Figure 1.

Kindlin-2 expression is enhanced in stiff mechanical environments in vivo and in vitro. (A) Sections of normal and hypertrophic rat hearts were stained for kindlin-2 (green), α-smooth muscle actin (α-SMA) (red), and vimentin (blue), and were observed with confocal microscopy; average intensity projections of z-stacks are displayed. Vimentin- and α-SMA-positive myofibroblasts in the hypertensive heart strongly express kindlin-2 in fibrotic lesions. (B) Human fibroblasts were cultured on tissue culture plastic (TCP) for 5 day and treated for 1–4 day with transforming growth factor (TGF)-β1 (2 ng/mL) to assess expression of kindlin-2 and α-SMA by quantitative immunoblotting with indicated molecular weights. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control and Smad2/3 to assess TGF-β1 downstream signaling. (C) After 4 day treatment with TGF-β1, cells were immunostained for kindlin-2 (green), α-SMA (red), and nuclei (4′,6-diamidino-2-phenylindole, DAPI, blue) and confocal images were taken. (D,E) Likewise, human cardiac fibroblasts (hCF) were cultured on silicone culture substrates with elastic modulus of 3, 26, and 65 kPa and GPa-stiff TCP for 5 d, followed by analysis using (B) immunoblotting, densitometry and (C) immunofluorescence for kindlin-2 (green) and vinculin (red). All immunoblot bands were quantified by densitometry, first normalized to GAPDH loading control and then to TCP control. Shown are mean values from at least three independent experiments (data points) ±SD (* p < 0.05, ** p <0.01, *** p < 0.005, using ANOVA followed by a post-hoc Tukey’s multiple comparison test). All scale bars: 20 µm.