Uncoupling Protein 2 Alleviates Myocardial Ischemia/Reperfusion Injury by Inhibiting Cardiomyocyte Ferroptosis

Figures

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  Fig. 1.. Upregulation of UCP2 in I/R-induced mouse hearts. a Experimental process. b ECGs before ischemia, after ischemia, and after reperfusion (n = 6 per group). c Protein level of UCP2 in myocardial tissue after I/R injury in WT mice (n = 3 per group). Top: optical density of the protein blot showing UCP2 versus GAPDH; bottom: representative image of the Western blotting results. d Relative mRNA expression of Ucp2 in myocardial tissue after I/R injury in WT mice (n = 6 per group). e Protein levels of UCP2 in the myocardial tissue of WT and Ucp2−/− mice (n = 3 per group). f mRNA expression of Ucp2 in the myocardial tissue of WT and Ucp2−/− mice (n = 6 per group). The values are presented as the mean ± SD. **p < 0.01 versus sham.

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  Fig. 2.. Knockout of Ucp2 exacerbates myocardial I/R injury. a–c Cardiac function was evaluated by ECG after I/R injury. d, e LDH and CK activities after I/R injury. The values are presented as the mean ± SD (n = 6 per group). *p < 0.05 versus WT + I/R group; #p < 0.05, ##p < 0.01 versus Ucp2−/− + I/R group; +p < 0.05, ++p < 0.01 versus WT.

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  Fig. 3.. Knockout of Ucp2 exacerbates myocardial I/R injury. a, b Representative images of TTC staining 2 h after reperfusion. c Representative images of H&E staining after I/R injury (scale bar = 40 μm). d, e Representative images of Masson’s trichrome staining of heart tissues after 4 weeks of reperfusion (scale bar = 40 μm). f, g Representative images of Sirius red-stained heart tissues after 4 weeks of reperfusion (scale bar = 40 μm). The values are presented as the mean ± SD (n = 6 per group). *p < 0.05, **p < 0.01 versus WT + I/R group; #p < 0.05, ##p < 0.01 versus Ucp2−/− + I/R group; +p < 0.05, ++p < 0.01 versus WT.

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  Fig. 4.. Knockout of Ucp2 exacerbates ferroptosis in I/R-induced mouse hearts. a, b Total iron and Fe2+ levels (n = 6 per group). c–f Detection of LPO, MDA, SOD, and GSH (n = 6 per group). g–k Protein expression levels of the ferroptosis-related biomarkers FTH1, TF, ACSL4, and GPX4 (n = 3 per group) (the internal reference was GAPDH). l–o Gene expression levels of the ferroptosis-related biomarkers Fth1, Tf, Acsl4, and Gpx4 (n = 6 per group). The values are presented as the mean ± SD. *p < 0.05, **p < 0.01 versus WT + I/R group; #p < 0.05, ##p < 0.01 versus Ucp2−/− + I/R group; +p < 0.05, ++p < 0.01 versus WT.

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  Fig. 5.. Knockout of Ucp2 exacerbates ferroptosis in I/R-induced mouse hearts. a, b The protein expression of GPX4 was assessed by immunofluorescence analysis. c, d The protein expression of ACSL4 was assessed by immunofluorescence analysis. e, f ROS analysis. Scale bar, 50 μm for all images. The values are presented as the mean ± SD (n = 6 per group). *p < 0.05, **p < 0.01 versus the WT + I/R group; #p < 0.05, ##p < 0.01 versus the Ucp2−/− + I/R group; +p < 0.05, ++p < 0.01 versus the WT group.

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  Fig. 6.. Knockout of Ucp2 activates p53 and TfR1 protein and mRNA expression in I/R-induced mouse hearts. a–c The protein expression of p53 and TfR1 was assessed by Western blotting (n = 3 per group) (the internal reference is GAPDH). d, e The gene expression of p53 and Tfr1 was determined by RT-qPCR (n = 6 per group). The values are presented as the mean ± SD. *p < 0.05, **p < 0.01 versus the WT + I/R group; #p < 0.05, ##p < 0.01 versus the Ucp2−/− + I/R group; +p < 0.05, ++p < 0.01 versus the WT group.