells but both time to peak shortening and time to 50% relengthening were prolonged. After culture, the concentration-response curve for ISO was significantly shifted to the left (EC(50) 5.16 x 10(-8) vs. 1.12 x 10(-8) M, p<0.05). LacZ gene transfer caused efficient beta-Gal expression without affecting the inotropic responses to ISO or stimulation rate but impaired the contractile amplitude. SERCA1 gene transfer increased FS by 68% vs. LacZ and accelerated relengthening kinetics (+dL/dt 93+/-13 vs. 61+/-8 mum/s, p<0.05 vs. LacZ).
Contractile responses of isolated human myocytes are comparable to multicellular preparations. The use of primary cell culture and adenovirus infection with CMV-promoter-mediated LacZ expression per se modulates contractile behavior in failing human myocytes. SERCA1 expression markedly improves contractile function. The method-related changes in contractile behavior observed here need to be taken into account in further studies.
腺病毒介导的基因转移至心肌细胞已成为研究单一蛋白质功能效应的一种有趣工具。然而，细胞分离、细胞培养本身以及腺病毒介导的LacZ或SERCA1基因转移在衰竭的人类心肌细胞中的功能后果仍需进一步研究。
在腺病毒介导的LacZ或SERCA1基因转移之前或之后进行原代细胞培养。在基础条件下（场刺激，0.5Hz，37℃）以及用异丙肾上腺素（ISO；10(-9)-10(-5)M）、Ca(2+)（1.5-15mM）进行变力刺激或增加刺激频率（0.25-2.5Hz）期间评估心肌细胞的功能行为。将结果与来自同一心脏的小梁进行比较。
与多细胞制剂相比，新鲜分离的心肌细胞显示出完全的变力能力。对ISO和Ca(2+)刺激的反应以及刺激频率的变化导致细胞缩短分数（FS）分别最大增加至215±24%和291±34%，以及FS随频率依赖性下降至基础值的46±5%。细胞培养48小时后，与新鲜细胞相比，基础FS没有显著变化，但缩短至峰值的时间和恢复至50%的时间均延长。培养后，ISO的浓度-反应曲线显著向左移动（EC(50) 5.16×10(-8) 对 1.12×10(-8)M，p<0.05）。LacZ基因转移导致有效的β-半乳糖苷酶表达，而不影响对ISO或刺激频率的变力反应，但损害了收缩幅度。与LacZ相比，SERCA1基因转移使FS增加了68%，并加速了恢复动力学（+dL/dt 93±13对61±8μm/s，与LacZ相比p<0.05）。
分离的人类心肌细胞的收缩反应与多细胞制剂相当。原代细胞培养和腺病毒感染以及CMV启动子介导的LacZ表达本身会调节衰竭人类心肌细胞的收缩行为。SERCA1表达显著改善收缩功能。在进一步研究中需要考虑此处观察到的与方法相关的收缩行为变化。
