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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session B

Poster I8 - Exploring the time and size dependent actin organization of cardiomyocytes at the single cell scale

Thursday, October 24, 2024

2:45 PM – 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

LH

Lillian Hendrey

Student
Rensselaer Polytechnic Institute
Troy, New York, United States

Co-Author(s)

FP

Frank Peters

Graduate Student
Rensselaer Polytechnic Institute
Holmes, New York, United States

Primary Investigator(s)

LW

Leo Wan, PhD

Professor
Rensselaer Polytechnic Institute `, United States

Introduction: Chirality is an intrinsic property of cells that is believed to be essential in the development of asymmetric tissue morphology. Of particular interest is the left-right asymmetrical development of the heart. In previous work from this group, reversal of cell chirality was found to reverse the direction of cardiac c-looping in chick embryos (Ray et al., PNAS 2018). Actin cytoskeleton dynamics has recently been studied as a driver of chirality at the cellular level. Previous work has shown that human foreskin fibroblasts (HFF) display strong counterclockwise (CCW) multicellular chirality and that their radial fibers tilt into a chiral pattern (Tee et al., Nature Communications 2023). When HFFs are treated with actin polymerization and crosslinking reagents, the chirality can be reversed from CCW to clockwise (CW) with a reversed direction in radial fiber tilting in single cells also reversed. However, more investigation is needed into whether and how actin dynamics may differ between cell types, especially those that normally display CW chirality and the role they play in tissue and organ morphology.
We aim to enhance our understanding of the mechanisms of chirality in cardiomyocyte cells, which may provide insight into cardiac development and regeneration. We seed AC16 cardiomyocytes on single cell circular patterns to visually assess actin dynamics at different time points. Overall, this study aims to provide new information on the actin dynamics of cardiomyocytes. Particularly, we would like to determine if cardiomyocytes follow the same trends of actin chirality described in other cell types.

Materials and

Methods: Cell Culture:
AC16 cardiomyocyte cells were cultured with F-12 DMEM (Sigma) supplemented to 10% FBS (Seradigm) and 1% penicillin/streptomycin (Sigma). Each pattern was seeded with 100,000 cells in 1.5 mL of media (0.667 x 105 cells/mL) and incubated for 20 - 30 minutes with agitation every 5 minutes before transfer to fresh media. Slides were fixed with 4% paraformaldehyde at 3, 4, 5, and 6 hours after initial seeding.
Microcontact Printing:
30 µm and 50 µm diameter circular patterns were fabricated using the microcontact printing technique, as previously described (Zhang et al., JoVE 2022). 1-octadecanethiol (C18; Sigma) was applied to titanium gold coated slides using patterned PDMS stamps. Slides were placed into EG3 (Prochimia) for a minimum of 6 hours, coated with 50 µg/µL fibronectin for 30 minutes, and stored in PBS.
Staining and Imaging:
Fixed cells were permeabilized with 0.2% Triton X-100/PBS for 10 minutes, blocked with 10% normal goat serum in 0.1% Triton X-100/PBS for 1 hour at room temperature, and stained with Alexa Fluor 488 or Alexa Fluor 568 phalloidin for 4 hours at RT or overnight at 4°C. Samples were mounted onto coverslips with DAPI Fluoromount. Cells were imaged at 40x magnification using the Keyence BZX microscope. Only cells that had spread to fill the majority of the pattern were analyzed.
Statistics:
For comparison of strong presence of structures between two groups, a proportionality test was performed with a confidence interval of 0.05.

Results, Conclusions, and Discussions: Following fixation, staining, and imaging for each group, it was found that there were many cells that did not cover the entirety of the fibronectin pattern. The spreading of cells may be affected by the quality of the patterns themselves or issues with the fibronectin coating process.
In general, cells on the single-cell patterns displayed bright rings of actin close to the cell boundary (Fig 1). For the 3-hour seeding group, 30 µm diameter patterns were more likely to display peripheral actin rings and less likely to have radial fibers than cells seeded onto 50 µm patterns (Fig. 2). This suggests that large cell spreading is necessary for AC16 cells to develop radial fibers and for proper transverse arc migration to occur. We also note that there were no significant differences in the percentage of cells with radial fibers between groups of the same size fixed at different time points (i.e., hour 3 vs. hour 5).
In general, in comparison to other cell types previously studied, such as HFFs, radial fibers do not seem to have a strong presence in AC16 cells. The AC16 cells have fewer radial fibers and less migratory transverse arc. This is likely due to the CW bias nature of AC 16 cells in contrast to the CCW bias of HFFs.
In conclusion, we successfully patterned AC16 cardiac cells on circular micropatterns of different sizes and found that the display of peripheral actin rings depends on the pattern size, and hours 3 and 5 after cell seeding made little difference in radial and transverse actin structures. Future work involves live actin imaging to study actin dynamics and symmetry breaking.

Acknowledgements (Optional): This work is supported by NIH NIGMS 141387 to L.Q.W.