Amylin deposition activates HIF1α and 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3) signaling in failing hearts of non-human primates

Figures

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  Fig. 1. Cardiac pathological changes in non-human primates (NHPs) with control (CTL) and heart failure (HF). a–e Histopathological evaluation of heart tissue from NHPs with CTL (n = 7) and HF (n = 6). a CTL hearts showed no abnormality. b–d HF hearts showed myocardial interstitial fibrosis, hypertrophy of cardiomyocytes with atypical karyomegaly, infiltrations of mixed inflammatory cells, and randomly distributed, multifocal degeneration and necrosis of cardiomyocytes. Arteriopathy with thickening of the tunica media of the vascular smooth muscles and narrowing of the lumen was also observed in NHPs with HF. e Histopathological grades of CTL and HF were determined using the semi-quantification scoring system (0-not apparent change,1-minimal, 2-moderate, 3-marked, 4-severe). f–k Masson’s trichrome staining and Picro Sirius red staining of heart tissue from NHPs with CTL (n = 7) and HF (n = 6). f, i No abnormality was observed in the CTL. g, j The HF hearts showed diffused blue-stained (g, Masson’s trichrome stain) and red-stained (j, Picro Sirius red stain). h, k Quantification of fibrotic area was shown in histogram. Scale bar, 100 μm. Data represent mean ± SEM, **P < 0.01 by student’s t…

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  Fig. 2. Amylin deposition in the heart sections of NHPs with HF. a–f Amylin staining visualized with DAB chromogen. a Positive staining for amylin in pancreas from CTL NHPs. b No amylin deposits were observed in the CTL heart (n = 7). c–f Amylin deposition in the nucleus (c), hypertrophic cardiomyocyte sarcolemma (c and d), inflammatory cells (e), and blood cells (f) of the HF group (n = 6). g Bar graph presented the relative positive signal intensity for amylin in a 284 × 214 μm field of view of heart sections from NHPs with CTL (n = 7) and HF (n = 6). Scale bar, 100 µm. Data represent mean ± SEM, *P < 0.05 by student’s t test.

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  Fig. 3. Circulating amylin and HF markers levels in NHPs. a Amylin concentration in serum from a parallel set of NHPs (CTL, n = 10; HF, n = 7). b Protein levels of ST2, GDF-15, and cTnI in serum as in panel (a). c–e The correlations between serum amylin level and ST2 (c), cTnI (d), and GDF-15 (e), respectively, in serum as in panel (a) and (b). Amylin levels in matched plasma (f) and RBC lysates (g) from the same animals whose hearts were used for histopathological evaluation in Fig. 1 and amylin assessment in Fig. 2 (CTL, n = 7; HF, n = 6). Data represent mean ± SEM. *P < 0.05; **P < 0.01 by Student’s t test. The correlations between serum amylin level and ST2 (c), cTnI (d), and GDF-15 (e), respectively, were analyzed by Spearman nonparametric correlation analysis in GraphPad 7.04 (GraphPad Inc., San Diego, CA), and the values for the Spearman r were indicated on the plots.

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  Fig. 4. Activation of HIF1α, PFKFB3, and PFKFB3 phosphorylation at Ser461 in the myocardium of NHPs with HF. Expression of HIF1α (a–e), PFKFB3 (f–j), and pSer461-PFKFB3 (k–l) in hearts from CTL (n = 7) and HF (n = 6) NHPs. qRT-PCR for HIF1α mRNA (a) and PFKFB3 (h) in hearts from NHPs with HF (n = 6) and CTL (n = 7). Immunohistochemistry of HIF1α (b) and PFKFB3 (f) (brown color) in heart sections from NHPs with HF and CTL as used in panel (a). Quantifications of the relative positive signal intensity for HIF1α (c) and PFKFB3 (g) in a 284 × 214 μm field of view of heart sections from NHPs as used in panels (a). Western blot analysis with anti-HIF1α (d), anti-PFKFB3 (i), and anti-pSer461-PFKFB3 (k) antibodies on heart lysates as used in panels (a). Quantifications of protein expression in western blot were shown in bar graphs (e, j, and l). Scale bar, 100 µm. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test.

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  Fig. 5. Cardiac amylin accumulation activated HIF1α and PFKFB3 in hiPSC-CMs. a Immunostaining of α-actinin (a, green color, mouse anti-α-Actinin antibody), positive signal of human amylin (a, red color, rabbit anti-human amylin antibody) and Hoechst 33342 (blue color) in cells treated with H-amy and b quantification of amylin positive signal was shown in bar graph (n = 6/group). c–l Expression of HIF1α (c–g) and PFKFB3 (h–l) in hiPSC-CMs treated with CTL and H-amy. Positive signal of HIF1α (c, green color, mouse anti-HIF1α antibody), PFKFB3 (h, red color, rabbit anti-PFKFB3 antibody) and Hoechst 33342 (c and h, blue color) levels were quantified as shown in bar graph (d, HIF1α; i, PFKFB3, n = 9/group). Western blot analysis of HIF1α (e, f) and PFKFB3 (j, k) in hiPSC-CMs incubated under the two conditions described in panel (c) (n = 3/group). qRT-PCR of HIF1α (g) and PFKFB3 (l) in myocytes incubated with CTL and H-amy (n = 5/group). Scale bar, 100 µm. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.01 by Student’s t test.

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  Fig. 6. Amylin drives the expression of HIF1α and PFKFB3 in hiPSC-CMs. a, b Effect of pre-treatment of hiPSC-CMs with a human amylin antibody for 24 h on amylin-induced HIF1α (a) and PFKFB3 (b) mRNA levels (n = 6/group). c Effect of pre-treatment of hiPSC-CMs with siRNA for HIF1α (siHIF1α) or for vehicle control (siCTL) on amylin-induced HIF1α (n = 4/group). d Effect of pretreatment of hiPSC-CMs with siHIF1α, siRNA for PFKFB3 (siPFKFB3), or siCTL on amylin-induced PFKFB3 (n = 4/group). e, f Effect of HIF1α inhibitor YC-1 on the upregulation of HIF1α (e) and PFKFB3 (f) induced by amylin (n = 6/group). Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by One-way ANOVA with Tukey’s post-test.

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  Fig. 7. Cardiac amylin accumulation activated HIF1α and PFKFB3 in isolated RVCMs. a–j Increased expression of HIF1α (a–d, i) and PFKFB3 (e–h, j) in RVCMs treated with or without H-amy for 2 h. a, b Immunofluorescence staining (a, green color, mouse anti-HIF1α antibody; blue color, Hoechst 33342) in cells treated under the above conditions and b quantification of HIF1α positive signal was shown in bar graph (n = 10/group). c, d Western blot analysis (blot, c and bar graph, d) of HIF1α (mouse anti-HIF1α antibody) in RVCMs treated as in panel (a) (n = 3/group). e, f Immunofluorescence staining (e, red color, rabbit anti-PFKFB3 antibody; blue color, Hoechst 33342) in cells under the above conditions and f quantification of PFKFB3 positive signal was shown in bar graph (n = 10/group). g, h Western blot analysis (blot, g and bar graph, h) of PFKFB3 (rabbit anti-PFKFB3 antibody) in RVCMs treated as in panel (a) (n = 3/group). i, j Increased mRNA levels of HIF1α (i) and PFKFB3 (j) in RVCMs incubated with amylin (n = 10/group). Scale bar, 100 µm. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test.

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  Fig. 8. Cardiac amylin accumulation induced changes in Ca2+ level, oxidative stress, mitochondrial function and lactate production in hiPSC-CMs. a Steady-state intracellular Ca2+ concentration was measured in hiPSC-CMs incubated with or without amylin for 2 h (n = 4/group). b ROS production was compared in hiPSC-CMs treated as in panel (a) (n = 6/group). c Mitochondrial membrane potential (MMP) (n = 6/group) and d ATP level (n = 8/group) were measured in hiPSC-CMs treated with amylin for 2 h. e Effect of pre-treatment with HIF1α inhibitor YC-1 on lactate production induced by amylin (n = 7/group). Data represent mean ± SEM. *P < 0.05; ***P < 0.001 by Student’s t test (a–d) and One-way ANOVA with Tukey’s post-test (e).