Vaspin Mediates the Intraorgan Crosstalk Between Heart and Adipose Tissue in Lipoatrophic Mice

Figures

• 

  FIGURE 1. Cardiac dysfunction is negatively correlated with serum level of vaspin in A-ZIP/F-1 lipoatrophic mice. (A) The ratio of heart weight/tibia length of 4-, 12-, and 24-week-old A-ZIP/F-1 mice. The C57BL/6J (C57) mice were the control group. (B,C) Representative images of whole hearts and cardiac slides were stained with hematoxylin and eosin solution (B), and quantitative analysis of average cardiomyocyte size (C). Scale bar = 20 μm. (D,E) Echocardiographic analysis of mouse cardiac ejection fraction (EF) (D) and fractional shortening (FS) (E). (F) Enzyme-linked immunosorbent assay (ELISA) analysis of mouse serum levels of vaspin. (G,H) The correlation between serum levels of vaspin and EF and FS in A-ZIP/F-1 mice. Results are shown as mean ± SEM, and n = 6–8 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001.

• 

  FIGURE 2. Administration of recombinant vaspin protein attenuates cardiac hypertrophy and fibrosis in A-ZIP/F-1 lipoatrophic mice. Sixteen-week-old A-ZIP/F-1 mice were treated with recombinant vaspin protein (100 μg/mouse) by an osmatic pump for 8 weeks. (A) The ratio of heart weight/tibia length. (B,C) Representative images of whole hearts, and cardiac slides were stained with hematoxylin and eosin solution (B), and quantitative analysis of average cardiomyocyte size (C). Scale bar = 20 μm. (D,E) Western blot analysis of cardiac atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) (D), and quantitative analysis of relative density of ANP/tubulin and BNP/tubulin (E). (F,G). Cardiac slides were stained with Sirius red solution (F) and quantitative analysis of relative density of collagen (G). Scale bar = 40 μm. (H,I) Western blot analysis of cardiac TGF-β1 and SMAD3 (H) and quantitative analysis of relative density of TGF-β1/tubulin and SMAD3/tubulin (I). Results are shown as mean ± SEM, and n = 8 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001.

• 

  FIGURE 3. Vaspin increases cardiac mitochondrial biogenesis and activities in A-ZIP/F-1 lipoatrophic mice. Sixteen-week-old A-ZIP/F-1 mice were treated with recombinant vaspin protein (100 μg/mouse) by an osmatic pump for 8 weeks. (A,B) The cardiac levels of triglyceride (A) and free fatty acid (B). (C,D) The representative images of cardiac mitochondria (C) and quantitative analysis of lipid droplets (D). (E,F) Western blot analysis of cardiac α-porin and PGC-1α (E), and quantitative analysis of relative density (F). (G) Real-time quantitative analysis of cardiac mitochondrial genes, including Pgc-1α, Nrf1, Nrf2, and Tfam. (H–K) Measurement of relative ATP production (H), oxygen consumption (I), citrate synthase activity (J), and ratio of complex I/CS (K) in extracted cardiac mitochondria. Results are shown as mean ± SEM, and n = 6–8 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001.

• 

  FIGURE 4. Blockage of vaspin reverses the cardioprotective benefits of fat transplantation in A-ZIP/F-1 lipoatrophic mice. Sixteen-week A-ZIP/F-1 mice were subcutaneously transplanted with 0.9 g of visceral fat from C57BL/6J mice and locally injected with 1 109 lentivirus-encoding vaspin siRNA or control siRNA for 8 weeks. (A) The serum levels of vaspin in mice treated with recombinant vaspin protein for 0, 1, 4, and 8 weeks. (B) The ratio of heart weight/tibia length. (C–E) Representative images of whole hearts, and cardiac slides were stained with hematoxylin and eosin or Sirius red staining solution (C), and quantitative analysis of average cardiomyocyte size (D) and relative density of collagen (E). Scale bar = 20 μm. (F,G) Echocardiographic analysis of mouse cardiac EF (F) and FS (G). (H,I) The representative images of cardiac mitochondria (H) and quantitative analysis of lipid droplets (I). Results are shown as mean ± SEM, and n = 7 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001.

• 

  FIGURE 5. Vaspin improves cardiomyocyte mitochondrial function by upregulating phosphorylated AKT. (A,B) Sixteen-week-old A-ZIP/F-1 mice were treated with recombinant vaspin protein (100 μg/mouse) by osmatic pump for 8 weeks. Western blot analysis of cardiac phosphorylated AKT and AKT (A), and quantitative analysis of relative density (B). (C) The measurement of cardiac AKT activity by commercial kit. (D–K) Primary cardiomyocytes (1 106) were incubated with 1 105 viral particles encoding siAkt for 48 h (#1 and #2), then treated with recombinant vaspin protein (2.5 μg/ml) for 24 h. Western blot analysis of phosphorylated GSK3β and GSK3β (D), and quantitative analysis of relative density (E). Western blot analysis of α-porin and PGC-1α (F), and quantitative analysis of relative density (G). (H–K) Measurement of relative ATP production (H), oxygen consumption (I), citrate synthase activity (J), and ratio of complex I/CS (K) in extracted cardiomyocyte mitochondria. Results are shown as mean ± SEM, and n = 6–8 mice/group or five independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.