DNA Tension Probes Show that Cardiomyocyte Maturation Is Sensitive to the Piconewton Traction Forces Transmitted by Integrins

Abstract

Cardiac muscle cells (CMCs) are the unit cells that comprise the heart. CMCs go through different stages of differentiation and maturation pathways to fully mature into beating cells. These cells can sense and respond to mechanical cues through receptors such as integrins which influence maturation pathways. For example, cell traction forces are important for the differentiation and development of functional CMCs, as CMCs cultured on varying substrate stiffness function differently. Most work in this area has focused on understanding the role of bulk extracellular matrix stiffness in mediating the functional fate of CMCs. Given that stiffness sensing mechanisms are mediated by individual integrin receptors, an important question in this area pertains to the specific magnitude of integrin piconewton (pN) forces that can trigger CMC functional maturation. To address this knowledge gap, we used DNA adhesion tethers that rupture at specific thresholds of force (∼12, ∼56, and ∼160 pN) to test whether capping peak integrin tension to specific magnitudes affects CMC function. We show that adhesion tethers with greater force tolerance lead to functionally mature CMCs as determined by morphology, twitching frequency, transient calcium flux measurements, and protein expression (F-actin, vinculin, α-actinin, YAP, and SERCA2a). Additionally, sarcomeric actinin alignment and multinucleation were significantly enhanced as the mechanical tolerance of integrin tethers was increased. Taken together, the results show that CMCs harness defined pN integrin forces to influence early stage development. This study represents an important step toward biophysical characterization of the contribution of pN forces in early stage cardiac differentiation.

All-Covalent Nuclease-Resistant and Hydrogel-Tethered DNA Hairpin Probes Map pN Cell Traction Forces

“…However, in our hands, the probe density decreased dramatically (>75% loss) over 48 h (Figure S4), and seeded cells were poorly attached and spread. 45 The lack of cell adhesion and tension signal is likely due to the k off rate of biotin-streptavidin at 37 °C, which is 2.6 × 10 −5 s −1 . 44,57 This led us to develop a covalent anchoring strategy using conventional TGT probes onto hydrogel; however, these probes showed significant nuclease-mediated probe degradation over 6 h of cell seeding (Figure S5).…”

“…Additionally, the studies primarily utilized biotin-streptavidin as the gel tethering group, which has an appreciable dissociation rate (2.6 × 10 −5 s −1 ) at physiological temperatures and is prone to degradation due to proteases released by cells and present in the cell culture medium. 44,45 To overcome these past barriers in integrin force measurement on soft substrates, we designed a hydrogel-tethered HP tension probe. Although our strategy is modular and can be adapted to work with virtually any hydrogel polymer, for our proof-of-concept, we decided to use PEG-based hydrogels because of their biocompatibility and minimal cytotoxicity of such gels.…”

“…However, these are irreversible and cannot capture the dynamic ECM–receptor interactions. Additionally, the studies primarily utilized biotin-streptavidin as the gel tethering group, which has an appreciable dissociation rate (2.6 × 10 –5 s –1 ) at physiological temperatures and is prone to degradation due to proteases released by cells and present in the cell culture medium. , …”

Recent advances in label-free imaging of cell–matrix adhesions

“…qTGT-based MTFM was used to address this knowledge gap. 103 It has been shown that adhesion tethers with greater force tolerance ( T tol > 56 pN) can lead to functionally mature CMCs.…”

Unbreakable DNA tension probes show that cell adhesion receptors detect the molecular force-extension curve of their ligands

“…These probes are useful in manipulating the maximum integrin tension and recording the ensuing cell signaling state. 16,17,18,19,20 Indeed, previous studies indicated that > 43 pN forces are required for initial cell adhesion. 15,21 Because DNA duplex probes rupture, mechanotransduction is terminated and hence such probes are poorly suited for measurement of forces.…”