Cardiac fibrosis inhibitor CTPR390 prevents structural and morphological changes in human engineered cardiac connective tissue

Figures

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  Figure 1. Administering CTPR390 to TGFβ1-activated ECCT resulted in a reversal of its physical and biomechanical characteristics to those observed in control ECCT (A) Representative images (bright field image, en face view, side views, and 3D rendering) of an ECCT using PS-OCT technique that showed the morphology of the engineered tissue around the two flexible poles. The white scale bar indicated 1.3 mm. (B) Three panels showing the correlative behavior observed within a sigma area between the Stokes U and the attenuation coefficient (μm−1), considering the quantified overlap in distributions in Control, TGFβ1, and TGFβ1-CTPR390 ECCT samples. n = 3 biological samples (ECCT) measuring 108 pixels in triplicate distributed in layers of 3 3.6 μm/px thick, assuming a refractive index of n = 1.38, typical of biological samples, evaluated up to the first 100 micrometers. White scale bar indicated 1 mm. (C) Representative ECCT images of the angle of polarization (AoP) orientation aligned with the fibers within the ECCT using the same color-coding as in panel. n = 3 biological samples (ECCT) measuring 108 pixels in triplicate distributed in layers of 3 3.6 μm/px thick, assuming a refractive index…

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  Figure 2. Restoration of Control ECCT gene and protein expression following the introduction of CTPR390 to TGFβ1-activated ECCT (A) Bright-field and epifluorescence imaging of control representative images of ECCT, TGFβ1-activated ECCT, and TGFβ1-activated ECCT subjected to CTPR390 treatment (Control, TGFβ1 and TGFβ1-CTPR390). Both white scale bars in the two panels indicated 5 mm. (B) Diagram illustrating the detection of CTPR390-green fluorescence (resulting from Alexa 488 fluorophore conjugated to CTPR390) incorporated into ECCT. It included a close-up view of the fluorescent fibroblasts within the ECCT and the subsequent extraction of fibroblasts from the ECCT, transferred to a tube, and later seeded onto a culture plate, revealing both fluorescent and non-fluorescent fibroblasts. Diagram generated with Biorender. (C) CTPR390 fluorescence captured directly from the surface of the TGFβ1 ECCT following a 13-day treatment with CTPR390 (TGFβ1-CTPR390 group). The white scale bar indicated 10 μm. (D) Bar graphs presenting the percentage and total cells retaining CTPR390 over a 13-day period in TGFβ1-CTPR390 ECCT, n = 4 ECCT per group, with three technical replicates. (E) Visualization of…

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  Figure 3. Attenuation of TGFβ1-activated ECCT pro-fibrotic cell features after CTPR390 treatment (A) Representative images of ECCT surface with fibroblasts stained with hematoxylin-eosin from cross-sections of Control, TGFβ1 and TGFβ1-CTPR390 ECCTs and representative image of inner fibroblast from an ECCT; the white scale bars indicated 25 μm. (B) Violin plots depicting significant differences in the number of cell surface layers of TGFβ1 ECCT compared to Control and TGFβ1-CTPR390 ECCT, n = 6 independent samples measured in triplicate. (C) Three panels featuring a depiction with a front view of fibroblasts on the surface of Control, TGFβ1 and TGFβ1-CTPR390 ECCTs by scan microscopy of 1 ECCT per group. Yellow arrows indicate surface blebs, and white arrows indicate filopodia in Control, TGFβ1 and TGFβ1-CTPR390 ECCTs; the white scale bars indicated 6 μm. (D and E) (D) Cell area count and, e) Cell perimeter count in the three groups of ECCT (Control, TGFβ1 and TGFβ1-CTPR390) performed in n = 12 ECCT per group, n of cells = 400–510 cells per group. (F) Confocal images showcasing the 3D reconstruction of cellular surfaces within 100 μm depth utilizing lysotracker (in red) as a cell dye for…

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  Figure 4. Characterization of distinct fibroblast subtypes in the ECCT (A and C) Hematoxylin/eosin staining on day 1 (A) and day 13 (C) of cross-sections of Control, TGFβ1, and TGFβ1-CTPR390 groups including insets of the whole cross section (C). The white bars of all panels indicated 50 μm, including inset panels showing the whole ECCTs. (B and D) Quantification of total cell numbers on day 1 (B) and day 13 (D) of Control, TGFβ1, and TGFβ1-CTPR390 groups. Cell counts were performed in 3–5 biological samples per group including 3 technical replicates per sample. (E–G) Electron microscopy images of superficial fibroblasts showing representative ultrastructural features of Control (E), TGFβ1 (F), and TGFβ1-CTPR390 (G) ECCT on day 13. (F) The blue arrow marks a depression of the plasma membrane; the red arrow points to a sac of rough endoplasmic reticulum (RER); and the green arrow points to one of the vesicles with very electron-dense fibrous elements. (G) The red arrow points to a normal-shaped RER sac; and the green arrow indicates a vacuole. Black scale bars indicated 2 μm (E, G) and 3 μm (F). (H and I) Bar graphs showing the percentage of superficial and inner fibroblasts per area on day…

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  Figure 5. Localization of organized collagen produced by human fibroblasts on the surface of TGFβ1-activated ECCT (A) Representative cross-sectional scanning microscopy images of Control, TGFβ1 and TGFβ1-CTPR390 long arms of the ECCTs. The white scale bars indicated 100 μm. (B) Representative magnified scanning microscopy images of inner-sections of Control, TGFβ1 and TGFβ1-CTPR390 ECCTs. The white scale bars indicated 10 μm. (C and D) Violin graph illustrating a significant reduction of the TGFβ1 ECCT cross section area (CSA in mm2) (C), and a reduction of the TGFβ1 ECCT perimeter (in mm) (D), indicating a higher degree of compaction in TGFβ1 samples in comparison to Control or TGFβ1-CTPR390 ECCTs (n = 15–33 independent ECCTs analyzed per group with 3 technical replicates per sample). (E) Positive and significant correlation (∗∗∗∗p < 0.0001) between CSA and perimeter of Control ECCTs. The other two groups (TGFβ1, and TGFβ1-CTPR390) did not show a correlation between the CSA and their respective perimeters (n = 25–33 ECCTs analyzed per group with 3 technical replicates per sample). (F) Representative images showing distinctively colored structured collagen at the periphery of the…

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  Figure 6. Differences in organization of collagen produced by human fibroblasts on the surface of TGFβ1-activated ECCT (A–C) Maps of the preferential orientation and degree of orientation of collagen in control (A), TGFβ1(B), and TGFβ1-CTPR390 (C) ECCTs. The white scale bars indicated 200 μm. Preferential of orientation in each 20 × 20 μm2 pixel is indicated by the direction of the arrow in each of the pixels of the figure and the color-coded discs. Degree of orientation in each 20 × 20 μm2 pixel is indicated by the thickness of the arrows in each of the pixels of the figure. Maps from individual ECCTs are found expanded in Figures S5A–S5C. (D) Violin graph illustrating significant changes in the degree of orientation of collagen in TGFβ1 (increase) and TGFβ1-CTPR390 (decrease) ECCTs. Graph was obtained considering the values from all of the pixels from each ECCT analyzing one complete ECCT per group including 3000–4000 pixels analyzed per ECCT. The dataset for panel (D) did not pass the Shapiro-Wilk test for normality. p-values (∗∗∗∗p < 0.0001) were determined using the non-parametric Mann-Whitney test, as the data did not follow a normal distribution. N = 5191-2051 measurements of 1 ECCT…