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.

Effects of cell state and staining on femtosecond laser nanosurgery.

Femtosecond laser nanosurgery enables precise manipulation of subcellular elements to study regeneration. However, currently it is not frequently employed-probably because of its unknown consequences on the whole cell level. To better understand the associated biological response of the cell, especially in the context of different cell states and cell staining, we manipulated C2C12 myoblasts and myotubes, which were either unstained (nicotinamide adenine dinucleotide signal) or stained with MitoTracker Red. Both signals overlap well and stain similar areas in untreated cells. We chose 3 different cutting lengths and performed surgery in the cytosol along the major cell axis. The cuts resealed within several minutes independent of the cutting length. We analyzed cell area, perimeter, major and minor axis on long term. We observed significant changes in the cell area and perimeter, dependent on the staining and more pronounced in differentiated myotubes. We conclude, that laser parameters must be chosen carefully, depending on the staining of the cell, its (differentiation) state, and the extent of the cut region, such that unwanted cell responses can be avoided. Laser manipulation of C2C12 myotubes with small ablation (0.8 µm) and large ablation (3.0 µm). While small damages recover, larger damages lead to elimination from the syncytium. Scale bar: 20 µm.