Cardiomyocyte sarcomere length variability: Membrane fluorescence versus second harmonic generation myosin imaging

Figures

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  Figure 1.. Representative confocal images of an isolated cardiomyocyte obtained by SHG microscopy and by membrane fluorescent staining with di-4-ANEPPS (ANEPPS). (A) SHG (top) and ANEPPS (bottom) images with five ROIs as indicated (colored lines) following the paths of single myofibrils. The insets show the sarcomeric striation of a single ROI (cf. yellow arrows). (B) Sarcomere striation profiles taken from a single ROI using SHG and ANEPPS images. The arrows show the positions of local maxima in the intensity profiles (note their relative shift by a half sarcomere because SHG visualizes the center of sarcomeres, while ANEPPS visualizes the areas near the sarcomeric Z-disks).

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  Figure 2.. The average SL value, but not the SL variability, is independent of the optical method used to derive SL striations. Confocal measurements were made either by using SHG microscopy or by fluorescent membrane staining with ANEPPS in n = 26 isolated cardiomyocytes from N = 3 hearts. (A) The median SL value and the following absolute and relative quantitative measures of SL variability were analyzed. (B–E) Inter-quartile range (IQR; B), median absolute deviation (MAD; C), IQR divided by median SL (D), and MAD divided by median SL (E). The data in A–E are shown as mean ± SD. Differences between SHG and ANEPPS were significant at P < 0.0001 (Wilcoxon matched-pairs signed rank test). (F) Averaged distribution plots for individual SL values obtained by SHG or ANEPPS. The peaks of the plots correspond to averaged median SL and the distributions are shown as a percentage of the corresponding median SL.

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  Figure 3.. The use of either local maxima (max) or local minima (min) of the sarcomere striation profile generates non-discriminated quantitative measures of SL median value and the variability in the individual SLs. Confocal measurements were by SHG microscopy or by fluorescent membrane staining with ANEPPS in n = 26 isolated cardiomyocytes from N = 3 hearts. (A and B) Median SL (A) and median absolute deviation (MAD; B) divided by median SL (MADM). Differences between SHG(max) and SHG(min) or between ANEPPS(max) and ANEPPS(min) were not significant (Wilcoxon matched-pairs signed rank test).

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  Figure 4.. The applicability of SHG measurements for evaluation of sarcomeric A-band 3-D structure. (A) A series of five SHG images of A-bands in an isolated cardiomyocyte obtained at different positions along the Z-axis, i.e., from the top (slice #1) to the bottom of the cell (slice #5). The yellow-colored ROI on each frame is kept fixed in size and X/Y positions and used to retrieve A-band axial regularity. Scale bar on the top image is common for all images. (B) Curves of A-band regularity patterns obtained from the corresponding ROIs shown in the images in A. (C) The shifts of individual A-band X position across the slices as calculated relative to the X-position in the center of the cell (i.e., slice #3). The plots are obtained for four frames (two below and two above the middle frame). The magnitude of the longitudinal shifts, their variation along the x axis, and the actual direction of the change during the axial slicing across the cell depth may be used to estimate z-axis A-band (mis)alignment.