TRPV4-mediated Mechanotransduction Regulates the Differentiation of Valvular Interstitial Cells to Myofibroblasts: Implications for Aortic Stenosis

Figures

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  Figure 1.. Primary mouse VIC express functional Trpv4 channels/receptors. A. VICs from WT mouse was immunostained with Trpv4 antibody (red). Trpv4 antibody pre-incubated with Trpv4 blocking peptide (right panel) was used to show specificity of Trpv4 IgG. B. VICs (~20,000 cells per well) were seeded on collagen-coated (10 μg/ml) 96-well plastic plate. Ca2+ influx is shown by are under curve measuring ΔF/F (Max-Min). FlexStation 3 recording of Calcium 6 dye-loaded WT VIC monolayers with or without TGFβ1 treatment show effect of Trpv4 agonist GSK101 or antagonist GSK219 on Ca2+ influx. **p < 0.01; ***p < 0.001; Student’s t-test. C. Fluorescence microscopy images show overexpression of Trpv4 proteins in WT and Trpv4 KO VICs transfected with GFP tagged Ad-Trpv4 for 48 hours. D. Spinning disc confocal microscopy images show recording of Ca2+ influx (red signals) in GSK101-exposed WT and Trpv4 KO VICs transfected with control Ad-Vec or Ad-Trpv4 constructs. E. Bar graph show quantitation of results (mean ± SEM) from Figure 1D. n = 50 cells per condition. ***p < 0.001; Student’s t-test.

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  Figure 2.. Trpv4 is required for both TGFβ1- and matrix stiffness-induced myofibroblast differentiation in VICs. A. WT and Trpv4 KO VICs were plated on collagen-coated plastic plates and incubated with or without TGFβ1 (5 ng/ml) for 48 h. Representative images from five different fields per condition shows morphological changes and co-localization of ɑ-SMA (green) and F-actin (red) with DAPI (blue) staining in VICs. B & C. Quantitation of results from Figure 2A. Data are expressed as mean ± SEM, n = 20 cells/condition; UT = untreated; ***p < 0.001, 1-way ANOVA. D. WT and Trpv4 KO VICs were plated on collagen-coated soft (1 kPa) and stiff (50 kPa) polyacrylamide hydrogels and incubated with or without TGFβ1 (5 ng/ml, 48 h). Representative images from five different fields per condition showing morphological changes and co-localization of ɑ-SMA (green) and F-actin (red) with DAPI (blue) staining in WT and Trpv4 KO VICs in response to increasing matrix stiffness and TGFβ1 treatment. E & F. Quantitation of results from Figure 2D. Data are expressed as mean ± SEM, n = 20 cells/condition; UT = untreated; **p < 0.01; ***p < 0.001, ****p < 0.0001, 1-way ANOVA.

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  Figure 3.. Differential requirement of Trpv4 domains for TGFβ1-induced VIC-to-myofibroblast differentiation. A. Schematic diagram of full-length Ad-TRPV4-WT and three Trpv4 constructs with deletions in N-terminal residues (Adeno-TRPV4-Δ–1-30, Adeno-TRPV4-Δ–1-130 and Adeno-TRPV4-Δ–100-130). PRD: proline rich domain. B. Representative images from five different fields per condition showing morphological changes and co-localization of ɑ-SMA (green) and F-actin (red) with DAPI (blue) staining in TGFβ1 (5 ng/ml)-stimulated Trpv4 KO VICs transfected with Ad-TRPV4-WT (with or without GSK219), Ad-TRPV4-Δ1-30, Ad-TRPV4-Δ1-130, Ad-TRPV4-Δ100-130, or Ad-Vec. C & D. Quantitation of results from Figure 3B. Data are expressed as mean ± SEM, n = 20 cells/condition; ***p < 0.001, 1-way ANOVA.

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  Figure 4.. N-terminal residues 30 to 130 in Trpv4 is critical in regulation of TGFβ1-induced traction force generation in VICs. A. WT and Trpv4 KO VICs were plated on collagen-coated (10 μg/ml) 25 kPa PA hydrogels containing 0.2 μm fluorescent beads in 24-well plates with or without 5 ng/ml of TGFβ1 for 24 h. Color-coded traction vector maps indicate the magnitude of the traction vector and corresponding phase images show the cells. B. Quantitation of results from traction force microscopy assay as shown in Figure 4A. Data are expressed as mean ± SEM, n = 10 cells/condition; ***p < 0.001, ****p < 0.0001, Student’s t-test; RMS: root mean square. C. Trpv4 KO VICs transfected with Ad-TRPV4-WT (with or without GSK219), Ad-TRPV4-Δ1-30, Ad-TRPV4-Δ1-130, Ad-TRPV4-Δ100-130, or Ad-Vec were seeded on collagen-coated (10 μg/ml) 25 kPa PA hydrogels containing 0.2 μm fluorescent beads in 24-well plates and were stimulated by 5 ng/ml of TGFβ1 for 24 h. D. Bar graph shows quantitation of results from Figure 4C. Data are expressed as mean ± SEM, n = 10 cells/condition; ***p < 0.001, ****p < 0.0001, Student’s t-test; UT: untreated.

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  Figure 5.. Deficiency of Trpv4 function blocks TGFβ1-induced phosphorylation of AKT in VICs. A. VICs were plated on plastic plates coated with collagen (10 μg/ml). Immunoblots show the expression levels of p-Smad2, total Smad2, p-Smad3, total Smad3, 𝛼-SMA, p-AKT, total AKT, p-Erk, total Erk, p-P38, total-P38. p-Jnk, total Jnk, and actin 48 h after treatment of WT and Trpv4 KO VICs with or without TGFβ1 (5 ng/ml) stimulation. All experiments were repeated at least three times. Graph shows quantification of results from Figure A for 𝛼-SMA (B) and p-AKT (C). **p < 0.01, ***p < 0.001, Student’s t-test. D. Immunoblots show the expression level of p-Smad2, total Smad2, 𝛼-SMA, p-AKT, total AKT, and actin 48 h after treatment of Trpv4 KO VICs transfected with Ad-TRPV4-WT (with or without GSK219) or control Ad-Vec in the presence or absence of TGFβ1 (5 ng/ml). All experiments were repeated three times. Graph shows quantification of results from Figure D for 𝛼-SMA (E) and p-AKT (F). **p < 0.01, ***p < 0.001.

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  Figure 6.. Trpv4-dependent phosphorylation of AKT by matrix stiffening is required for VIC-to-myofibroblast differentiation and traction force generation. A. Immunofluorescent microscopy images show the level of p-AKT (red) in WT and Trpv4 KO VICs plated on soft (1 kPa) and stiff (50 kPa) PA hydrogels with or without TGFβ1 (5 ng/ml) treatment for 24h. B. Bar graph shows quantification of results from Figure 6A for p-AKT. n = 20 cells/condition; UT = untreated; **p < 0.01, ***p < 0.001, Student’s t-test. C. Immunofluorescent microscopy images show the level of p-AKT (red) and ɑ-SMA (green) in Trpv4 KO VICs transfected with Ad-TRPV4-WT (with or without LY) or control Ad-Vec and grown in the presence of TGFβ1 (5 ng/ml) for 24h on stiff (50 kPa) PA hydrogels. D & E. Bar graph shows quantification of results from Figure 6C for ɑ-SMA (D) and p-AKT (E). n = 20 cells/condition; ***p < 0.001, Student’s t-test. F. Color-coded traction vector maps indicate the magnitude of the traction vector and corresponding phase images show the cells. G. Quantitation of results from traction force microscopy analysis. Data are expressed as mean ± SEM, n = 10 cells/condition, ***p < 0.001, Student’s t-test. H.…