High Time Resolution Analysis of Voltage-Dependent and Voltage-Independent Calcium Sparks in Frog Skeletal Muscle Fibers

Figures

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  FIGURE 1. Identification of the cell structure in a representative x-y image. (A) An original x-y image of an intact frog skeletal muscle fiber loaded with Fluo-4 displaying calcium release events. (B) The picture of the basic fluorescence values of each pixel showing good signal-to-noise ratio for static image elements, such as Z-lines. (C) Fast Fourier transform showing the frequency spectrum of the image in panel (B). At 0.5 μm–1 (and at its inverse, red rectangles) the sharp band corresponding to one sarcomere is clearly visible. (D) The result of the inverse Fourier transformation, where the Z-lines are excellently traceable. White lines perpendicular (X) and parallel (Y) to the Z-lines determine the directions of FWHMs. Scale of panels (B,D) is identical to that in panel (A).

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  FIGURE 2. Generation of structure-free normalized image for spark identification and localization. (A) Original x-y image. (B) F/F0 image to show that the structure was removed and only the sparks are visible. (C) Fiber structure superimposed with areas of sparks. (D) Part of the image in (B) showing four events at high magnification. White curves encircle the areas corresponding to the identified events. Yellow dots present the position of the maxima, while the four blue dots in each event show the orientation of the two axis (perpendicular and parallel to the Z-lines) within the spark. Red pixels represent the position of the fluorescence maximum of Fluo-4, and thus the distance between yellow and red pixels was used to calculate the distance of the center of the spark from the nearest Z-line. (E) Same area as in panel (B) enlarged from panel (C) showing the superimposed Z-lines. The dark bands mark the triads at the Z-lines. (F) A spark enlarged from panel (E) showing the location of color dots described above. Light blue lines show the fluorescence maximum (i.e., the middle of a sarcomere).

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  FIGURE 3. Normalized (F/F0) full size x-y images of intact frog skeletal muscle fibers bathed in Normal Ringer (NR), in the presence of 1 mM Caffeine (Caffeine), in depolarizing solutions corresponding to a calculated resting membrane potential of -70 and -65 mV, and supplemented with 1 mM Caffeine. All images were recorded with 30 FPS. Note how the number of sparks is changing with the different conditions.

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  FIGURE 4. (A) Individual sparks on a larger scale. All images were recorded with 30 FPS. Next to the spark the spatial profile of F/F0 in X & Y direction (parallel and perpendicular to fiber axis) are presented. Panels (B–D) present the distribution histograms for the amplitude (B), FWHM-X (C), and FWHM-Y (D) under control conditions (black circles), on fibers depolarized to a calculated value of -65 mV (green squares), and in the presence of 1 mM caffeine (red triangles). Note how both depolarization and caffeine has shifted the distribution the higher values, especially visible in case of FWHM-X and FWHM-Y in the presence of caffeine from 2.5 μm to 4 μm.

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  FIGURE 5. Identifying positions of spark occurrence in images. (A) Mesh plot of frequencies of spark occurrence (i.e., the number of sparks in the given region) in a 16 × 16 pixel voxels (generated from 60 consecutive images). All images were recorded with 30 FPS. (B) Frequency distribution of regions with different number of sparks. Solid line superimposed is the fitted Poisson distribution, while dashed black curves show the Poisson distribution generated with λ being equal to the average event numbers. The values for λ used to generate the dashed curves were 0.349, 1.42, and 1.12 while those obtained from the fits were 0.281, 1.19, and 1.04 for control conditions, for depolarized fibers, and in the presence of caffeine, respectively.

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  FIGURE 6. Distribution of the distances between the centers of two sparks. For each spark concentric squares were constructed around the center of the selected spark. The number of sparks with centers falling onto the side of the given square were then counted. The relative frequency of these neighboring sparks are presented for control conditions (A), for depolarized (B), and caffeine-treated fibers (C). Note that in this representation the number of pixels increases linearly with the length of the side of the square, thus if sparks appear randomly a linearly increasing distribution is expected. To avoid the problem of bringing pixels from outside of the fiber into the counting 1./only those sparks were selected that lay close to the center of the fiber and 2./the side of the square was restricted to being less the 120 pixels (appr. 25 μm). Thus in the panels (A–C) the ordinate is in pixels. Dashed lines represent least-squares fits of a straight line to the data points in each graph with R2 being 0.829, 0.964, and 0.918, for panels (A–C), respectively.

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  FIGURE 7. Localization of spark centers relative to the Z-line. (A) Two samples from an image showing the fiber structure superimposed with sparks (indicated by the areas encircled by the white lines). White dots present the position of the center of the sparks. (B) Distribution histograms of the distance between center of sparks and the nearest Z-line for control conditions (NR, black circles), on depolarized fibers (green squares), and in the presence of caffeine (red triangles). Note that the distributions are essentially symmetric to the Z-line. (C) Mean distances of spark centers under the three conditions. Both the depolarization and the presence of caffeine shifted the center of the spark to positions further away from the Z-line as compared to the control conditions. Scale in panel A corresponds to 40 μm for the middle image and 4.3 μm for the enlarged images on left and right side. The middle image was recorded with 30 FPS. Here and in subsequent figures *** denotes significant difference versus NR at p < 0.001, ### denotes significant difference vs. Depol at p < 0.001.

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  FIGURE 8. Modified eccentricity of the sparks (A). Distribution of the modified eccentricity calculated using Eqn. 2 for control conditions (NR, black circles), for depolarized fibers (green squares), and in the presence of caffeine (red triangles). Note that albeit negative values were present, the distribution is not symmetric, it is shifted toward positive values indicating that FWHM-X was, in most cases, greater than FWHM-Y. (B) Average modified eccentricity for the three experimental conditions calculated using only the positive values from panel (A). Note that both the depolarization and caffeine treatment increased the modified eccentricity, and, furthermore, its value was far greater even under control conditions than what is expected from the skewing introduced by the scanning.

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  FIGURE 9. Traveling events on consecutive x-y images. (A) Five consecutive F/F0 images with the traveling sparks identified. The sampling rate was 17 ms/image (60 FPS). (B–D) Averages of distances traveled. (B,C) report distances in X and Y direction, respectively, while panel (D) presents the total distance traveled. The numbers of events analyzed are presented in Table 2.