Harding S E, Vescovo G, Kirby M, Jones S M, Gurden J, Poole-Wilson P A
Cardiothoracic Institute, London, UK.
J Mol Cell Cardiol. 1988 Jul;20(7):635-47. doi: 10.1016/s0022-2828(88)80121-4.
Myocytes were isolated by Langendorff perfusion of rat or rabbit hearts with low calcium solution followed by collagenase and hyaluronidase, or by incubation of chunks of rat ventricular tissue in similar media. Cells were then placed in a bath on a microscope stage, superfused and electrically stimulated. Contraction amplitude and rate of change of length during contraction were measured using a video camera and edge detection monitor. Cells were selected for study using a number of criteria developed to identify and define a cell population able to give consistent inotropic responses over a long period. The maximum contraction amplitude with isoproterenol in rabbit cells was 0.244 micron (sarcomere length change) or 13.1% (percentage change in cell length), and the EC50 was 12.8 nM. The maximum contraction amplitude with isoproterenol did not differ significantly between rat and rabbit, between cells prepared by perfusion and those made from chunks, or when determined from non-cumulative rather than cumulative curves. The EC50 for isoproterenol in rat cells made by the perfusion method (cumulative curves) was 3.81 nM, significantly lower than in rabbit. The maximum amplitude obtained with increasing concentrations of calcium was not significantly different from that with isoproterenol under any condition. The EC50 for calcium averaged 2.78 mM in rat cells made by the perfusion method (cumulative curves) and was significantly greater than that in rabbit (1.4 mM). Maximum rates of contraction for rat cells averaged 4.59 micron/s in 8 mM calcium. Rat cells contracted faster than they relaxed, whereas rabbit cells in 8 mM calcium relaxed faster than they contracted. Rat cells, maximally activated by either calcium or isoproterenol, contracted significantly faster than rabbit. There was no difference in rates of contraction (or relaxation) between rat cells prepared by perfusion and those made from chunks of tissue.
通过用低钙溶液对大鼠或兔心脏进行Langendorff灌注，随后使用胶原酶和透明质酸酶，或者通过将大鼠心室组织块在类似培养基中孵育来分离心肌细胞。然后将细胞置于显微镜载物台上的浴槽中，进行灌流并给予电刺激。使用摄像机和边缘检测监测器测量收缩幅度和收缩期间长度的变化率。使用为识别和定义能够长期给出一致变力反应的细胞群体而制定的一些标准选择细胞进行研究。兔细胞中异丙肾上腺素的最大收缩幅度为0.244微米（肌节长度变化）或13.1%（细胞长度变化百分比），EC50为12.8 nM。大鼠和兔之间、通过灌注制备的细胞与由组织块制备的细胞之间，或者从非累积曲线而非累积曲线确定时，异丙肾上腺素的最大收缩幅度没有显著差异。通过灌注法制备的大鼠细胞（累积曲线）中异丙肾上腺素的EC50为3.81 nM，显著低于兔细胞。在任何条件下，随着钙浓度增加获得的最大幅度与异丙肾上腺素的最大幅度没有显著差异。通过灌注法制备的大鼠细胞（累积曲线）中钙的EC50平均为2.78 mM，显著高于兔细胞（1.4 mM）。在8 mM钙中，大鼠细胞的最大收缩速率平均为4.59微米/秒。大鼠细胞收缩比舒张快，而在8 mM钙中的兔细胞舒张比收缩快。被钙或异丙肾上腺素最大激活的大鼠细胞收缩明显比兔细胞快。通过灌注制备的大鼠细胞与由组织块制备的大鼠细胞之间的收缩（或舒张）速率没有差异。
