Temperature and transmural region influence functional measurements in unloaded left ventricular cardiomyocytes.
Temperature and transmural region influence functional measurements in unloaded left ventricular cardiomyocytes.
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DOI：
10.1002/phy2.158
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发表时间：
2013-11
影响因子：
2.5
通讯作者：
Campbell, Kenneth S
中科院分区：
文献类型：
--
作者：
Chung, Charles S;Campbell, Kenneth S
完整的心肌细胞越来越多地被用于研究收缩的分子机制和筛选新的治疗化合物。心肌细胞的功能通常通过钙瞬变和肌节长度分布来衡量。我们研究了实验温度和跨壁区域对新鲜分离的、卸载的心肌细胞功能指标的影响。从3月龄SD大鼠的心内膜下、中层心肌和心外膜下分离出完整的心肌细胞。在25°C、31°C和37°C下对每个区域的肌细胞进行研究，用Fura-2荧光法测量胞浆钙瞬变，而用高速视频捕获测量肌节长度缩短和重新加长的轮廓。随着温度的升高，钙瞬变时间和肌节长度曲线的峰值时间和半松弛时间均显著缩短。升高温度也提高了所有细胞的最低和最高钙水平。值得注意的是，在较高的温度下，钙荧光幅度、达到钙峰值的时间以及肌节缩短和再加长的速度的变异系数(标准差除以平均值)降低。钙瞬变的幅度和最小值明显依赖于跨壁区域，一些肌节长度参数显示出温度和跨壁区域之间的统计交互作用。总之，这些结果表明，通过在37℃而不是室温下进行实验，以及从特定的跨壁区域分离细胞，可以减少生物变异性。采用这些程序将提高后续分析的统计能力，并提高未来实验的效率。
Intact cardiomyocytes are increasingly being used to investigate the molecular mechanisms of contraction and to screen new therapeutic compounds. The function of the cardiomyocytes is often measured from the calcium transients and sarcomere length profiles. We studied the role of experimental temperature and transmural region on indices of function in freshly isolated, unloaded cardiomyocytes. Intact cardiomyocytes were isolated from the subendocardium, midmyocardium, and subepicardium of 3-month-old Sprague-Dawley rats. Myocytes from each region were studied at 25°C, 31°C, and 37°C. Cytosolic calcium transients were measured using Fura-2 fluorescence, whereas sarcomere length shortening and relengthening profiles were measured using high-speed video capture. For both the calcium transients and sarcomere length profiles, the time to peak and the time to half relaxation decreased significantly with increasing temperature. Increasing temperature also raised the minimum and maximum calcium levels of all cells. Of note, there was a reduced coefficient of variation (standard deviation divided by the mean) at higher temperatures for calcium fluorescence amplitudes, time to peak calcium, and rates of sarcomeric shortening and relengthening. The amplitudes and minimum of the calcium transients were significantly dependent on transmural region, and several sarcomere length parameters exhibited statistical interactions between temperature and transmural region. Together, these results show that biological variability can be reduced by performing experiments at 37°C rather than at room temperature, and by isolating cells from a specific transmural region. Adopting these procedures will improve the statistical power of subsequent analyses and increase the efficiency of future experiments.