Fig 1.
SNAIL1 expression increases in mouse heart post LAD ligation. (A) Schematic representation of the SNAIL1-CBR targeting construct. E1,E2,E3 are the three exons of SNAIL1-CBR is clic beetle red bioluminescent protein. (B) The timeline of the experiment. (C) SNAIL1-CBR bioluminescence signal in sham (n = 3) and infarcted hearts (n = 4) after Left Anterior Descending Artery Occlusion (LAD). (D) Quantification of biolumincence signal in (C). (E) Relative level of SNAIL1 mRNA in LAD ventricles, normalized to SNAIL1 mRNA from sham treated ventricles, as determined by Q-PCR. SNAIL1 mRNA level in sham treated hearts was arbitrarily set to 1. (F) Relative mRNA levels for select fibrogenic genes, as indicated, in LAD ventricles normalized to sham treated ventricles. Determined by Q-PCR. SNAIL1 mRNA level in sham treated hearts was arbitrarily set to 1. Representative of 3 hearts. (G) Representative images of trichrome staining for collagen in Sham and LAD sections. All experiments were repeated three times with three mice (minimum) in each group.
Fig 2.
SNAIL1 expression in infarct zone of the heart. Immunofluoresecnce staining of SNAIL1 and cardiac cell type markers in the ventricular infarct zone of LAD surgery mice. Cardiac cell type markers: (A) αSMA (alpha smooth muscle actin)–myofibroblast marker (B) periostin–myofibroblast marker, (C) CD31 –endothelial cell marker, (D) CD45 –leukocyte cell marker, (E) α-actinin–cardiomyocyte marker. Scale bars: 50 microns. Infarcted hearts from six mice were analyzed. There were 4–5 images per cell type per heart. (F) Table quantifying marker staining in SNAIL1 positive and SNAIL1 negative cells within the infarcted heart.
Fig 3.
Pro-fibrotic factors increase SNAIL1-CBR level in primary cardiac fibroblasts. Primary fibroblasts were isolated from the hearts of un-infarcted, normal SNAIL1-CBR/+ mice. Bioluminescence intensity after treatment with TGFβ (2 ng/uL) (A), PDGF (10 ng/uL) (B), Hypoxia (400 uM CoCl2) (C) and Angiotensin II (1 uM) (D). Representative data from one of 4 experiments are shown. (E) Relative fold change in SNAIL1-CBR bioluminescence intensity in cardiac fibroblasts exposed to increasing collagen I concentration. In each condition, values at t = 0 were arbitrarily set to equal 1. (F) SNAIL1-CBR bioluminescence intensity in cardiac fibroblasts plated on 4mg/mL collagen I for increasing lengths of time. A representative example from 3 experiments is shown. (G) Relative SNAIL1 mRNA fold change in primary cardiac fibroblasts 4 hours post stimulation with indicated factors, as determined by Q-PCR. All values were normalized to GAPDH and compared to non-treated cells as control. SNAIL1 mRNA level in nontreated cells was arbitrarily set to 1. Representative example of one of 3 experiments is shown.
Fig 4.
SNAIL1-deleted cardiac fibroblasts have decreased collagen I mRNA expression and collagen matrix deposition. (A) Relative SNAIL1 mRNA level in isolated SNAIL1f/f; ROSA-LSL-tdTomato cardiac fibroblasts treated with Adeno-LacZ (CTL) or Adeno-Cre (+ Cre), with and without TGFβ stimulation (2ng/ml for 2 hours), as determined by Q-PCR. SNAIL1 mRNA level in CTL unstimulated cells was arbitrarily set to 1. (B) Western blot analysis, with the indicated antibodies, of extracts from cardiac fibroblasts isolated from SNAIL1f/f; ROSA-LSL-tdTomato mice and treated with Adeno-LacZ (CTL) or Adeno-Cre (+ Cre), with and without TGFβ stimulation (2 ng/ml for 2 hours). Col1α1 (C) and Col3α1 (D) mRNA levels at baseline and following TGFβ stimulation (2 ng/ml for 2 hours) in control (CTL) and SNAIL1-/- (+ Cre) cardiac fibroblasts. Shown is a representative result of one of 4 independent experiments. (E) Collagen I immunofluorescence of cell free matrix deposited by control (CTL) and SNAIL1-/- (+ Cre) cardiac fibroblasts. (n = 3). (F) Quantification of results in (E). (G) Detection of Collagen 1 by Sirius Red Staining of cell free matrix deposited by control (CTL) and SNAIL1-deleted (+ Cre) cardiac fibroblasts. (n = 4). (H) Birefringence imaging of Sirius red stained matrix produced by control (CTL) or SNAIL-/- (+ Cre) cardiac fibroblasts. (I) Quantification of birefriengence imaging (n = 4, 20 images counted for each experiment). Scale bars: 50 microns.
Fig 5.
SNAIL1-deleted cardiac fibroblasts have decreased αSMA, LoxL2 & Periostin expression. (A) αSMA immunofluorescence of control (CTL) and SNAIL1-deleted (+ Cre) cardiac fibroblasts. (B) Quantification of results in (A). 30 fields were counted in each group. Shown is a representative result of one of 3 independent experiments. (C) Gel contraction assay of control (CTL) and SNAIL1-/- (+ Cre) cardiac fibroblasts. (D) Quantification of (C). Shown is pooled data from 3 independent experiments, done in triplicates. (E-J) Relative fibrogenic gene mRNA expression in SNAIL1-/- cardiac fibroblasts following TGFβ stimulation: IL6 (E), TGFβ (F), CTGF (G), LoxL2 (H), Periostin (I and J). Representative example of 3 separate experiments. (K) Western blot analysis with periostin antibody of extracts from control (CTL) and SNAIL1-/- cardiac fibroblasts. Representative example of one of three separate experiments.