PFKFB3 Inhibitor 3PO Reduces Cardiac Remodeling after Myocardial Infarction by Regulating the TGF-β1/SMAD2/3 Pathway

Abstract

Adverse cardiac remodeling, including cardiac fibrosis, after myocardial infarction (MI) is a major cause of long-term heart failure. 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), an enzyme that regulates glucose metabolism, also plays an important role in various fibrotic and cardiovascular diseases. However, its effects on MI remain unknown. Here, PFKFB3 inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) and a permanent left anterior descending ligation mouse model were used to explore the functional role of PFKFB3 in MI. We showed that PFKFB3 expression increased significantly in the area of cardiac infarction during the early phase after MI, peaking on day 3. 3PO treatment markedly improved cardiac function, accompanied by decreased infarction size and collagen density in the infarct area. Meanwhile, 3PO attenuated cardiac fibrosis after MI by reducing the expression of collagen and fibronectin in murine hearts. Notably, 3PO reduced PFKFB3 expression and inhibited the transforming growth factor-beta 1/mothers against the decapentaplegic homolog 2/3 (TGF-β1/SMAD2/3) signaling pathway to inhibit cardiac fibrosis after MI. Moreover, PFKFB3 expression in neonatal rat cardiac fibroblasts (NRCFs) increased significantly after MI and under hypoxia, whereas 3PO alleviated the migratory capacity and activation of NRCFs induced by TGF-β1. In conclusion, 3PO effectively reduced fibrosis and improved adverse cardiac remodeling after MI, suggesting PFKFB3 inhibition as a novel therapeutic strategy to reduce the incidence of chronic heart failure following MI.

Keywords: PFKFB3; cardiac fibroblast; cardiac remodeling; fibrosis; myocardial infarction (MI).

Figure 1

PFKFB3 expression increases in cardiac tissue after myocardial infarction (MI). (A,B) Protein level detection of Pfkfb3 of murine hearts at days 1, 3, 7, 14, and 28 after MI in both infarct and non-infarct areas (n = 4). (C) The mRNA level of Pfkfb3 of murine hearts at days 1, 3, 7, 14 and 28 after MI in both infarct and non-infarct areas (n = 4–9). (D,E) Representative immunofluorescence staining of Pfkfb3 (green) and DAPI (blue) in murine heart at day 3 after MI, including the infarct area (IA), border area (BA), and non-infarct area (n = 4; scale bar, 50 μm). Data were expressed as mean ± SEM. Data presented in (B,C,E) were analyzed by one-way ANOVA tests. NS indicates not significant. * p < 0.05. ** p < 0.01. *** p < 0.001. DAPI indicates 4′,6-diamidine-2′-phenylindole dihydrochloride; and ROI, region of interest.

Figure 2

PFKFB3 inhibitor 3PO ameliorates cardiac remodeling induced by myocardial infarction (MI). (A) Schematic diagram of the experiment design. (B–E) Echocardiographic analysis of left ventricular (LV) ejection fraction (EF), LV fractional shortening (FS), LV end-diastolic volume (vol; d), LV end-systolic volume (vol; s), LV posterior wall thickness at diastole (LVPW; d) and interventricular septum thickness at diastole (IVS; d) at days 14 and 28 after MI (n = 9–13) in DMSO and 3PO mice, together with representative B-mode and M-mode echocardiographic images. (F) Representative Masson trichrome (TC, top and middle) and picrosirius red (PSR, lower) staining of heart transverse sections obtained from DMSO and 3PO mice at day 28 after MI (scale bar, 500 μm in the top images and 50 μm in the middle and lower images). (G) Quantitative analysis of infarction size (left) and collagen density (middle and right) in DMSO and 3PO mice at day 28 after MI (n = 4–6). Data were expressed as mean ± SEM. Data presented in (C–E,G) were analyzed by two-way ANOVA tests. NS indicates not significant. * p < 0.05. ** p < 0.01. *** p < 0.001. DMSO indicates Dimethylsulfoxide; 3PO, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one; and WT, wild type.

Figure 3

PFKFB3 inhibitor 3PO attenuates cardiac fibrosis induced by myocardial infarction (MI). (A) Total mRNA of whole murine hearts was isolated to compare the expression of Col1a1, Col3a1, and fibronectin between DMSO and 3PO mice at day 28 after MI (n = 4–7). (B–E) Representative immunofluorescence staining of col-1, col-3, fibronectin (all indicated in green), and DAPI (blue) in hearts of DMSO and 3PO mice at day 28 after MI (n = 3–4; scale bar,50 μm). Data were expressed as mean ± SEM. Data presented in (A,E) were analyzed by two-way ANOVA tests. NS indicates not significant. ** p < 0.01. *** p < 0.001. DMSO indicates Dimethylsulfoxide; 3PO, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one; Col-1, collagen I; Col-3, collagen III; FN, fibronectin; DAPI, 4′,6-diamidine-2′-phenylindole dihydrochloride; and ROI, region of interest.

Figure 4

3PO decreases expression of PFKFB3 and inhibits TGF-β1/Smad2/3 pathway after MI. (A) Schematic diagram of the experiment design. (B,C) Western blot analysis of PFKFB3, TGF-β1, P-SMAD2, SMAD2, P-SMAD3, SMAD3, and SMAD7 expression in the DMSO and 3PO mouse hearts at day 3 after MI (n = 3–4). Data were expressed as mean ± SEM. Data presented in (C) were analyzed by two-way ANOVA tests. NS indicates not significant. * p < 0.05. ** p < 0.01. *** p < 0.001. DMSO indicates Dimethylsulfoxide; 3PO, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one; and TGF-β1, transforming growth factor-β1.

Figure 5

PFKFB3 is abundantly expressed in cardiac fibroblast after MI and under hypoxia. (A) Representative dual-immunofluorescence staining of PFKFB3 (green) and Vimentin (red) in murine hearts at day 3 after MI (scale bar, 50 μm in the top and middle images, 12.5 μm in the lower left image, and 25 μm in the lower right image). (B,C) The mRNA level and protein level of PFKFB3 expression of cardiac fibroblasts treated under hypoxia for 6, 12, and 24 h, respectively (n = 3). (D) Representative immunofluorescence staining of Pfkfb3 (green) and DAPI (blue) of cardiac fibroblasts treated under hypoxia for 6, 12, and 24 h, respectively (n = 3–4, scale bar, 50 μm). (E) Cardiac fibroblasts were under hypoxia for 6 h after being treated with 3PO (30 μM) or DMSO for 1 h, and the protein levels of PFKFB3 expression were demonstrated by Western blot analysis (n = 4). Data were expressed as mean ± SEM. Data presented in (B–D) were analyzed by one-way ANOVA tests. Data presented in (E) was analyzed by Mann–Whitney U tests. NS indicates not significant. * p < 0.05. ** p < 0.01. *** p < 0.001. DMSO indicates Dimethylsulfoxide; 3PO, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one; DAPI, 4′,6-diamidine-2′-phenylindole dihydrochloride; and ROI, region of interest.

Figure 6

3PO mitigates the migration, proliferation, and activation of CFs induced by TGF-β1. (A,B) Cardiac fibroblasts were treated with 3PO (30 μM) or DMSO for 1 h and stimulated with TGF-β1 (20 ng/mL) or vehicle for 24 h, and the relative speed of migration was measured by wound healing assay (n = 3–4; scale bar, 1 mm). (C,D) Cardiac fibroblasts were treated with 3PO (30 μM) or DMSO for 1 h and stimulated with TGF-β1 (20 ng/mL) or vehicle for 24 h, and cell migration was measured by Boyden chamber migration assay (n = 5; scale bar, 100 μm). (E,F) Cardiac fibroblasts were treated with 3PO (30 μM) or DMSO for 1 h and stimulated with TGF-β1 (20 ng/mL) or vehicle for 24 h, and the proliferation capacity was measured by EDU staining assay (n = 4; scale bar, 100 μm). (G,H) Cardiac fibroblasts were treated with 3PO (30 μM) or DMSO for 1 h and stimulated with TGF-β1 (20 ng/mL) or vehicle for 24 h, and expression of α-SMA (green) was demonstrated by immunofluorescence staining to indicate fibroblast activation (n = 4; scale bar, 50 μm). Data were expressed as mean ± SEM. Data presented in (B,D,F,H) were analyzed by the two-way ANOVA tests. NS indicates not significant. ** p < 0.01. *** p < 0.001. DMSO indicates Dimethylsulfoxide; 3PO, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one; TGF-β1, transforming growth factor β1; EDU, 5-Ethynyl-2’-deoxyuridine; α-SMA, α-smooth muscle actin; DAPI, 4′,6-diamidine-2′-phenylindole dihydrochloride; and ROI, region of interest.