Evaluation of laser induced sarcomere micro-damage: Role of damage extent and location in cardiomyocytes

Figures

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  Fig 1. Relationship between CMs viability and the extent of Z-disc damage within 2 h after damage. Femtosecond laser-based nanosurgery was used to ablate 10, 5, or 3 Z-discs per CM. Neighboring Z-discs were ablated in a longitudinal set (A, C) or randomly selected Z-discs were ablated (B, D) over the cell. A representative turboRFP linked α-actinin expressing rat CM before and directly after the ablation of 3 Z-discs in a set (yellow box) is depicted in (A). A merged image before (red) and after (cyan) random ablation of 3 Z-discs is shown in (B). Scale bars 20 μm. Bar charts represent mean viabilities + standard deviation. Treated CMs were statistically compared with untreated, control CMs (individual determined for each experiment) using a One-Way ANOVA followed by Tukey test (**p < 0.01, and *p < 0.05).

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  Fig 2. Relationship between CMs cell areas and total Z-disc counts with cell survival. Violin plots are displayed to visualize the distribution, median (dotted line) and mean (solid line) of CMs in terms of cell area (upper panel) and total Z-disc counts (lower panel) pre (grey) and 2 h post (blue) multiple Z-discs ablations. Each dot represents a single cell. Neighboring Z-discs in a longitudinal set: 10 Z-discs n = 26, 5 Z-discs n = 32, 3 Z-discs 27; Randomly selected Z-discs: 10 Z-discs n = 39, 5 Z-discs n = 29, 3 Z-discs n = 33.

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  Fig 3. Comparison of CM survival after 2 h and 24 h post ablation of 3 Z-discs. Bar charts represent the mean viabilities + standard deviation of CMs. In addition, either 3 neighboring Z-discs or 3 Z-discs randomly selected over the cell were ablated. Violin plots contrasting the distribution, median (dotted line), and mean (solid line) of CMs in terms of cell area (middle panel) and total Z-disc counts (lower panel) pre (grey) and post (blue) multiple Z-disc ablations. Each dot represents a single cell. Neighboring Z-discs in a longitudinal set: viability after 2 h area n = 27, Z-disc count n = 27, viability after 24 h area n = 29, Z-disc count n = 30; Randomly selected Z-discs: viability after 2 h area n = 33, Z-disc count n = 33, viability after 24 h area n = 34, Z-disc count n = 36.

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  Fig 4. Relationship between damage position and CM survival. The viability of CMs 24 h after single Z-disc ablation (A) was determined using Calcein-AM staining. Bar chart represents mean viabilities + standard deviation. Violin plots contrasting the distribution, median (dotted line), and mean (solid line) Z-disc distance to the nucleus of CMs pre (grey) and post (blue) ablation of a single Z-disc per CM. Each dot represents a single cell. 1 nucleus n = 32, 2 nuclei n = 21, (3 nuclei n = 1 not shown). No statistical differences were observed between the pre and post ablation groups for each setting (p ≥ 0.48).

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  Fig 5. Sarcomere orientation was analyzed pre and post Z-disc ablation using Scanning Gradient Fourier Transform (SGFT) method. From a representative Z-disc pattern of a GFP-actinin expressing CM before nanosurgery (A), the angular orientation distribution, the sarcomere direction (quiver plot), and the sarcomere pattern strength were examined. The pattern strength heat map visualized regions with high levels of repeating patterning in yellow/white and less repeating structures in darker red. Following, 5 neighboring Z-discs were ablated (yellow arrow). An overlay of the Z-disc pattern pre (red) and post-ablation (cyan) is shown in (B). The SGFT analysis revealed a non-significant change in the angular distribution (p = 0.45). In contrast, a significant change was found 2 h post-ablation (p = 0.006). Data were statistically compared using a Watson-Williams-test for circular distributions. Scale bar 20 μm.

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  Fig 6. Quantitative analysis of sarcomere orientation pre and post Z-disc ablation using Scanning Gradient Fourier Transform (SGFT) method. The sarcomere pattern of GFP-actinin expressing CMs pre, 1 min, and 2 h post nanosurgery were analyzed. The resulting absolute changes in sarcomere orientations within 20° angle of the vertical axis are depicted for the relaxed (A) and for the contracted cell state (B). Each dot represents a single cell. Charts represent mean values ± SEM. The percentage of CMs, which differed significantly (p < 0.05) in their angular sarcomeric orientation on a single cell level, are listed for the relaxed (C) and for the contracted cell state (D). Data were statistically compared using a Watson-Williams-test for circular distributions. 1 Z-disc n = 15, 3 Z-discs n = 12, 5 Z-discs n = 13.

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  Fig 7. SarcTrack assessment of CMs pre and post Z-disc ablation. The sarcomere pattern of GFP-actinin expressing CMs pre, 1 min, and 2 h post nanosurgery were analyzed. CM contractility was compared pre and post ablation of 1, 3, or 5 Z-discs in a longitudinal set in terms of contraction time (A) and contraction period (B). In addition, the relaxation duration (C, time from max contraction to max relaxation) and the sarcomere shortening as a percentage of the initial sarcomere length (D) were plotted. Each dot represents a single cell. Charts represent mean values ± SEM. Data were tested for significance with a Two-Way-ANOVA and Holm-Bonferroni test. *p < 0.05. 1 Z-disc n = 14, 3 Z-discs n = 12, 5 Z-discs n = 11.