Extracorporeal shock waves protect cardiomyocytes from doxorubicin-induced cardiomyopathy by upregulating survivin via the integrin-ILK-Akt-Sp1/p53 axis

Figures

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  Figure 1. Extracorporeal shock waves (ESW) upregulates survivin through the integrin/ILK/Akt signaling pathway in cardiomyocytes. (a) After treatment with 1,000 shots of ESW (0.04 mJ/mm2), H9c2 cells were incubated in a 5% CO2 incubator at 37 °C for the indicated time periods. The protein levels of p-Akt, Akt, survivin, and GAPDH were measured by Western blot. The protein expression levels were normalized to GAPDH (internal control), and are indicated relative to the control. The full-length blots are presented in Supplementary Fig. S2a. n = 5, Mean ± SEM. *Significant difference compared to control (p < 0.05). †Significant difference compared to the DOX condition (p < 0.05). (b–d) and (e) The cells were exposed to ESW after treatment with LY294002, cilengitide, or Cpd22 at the indicated concentrations for 2 h or left untreated. The cells were harvested after a 30 min or 24 h incubation under a static condition. The protein expression levels of p-Akt, Akt, survivin, and GAPDH were measured by Western blot and normalized to GAPDH (internal control), and are indicated relative to those of the control. Representative images from at least three independent results are shown. The full-length…

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  Figure 2. ESW protects cardiomyocytes from doxorubicin (DOX)-induced cell death. H9c2 cells were left untreated or treated with 1,000 shots of ESW (0.04 mJ/mm2), and then statically incubated in a 5% CO2 incubator for 1 h. After the incubation, cells were treated with 1 μM DOX for 24 h. (a) Cell viability under each condition was measured by the MTT assay. The values of all conditions are indicated relative to the control. n = 5, Mean ± SEM. *Significant difference compared to control (p < 0.05). †Significant difference compared to the DOX condition (p < 0.05). (b) The bar graph shows the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cell ratio (%) as a cellular apoptotic index. The TUNEL-stained cells (green) were counted and normalized by the DAPI-stained cells (blue): control (0.08833 ± 0.009458, means ± standard error), DOX (20.17 ± 0.6499, means ± standard error) and ESW + DOX (6.516 ± 2.644, means ± standard error). n = 5, Mean ± SEM. *Significant difference compared to control (p < 0.05). †Significant difference compared to DOX condition (p < 0.05). (c) Representative fluorescence microscopic TUNEL images from at least three independent experiments…

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  Figure 3. ESW inhibits DOX-induced downregulation of survivin via the integrin/ILK/Akt pathway in cardiomyocytes. (a,b) H9c2 cells were cultured in 1 μM DOX for 8 or 24 h 1 h after ESW. The protein expression levels of p-Akt, Akt, survivin, and GAPDH were measured by Western blot and normalized to GAPDH (internal control). The expression levels are indicated relative to those of the control. The full-length blots are presented in Supplementary Fig. S7a,b. (c) After treatment with 50 μM LY294002 for 2 h, the cells were exposed to ESW or left unexposed. After a 1 h incubation, the cells were treated with 1 μM DOX for 24 h. The full-length blots are presented in Supplementary Fig. S7c. (d,e) After treatment with 2 μM cilengitide or Cpd22 for 2 h, the cells were exposed to ESW or left unexposed. After a 1 h incubation, the cells were cultured in 1 μM DOX for 24 h. The protein expression levels of survivin and GAPDH were measured by Western blot and normalized to GAPDH (internal control). The expression levels are indicated relative to the control. The full-length blots are presented in Supplementary Fig. S8. n = 5, Mean ± SEM. *Significant difference compared to control (p < 0.05).…

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  Figure 4. Knockdown of survivin alleviates the cardioprotective effect of ESW in DOX-induced cell death. After transfection with 50 nM siRNA targeting survivin for 24 h, H9c2 cells were subjected to ESW 1 h prior to the DOX treatment. The cells were harvested after a 24 h incubation. (a) The protein expression levels of survivin, Bcl2, and GAPDH were measured by Western blot and normalized to GAPDH (internal control). The expression levels are indicated relative to those of the control. The full-length blots are presented in Supplementary Fig. S10. (b) Cell viability of each condition was measured by the MTT assay. The values for all conditions are indicated relative to those of the control. (c) The bar graph shows the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cell ratio (%) as a cellular apoptotic index. The TUNEL-stained cells (green) were counted and normalized by the DAPI-stained cells (blue): control (0.05893 ± 0.03628, means ± standard error), DOX (6.212 ± 0.6617, means ± standard error) and ESW + DOX (3.331 ± 0.6028, means ± standard error), siRNA + ESW + DOX (6.149 ± 0.7767, means ± standard error). n = 5, Mean ± SEM. *Significant difference…

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  Figure 5. Phosphorylation of Akt by ESW induces survivin by downregulating p53 and upregulating Sp1. The cells were cultured in 1 μM DOX for 24 h after being subjected or not to ESW. (a) The protein expression levels of Sp1, p53, and GAPDH were measured by Western blot and normalized to GAPDH (internal control). The expression levels are indicated relative to those of the control. The full-length blots are presented in Supplementary Fig. S14a. (b) Cytoplasmic and nuclear extracts isolated from H9c2 cells under each condition were separated by Western blot with antibodies against Sp1, p53, GAPDH, and lamin A/C and are indicated relative to those of the control. GAPDH and lamin A/C were used as the loading controls for the cytosolic and nuclear fractions, respectively. The full-length blots are presented in Supplementary Fig. S14b. (c) After treatment with 50 μM LY294002 for 2 h, the cells were exposed to ESW or left unexposed. After a 1 h incubation, the cells were treated with 1 μM DOX for 24 h. The full-length blots are presented in Supplementary Fig. S15. n = 5, Mean ± SEM. *Significant difference compared to the control (p < 0.05). †Significant difference compared to the DOX condition…

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  Figure 6. ESW improves acute DOX-induced cardiomyopathy, while inhibition of survivin reverses the cardioprotective effect of ESW in an in vivo mouse model. (a) Representative M-mode images from the control, YM155, DOX ESW + DOX, and YM155 + ESW + DOX groups. (b) Quantitative echocardiographic group data: LVEDD (mm), LVESD (mm), FS (%) and EF (%). n = 8/group, Mean ± SEM. *Significant difference compared to control (p < 0.05). †Significant difference compared to the DOX condition (p < 0.05). ‡Significant difference compared to the ESW + DOX group (p < 0.05).

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  Figure 7. ESW attenuates cardiomyocyte apoptosis by upregulation of phosphorylation of Akt and survivin followed by upregulation of Bcl2 in an in vivo mouse model. (a) Immunofluorescence staining to detect Bcl2 and survivin in various regions of the mice myocardium. After fixation, the tissues were stained with anti-Bcl2 antibody (yellow) and anti-survivin antibody (red), while nuclei were stained with DAPI (blue). The levels of Bcl2 and survivin expression were compared among the three groups (control, YM155, DOX, ESW + DOX, and YM155 + ESW + DOX) using confocal microscopy. Representative images are shown (magnification 400×; scale bars, 20 μm). (b) The hearts were harvested at 14 days after the DOX injection and examined by Western blot for p-Akt, Akt, survivin, Bcl2, and GAPDH. The expression levels were normalized to GAPDH (internal control) and are indicated relative to those of the control (n = 8/group). Representative images from at least three independent results are shown. *Significant difference compared to control (p < 0.05). †Significant difference compared to the DOX condition (p < 0.05). ‡Significant difference compared to the ESW + DOX group (p < 0.05). The English in this…