Matrix metalloproteinase-28 deletion exacerbates cardiac dysfunction and rupture after myocardial infarction in mice by inhibiting M2 macrophage activation

Abstract

Rationale: Matrix metalloproteinase (MMP)-28 regulates the inflammatory and extracellular matrix responses in cardiac aging, but the roles of MMP-28 after myocardial infarction (MI) have not been explored.

Objective: To determine the impact of MMP-28 deletion on post-MI remodeling of the left ventricle (LV).

Methods and results: Adult C57BL/6J wild-type (n=76) and MMP null (MMP-28((-/-)), n=86) mice of both sexes were subjected to permanent coronary artery ligation to create MI. MMP-28 expression decreased post-MI, and its cell source shifted from myocytes to macrophages. MMP-28 deletion increased day 7 mortality because of increased cardiac rupture post-MI. MMP-28(-/-) mice exhibited larger LV volumes, worse LV dysfunction, a worse LV remodeling index, and increased lung edema. Plasma MMP-9 levels were unchanged in the MMP-28((-/-)) mice but increased in wild-type mice at day 7 post-MI. The mRNA levels of inflammatory and extracellular matrix proteins were attenuated in the infarct regions of MMP-28(-/-) mice, indicating reduced inflammatory and extracellular matrix responses. M2 macrophage activation was impaired when MMP-28 was absent. MMP-28 deletion also led to decreased collagen deposition and fewer myofibroblasts. Collagen cross-linking was impaired as a result of decreased expression and activation of lysyl oxidase in the infarcts of MMP-28(-/-) mice. The LV tensile strength at day 3 post-MI, however, was similar between the 2 genotypes.

Conclusions: MMP-28 deletion aggravated MI-induced LV dysfunction and rupture as a result of defective inflammatory response and scar formation by suppressing M2 macrophage activation.

Figure 1. Expression patterns and cellular source of MMP-28 at baseline and post-MI

A, MMP-28 expression decreased in both remote and infarct regions post-MI. MMP-28^−/− day 0 hearts were used as negative controls. n=4–6/group. *p<0.05 vs. day 0. B, MMP-28 co-localized with phalloidin, a marker of myocytes, at baseline (day 0). C, MMP-28 from macrophages increased in the infarct region post-MI. MMP-28 co-localized with Mac-3, a marker of macrophages, at day 5 post-MI. Representative images from n=3 stained LVs for B and C. Blue=DRAQ5, Green=MMP-28, Red=phalloidin or Mac-3.

Figure 2. MMP-28 deletion decreased survival, increased cardiac rupture, and exacerbated LV dysfunction post-MI

A, Infarct areas were similar between WT and MMP-28^−/− mice. n=8–14/group. B, MMP-28 deletion reduced 7 day post-MI survival. n=6–26/group. C, Cardiac rupture post-MI was increased in MMP-28^−/− mice. n= 10–24/group. D and E, LV end systolic and diastolic volumes at day 7 post-MI were higher in MMP-28^−/−, compared with WT. F, Ejection fraction in MMP-28^−/− mice was attenuated at day 1, but exacerbated at day 7 post-MI, compared with WT. G, The ratio of lung weight to tibia length (edema index) at day 7 post-MI was higher in MMP-28^−/− than in WT. H, MMP-28 deletion showed higher EDV/ LV mass (LV remodeling index) at day 7 post-MI. n=8–14/group for D through H. *p<0.05 vs. day 0 and #p<0.05 vs. WT.

Figure 3. M2 macrophage activation was impaired with MMP-28 deletion

A, Galectin-3 expression was similarly induced in WT and MMP-28^−/− mice post-MI. Isolated peritoneal macrophages were used as positive controls. n=4/group. B, Macrophage numbers infiltrated into infarct area at day 5 post-MI showed no difference between WT and MMP-28^−/− mice. n=5–7/group. C, MMP-28 deletion did not affect M1 macrophage markers (except IL-1β) in day 7 infarct. D, MMP-28 deletion attenuated M2 macrophage markers in day 7 infarct. n=6/group for C and D. *p<0.05 vs. day 3 and #p<0.05 vs. WT.

Figure 4. MMP-28 deletion attenuated macrophages polarization towards M2 subtype

Peritoneal macrophages were stimulated with IFN-γ+LPS or IL-4. A, MMP-28 expression was similar among different macrophage subtypes. B, MMP-28 deletion did not affect macrophage activation towards M1 subtype. C, MMP-28 deletion attenuated macrophage polarization towards M2 phenotype. n=4/group for A through C. *p<0.05 vs. unstimulated and #p<0.05 vs. WT.

Figure 5. Collagen deposition, lysyl oxidase content, and collagen cross-linking were reduced with MMP-28 deletion

A and B, MMP-28 deletion led to reduced collagen I and collagen III accumulation at day 7 post-MI. C, Both precursor and active forms of lysyl oxidase at day 7 post-MI were suppressed with MMP-28 deletion. n=4/group for A through C. Dand E, Hydroxylysyl pyridinoline and lysyl pyridinoline in day 7 infarct were reduced with MMP-28 deletion. n=3/group. *p<0.05 vs. day 3, ^p<0.05 vs. day 5, and #p<0.05 vs. WT day 7.

Figure 6. MMP-28 deletion reduced myofibroblast numbers at day 7 post-MI and affected myofibroblast function

A, α-SMA expression at day 7 post-MI was reduced in MMP-28^−/− mice, compared to WT. Isolated cardiac fibroblasts were used as positive controls. n=4/group. B, Immunofluorescence staining showed that MMP-28^−/− mice had fewer myofibroblast numbers in the non-vessel interstitial area at day 7 after MI. Blue=DAPI, red=α-SMA. Representative images from n=3 stained LVs. C and D, TGF-β1 induced similar upregulation in α-SMA in both WT and MMP-28^−/−cardiac fibroblasts. E, Fibronectin 1 was lower in MMP-28^−/− fibroblasts stimulated with TGF-β1, compared to WT. F, TGF-β1 induced similar upregulation in CTGF in both genotypes. n=4/group for C through F. *p<0.05 vs. unstimulated and #p<0.05 vs. WT.