How Localized Z-Disc Damage Affects Force Generation and Gene Expression in Cardiomyocytes

Figures

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  Figure A1. Maximal measured traction force in relation to cell size and aspect ratio (long to short axis) before (grey dot) and after (blue dot) laser ablation for each cell. The size and aspect ratio do not change strongly, which is indicated by the nearly vertical lines.

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  Figure 1. We have developed a workflow to investigate cellular traction forces and gene expression after ablation of a single Z-disc in cardiomyocytes despite low cell numbers.

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  Figure 2. Traction force microscopy of hPSC-derived CMs before and after laser ablation of a single Z-disc. (A) Microscopic presentation of the same cell before and after ablation of a single Z-disc (arrow) in the relaxed and the contracted state. Left images show the Z-disc pattern in a relaxed cell, the small magnified image in the right corner highlights the removed Z-disc in a colored overlay of the pattern before (red) and after (cyan) Z-disc removal. Right images present force vectors from traction force microscopy in relaxed and contracted state. Scale bar 50 µm. (B) Outline of relative force determined via traction force microscopy. The measured curves were obtained by averaging twitches of a single cell and are normalized to the force of the relaxed state before ablation. A lower magnitude of the maximum contractile force after ablation is visible. (C) Analysis of the force produced by each cell before and after Z-disc removal in the relaxed and state of maximal force. Data are normalized to the mean maximal force before laser ablation (mean of grey box (open circle)). Data are based on 16 cells and at least 10 contractions per cell were analyzed. A significant lower force (**)…

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  Figure 3. RT-qPCR analysis of stress-related gene expression comparing control CMs and CMs, which were laser manipulated (n ≥ 3 experiments). NF-kB was significantly upregulated (***) after Z-disc removal. All data were calculated according to Taylor et al. [39] and are normalized to the mean of the control group, small red dots present raw data.

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  Figure 4. RT-qPCR analysis of injury and contraction related gene expression comparing control CMs and CMs, which were laser manipulated (n ≥ 3 experiments). TNNI3 and TNNT2 were significantly (*) downregulated. All data were calculated according to Taylor et al. [39] and are normalized to the mean of the control group, small red dots present raw data.

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  Figure 5. RT-qPCR analysis of sarcomeric cytoskeleton related gene expression comparing control CMs and CMs, which were laser manipulated (n ≥ 3 experiments). ACTN2 and FLNc were significantly (ACTN: *, FLNC: **) upregulated. All data were calculated according to Taylor et al. [39] and are normalized to the mean of the control group, small red dots present raw data.