Two-photon molecular excitation imaging of Ca2+ transients in Langendorff-perfused mouse hearts
Two-photon molecular excitation imaging of Ca2+ transients in Langendorff-perfused mouse hearts
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DOI：
10.1152/ajpcell.00469.2002
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发表时间：
2003-06-01
影响因子：
5.5
通讯作者：
Field, LJ
中科院分区：
文献类型：
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作者：
Rubart, M;Wang, EX;Field, LJ
在完整的去极化心脏内对亚细胞水平的钙信号进行成像的能力可以为更全面地了解心脏功能提供有价值的信息。因此，开发了一种将双光子激发与激光扫描显微镜相结合的系统，用于监测非收缩 Langendorff 灌注小鼠心脏内单个心肌细胞中电诱发的 [Ca2+](i) 瞬变。在存在兴奋收缩解偶联剂细胞松弛素 D 的情况下，使用荧光指示剂 rhod-2 或 fura-2 在距离心外膜表面小于或等于 100 μm 的深度处记录 [Ca2+](i) 瞬变。诱发的 [Ca2+](i) 瞬变在相邻心肌细胞之间高度同步。在 1 Hz 时，峰值 [Ca2+](i) 从 90 至 50% (t(90-50%)) 和从 50 至 10% (t(50-10%)) 的时间分别为（平均值 +/- SE）73 +/- 4 和 126 +/- 10 ms，而在 2 Hz 时，为 62 +/- 3 和 94 +/- 6 ms（n = 19，负载 rhod-2 的心肌细胞中，P < 0.05 vs. 1 Hz）。负载 fura-2 的心脏中 [Ca2+](i) 衰减明显较慢 (t(90-50%) 在 1 Hz、128 +/- 9 ms 和 2 Hz、88 +/- 5 ms 时；t(50-10%) 在 1 Hz、214 +/- 18 ms 和 2 Hz、163 +/- 7 ms 时；n = 19，P < 0.05罗德-2）。 Fura-2 诱导的 [Ca2+](i) 下降减速是由于胞质 Ca2+ 缓冲增加所致，因为 rhod-2 衰变动力学类似于掺入 Ca2+ 螯合剂 BAPTA 后使用 fura-2 获得的动力学。在起搏的心脏中很容易检测到传播的钙波和 [Ca2+](i) 振幅交替。这种方法应该具有普遍的实用性，可以监测单光子成像无法到达的整个小鼠心脏内的细胞钙信号传导的遗传和/或功能异质性的后果。
The ability to image calcium signals at subcellular levels within the intact depolarizing heart could provide valuable information toward a more integrated understanding of cardiac function. Accordingly, a system combining two-photon excitation with laser-scanning microscopy was developed to monitor electrically evoked [Ca2+](i) transients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [Ca2+](i) transients were recorded at depths less than or equal to100 mum from the epicardial surface with the fluorescent indicators rhod-2 or fura-2 in the presence of the excitation-contraction uncoupler cytochalasin D. Evoked [Ca2+](i) transients were highly synchronized among neighboring cardiomyocytes. At 1 Hz, the times from 90 to 50% (t(90-50%)) and from 50 to 10% (t(50-10%)) of the peak [Ca2+](i) were (means +/- SE) 73 +/- 4 and 126 +/- 10 ms, respectively, and at 2 Hz, 62 +/- 3 and 94 +/- 6 ms (n = 19, P < 0.05 vs. 1 Hz) in rhod-2-loaded cardiomyocytes. [Ca2+](i) decay was markedly slower in fura-2-loaded hearts (t(90-50%) at 1 Hz, 128 +/- 9 ms and at 2 Hz, 88 +/- 5 ms; t(50-10%) at 1 Hz, 214 +/- 18 ms and at 2 Hz, 163 +/- 7 ms; n = 19, P < 0.05 vs. rhod-2). Fura-2-induced deceleration of [Ca2+](i) decline resulted from increased cytosolic Ca2+ buffering, because the kinetics of rhod-2 decay resembled those obtained with fura-2 after incorporation of the Ca2+ chelator BAPTA. Propagating calcium waves and [Ca2+](i) amplitude alternans were readily detected in paced hearts. This approach should be of general utility to monitor the consequences of genetic and/or functional heterogeneity in cellular calcium signaling within whole mouse hearts at tissue depths that have been inaccessible to single-photon imaging.