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.

Keywords: DNA sensors; cardiomyocyte’s maturation; integrin forces; integrin mechanotransduction; pN forces; rupture probes; substrate stiffness.

Figure 1.

DNA probes to control CMC integrin tension. (a) Schematic representation of cardiac muscle cells (CMCs) pulling on the varied threshold of rupture probe surfaces 12, 56, and 160 pN. The scheme shows the oligonucleotides used in the work where 12 and 56 pN probes have the same chemical composition but different orientations of the biotin group anchoring the probe to the surface. For 160 pN probes, the same strand presents the biotin and RGD groups. (b) Chemical structures of modification to oligonucleotides used to construct rupture probes and (c) rupture probability of probes under different loading times.

Figure 2.

Cardiomyocytes display elongated morphology with integrin–ligand tension > 12pN. (a) Representative images of CMCs (live) on 12, 56, and 160 pN rupture probe surfaces; the leftmost panel (RICM) channel shows the outline of the attached cells, and the middle panel (bright field), TRITC channel, shows the signal increase of Cy3B fluorescence due to probe rupture for 12 and 56 pN probes and the inverted fluorescence loss for 160 pN probes (t = 6–8 h. (b–f) Bar graphs showing the spread area, aspect ratio (x/y), circularity, and mean fluorescence under the cells and integrated fluorescent density obtained from CMCs that were cultured on 12, 56, and 160 pN probes. Each data point represents a single cell while the bar shows the average. ****, ***, **, *, and ns indicate p < 0.0001, p < 0.001, p < 0.01, p < 0.05, and not significant, respectively, as determined from one-way ANOVA. Error bars show standard deviation for n > 3, where each experiment was averaged from three or more different cell isolations with three different sets of surface preparations. Scale bar = 20 μm.

Figure 3.

Cardiomyocytes displayed a contractile profile consistent with greater maturation on surfaces with T_tol = 56 and 160 pN. (a) Brightfield images from time-lapse of cardiomyocytes twitching on different probes (t = 8–10 h); the dashed yellow line shows the major axis of the kymograph. (b) Kymographs of representative cells along the yellow dashed line. The y-axis of the graphs is elapsed time (t), and x shows the displacement, t = 2 s, x = 10 μm. (c) Bar graphs showing the average number of twitches per cell for each replicate. Ten 1 min long videos were analyzed in BF; the error bar shows the standard deviation for n = 3. The error bar shows the standard deviation for n = 3. ****, ***, **, *, and ns indicate p < 0.0001, p < 0.001, p < 0.01, p < 0.05, and not significant, respectively, as determined from one-way ANOVA. (d) Bar graph showing the percentage of attached cells measured from 11 videos per replicate, calculated by the percentage of cells that had contractility; the error bar shows the standard error of the mean for n = 5. Scale bar = 10 μm.

Figure 4.

Cardiomyocytes displayed a calcium profile consistent with greater maturation on surfaces with T_tol = 56 and 160 pN. (a–c) FITC images of CMCs on 12, 56, and 160 pN rupture probes stained with Fluo-4. Images were taken from the time-lapse to measure the transient Ca²⁺ sparks (t = 12–14 h). (d–f) Transient Ca²⁺ flux profile of the outlined CMCs on 12, 56, and 160 pN probes. The dotted yellow line shows if the Ca²⁺ sparks have the same τ for the outlined cells. (g–j) Bar graph showing spike intensity, percent active, the time difference between two spikes (each data point represents a video were n > 10 cells were analyzed for each data point), and spike frequency obtained from Fluo-4-stained CMCs on 12, 56, and 160 pN probes. ****, ***, **, *, and ns indicate p < 0.0001, p < 0.001, p < 0.01, p < 0.05, and not significant, respectively, as determined from one-way ANOVA. Scale bar = 10 μm.

Figure 5.

Cardiomyocytes having aligned sarcomeres at higher integrin force. (a) RICM of representative cells on the 12, 56, and 160 pN surfaces along with overlay immunostaining images showing SERCA2a (red) and DAPI (blue) staining (t = 10–12 h). Plots showing (b) the average SERCA2a expression and (c) total SERCA2a expression, as measured from immunostaining images. (d) RICM and sarcomeric α-actinin immunostaining of representative cells on the 12, 56, and 160 pN surfaces. The inset highlights the myofibril widths (z-band), and the yellow dashed line shows the length of individual z bands. Plots of (e) sarcomere length and (f) myofibril width for CMCs cultured on 12, 56, and 160 pN probes. For (a) through (f), each data point represents one cell, and data was obtained from three biological replicates where each replicate included analysis of n > 15 cells. (g) and (h) show plots of percentage of cells containing multiple nuclei and average nuclear diameter, respectively. n = 5 independent replicates. ****, ***, **, *, and ns indicate p < 0.0001, p < 0.001, p < 0.01, p < 0.05, and not significant, respectively, as determined from one-way ANOVA. Error bars show the standard deviation, where each independent experiment used cells obtained from cells pooled from >20 animals with three different sets of surface preparations. Scale bar = 20 μm.

Figure 6.

YES, associated protein (YAP), is upregulated when integrin F > 12 pN. (a) RICM, YAP, and DAPI immunostaining of representative cells on the 12, 56, and 160 pN surfaces (t = 12–14 h). (b) Total expression of YAP measured by immunostaining (t = 8–10 h). (c) Average YAP expression (t = 8–10 h). (d) Fraction of nuclear YAP expression over total cellular YAP expression (t ≥ 12–14 h); each data point represents a single cell while the bar shows the average. ****, ***, **, *, and ns indicate p < 0.0001, p < 0.001, p < 0.01, p < 0.05, and not significant, respectively, as determined from one-way ANOVA. Error bars show standard deviation for n > 3, where each experiment was averaged from three or more different cell isolations with three different sets of surface preparations. Scale bar = 10 μm. (e) Proposed mechanism of YAP upregulation due to integrin activation.

Figure 7.

Podosomes/podosome-like organelles as an early maturation marker in CMCs. (a) Representative images of CMCs (fixed) having podosome-like structure on the 12 pN surface (t = 8–10 h), RICM showing the cell of interest, TRITC channel showing the podosome tension ring, and Phalloidin staining confirming colocalization of actin in the structure. Scale bar = 20 μm (first column) and 6.4 μm (third column). (b) Number of podosomes in rupture probe surfaces. (c) Percentage of cells having podosomes (where each dot represents one biological replicate, n = 5). (d) Diameter distribution of podosomes. Each data point represents a single cell, while the bar shows the average. ****, ***, **, *, and ns indicate p < 0.0001, p < 0.001, p < 0.01, p < 0.05, and not significant, respectively, as determined from one-way ANOVA. Error bars show standard deviation for n > 3, where each experiment was averaged from three or more different cell isolations with three different sets of surface preparations.