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. 2010 May 7;9(5):2649-57.
doi: 10.1021/pr100147r.

In vivo matrix metalloproteinase-7 substrates identified in the left ventricle post-myocardial infarction using proteomics

Ying Ann Chiao  1 , Rogelio Zamilpa, Elizabeth F Lopez, Qiuxia Dai, Gladys P Escobar, Kevin Hakala, Susan T Weintraub, Merry L Lindsey
Affiliations

In vivo matrix metalloproteinase-7 substrates identified in the left ventricle post-myocardial infarction using proteomics

Ying Ann Chiao et al. J Proteome Res. .

Abstract

Matrix metalloproteinase-7 (MMP-7) deletion has been shown to improve survival after myocardial infarction (MI). MMP-7 has a large array of in vitro substrates, but in vivo substrates for MMP-7 following MI have not been fully identified. Accordingly, we evaluated the infarct regions of wild-type (WT; n = 12) and MMP-7 null (null; n = 10) mice using a proteomic strategy. Seven days post-MI, infarct regions of the left ventricles were excised, homogenized, and protein extracts were analyzed by two-dimensional gel electrophoresis and mass spectrometry. Of 13 spots that showed intensity differences between WT and null, the intensities of eight spots were higher and those of five spots were lower in the null group (p < 0.05). Fibronectin and tenascin-C, known in vitro substrates of MMP-7, were identified in spots that showed lower intensity in the null. Immunoblotting and in vitro cleavage assays confirmed reduced fibronectin and tenascin-C fragment generation in the null, and this effect was restored by exogenous administration of MMP-7. Lower levels of full-length peroxiredoxin-1 and -2 and higher levels of the full-length peroxiredoxin-3 were detected in the null group, suggesting MMP-7 deletion may also indirectly regulate protein levels through nonenzymatic mechanisms. In conclusion, this is the first study to identify fibronectin and tenascin-C as in vivo MMP-7 substrates in the infarcted left ventricle using a proteomic approach.

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Figures

Figure 1
Figure 1
Representative 2-DE gels of WT infarct extract (left) and MMP-7 null infarct extract (right). Spot numbers are indicated in the MMP-7 null gel.
Figure 2
Figure 2
MMP-7 null infarcted LV showed lower levels of fibronectin by 2-DE and immunoblotting. (A) Fibronectin was identified in spot 17 and spot 23 of the 2-DE gels. Both spot 17 (B) and spot 23 (C) showed significant lower intensity in infarct LV of MMP-7 null mice compared to WT. (D) Immunoblotting of fibronectin in WT and MMP-7 null LV extracts were performed. The densitometry of the 273 kDa full-length (E) and the 166 kDa fragments (F) of fibronectin indicated both bands showed significantly lower intensity in MMP-7 null LV infarct when compared with the wt (p<0.05).
Figure 3
Figure 3
Immunoblotting showed lower levels of full-length and fragments of tenascin-C in MMP-7 null LV infarct. (A) 4 major bands of 133 kDa, 170 kDa, 200 kDa and 260 kDa were observed by immunoblotting of TN-C in WT and MMP-7 null LV. (B) Densitometry showed significantly lower levels of full length (260 kDa) TN-C in MMP-7 null LV infarct when compared to WT. (C) Although the 200 kDa form did not show a significant difference, both 170 kDa (D) and 130kDa (E) fragments of TN-C showed significantly lower intensity in the MMP-7 null group.
Figure 4
Figure 4
Levels of different Prx isoforms were differentially regulated by MMP-7 deletion. Immunoblotting showed significantly lower intensity of Prx-1 (A) and Prx-2 (B) in the MMP-7 null LV infarct when compared to WT. (C) However, higher levels of Prx-3, a mitochondrial Prx, were detected in the MMP-7 null group. (D) No significant difference of Prx-6 between WT and MMP-7 null groups were observed.
Figure 5
Figure 5
The addition of exogenous MMP-7 generated Fn and TN-C fragments in MMP-7 null infarct extract. (A) MMP-7 null extract treated with 100 pg or 10 ng of recombinant MMP-7 showed reduced levels of full-length Fn (273 kDa) and increased levels of the 166 kDa Fn fragment compared to the untreated control. (B) MMP-7 null extract treated with 100 pg or 10 ng of MMP-7 showed reduced levels of full-length TN-C (260 kDa and 200 kDa) in the null extract, but increased the levels of 65 kDa fragment in the MMP-7 null extract.
Figure 6
Figure 6
Schematic diagram of the direct and indirect effects of MMP-7 deletion in infarcted left ventricles. When MMP-7 plays a proteolytic role and cleaves the full-length protein and to fragments, the deletion of MMP-7 is expected to result in lower levels of fragments. If the fragments can feedback induce the expression of the protein, lower levels of fragments due to MMP-7 deletion will reduce expression and therefore, result in lower levels of full length proteins. MMP-7 also indirectly regulates the expression of proteins, including Prx-1 and -3. If MMP-7 indirectly suppresses the expression of one protein, MMP-7 deletion will result in increased expression of that protein and result in higher levels of full-length protein. On the other hand, if MMP-7 indirectly induces the expression of one protein, MMP-7 deletion will result in reduced expression of that protein and result in lower levels of full-length protein.
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