Fig 1.
Cardiac fibroblast differentiation during culture on plastic.
Actin alpha 2 smooth muscle (Acta2) mRNA (A) and smooth muscle α-actin (SMA) staining intensity (B) in cardiac fibroblasts plated on plastic for different times. C) Representative immunostaining images for SMA. Scale bar 100μm. D) mRNA expression of collagen (Col) 1a1 and lysyl oxidase (Lox) in cardiac fibroblast cultured on plastic for different durations of time. One-way ANOVA with Dunnett’s multiple comparisons test as indicated for significant differences compared to the 1 day time point. N = 3 (2h, 1d, 2d, 3d, 9d, 12d and 15d), N = 5 (5d), N = 9 (7d). mRNA was normalized to ribosomal 18S.
Fig 2.
Relieving cells of high stiffness delays, but does not prevent, myofibroblast differentiation.
A) Schematic illustration of protocol used for experiments in B and C. Cardiac fibroblasts were cultured on plastic or soft gels for 3, 9 or 15 days (d) and then analysed. B) Immunostaining for smooth muscle α-actin (SMA; green) in cardiac fibroblasts. Nuclei were stained with DAPI (blue). Scale bar 100μm. C) mRNA expression of myofibroblast markers and Tcf21 at 13 days after seeding. Y-axis value, 2-ΔCt, represents mRNA levels for each gene relative to 18S rRNA. ΔCt is the Ct value of the gene of interest minus the Ct value of 18S rRNA. D) Schematic illustration of protocol used for experiments in E and F. Cardiac fibroblasts were cultured on plastic for 9d where after they were either transferred to soft gels of kept on plastic for an additional 4d. E) Immunostaining for SMA (green) in cardiac fibroblasts. Nuclei were stained with DAPI (blue). Scale bar 100μm. F) mRNA expression of myofibroblast markers and Tcf21. Significant differences were determined by Student’s t-test corrected for multiple comparisons using Holm-Sidak test. N = 8 (C, plastic and 3d N = 4 (C, 9d), N = 3 (C, 15d) and N = 3 (F).
Fig 3.
Effect of different concentration of ROCK and TGFβRI inhibitors on smooth muscle α-actin fibers.
A) Schematic diagram (modified from Herum et al., JCM, 2017) of the three main mechanically activated signalling pathways that lead to myofibroblast differentiation and pro-fibrotic gene expression. B) Inhibition points using the inhibitors Y27632, SB431542 and cyclosporine A (CsA). Immunostaining of cardiac fibroblasts for smooth muscle α-actin (SMA) in cardiac fibroblasts treated with different concentrations of Y27632 (C) and SB431542 (D and E). The micrograph in E is the same as in D. Phalloidin was used to stain F-actin (C) and DAPI to stain nuclei (D and E). Scale bar 100μm (C) and 200μm (D and E).
Fig 4.
Inhibiting ROCK and TGFβRI is most efficient for preventing and reversing the myofibroblast phenotype.
A and B) Schematic illustration of treatment regimen. Red arrows indicate the addition of fresh inhibitor solution every 24h. C and D) Immunostaining for smooth muscle α-actin (SMA) in cardiac fibroblasts treated with blockers of ROCK (Y27), TGFβRI (SB) and calcineurin (CsA). Scale bar 100μm. E and F) Quantification of SMA staining intensity. G and H) Expression of myofibroblast markers actin alpha 2 smooth muscle (Acta2), connective tissue growth factor (Ctgf), collagen (Col) 1a1, Col1a2, Col3a1, lysyl oxidase (Lox) and periostin (Postn), and the marker of resting cardiac fibroblasts, transcription factor 21 (Tcf21). One-way ANOVA as indicated beside gene name and Dunnett’s multiple comparisons test for significant differences compared to the “no treatment” control as indicated in the heat map. N = 3 (E and F), 4 (G) 4–7 (H).
Fig 5.
Reversibility of myofibroblasts depends on the duration of prior culturing time on plastic.
A and B) Schematic illustrations of protocols used in C and D, respectively. Cardiac fibroblasts were cultured on plastic for 6 (A) or 13 (B) days (d) and then treated with blockers of ROCK (Y27) and TGFβRI (SB) for 3d. C and D) mRNA expression of myofibroblast markers actin alpha 2 smooth muscle (Acta2), connective tissue growth factor (Ctgf), collagen (Col) 1a1, Col3a1, lysyl oxidase (Lox) and periostin (Postn) in cardiac fibroblasts treated with Y27 and SB (reversed; grey bars) relative to untreated cardiac fibroblast (control; black bars). The myofibroblast marker gene measurements in D are also shown in the heat map of Fig 4H. Significant differences were determined by Student’s t-test. N = 6 (C) and N = 3 (D).
Fig 6.
Reversed cardiac fibroblasts re-differentiate on plastic.
A) Schematic illustration of re-differentiation protocol. Cardiac fibroblasts were cultured on plastic for 6 days (d) and then treated with blockers of ROCK (Y27) and TGFβRI (SB) for 3d, where after blockers were removed and cells transferred to soft gels for 4d. B) Immunostaining for smooth muscle α-actin (SMA; green). Nuclei are stained with DAPI (blue). Scale bar 100μm. C) mRNA expression of actin alpha 2 smooth muscle (Acta2), connective tissue growth factor (Ctgf), collagen (Col) 1a1, Col1a2, Col3a1, lysyl oxidase (Lox) and periostin (Postn) relative to reversed cells (grey bars), following re-differentiation on plastic (black bars) or soft gels (white bars). Significant differences were determined by Student’s t-test with Holm-Sidak correction for multiple comparisons. N = 3.