TIMP-2 mutant decreases MMP-2 activity and augments pressure overload induced LV dysfunction and heart failure

Abstract

Pressure overload induces cardiac extracellular matrix (ECM) remodelling and results in heart failure. ECM remodelling by matrix metalloproteinases (MMPs) is primarily regulated by their target inhibitors, tissue inhibitor of matrix metalloproteinases (TIMPs). It is known that TIMP-2 is highly expressed in myocardium and is required for cell surface activation of pro-MMP-2. We and others have reported that imbalance between angiogenic growth factors and anti-angiogenic factors results in transition from compensatory cardiac hypertrophy to heart failure. We previously reported the pro-angiogenic role of MMP-2 in cardiac compensation, however, the specific role of TIMP-2 during pressure overload is yet unclear. We hypothesize that genetic ablation of TIMP-2 exacerbates the adverse cardiac matrix remodelling due to lack of pro-angiogenic MMP-2 and increase in anti-angiogenic factors during pressure overload stress and results in severe heart failure. To verify this, ascending aortic banding (AB) was created to mimic pressure overload, in wild type C57BL6/J and TIMP-2-/- (model of MMP-2 deficiency) mice. Left ventricular (LV) function assessed by echocardiography and pressure-volume loop studies showed severe LV dysfunction in TIMP-2-/- AB mice compared to controls. Expression of MMP-2, vascular endothelial growth factor (VEGF) was decreased and expression of MMP-9, anti-angiogenic factors endostatin and angiostatin was increased in TIMP-2-/- AB mice compared with wild type AB mice. Connexins (Cx) are the gap junction proteins that are widely present in the myocardium and play an important role in endothelial-myocyte coupling. Our results showed that expression of Cx 37 and 43 was decreased in TIMP-2-/- AB mice compared with corresponding wild type controls. These results suggest that genetic ablation of TIMP-2 decrease the expression of pro-angiogenic MMP-2, VEGF and increases anti-angiogenic factors that results in exacerbated abnormal ventricular remodelling leading to severe heart failure.

Figure 1

A. Genotyping of wild type and TIMP-2-/- mice. Wild type band shown at 250 Kbp and TIMP-2-/- at K280 bp. B. Zymography data showing the activity of MMP-2 in sham (wt), Wt AB 8 weeks, TIMP-2-/- Ctrl and TIMP-2-/- AB 8 weeks mice. Bar graph represents densitometry values in arbitrary units. *p<0.05 was considered significant compared with wt ctrl and wt AB and ^#p<0.05 compared with wt AB 8 week group.

Figure 2

Representative M-mode echocardiography images in sham (wt), Wt AB 8 weeks, TIMP-2-/- Ctrl and TIMP-2-/- AB 8 weeks. Percentage ejection fraction (%EF) was obtained from Pressure-volume loop study. Bar graphs represent % FS, LVEDd (mm) and % EF values. *p<0.05 was considered significant compared with sham and ^#p<0.05 compared with wt AB 8 week group.

Figure 3

IHC staining of heart sections with connexins 37 and 43 antibodies in sham (wt), Wt AB 8 weeks, TIMP-2-/- Ctrl and TIMP-2-/- AB 8 weeks. Expression of Cx 37 is seen as green fluorescence intensity and Cx 43 as red fluorescence intensity. Scale bar – 20 μm. *p<0.05 was considered significant compared with sham and ^#p<0.05 compared with wt AB 8 week group.

Figure 4

Masson’s trichrome staining of heart sections in sham (wt), Wt AB 8 weeks, TIMP-2-/- Ctrl and TIMP-2-/- AB 8 weeks. Bar graphs represent percentage collagen. *p<0.05 was considered significant compared with sham and ^#p<0.05 compared with wt AB 8 week group.

Figure 5

mRNA expressions of MMP-2 and VEGF in sham (wt), Wt AB 8 weeks, TIMP-2-/- Ctrl and TIMP-2-/- AB 8 wks.

Figure 6

Immunohistochemistry staining for CD 31 (angiogenesis marker) in sham (wt), Wt AB 8 weeks, TIMP-2-/- Ctrl and TIMP-2-/- AB 8 weeks. Scale bar – 20 μm. Bar graph depicts the number of CD 31+ve capillaries. *p<0.05 was considered significant compared with sham and ^#p<0.05 compared with wt AB 8 week group.

Figure 7

mRNA expressions of MMP-9 and angiostatin in sham (wt), Wt AB 8 weeks, TIMP-2-/- Ctrl and TIMP-2-/- AB 8 weeks.

Figure 8

Protein expression of MMP-2, MMP-9, MMP-14 and angiostatin by western blot in sham (wt), Wt AB 8 weeks, TIMP-2-/- Ctrl and TIMP-2-/- AB 8 weeks. *p<0.05 was considered significant compared to sham and ^#p<0.05 compared with wt AB 8 week group.

Figure 9

Immunostaining of heart sections with MMP-2 and MMP-9 antibodies in sham (wt), Wt AB 8 weeks, TIMP-2-/- Ctrl and TIMP-2-/- AB 8 weeks. Expression of MMP-2 is seen as red fluorescence intensity and MMP-9 as green fluorescence intensity. Scale bar – 50 μm. *p<0.05 was considered significant compared with sham and ^#p<0.05 compared with wt AB 8 week group.

Figure 10

IHC staining of heart sections with angiostatin and endostatin antibodies in sham (wt), Wt AB 8 weeks, TIMP-2-/- Ctrl and TIMP-2-/- AB 8 weeks. Expression of angiostatin is seen as green fluorescence intensity and endostatin as red fluorescence intensity. Scale bar – 50 μm. *p<0.05 was considered significant compared with sham and ^#p<0.05 compared with wt AB 8 week group.