Relationship among LRP1 expression, Pyk2 phosphorylation and MMP-9 activation in left ventricular remodelling after myocardial infarction

Abstract

Left ventricular (LV) remodelling after myocardial infarction (MI) is a crucial determinant of the clinical course of heart failure. Matrix metalloproteinase (MMP) activation is strongly associated with LV remodelling after MI. Elucidation of plasma membrane receptors related to the activation of specific MMPs is fundamental for treating adverse cardiac remodelling after MI. The aim of current investigation was to explore the potential association between the low-density lipoprotein receptor-related protein 1 (LRP1) and MMP-9 and MMP-2 spatiotemporal expression after MI. Real-time PCR and Western blot analyses showed that LRP1 mRNA and protein expression levels, respectively, were significantly increased in peri-infarct and infarct zones at 10 and 21 days after MI. Confocal microscopy demonstrated high colocalization between LRP1 and the fibroblast marker vimentin, indicating that LRP1 is mostly expressed by cardiac fibroblasts in peri-infarct and infarct areas. LRP1 also colocalized with proline-rich tyrosine kinase 2 (pPyk2) and MMP-9 in cardiac fibroblasts in ischaemic areas at 10 and 21 days after MI. Cell culture experiments revealed that hypoxia increases LRP1, pPyk2 protein levels and MMP-9 activity in fibroblasts, without significant changes in MMP-2 activity. MMP-9 activation by hypoxia requires LRP1 and Pyk2 phosphorylation in fibroblasts. Collectively, our in vivo and in vitro data support a major role of cardiac fibroblast LRP1 levels on MMP-9 up-regulation associated with ventricular remodelling after MI.

Keywords: LRP1; MMP-9; cardiac remodelling; myocardial infarction; pPyk2.

Figure 1

Changes in Lrp1 mRNA and LRP1 protein levels after MI. Frozen myocardial tissue samples (≈ 40 mg) from the remote, peri‐infarct and infarct zones were homogenized in TriPure™ Reagent. RNA and protein were isolated as explained in the Materials and Methods. (A) RT‐PCR showing Lrp1 mRNA expression levels. Data were processed using a specially designed software program based on the Ct values of each sample and normalized to 18s rRNA, which served as an endogenous control. (B) Representative Western blot analysis results showing LRP1 and troponin T protein expression and bar graphs showing the mean ± S.D. of LRP1 protein bands normalized to troponin T bands. n = 8. *P < 0.05 versus remote; ^# P < 0.05 versus peri‐infarct; **P < 0.01 versus remote; ^## P < 0.01 versus peri‐infarct; ***P < 0.005 versus remote. (C) Representative immunostaining of heart cross sections showing the temporal evolution of LRP1 levels after MI. LRP1 is shown in red, and cTnI, in green. Cell nuclei were counterstained with DAPI (blue). Scale bars, 20 μm.

Figure 2

Identification of LRP1‐expressing cells in infarcted heart cross sections. LRP1 is shown in red; vimentin, in green; cTnI, in grey; and Lrp1 and vimentin colocalization, in yellow. Cell nuclei were counterstained with DAPI (blue). Scale bars, 20 μm.

Figure 3

Temporal and spatial evolution of myocardial phosphorylated Pyk2 and ERK1,2 levels after MI. (A) Representative Western blot analysis showing pPyk2, total Pyk2, pERK1,2 and total ERK1,2 protein expression. Bar graphs showing the mean ± S.D. of the pPyk2/total Pyk2 ratio and the pERK1,2/total ERK1,2 ratio in the remote, peri‐infarct and infarct zones at 1 (B, E), 10 (C, F) and 21 (D, G) days after MI. n = 8. *P < 0.05 versus remote; ^# P < 0.05 versus peri‐infarct; **P < 0.01 versus remote; ^## P < 0.01 versus peri‐infarct; ***P < 0.005 versus remote; ^### P < 0.005 versus peri‐infarct.

Figure 4

Temporal and spatial evolution of myocardial MMP‐9 and MMP‐2 activation and expression after MI. (A) Representative zymography analysis results showing myocardial MMP‐9 and MMP‐2 activity. Bar graphs showing the mean ± S.D. of MMP‐9 and MMP‐2 activity in the remote, peri‐infarct and infarct zones at 1 (B and E), 10 (C and F) and 21 (D and G) days after MI. (H–M) Real‐time PCR showing MMP‐9 and MMP‐2 mRNA expression levels. Data were processed using a specially designed software program based on the Ct values of each sample and normalized to 18s rRNA, which served as an endogenous control. The results are shown as the mean ± S.D. of MMP‐9 and MMP‐2 mRNA expression levels in the remote, peri‐infarct and infarct zones at 1 (H and K), 10 (I and L) and 21 (J and M) days after MI. n = 8. *P < 0.05 versus remote; **P < 0.01 versus remote; ^## P < 0.01 versus peri‐infarct; ***P < 0.005 versus remote; ^### P < 0.005 versus peri‐infarct.

Figure 5

Representative confocal microscopy images showing LRP1, MMP‐9 and pPyk2 levels in the remote, peri‐infarct and infarct zones after MI. Immunostaining of heart cross sections showing the temporal evolution of pPyk2, MMP‐9 and LRP1 expression levels after MI. LRP1 is shown in red; MMP‐9, in green; and pPyk2, in grey. A and B inserts are amplified in A’ and B’, respectively, to show colocalization of the three markers in light yellow. Cell nuclei were counterstained with DAPI (blue). Scale bars, 50 μm.

Figure 6

Representative confocal microscopy images showing LRP1, MMP‐9 and pERK1,2 expression levels in the remote, peri‐infarct and infarct zones after MI. (A) Immunostaining of heart cross sections showing the temporal evolution of pERK1,2 (grey), MMP‐9 (green) and LRP1 (red) expression levels after MI. (B) Immunostaining of heart cross sections showing the temporal evolution of pERK1,2 (grey), MMP‐2 (green) and LRP1 (red) expression levels after MI. Cell nuclei were counterstained with DAPI (blue). Scale bars, 50 μm.

Figure 7

Comparison of the hypoxia impact on LRP1, pPyk2 levels and MMP‐9/MMP‐2 activation in macrophages and fibroblasts. Quiescent macrophages and fibroblasts were exposed to normoxic or hypoxic conditions for 18 hrs. Representative Western blot analysis showing LRP1, pPyk2, total Pyk2 and β‐tubulin bands (A&G). Bar graphs showing the mean ± S.D. of LRP1 normalized to β‐tubulin levels (B&H) and pPyk2/total Pyk2 ratio (C&I). (D&J) Representative zymography analysis showing MMP‐9 and MMP‐2 activity and bar graphs showing the mean ± S.D. of MMP‐9 (E&K) and MMP‐2 (F&L) activity levels. Results are shown as the mean ± S.E.M. of three independent experiments performed in triplicate. ***P < 0.001 versus normoxia. NO: normoxia; HO: hypoxia.

Figure 8

Comparison of the hypoxia effect on pPyk2 levels and MMP‐9/MMP‐2 activation in control (Lrp1 ^+/+) and LRP1‐deficient fibroblasts (Lrp1 ^−/−). Quiescent Lrp1 ^+/+ and Lrp1 ^−/− fibroblasts were exposed to normoxic or hypoxic conditions for 18 hrs. (A) Representative Western blot analysis showing LRP1, pPyk2, total Pyk2 and β‐tubulin levels and bar graphs showing the mean ± S.D. of LRP1 levels normalized to β‐tubulin (B) and pPyk2/total Pyk2 ratio (C). (D) Representative zymography analysis showing MMP‐9 and MMP‐2 activity levels and bar graphs showing the mean ± S.D. of MMP‐9 (E) and MMP‐2 activity levels (F). Results are shown as the mean ± S.E.M. of three independent experiments performed in triplicate. ***P < 0.001 versus normoxia. (G) Representative scheme showing the crucial role of LRP1 up‐regulation in hypoxic cardiac fibroblasts in cardiac remodelling.