Immunostaining of cardiomyocytes monolayers grown on fibrous rS2/12-Linker-RGDS spidroin substrate.

<p>A: Photo of fibrous substrate, scale bar 20 μm.B: α-actinin (green), connexin (red), nuclei (blue) labeling, scale bar 20 μ.m (C-F) Close-ups of segments on image B, scale bar—5 μm.</p

Fluorescent image of α-actin (green) stained with Alexa Fluor-488 Phaloidinin conjugate and nuclei (blue) stained with DAPI of cultured rat cardiomyocytes monolayers grown on fibrous rS2/12-Linker-RGDS spidroin substrate (fibers are yellow).

<p>Scale bar—50 μm.</p

Pseudocardiograms of wave propagation on various types of substrates.

<p>Arrows depict starting moments of an electrical stimulation. Decreased frequency or transformation rhythm responses are marked by the asterisks. A—PCL+fibronectin pseudocardiogram for fibronectin coated PCL fibrous substrate. B—pseudocardiogram for rS1/9 fibrous substrate. C—pseudocardiogram for rS2/12-Linker-RGDS fibrous substrate. D—pseudocardiogram for rS1/9+rS2/12-Linker-RGDS fibrous substrate. The calculated critical frequencies for various substrates are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121155#pone.0121155.t001" target="_blank">Table 1</a>.</p

Verification of suitability of monolayers grown on rS2/12-Linker-RGDS spidroin fibrous substrate for excitation wave propagation.

<p>A, D: isochrone maps for point stimulation from rS2/12-Linker-RGDS fibers and fibronectin coated side, respectively. Line interval: 60 ms. Arrows show direction of wave propagation in each case. B, C—freezed frames of processed optical mapping videos represented by A,D pictures, correspondingly. E, F—time-space plots for white lines shown on pictures B, C with marked angles for velocity calculation.</p

Comparison of functional properties of cardiac monolayers on different substrates.

<p>Comparison of functional properties of cardiac monolayers on different substrates.</p

Quantitative analysis of an adhesion assay of fibronectin-coated common substrates and silk polymer substrates.

<p>1-fibronectin, 2- PCL coated with fibronectin (thickness 0.3 μm), 3- fibroin Bombyx mori coated with fibronectin (thickness 0.25μm), 4-fibroin Bombyx mori (thickness 0.25 μm), 5- rS1/9 (thickness 0.4 μm), 6- rS2/12 (thickness 0.4 μm), 7- rS2/12-Linker-RGDS(thickness 0.35 μm), 8- rS2/12-Linker-RGDS (thickness 0.25 μm), 9- rS1/9+rS2/12-Linker-RGDS 1:1(thickness 0.6μm)</p

Gene construction for Linker-RGDS.

<p>Gene construction for Linker-RGDS.</p

Representation of coordinated contractile activity of cardiomyocytes monolayer grown on rS2/12-Linker-RGDS spidroin fibrous substrate.

<p>Video freeze-frame of contractile activity, scale bar 20 μm. (A-E): Graphs of intensity variation over time on corresponding outlined regions of interest.</p

SEM micrographs of recombinant spidroin and fibroin <i>Bombyx mori</i> fibrous substrates.

<p>(a) low magnification of rS1/9 spidroin substrate (frame size 257 μm × 192 μm), (b) high magnification micrograph of rS1/9 spidroin fibers (frame size 5.6 μm × 4.2 μm), (c) high magnification micrograph of rS2/12-Linker-RGDS spidroin fibers (frame size 7.7 μm × 5.8 μm), (d) high magnification micrograph of rS1/9+rS2/12-Linker-RGDS spidroin (1:1) mixture fibers (frame size 6.3 μm × 4.7 μm), (e) high magnification micrograph of fibroin <i>Bombyx mori</i> fibers (frame size 11.4 μm × 8.5 μm).</p