Hsp20-Mediated Activation of Exosome Biogenesis in Cardiomyocytes Improves Cardiac Function and Angiogenesis in Diabetic Mice
Article Figures & Tables
Figures
- Figure 1
Hsp expression profiles in acute and chronic STZ-treated murine hearts. A: A diagram of STZ treatment in mice and sample collection for biochemical assays. B: Blood glucose concentration was measured in mice at 10 days or 3 months after the first injection of STZ or buffer. C: Body weight increases were measured in mice at 3 months after the first injection of STZ or buffer (n = 4 for buffer-treated group and n = 7 for STZ-treated group). *P < 0.05. Representative Western blots (D) and quantitative results (E) showed that the distinct expression pattern of various Hsps in acute and chronic STZ-treated mouse hearts. α-Actin was used as a loading control (n = 4). *P < 0.05 vs. controls. Ctl, control; wk, week.
- Figure 2
Cardiac-specific overexpression of Hsp20 attenuates STZ-induced cardiac dysfunction and remodeling. A and B: Western blotting results showed that Hsp20 was overexpressed by 10-fold in TG mouse hearts. α-Actin was used as a loading control (n = 4). *P < 0.05 vs. WT. C–F: STZ-induced cardiac depression in WT mice, measured by echocardiography (C), was significantly improved in Hsp20-TG mice (n = 5 for buffer-treated WT or TG group and n = 9 for STZ-treated WT or TG). *P < 0.05 vs. WT/buffers; #P < 0.05 vs. WT/STZ. G–K: Hsp20 overexpression protected mice against STZ-induced adverse remodeling. Cardiomyocyte hypertrophy, determined by WGA staining, was exhibited in STZ-treated WT hearts, but not in TG (G and H). Triple staining with anti–α-sarcomeric actin antibody, DAPI, and TUNEL to determine cardiomyocyte apoptosis in STZ-treated WT or TG hearts (arrows indicate TUNEL-positive nuclei) (G and I). iB4 staining (G and J) and RT-PCR analysis of CD31 (K) to detect myocardial microvessel density (F and J) (n = 5 hearts, with two sections from each heart). Scale bars: 100 μm. *P < 0.05 vs. WT/buffers; #P < 0.05 vs. WT/STZ. Ctl, control.
- Figure 3
Hsp20 promotes exosome biogenesis via interacting with Tsg101. A: Diagram of exosome biogenesis/release pathway. B: Overexpression of Hsp20 in the heart increased the expression levels of major mediators involved in exosome generation (n = 3). *P < 0.05 vs. WT. α-Actin was used as a loading control. C: Cardiomyocytes isolated from adult WT or TG mouse hearts were successfully cultured. D: The exosome concentration was increased in the culture supernatants of TG cardiomyocytes (n = 3 wells). *P < 0.05 vs. WT. Similar results were observed from three different hearts. E: A scheme for protein-protein interaction analysis. F: Recombinant protein Tsg101 coated on a plate dose-dependently captured the Hsp20 protein, whereas Rab11a/b, Rab35, and control protein BSA coated did not effectively arrest the Hsp20 protein. G and H: Coimmunoprecipitation of heart homogenates with Hsp20 antibody or Tsg101 antibody showed that Hsp20 directly interacted with Tsg101. Ab, antibody; CM, cardiomyocyte; IP, immunoprecipitation; OD, optical density.
- Figure 4
Exosomes derived from Hsp20-overexpressing cardiomyocytes protect myocardial endothelial cells and cardiomyocytes against HG-induced stress conditions. The size of exosomes derived from WT cardiomyocytes (A) and Hsp20-TG cardiomyocytes (B), measured using a Zetasizer Nano ZS instrument. C: Protein levels of CD63 and CD81, two exosome markers, were similarly encased in WT-Exo and TG-Exo. D: Effects of WT-Exo (collected from the supernatants of WT cardiomyocytes) and TG-Exo (collected from the supernatants of TG cardiomyocytes) on HG-induced endothelial cell growth (n = 6 wells). *P < 0.05 vs. control NG conditions; #P < 0.05 vs. cells treated with WT-Exo under NG conditions; $P < 0.05 vs. control HG conditions; &P < 0.05 vs. cells treated with WT-Exo under HG conditions. Similar results were observed in three additional, independent experiments. Representative endothelial cells that were transwelled and formed tube-like structure when exposed to WT-Exo or TG-Exo (E) and the quantitative results of migrated endothelial cells (F) and tube length formed (G) (n = 3 wells). *P < 0.05 vs. control; #P < 0.05 vs. WT-Exo group. Similar results were observed in three additional, independent experiments. H: Effects of WT-Exo and TG-Exo on HG-induced cardiomyocyte death (n = 3 wells). *P < 0.05 vs. NG conditions; #P < 0.05 vs. cells treated with WT-Exo under HG conditions. Similar results were observed in two additional, independent experiments. CM, cardiomyocyte; Ctl, control; dm, diameter; EC, endothelial cell.
- Figure 5
Altered exosomal contents by overexpression of Hsp20 in cardiomyocytes could be delivered to endothelial cells and native cardiomyocytes. Representative Western blots (A) and their quantitative results (B) showed that higher levels of Hsp20, survivin, and p-Akt encased in TG-Exo than WT-Exo. C: SOD1 levels were increased in TG-Exo, compared with WT-Exo (n = 4 for A–C). *P < 0.05 vs. WT-Exo. D: Green dye–labeled exosomes were detectable in the cytosol of MCECs after exposure to green dye PKH67-labeled WT- and TG-Exo, respectively. E and F: Exosomal Hsp20, p-Akt, and survivin were effectively transported to endothelial cells, compared with control conditions (n = 4). *P < 0.05 vs. control conditions; #P < 0.05 vs. WT-Exo–treated samples. β-Actin was used as a loading control. EC, endothelial cell.
- Figure 6
Hsp20 exosomes suppress HG-induced oxidative stress in endothelial cells and cardiomyocytes. The amount of SOD1 was increased in exosome-treated endothelial cells (A) and cardiomyocytes (B) under normal conditions. HG-induced decrease in the amount of SOD1 was improved to a greater degree in TG-Exo–treated cells than WT-Exo–treated samples (A and B). By contrast, HG-triggered increase of ROS levels was attenuated to a greater degree in TG-Exo–treated endothelial cells (C)/cardiomyocytes (D) than WT-Exo–treated samples (C and D) (n = 3 wells). *P < 0.05 vs. control NG conditions; #P < 0.05 vs. cells treated with WT-Exo under NG conditions; $P < 0.05 vs. control HG conditions; &P < 0.05 vs. cells treated with WT-Exo under HG conditions. Data shown are representative of three separate experiments. CM, cardiomyocyte; EC, endothelial cell.
- Figure 7
Hsp20-mediated cardioprotective effects are largely offset by blockade of the exosome generation. A: A scheme of the experimental procedure for the treatment of mice with STZ and GW4869. B: Exosome concentration was measured in the serum of mice treated with GW4869 (n = 5). *P < 0.05 vs. WT; #P < 0.001 vs. DMSO-treated control groups. C: Representative echocardiographic images. LVIDd (D) and LVEF% (E) were calculated in various groups as indicated (n = 5 for buffer-treated groups and n = 7 for STZ-treated groups). *P < 0.05 vs. STZ/DMSO-treated WT. Hsp20-mediated attenuation of cardiomyocyte hypertrophy (F and G), cardiac apoptosis (H and I, arrows indicate TUNEL-positive nuclei), and Hsp20-mediated promotion of myocardial angiogenesis (J and K) were significantly limited by the GW4869 treatment (n = 5 hearts, with two sections from each heart). Scale bars: 100 μm. *P < 0.05 vs. STZ/DMSO-treated WT. Ctl, control; Mon, month.
- Figure 8
Injection of Hsp20-enriched exosomes collected from TG cardiomyocytes protects mice against STZ-induced cardiac adverse remodeling. A: DiR-labeled cardiomyocyte-derived exosomes were detectable in mouse cardiomyocytes 1 h after the tail-vein injection in vivo. The right image is magnified from insert square of A. B and C: Exosomal Hsp20 was dose-dependently delivered to the mouse heart after the tail-vein injection. D and E: The time course determination of cardiac Hsp20 levels in TG-Exo–injected mice. F: A scheme of the experimental procedure for the exosome injection in STZ-treated mice. G and H: LVIDd and LVEF% were significantly improved in TG-Exo–injected diabetic mice (n = 5–8). *P < 0.05 vs. buffer/PBS group; #P < 0.05 vs. STZ/PBS group. Full echocardiographic data are listed in Supplementary Table 3. I: Representative TUNEL staining images (red, α-sarcomeric actin for cardiomyocytes; blue, DAPI for nuclear staining; green, apoptotic nuclear; arrows indicate TUNEL-positive nuclei) and quantification results (J) (n = 5 hearts, two sections per heart). *P < 0.05 vs. buffer/PBS group; #P < 0.05 vs. STZ/PBS group. K: Representative merged images of WGA staining (cardiomyocytes, green) and iB4 staining (blood vessels, red). L: The quantified density of myocardial blood microvessels (n = 5 hearts, two sections per heart, detected at low magnification, ×200) (L) and further confirmed by RT-PCR analysis for CD31 expression (n = 5) (M). *P < 0.05 vs. buffer/PBS group; #P < 0.05 vs. STZ/PBS group. D, day; W and wk, week.
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