Loss of Bone Morphogenetic Protein-9 Reduces Survival and Increases MMP Activity After Myocardial Infarction

Figures

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  Figure 1. BMP-9 Loss Leads to Decreased Survival and Increased Incidence of Cardiac Rupture and Fibrosis Wild-type (WT) or BMP-9−/− mice were subjected to left anterior descending artery ligation to induce myocardial infarction (MI) and were allowed to recover over 14 days to assess their survival. (A) Survival plot showing the percentage of mice survived over 14 days after MI and incidences of cardiac ruptures among BMP-9−/− deaths. ∗ P < 0.05, log-rank test, n = 16 to 18. (B) Images showing MI with intact myocardium (WT MI) and ruptured myocardium (BMP-9−/−) observed at necropsy after MI. (C) Relative bone morphogenic protein-9 (BMP-9) protein levels in the left ventricle (LV) of mice from indicated groups corrected for GAPDH protein levels. (D) Fold change in BMP-9 messenger RNA (mRNA) levels in LV tissues corrected for 18s rRNA expression in indicated groups. (E) BMP-9 protein levels in LV tissue lysates detected by enzyme-linked immunosorbent assay in indicated groups. ∗P < 0.05, ∗∗∗P < 0.001, 1-way analysis of variance followed by Tukey’s honest significant difference, n = 5 to 7. (F, G) Picrosirius staining of an infarcted LV cross-section showing the extent of fibrosis along with a…

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  Figure 2. Loss of BMP-9 Increased Profibrotic Signaling, Collagen, and Endoglin Levels in the LV 2 Weeks After MI (A) Transforming growth factor β1 (TGF-β1) messenger RNA levels measured by real-time quantitative polymerase chain reaction and (B) TGF-β1 protein levels measured by enzyme-linked immunosorbent assay in LVs of indicated groups. Western blot images and corresponding quantification graphs for relative protein expression of (C) type 1 collagen, (D) endoglin, and (E) α-smooth muscle actin (α-SMA) corrected for GAPDH protein levels. Relative protein levels of (F) pSMAD1/5, (H) pSMAD3, and (H) pSMAD2 corrected for corresponding total SMADs. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, 1-way analysis of variance followed by Tukey’s honest significant difference, n = 5 to 10. rRNA = ribosomal RNA; abbreviations as in Figure 1.

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  Figure 3. BMP-9−/− Mice Have Increased Levels of MMP-9 Protein and Enzymatic Activity 2 Weeks After MI Immunoblots, zymographs, and corresponding quantification graphs for (A) Western blot images showing relative matrix metalloproteinase-9 (MMP-9) protein levels corrected for GAPDH protein levels and (B) zymography gel image and corresponding quantification showing relative MMP-9 enzyme activity levels in infarcted and noninfarcted regions of LVs of mice from indicated groups 2 weeks after MI. (C) Western blot images showing relative MMP2 protein levels corrected for GAPDH protein levels in LVs of indicated groups and (D) zymography gel image and corresponding quantification showing relative MMP2 enzyme activity levels in infarcted and noninfarcted regions of LVs of mice from indicated groups 2 weeks after MI. ∗P < 0.05, ∗∗P < 0.01 ∗∗∗P < 0.001, 1-way analysis of variance followed by Tukey’s honest significant difference, n = 5 to 7. Abbreviations as in Figure 1.

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  Figure 4. Loss of BMP-9 Is Associated With Increased Levels of Type 1 Collagen Degradation Marker Serum levels of (A) procollagen type I N-terminal propeptide (PINP) and (B) type 1 collagen C-terminal telopeptide (ICTP) 2 weeks after MI. (C) Bar graph showing increased serum ICTP/PINP ratio in BMP-9−/− mice 2 weeks after MI indicating increased type 1 collagen degradation. ∗P < 0.05, 1-way analysis of variance followed by Tukey’s honest significant difference, n = 4 to 5. Abbreviations as in Figure 1.

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  Figure 5. siRNA-Mediated Knockdown of BMP-9 Leads to Increased MMP-9 Protein Expression and Enzymatic Activity in H-CFbs (A) Bar graph showing fold changes in BMP-9 and ID1 (inhibitor of DNA binding 1) Messenger RNA expression levels in human cardiac fibroblasts (H-CFbs) transfected with control (siControl) or BMP-9 small interfering RNA (siBMP-9). (B) Graphs showing relative protein expression of BMP-9 C type 1 collagen. (D) Graph showing relative MMP-9 activity assessed by zymography. Graphs showing relative protein expression of (E) MMP-9, (F) pSMAD3 corrected for tSMAD3, (G) pSMAD1/5 corrected for tSMAD1, (H) endoglin, and (I) α-smooth muscle actin in H-CFbs transfected with scrambled siRNA (siControl) or siRNA against BMP-9. (J) Corresponding zymography gel image and Western blot images. ∗P < 0.05, ∗∗P < 0.01 ∗∗∗P < 0.001. Student’s t test, n = 4 to 6. Abbreviations as in Figures 1, 2, and 3.

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  Figure 6. BMP-9 Is Required for Survival After MI and Its Loss Is Associated With Increased Incidences of Myocardial Rupture and Death Central figure depicting the mechanism of the TGF-β/BMP-9-mediated wound healing process after MI. The intact BMP-9 axis preserves physiological scar formation while, loss of BMP-9-medicated signaling increases fibrosis and myocardial rupture along with collagen degradation. AMI = acute myocardial infarction; other abbreviations as in Figures 1, 2, and 3.