USP16 S-nitrosylation aggravates coronary microembolization-induced myocardial injury via repressing KDM1A-mediated glutathione homeostasis

Figures

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  Fig. 1. Cardiac dysfunction and GSH imbalance in CME rats. A The flowchart for the establishment of the CME model in rats. B Cardiac function was evaluated using echocardiography. P = 0.0010 for EF, P = 0.0012 for FS, P = 0.0012 for LVIDd, P = 0.0006 for LVIDs. C Serum CK-MB level was assessed using ELISA. P = 0.0010. D The myocardial microinfarct size was determined using HBFP staining (scale bar = 200 μm, 50 μm). The black arrow indicates a microsphere. P < 0.0001. E The pathological alterations in the hearts were evaluated using HE staining (scale bar = 200, 50 μm). The black arrow indicates a microsphere, and the red arrow points out aggregated inflammatory cells (leukocytes), which are manifested as inflammatory infiltration. P < 0.0001. F Apoptosis in the myocardial tissues of CME rats was observed by TUNEL (scale bar = 100, 50 μm). P < 0.0001. G The flowchart for the process of GSH synthesis. H The expression of GCLM, GLS, GSR, GSS, and GDH in the myocardial tissues was assessed using immunohistochemical staining (scale bar = 100, 50 μm). P = 0.0001 for GCLM and P = 0.0001 for GLS. I GSH and GSSG contents in heart tissues were measured to calculate the GSH/GSSG ratio. P = 0.0001.…

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  Fig. 2. GCLM/GLS overexpression maintains GSH homeostasis in hypoxia-challenged cardiomyocytes. A The GSH/GSSG ratio in cardiomyocytes exposed to hypoxia for 12, 24, and 48 h was measured. Detailed P values see (A). B ROS level in cardiomyocytes after stimulation with hypoxia for 12, 24, and 48 h was detected using DCFH-DA staining. Detailed P values see (B). C The protein levels of GCLM, GLS, GSR, GSS, and GDH in hypoxia-stimulated cardiomyocytes were determined using western blotting. Detailed P values see (C). D Overexpression efficiency of GCLM and GLS in cardiomyocytes was confirmed using western blotting. GCLM or GLS-overexpressed cardiomyocytes were subjected to hypoxia for 24 h. P = 0.0022 for GCLM and P = 0.0016 for GLS in H9c2 cells, and P = 0.0022 for GCLM and P = 0.0125 for GLS in NRVM cells. E The GSH/GSSG ratio was calculated. Detailed P values see (E). F ROS level was measured using DCFH-DA staining. Detailed P values see (F). G Western blotting analysis of GCLM and GLS protein abundance. Detailed P values see (G). Data are presented as mean ± standard deviation (SD). n = 4 represents four independent biological repetitions, and each biological repetition contains three…

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  Fig. 3. KDM1A overexpression improves cardiac dysfunction and GSH imbalance in CME rats in vivo. A KDM1A expression in myocardial tissues was evaluated using immunohistochemical staining (scale bar = 100, 50 μm). P < 0.0001. B Cardiac function was determined using echocardiography. Detailed P values see (B). C Serum CK-MB level was measured using ELISA. Detailed P values see (C). D HBFP staining analyzed the microinfarct size in the hearts (scale bar = 100, 50 μm). The black arrow indicates a microsphere. Detailed P values see (D). E The pathological alterations in the myocardial tissues were observed by HE staining (scale bar = 100, 50 μm). The black arrow indicates a microsphere, and the red arrow indicates inflammatory cell infiltration. Detailed P values see (E). F Apoptosis in the myocardial tissues was assessed using TUNEL (scale bar = 100, 50 μm). Detailed P values see (F). G The expression of KDM1A, GCLM, and GLS in the myocardial tissues was measured using immunohistochemical staining (scale bar = 100, 50 μm). Detailed P values see (G). H The GSH/GSSG ratio in the heart tissues was calculated. Detailed P values see (H). I DCFH-DA staining detected the ROS level in the myocardial…

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  Fig. 4. KDM1A overexpression alleviates CME-induced GSH imbalance in cardiomyocytes. A KDM1A expression in cardiomyocytes stimulated with hypoxia for 12, 24, and 48 h was assessed using western blotting. Detailed P values see (A). B Western blotting analysis of KDM1A, GCLM, and GLS protein levels in cardiomyocytes after KDM1A overexpression. KDM1A-overexpressed cardiomyocytes were stimulated with hypoxia for 24 h. P = 0.0004 for KDM1A, P = 0.0069 for GCLM, and P = 0.0110 for GLS in H9c2 cells, and P = 0.0002 for KDM1A, P = 0.0061 for GCLM, and P = 0.0200 for GLS in NRVM cells. C The GSH/GSSG ratio was calculated. Detailed P values see (C). D ROS production was assessed using DCFH-DA staining. Detailed P values see (D). E Western blotting analysis of KDM1A, GCLM, and GLS protein levels. Detailed P values see (E). Data are presented as mean ± standard deviation (SD). n = 4 represents four independent biological repetitions, and each biological repetition contains three technological repetitions. In A, B, D, E, experiments were repeated independently at least 4 times with similar results. Representative images from one experiment are shown. Unpaired two-tailed Student’s t-test (for B) and…

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  Fig. 5. KDM1A facilitates transcriptional activation of GCLM and GLS. A Western blotting analysis of KDM1A, GCLM, and GLS protein levels in shKDM1A-transfected cardiomyocytes. P = 0.0006 for KDM1A, P = 0.0175 for GCLM, and P = 0.0074 for GLS in H9c2 cells, and P = 0.0042 for KDM1A, P = 0.0061 for GCLM. and P = 0.0204 for GLS in NRVM cells. B AnimalTFDB database predicted the binding sites of KDM1A to GCLM and GLS promoters. C The interplay between KDM1A to GCLM and GLS promoters was verified using ChIP. P < 0.0001 for BS2 in GCLM promoter, P = 0.0006 for BS1 in GCLM promoter from H9c2 cells, and P = 0.0002 for BS2 in GCLM promoter, P = 0.0052 for BS1 in GCLM promoter from NRVM cells; P < 0.0001 for BS2 in GLS promoter from H9c2 cells and P = 0.0038 for BS2 in GLS promoter from NRVM cells. D The binding of KDM1A to GCLM and GLS promoters in shKDM1A-transfected cardiomyocytes was evaluated using ChIP. P = 0.0008 for BS2 in GCLM promoter, P = 0.0087 for BS1 in GCLM promoter from H9c2 cells, and P = 0.0030 for BS2 in GCLM promoter, P = 0.0103 for BS1 in GCLM promoter from NRVM cells; P = 0.0024 for BS2 in GLS promoter from H9c2 cells and P = 0.0015 for BS2 in GLS promoter from NRVM cells.…

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  Fig. 6. USP16 stabilizes KDM1A in the in vitro CME model via deubiquitination. A Hypoxia-stimulated cardiomyocytes were treated with MG132 or CQ, and KDM1A protein abundance was detected using western blotting. Detailed P values see (A). B Co-IP analyzed the ubiquitination level of KDM1A in hypoxia-exposed cardiomyocytes. C The ubiquitination level of KDM1A in the heart tissues of CME rats was evaluated using Co-IP. D E3 ligases and deubiquitinases interacting with KDM1A were analyzed using mass spectrometry. E The protein abundance of USP16 and RNF138 in hypoxia-challenged cardiomyocytes was assessed using western blotting. Detailed P values see (E). F Western blotting analysis of KDM1A and USP16/RNF138 expression in cardiomyocytes transfected with shUSP16-1/2 or shRNF138-1/2. Detailed P values see (F). G The interplay between KDM1A and USP16/RNF138 was validated by Co-IP. Data are presented as mean ± standard deviation (SD). n = 4 represents four independent biological repetitions, and each biological repetition contains three technological repetitions. In A–C, E–G, experiments were repeated independently at least 4 times with similar results. Representative images from one experiment…

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  Fig. 7. KDM1A is ubiquitinated at the K355 site in hypoxia-exposed cardiomyocytes. A The potential ubiquitination sites for KDM1A were determined using mass spectrometry. B H9c2 cells were transfected with KDM1A-WT or KDM1A-K355R and then subjected to hypoxia for 24 h in the presence of MG132. Co-IP analyzed KDM1A ubiquitination level. H9c2 cells were transfected with KDM1A-WT or KDM1A-K355R together with shNC or shUSP16, followed by hypoxia for 24 h. C KDM1A ubiquitination level was evaluated using Co-IP. D The GSH/GSSG ratio was calculated. Detailed P values see (D). E ROS production was analyzed using DCFH-DA staining. Detailed P values see (E). F Western blotting analysis of KDM1A, GCLM, and GLS protein levels. Detailed P values see (F). Data are presented as mean ± standard deviation (SD). n = 4 represents four independent biological repetitions, and each biological repetition contains three technological repetitions. In B, C, E, F, experiments were repeated independently at least 4 times with similar results. Representative images from one experiment are shown. One-way ANOVA followed by Tukey’s test for multiple group comparison (for D–F) was performed to analyze data. Source data…

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  Fig. 8. Repression of SNO-USP16 at the C731 site restores GSH homeostasis in hypoxia-stimulated cardiomyocytes. A GPS-SNO database predicted the SNO sites for USP16. H9c2-KO cells with sgUSP16 under normal or hypoxic conditions were transfected with USP16-WT or USP16-C731A. B Biotin-switch assay detected SNO-USP16 level in H9c2 cells. Detailed P values see (B). C The GSH/GSSG ratio was calculated. Detailed P values see (C). D ROS level was determined using DCFH-DA staining. Detailed P values see (D). E Western blotting analysis of KDM1A, GCLM, and GLS protein levels. Detailed P values see (E). Data are presented as mean ± standard deviation (SD). n = 4 represents four independent biological repetitions, and each biological repetition contains three technological repetitions. In B, D–E, experiments were repeated independently at least 4 times with similar results. Representative images from one experiment are shown. One-way ANOVA followed by Tukey’s test for multiple group comparison (for B–E) was performed to analyze data. Source data are provided as a Source data file. SNO S-nitrosylation, USP16 ubiquitin-specific peptidase 16, GSH glutathione, GCLM glutamate-cysteine ligase modifier…

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  Fig. 9. Inhibition of SNO-USP16 at the C731 site improves cardiac dysfunction and GSH imbalance in CME rats. A Cardiac function was evaluated using echocardiography. Detailed P values see (A). B Serum CK-MB level was measured using ELISA. Detailed P values see (B). C HBFP staining evaluated the microinfarct size in the heart tissues (scale bar = 100, 50 μm). The black arrow indicates a microsphere. Detailed P values see (C). D The pathological changes in the myocardial tissues were determined using HE staining (scale bar = 100, 50 μm). The black arrow indicates a microsphere, and the red arrow indicates inflammatory cell infiltration. Detailed P values see (D). E Apoptosis in the myocardial tissues was measured using TUNEL (scale bar = 100, 50 μm). Detailed P values see (E). F The expression of USP16, KDM1A, GCLM, and GLS in the myocardial tissues was analyzed using immunohistochemical staining (scale bar = 100, 50 μm). Detailed P values see (F). Data are presented as mean ± standard deviation (SD). n = 9 rats per group, indicating nine biological repetitions. In A, C–F, experiments were repeated independently at least 9 times with similar results. Representative images from one…

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  Fig. 10. SNO-USP16 at the C731 site is responsible for hypoxia-induced KDM1A ubiquitination. H9c2-KO cells with sgUSP16 under normal or hypoxic conditions were transfected with USP16-WT or USP16-C731A. A Co-IP evaluated the interaction between USP16 and KDM1A proteins. B The protein level of KDM1A was assessed using western blotting. Detailed P values see (B). C The stability of KDM1A in H9c2 cells was detected using western blotting, P = 0.0062 for 0 min, P = 0.0009 for 30 min, P = 0.0004 for 60 min, P = 0.0004 for 120 min, P = 0.0322 for 240 min. D The ubiquitination level of KDM1A was determined using Co-IP. Data are presented as mean ± standard deviation (SD). n = 4 represents four independent biological repetitions, and each biological repetition contains three technological repetitions. In A–D, experiments were repeated independently at least 4 times with similar results. Representative images from one experiment are shown. One-way ANOVA followed by Tukey’s test for multiple group comparison (for B) and unpaired two-tailed Student’s t-test (for C) and were performed to analyze data. Source data are provided as a Source data file. SNO S-nitrosylation, USP16 ubiquitin-specific…