Chiral Orientation of Skeletal Muscle Cells Requires Rigid Substrate

Abstract

Reconstitution of tissue morphology with inherent left–right (LR) asymmetry is essential for tissue/organ functions. For skeletal muscle, the largest tissue in mammalian organisms, successful myogenesis requires the regulation of the LR asymmetry to form the appropriate muscle alignment. However, the key factor for reproducing the LR asymmetry of skeletal tissues in a controllable, engineering context remains largely unknown. Recent reports indicate that cell chirality may underlie the LR development in tissue morphogenesis. Here, we report that a rigid substrate is required for the chirality of skeletal muscle cells. By using alternating micropatterned cell-adherent and cell-repellent stripes on a rigid substrate, we found that C2C12 skeletal muscle myoblasts exhibited a unidirectional tilted orientation with respect to the stripe boundary. Importantly, such chiral orientation was reduced when soft substrates were used instead. In addition, we demonstrated the key role of actin stress fibers in the formation of the chiral orientation. This study reveals that a rigid substrate is required for the chiral pattern of myoblasts, paving the way for reconstructing damaged muscle tissue with inherent LR asymmetry in the future.

Remnant Effects of Culture Density on Cell Chirality After Reseeding

“…It is based on our previous finding that cells can accumulate their actomyosin stress fibers when encountering the interface between cell-adherent and cell-repellent substrates, which allows cells to migrate and orient with LR bias, or chirality . Thus, the requirement of actomyosin stress fibers suggested a possible dependence of chiral mechanics on substrate stiffness, as discussed previously . C2C12 myoblasts were seeded with low (seeding density at 2200 cells/cm 2 at each passage) or high cell density (seeding density at 22 000 cells/cm 2 at each passage) from P9 to P12 (Figure a).…”

“…In recent years, cells on micropatterns were found with particular chiral mechanics . When cultured on micropatterns, adherent cells , were reported with specific chirality in cell polarity, , cell–cell alignment , and multicellular organization. ,, C2C12 myoblasts were also found with chiral orientation on micropatterns, especially on rigid substrate such as glass . Remarkably, such chiral mechanics are associated with actomyosin activity.…”

“…7,10,11 C2C12 myoblasts were also found with chiral orientation on micropatterns, 8 especially on rigid substrate such as glass. 12 Remarkably, such chiral mechanics are associated with actomyosin activity. That is, chirality in cell migration was activated with accumulation of actomyosin stress fibers, 7,8 but suppressed or even reversed when actin was inhibited.…”

Outline‐etching image segmentation reveals enhanced cell chirality through intercellular alignment

“…We next applied this imaging technique to investigate the dependence of the chiral orientation of C2C12 myoblasts on the substrate material, cell density, and intercellular alignment. Previously we reported that cell chirality is enhanced on glass substrate but not on PDMS substrate (Zhu et al, 2017), as indicated by more cells oriented with negative angles (Figure 4). While we previously concluded that the difference in substrate rigidity is the primary reason for this finding, how substrate rigidity can enhance cell chirality remained unclear.…”

“…Moreover, accompanying cell assembly, the chiral orientation later causes the formation of a tissue‐like structure with a specific, left–right biased pattern (Chen, Hsu, et al, ; Chen, Zhu, et al, ). Importantly, the chiral orientation is not only observed in vascular mesenchymal cells but in other cell types (Zhu, Kwong, Bao, & Chen, ). As the formation of coherent cell orientation is driven by autocatalytic feedback based on intercellular alignment (Junkin, Leung, Whitman, Gregorio, & Wong, ), it is believed that intercellular alignment may play a role in amplifying the cell chirality.…”