Professor Rensselaer Polytechnic Institute `, United States
Introduction: Approximately 1 in 110 infants are born with congenital heart disease (CHD) in the United States [1], but prevention is difficult as the underlying causes of these CHDs are often unknown. The initiation of CHDs may occur during early heart development when the initial embryonic linear heart tube grows and twists as it loops towards the right side of the body in a process called c-looping. This is the first left-right symmetry-breaking structure in the embryo [2]. Disturbing this directionality can lead to heart defects and severe health consequences. Recently, our lab showed that it is possible to disturb the directionality of c-looping in chick embryos by disrupting the cell chirality, or the intrinsic left- or right-ward bias of cells, of the cardiomyocytes that make up the embryonic heart tube [3]. However, the biomechanics of the link between cell chirality and cardiac c-looping direction are still unknown, and current in vitro cardiac morphogenesis models do not model the asymmetric bending process of c-looping. This study proposes a new in vitro platform of helical cell sheets to study the effects of chiral cell alignment on asymmetric force generation by cells on their substrate. In the future, this platform can be used to determine the effects of cell chirality on the biomechanics of c-looping and potential mechanisms in the formation of CHDs.
Materials and
Methods: To generate PDMS ribbons, 4.75 mm wide lines of Scotch Super-Hold tape were placed about 5 mm apart on cleaned glass slides prior to spin coating 10% w/v Poly(N-isopropylacrylamide) (PIPAAm) in butanol at 5000 rpm for 1 minute. After tape removal, Sylgard 184 PDMS at a 19:1 base to curing agent ratio was spin coated at 5000 rpm for 5 minutes over the entire slide surface and then cured for 2 hours at 100°C. Coated slides were UV/ozone treated for 15 minutes, coated with 50 μg/mL fibronectin for 15 minutes on a 50°C hot plate, rinsed in 37°C PBS, and stored in 37°C cell culture medium prior to cell seeding. Samples were seeded with approximately 45,000 cells/cm^2 C2C12 mouse myoblast cells and cultured overnight. Microtome blades were used to cut lines 200 μm apart perpendicular and approximately 500-1000 μm apart parallel to the boundary of PIPAAm regions, creating rectangular areas that overlapped regions both with and without PIPAAm. The PIPAAm layer under PDMS ribbons was dissolved by cooling the sample below the 32°C lower critical solution temperature of PIPAAm. After overnight culture, the ribbons were treated with 20 μM blebbistatin and phase contrast imaged every 2.5 minutes for 3 hours with a Keyence BZX microscope.
Results, Conclusions, and Discussions: Results and
Discussion: C2C12 mouse myoblast cells, which show strong counterclockwise chiral bias similar to chick embryonic heart tube cardiomyocytes [3,4], were used for prototyping purposes on the platform. Rectangular ribbons of varying widths with C2C12 cells detached from the glass substrate after the underlying PIPAAm was dissolved and grew to confluency (Fig. 1A). Most of the ribbons in Figure 1A bent to form a left-handed helical shape (8) versus a right-handed helix (2) or a neutral bending direction (1) (Fig. 1B). To test if the C2C12 cytoskeletal forces were affecting ribbon bending, the cells were treated with 20 µM blebbistatin, a known inhibitor of actin-based contractility. Immediately after blebbistatin addition, the helical bending remained (Fig. 1C(i)), and after t=30 minutes the ribbons became planar and lost their handedness as indicated by arrows (Fig. 1C(ii)). The C2C12 cells showed chiral alignment on narrow ( < 300 μm) width ribbons, with most cells aligned with a positive angle of alignment and few cells aligned perpendicular to the length of the ribbon (Fig. 1D), as expected based on previous work showing counterclockwise biased alignment of C2C12 cells.4 This chiral cell alignment did not appear to be affected by the addition of blebbistatin.
Conclusions: Loss of bending shape during inhibition of actin contractility by blebbistatin indicates forces from the actin cytoskeletal network are involved in bending the PDMS ribbons. The counter-clockwise alignment of C2C12 cells on the ribbons agrees with previous studies on the characteristic chiral direction of C2C12 cells [4]. This biased alignment of cells may be the cause of the preferred left helical bending direction of the ribbons. Further work will be done to analyze the relationship between ribbon width, handedness of helical bending, and the cell chiral cell alignment, as well as optimization for use with stem cell-derived cardiomyocytes. In the future, these helical cell sheets could be used to study the biomechanics of helical looping during heart development and screening possible causes of CHDs.
Acknowledgements (Optional): The work is supported by National Institutes of Health (OD/NICHD DP2HD083961 and NHBLI R01HL148104) and American Heart Association Grant# 24PRE1240154/Madison Stiefbold/2024.
