GDF11 inhibits cardiomyocyte pyroptosis and exerts cardioprotection in acute myocardial infarction mice by upregulation of transcription factor HOXA3

Figures

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  Fig. 1. Downregulation of GDF11 expression in ischemic heart and hypoxic cardiomyocytes. A Western blot analysis of GDF11 protein level in mice hearts from sham and MI models. ***p < 0.001 vs sham; n = 10. B Real-time PCR results showing the GDF11 mRNA expression in mice hearts from sham and MI models. ***p < 0.001 vs sham; n = 5. C Western blot analysis of GDF11 protein level from NMCMs treated with and without hypoxia for 12 h. ***p < 0.001 vs control; n = 8. D Real-time PCR results showing the GDF11 mRNA expression from NMCMs treated with and without hypoxia for 12 h. ***p < 0.001 vs control; n = 8.

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  Fig. 2. GDF11 improves heart function in MI mice. A Echocardiogram and EF%, FS%, LVIDd, and LVIDs in the sham, MI, MI + AAV9-GDF11, and MI + AAV9-NC groups. **p < 0.01, ***p < 0.001 vs sham; #p < 0.05, ##p < 0.01 vs MI; &p < 0.05, &&p < 0.01 vs MI + AAV9-GDF11; n = 3 for EF% and FS%, n = 4 for LVIDd, n = 5 for LVIDs. B HE staining assesses histology of heart tissue. Scale bar indicates 50 μm; n = 3.

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  Fig. 3. GDF11 inhibits pyroptosis in ischemic heart. A Representative western blot bands for pyroptosis-associated proteins (NLRP3, ASC, c-caspase-1, GSDMD-N). B–E Western blot analysis of NLRP3 (B), ASC (C), c-caspase-1 (D), GSDMD-N (E) protein in the sham, MI, MI + AAV9-GDF11, and MI + AAV9-NC groups. GAPDH was used as an internal control. **p < 0.01 vs sham; ##p < 0.01 vs MI; &p < 0.05, &&p < 0.01, &&&p < 0.001 vs MI + AAV9-GDF11; n = 5. F, G IL-1β and IL-18 concentrations in serum. ***p < 0.001 vs sham; ##p < 0.01, ###p < 0.001 vs MI; &&&p < 0.001 vs MI + AAV9-GDF11; n = 4 for IL-1β, n = 7 for IL-18. H TEM micrograph of adult murine heart. Scale bar indicates 200 μm for the upper panels and 100 μm for the lower panels; n = 3.

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  Fig. 4. GDF11 inhibits pyroptosis in hypoxic cardiomyocytes. A Representative western blot bands for pyroptosis-associated proteins (NLRP3, ASC, c-caspase-1, GSDMD-N). Western blot analysis of NLRP3 (B), ASC (C), c-caspase-1 (D), GSDMD-N (E) protein in NMCMs. GAPDH was used as an internal control. **p < 0.01, ***p < 0.001 vs control; ##p < 0.01, ###p < 0.001 vs hypoxia; &p < 0.05, &&p < 0.01 vs hypoxia+GDF11; n = 5 for NLRP3, ASC and c-caspase-1, n = 6 for GSDMD-N. F Double-fluorescent staining with Hoechst 33342 (blue) and PI (red). Scale bar indicates 500 μm; n = 5. G CCK-8 examined GDF11-treated NMCMs cell viability. ***p < 0.001 vs control; ###p < 0.001 vs hypoxia; &&&p < 0.001 vs hypoxia+GDF11; n = 5.

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  Fig. 5. HOXA3 transcriptionally inactivates NLRP3 expression and indirectly regulates pyroptosis of cardiomyocytes. A Western blot analysis of HOXA3 protein level in mice hearts from sham and MI model. ***p < 0.001 vs sham; n = 10. B Real-time PCR results showing the HOXA3 expression in mice hearts from sham and MI model. ***p < 0.001 vs sham; n = 5. C Western blot analysis of HOXA3 protein level from NMCMs treated with and without hypoxia for 12 h. ***p < 0.001 vs control; n = 10. D Real-time PCR results showing the HOXA3 mRNA expression from NMCMs treated with and without hypoxia for 12 h. ***p < 0.001 vs control; n = 6. E ChIP assay was performed to analyze the binding of HOXA3 to the NLRP3 promoter; n = 3. F, G Western blot analysis of NLRP3 protein. **p < 0.01 vs control; ###p < 0.001 vs hypoxia; &&&p < 0.001 vs hypoxia+GDF11; n = 5. Western blot analysis of ASC (H), c-caspase-1 (I), and GSDMD-N (J) protein transfected with HOXA3 overexpression plasmid in the NMCMs with hypoxia. *p < 0.05, **p < 0.01 vs control; #p < 0.05, ##p < 0.01 vs hypoxia; &p < 0.05, &&p < 0.01, &&&p < 0.001 vs hypoxia+GDF11; n = 4 for c-caspase-1, n = 5 for ASC, n = 6 for GSDMD-N.

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  Fig. 6. GDF11 increases HOXA3 expression via TGF-β/Smad2/3 signaling. A Western blot analysis of HOXA3 protein. ***p < 0.001 vs hypoxia; ##p < 0.01 vs hypoxia+GDF11; &&&p < 0.001 vs hypoxia+GDF11 + SB505124; n = 5. B Western blot analysis of HOXA3 protein. **p < 0.01 vs GDF11; #p < 0.05 vs GDF11; n = 5.