Femtosecond laser-based nanosurgery reveals the endogenous regeneration of single Z-discs including physiological consequences for cardiomyocytes

A highly organized cytoskeleton architecture is the basis for continuous and controlled contraction in cardiomyocytes (CMs). Abnormalities in cytoskeletal elements, like the Z-disc, are linked to several diseases. It is challenging to reveal the mechanisms of CM failure, endogenous repair, or mechanical homeostasis on the scale of single cytoskeletal elements. Here, we used a femtosecond (fs) laser to ablate single Z-discs in human pluripotent stem cells (hPSC) -derived CMs (hPSC-CM) and neonatal rat CMs. We show, that CM viability was unaffected by the loss of a single Z-disc. Furthermore, more than 40% of neonatal rat and 68% of hPSC-CMs recovered the Z-disc loss within 24 h. Significant differences to control cells, after the Z-disc loss, in terms of cell perimeter, x- and y-expansion and calcium homeostasis were not found. Only 14 days in vitro old hPSC-CMs reacted with a significant decrease in cell area, x- and y-expansion 24 h past nanosurgery. This demonstrates that CMs can compensate the loss of a single Z-disc and recover a regular sarcomeric pattern during spontaneous contraction. It also highlights the significant potential of fs laser-based nanosurgery to physically micro manipulate CMs to investigate cytoskeletal functions and organization of single elements.


Supplementary
. Viability staining of a CM after single Z-disc ablation. In this representative image, an turboRFP linked α-actinin expressing hPSC-CM is shown 24 h after laser treatment (A). The metabolic activity was visualized using Calcein-AM staining (B) and a spherical morphology with blebs (arrow heads) on the cell membrane was observed. Scale bar 5 µm. Figure S2. Fluorescence recovery after photobleaching of a Z-disc. FRAP was performed in neonatal rat CMs after bleaching of turboRFP linked α-actinin in a single Z-disc. An exposure time of 5 ms at 100 % laser intensity on a Leica TCS SP5 confocal microscope was used. Cells were imaged afterwards using an excitation wavelength of 543 nm until the intensity of the bleached area saturated. (A) Image series representing the Z-disc pattern of an turboRFP linked αactinin expressing neonatal rat CM before bleaching and at four time points post bleaching. A region of interest (dotted circle) was bleached. Post-bleach images were recorded at a time interval of 20 s. Scale bar 5 µm. (B) The fluorescence in the bleached region of interest was measured and normalized to the fluorescence intensity of an untreated Z-disc in the cell, including background subtraction. The intensity I(t) at every time point was averaged over nine measurements and is depicted with SEM. A single exponential growth ( ) = 0 + 1 • (1 − − ) with a bleaching of k indicated a half-recovery time of 1/2 = ln(2) = 200 s.

Supplementary Figure S3. X-expansion changes in CMs after single Z-disc ablation.
Multiphoton images of CMs were recorded before and frequently after single Z-disc ablation for a time period of 24 h. The x-expansion was determined using a self-written ImageJ macro. Untreated CMs served as a control group. A significant decrease in cell's x-expansion was observed for 6 DIV old neonatal rat CMs compared to the control (*) and for 14 DIV old hPSC-CMs compared to earlier points in time (+). Upper line of box, 75th percentile; lower line of box, 25th percentile; horizontal bar within box, median; upper bar outside box, 90th percentile; lower bar outside box, 10th percentile. Dots represent outliers. *P<0.05, +P<0.05, ++P<0.01, +++P<0.001.

Supplementary Figure S4. Y-expansion changes in CMs after single Z-disc ablation.
Multiphoton images of CMs were recorded before and frequently after single Z-disc ablation for a time period of 24 h. The y-expansion was determined using a self-written ImageJ macro. Untreated CMs served as a control group. A significant decrease in cell's y-expansion was observed for 14 DIV old hPSC-CMs compared to earlier points in time. Upper line of box, 75th percentile; lower line of box, 25th percentile; horizontal bar within box, median; upper bar outside box, 90th percentile; lower bar outside box, 10th percentile. Dots represent outliers. +P<0.05, ++P<0.01, +++P<0.001.

Supplementary Figure S5. Perimeter changes in CMs after single Z-disc ablation.
Multiphoton images of CMs were recorded before and frequently after single Z-disc ablation for a time period of 24 h. The perimeter was determined using a self-written ImageJ macro. Untreated CMs served as a control group. Non-significant fluctuations in cell perimeter were observed for neonatal rat and hPSC-CMs. Upper line of box, 75th percentile; lower line of box, 25th percentile; horizontal bar within box, median; upper bar outside box, 90th percentile; lower bar outside box, 10th percentile. Dots represent outliers.
Supplementary Figure S6. Calcium oscillations in CMs after single Z-disc ablation in neighboring CMs. For the determination of calcium oscillations in CMs, adjacent to treated CMs, the previous described time series of recorded Fluo 4 images were analyzed. The fluorescence intensity of a selected region of interest in a CM, adjacent to the treated CM, was determined and plotted as described before. The relative changes in calcium oscillations over time are visualized in box plot graphs. Calcium oscillations in CMs before single Z-disc ablation in the neighboring CMs served as reference value. A significant increase in cell's calcium oscillations were found for 14 DIV old neonatal rat CMs compared to the control (*). Upper line of box, 75th percentile; lower line of box, 25th percentile; horizontal bar within box, median; upper bar outside box, 90th percentile; lower bar outside box, 10th percentile. Dots represent outliers. 7 DIV n=27, 14 DIV n=25, 15 DIV n=20, 22 DIV n=17. *P<0.05.