Soluble RAGE attenuates myocardial I/R injuries via FoxO3-Bnip3 pathway

Abstract

Soluble receptor for advanced glycation end-products (sRAGE) was reported to inhibit cardiac apoptosis through the mitochondrial pathway during myocardial ischemia/reperfusion (I/R) injury. Meanwhile, the proapoptotic protein Bcl2 and adenovirus E1B 19-kDa-interacting protein 3 (Bnip3) was reported to mediate mitochondrial depolarization and be activated by the Forkhead box protein O3 (FoxO3a). Therefore, it is supposed that FoxO3a-Bnip3 pathway might be involved in the inhibiting effects of sRAGE on mitochondrial apoptosis during I/R. I/R surgery or glucose deprivation/reoxygenation was adopted to explore mitochondrial depolarization, apoptosis and related signaling pathways in mice hearts and cultured cardiomyocytes. The results showed that overexpression of sRAGE in cardiomyocytes dramatically improved cardiac function and reduced infarct areas in I/R treated mice. sRAGE inhibited mitochondrial depolarization and cardiac apoptosis during I/R, which correlated with reduced expression of Bnip3, Sirt2, phosphorylation of Akt and FoxO3a which translocated into nucleus in cultured cardiomyocytes. Either Sirt2 or FoxO3a silencing enhanced the inhibiting effects of sRAGE on mitochondrial depolarization induced by I/R in cultured cardiomyocytes. Meanwhile, overexpression or silencing of FoxO3a affected the inhibiting effects of sRAGE on Bnip3 and cleaved caspase-3 in cultured cardiomyocytes. Therefore, it is suggested that sRAGE inhibited I/R injuries via reducing mitochondrial apoptosis through the FoxO3a-Bnip3 pathway.

Keywords: Inflammatory cytokines; Mitochondrial damages; Myocardium; Sirtuins.

Fig. 1

sRAGE inhibited cardiac dysfunction and reduced infarct area induced by I/R in mice. a The representative M-mode echocardiography images of long-axis. The red line segments indicated the systolic position of the endocardium, green line segments indicated the diastolic position of the endocardium and the white arrows indicated the amplitude of the left ventricular anterior wall motion. b Quantification of left ventricular ejection fraction (LVEF, %) for each group. c Quantification of fractional shortening (FS, %) for each group. d Quantification of left ventricular cardiac output (CO, mL/min) for each group. e Quantification of left ventricular end-diastolic volume (LVEDV, μL) for each group. f Quantification of left ventricular end-systolic volume (LVESV, μL) for each group. g Quantification of heart rate (HR, bpm) for each group. h The representative TTC staining images for each group. i Quantification of the infarct area/total left ventricular area for each group. n = 6 per group. The data are expressed as the mean ± SEM. **p < 0.01; ns nonsignificant (p ≥ 0.05)

Fig. 2

sRAGE inhibited cardiac apoptosis during I/R. a Representative images of TUNEL staining in each group. TUNEL-positive spots were shown as red. The green fluorescence indicated α-actinin antibody staining. The blue (DAPI) fluorescence indicated the nuclei. The white arrows indicated apoptosis positive cardiomyocyte. The scale bar is 50 μm. b Representative images of immunochemistry staining for cleaved caspase-3 in each group. The target protein was shown as brown. The scale bar is 50 μm. c Quantitative data of TUNEL staining. n = 6 per group. d Quantitative data of immunochemistry staining images for cleaved caspase-3. n = 6 per group. The data are expressed as the mean ± SEM. **p < 0.01

Fig. 3

sRAGE inhibited mitochondrial damages during I/R. a Representative images of TMRE staining in sRAGE treated cardiomyocytes. The orange–red fluorescence (TMRE) intensity indicated ΔΨm changes. The blue fluorescence (Hoechst 33342) indicated nuclei. The scale bar is 50 μm. b Quantification of TMRE fluorescence intensity in each group. n = 6 per group. c Representative images of immunochemistry staining for Bnip3 in each group. The target protein was shown as brown. The scale bar is 50 μm. d Quantitative data of immunochemistry staining images for Bnip3. n = 6 per group. e Representative images of Western blotting for Bnip3. f Quantitative analysis of the Western blotting results for Bnip3. n = 3 per group. g Representative images of Bnip3 and VDAC double staining. Bnip3 was shown as red, VDAC was shown as brown and the black arrows indicated Bnip3 translocation into the mitochondria. The scale bar is 50 μm or 10 μm. n = 6 per group. h Representative images of Western blotting for Bnip3. i Quantitative analysis of the Western blotting results for Bnip3 in cytoplasm. n = 3 per group. j Quantitative analysis of the Western blotting results for Bnip3 in mitochondria. n = 3 per group. The data are expressed as the mean ± SEM. *p < 0.05; **p < 0.01

Fig. 4

sRAGE inhibited FoxO3a translocation to nucleus during I/R. a Representative images of immunochemistry staining for FoxO3a. The target protein was shown as brown and the black arrows indicated FoxO3 in the nucleus. The scale bar is 50 μm or 10 μm. b Quantitative data of immunochemistry staining images for FoxO3a. n = 6 per group. c Representative images of Western blotting for FoxO3a. d Quantitative analysis of the Western blotting results for FoxO3a in cytoplasm. n = 5 per group. e Quantitative analysis of the Western blotting results for FoxO3a in nucleus. n = 5 per group. The data are expressed as the mean ± SEM. *p < 0.05; **p < 0.01

Fig. 5

Increased or Silenced FoxO3 affected the inhibiting effects of sRAGE on I/R-induced cardiomyocytes apoptosis. a Representative images of Western blotting for Bnip3 and cleaved caspase-3. b Quantitative analysis of the Western blotting results for Bnip3, n = 3 per group. c Quantitative analysis of the Western blotting results for cleaved caspase-3, n = 4 per group. d Representative images of Western blotting for Bnip3 and cleaved caspase-3. e Quantitative analysis of the Western blotting results for Bnip3, n = 5 per group. f Quantitative analysis of the Western blotting results for cleaved caspase-3, n = 4 per group. The data are expressed as the mean ± SEM. *p < 0.05; **p < 0.01

Fig. 6

sRAGE inhibited Akt and FoxO3a phosphorylation, and Sirt2 expression during I/R in cardiomyocytes. a Representative images of Western blotting for Akt, FoxO3a and Sirt2 of each group. b Quantitative analysis of the Western blotting results for phosphorylated Akt/Akt, n = 4 per group. c Quantitative analysis of the Western blotting results for phosphorylated FoxO3a/FoxO3a, n = 5 per group. d Quantitative analysis of qRT-PCR results for Sirt1, Sirt2 and Sirt3 of each group, n = 3 per group. e Quantitative analysis of the Western blotting results for Sirt2, n = 8 per group. f Representative images of TMRE staining in AK-7 treated cardiomyocytes. The orange–red fluorescence (TMRE) intensity indicated ΔΨm changes. The blue fluorescence (Hoechst 33342) indicated nuclei. The scale bar is 50 μm. g Quantification of TMRE fluorescence intensity in each group. n = 6 per group. h Representative images of TMRE staining in si-FoxO3a treated cardiomyocytes. i Quantification of TMRE fluorescence intensity in each group. n = 6 per group. The data are expressed as the mean ± SEM. *p < 0.05; **p < 0.01; ns nonsignificant (p ≥ 0.05)

Fig. 7

sRAGE selectively decreased plasma levels of inflammatory cytokines during I/R. a Quantification of plasma IL-6 level. b Quantification of plasma IL-17 level. c Quantification of plasma KC level. d Quantification of plasma MIP-2 level. e Quantification of plasma GM-CSF level. n = 8–9 per group. The data are expressed as the mean ± SEM. *p < 0.05; **p < 0.01; ns nonsignificant (p ≥ 0.05)