Sarcomeric Ca2+ gradients during activation of frog skeletal muscle fibresimaged with confocal and two-photon microscopy

1. Intra-sarcomeric gradients of [Ca2+] during activation of action potenti al stimulated frog single fibres were investigated with the Ca2+ indicator fluo-3 and confocal and two-photon microscopy. The object of those experime nts 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-compartm ent 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 b e compared directly to experimental data. The model which reproduced our ex perimental findings most accurately included Ca2+ binding by ATP, as well a s indicator binding to immobile sarcomeric proteins. After taking sarcomeri c misregistration within the fibre into account, there was very good agreem ent between the model and experimental results. 4. We conclude that there is no experimental evidence fur Ca2+ release at l ocations other than at z-lines. In addition, our calculations support the c onclusion that rapidly diffusing Ca2+ buffers (such as ATP) are important i n shaping the Ca2+ transient and that the details of intracellular indicato r binding need to be considered to explain correctly the time course of flu orescence change in the fibre.