Matrix metalloproteinase-9 deletion attenuates myocardial fibrosis and diastolic dysfunction in ageing mice

Abstract

Aims: Age-related diastolic dysfunction has been attributed to an increased passive stiffness, which is regulated by extracellular matrix (ECM). We recently showed that matrix metalloproteinase (MMP)-9, an ECM mediator, increases in the left ventricle (LV) with age. The aim of this study, accordingly, was to determine the role of MMP-9 in cardiac ageing.

Methods and results: We compared LV function in young (6-9 months), middle-aged (12-15 months), old (18-24 months) and senescent (26-34 months) wild-type (WT) and MMP-9 null mice (n ≥ 12/group). All groups had similar fractional shortenings and aortic peak velocities, indicating that systolic function was not altered by ageing or MMP-9 deletion. The mitral ratios of early to late diastolic filling velocities were reduced in old and senescent WT compared with young controls, and this reduction was attenuated in MMP-9 null mice. Concomitantly, the increase in LV collagen content was reduced in MMP-9 null mice (n = 5-6/group). To dissect the mechanisms of these changes, we evaluated the mRNA expression levels of 84 ECM and adhesion molecules by real-time qPCR (n = 6/group). The expression of pro-fibrotic periostin and connective tissue growth factor (CTGF) increased with senescence, as did transforming growth factor-β (TGF-β)-induced protein levels and Smad signalling, and these increases were blunted by MMP-9 deletion. In senescence, MMP-9 deletion also resulted in a compensatory increase in MMP-8.

Conclusion: MMP-9 deletion attenuates the age-related decline in diastolic function, in part by reducing TGF-β signalling-induced periostin and CTGF expression and increasing MMP-8 expression to regulate myocardial collagen turnover and deposition.

Figure 1

MMP-9 expression increases with ageing in the LV of WT mice. (A) By real-time qPCR, MMP-9 levels increase in old and senescent LV compared with young and MA controls. (B) A representative immunoblot showing that LV MMP-9 protein levels increase with senescence. (C) A graph showing the densitometry results of the MMP-9 immunoblot. Sample sizes are n = 6 per group for real-time qPCR and n = 12 per group for immunoblotting *P < 0.05 compared with the young control; +P < 0.05 compared with the MA control; #P < 0.05 compared with the old control.

Figure 2

MMP-9 deletion attenuates the age-related decline in diastolic function. (A) At 30 min after dobutamine injection, FS of MMP-9 null mice are similar to their age-matched WT, whereas senescent WT mice have reduced FS compared with old WT mice. Sample sizes are n ≥ 11/group. (B) Aortic peak velocities are not statistically different with age and are similar between WT and MMP-9 null mice (all P = not significant). (C) E/A ratios reduce with age in WT but not MMP-9 null mice, and old and senescent MMP-9 null mice have higher E/A ratios compared with age-matched WT mice. *P < 0.05 compared with the young control; +P < 0.05 compared with the MA control; #P < 0.05 compared with the old control; ^P < 0.05 compared with age-matched WT controls. Sample sizes are n ≥ 12/group.

Figure 3

MMP-9 deletion attenuates the increase in the LV collagen content with senescence. (A) Representative pictures of picrosirius red staining of LV mid-cavity sections of young and senescent WT and MMP-9 null mice. (B) A graph of percentages of the collagen area showing an increased collagen content in the senescent WT and MMP-9 null LV compared with corresponding young controls. Additionally, senescent MMP-9 null LV have less collagen content than senescent WT LV. Sample sizes are n = 5 or 6/group. *P < 0.05 compared with the young control; ^P < 0.05 compared with age-matched WT control. Sample sizes are n = 6/group.

Figure 4

MMP-9 deletion attenuates the age-related increases in periostin and CTGF mRNA expression. (A) Type I collagen mRNA expression levels decrease, rather than increase, with senescence in WT mice. (B) Senescent MMP-9 null mice express reduced levels of type III collagen mRNA compared with the corresponding young, MA and old controls. (C and D) Senescent WT mice express increased levels of periostin (C) and CTGF (D) compared with young WT mice, and senescent MMP-9 null mice express reduced levels of periostin and CTGF compared with senescent WT mice. *P < 0.05 compared with the young control; +P < 0.05 compared with the MA control; #P < 0.05 compared with the old control; ^P < 0.05 compared with age-matched WT control. Sample sizes are n = 6/group.

Figure 5

(A) By real-time qPCR, the gene expression of tgfbi increased with senescence in WT mice, but this increase was attenuated in MMP-9 null mice. (B) The increase of tgfbi protein levels in WT senescent mice was attenuated in the null senescent mice. (C) By immunoblotting, the psmad2/smad2 ratio increased in the WT senescent group, and this increase was attenuated in the null senescent LV. *P < 0.05 compared with the young control; +P < 0.05 compared with the MA control; #P < 0.05 compared with the old control; ^P < 0.05 compared with age-matched WT control. Sample sizes are n = 6/group.

Figure 6

MMP-9 deletion led to a compensatory increase in MMP-8 expression in senescence. (A) MMP-8 mRNA levels increased with senescence in MMP-9 null mice, and senescent MMP-9 null mice had higher MMP-8 mRNA levels than age-matched WT control. (B) Immunoblotting comparing MMP-8 protein levels in senescent WT and MMP-9 null mice. (C) Densitometry results showed higher MMP-8 levels in senescent MMP-9 null LV compared with WT senescent LV. Sample sizes are n = 12/group. (D) Ly-6G immunoblotting showed no significant changes in neutrophil levels with age (WT P = 0.78; Null P = 0.09). Sample sizes are n = 6/group. *P < 0.05 compared with the young control; +P < 0.05 compared with the MA control; #P < 0.05 compared with the old control; ^P < 0.05 compared with age-matched WT controls.

Figure 7

A proposed simplified overview of MMP-9 involvement in cardiac ageing. Cardiac ageing is associated with increased LV collagen deposition and a decline in diastolic function. With ageing, MMP-9 expression increases in the LV. The increased MMP-9 levels increases TGF-β activation, and therefore, increases transcription of TGF-β inducible genes. Periostin and CTGF are two TGF-β inducible genes that are shown to be pro-fibrotic. The increased periostin and CTGF expressions promote collagen deposition in the ageing LV, in turn causing a decline in diastolic function. MMP-9 deletion reduces TGF-β activation and TGF-β inducible transcription, resulting in decreased levels of periostin and CTGF in senescent null mice. Additionally, there is a compensatory increase in MMP-8 levels in senescent MMP-9 null mice, which promotes collagen degradation. The reduced pro-fibrotic signals and increased collagen degradation explain the lower LV collagen content and improved diastolic function in senescent MMP-9 null mice compared with age-matched WT mice.