Matrix metalloproteinase-28 deletion amplifies inflammatory and extracellular matrix responses to cardiac aging

Abstract

To determine if matrix metalloproteinase (MMP)-28 mediates cardiac aging, wild-type (WT) and MMP-28-/- young (7 ± 1 months, n = 9 each) and old (20 ± 2 months, n = 7 each) female mice were evaluated. MMP-28 expression in the left ventricle (LV) increased 42% in old WT mice compared to young controls (p < 0.05). By Doppler echocardiography, LV function declined at 20 ± 2 months of age for both groups. However, dobutamine stress responses were similar, indicating that cardiac reserve was maintained. Plasma proteomic profiling revealed that macrophage inflammatory protein (MIP)-1 α, MIP-1β and MMP-9 plasma levels did not change in WT old mice but were significantly elevated in MMP-28-/- old mice (all p < 0.05), suggestive of a higher inflammatory status when MMP-28 is deleted. RT2-PCR gene array and immunoblotting analyses demonstrated that MIP-1α and MMP-9 gene and protein levels in the LV were also higher in MMP-28-/- old mice (all p < 0.05). Macrophage numbers in the LV increased similarly in WT and MMP-28-/- old mice, compared to respective young controls (both p < 0.05). Collagen content was not different among the WT and MMP-28-/- young and old mice. In conclusion, LV inflammation increases with age, and MMP-28 deletion further elevates inflammation and extracellular matrix responses, without altering macrophage numbers or collagen content.

Figure 1

MMP-28 protein expression in the LV increased with age. MMP-28 levels were up-regulated in WT old mice, compared to WT young mice. All values are mean ± SEM. n = 6/group. *p < 0.05 versus WT young. The data were analyzed by unpaired t-test.

Figure 2

MMP-28 deficiency increased LV MMP-9 expression with aging. MMP-9 protein levels in WT old mice did not change, compared to WT young levels. In contrast, MMP-9 levels significantly increased in MMP-28^−/− old mice, compared with both young controls and WT old mice. All values are mean ± SEM. n = 6/group. *p < 0.05 versus MMP-28^−/− young; ^†p < 0.05 versus WT old. The data were analyzed by ANOVA.

Figure 3

MMP-28 deletion did not affect LV macrophage numbers. Immunohistochemical staining for mac-3 revealed that LV macrophage numbers increased with age similarly in both WT and MMP-28^−/− mice. A: Representative images showed the positively stained macrophages (black staining). Scale = 200 µm. B: Quantitative analysis of positive cell numbers per high magnification field (magnification 400×). All values are mean ± SEM. n = 7/group. *p < 0.05 versus respective young control. The data were analyzed by ANOVA.

Figure 4

Total collagen content did not change between young and old mice. Total collagen quantity assessed by PSR staining was not different among the four groups. A: Representative images of PSR staining. B: Quantitative analysis of the ratio of collagen area to total area (magnification 200×). All values are mean ± SEM. n = 5/group. The data were analyzed by ANOVA.