Cardiac-specific over-expression of TGFBI.

<p>(A) Schematic representation of the double transgenic (DTG) system used to generate cardiac-specific, doxycycline inducible expression of TGFBI. (B) Western blot analysis of TGFBI in hearts of DTG mice. GAPDH was used as a control. (C) Immunohistochemistry of heart histological sections at 6 months of age from WT or TGFBI DTG mice. Sections were imaged for the proteins or markers shown. Scale = 10 μm. (D) Heart-weight (HW) normalized to body-weight (BW) in WT and DTG mice at 6 months of age. *p<0.05 vs WT using an unpaired Student’s T-test. (E) Percent fractional shortening (FS%) in WT and DTG mice at 6 months of age. *p<0.05 vs WT using an unpaired Student’s T-test. (F) Left ventricular dimension in diastole (LVED) in WT and DTG mice at 6 months of age as measured by echocardiography. (G) Percent fibrosis measured from Masson’s trichrome-stained cardiac histological sections from WT and DTG mice at 1 year of age. (H) Assessment of FS% in the indicated groups of mice over 12 weeks after sham or TAC surgery. (I) HW normalized to BW in the indicated groups of mice after 12 weeks after TAC. *p<0.05 vs corresponding sham group. (J) Assessment of lung-weight (LW) normalized to BW in the indicated groups of mice after 12 weeks of TAC. *p<0.05 vs corresponding sham group analyzed by parametric one way ANOVA with a Newman-Keuls post-hoc test.</p

Loss of <i>Tgfbi</i> does not alter disease progression after MI.

<p>(A) Percent survival in the groups of mice shown over 7 days following MI surgery. No mortality was observed in sham-operated WT or <i>Tgfbi</i>^<i>-/-</i> mice. No differences in survival between WT and <i>Tgfbi</i>^<i>-/-</i> after MI was detected using the log rank Mantel-Cox statistical test. (B) Western blot analysis for TGFBI and periostin from isolated infarct areas of the hearts of the indicated groups of mice 7 days after MI. GAPDH was used as a loading control. (C and D) Quantification of protein levels normalized to GAPDH using densitometry for periostin in WT and <i>Tgfbi</i>^<i>-/-</i>-operated hearts and for TGFBI in WT and <i>Postn</i>^<i>-/-</i>-operated hearts. *p<0.05 vs WT using an unpaired Student’s T-test. (E) Percent survival in the indicated groups of mice over 7 days after sham or MI surgery. p<0.05 vs WT MI-operated animals using the log rank Mantel-Cox test (F) Percent ventricular fractional shortening (FS%) as measured by echocardiography in the indicated genotypes of mice 8 weeks after MI. *p<0.05 vs sham. (G) Left ventricular end diastolic dimension (LVED) as measured by echocardiography in the indicated groups of mice 8 weeks after MI. *p<0.05 vs sham using a parametric one way ANOVA with a Newman-Keuls post-hoc test.</p

TGFBI and periostin are induced in the heart after injury.

<p>(A) Western blot analysis of periostin and TGFBI in isolated adult cardiomyocytes and fibroblasts. Sarcomeric α-actin was used as a control for cardiomyocyte purity and GAPDH was used as a loading control. (B) Quantitative real time PCR for <i>Postn</i> and <i>Tgfbi</i> from 1 week sham or MI-operated hearts. mRNA levels were normalized to 18s ribosomal RNA. *p<0.05 for both genes in MI-operated animals compared to sham animals using an unpaired Student’s T-test. n = 3 animals. (C) Western blot analysis for periostin and TGFBI in the infarcted areas isolated from hearts 24 hours, 7 and 14 days after MI surgery. GAPDH was used as a loading control. Each lane corresponds to protein from one mouse. (D and E) Quantification of TGFBI (D) and periostin (E) protein levels from the conditions shown in panel C except that 3 hearts were analyzed in total each. (F) Immunohistochemistry for the indicated markers/proteins on sham or MI-operated hearts after 1 week. Images were taken at 400x magnification. Scale = 20 μm.</p

Loss of <i>Tgfbi</i> does not appreciably alter disease progression after long-term TAC.

<p>(A) Percent ventricular fractional shortening as measured by echocardiography 12 weeks after TAC in the indicated groups. *p<0.05 vs sham-operated controls. (B) Heart-weight normalized to body-weight in the indicated groups of mice after 12 weeks of TAC. *p<0.05 vs sham-operated controls. (C) Cardiomyocyte cross-sectional area from ventricular histological sections in the indicated groups of mice 12 weeks after MI. *p<0.05 vs sham-operated controls. Four to six hearts were processed from each group, with at least 3 fields analyzed per histological section. (D) Lung-weight normalized to body-weight in the indicated groups of mice 12 weeks after TAC. *p<0.05 vs sham-operated controls. (E) Ventricular fibrosis from Masson's trichrome-stained histological images in the indicated genotypes of mice after 12 weeks of TAC. *p<0.05 vs sham-operated controls. Four to six hearts were processed from each group, with at least 3 fields analyzed per histological section. (F) Representative Masson’s trichrome-stained histological heart sections from sham and TAC hearts after 12 weeks of MI. Images were taken at 100x magnification. Scale bar = 100 μm. All statistical analyses were performed using a parametric one way ANOVA with a Newman-Keuls post-hoc test.</p