Fig. 5

Scleraxis null mice exhibit reduced cardiac extracellular matrix and fibroblast marker gene expression. a Cross sections of hearts from wild type (WT) or Scx null (KO) hearts stained for fibrillar collagen using picrosirius red (BF, bright-field; POL, polarized light), Masson’s trichrome or via immunolabeling for total collagen I (red; plus DAPI nuclear staining in blue) revealed matrix loss in KO hearts; collagen I: 20× objective, scale bar = 35 μm; picrosirius/trichrome: 40× objective, scale bar = 66 μm. The blue color channel was extracted from the Masson’s trichrome sections for improved visualization. Samples are representative of at least three individual animals of each genotype. b Decellularized and dehydrated cardiac ventricular ECM from WT or KO mice was normalized to cardiac ventricular wet mass or tibia length and revealed ECM loss in KO hearts; n = 7 animals of each genotype. c–d Fibrillar collagen gene expression by qPCR (c) or western blotting normalized to Gapdh or β-actin (d) was decreased in Scx KO hearts compared to WT; n = 3 ((c) or collagen I blot in (d)) or n = 4 (collagen III and IV blots in (d)). Collagen I protein expression was the sum of the α1 and α2 isoforms. e–f The expression of mRNAs encoding several proteoglycans (e) and matrix metalloproteinases (f) (assessed by qPCR) was lost in Scx KO hearts; n = 3 animals of each genotype. g MMP2 and MMP9 activity (latent pro-form and mature, assayed by gel zymography) of protein lysates from WT or KO hearts was reduced in null animal hearts; n = 3 animals of each genotype. *P < 0.05 vs WT. h–i Fibroblast/myofibroblast marker gene mRNA (h) and protein (i) expression was assayed in WT and Scx KO mice, assayed by qPCR or western blot; n = 3 ((h) and vimentin in (i)) or n = 4 (DDR2 and αSMA in (i)). Scx loss induced similar down-regulation of fibroblast and myofibroblast markers. *P < 0.05 vs WT