Contractile State Dependent Sarcomere Length Variability in Isolated Guinea-Pig Cardiomyocytes

Figures

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  FIGURE 1. Sarcomere striation profile in a single isolated membrane intact guinea-pig cardiomyocyte. (A) Whole-cell image of a single cardiomyocyte with two ROIs selected for the analysis of sarcomere striation profiles in two different regions: central (green) and peripheral (yellow) regions of the same cell. (B) Example of the sarcomere striation profile observed in one ROI and one frame. The grey curve follows the sarcomere striation profile, and black rectangles show the positions of individual sarcomeres along the ROI beginning from the left edge. (C) Plots of individual SL values (here—end-diastolic) as a function of their position along the ROI. The SL values of each cardiomyocyte were derived from corresponding frames for end-diastole or end-systole, where several consecutive twitches were superimposed to obtain the SL values. The black box demonstrates where an error of SL measurement occurred because of the instability in the sarcomere detection in some frames. Such fragments were excluded from further analysis. Dashed lines show lower and upper SL bounds (1.4 and 2.2 µm for end-diastolic SL values, respectively). (D) SL distribution obtained from data displayed in “C”. See text…

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  FIGURE 2. Sarcomere length distribution in ENDO and EPI myocytes during the contraction. (A,C) Representative SL changes in time, averaged from one ROI in a single paced ENDO (A) and EPI (C) cardiomyocyte at 1 Hz. (B,D). Representative distributions, obtained in end-diastole (EDSL, blue) and end-systole (ESSL, red), in ENDO (B) and EPI (D) cardiomyocytes. Top panels show plots of individual SL values as function of their position along the ROI. The SL values of each cardiomyocyte were derived from frames recorded during end-diastole or end-systole; several consecutive twitches were superimposed. The distributions of individual SL sets are plotted with a bin size of 0.04 µm.

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  FIGURE 3. The percentage of ENDO (A) and EPI (B) cardiomyocytes in which distributions of individual end-diastolic SL (EDSL) and end-systolic (ESSL) passed different normality tests. S-W: Shapiro-Wilk one-sample test, A-D: Anderson-Darling one-sample test; S-W + A-D: Shapiro-Wilk one-sample test together with Anderson-Darling one-sample test (both tests passed). The number of ENDO cardiomyocytes n = 40, the number of EPI cardiomyocytes n = 43 (from 8 hearts). Dotted horizontal lines correspond to the level where all cells passed a test (100%).

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  FIGURE 4. Median SL values obtained from individual SL sets in central and peripheral regions of ENDO (n = 40) and EPI (n = 43) cardiomyocytes (from 8 hearts). (A): Median end-diastolic SL (EDSL) values. (B) Median end-systolic SL (ESSL) values. Two-way ANOVA was used for all comparisons. None of the comparisons revealed any statistically significant differences.

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  FIGURE 5. Median absolute deviation divided by median value (MADM, %) obtained for individual SL sets measured at different contraction states (end-diastole, EDSL, or end-systole, ESSL) in central and peripheral intracellular regions of ENDO (n = 40) and EPI (n = 43) cardiomyocytes (from 8 hearts). (A): MADM for SL distributions in ENDO cardiomyocytes. (B): MADM for SL distributions in EPI cardiomyocytes. (C): MADM for SL distributions in the central intracellular region of ENDO and EPI cardiomyocytes. (D): MADM for SL distributions in the peripheral intracellular region of ENDO and EPI cardiomyocytes. p < 0.0001, three-way ANOVA was used for all comparisons. Note that the top and bottom panels contain the same data sets, but displayed in different compositions depending on the comparison factor (top panels = ENDO vs. EPI, bottom panels = centre vs. periphery).

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  FIGURE 6. Correlation plots between actual median value of sarcomere length and the extent of sarcomere-to-sarcomere SL variability (evaluated by median absolute deviation divided by median, MADM) for end-diastole (higher SL value for each individual line) and end-systole (lower SL value for each individual line). (A) Pool of ENDO cells from central and peripheral intracellular regions (n = 40). (B) Pool of EPI cells from central and peripheral intracellular regions (n = 43). Red lines show cells with inverted relationship between median SL value and MADM value [∼15% of total number of cells for pooled ENDO and EPI cardiomyocytes (from 8 hearts)].