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

Abstract

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.

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

“…To do this, we used an fs laser-based setup to precisely ablate Z-disc elements in CMs. This setup allowed us to introduce different damage patterns in CMs in a spatio-temporal confined and reliable fashion [ 11 , 12 ].…”

“…These findings are in agreement with earlier studies by Leber et al . [ 41 ] and with calcium measurements after ablation of a single Z-disc in CMs [ 11 ]. Continuous contraction has also been reported as a critical factor for proper myofibril assembly [ 15 ], which is required during sarcomere repair.…”

“…Rat cardiomyocytes were isolated from postnatal Spraque-Dawley rats (P2-P5) of both sexes as previously described [ 11 ]. One million cardiomyocytes were seeded into 35 mm glass bottom dishes (Ibidi, Germany) for imaging and ablation experiments.…”

“…A Ti:Sapphire laser system with a pulse length of 140 fs and a repetition rate of 80 MHz was used for multiphoton imaging and Z-disc ablation [ 11 ]. The custom-build setup was further equipped with a High-Power LED (SOLIS-3C, Thorlabs, USA) and a CCD camera (ProgRes® MF cool , Jenoptik, Germany) to allow epi-illumination and recording of CMs using fluorescence microscopy.…”

“…For nanosurgery, Z-discs were ablated at a wavelength of 730 nm, a scanning velocity of 100 μm/s, and a laser power of 0.9 nJ as revealed in our recent study. These parameters were validated to result in Z-disc loss via subsequent immunostaining against α-actinin [ 11 ]. A minimum of three treated and untreated CMs were recorded per trial.…”