Fig. 4

Scleraxis regulates cardiac myofibroblast α-smooth muscle actin expression and cell contraction. a–b αSMA mRNA (a) and protein (b) expression were assayed by qPCR or western blot, respectively, following knockdown of Scx (AdshScx) in primary cardiac proto-myofibroblasts, showing attenuated αSMA expression compared to control (AdshLacZ); n = 3. c Scx over-expression (AdScx) up-regulated αSMA expression in primary rat cardiac proto-myofibroblasts as determined by mRNA or protein expression (qPCR or western blot, respectively); n = 3. d Induced αSMA was incorporated into stress fibers, as demonstrated by immunocytochemistry, indicating promotion of the myofibroblast phenotype by Scx; 20× objective, scale bar = 65 μm. e Primary cardiac proto-myofibroblasts seeded onto compressible collagen gel matrices were assayed for gel contraction following Scx over-expression or knockdown, with or without concomitant 10 ng/mL TGFβ₁ to induce contraction. Crosses denote the detected margin of the collagen gel. f–g Quantification of gel contraction images, reported as the percentage of the maximum contraction induced by 10 % FBS, demonstrates that Scx over-expression induces cell contraction (f), while Scx knockdown attenuates TGFβ₁-induced contraction (g); n = 6 ((f) except AdGFP + TGFβ and AdScx + TGFβ; n = 3 each) or n = 3 (g). *P < 0.05 vs AdshLacZ (a–b), vs AdGFP (c, f) or vs AdshLacZ + vehicle (g), #P <0.05 vs vehicle controls (f) or vs AdshLacZ + TGFβ (g). h Alignment of putative Scx-binding E-boxes (E1 and E2; dashed lines) in the proximal rat, mouse and human αSMA promoters. Asterisks and periods denote nucleotides conserved across three or two species, respectively. i Mutation of one or both E-boxes (mE1, mE2) attenuates transactivation of the rat αSMA proximal promoter by Scx as determined by luciferase reporter assay; n = 3. j Electrophoretic mobility shift assays demonstrate Scx-binding to E-boxes E1 and E2; this binding is abolished when the E-boxes are mutated (mE1 and mE2). NS, non-shifted lane; S, shifted lane; CC, 500× cold competition. The arrow denotes the shifted complex. k Scx-binding to the αSMA gene promoter is significantly enriched in cardiac myofibroblasts (MyoFb) compared to fibroblasts (Fb) as determined by chromatin immunoprecipitation assay with qPCR quantification of Scx-bound DNA; n = 3. *P < 0.05 vs control empty vector (i) or vs Fb (k), #P < 0.05 vs scleraxis + non-mutated promoter (i)