Fig 1.
Fibrosis and increase abundance of collagen and periostin in heart with increased expression of calreticulin.
(A) Gomori’s trichrome staining for collagen depositions in control and HeartCRT+ myocardium. The arrows indicate the location of the blue staining for collagen. Quantitative analysis of the percentage of areas with collagen deposition in control and HeartCRT+ hearts is shown to the right. **p<0.01. Data are representative of 6 biological replicates. (B) Abundance of fibrillar collagen mRNA was analyzed by Q-PCR. Values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. Col1A1, collagen type I, alpha 1; Col1A2, collagen type I, α2; Col3A1, collagen type III, α1; Col5A1, collagen type V, α1; Col5A2, collagen type V, α2. ** p<0.01. Data are representative of 9 biological replicates. (C) Immunoblot analysis and quantification of the abundance of collagen type I in control and HeartCRT+ hearts. Anti-GAPDH were used as a loading control. **p<0.01. Data are representative of 6 biological replicates. (D) Abundance of collagen type III in control and HeartCRT+ hearts. Anti-GAPDH antibodies were used as a loading control. *P<0.05. Data are representative of 6 biological replicates. (E) Subcellular distribution of type I collagen by immunohistochemistry in cardiac tissue cross-sections from control and HeartCRT+ hearts. Green color represents staining for type I collagen. (F) Abundance of periostin mRNA in control and HeartCRT+ hearts. **p<0.01. Data are representative of 9 biological replicates. Immnoblot and quantitative analyses of periostin in HeartCRT+ and control transgenic hearts. *p<0.05. Anti-GAPDH antibodies were used as a loading control. Data are representative of 6 biological replicates. (G) Cellular distribution of periostin in cardiac tissue cross-sections from control and HeartCRT+ hearts. Nuclei were visualized with DAPI staining. 3–5 animals were used for each analysis.
Fig 2.
TGFβ1 and unfolded protein response (UPR) in calreticulin expressing hearts.
(A) Immnoblot analysis and quantification of TGF-β1 protein in control and HeartCRT+ hearts. Antibodies to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as a loading control. **p<0.01. Data are representative of 6 biological replicates. (B) Real-time Q-PCR analysis for TGF-β1, β2 and β3 transcripts. **p<0.01. Data are representative of 6 biological replicates. (C) Immunoblot analysis of Smad2 and phospho-Smad2 (P-Smad2) at Ser465 and Ser467 antibodies from control and HeartCRT+ hearts. Quantitative analysis of P-Smad2/Smad2 expression ratio shown in. Anti-GAPDH antibodies were used as a loading control. * p<0.05. Data are representative of 6 biological replicates. (D) Immnuoblot analysis of Smad3 and phospho-Smad3 (P-Smad3) at Ser423 and Ser425 antibodies from control and HeartCRT+ hearts. Quantitative analysis of P-Smad3/Smad3 ratio. Anti-GAPDH antibodies were used as a loading control. NS, not significant. Data are representative of 6 biological replicates. (E) Real-time Q-PCR analysis of spliced XBP1 (sXBP1) mRNA abundance. The abundance of the sXBP1 transcripts in HeartCRT+ was slightly decreased (0.73±0.06 vs. control). **p<0.01. Data are representative of 6 biological replicates. NIH3T3 cells treated with tunicamycin (TM), an activator of unfolded protein response, were used as a positive control for the XBP1 splicing analysis. (F) Immnunoblot analyses of UPR markers were carried out with specific anti-ATF6 p50 fragment, anti-P-elF2α, anti-BiP and anti-CHOP antibodies. Anti-GAPDH antibodies were used as a loading control. Data are representative of 6 biological replicates.
Fig 3.
XBP1 splicing and abundance of ATF4, CHOP and BiP mRNA in the HeartCRT+ myocardium.
(A) Real-time Q-PCR analysis of XBP1 splicing as a function of treatment time. Mice were fed tamoxifen (TAM) to induced overexpression of calreticulin in the hearts of transgenic calreticulin mice. Immnunoblot probed with anti-calreticulin antibodies shows the abundance of the calreticulin protein (CRT, arrow) in transgenic calreticulin hearts. *, non-specific immunoreactive protein band. The graph below shows the abundance of spliced XBP1 (sXBP1) in the hearts of control animals (gray bars), and the TAM fed animals (black bars) and animals fed TAM plus tauroursodeoxycholic acid (TUDCA) (white bars). Day 7 *p = 0.0233, Day 14 **p = 0.0028. Data are representative of 6 biological replicates. 3–5 animals were used for each analysis. (B,C,D) Real-time Q-PCR analysis of abundance of ATF4 (B), CHOP (C) and BiP (D) mRNA as a function of treatment time. Mice were fed tamoxifen (TAM) to induced expression of calreticulin in the hearts of transgenic calreticulin mice. TUDCA, tauroursodeoxycholic. For day14 BiP mRNA graph *p = 0.0467, NS, not significant.
Fig 4.
Tauroursodeoxycholic acid (TUDCA) prevents cardiac fibrosis in HeartCRT+ mice.
(A) Trichrome staining for collagen deposition in the myocardium of control, transgenic and HeartCRT+ mice fed TUDCA. The arrows indicate the location of blue staining for collagen. (B) Quantitative analysis of the percentage of area with collagen deposition in control (ctrl), and HeartCRT+ animals fed tamoxifen (TAM) or TAM+ TUDCA at day 14 and day 21 *p<0.01. Data are representative of 6 biological replicates. (C) Immnunoblot analysis and quantification of TGFβ1 protein in hearts from HeartCRT+ mice fed TAM or TAM+TUDCA. Antibodies to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as a loading control. Data are representative of 6 biological replicates. (D) Real-time Q-PCR analysis for TGFβ1 transcript in hearts from HeartCRT+ animals fed TAM or TAM+ TUDCA. * p = 0.0254 ** p = 0.0148. Data are representative of 6 biological replicates. TAM samples were normalized to Day 0 and TAM+TUDCA samples were normalized to the corresponding day in TAM treatment to highlight the TUDCA effect. (E) Abundance of collagen type 1A1 in wild-type (wt) and transgenic mice fed TAM+TUDCA for 14 or 21 days. Antibodies to human influenza hemagglutinin (HA) were used to probe for the abundance of HA-tagged recombinant calreticulin (CRT). Anti-GAPDH antibodies were used as a loading control. (F) Abundance of collagen 1A1 mRNA in hearts from HeartCRT+ animals fed TAM or TAM+TUDCA. NS, not significant. *p = 0.0424. Data are representative of 6 biological replicates. TAM samples were normalized to Day 0 and TAM+TUDCA samples were normalized to corresponding day in TAM treatment to highlight the TUDCA effect. (G) Western blot analysis of periostin in hearts from TAM+TUDCA fed mice. Anti-HA antibodies were used to probe for abundance of HA-tagged recombinant calreticulin (CRT). Anti-GAPDH antibodies were used as a loading control. (H) Abundance of periostin mRNA in hearts from HeartCRT+ animals fed TAM or TAM+TUDCA. Data are representative of 6 biological replicates. TAM samples were normalized to Day 0 and TAM+TUDCA samples were normalized to corresponding day in TAM treatment to highlight the TUDCA effect. 3–5 animals were used for each analysis.