Figure 1.
ILK and its associated protein levels in F and NF hearts.
A Expression of ILK, PINCH-1, PINCH-II, α-Parvin, β-Parvin, and ILK in NF and F human left ventricular lysates. GAPDH was the loading control. Six NF hearts and nine F hearts were used for all proteins except for PINCH-1 (12 F hearts). Shown here are representative examples of these samples. Note that two representative gels from two different runs were shown for PINCH-1 and PINCH-II respectively. Also, in some of the F hearts, there exists a higher molecular weight band above PINCH-II. The origin of the band is not clear. B–F Quantitation of protein expression in the F and NF hearts. (B) PINCH-1, (C) PINCH-II, (D) ILK), (E) α-Parvin, and (F) β-Parvin. *p<0.001 F vs. NF (paired t-test), n = 6 for NF hearts and N = 9 for DCM hearts for PINCH-1 ILK and α-Parvin protein expression. p = n.s for PINCHII and β-Parvin protein.
Figure 2.
The interaction of ILK/PINCH-1 in failing (F) human hearts.
A Immunoprecipitation of the α-Parvin/ILK/PINCH-1 complex in the F and NF human hearts. Extracts were immuno-precipitated with anti-PARVA antibody 1D4 (α-P). Immuno-precipitated proteins were immunoblotted with monoclonal anti-PARVA clone 3B5 antibody or with monoclonal anti-ILK antibody and monoclonal anti-PINCH antibody (Cell signaling, Inc). B. Phosphorylation of Akt Ser473 in representative F and NF hearts. The total Akt level is shown below. Six NF hearts and nine F hearts were used for all proteins except for PINCH-1 (12 F hearts). Shown here are representative examples of these samples. Results are presented as five different mice (n = 5).
Figure 3.
Expression profile for PIP in TAC and MI.
A. Individual PIP proteins were analyzed by Western blotting in mice 4 weeks after aortic banding (AB) Upper panel shows the protein expression of Pinch-1, the middle panel shows ILK and the bottom panel shows α-Parvin. Actin is used as a loading control. B. Quantification of panel A is shown on right side of panel A. The error bars represent the average ± S.E. of five different mice repeated in an independent experiments (n = 5, *p<0.001 compared to sham group, student t-test). Expression profile of PIP in MI and the effect of Tβ4 on MI. C. Western blot analysis of ILK, Pinch-1 and α- Parvin after 7 days post-MI. D. Western blot analysis of ILK, Pinch-1 and α- Parvin in Tβ4 treated MI mice. E. Right panel shows western blotting analysis of Akt 473 phosphorylation as well as total AKT level in 7 days post-MI mice. F. Quantification of blots from 3C, 3D and 3E are shown on right side of panel of 3E. Results are presented in five different mice (n = 5,* p<0.01 compared to sham or MI group, student t-test). The one way ANOVA test among the groups of MI and MI+Tβ4 also showed significant at p<0.05 level.
Figure 4.
Infarct size and Echocardiographic analysis of Tβ4 and Tβ4+ wortmannin treated MI mice.
A. The percent infarct size 7 days post-MI as determined by the epicardial infarct length divided by the epicardial left ventricle circumference in axial sections prepared with Masson's trichrome. The error bars represent the average ± S.E. of five different mice in an independent experiments (#p = 0.02 and was significant when compared MI vs. MI+Tβ4, *p = 0.69 and considered as non-significant in one way ANOVA analysis when compared to MI vs. MI+Tβ4+ wortmannin) B. Representative is the images of hearts after MI in the presence and absence of Tβ4 and wortmannin. C. Western blots analysis of phosphorylation of Akt Ser473 in of Sham (WT), MI, Tβ4 treated MI and MI+Tβ4+ wortmannin treated mice of 7 days post-MI. The total Akt level is shown below. D. The echocardiogram picture of Sham, MI, Tβ4 treated MI and MI+Tβ4+ wortmannin treated mice after 7 days of Tβ4 treatment. E–H Quantification of LVH, FSH, LVEDd and EF of the above mice after 7 days of MI. Results are presented in five different mice (n = 5, *p<0.001 compared to sham or MI group at 7 days post-op, student's t-test). The above parameters were not significant at day zero. The one way ANOVA test among the groups of MI and MI+Tβ4 also showed significant at p<0.05 level. The EF and FS in wortmannin +MI+Tβ4 treated group showed non- significant (N.S) when compared to MI+Tβ4 group.
Figure 5.
Tβ4 inhibits NF-κB translocation in MI mice.
A. Mice were subjected to MI and Tβ4 was injected and mice were kept for 7 days. NF-κB activation was measured by gel mobility shift assay using 32p NF-κB DNA as a probe. Cold NF-κB DNA was used for competition analysis. A p65 antibody was used for super shift analysis. B. Semi-quantification of panel A is shown on right side of panel A. Results are presented as five different mice (n = 5, *p<0.001 compared to sham or MI group, student's t-test). The one way ANOVA test among the groups of MI and MI+Tβ4 also showed significant at p<0.05 level.
Figure 6.
Tβ4 attenuates cardiac fibrosis.
A. Collagen type I and type III mRNA Expression was performed using their respective cDNA probes. Quantification of panel A is shown on right side of panel A. Results are presented as five different mice (n = 5, *p<0.001 compared to sham or MI group, student's t-test). The one way ANOVA test among the groups of MI and MI+Tβ4 also showed significant at p<0.05 level. B. Immuno-histological analysis was performed by Masson's trichome staining in MI and Tβ4 treated MI mice.