Neuropeptide Y attenuates cardiac remodeling and deterioration of function following myocardial infarction

Abstract

Plasma levels of neuropeptide Y (NPY) are elevated in patients with acute myocardial infarction (AMI), but its role in AMI remains unclear, which was examined here in NPY wild-type/knockout (WT/KO) mice treated with/without exogenous NPY and its Y1 receptor antagonist (Y1Ra) BIBP 3226. We found that AMI mice lacking NPY developed more severe AMI than WT mice with worse cardiac dysfunction, progressive cardiac inflammation and fibrosis, and excessive apoptosis but impairing angiogenesis. All of these changes were reversed when the NPY KO mice were treated with exogenous NPY in a dose-dependent manner. Interestingly, treatment with NPY also dose dependently attenuated AMI in WT mice, which was blocked by BIBP 3226. Phenotypically, cardiac NPY was de novo expressed by infiltrating macrophages during the repairing or fibrosing process in heart-failure patients and AMI mice. Mechanistically, NPY was induced by transforming growth factor (TGF)-β1 in bone marrow-derived macrophages and signaled through its Y1R to exert its pathophysiological activities by inhibiting p38/nuclear factor κB (NF-κB)-mediated M1 macrophage activation while promoting the reparative M2 phenotype in vivo and in vitro. In conclusion, NPY can attenuate AMI in mice. Inhibition of cardiac inflammation and fibrosis while enhancing angiogenesis but reducing apoptosis may be the underlying mechanisms through which NPY attenuates cardiac remodeling and deterioration of function following AMI.

Keywords: AMI; NPY; Y1 receptor; cardiac remodeling; macrophage.

Graphical abstract

Figure 1

NPY is largely expressed in areas with cardiac repairing after ischemic injury in patients with chronic heart failure and in a mouse model of AMI (A and B) Representative immunohistochemical staining of NPY from a normal, unmatched heart or patients with failing heart. Scale bar, 50 μm. (C and D) Representative immunohistochemical staining of NPY cardiac tissues from sham and AMI mice over a 28-day period after AMI, Scale bar, 50 μm. Note that NPY expression is peaking at day 7 and declined afterward over 28 days during AMI. (E) Plasma levels of NPY over the 28-day period after AMI. Note that NPY is rapidly elevated and peaked at day 1 after AMI. (F and G) Two-color immunofluorescence shows that most NPY-expressing cells within the infarct area at day 7 after AMI are macrophage phenotypes by co-expressing the F4/80 antigen. The above panels are general views (scale bar, 50 μm) the infarct area of heart, and the bottom panels are partial infarct region (scale bar, 25 μm). (H) Three-color flow cytometry analysis of sorted CD45⁺ cells infiltrating the infarcted heart at day 7 after AMI. Results show that about 70% of CD45⁺ cells are co-expressing NPY (CD45⁺NPY⁺). Of them, 90% are of macrophage phenotype (CD68⁺NPY⁺). Among NPY-expressing macrophages, most of them (80%) are M2 macrophages by co-expressing CD206 (CD68⁺NPY⁺CD206⁺). Each dot represents one patient or mouse. Data represent mean ± SEM for groups of 3−10 patients or 4−6 mice. ∗∗p < 0.01, ∗∗∗p < 0.001 versus normal (B) or sham groups (C) or day 0 (E); #p < 0.05, ##p < 0.01 versus day 1 (E) or day 3 (C); and &p < 0.05 versus day 3 (E) or day 7 (C).

Figure 2

Deletion of NPY aggravates cardiac dysfunction and increases infarcted size and cardiac remodeling after AMI (A and B) Representative echocardiography (M-mode) and LV function at 7 days after AMI. (C) The survival curve of AMI mice over 7 days after AMI (n = 11, p = 0.3561). (D) Representative images and quantitative analysis of infarcted size by TTC and Masson’s trichrome staining at 7 days after AMI. (E and F) Immunohistochemical staining and quantitative analysis of α-SMA⁺ myofibroblasts and collagen I accumulation at the infarcted site on day 7 after AMI. Each dot represents one mouse, and data represent mean ± SEM for groups of 6−8 mice. ∗p < 0.05, ∗∗p < 0.01 versus corresponding sham (B) or NPY WT (D) or sham (F) and #p < 0.05 versus AMI WT mice (B and F). Scale bar, 200 μm.

Figure 3

Effect of NPY on angiogenesis at day 7 after AMI induced in NPY WT/KO mice treated with/without exogenous NPY and/or Y1 receptor antagonist (Y1Ra; BIBP) The angiogenesis is identified by CD31-expressing endothelial cells. (A and D) Representative immunohistochemical staining of CD31 expression of NPY WT and KO mice. (B and E) Representative immunohistochemical staining of CD31 expression in NPY KO mice received different dosages of exogenous NPY (micrograms per kilogram per day for 7 days). (C and F) Representative immunohistochemical staining of CD31 expression in NPY WT mice treated with NPY (25 μg/kg/day) with/without BIBP 3226 (300 μg/kg/day) for 7 days. Data represent mean ± SEM for groups of 6 mice. ∗p < 0.05, ∗∗∗∗p < 0.0001 versus corresponding sham groups; #p < 0.05, ##p < 0.01 versus WT-MI (D) or saline (E and F); &p < 0.05 versus NPY 25 (E) or BIBP (F); and @p < 0.05 versus NPY 50 (E) or NPY (F). Scale bar, 50 μm.

Figure 4

Exogenous delivery of NPY significantly attenuates cardiac dysfunction and fibrosis in NPY KO mice with AMI (A and B) Representative echocardiography (M-mode) and LV function at 7 days after AMI. (C) Representative images and quantitative analysis of infarcted size by TTC and Masson’s trichrome staining at 7 days after AMI. (D) Representative images and quantitative analysis of α-SMA and collagen I by western blot at 7 days after AMI. (E) Immunohistochemical staining and quantitative analysis of α-SMA⁺ myofibroblasts and collagen I accumulation at the infarcted site on day 7 after AMI. Each dot represents one mouse, and data represent mean ± SEM for groups of 6−12 mice. ∗p < 0.05, ∗∗p < 0.01 versus sham and #p < 0.05 versus saline. Scale bar, 200 μm.

Figure 5

Mice treated with NPY significantly attenuated cardiac remodeling 7 days after AMI (A and B) Representative echocardiography (M-mode) and LV function at 7 days after AMI. (C) Representative images and quantitative analysis of infarcted size by TTC and Masson’s trichrome staining at 7 days after AMI. (D) Representative images and quantitative analysis of α-SMA and collagen I by western blot at 7 days after AMI. (E) Immunohistochemical staining and quantitative analysis of α-SMA⁺ myofibroblasts and collagen I accumulation at the infarcted site on day 7 after AMI. Each dot represents one mouse, and data represent mean ± SEM for groups of 6−12 mice. ∗p < 0.05, ∗∗p < 0.01 versus sham and #p < 0.05 versus saline. Scale bar, 200 μm.

Figure 6

BIBP 3226 can abrogate the function of NPY that attenuated cardiac remodeling at day 7 after AMI (A and B) Representative echocardiography (M-mode) and LV function at 7 days after AMI. (C) Representative images and quantitative analysis of infarcted size by TTC and Masson’s trichrome staining at 7 days after AMI. (D) Representative images and quantitative analysis of Y1R, CD206, and iNOS by western blot at 7 days after AMI. (E−G) Two-color immunofluorescence shows that NPY switches M1 macrophages to M2 phenotype within the infarcted area at day 7 after AMI by co-expressing iNOS and CD206 antigen, which is abrogated by NPY Y1Ra BIBP. (H) Plasma levels of IL-6 at 7 days after AMI. Each dot represents one mouse, and data represent mean ± SEM for groups of 6−12 mice. ∗p < 0.05, ∗∗p < 0.01 versus sham; #p < 0.05, ##p < 0.01 versus saline; &p < 0.05 versus BIBP; and @p < 0.05 versus NPY. Scale bar, 50 μm.

Figure 7

NPY switches M1 macrophages to M2 phenotype, which is abrogated by NPY Y1Ra BIBP in a mouse model of AMI and BMDMs (A) Three-color flow cytometry shows the M1 (CD68⁺iNOS⁺) and M2 (CD68⁺CD206⁺) macrophages in the infarcted site at 7 days after AMI. (B) Two-color immunofluorescence shows M1 (F4/80⁺iNOS⁺) and M2 (F4/80⁺CD206⁺) macrophages in NPY WT and KO mice at 7 days after AMI. (C) Representative images and quantitative analysis of CD206 and iNOS by western blot in the infarct area of NPY WT and KO mice at 7 days after AMI. (D) Representative images and quantitative analysis of Y1R, CD206, and iNOS by western blot in BMDMs derived from NPY WT and KO mice after stimulated for 24 h by TNF-α. (E) Representative images and quantitative analysis of Y1R, CD206, and iNOS by western blot in BMDMs derived from NPY WT mice stimulated with TNF-α for 24 h under administration of NPY or BIBP. Each dot represents one mouse, and data represent mean ± SEM for groups of 6−12 mice or three independent experiments. ∗p < 0.05, ∗∗p < 0.01 versus sham (A) or WT (B) or corresponding sham (C) or corresponding cytotoxic T lymphocyte (CTL; D) or CTL (E); #p < 0.05 versus saline (A) or WT AMI (C) or WT TNF-α (D) or TNF-α (E); &p < 0.05 versus BIBP; and @p < 0.05 versus NPY. Scale bar, 50 μm.

Figure 8

NPY is functional with Y1R to inhibit cardiac remodeling via regulating macrophage polarization by targeting the p38 MAPK and NF-κB pathways (A and B) Representative images and quantitative analysis of Y1R, P-p38, p38, P-p65, and p65 by western blot in the infarct area of NPY WT and KO mice at 7 days after AMI. Note that mice lacking NPY resulted in inhibition of Y1R expression but largely promoted phosphorylation of p38 and NF-κB/p65. (C and D) Representative images and quantitative analysis of p-p38, p38, p-p65, and p65 by western blot in the infarct area of NPY WT mice treated with NPY or BIBP at 7 days after AMI. (E and F) Representative images and quantitative analysis of p-p38, p38, p-p65, and p65 by western blot in BMDMs derived from NPY WT mice stimulated with TNF-α for 24 h under NPY treatment. Note that NPY can dose dependently inhibit the p38 MAPK and NF-κB signaling pathways, and the 10⁻⁹ M dose is the best. (G and H) Representative images and quantitative analysis of p-p38, p38, p-p65, and p65 by western blot in BMDMs derived from NPY WT mice stimulated with TNF-α for 24 h under administration of NPY or BIBP. Each dot represents one mouse, and data represent mean ± SEM for groups of 6 mice or three independent experiments. ∗p < 0.05, ∗∗p < 0.01 versus corresponding sham (B) or sham (D) or CTL (F and H); #p < 0.05, ##p < 0.01 versus WT MI (B) or saline (D) or TNF-α (F and H); &p < 0.05 versus BIBP (D) or TNF-α + BIBP (H); and @p < 0.05 versus NPY (D) or TNF-α + NPY (H).