Sarcomeric Ca2+ gradients during activation of frog skeletal muscle fibres imaged with confocal and two-photon microscopy
Sarcomeric Ca2+ gradients during activation of frog skeletal muscle fibres imaged with confocal and two-photon microscopy
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DOI：
10.1111/j.1469-7793.2000.t01-1-00551.x
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发表时间：
2000-08-01
影响因子：
5.5
通讯作者：
Cannell, MB
中科院分区：
文献类型：
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作者：
Hollingworth, S;Soeller, C;Cannell, MB
1. 用Ca2+指示剂fluo-3和共聚焦和双光子显微镜研究了动作电位刺激蛙单纤维激活过程中[Ca2+]的肌内梯度。这些实验的目的是寻找关节外Ca2+释放的证据，并检查Ca2+在肌角内的微观扩散。通过利用纤维内肌瘤的空间周期性，我们可以实现高的有效线扫描率（类似于8000线s（-1)），尽管激光扫描显微镜被限制在< 1000线s(-1)）在这种高时间分辨率下，荧光变化的时间过程在z线和m线上是非常不同的，在z线和m线荧光的上升之间有明显的延迟（类似于1 ms； 22℃）。为了计算预期的荧光变化，我们使用了半肌节中Ca2+运动的多室室模型，其中Ca2+释放仅限于三联结（位于z线）。模拟了显微镜下的光学模糊现象，产生的荧光信号可以直接与实验数据进行比较。该模型最准确地再现了我们的实验结果，包括Ca2+与ATP的结合，以及指示剂与固定化肌合成蛋白的结合。在考虑了纤维内的肌体错配后，模型与实验结果吻合得非常好。我们得出结论，没有实验证据表明Ca2+释放在其他位置的z线。此外，我们的计算支持这样的结论，即快速扩散的Ca2+缓冲液（如ATP）对形成Ca2+瞬态很重要，并且需要考虑细胞内指示剂结合的细节，以正确解释纤维中荧光变化的时间过程。
1. Intra-sarcomeric gradients of [Ca2+] during activation of action potential stimulated frog single fibres were investigated with the Ca2+ indicator fluo-3 and confocal and two-photon microscopy. The object of those experiments tvas to look for evidence of extra-junctional Ca2+ release and examine the microscopic diffusion of Ca2+ within the sarcomere.2. By exploiting the spatial periodicity of sarcomeres within the fibre, we could achieve a high effective line-scanning rate (similar to 8000 lines s(-1)), although the laser scanning microscope was limited to < 1000 lines s(-1) At this high time resolution, the time course of fluorescence changes was very different at the z- and m-lines, with a significant delay (similar to 1 ms; 22 degrees C) between the rise of fluorescence at the z-line and the m-line.3. To calculate the expected fluorescence changes, we used a multi-compartment model of Ca2+ movements in the half-sarcomere in which Ca2+ release was restricted to triadic junctions (located at z-lines). Optical blurring by the microscope was simulated to generate fluorescence signals which could be compared directly to experimental data. The model which reproduced our experimental findings most accurately included Ca2+ binding by ATP, as well as indicator binding to immobile sarcomeric proteins. After taking sarcomeric misregistration within the fibre into account, there was very good agreement between the model and experimental results.4. We conclude that there is no experimental evidence fur Ca2+ release at locations other than at z-lines. In addition, our calculations support the conclusion that rapidly diffusing Ca2+ buffers (such as ATP) are important in shaping the Ca2+ transient and that the details of intracellular indicator binding need to be considered to explain correctly the time course of fluorescence change in the fibre.